Decoy insulin receptor controls Drosophila body size

(Front) Yuki YAMAUCHI, Rinna NAKAMORI(Middle) Junko SHINNO, Tomoka MURAI, Kanako HIGUCHI, Noriko NISHIMURA(Back) Takashi NISHIMURA, Yuichiro KASHIHARA, Tomoyuki TAKAHASHI, Naoki OKAMOTO, Takayuki YAMADA

Insulin and insulin-like growth factors (IGFs) are known toplay important roles in growth and metabolism. These hormones, which are conserved from insects to mammals, circulate in the blood and bind with specific cell surface receptors, triggering downstream signals within the cell.
Although Insulin and IGFs are essential for regulating growthand metabolism, how these secreted molecules are regulated in the circulating blood has remained something of apuzzle.
Naoki Okamoto and colleagues in the Lab for Growth Control Signaling (Takashi Nishimura, Team Leader) have fitted
a major new piece into this jigsaw, showing how a secreted decoy insulin receptor acts as a negative regulator of in-10

sulin/IGF signaling in Drosophila, thus controlling growth.
This work is published in Genes & Development.
In vertebrates, secreted IGFs associate with IGF-binding
proteins (IGFBPs) in the bloodstream, which stabilizes the
IGFs and prevents them from binding with cell surface receptors. In the fly, Drosophila insulin-like peptides (Dilps)
play the roles of both insulin and the IGFs in vertebrates.
Previous reports have shown that the IGFBP-like protein
Imp-L2 binds directly to several Dilps in the hemolymph,
thereby regulating insulin/IGF signaling. But despite theneed to keep such a crucial pathway under tight control, noother molecules capable of regulating Dilps have been reported in Drosophila.

Downloaded from genesdev.cshlp.org on February 3, 2013 - Published by Cold Spring Harbor Laboratory Press©2013 Cold Spring Harbor Laboratory Press
Okamoto N, et al. A secreted decoy of InR antagonizes insulin/IGF signaling to
restrict body growth in Drosophila. Genes Dev 27.87-97 (2013)

A secreted decoy of InR antagonizes insulin/IGF signaling to
restrict body growth in Drosophila
Naoki Okamoto, Rinna Nakamori, Tomoka Murai, et al.
Genes Dev. 2013 27: 87-97
Access the most recent version at doi:10.1101/gad.204479.112

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The Nishimura lab set out to identify novel molecules involved in developmental growth regulation by screening secreted protein RNAi knockdown phenotypes for effects onbody size, and identified a gene whose loss of function resulted in a higher growth rate in larval stages and larger
adult body sizes. This new gene included a sequence closely
similar to that of the insulin receptor (InR) extracellular domain, which, along with its functional attributes, led them toname it secreted decoy of InR (SDR). SDR mutants showedno defects in survival or fertility, but grew about 20% larger
than wildtype flies. Close observation of wings in adult mutants revealed that the change in overall size was a result
both of greater numbers of cells and increased cell size.
SDR has neither a transmembrane nor a cytoplasmic domain, pegging it as a likely secreted protein, which Okamoto et al confirmed by detecting high levels of the factor inhemolymph. The team next looked at gene expression patterns and conditional RNAi phenotypes in various tissues
and found that it was highly expressed in the glial cells of
the brain. After deciphering where SDR was made, the next
step was to find out how it worked. Using the localization ofdFOXO, which responds to insulin/IGF signaling on/off
cues, as an index, they found that insulin/IGF signaling was
accelerated in SDR mutants, and conversely inhibited on its
overexpression. In vitro assays further showed direct bind-ing between SDR and several Dilps, suggesting that its
mode of action involves antagonistic binding with these Dilps, preventing their association with receptors.
Other insulin-linked molecules are known to respond tochanges in nutritional status. Looking at starved flies, inwhich the expression of some Dilps is down-regulated andthat of Imp-L2 increased, a mechanism though to allow the
body to sacrifice growth in favor of storing nutrients, theyfound that SDR expression and secretion remained unexpectedly stable under low-nutrition conditions. But when
SDR mutants were bred under extremely adverse conditions where food availability was only one-tenth normal, thefailure of growth inhibition led to poor survival, with over halfof the flies dying during metamorphosis, suggesting that
SDR is an essential safety mechanism for controlling insulin/IGF signaling under poor nutritional conditions.
“Although the SDR gene is found only in insects, similar decoy systems for Insulin/IGF signaling are likely present inother species. Recent observations have indicated thatmammalian insulin receptor can potentially produce a soluble decoy,” says Nishimura. “Our initial focus was on the roleof SDR in growth regulation, but from now we will be interested to learn more about its functions in other phenomena, such as metabolism and aging.”

Cartoon of SDR mode of action (left), shows binding to Dilps in hemolymph, preventing association with its membrane
receptors. SDR mutant phenotypes (right) showing changes in body size; loss of function leads to larger sizes,
overexpression to smaller.

11Mitotic cell rounding as a driver of morphogenesis

Takefumi KONDO

The inward folding, or invagination, of epithelial tissues is an
important morphogenetic phenomenon, converting flat sheetsof cells into three-dimensional tissues. This process is often, but not always, associated with apical constriction of
the actin cytoskeleton and produces indented hollows inepithelial surfaces, such as the placodes that give rise to
sensory and other organs. Cells entering mitosis have generally been thought to be incapable of undergoing the intensive cell shape remodeling that underlies these processes, but as some tissues in which invagination takes placeshow signs of cell cycle activity, leaving open the questionof how these two phenomena might interact.
Takefumi Kondo of the Laboratory for Morphogenetic Signaling (Shigeo Hayashi, Group Director) has now shown

12

that a process known as mitotic rounding drives invagination of the tracheal placode in the fruit fly, Drosophila melanogaster. This work, published in Nature, shows that mitosis plays an unsuspected role in this form of developmental
tissue remodeling.
The apical constriction of epithelial cells, in which the apicalregions of cells shrinks giving them a roughly conic structure, has long been thought to be the driving force behindinvagination. But a number of tantalizing exceptions inwhich inward folding occurs without such constriction suggested that some other dynamic must also be involved. Using live imaging of the tracheal placode in Drosophila embryos, the group sought to answer this question. This
placode begins as a flat sheet of about 40 cells, which

LETTER©2013 Nature Publishing Group
Kondo T. and Hayashi S. Mitotic cell rounding accelerates epithelial invagination.
Nature 494.125-9 (2013)

doi:10.1038/nature11792

Mitotic cell rounding accelerates epithelial
invagination
Takefumi Kondo1 & Shigeo Hayashi1,2Mitotic cells assume a spherical shape by increasing their surface
tension and osmotic pressure by extensively reorganizing theirinterphase actin cytoskeleton into a cortical meshwork and theirmicrotubules into the mitotic spindle1,2. Mitotic entry is known tointerfere with tissue morphogenetic events that require cell-shapechanges controlled by the interphase cytoskeleton, such as apicalconstriction3–5. However, here we show that mitosis plays an activerole in the epithelial invagination of the Drosophila melanogastertracheal placode. Invagination begins with a slow phase under thecontrol of epidermal growth factor receptor (EGFR) signalling; inthis process, the central apically constricted cells, which are surrounded by intercalating cells6,7, form a shallow pit. This slow
phase is followed by a fast phase, in which the pit is rapidly
depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. We found that mitotic cellrounding, but not cell division, of the central cells in the placode isrequired to accelerate invagination, in conjunction with EGFRinduced myosin II contractility in the surrounding cells. We propose that mitotic cell rounding causes the epithelium to buckle
under pressure and acts as a switch for morphogenetic transitionat the appropriate time.
The invagination of epithelial placodes converts flat sheets into thethree-dimensional structures that form complex organs, and it is a key
morphogenetic process in animal development8. A major mechanismof invagination is apical constriction, which is driven by actomyosin
contraction9. However, not all constricted cells invaginate10,11, andsome cell internalization occurs without apical constriction6,7,12,13, suggesting that additional mechanisms of inward cell movement contribute to invagination.
To obtain three-dimensional information about cell behaviour during invagination, we performed live imaging of the Drosophila tracheal
placode. Ten pairs of tracheal placodes, each of which is composed ofabout 40 cells, are formed in the ectoderm at mid-embryogenesis, andeach placode initiates invagination simultaneously14. Using an adherens junction marker, DE-cadherin–green fluorescent protein (E-cad–
GFP)15, we found that the adherens junctions of the central placodecells slowly created a depression by apical constriction, which became
the tracheal pit7 (Fig. 1a). After 30 to 60 min of slow movement (slowphase), the tracheal pit was suddenly enlarged, and the tracheal cells
were rapidly internalized (fast phase) and eventually formed L-shaped
tube structures (Fig. 1a, b, e, Supplementary Fig. 1 and Supplementary
Video 1).
After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis16.
We noticed that the fast invagination was always associated with themitotic entry of central cells that were frequently the first to enter
mitosis 16 (10 out of 14 cases) (Fig. 1b, c and Supplementary Fig. 1a).
Intriguingly, mitotic rounding of the central constricted cells occurred
simultaneously with the rapid depression of their apices, followed bychromosome condensation 10 min later (Fig. 1d and Supplementary
Video 2). In this study, we call this atypical mitotic rounding associatedwith apical depression ‘internalized cell rounding’, to distinguish it fromcanonical surface mitosis (surface cell rounding).
To determine whether cell rounding is required for invagination, we
analysed zygotic mutants of the cell-cycle gene Cyclin A (CycA), which
fail to enter mitosis 16 (ref. 17), and double parkeda3 (dupa3), whichshow a prolonged S phase 16 and delayed entry into mitosis 16(ref. 18). Tracheal invagination was initiated normally in the CycA
and dupa3 mutants, but proceeded more slowly than in controls(Fig. 2a, d, Supplementary Fig. 2 and Supplementary Video 3), indicating that entry into mitosis 16 is required for proper timing of the fastphase.
Although delayed, the accelerated invagination in the CycA or dupa3
mutants eventually occurred, allowing the formation of tube structures (Fig. 2d and Supplementary Fig. 2b) and suggesting that additional mechanisms are involved. After invagination, fibroblast growth
factor (FGF) signalling is activated in the tracheal cells to induce
branching morphogenesis through chemotaxis19,20. To examine thecontribution of FGF signalling to invagination, we analysed mutantsof the FGF ligand branchless (bnl) or the FGF receptor breathless (btl)(Fig. 2b, e and Supplementary Fig. 3a, c). These mutants invaginatednormally, indicating that chemoattraction to FGF is dispensable forinvagination.
Next, to assess FGF’s role in the mitosis-defective condition, we
analysed double mutants for CycA and bnl or CycA and btl, and foundthat they showed slower invagination than CycA single mutants(Fig. 2c, f and Supplementary Fig. 3b, d). Furthermore, the invagination in these double mutants was incomplete, in that the cells failed toform L-shaped tubular structures (Fig. 2c, Supplementary Fig. 3b andSupplementary Video 4, see 120-min time point). Therefore, FGF
signalling is critical for invagination when mitosis is blocked, serving
a back-up role. Tracheal-specific CycA expression rescued the defects
in invagination speed and tube structure in the CycA btl mutants(Supplementary Fig. 4). In addition, we occasionally observed mitosisof cells outside the pit that occurred before the mitosis of the central
apically constricted cells and was not correlated with the fast invagination phase (Supplementary Fig. 1c). Thus, mitosis of the surrounding
epidermal cells is dispensable for tracheal invagination. Taken together,
we conclude that mitotic entry of central cells is a major mechanism foraccelerating tracheal invagination.
To distinguish the role of cell rounding from that of cell division in
the fast phase, we used the microtubule inhibitor colchicine to arrest
the cell cycle after cell rounding. Colchicine treatment after mitosis 15
induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected (Fig. 3 andSupplementary Video 5). This result indicates that cell rounding,but not cell division, is responsible for the acceleration phase of thetracheal invagination.
Mitosis of cells in the columnar epithelium normally occurs at theapical surface after surface rounding21,22. We next asked how the apicalsurface of the central cells becomes depressed during internalized cellrounding. One possible model explains internalized cell rounding as

1

Laboratory for Morphogenetic Signaling, RIKEN Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. 2Department of Biology, Kobe University
Graduate School of Science, Kobe, Hyogo 657-8501, Japan.
0 0 M O N T H 2 0 1 3 | VO L 0 0 0 | N AT U R E | 1©2013 Macmillan Publishers Limited. All rights reserved

transforms into an L-shaped tubular structure. Watching
the process with an eye to detail, Kondo observed that thisinvagination played out in two distinct phases. In the first,
slow phase, cells in the center of the placode undergo apical constriction, and gradually move toward the embryo interior. A second, fast phase followed in which the trachealpit grew and cells were more rapidly internalized.
Interestingly, this fast phase was consistently associatedwith the mitotic entry of cells in the placode center. As cells
enter the mitotic phase, they ball up. In orderly epithelial
sheets, this ‘rounding’ behavior can destabilize the tissue,
meaning that its site and timing need to be closely controlled, and indeed studies of mutants have shown thatmorphogenesis is disturbed when this program is dysregulated. Knowing this, Kondo used mutants of a cell cycle
gene (CycA), which fail to enter the final stage of embryonic mitosis, and watched for effects on invagination. Although the slow phase proceeded as usual, the pace of thefast phase was appreciably decreased. Despite the delay,
however, tracheal invagination did occur, suggesting that
other forces might be at work as well.
The FGF signaling pathways is involved in inducing branching morphogenesis in the fly trachea, so Kondo tested next
looked for a role for this molecule, but found that in single
FGF mutants invagination was unaffected. In double mu-tants for the cell cycle gene and FGF, however, invagination
was slowed and incomplete, suggesting FGF might function as a backup mechanism in this process. In a separate
experiment, chemical inhibition of microtubules showedthat invagination can take place even when rounding occurs in the absence of mitotic spindles, indicating that it is themorphogenetic changes, not the cell division, that are required for the involution of the epithelial sheet.
So how then do cells in the placodal center know when andwhere to begin invaginating? Kondo turned back to a previous study by the Hayashi lab, in which it was shown thatthe motor protein myosin confers constrictive force to actin
fibers in cells peripheral to the tracheal placode center,
forming a pressure front that may drive cells at the center
inward. As this mechanism relies on the EGF receptor protein (EGFR), the group generated EGFR mutants andwatched for effects. When this gene was ablated along with
CycA and FGF, invagination failed completely.
“This work shows how multiple independent mechanisms
can play overlapping and complementary roles in epithelial
invagination,” says Hayashi. “It is these kinds of elegant, robust programs that enable development to proceed in suchas stable fashion. It seems that in order to give rise to thebeauty of the animal form, embryos need to follow beautiful
rules.”

Tracheal invagination begins with a slow phase, later transitioning to a fast phase in which mitotic cells near the center of the internalizing epithelium undergorounding and drive the formation of an L-shaped tube.

13

Retrograde recycling keeps tubules in shape

Bo DONG

Networks of tubular structures form the basis for many important organs and tissues in the animal body, such as thevasculature and airways. The need for such luminal structures developed as bodies grew larger over the course ofevolution, necessitating conduits for the life-sustaining circulation of nutrients, gases and wastes throughout the
body. Animals and their organs come in many different sizes and shapes, of course, so it is also necessary for lumens
to fit the surrounding morphology. Width and length are thecritical parameters in this equation, but how does the developing embryo make these measurements?
New work by Bo Dong and others in the Lab for Morphogenetic Signaling (Shigeo Hayashi, Group Director) now
shows how an intracellular mechanism known as retro-

14

grade trafficking establishes the length of the developing
trachea in fruit fly embryos. Published in Nature Communications, this study sheds light on the morphological regulation of an important class of tissue structures.
The formation of the Drosophila trachea is under the control
of extracellular matrix proteins such as chitin secreted within its lumen. It has previously been shown that the diameterof this structure is regulated by an intracellular protein trafficking pathway mediated by actin complexes, while its
length is controlled by a chitin deacetylase known as Serpentine (Serp). Loss of Serp results in an abnormally longtrachea, but just how this mechanism is tied to the protein
trafficking that regulates intraluminal secretion was poorly
understood.

©2013 Nature Publishing GroupARTICLE
Received 22 Jun 2012 | Accepted 30 Nov 2012 | Published 15 Jan 2013

Dong B, et al. Rab9 and retromer regulate retrograde trafficking of luminal protein
required for epithelial tube length control. Nat Commun 4.1358 (2013)

DOI: 10.1038/ncomms2347

OPEN

Rab9 and retromer regulate retrograde
trafficking of luminal protein required
for epithelial tube length control
Bo Dong1, Ken Kakihara1,2,w, Tetsuhisa Otani1, Housei Wada1 & Shigeo Hayashi1,2Apical extracellular matrix filling the lumen controls the morphology and geometry of epithelial tubes during development, yet the regulation of luminal protein composition and its
role in tube morphogenesis are not well understood. Here we show that an endosomalretrieval machinery consisting of Rab9, retromer and actin nucleator WASH (Wiskott–Aldrich
Syndrome Protein and SCAR Homolog) regulates selective recycling of the luminal protein
Serpentine in the Drosophila trachea. Secreted Serpentine is endocytosed and sorted into thelate endosome. Vps35, WASH and actin filaments differentially localize at the Rab9-enrichedsubdomains of the endosomal membrane, where Serpentine containing vesicles bud off. In
Rab9, Vps35 and WASH mutants, Serpentine was secreted normally into the tracheal lumen,
but the luminal quantities were depleted at later stages, resulting in excessively elongatedtubes. In contrast, secretion of many luminal proteins was unaffected, suggesting that
retrograde trafficking of a specific class of luminal proteins is a pivotal rate-limiting
mechanism for continuous tube length regulation.

1 Laboratory for Morphogenetic Signaling, Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Hyogo, Kobe 650-0047, Japan.
2 Department of Biology, Kobe University Graduate School of Science, 1-1 Rokkodai-cho, Nada-ku, Hyogo, Kobe 657-8051, Japan. w Present address: CTC

Laboratory Systems Corporation, 16-7, 1-chome Komazawa, Setagaya-ku, Tokyo, 154-0012, Japan. Correspondence and requests for materials should be
addressed to S.H. (email: shayashi@cdb.riken.jp).
NATURE COMMUNICATIONS | 4:1358 | DOI: 10.1038/ncomms2347 | www.nature.com/naturecommunications

1& 2013 Macmillan Publishers Limited. All rights reserved.

After searching for proteins showing specific localization
patterns in the cells of the trachea, Dong et al. came to focus on Rab9, a protein known to be involved in retrograde
trafficking, a kind of intracellular recycling process in which
secreted molecules in the cell’s environment are endocytosed and carried in endosomes to the Trans-Golgi network, where they are modified and readied to be secreted
again.
Generating a rab9 mutant, the group noted that while thewidth of the trachea and embryogenesis as a whole appeared normal, the trachea grew to unusual length, a phenotype resembling that seen in Serp mutants. Looking forco-localization of the two molecules, they found that after
about 12 hours of development, the levels of Serp expression within the tracheal lumen dropped sharply, while chitin
and other proteins were unaffected. In mutants of anotherknown retrograde trafficking protein, Vps35, the over-longtrachea phenotype was also observed, and biochemistry
assays indicated that Rab9 and Vps35 bind each other directly.

Using a live imaging system, Dong watched the dynamics
of these three proteins and found that they co-localize asassemblies in an endosomal subtype known as late endosomes. Knowing that actin is crucial for the budding of vesicles from endosomes, they next looked for a spatial relationship between actin and the Rab9 endosomes. They
found that actin and a newly identified actin-associated factor, WASH, localized with Vsp35 at budding sites and are
involved in the endosomal recycling of Serp.
“We think it is very interesting that loss of retrograde trafficking does not affect the tracheal diameter, which suggests that length and width are under the control of separate trafficking pathways,” says Hayashi. “Retrograde
trafficking is widely conserved in animals, including humans, so we will be eager to discover whether it plays amore general role in morphogenesis.”

Formation of abnormally long trachea (top right), and reduced intraluminal expression of Serp (bottom right) in Rab9 mutant.

15

Cutting the crosstalk: New confocal technique provides
deeper look inside biological samples

Togo (and Yuh) SHIMOZAWA

Confocal microscopy is a common tool for looking at the interiors of living samples in three dimensions. To increase
the speed of image acquisition, multi-point scanning methods using a spinning disc embedded with thousands of
pinholes that remove out-of-focus background light havebeen developed. But while spinning disk confocal microscopes cause less radiation damage to samples and achieve
higher temporal resolution than conventional alternatives,the conventional model was limited in its ability to probe theinteriors of large specimens due to a phenomenon known
as ‘pinhole crosstalk,’ in which out-of-plane and scatteredemissions bleed through inappropriate neighboring pinholes and increase the background haze that obscures images.

16

A new development by Togo Shimozawa and others in the
Optical Image Analysis Unit (Yuko Mimori-Kiyosue, Unit
Leader) shows a way out of the crosstalk by adjustments topinhole placement and the use of two-photon excitation to
reduce out-of-focus light. Published in the Proceedings of
the National Academy of Sciences, these enhancements
extend the range of applications of spinning disk confocal
microscopy in the live imaging of larger specimens, including whole embryos. Shimozawa has since moved to Gakushuin University.
Expecting, based on theory, that the problematic pinholecrosstalk might be eliminated by increasing the space between these apertures, the lab modified a standard Confocal Scanner Unit (CSU), doubling the distance between pin-Shimozawa T, et al. Improving spinning disk confocal microscopy by preventing pinholecross-talk for intravital imaging. Proc Natl Acad Sci USA 110.3399-404 (2013)

Improving spinning disk confocal microscopy by
preventing pinhole cross-talk for intravital imaging
Togo Shimozawaa, Kazuo Yamagatab, Takefumi Kondoc, Shigeo Hayashic, Atsunori Shitamukaid, Daijiro Konnod,
Fumio Matsuzakid, Jun Takayamae, Shuichi Onamie, Hiroshi Nakayamaf, Yasuhito Kosugif, Tomonobu M. Watanabeg,h,Katsumasa Fujitai, and Yuko Mimori-Kiyosuea,1aOptical Image Analysis Unit, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; bResearch Institute for Microbial Diseases, Osaka
University, Osaka 565-0871, Japan; Laboratories for cMorphogenetic Signaling, dCell Asymmetry, and eDevelopmental Dynamics, RIKEN Center for
Developmental Biology, Kobe 650-0047, Japan; fLife Science Headquarters, Product Marketing Section, Yokogawa Electric Company, Kanazawa 920-0177,
Japan; gLaboratory for Comprehensive Bioimaging, RIKEN Quantitative Biology Center, Osaka 565-0874, Japan; hSingle Molecule Imaging, World PremierInitiative, Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan; and iDepartment of Applied Physics, Osaka University, Osaka 5650871, Japan

Edited by Jennifer Lippincott-Schwartz, National Institutes of Health, Bethesda, MD, and approved January 7, 2013 (received for review September 25, 2012)

A recent key requirement in life sciences is the observation of
biological processes in their natural in vivo context. However,imaging techniques that allow fast imaging with higher resolution
in 3D thick specimens are still limited. Spinning disk confocal
microscopy using a Yokogawa Confocal Scanner Unit, which offers
high-speed multipoint confocal live imaging, has been found to havewide utility among cell biologists. A conventional Confocal Scanner
Unit conﬁguration, however, is not optimized for thick specimens,
for which the background noise attributed to “pinhole cross-talk,”
which is unintended pinhole transmission of out-of-focus light, limits
overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy byeliminating pinhole cross-talk. First, the amount of pinhole cross-talkis reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitationthat achieves selective-plane illumination. We evaluate the effect ofthese modiﬁcations and test the applicability to the live imaging ofgreen ﬂuorescent protein-expressing model animals. As demonstrated by visualizing the ﬁne details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies
dramatically improve higher-resolution intravital imaging.
GFP imaging| cytoskeleton | EB1 | morphology | development

W

hen imaging the 3D interior of living organisms, fast confocal ﬂuorescence microscopy is essential in monitoring
dynamic life processes by collecting optically sectioned images
over time. One of the main challenges to achieving high-speed
acquisition has been to use a multipoint scanning strategy usinga spinning disk scanner containing a set of confocal pinholes. This
overcomes the speed-limiting disadvantage in single-beam laser
scanning microscopy (LSM). The Confocal Scanner Unit (CSU)
developed by Yokogawa Electric is one of the most advanced
spinning disk systems (1, 2) (Fig. S1A).
The Yokogawa CSU consists of two disks, each containing ∼20,000 microlenses and pinholes, arranged in a spiral arrayof equal pitch (3) (Fig. 1 A and B). The microlenses are positioned to increase the amount of light transmitted through the
disk. The light shone from a beam-expanded laser onto the ﬁrst
disk is focused by each microlens onto the corresponding pinholeof the second disk to generate a parallelized multibeam (Fig.
S1A). During imaging of emitted ﬂuorescence at each illumination point, each pinhole then acts as a detection pinhole andrejects out-of-focus signals, and the points of ﬂuorescence merge
into a 2D image that can be collected with a digital camera as thedisk rotates.
Cell biologists have noted that spinning disk confocal (SDC)
microscopy with CSU signiﬁcantly reduces radiation damage,compared with conventional point-scanning LSM and allows
high-resolution imaging using a low-noise and high-dynamicrange detector such as a charge-coupled device (CCD) camera, as
opposed to the noisier photomultiplier tubes (PMTs) used for

LSM (2, 4–7). As a result, this system has been found to be indispensable for biological cell studies. Recent progress in lightmicroscopy has included the achievement of imaging large
specimens such as a whole embryo and separating nanostructuresat resolution of less than 100 nm by combining optics technologies
including planar illumination and superresolution techniques,
respectively (8–11; reviewed in refs. 12–14). However, light-sheet
microscopy for large specimens is not intended for higher-resolution imaging and not yet available for routine laboratory use,
whereas satisfactory results obtained using superresolution techniques are limited to very thin specimens. The SDC method using
a CSU is, on the other hand, well suited to a wide range ofapplications in biological studies using a variety of sample formats. The simple interface can be easily afﬁxed to any upright orinverted microscope in combination with a variety of devices, suchas separate stimulation lasers, without special expertise.
The multipoint scanning strategy, however, suffers an intrinsic
problem. Fluorescence emissions originating from remote focal
planes and out-of-focus scattering pass through adjacent pinholes, increasing the background signal haze that obscures theimage (Fig. S2). This phenomenon, called “pinhole cross-talk”(1, 2, 15), makes it difﬁcult to apply the SDC to thick specimenssuch as tissues and animals, which produce a large amount ofout-of-focus background noise.
In this study, we sought to improve SDC microscopy by eliminating pinhole cross-talk (Fig. S2). It is expected that, if theinterpinhole distance is increased, the amount of pinhole crosstalk will be reduced, in proportion to the distance, but at the costof overall power. If the generation of out-of-focus light is prevented by two-photon excitation (2PE), which excites ﬂuorophores only in the focal plane (16, 17), the major contributor of
background noise will be removed. Here, we evaluate the effect ofincreased interpinhole distance and the use of 2PE and test its
applicability to the live-imaging of green ﬂuorescent protein(GFP)-expressing model animals.
Results
Construction and Evaluation of a 2PE SDC Microscope. The current
CSU model, CSU-X1, contains 250-μm-diameter microlenses
and 50-μm-diameter pinholes with 250-μm pitch (Fig. 1 A and B
and Fig. S1B). The pinhole size has been designed for highmagniﬁcation with high numerical aperture (NA) objectives set
close to 1 Airy unit. To adapt 2PE, we modiﬁed the CSU

Author contributions: Y.M.-K. designed research; T.S., K.Y., T.K., S.H., A.S., D.K., F.M., J.T.,
S.O., H.N., Y.K., T.M.W., K.F., and Y.M.-K. performed research; T.S. and Y.M.-K. analyzed
data; and Y.M.-K. wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission.
1

To whom correspondence should be addressed. E-mail: y-kiyosue@cdb.riken.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1216696110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1216696110

holes as well as the size of the microlenses that focus lightonto them. The doubling of microlens size increases theconcentration of light to the pinholes, boosting two-photon
absorption efficiency, which is crucial to eliminate background light generated in out-of-focus planes, a key contributor to pinhole crosstalk.
A comparison of the two-photon excitation (2PE) setup withthe standard CSU revealed the benefits of the new approach. While in the conventional technology backgroundnoise due to crosstalk can be as high as 60% of the total
detected signals when imaging a thick specimen, with 2PE
this dropped to as low as 0.5%. When they used the new
apparatus to image a bulky (diameter 30 μm) pollen grain,
not only was background light reduced, but 2PE also revealed rich new features of the particle’s interior. And by assembling multiple images taken in this fashion, the lab wasable to reconstruct its structure as a 3D image, puling out
details from even its far side that remained obscured in the
background of single-photon images.

CELL BIOLOGY©2013 National Academy of Sciences

PNAS | February 26, 2013 | vol. 110 | no. 9 | 3399–3404

For their next demonstration, Shimozawa et al. used the2PE system to examine GFP-expressing model organismsof the sort typically used in embryology studies. In mouseembryos, where traditional spinning disk confocal microscopes can only visualize to depths of around 30 μm, the
new system could image three times as deep, to over 100
μm. The 2PE unit also showed the ability to visualize submicron structures in vivo. Looking at Drosophila embryos,
they were able to follow the movements of a microtubule
plus end-associated protein, EB1, by time-lapse imaging.
“We’re very hopeful that this new technology will find applications across the life sciences and in other disciplines as
well,” says Mimori-Kiyosue. “But as the power of the lasersthat generate the multiple beams needed for this approachis the limiting factor, we are looking to develop higher-power laser sources that will allow us to look even deeper andmore clearly into microscopic specimens.”

Imaging of sectioned mouse embryo expressing H2B-GFP which is distributing in nuclei. In the conventional image (left), pinholecrosstalk causes low contrast and prevents the visualization of nuclei outlines deeper than 30 μm, but in the 2PE image (right),
contrast is high and nuclear outlines can be seen as deep as 100 μm.

17Clones of clones can make clones

Sayaka WAKAYAMA

Since the first reports of successful cloning of mammals bysomatic cell nuclear transfer, concerns have been raised
about the efficiency and repeatability of cloning techniques,
and the health of cloned offspring. Although it has been
showed since the early 2000s that cloned animals canthemselves be cloned, the efficiency of SCNT appeared totaper with each successive generation, leading some to
speculate that there might be an inherent limit to cloning
from clones due to the accumulation of genetic errors.
Now, however, a new report by the Laboratory for Genomic Reprogramming (Teruhiko Wakayama, Team Leader)shows that mice can be cloned successively for at least 25
generations. Published in Cell Stem Cell, this work by
Wakayama, now at the University of Yamanashi, suggests

18

that there is no inherent limit to SCNT repeatability, given
sufficiently efficient methods.
The team used four genotyped female mice as its starting
set of nuclear donors, removing the genetic material fromcumulus cells for use in SCNT attempts, giving rise to anumber of cloned offspring. Donor mice that showed thegreatest ability to give rise to first generation clones was selected to serve as the donor for the subsequent serial cloning experiment, which began in late 2005. For each successive generation, cumulus cells were obtained from
3-month-old mice and used as donor cells for the next
round. Wakayama found that the efficiencies varied by generation, ranging from 4–5% in early rounds to more than
20% in later generations, including the 25th (G25), the last

Cell Stem Cell

Brief Report©2013 Elsevier B.V.
Wakayama S, et al. Successful Serial Recloning in the Mouse over Multiple
Generations. Cell Stem Cell 12.293-7 (2013)

Successful Serial Recloning in the Mouseover Multiple Generations
Sayaka Wakayama,1 Takashi Kohda,2 Haruko Obokata,1,3 Mikiko Tokoro,1,4 Chong Li,1,5 Yukari Terashita,1,6
Eiji Mizutani,1,7 Van Thuan Nguyen,1,8 Satoshi Kishigami,1,9 Fumitoshi Ishino,2 and Teruhiko Wakayama1,7,*
1RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
2Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-0034, Japan
3Laboratory for Tissue Engineering and Regenerative Medicine, Brigham and Women’s Hospital/Harvard Medical School,
Boston 02115, USA
4Asada Ladies’ Clinic, Aichi 486-0931, Japan
5Department of Regenerative Medicine, Tongji University School of Medicine, Shanghai 200092, China
6Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
7Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan
8Department of Biotechnology, School of Biotechnology, International University, Vietnam National University, Ho Chi Minh City 70000,
Vietnam
9Department of Genetic Engineering, Kinki University, Wakayama 649-6493, Japan*Correspondence: teru@cdb.riken.jp
http://dx.doi.org/10.1016/j.stem.2013.01.005

SUMMARY

Previous studies of serial cloning in animals showeda decrease in efficiency over repeated iterations
and a failure in all species after a few generations.
This limitation led to the suggestion that repeatedrecloning might be inherently impossible because of
the accumulation of lethal genetic or epigenetic
abnormalities. However, we have now succeeded incarrying out repeated recloning in the mouse througha somatic cell nuclear transfer method that includes
a histone deacetylase inhibitor. The cloning efficiency
did not decrease over 25 generations, and, to date, wehave obtained more than 500 viable offspring froma single original donor mouse. The reprogramming
efficiency also did not increase over repeated roundsof nuclear transfer, and we did not see the accumulation of reprogramming errors or clone-specific abnormalities. Therefore, our results show that repeatediterative recloning is possible and suggest that, withadequately efficient techniques, it may be possible
to reclone animals indefinitely.
Animals have been cloned from a number of species and organs(Thuan et al., 2010; Wakayama et al., 1998; Wilmut et al., 1997)
and even from frozen cadavers (Wakayama et al., 2008). In
some mammalian species, it is also possible to produce recloned animals with somatic cell nuclei derived from previously
cloned animals (Cho et al., 2007; Kubota et al., 2004; Kuromeet al., 2008; Wakayama et al., 2000; Yin et al., 2008). In principle,
this type of approach could be useful for the large-scale production of superior-quality domesticated animals and for research
into genomic reprogramming (Graf, 2011). Previously, we
proposed that repeated rounds of genomic reprogramming via
serial cloning might lead to an increase in efficiency oversuccessive generations because of the selection of easily re-programmable cells. Disappointingly, however, it has beenfound that the success rate in fact decreased with each iteration. In one study, only one cloned mouse was produced in
the sixth generation from more than 1,000 nuclear transfer
attempts—but it was cannibalized by its foster mother (Wakayama et al., 2000). We have never succeeded in understanding the reason for this failure of recloning over successive
generations. Similar results have been reported in cattle, where
serial nuclear transfer failed to produce a third generation (Kubota et al., 2004). The recloning of cats (Yin et al., 2008) andpigs (Cho et al., 2007; Kurome et al., 2008) has also beenstudied, but those attempts reached only the second and third
generations, respectively.
One possible explanation for this limit on the number ofrecloning attempts is an accumulation of genetic or epigenetic
abnormalities over successive generations. It is well known
that cloned animals frequently show several abnormal phenotypes (Inoue et al., 2002; Ogonuki et al., 2002; Wakayama andYanagimachi, 1999) caused by genomic reprogramming errors
at the time of somatic cell nuclear transfer (Inoue et al., 2010;
Yang et al., 2007). Thus, if a donor nucleus from a cloned animal
is already epigenetically abnormal, the additional abnormalities
introduced during a subsequent round of reprogramming mightlead to embryo failure. Another possibility is that cloned animals
contain only a few normal or reprogrammable somatic cells andthat recloning was successful only when those cells wereselected by chance, but the number of such cells drops oversuccessive generations. A more straightforward explanation
would simply be that the inherent success rate of cloning was
too low for it to be reliable over repeated generations. In the priorstudies, this was the case, and we were unable to investigate
these possibilities (Wakayama et al., 1998), and the reason,
therefore, remained unclear. Recently, we were able to improve
the success rate of mouse cloning up to 5-fold by limiting theaccumulation of epigenetic abnormalities by using a histone
deacetylase inhibitor, trichostatin A (TSA) (Kishigami et al.,2007; Kishigami et al., 2006; Thuan et al., 2010). In the present
study, we attempted serial mouse cloning again with the
Cell Stem Cell 12, 293–297, March 7, 2013 ª2013 Elsevier Inc. 293

generation reported. Some of this variation may be attributable to improvements in cloning technology that emerged
during the study period.
During this lengthy investigation, which gave rise to morethan 500 viable cloned offspring, the team examined whether
complete matching of donor nuclei with recipient oocytes
would boost efficiency, but they found that even microinjection of donor somatic nuclei into oocytes from the same
animal did not result in a higher success rate, suggesting
that, in principle, heterogeneous donors are equally capable of giving rise to clones.
Cloned offspring in some species have shown a range ofdevelopmental and health defects. In mice, clones havebeen observed to have enlarged placentas (placentomegaly). In the 25-generation series, however, the body weightsof the cloned offspring remained consistently within thenormal range for wildtype animals, while placentas weretwo- to three-fold heavier. This effect did not increase withhigher generations suggesting that the abnormality that underlies this phenomenon is not cumulative.
Fertility is another measure of the health status of cloned
animals. Wakayama randomly selected four animals from

Recloned mouse (black) and surrogate mother (white)the 20th generation of the series and mated them with micethat had not been cloned, and found that all four were fertile and gave birth to healthy litters of pups. The lab also
conducted tests of telomere lengths and gene expression
patterns in the cloned mice, While no shortening of telomeres was observed even in later generation clones, there
were differences in the expression of some genes. But
again, these resembled differences observed in non-serially cloned control animals, and did not appear to change
from generation to generation.
One key element of the cloning technique appears to be
the addition of a reagent known as trichostatin A to the cell
medium, which has previously been shown to raise SCNT
efficiency by approximately double. In G20 cloning efforts,
the team compared SCNT with and without trichostatin,
and found that the effect on reprogramming remained thesame.
“This series of experiments is exciting because it reveals
how, with the right techniques, clones can continue to bemade even after the death of the original donor animal,”says Wakayama.

Cloning efficiencies over 25 generations

19

CandyFISH lights up gene expression

Satoko MORIWAKI, Lars Martin JAKTTechniques for imaging the inner workings of cells are abackbone of modern biological investigations. In developmental biology studies, information on the behavior andproperties, such as gene expression, of interacting cells is
fundamentally important for gaining insight into their respective spatial arrangements and dynamics. Fluorescencein situ hybridization (FISH) is a well-known method for visualizing the localization of individual RNA molecules withincells, providing insight into the expression of genes of interest, but this technique is limited in the number of different
genes it can be applied to simultaneously in a single cell.
In a new report, Lars Martin Jakt and colleagues in the Lab
for Stem Cell Biology (Shin-Ichi Nishikawa, Group Director)
describes a new variant of this technique that uses large

20

numbers of short probes making it possible to target transcripts by multiple fluorescent molecules, dramatically expanding the number of genes that can be visualized in situ.
Published in Development, this new CandyFISH method
may open the way to the simultaneous visualization of transcripts from 10 or more genes. In a proof of concept demonstration Jakt, now at Keio University, put the techniqueto use, revealing changes in gene expression in individual
cells during the differentiation of mesoderm into endothelium.
The original FISH method involves labeling mRNA molecules with fluorescent RNA probes, enabling the visualization of the types and amounts of expressed genes in living
cells. A recent improvement to this method have fused mul-

216

©2013 The Company of Biologists

RESEARCH ARTICLETECHNIQUES AND RESOURCES

Development 140, 216-225 (2013) doi:10.1242/dev.086975© 2012. Published by The Company of Biologists Ltd

A continuum of transcriptional identities visualized by
combinatorial fluorescent in situ hybridization
Lars Martin Jakt*, Satoko Moriwaki and Shinichi Nishikawa

Jakt L. M, et al. A continuum of transcriptional identities visualized by combinatorial
fluorescent in situ hybridization. Development 140.216-25 (2013)

SUMMARY
Oligonucleotide-based fluorescent in situ hybridization (FISH) coupled with high-resolution high-sensitivity microscopy allows thevisualization of single RNA molecules within fixed cells and tissues as distinct foci. We show here that combinatorial labeling of RNA
molecules with several fluorescent dyes extends the number of genes that can be targeted simultaneously beyond the number offluorophores used. This approach also inherently validates the identification of transcripts reducing false positive counts. We haveused combinatorial FISH and image analysis to measure the transcript densities of six genes using three fluorophores. This has allowed
us to visualize the endothelial maturation of lateral mesoderm in an in vitro ES differentiation assay from a single snapshot ofmolecular identities. Our observations show that, under these specific conditions, endothelial maturation follows a homogeneouscourse with a gradual increase in expression of Cdh5 and a concomitant loss of early transcription factors, arguing that maturationis governed in a generally deterministic manner. This methodology is limited by the number of fluorophores that can be used andby the available microscopic resolution, but currently available equipment should allow the visualization of transcripts from 10 or moregenes simultaneously.

INTRODUCTION
The primary unit of biological control is the cell. Measurement ofcellular properties using bulk populations of cells is thus
problematic unless a reasonable extent of homogeneity of identity
or behavior exists within the population. As the output ofdevelopment or cellular differentiation is the generation of cellular
heterogeneity, the study of such processes requires simultaneous
measurements at the single cell level of a sufficient number ofparameters to describe the internal variance. As cell interactions arefundamental to developmental processes, the description of spatial
arrangements is also desirable.
We therefore sought to develop methods for in situ
multiparametric quantification of gene expression at the single celllevel in large numbers of cells. Currently, existing methods for
single cell multiparametric measurements can be broadly divided
into cytometry and RT-PCR-based assays. Modern RT-PCR-based
methods allow the simultaneous measurements of large numbers ofparameters in intermediate cell numbers (~100); however,measurements rely on exponential amplification and expression
estimates can vary by orders of magnitude from linear-based
methods (Hayashi et al., 2008; Schwanhäusser et al., 2011) making
data interpretation difficult.
Cytometry (e.g. fluorescence activated cell sorting; FACS) allows
rapid multiparameter (up to 10) measurements on very large numberof cells; recently, the combination of cytometry with mass
spectrometry has allowed the large scale characterization of 31
antigens simultaneously (Bendall et al., 2011). Cytometry is,however, limited by the availability of suitable antibodies and its
accuracy is difficult to assess.
In situ-based methods allow the localization of expressing cells,
but have historically not provided quantitative expression
Stem Cell Biology Group, Riken Center for Developmental Biology, 2-2-3
Minatojima-minamimachi, Chuo-ku, Kobe, Japan.
*Author for correspondence (mjakt@cdb.riken.jp)
Accepted 25 September 2012

tiple short probes to single genes, yielding higher fluorescence and comparatively low cost, but with the limitation of
being applicable to a maximum of only three genes. Jakt’s
refinement shows that using many short probes allows
transcripts to be targeted by multiple fluorophores, allowing
the quantification of gene expression from more than double that number of genes.
In a proof of principle experiment, the group next used the
new method to examine changes in gene expression profile
of cells during the differentiation of vascular endothelium
from embryonic mesoderm. Previous in vitro studies had
shown Etv2 to be an important gene in this process, involved in the induction of endothelial genes, as well as a direct inducer of genes involved in stabilizing the hematopoietic state. This diversity of downstream targets made for avery fuzzy picture of the order in which Etv2 activates othergenes.

measurements. Although it was shown in 1998 that oligonucleotidebased fluorescent in situ hybridization (FISH) combined with highresolution microscopy can be used to visualize individual mRNA
molecules (Femino et al., 1998), this route to quantification has only
been used to address very specific questions (e.g. Raj et al., 2006).
More recently, Raj et al. (Raj et al., 2008) proposed the use ofnumerous short (20 nucleotide) probes labeled with single
fluorophores for the identification of individual transcripts. Thismethod has two primary benefits over previous methods: (1) the useof large numbers of probes targeted to individual transcript regions
provides an amplification of the signal that is dependent on thesequence targeted; (2) a lower cost of probe synthesis allowing
routine use. Such methods provide unambiguous quantification oftranscripts but have been limited in mammalian cells to themeasurement of three genes simultaneously (Itzkovitz and vanOudenaarden, 2011).
The use of multiple probes per transcript allows the encoding oftranscript identities using a combination of fluorophores and it wasdemonstrated in 2002 that this can be used to detect the sites oftranscription of more than 10 genes simultaneously (Levsky et al.,2002). This and subsequent work (Raj et al., 2006) demonstrated
the stochastic nature of transcription, showing that active
transcription cannot be used to describe cell state, as gene activation(a change in state) implies not continuous transcription but anincrease in the probability of transcriptional events. Conceptually,
the methods used by Levsky et al. (Levsky et al., 2002) can be used
to detect individual transcripts; however, the use of a small numberof probes per transcript (up to five used) do not provide an
amplification of the signal in the absence of colocalization of large
numbers of transcripts (observed at sites of transcription) resulting
in a poor signal. Here, we show that the use of large numbers ofshort probes allows transcripts to be targeted by several
fluorophores, allowing the simultaneous quantification of gene
expression from more than six genes in cultures of mammalian cells.
We also present a workflow for the development and verificationof probe sets, FISH, microscopy and analyses of both raw image
data and subsequent transcript counts.

DEVELOPMENT

KEY WORDS: RNA visualization, Cell state, Differentiation, Fluorescent in situ hybridization, Single cell, Single molecule

Jakt studied ES cells induced to differentiate into endothelium using six genes of interest marked using the CandyFISH technique, enabling him to visualize the intracellular
localization of their RNA transcripts. By plotting changes intranscript density in individual cells over time, the group
was able to work out a progression of the switching on andoff of Etx2 and its targets, yielding a much clearer image ofhow gene expression changes in single cells during endothelial differentiation.
CandyFISH represents an important new step in simultaneously measuring the abundances of multiple RNAs at thesingle-cell level, and consequently tracking changes in cellular identity. Additional refinements in microscopy and fluorescent labels may enable the tracking of the expressionof as many as 10 genes in individual cells, and to analyze
deeper tissue strata.

Combinations of three different-colored fluorescent tags enable labeling of six genes.

FISH analysis of six genes yields images resembling coloredcandy in a bowl.

21

Vertebrate Body Plan
Pre-gastrular developmental routines have changed dramatically over the course of vertebrate
evolution. The yolk, for example, has been stored in various amounts, and cleavage development has changed from the holoblastic cleavage seen in amphibian, bichir, lamprey, and mammal to the meroblastic form in teleost, reptile, and avian. Extraembryonic structures in amniotes,
especially those in mammal, were acquired later in vertebrate evolution, and were formed by altering post-cleavage development. In light of these alterations, it is obscure how the mechanisms of anterior-posterior (A-P) axis formation and head development are related and have diverged in each family of vertebrates; these are among the most fundamental events in vertebrate
development. Our aim is to assess A-P axis formation in an ancestral amniote and its divergencein each amniote lineage by comparative studies in extant amniote animals: mouse, rabbit, pig,suncus, chick, quail, soft-shelled turtle, gecko, and gray short-tailed opossum.

Shinichi AIZAWA Ph.D.
http://www.cdb.riken.go.jp/en/aizawa
Shinichi Aizawa received his Ph.D. in biochemistryfrom the Tokyo Kyoiku University Department of
Zoology in 1973. He spent the period from 1974 to
1979 as an investigator at the Tokyo Metropolitan
Institute of Gerontology, then two years as a research fellow at the University of Washington. He
returned to the Tokyo Metropolitan Institute of Gerontology in 1982, where he remained until 1986,
when he moved to the RIKEN Tsukuba Life Science
Center. He was appointed professor in the Kumamoto University School of Medicine in 1994, andserved in that position until 2002. Since 2000 hehas served as CDB deputy director and group director of the Vertebrate Body Plan lab, as well ashead of the Animal Resources and Genetic Engineering Laboratory. He has left these positions at
March 2013, and is now the Unit Leader of Laboratory for Vertebrate Body Plan.

Staff
Group Director

Technical Staff

Shinichi AIZAWA

Eriko KAJIKAWA
Tomomi OHMURA
Daisuke YAMAMOTO

Research Specialist

Yoko SUDA
Michio YOSHIDA
Research Scientist

Part-Time Staff

Michiko INOUE

Hideharu HOSHINO
Visiting Scientist

Daisuke KUROKAWA
Masaki TAKEUCHI

Recent Publications
Huilgol D., et al. Dual origins of the mammalian accessory olfactory bulb revealed by an evolutionarily conserved migratory stream. Nat Neurosci 16.157-65 (2013)

Sunc

Gray short-tailed opossum

Abe T, et al. Visualization of cell cycle in mouse embryos with
Fucci2 reporter directed by Rosa26 promoter. Development
140.237-46 (2013)
Inoue F, et al. Gbx2 directly restricts Otx2 expression to forebrain and midbrain, competing with class III POU factors.
Mol Cell Biol 32.2618-27 (2012)
Kurokawa D, et al. A lineage specific enhancer drives Otx2
expression in teleost organizer tissues. Mech Dev 128.65361 (2012)
Shibata M, et al. MicroRNA-9 regulates neurogenesis inmouse telencephalon by targeting multiple transcription factors. J Neurosci. 31.3407-22 (2011)
Kurokawa D, et al. Evolutionary origin of the Otx2 enhancer
for its expression in visceral endoderm. Dev. Biol. 342.11020 (2010)

Soft-shelled turtle

24

Gecko

Reconstitutive Developmental Biology
When examining a developmental phenomenon of interest, researchers often seek to identify
the basic underlying elements (such as genes), and from them infer the mechanism. But after a certain degree of understanding has been achieved, approaches based on testingwhether it is possible to reconstitute the phenomena from the inferred mechanisms become
increasingly effective. In our lab, we seek to create artificial networks of around five genes incultured cells to investigate whether it is possible to reconstitute the essence of developmental phenomena in vitro.
We are particularly focused on reconstituting mechanisms of intercellular communications.
In metazoan development, such communications induce the spontaneous differentiation ofgroups of cells that underlies the cellular patterns seen in various tissues (known as self-organized cell differentiation patterns). We use simplified artificial networks of the Delta-Notch
pathway, which is responsible for signaling between adjacent cells, in an effort to reconstitute the essence of self-organized cell differentiation patterns in mammalian cells in vitro.
This may allow us to identify minimal gene networks and sufficient conditions underlying
such phenomena as well as discover unexplained or unexpected elements through observation.
Such artificially reconstituted systems also have the advantage of facilitating measurement
and the modification of parameters, which we hope will contribute to the quantitative understanding of communication between adjacent cells.

Miki EBISUYA Ph.D.
http://www.cdb.riken.go.jp/en/ebisuya
Miki Ebisuya received her Ph.D. from the Graduate
School of Biostudies, Kyoto University under thesupervision of Prof. Eisuke Nishida in 2008. She
worked briefly as a postdoctoral fellow in the samelaboratory before being appointed a group leader of
the Career-Path Promotion Unit at Kyoto University
in 2009, where she began research in synthetic biology. In 2013, she was selected as a PRESTO researcher in the area of design and control of cellular functions, and was also appointed to her current
position as unit leader of the Laboratory for Reconstitutive Developmental Biology at the CDB.

Staff

Signal propagation

Research Unit Leader

Technical Staff

Miki EBISUYA

Yuko SAKAIDE

Research Scientist

Student Trainee

Mitsuhiro MATSUDA
Ryoji SEKINE

Makito KOGA

Synthetic signal propagation
Recent Publications
Koga M, et al. Foxd1 is a mediator and indicator of the cell
reprogramming process. Nat Commun 5.3197 (2014)

Pattern formation

Takashima S, et al. Regulation of pluripotency in male germline stem cells by Dmrt1. Genes Dev 27.1949-58 (2013)
Matsuda M, et al. Synthetic signal propagation through directcell-cell interaction. Sci Signal 5.ra31 (2012)

Reconstitution of signal propagation. The gene
expression signal (green) propagated to theneighboring cells along a line.

Ebisuya M, et al. Ripples from neighbouring transcription.
Nat Cell Biol 10.1106-13 (2008)

Examples of gene networks for cell-cell communication. Mutual activation between adjacent cells results in signal propagation (upper),while mutual inhibition results in cell patternformation (lower).

25

Neuronal Differentiation and Regeneration
The architecture of the nervous system, which underlies much of the dynamism of animal
life, is frequently described as complex, but closer examination reveals great beauty as well.
The construction of this exquisite and elaborate structure involves the birth, growth anddeath of countless individual cells and is regulated by intercellular communication and cooperative activity.

Hideki ENOMOTO M.D., Ph.D.
http://www.cdb.riken.go.jp/en/enomoto
Hideki Enomoto received his M.D. from the Chiba
University School of Medicine in 1988, and his
Ph.D. from the same institution in 1996. After serving as the Chief of the Department of Pediatric Surgery, Matsudo Municipal Hospital in Chiba, Japan,
he moved to the Washington University School of
Medicine, St. Louis in 1997, to work as a research
associate studying the physiological roles of the
GDNF Family of Ligands in neural development andfunction. He returned to Japan to take a position asa team leader at the CDB in 2002.

Our laboratory is interested in questions fundamental to the development of the nervous
system: determining the specific extracellular signals and cell autonomous routines that participate in the processes of neural development, and resolving the means by which diverse
families of signaling molecules coordinate and interact at the molecular and cellular levels.
To address these questions, we focus on a class of signaling molecules known as neurotrophic factors, most particularly the GDNF Family Ligands (the GFLs). This family of neurotrophic factors includes four known members – GDNF (Glial cell line Derived Neurotrophic
Factor), Neurturin, Artemin, and Persephin. The GFLs signal via receptor complexes formedby the RET receptor tyrosine kinase and one of four co-receptors, GRFα1-4. In vitro, thesefour receptors show differential affinities for specific GFLs. GFL signaling has been shown toaffect neuronal growth, survival and migration as well as axon guidance, and defects in thissignaling system have been implicated in a number of congenital health problems.
Our efforts will focus on the analysis of the physiological roles of neurotrophic factors, which
we hope will lead to an improved understanding of the molecular bases of neural development, differentiation and pathology, and ultimately to potential clinical applications in thetreatment of nervous system trauma and disease.

Staff
Team Leader

Student Trainee

Hideki ENOMOTO

Sachie ONO

Research Scientist

Pilaiwanwadee HUTAMEKALIN
Keisuke ITOH
Toshihiro UESAKA
Technical Staff

Chihiro NISHIYAMA

Part-Time Staff

Azusa FUKUDA
Toko KONDO
Akira YAMAOKA
Assistant

Yuka NODA

Junior Research Associate

Mitsuhiro IWASAKI
Yohei YONEKURA

Recent Publications
Uesaka T et al. GDNF signaling levels control migration andneuronal differentiation of enteric ganglion precursors.
J Neurosci 33. 16372-82 (2013)
Nishiyama C, et al. Trans-mesenteric neural crest cells are
the principal source for the colonic enteric nervous system.
Nat Neurosci 15.1211-18 (2012)
Nagashimada M, et al. Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression.
J Clin Invest 122.3145-58 (2012)
Uesaka T and Enomoto H. Neural precursor death is central
to the pathogenesis of intestinal aganglionosis in Ret hypomorphic mice. J Neurosci 30.5211-8 (2010)
Uesaka T, et al. Diminished Ret expression compromises
neuronal survival in the colon and causes intestinal aganglionosis in mice. J Clin Invest 118.1890-8 (2008)
Gould T W, et al. The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes. J Neurosci 28.2131-46 (2008)

26

Developing enteric nervous system (ENS) in which GDNF receptor RET was conditionally inactivatedin a small population of ENS cells (mouse gut: embryonic day 14.5, Green: Ret-deficient cells, Red:
enteric neurons).

Tissue Microenvironment
There is a wide diversity of environments on earth, from the North Pole to the Sahara, inwhich well-adapted organisms live. The traits and behaviors of all such organisms havebeen developed and modified through exposure to their specific environments.
In our own bodies, we also have millions of different environments in which cells reside,which are known as cellular or tissue microenvironments. A series of recent studies has
shown that these specialized tissue microenvironments instruct the fate and behaviors ofcells. The aim of our lab is to gain a better understanding of the mechanisms underlying theways in which tissue microenvironments are regionally specialized, and how these specialized microenvironments then instruct cellular behavior and communication, and the formation of organs. We are particularly interested in the role of regional specialization of the extracellular matrix (ECM) in the formation of the stem cell microenvironment, or niche. A
deeper knowledge of this will provide a molecular basis to an improved understanding of theniche regulation of stem cells, and the development of tailor-made microenvironments fordifferent lineages of stem cells.
The ECM is divided in two major groups – the fibrillar interstitial matrix, which fills the interstitial connective tissues, and the basement membrane (BM), a thin sheet-like ECM located at
the borders of tissues. Stem cells in most tissues reside at this border, adhere to the BM andinteract with neighboring niche cells. By virtue of its remarkable heterogeneity in composition, the BM contributes to the spatial organization of niches, and modulates the local concentration of adhesive and soluble signalling molecules that are available to stem cells. A recent study by our team has shown that the molecular composition of the BM in the mousehair follicle stem cell niche, the bulge, is highly specialized. One stem cell-derived component, nephronectin, is important for the development and positioning of the bulge-residing
arrector pili muscles, which, among other functions, are responsible for goosebumps. This
was the first report to show that stem cells regulate the fate and positioning of surrounding
niche cells through the specialization of the BM. To gain further insight into fundamental aspects of the microenvironmental regulation of stem cells, we use mouse skin as a model andseek to better understand 1) the molecular landscape of BM specialization in the stem cellniche, 2) mechanisms by which the BM in the stem cell niche is regionally specialized, and3) how the specialized BM controls stem cell niche formation, stem cell behavior and theconversation between stem cells and their neighboring cells.

Hironobu FUJIWARA Ph.D.
http://www.cdb.riken.go.jp/en/fujiwara
Hironobu Fujiwara attained his baccalaureate from
Kyoto Pharmaceutical University, and went on toreceive his M.S. and Ph. D. from Osaka University,
the latter in 2003 for his work on the characterization of the human laminin-8 protein. From 2003 to
2007, he served as a postdoctoral researcher at
the Osaka University Institute for Protein research,with support from the ERATO Sekiguchi Biomatrix
Signaling Project (2003 – 2006). He then moved to
the Cancer Research UK Cambridge Research Institute, where he completed a second postdoctoral
fellowship, before returning to Japan to take a position as a Team Leader at the RIKEN CDB in 2012.

Staff
Team Leader

Technical Staff

Hironobu FUJIWARA

Noriko BAN-SANZEN

Research Scientist

Assistant

Chun-Chun CHENG
Ritsuko MORITA
Ko TSUTSUI

Asako NAKAGAWA

Recent Publications
Fujiwara H, et al. The basement membrane of hair follicle
stem cells is a muscle cell niche. Cell 144.577-89 (2011)
Watt F. M. and Fujiwara H. Cell-extracellular matrix interactions in normal and diseased skin. Cold Spring Harb Perspect Biol 3.(2011)
Ferreira M, et al. An activating beta1 integrin mutation increases the conversion of benign to malignant skin tumors.
Cancer Res 69.1334-42 (2009)
Fujiwara H, et al. Regulation of mesodermal differentiation ofmouse embryonic stem cells by basement membranes.
J Biol Chem 282.29701-11 (2007)
Fujiwara H, et al. Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8. Exp Cell Res
292.67-77 (2004)

The image shows arrector pili muscles anchored to the bulge of hair follicles in a wholemount preparation of dorsal skin in mouse, viewed from the dermal side. Arrector pilimuscles are visualized by staining for α-smooth muscle actin (green) and SM22α (red).
The whole-mount is labelled with a nuclear counter-stain (blue).

27

Neocortical Development

Carina HANASHIMA Ph.D.
http://www.cdb.riken.go.jp/en/hanashima
Carina Hanashima received her Ph.D. from Waseda University School of Science and Engineering in
1999. She worked as a postdoctoral fellow in thelaboratory of Eseng Lai at Memorial Sloan-Kettering
Cancer Center, Cell Biology Program from 1999 to
2002, where she was engaged in developmental
neuroscience. She moved to Skirball Institute Developmental Genetics Program in 2002 in Gord
Fishell’s laboratory where she extended her research to cell specification in the neocortex. She
was appointed team leader at the CDB in September 2007.

Staff
Team Leader

Technical Staff

Carina HANASHIMA

Chihiro NISHIYAMA

Research Scientist

Part-Time Staff

Yuko GONDA
Takuma KUMAMOTO

Chika KUMAMOTO
Reiko ODA

Foreign Postdoctoral Researcher

Assistant

Torsten BULLMANN

Eri KOJIMA
Miyuki MIURA

Visiting Researcher

The neocortex, by far the most complex structure of all vertebrate brain systems, is organized into cytoarchitectonic groups of neurons that process unique sensory inputs, such aspressure, light and sound. This highly ordered structure nonetheless is generated from a relatively simple sheet of neuroepithelium earlier during development. Research in our laboratory aims to understand how diverse arrays of cortical neurons are specified and coordinated into high-functional territories. Despite its well-defined anatomical character andfunctional significance, the mechanisms underlying the precise assembly of distinct functional areas of the cerebral cortex remains largely unknown. Progress has been impeded byour present lack of understanding of the molecular mechanisms by which neuronal subtypes within cortical layers and across function domains are specified. In our laboratory, we
address important questions concerning neocortical development: 1) What are the mechanisms by which diverse cell fate is determined in the neocortex? 2) How are neurons precisely arranged into distinct cortical areas and how do they establish cytoarchitectonic
boundaries? 3) To what extent does the refinement of functional areas rely on environmental inputs? To investigate these questions we utilize mouse as a model to understand the assembly of functional subdivisions in the mammalian neocortex.
Previous work has shown that the fate of neocortical neurons is at least in part pre-determined within the progenitor cells. We have found that specification of deep-layer projection
neurons is dependent on transcriptional repressor that function cell-autonomously to prevent neurons from acquiring an earlier neuronal phenotype. These results imply cortical intrinsic programs in which neuron fate is established by temporal changes in gene expression
may be co-opted. However, in the mature cortex, cortical areas differ in their types andnumbers of specific layer neurons along both the anteriorposterior (AP) and medial-lateral(ML) axes. We are exploring the extent to which intrinsic determinants control the specification of neuronal subtypes within discrete regions of the neocortex, as well as the extrinsic influences that refine the boundaries between functional areas of neocortex. To further thesestudies, we employ genetic manipulations in mice that will enable conditional loss of geneand cellular functions, recombination mediated cell-lineage tracing, and systematic approaches to identify novel molecules responsible for precise areal specification. Throughthese studies we wish to understand the mechanistic basis by which unique sensory perceptions and functional circuitries develop in the human neocortex.

Tien-Cheng WANG
Junior Research Associate

Ken-ichi TOMA

Recent Publications
Kumamoto T, et al. Foxg1 Coordinates the Switch from Nonradially to Radially Migrating Glutamatergic Subtypes in the
Neocortex through Spatiotemporal Repression. Cell Rep
3.931-45 (2013)
Gonda Y, et al. Robo1 regulates the migration and laminar
distribution of upper-layer pyramidal neurons of the cerebral
cortex. Cereb Cortex 23.1495-508 (2013)
Kasukawa T, et al. Quantitative expression profile of distinct
functional regions in the adult mouse brain. PLoS One 6.
e23228 (2011)
Fishell G and Hanashima C. Pyramidal neurons grow up andchange their mind. Neuron 57. 333-8 (2008)
Hanashima C, et al. The role of Foxg1 and dorsal midline signaling in the generation of Cajal-Retzius subtypes.
J Neurosci 27. 11103-11 (2007)
Hanashima C, et al. Foxg1 suppresses early cortical cell fate.
Science 303. 56-9 (2004)

Recruitment of late-born neocortical neurons expressing membrane-targeted
GFP (green) and nuclear lacZ (red) to the motor cortex.

28

Morphogenetic Signaling
The main research interest in my lab focuses on the mechanisms by which cell-cell and tissue-tissue interactions are modulated during embryonic morphogenesis. A range of vital developmental processes, including the ability to build complex structures through selective
cell adhesion, to move through the body, and to form epithelial tissues, rely on interactions
between cells, and between cells and the extracellular matrix. The means by which cells areable to communicate, congregate, and work together to build a body is a central questionin the study of morphogenesis. To tackle this problem, we use the model organism Drosophila which is suited for genetic dissection of biological processes and for high-resolution
imaging, and study the problem at three levels of cellular organization: single-cell, multiple
cells, and organ.
Our study is centered on the tracheal system, a respiratory organ that delivers air directly totissues through a network of finely branched epithelial tubes. Tracheal development begins byinvagination of ectodermal epithelium that subsequently forms branches with specific types ofcells migrating in stereotyped patterns. Cellular forces produce mechanical strain in the epithelium, and alleviation of that strain is essential for smooth-tissue movement. As a consequence of epithelial cells behaving as elastic bodies, their shape changes and movement
proceed with local fluctuations. We aim at elucidating (1) mechanisms that coordinate cell
movement, (2) mechanisms for alleviating tissue strain, and (3) mechanisms of cross-talkbetween these two mechanisms. To accomplish these goals, the mechanical state of cells
will be measured by combining techniques such as quantitative cell imaging, and various
cell perturbation techniques are being used to assess the mechanical states of cells. The results of these analyses will be used to construct epithelial cell models and simulations.
In addition, we study intracellular mechanisms of cell polarization and elongation using model systems of mechanosensory bristles.

Shigeo HAYASHI Ph.D.
http://www.cdb.riken.go.jp/en/hayashi
Shigeo Hayashi received his B. Sc. in Biology from
Kyoto University in 1982, and his Ph.D. in Biophysics from the same institution in 1987, for his workon lens-specific regulation of the chicken delta
crystallin gene. Inspired by the discovery of the homeobox, he changed his research focus to the developmental genetics of Drosophila and spent three
years as a postdoctoral research associate in Matthew Scott’s lab at the University of Colorado before returning to Japan to work in the National Institute of Genetics. He became an associate professor
at the same Institute in 1994, and professor in
1999. Also in 1999, he received the incentive prizeof the Genetics Society of Japan for Genetic Studies on Drosophila Development. He was named
group director of the Morphogenetic Signaling research group at the RIKEN CDB in May 2000. His
current research interests are dynamic aspects ofcell adhesion, cell migration and cell morphogenesis in Drosophila.

StaffGroup Director

Technical Staff

Shigeo HAYASHI

Akiyo KIMPARA
Housei WADA

Research Scientist

Toshiya ANDO
Aki HAYASHI
Takuya MAEDA
Tetsuhisa OTANIInternational Program
Associate

Foreign Postdoctoral
Researcher

Ikuko FUKUZYOUNoriko MORIMITU

Bo DONG
Special Postdoctoral
Researcher

Guangxia MIAO
Part-Time Staff

Assistant

Ryoko ARAKI

Takefumi KONDO

Recent Publications
Niwa N, et al. Homeogenetic inductive mechanism of segmentation in polychaete tail regeneration. Dev Biol 381.46070 (2013)
Dong B, et al. Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control. Nat Commun 4.1358 (2013)
Kondo T. and Hayashi S. Mitotic cell rounding accelerates
epithelial invagination. Nature 494.125-9 (2013)
Otani T, et al. IKKε regulates cell elongation through recycling
endosome shuttling. Dev Cell 20.219-32 (2011)
Tajiri R, et al. Joint morphology in the insect leg: evolutionary
history inferred from Notch loss-of-function phenotypes in
Drosophila. Development 138.4621-6 (2011)

Drosophila embryo at the beginning of tracheal placed invagination (magenta). Cell outlines labeledgreen.

Noguchi T, et al. Sustained elongation of sperm tail promoted by local remodeling of giant mitochondria in Drosophila.
Curr Biol 21.805-14 (2011)29

Developmental Epigenetics
We wish to clarify the molecular mechanisms underlying global facultative heterochromatin
formation during early mouse embryogenesis, with the belief that understanding the developmental regulation of higher-order chromosome organization will lead to a deeper understanding of cell differentiation.

Ichiro HIRATANI Ph.D.
http://www.cdb.riken.go.jp/en/hiratani
Ichiro Hiratani received his B. Sc. in Biological
Sciences from the University of Tokyo in 1998, and
his Ph.D. from the same institute in 2003, for his
work on Xenopus embryonic development with
Masanori Taira. In 2003, he moved to the UnitedStates for his postdoctoral training with David Gilbert
at the State University of New York Upstate Medical
University and studied the developmental regulationof DNA replication timing and nuclear genome
organization. He later moved with Gilbert to Florida
State University in 2006 and returned to Japan in2010 to take a position as Assistant Professor at
the National Institute of Genetics. In 2011, he wasawarded a grant from the JST PRESTO program in
Epigenetics. He was appointed to his current position
as Team Leader at the RIKEN CDB in October
2013.

The term facultative heterochromatin refers to chromosomal regions that condense, become
inactivated, and are stably maintained in this manner after a certain developmental stage. A
classic example is the inactive X chromosome in mammals, which becomes detectable immediately prior to the formation of germ layers and is stably maintained thereafter in all
downstream lineages. Intriguingly, we recently discovered that many autosomal domains
also undergo a similar process of facultative heterochromatin formation at the same developmental stage, which accounts for more than 6% of the genome. This suggests that facultative heterochromatin formation at this stage is not specific to the inactive X, but rather amore widespread phenomenon affecting the entire genome. Recent studies have also revealed low reprogramming efficiency of cells immediately after this developmental stage,already as low as downstream somatic cell types. Thus, this facultative heterochromatin is
a common epigenetic feature of all somatic cells beyond the germ layer formation stage,
and the reprogramming experiments imply a potential link to the cell's differentiated state.
For these reasons, we combine genome-wide approaches with molecular and cell biology
and imaging techniques to elucidate the molecular mechanisms underlying the facultative
heterochromatin formation process. In the future, we will address the biological significanceof this phenomenon and eventually wish to understand the fundamental implications ofhigher-order chromosome organization.

Staff
Team Leader

Ichiro HIRATANITechnical Staff

Akie TANIGAWA

Recent Publications

Early- and late-replicating DNA localize to theinterior (green) and periphery (red) of the nucleus, respectively. Because of this relationship,
genome-wide DNA replication profiling (graphs)
can be used to deduce the 3D genome organization at the sequence level.

Electron microscopy reveals a large-scale genome reorganization during pre- (left) to postepiblast (right) transition, consistent with predictions made by DNA replication profiling.

Ryba T, et al. Genome-scale analysis of replication timing:from bench to bioinformatics. Nat Protoc 6.870-95 (2011)
Hiratani I. and Gilbert D. M. Autosomal lyonization of replication domains during early Mammalian development. Adv Exp
Med Biol 695.41-58 (2010)
Ryba T, et al. Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res 20.761-70(2010)
Hiratani I, et al. Genome-wide dynamics of replication timing
revealed by in vitro models of mouse embryogenesis. Genome Res 20.155-69 (2010)
Hiratani I, et al. Replication timing and transcriptional control:
beyond cause and effect--part II. Curr Opin Genet Dev
19.142-9 (2009)
Hiratani I, et al. Global reorganization of replication domains
during embryonic stem cell differentiation. PLoS Biol 6.e245(2008)

Genome-wide DNA replication profiling during ES cell differentiation canreveal domains that show large-scale changes in nuclear organization.

30

Sensory Circuit Formation
The mammalian nervous system is composed of enormous numbers of neurons, but how
do these cells take on diverse fates and organize and array themselves during development? In recent years, it has become clear that the mouse olfactory system provides an
excellent platform for addressing these questions experimentally. In this system, there are
1,000 types of odorant receptors that are capable of detecting and discriminating between
odorant molecules. Each olfactory sensory neuron expresses a single type of odorant receptor, and the axons of neurons expressing the same receptor type converge on the same site
in the olfactory bulb. Olfactory sensory neurons connect axons to the dendrites of mitral andtufted (M/T) cells in the bulb, where each receives inputs from a single specific type of olfactory sensory neurons.
It has generally been thought that neuronal identities are genetically programmed, and thatneuronal connectivity is maintained by molecular “lock and key” mechanisms. The mouseolfactory system, however, is highly adaptive; olfactory neuronal identities are dependent on
peripheral inputs, and form the basis for a self-organizing olfactory map. A better understanding of this flexibility may provide new insights into the diversification of function thattook place during the evolution of the human brain.
Our lab will seek to develop a better understanding of odorant receptor-dependent axon
projection of olfactory sensory neurons, and the formation of neuronal circuitry in the olfactory bulb dependent on inputs from these neurons. We are also seeking to develop nextgeneration genetic tools and imaging techniques to aid in our developmental and functional
analyses of specific neuronal inputs in the brain. For example, we have recently developed
a novel optical clearing agent named SeeDB, which is quick, efficient, and enables three-dimensional imaging of biological tissues at millimeter-scale levels.

Takeshi IMAI Ph.D.
http://www.cdb.riken.go.jp/en/imai
Takeshi Imai completed his Ph.D. in the laboratoryof Prof. Hitoshi Sakano at the University of Tokyo
Graduate School of Science in 2006. He did consecutive postdoctoral fellowships in the CREST
Program and the University of Tokyo in 2006 and2007, before his appointment as Assistant Professor at the same university in 2009. He received independent funding under the JST PRESTO program that year, before joining the RIKEN CDB asteam leader in 2010. He also holds an adjunct position as Associate Professor at the Kyoto University
Graduate School of Biostudies.

Staff
Team Leader

Student Trainee

Takeshi IMAI

Shuhei AIHARA
Meng-Tsen KE

Research Scientist

Satoshi FUJIMOTO
Ryo IWATA
Marcus LEIWE

Assistant

Eri YAMASHITA

Technical Staff

Miwako NOMURA

Recent Publications
Nakashima A, et al. Agonist-Independent GPCR Activity
Regulates Anterior-Posterior Targeting of Olfactory Sensory
Neurons. Cell 154.1314-25 (2013)
Ke M. T, et al. SeeDB: a simple and morphology-preserving
optical clearing agent for neuronal circuit reconstruction. Nat
Neurosci 16.1154-61 (2013)
Imai T, et al. Topographic Mapping - The Olfactory System.
Cold Spring Harb Perspect Biol 2. A001776 (2010)
Imai T, et al. Pre-target axon sorting establishes the neuralmap topography. Science 325.585-90 (2009)
Imai T, et al. Odorant receptor-derived cAMP signals direct
axonal targeting. Science 314. 657-61 (2006)

Adult Thy1-YFP-H mouse brain was cleared with an optical clearing agent SeeDB and imaged using
two-photon microscopy. 3D rendering image of a volume of 4 mm x 5 mm x 2mm, encompassing
cerebral cortex and hippocampus, is shown.

31

Chromosome Segregation
In order to maintain genetic information across generations, cells must allocate chromosomes equally to daughter cells during mitosis. Meiotic divisions of the mammalian oocyte,
however, are known to exhibit a higher frequency of errors in chromosomal segregation
than in other cell types. Oocytes formed from such divisions are aneuploid, meaning they
have incorrect numbers of chromosomes; if these are fertilized and develop to term, the resulting individual may exhibit congenital anomalies, such as trisomy 21 (Down syndrome).
Such errors in chromosomal segregation are also known to increase with the age of themother, and this risk may be a contributing factor to the low birth rates seen in many developed nations.

Tomoya KITAJIMA Ph.D.
http://www.cdb.riken.go.jp/en/kitajima
Tomoya Kitajima received his Master’s and doctoral degrees from the University of Tokyo, for histhesis on genetic screens for meiosis-specific proteins regulating chromosome segregation in fission
yeast. After receiving his Ph.D. in 2004, he served
as research associate at the Institute of Molecular
and Cellular Biosciences at the same university,
before moving to the European Molecular Biology
Laboratory in Heidelberg, Germany as a postdoctoral researcher. He was appointed Team Leader at
the CDB in 2012.

Using the mouse as a model, we will seek to conduct detailed and comprehensive analysesof the dynamics of chromosomes and the molecular machinery that underlies chromosome
segregation during cell division. We plan to take advantage of the latest live imaging technologies to study the chromosome dynamics of the mouse oocyte at a level detail unprecedented in other cell types. Oocyte chromosomes behave in ways distinct from those in other cells, and these unique dynamics may provide insights into novel mechanisms forchromosome allocation. By combining live imaging with genetics techniques such as RNAiand gene knockouts, we hope to study the mechanisms underlying chromosomal segregation in oocyte meiosis, and identify the causes behind age-related increases in ploidy errors.

Staff
Team Leader

Technical Staff

Tomoya KITAJIMA

Masako KAIDO

Research Scientist

Student Trainee

Yogo SAKAKIBARA
Shuhei YOSHIDAYi DING

Visiting Scientist

Kaori HAMADA

Aurélien COURTOIS
Hirohisa KYOGOKU

Assistant

Recent Publications
Kitajima T. S, et al. Complete kinetochore tracking reveals
error-prone homologous chromosome biorientation in mammalian oocytes. Cell 146.568-81 (2011)
Lee J, et al. Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells. Nat Cell
Biol 10.42-52 (2008)
Kitajima T. S, et al. Shugoshin collaborates with protein
phosphatase 2A to protect cohesin. Nature 441.46-52(2006)

Tracking of kinetochores (green) and chromosomes

Kitajima T. S, et al. Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization. Curr Biol 15.353-9 (2005)
Kitajima T. S, et al. The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis. Nature 427.510-7 (2004)
Kitajima T. S, et al. Distinct cohesin complexes organize meiotic chromosome domains. Science 300.1152-5 (2003)

Prometaphase of meiosis I. Microtubules (green),kinetochores (red), chromosomes (blue).

32

Kinetochore tracking during anaphase of meiosis I.

Histogenetic Dynamics
The development of multicellular organisms involves the collective effect of multiple eventsat the level of the individual cell, such as proliferation, differentiation, adhesion, and migration. Programmed cell death, for example, is a process by which cells are selected for deathat set times in development, allowing for the sculpting of tissue, and is used in the adultorganism to maintain homeostasis by eliminating cells that have developed abnormalities.
Perturbations in cell death signaling can thus affect an organism’s physiological stability,
and result in developmental defects, tumorigenesis, or neurodegenerative disease. Cell deathplays an important role in maintaining the cellular society not only by eliminating unneeded
cells at given sites and stages, but in other functions, such as regulating the proliferation andmigration of neighboring cells, as well. Such cellular behaviors give rise to cell networks
capable of organizing into tissues, the study of which requires a experimental approach tospatiotemporal information in living systems, such as can be obtained through the real-time
live imaging of biological phenomena.
We have chosen the fruit fly Drosophila melanogaster as our primary research model, seeking
to take advantage of its utility in developmental studies and wealth of genetic data in studying the coordination of histogenesis through live imaging and genetic screens. To elucidate
the role of cell death in histogenetic processes, we will analyze caspase mutant phenotypes
in which the exterior male genitalia (terminalia) develops abnormally. In normal Drosophila
development, the terminalia rotates 360° as it forms, but in caspase mutants, this revolution
is incomplete. Image analysis reveals that in wildtype, the speed of this rotation is variable,with distinct initiation, acceleration, deceleration, and termination stages; caspase inhibition
results in loss of the acceleration phase, and failure in terminalia development. We will seek
to identify how caspase function and cell death control acceleration of the rotation throughsearching for associated genes and live imaging analysis. It has further been predicted thatcell death alone cannot account for rotation that maintains tissue area, suggesting othermechanisms are also at work. We will conduct single-cell analyses to determine whether
other behaviors such as proliferation or migration are also altered. Through the use of theextensive Drosophila genetics toolset and live imaging technologies, we hope to be able toaddress questions that have proven technically challenging in the past, and by visualizing
the activities of individual cells, develop a better understanding of how cellular network systems work in histogenesis.

Erina KURANAGA Ph.D.
http://www.cdb.riken.go.jp/en/kuranaga
Erina Kuranaga received her doctorate in medical
science from the Osaka University Graduate Schoolof Medicine in 2004, after which she moved to the
University of Tokyo Graduate School of Pharmaceutical Sciences as assistant professor in the Department of Genetics. In 2006, she was promoted
to associate professor in the same department,
where she remained until she joined the RIKEN
Center for Developmental Biology as team leader in
2011. She was awarded the Wiley-Blackwell Prize
2012.

Staff
Team Leader

Student Trainee

Erina KURANAGA

Yuhei KAWAMOTO
Shuji SHIMAMURA

Research Scientist

Kenichi KANAIEmi MAEKAWA
Technical Staff

Part-Time Staff

Arata KURANAGA
Yoko UMEGAKI

Ayako ISOMURA
Aimi TSUKIOKA

Recent Publications
Takeishi A, et al. Homeostatic epithelial renewal in the gut isrequired for dampening a fatal systemic wound response in
Drosophila. Cell Rep 3.919-930 (2013)
Sekine Y, et al. The Kelch repeat protein KLHDC10 regulatesoxidative stress-induced ASK1 activation by suppressing
PP5. Mol Cell 48.692-704 (2012)
Kuranaga E, et al. Apoptosis controls the speed of looping
morphogenesis in Drosophila male terminalia. Development
138.1493-9 (2011)
Koto A, et al. Apoptosis ensures spacing pattern formationof Drosophila sensory organs. Curr Biol 21.278-87 (2011)
Nakajima Y, et al. Nonautonomous apoptosis is triggered by
local cell cycle progression during epithelial replacement in
Drosophila. Mol Cell Biol 31.2499-512 (2011)
Kuranaga E, et al. Drosophila IKK-related kinase regulates
nonapoptotic function of caspases via degradation of IAPs.
Cell 126.583-96 (2006)

Dorsal (left) and ventral (right) views of Drosophila pupae that express fluorescent protein
in cells located posterior component of eachsegment. Location of male genitalia is pointed
in yellow square.

Caudal view of DE-Cadherin:GFP-expressing
Drosophila. This image was taken before rotation.

33

Transcriptional switch controls neuronal fate in neocortex

There is something to the notion that it is our neocortex thatmakes us human. This sheet of neurons covering the brain
is the seat of the senses, cognition, language and othercerebral faculties. Its development is thus a matter of intrinsic interest, but of elegant complexity as well. Cortical neurons of various types form in sequential order, with theolder neurons inhabiting deeper layers and younger neurons forming the upper strata. In total, there are six suchlayers in the neocortex, but the genetic controls that direct
the transitions from one neuronal subtype to another haveremained elusive.
New work by Takuma Kumamoto and others in the Laboratory for Neocortical Development (Carina Hanashima, Team
Leader) provides insight into how a transcription factor
known as Foxg1 functions as the earliest switch in regulating neuronal cell fate in the mouse neocortex. Published inCell Reports, this study opens new inroads into our understanding of the formation of neocortical neuronal diversity.
Cajal–Retzius (CR) cells, the earliest glutamatergic neurons
to emerge, play a central role in neocortical development.
These cells secrete factors that direct the cortical stratification and the radial projection of later generations of neurons, but are gradually lost after birth, disappearing altogether by adulthood. CR cells are rare in birds and reptiles,
leading to speculation that they are important to the formation of the comparatively massive mammalian cortex. It had
earlier been thought that these cells arose from a different
progenitor domain than the neocortex, but a previous study
by Hanashima found that Foxg1, which is expressed inneocortical progenitors, suppresses CR cell differentiation,
revealing that CR cells share a common progenitor withprojecting neurons.

Takuma KUMAMOTO

34

Kumamoto set out to build on that work, seeking the mechanism by which Foxg1 regulates neural stem cell differentiation. The first step was to create a knockout mouse lacking Foxg1, and analyze its phenotype. In these mutants, hefound that projection neurons of all layers failed to form,
while CR cells developed in greater than usual numbers.
Migration patterns of neurons that appear in a distinctive
radial fashion in wildtype were also aberrant in the knockout
embryos. The loss of Foxg1 function thus appeared toCell Reports

Article©2013 Cell Press
Kumamoto T, et al. Foxg1 Coordinates the Switch from Nonradially to Radially
Migrating Glutamatergic Subtypes in the Neocortex through Spatiotemporal
Repression. Cell Rep 3.931-45 (2013)

Foxg1 Coordinates the Switch from Nonradiallyto Radially Migrating Glutamatergic Subtypes
in the Neocortex through Spatiotemporal Repression
Takuma Kumamoto,1 Ken-ichi Toma,1,3 Gunadi,1 William L. McKenna,4 Takeya Kasukawa,2 Sol Katzman,5 Bin Chen,4and Carina Hanashima1,3,*
1Laboratory for Neocortical Development
2Functional Genomics Unit
RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
3Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan
4Department of Molecular, Cell and Developmental Biology
5Center for Biomolecular Science and Engineering
University of California, Santa Cruz, Santa Cruz, CA 95064, USA*Correspondence: hanashima@cdb.riken.jp
http://dx.doi.org/10.1016/j.celrep.2013.02.023

SUMMARY

The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however,
the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here,
we report that the forkhead box transcription factor
Foxg1 coordinates the production of neocorticalprojection neurons through the global repressionof a default gene program. The delayed activationof Foxg1 was necessary and sufficient to induce
deep-layer neurogenesis, followed by a sequential
wave of upper-layer neurogenesis. A genome-wide
analysis revealed that Foxg1 binds to mammalianspecific noncoding sequences to repress over 12transcription factors expressed in early progenitors,
including Ebf2/3, Dmrt3, Dmrta1, and Eya2. Thesefindings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at
a progressed stage during development and identify
Foxg1 as a critical initiator of neocorticogenesis
through spatiotemporal repression, a system thatbalances the production of nonradially and radiallymigrating glutamatergic subtypes during mammaliancortical expansion.
INTRODUCTION
The functional integrity of mammalian brain systems depends
on the precisely coordinated production of diverse neuron
populations during development. Specifically, in the cerebral
cortex, distinct neuronal subtypes are produced in a stereotypical temporal order (Angevine and Sidman, 1961). In recent
years, considerable progress has been made in the identification of genes that control the differentiation of each neuronal

type in the neocortex (Fame et al., 2011; Leone et al., 2008). In
contrast, little is known about the regulation of the transitions
between the sequentially generated subtypes.
Interestingly, although most cortical glutamatergic neurons
arise from local progenitors that migrate radially and differentiate
into projection neurons, some exceptions exist, in which earlyborn neurons originate within the surrounding pallial progenitors
and invade the neocortex through a distinct migration mode.
These cells have both mitogenic and patterning effects onlater-born projection neurons and are unique to mammalianvertebrates (Borello and Pierani, 2010; Puelles, 2011). By far
the most characterized neurons, Cajal-Retzius (CR) cells, which
express the glycoprotein Reelin (Reln), have emerged rapidly
both in number and molecular diversity over the course of
mammalian evolution (Meyer, 2010; Pollard et al., 2006).
Functionally, this is not surprising, given the specialized rolesof these cells in regulating both the radial migration and areal
expansion of later-born projection neurons, which are uniqueto the laminated neocortex system. Mechanistically, the regulation of the switch from the production of early signaling cells toradially migrating projection neurons requires a developmental
process in the broader context of cortical evolution, which
ultimately balances the numbers of these two functionally distinct subtypes. Hence, such mechanisms must utilize a system
adaptable to changes in cortical size during mammalianevolution.
Both mouse and human cortical progenitors faithfully recapitulate in vitro the sequential generation of principal glutamatergic
subtypes in vivo: preplate (ppl), deep-layer (DL), and upper-layer(UL) neurons (Eiraku et al., 2008; Gaspard et al., 2008; Shi et al.,2012). These studies imply that a common intrinsic program
regulating progenitor cell competence might regulate transitions
between nonradially and radially migrating mammalian corticalsubtypes. Indeed, CR cells, which represent the earliest
glutamatergic cell lineage in the developing neocortex (Hevner
et al., 2003b), differentiate prior to all projection neuron subtypes in vitro (Eiraku et al., 2008; Gaspard et al., 2008). The
unique differentiation capacity of CR cells raises the intriguing
Cell Reports 3, 931–945, March 28, 2013 ª2013 The Authors 931

cause a failure in the transition to generation of projection
neurons, resulting in overproduction of CR cells.
The team next made a conditional knockout (cK0) in which
Foxg1 expression could be switched off mid-development,
to test whether upper-layer progenitor cells retain the ability
to differentiate into CR cells following inactivation of thegene (something the lab had previously shown deep-layer
progenitors capable of doing). Interestingly, upper-layer
progenitor cells did not give rise to CR cells when Foxg1
was inactivated at embryonic day (E) 15, suggesting thatcompetence is progressively restricted during the transition
from deep- to upper-layer neurons, implying that corticalprogenitors use an intrinsic program to steer transitions between cortical subtypes.
To test that possibility, the team generated a conditional
mutant in which Foxg1 was induced at a later stage thannormal, the time of the transition from deep to upper-layer
progenitors in wildtype. When Foxg1 was induced at E14.5,
Kumamoto observed a nearly immediate generation of
deep-layer projection neurons after the excess CR cell production, indicating that Foxg1 works to direct cell identity
decisions. Intriguingly, upper-layer projection neurons also
subsequently appeared in these late Foxg1-induced em-

bryos, which ultimately developed a cortex with cells of thevarious types in numbers similar to those seen in wildtype.
Foxg1, it seems, works as a transcriptional switch ensuringthat the right types of cells emerge at the right timing during
neocortical development.
In an effort to hunt for genes downstream of Foxg1, theteam conducted microarray analyses of progenitor cells
taken at different timepoints from cK0 embryos in which its
expression was switched on at E14.5. Their examination ofgene expression patterns showed that Foxg1 strongly inhibited genes associated with CR cells; chromatin immunoprecipitation suggested this was the result of direct binding
between the factors. Interestingly, the majority of thesebinding domain sequences appears to have emerged after
mammalian evolution.
“The robust and elegant spatiotemporal cell fate switchingsystem mediated by Foxg1 maintains the right balance between projection neurons and CR cells in mouse and human alike,” says Hanashima. “We are excited to continue inour efforts to understand just what goes on in the buildingof the neocortex that underlies many of humanity’s mostfundamental traits.”

Induction of neocortical neurons by Foxg1. Images depicting Foxg1 and EGFP electroporated cells (shown in green) within Foxg1 knockout cells (blue), inducing Ctip2 expression and neocortical projection neuron fate (red).

35

FASTER makes sleep staging a cinch

Genshiro SUNAGAWA

Sleep is a crucial daily activity for an extraordinarily wide
range of animal species. Consciousness is typically divided
into waking, REM (rapid eye movement), non-REM states,reflecting patterns of activity recorded in electroencephalography (EEG) and electromyography (EMG) data. Analyses of sets of such data are indispensable for studies of themechanisms underlying sleep/wake states and sleep pathologies, but the need for inspection by individual humanraters has imposed limits on the scale and speed of assessment and introduced problems of standardization andpotential subjective biases.
A new automated approach to sleep staging developed by
Genshiro A. Sunagawa and colleagues in the Lab for Sys-

36

tems Biology (Hiroki R. Ueda, Project Leader) now potentially makes the process more scalable and objective. Published in Genes to Cells, this new FASTER (Fully AutomatedSleep sTaging method via EEG/EMG Recordings) method
was developed in collaboration with researchers from
Tokushima University, Osaka Bioscience Institute, and Nihon University College of Pharmacy.
The EEG and EMG waveforms observed during sleep differ
between individuals, and between strains of laboratory
mice. For this reason, sleep analyses have traditionally relied on the rating of data obtained from individual animals
over a fixed period (such as 24 hours) to establish standards for the three sleep/wake stages. This process re-©2013 John Wiley & Sons, Inc.
Sunagawa G. A, et al. FASTER: an unsupervised fully automated sleep staging
method for mice. Genes Cells 18.502-18 (2013)

FASTER: an unsupervised fully automated sleep staging
method for mice
Genshiro A. Sunagawa1, Hiroyoshi Sei2, Shigeki Shimba3, Yoshihiro Urade4 andHiroki R. Ueda1,5,6,7,8*
1

Laboratory for Systems Biology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School,
Tokushima 770-8503, Japan
Department of Health Science, College of Pharmacy, Nihon University, Funabashi 274-8555, Japan
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita 565-0874, Japan
Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Kobe 650-0047, Japan
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan
Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan
Department of Systems Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan

2

3
4
5
6
7
8

Identifying the stages of sleep, or sleep staging, is an unavoidable step in sleep research andtypically requires visual inspection of electroencephalography (EEG) and electromyography(EMG) data. Currently, scoring is slow, biased and prone to error by humans and thus is themost important bottleneck for large-scale sleep research in animals. We have developed an
unsupervised, fully automated sleep staging method for mice that allows less subjective andhigh-throughput evaluation of sleep. Fully Automated Sleep sTaging method via EEG/EMG
Recordings (FASTER) is based on nonparametric density estimation clustering of comprehensive EEG/EMG power spectra. FASTER can accurately identify sleep patterns in mice thathave been perturbed by drugs or by genetic modification of a clock gene. The overall accuracy is over 90% in every group. 24-h data are staged by a laptop computer in 10 min, which
is faster than an experienced human rater. Dramatically improving the sleep staging process inboth quality and throughput FASTER will open the door to quantitative and comprehensive
animal sleep research.

Introduction
Ever since the discovery of sleep/wake status relationship to electroencephalography (EEG) in the early
20th century, sleep staging based on EEG has been
the standard method to evaluate sleep/wake status inanimals. In mice, states of consciousness are classified
into at least three stages: nonrapid eye movement
sleep (NREM sleep), rapid eye movement sleep(REM sleep) and wake. Determination of an animal’s
sleep/wake stage is based on visual inspection of EEG
and electromyography (EMG) of the animal by welltrained human raters with some or no computational
support. The classical sleep scoring criteria for miceCommunicated by: Ryoichiro Kageyama*Correspondence: uedah-tky@umin.ac.jp

use the amplitude of selected frequency bands of
EEG and EMG (Fig. 1A). NREM sleep is characterized by large and slow EEG waves with low EMG
amplitude, REM sleep has lower and faster EEG withvery low EMG amplitude, and wake has an EEG pattern similar to REM sleep with very high amplitudeof EMG.
There are two problems scoring sleep/wake stages
by visual inspection: quality control and throughput.
Scoring sleep stages depends on human rater bias andwill differ between two human raters (inter-rater variance) and a human rater analyzing the same data multiple times (intrarater variance). The main reason why
visual inspection by a human rater is still used for sleep
staging is related to the fundamental difficulty of sleep
staging in drawing boundaries between different
stages. There are many factors causing this difficulty,

DOI: 10.1111/gtc.12053© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

quires individual raters to take into consideration inter-individual differences and eliminate noise, but also introduces
subjectivity and the risk of human error, as well as limiting
the scale of data that can be evaluated. Semi-automatedapproaches have been developed, but these still require
human supervision over the setting of scoring criteria.

1

The team next tested the FASTER algorithm against resultsfrom human rater evaluations of mouse sleep/wake stages
in wildtype animals in which these patterns were altered bydrugs that induced wakefulness or sleep. When they compared the results of human raters’ assessments with thosegenerated by the FASTER algorithm, they found greaterthan 90% agreement between the two, indicating the highlevel of precision of this unsupervised, fully automatedmethod. The FASTER approach was also...faster, taking
only 10 minutes to complete an analysis that would require
1–2 hours using conventional methods.

The FASTER method developed by Sunagawa and colleagues now provides a fully automated alternative. The algorithm is composed of three main steps: character extraction, clustering, and annotation. The first of these involves
breaking the observation period into epochs of fixed length(8 sec), converting the EEG/EMG outputs for each epoch
into frequency domain data, and then subjecting them toprincipal component analysis to extract characters of theirpower spectra. After clustering regions showing similar features as determined by nonparametric density estimation,
the annotation was performed without human supervision
as determined by statistical characteristics for each cluster.

Light phase

Genes to Cells (2013)“FASTER by itself won’t make it possible to automate thestaging of huge sleep data sets,” cautions Sunagawa.
“There is still quite a lot of manual work involved, such asattaching electrodes to mice, something we hope to tackle
in future work. We are also looking forward to applying thisnew approach to sleep staging in humans, which will require handling much larger amounts of data.”

Dark phase

Light phase

Dark phase↓ Drug administration

NREMS
REMS

0
20
0
60

Wake

Hourly mean time (min)

60

0

12:00

00:00

12:00

Subjective time
Sleep staging by human rater
Sleep staging by FASTER method
Sleep staging assessments made using visual inspection by human raters (dotted line)
and FASTER methods (orange, blue, green)

37

Genomes shed light on turtle evolutionary history

Juan PASCUAL ANAYA, Naoki IRIE, Shigeru KURATANIThe turtle is something of a monster of evolutionary developmental biology, not by virtue of its stern physiognomy orpowerful jaw, but for its utterly unique morphology. The turtle
carapace, for example, is made of up ribs that have protruded through the animal’s back and fused together in abony shell, while its scapulae have moved to the body interior. Turtles also lack temporal fenestrae, small holes foundin the skulls of other reptilians, further muddying the pictureof their phylogeny.
A study of a pair of turtle genomes by Naoki Irie and othersin the Laboratory for Morphological Evolution (Shigeru Kuratani, Group Director) now sheds new light on its evolutionary history, establishing turtles as a sister group of
crocodilians and birds (collectively, Archosauria), both of38

which diverged from the more ancient lizards and snakes(Lepidosauria). Published in Nature Genetics, this work reflects the collaborative efforts of an international turtle genome sequencing consortium with scientists from 11 institutions, including BGI and Wellcome Trust Sanger Institute.
A number of competing hypotheses have been proposed
to find the turtles’ true branch on the tree of life. These including placing turtles among early reptiles (anapsids), on
account of their missing fenestrae; grouping them together
with the snakes and lizards; and, categorizing them as archosaurs, along with crocodilians and birds. Using a massively parallel shotgun sequencing approach, the consortium generated and analyzed the genomes of the soft-shellturtle (Pelodiscus sinensis) and the green sea turtle (Chelo-

LETTERS©2013 Nature Publishing GroupThe draft genomes of soft-shell turtle and green sea turtle
yield insights into the development and evolution of theturtle-specific body plan
© 2013 Nature America, Inc. All rights reserved.

Wang Z, et al. The draft genomes of soft-shell turtle and green sea turtle yield insights
into the development and evolution of the turtle-specific body plan. Nat Genet
45.701-6 (2013)

OPEN

Zhuo Wang1,12, Juan Pascual-Anaya2,12, Amonida Zadissa3,12, Wenqi Li4,12, Yoshihito Niimura5, Zhiyong Huang1,
Chunyi Li4, Simon White3, Zhiqiang Xiong1, Dongming Fang1, Bo Wang1, Yao Ming1, Yan Chen1, Yuan Zheng1,
Shigehiro Kuraku2, Miguel Pignatelli6, Javier Herrero6, Kathryn Beal6, Masafumi Nozawa7, Qiye Li1, Juan Wang1,
Hongyan Zhang4, Lili Yu1, Shuji Shigenobu7, Junyi Wang1, Jiannan Liu4, Paul Flicek6, Steve Searle3, Jun Wang1,8,9,Shigeru Kuratani2, Ye Yin4, Bronwen Aken3, Guojie Zhang1,10,11 & Naoki Irie2
The unique anatomical features of turtles have raisedunanswered questions about the origin of their unique bodyplan. We generated and analyzed draft genomes of the softshell turtle (Pelodiscus sinensis) and the green sea turtle(Chelonia mydas); our results indicated the close relationshipof the turtles to the bird-crocodilian lineage, from which they
split ~267.9–248.3 million years ago (Upper Permian to Triassic).
We also found extensive expansion of olfactory receptor genes
in these turtles. Embryonic gene expression analysis identified anhourglass-like divergence of turtle and chicken embryogenesis,
with maximal conservation around the vertebrate phylotypic
period, rather than at later stages that show the amniotecommon pattern. Wnt5a expression was found in the growthzone of the dorsal shell, supporting the possible co-option oflimb-associated Wnt signaling in the acquisition of this turtlespecific novelty. Our results suggest that turtle evolution was
accompanied by an unexpectedly conservative vertebrate
phylotypic period, followed by turtle-specific repatterning ofdevelopment to yield the novel structure of the shell.
The unique anatomy of turtles has raised questions about theirevolution1. Their armor, even compared to other armored tetrapods (for example, the armadillo and Indian rhinoceros), is distinct
in that the dorsal part of the shell (carapace) represents transformed vertebrae and ribs. In addition, their shoulder blades or
scapulae1 display an inside-out topology against the rib cage
(Supplementary Fig. 1 and Supplementary Note), and the lack ofa temporal fenestra further complicates the reconstruction of their
phylogenetic position1,2.

Three major hypotheses have been proposed for the evolutionary
origin of turtles, including that they (i) constitute early-diverged reptiles, called anapsids3, (ii) are a sister group of the lizard-snake-tuatara(Lepidosauria) clade4 or (iii) are closely related to a lineage that
includes crocodilians and birds (Archosauria)5–8. Even using molecularapproaches, inconsistency still remains6–9. To clarify the evolution of
the turtle-specific body plan, we first addressed the question of evolutionary origin of the turtle by performing the first genome-wide phylogenetic analysis with two turtle genomes sequenced in this project (thegreen sea turtle, C. mydas, and the Chinese soft-shell turtle, P. sinensis;
Fig. 1a). In brief, the fragmented genomic DNA libraries of the two
turtles were independently shotgun sequenced using the HiSeq 2000
sequencer and assembled using the SOAPdenovo assembler (Online
Methods). The generated turtle genomes were both around 2.2 Gb in
size, with the N50 lengths of scaffolds longer than 3.3 Mb (Table 1,
Supplementary Figs. 2–5 and Supplementary Tables 1–9).
On the basis of the largest turtle data set so far, our phylogenetic analysis, with an orthologous set of 1,113 single-copy coding
genes, robustly indicated that turtles are likely to be a sister groupof crocodilians and birds (Fig. 1b, Supplementary Figs. 6 and 7 andSupplementary Tables 10–13), implying that the temporal fenestrae
in the turtle skull were most likely secondarily lost in the turtle lineage1. A molecular evolutionary clock analysis with time constraints
based on the fossil records estimated that turtles diverged from archosaurians approximately 257.4 million years ago, with a 95% credibility interval between 267.9 and 248.3 million years ago (Fig. 1b,
Supplementary Fig. 7 and Supplementary Table 12). These results
are consistent with the oldest turtle fossil (from 220 million yearsago), named Odontochelys10. The estimated time range corresponds

1BGI-Shenzhen, Shenzhen, China. 2RIKEN Center for Developmental Biology, Kobe, Japan. 3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus,
Hinxton, Cambridge, UK. 4BGI-Japan, Kobe, Japan. 5Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. 6European Bioinformatics
Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. 7National Institute for Basic Biology (NIBB) Core Research Facilities, NIBB, Okazaki, Japan.
8Department of Biology, University of Copenhagen, Copenhagen, Denmark. 9King Abdulaziz University, Jeddah, Saudi Arabia. 10China National GeneBank,
BGI-Shenzhen, Shenzhen, China. 11Center for Social Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark. 12These authors
contributed equally to this work. Correspondence should be addressed to N.I. (asurain@cdb.riken.jp), G.Z. (zhanggj@genomics.org.cn), B.A. (ba1@sanger.ac.uk)
or Y.Y. (yinye@genomics.cn).

Received 4 November 2012; accepted 27 March 2013; published online 28 April 2013; doi:10.1038/ng.2615

NATURE GENETICSADVANCE ONLINE PUBLICATION

1

nia mydas). Both genomes weighed in at about 2.2 billion
base pairs, encoding around 19,000 genes. Comparisonswith 10 other vertebrate genomes (including those of thezebrafish, frog, alligator, chicken and human) identified
1,113 orthologous genes, phylogenetic analysis of which
revealed that turtles are most closely allied with the archosaurian birds and crocs. Using this data supplemented, theauthors place the turtle divergence to around 268–248 million years ago. This conclusion agrees nicely with the fossil
record, as the oldest known turtle dates back 220 million
years. The apparent timing of their emergence, which
would have coincided with the transitional period known as
the Permian–Triassic Transition, characterized by largescale extinctions, raises intriguing questions about possible
linkage between the two events.

model, that in which homology is highest at a midpoint indevelopment (called the vertebrate phylotypic period, during which the basic body plan is being established), anddiffers more widely before and after. To confirm whether
turtles would also fit this scheme, the group used RNA sequencing to compare turtle and chicken, and found hourglass-like divergence in the embryogenesis of these taxa,
indicating that molecularly conserved turtle embryos exhibit a common vertebrate developmental plan, after which
turtle-specific patterning is likey to begin in earnest. The
group not only identified 233 genes that showed turtlespecific expression patterns after the phylotypic stage, butalso found the Wnt5a gene expressed both in the limbs andthe carapacial ridge, suggesting possible involvement of
Wnt signaling in carapace evolution.

Looking next to uncover details of the turtle genome itself,
Irie et al. found an unexcpectedly large number of olfactory
receptor genes, as many as 1,137 in the soft-shell turtle, anumber similar to that possessed by mammals. Of these,
many coded for group α olfactory receptors, used to detect
hydrophilic odorant molecules, which may indicate that
turtles have an excellent ability to sniff out variety of smells.
A number of genes involved in taste, appetite, and metabolic regulation, however, were missing, which may reflect
these animlas’ low-metabolic strategy.

“Species make wide use of a shared toolbox of developmental processes and genetic information, but also generate new morphologies that could truly be called inventions.
These new findings should be useful in guiding predictions
about the course of vertebrate evolution,” says Kuratani.
“But while these genomes are now sequenced, knowing
the order of base pairs is not much more than being able toscan lines of text on a page. From now, we will need to
explore how these genes are actually used during development to build body plans if we hope to decipher their true
meaning.”Previous work by Irie had focused on the developmental
hourglass model that explains the evolutionary diversity ofvertebrate embryos, and found support for the “hourglass”

Phyogenetic history based on comparison of 12 vertebrate genomes and data from the fossil record. Turtles (red) diverged from crocodilian-avian archosaur lineage around the time of
the massive Periman extinction event approximately 250 million years ago.

39

The remarkable tail of polychaete regeneration

Nao NIWA

Segmentation is a highly successful patterning strategy in awide range of animals, including annelids, arthropods, andvertebrates, providing both flexibility and diversity in axial
subdivisions of the body. While in many taxa, the number ofbody segments is strictly fixed, with anterior segments
specified early, and more posterior segments added from aposterior segment addition zone (SAZ) as development
progresses. This zone is lost in most post-embryonic animals,
meaning that segments in general cannot regenerate. But
certain annelids known as polychaetes have been shown tobe able to regenerate amputated tail segments any numberof times. However, previous studies have indicated that the
SAZ in these species lack an identifiable popularion of stem
cells, leaving the mechanisms behind this apparently limitless regenerative capacity an open question.

40

Nao Niwa and colleagues in the Lab for Morphogenetic
Signaling (Shigeo Hayashi, Group Director) now report thatposterior segment regeneration in the polychaete Perinereis nuntia relies on the provision of inductive signals fromthe pre-existing segment adjacent to the newly forming
segment. Published in Developmental Biology, these new
findings help to explain the mysterious ability of these animals to regrow body segments indefinitely.
The study began with observations of segment proliferation
during the development of P. nuntia. Niwa and colleagues
found that cells in the segment preceding the posteriormost region (pygidium) were in active cell cycle, while thosein the terminal region remained at rest. The active cells expressed high levels of PCNA, and formed what the group

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎©2013 Elsevier B.V.
Niwa N, et al. Homeogenetic inductive mechanism of segmentation in polychaete
tail regeneration. Dev Biol 381.460-70 (2013)

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Contents lists available at SciVerse ScienceDirect

Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Homeogenetic inductive mechanism of segmentation in polychaete
tail regeneration
Q1

Nao Niwa a, Ai Akimoto-Kato a, Masashi Sakuma b, Shigehiro Kuraku c, Shigeo Hayashi a,d,nabcdLaboratory for Morphogenetic Signaling, RIKEN Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Institute of Biological Sciences, Sugadaira Montane Research Center, University of Tsukuba, Sugadaira Kogen Ueda, Nagano 386-2204, Japan
Genome Resource and Analysis Unit, RIKEN Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Department of Biology, Kobe University Graduate School of Science, Kobe, Hyogo 657-8501, Japan

art ic l e i nf o

a b s t r a c t

Article history:
Received 12 February 2013
Received in revised form
24 March 2013
Accepted 4 April 2013

Segmentation is a body-patterning strategy in which new segments are speciﬁed from a segmentaddition zone containing uncommitted cells. However, the cell-recruitment process is poorly understood.
Here we investigated in detail the segmentation in a polychaete annelid, Perinereis nuntia (Lophotrochozoa), in which new segments emerge at the boundary between the posterior end of the segmented
region and the terminal pygidium. Cells at this border synchronously remodel their chromatin, enter thecell cycle, and undergo oriented cell division, before being added to new segments. wingless is expressedat the posterior edge of the pre-existing segment, abutted by hedgehog in the ﬁrst row of the new
segment. Overstimulation of Wingless signaling caused excess cells to enter the cell cycle, prolonging
segmentation and widening the new segment. Thus, segment addition may occur by a homeogenetic
mechanism, in which Wingless expressed in the differentiated segment coordinates the stepwise
recruitment of undifferentiated cells from the segment/pygidium boundary.
& 2013 Elsevier Inc. All rights reserved.

Keywords:
Segmentation
Regeneration
Wingless
Polychaete

Introduction
Segmentation, allows axis-length ﬂexibility and pattern diversity, and has been one of the most successful body-patterning
strategies in bilaterians. Segments are speciﬁed from uncommittedprogenitor cells partitioned into equal-sized groups arranged
along the anterior-posterior axis. The segment speciﬁcation mustbe highly reproducible and precise, and various mechanisms areused to accomplish it by different taxa (Peel et al., 2005). In
vertebrates (deuterostomes), somite segmentation is promoted by
Notch signals that propagate as an anteriorly directed wave in theundifferentiated presomitic mesoderm (Palmeirim et al., 1997;
Saga and Takeda, 2001).
Segmentation in arthropods has been most extensively studied
in the insect Drosophila melanogaster. In Drosophila, a transcription
factor of maternal origin, Bicoid (Bcd), forms an anterior–posterior
gradient in early syncytial embryos and regulates the expression of
zygotic gap class transcription factors (Driever and NüssleinVolhard, 1988; Small et al., 1991). The Bcd and gap protein
gradients in the syncytium together regulate the expression ofprimary pair-rule genes, which collectively specify the locations ofnCorresponding author at: Laboratory for Morphogenetic Signaling, RIKEN
Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku,
Kobe, Hyogo 650-0047, Japan. Fax: +81 78 306 3183.
E-mail address: shayashi@cdb.riken.jp (S. Hayashi).

0012-1606/$ - see front matter & 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ydbio.2013.04.010

67
68
69
70
14 segment anlagen (Lawrence, 1992; Mito et al., 2011; Wilson
71
et al., 2010). Thus, gradients of Bcd and its subordinate gene72
products subdivide the entire egg into segment primordia prior to73
the onset of cell proliferation. However, this mechanism is limited
74
to the relatively small group of insect species that undergo75
segmentation at the syncytium stage (Palmeirim et al., 1997).
76
Most insects and arthropods undergo segmentation in a cel77
lular environment. Typically anterior most segments are speciﬁed78
in early stages, followed by sequential addition of new segments
79
from a posterior segment-addition zone (SAZ), similar to verte80
brate somites. In spiders (Chelicerates), Notch is reported to be81
required for proper segment formation (Stollewerk et al., 2003).
82
However, studies in model insects (Gryllus, Apis) have shown that
83
Notch is mainly involved in insect segment patterning and not
84
segment formation (Mito et al., 2011; Wilson et al., 2010). In
85
Gryllus, wingless (wg) and its downstream regulator caudal are
86
expressed in the growth zone, and their signaling is required for87
segment formation (Bolognesi et al., 2008; Miyawaki et al., 2004;
88
Shinmyo et al., 2005). During insect segmentation, wg and hedge89
hog (hh) are expressed in stripes that abut each side of segment
border and regulate cell patterning and segment border formation Q2 90
91(Dougan, 1992; Farzana and Brown, 2008; Heemskerk and92
DiNardo, 1994). However, although the genes responsible for93
segmentation in non-Drosophila insects are rapidly being identi94
ﬁed, many fundamental features of segmentation remain elusive.
95
Key questions include how the initial segment width and its
96
97

Please cite this article as: Niwa, N., et al., Homeogenetic inductive mechanism of segmentation in polychaete tail regeneration. Dev.
Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.04.010i

called the zone of cell cycle synchronization (ZCS). As theprocess of segment proliferation progressed slowly during
development, the group decided to study it more closely inregenerating animals, which add segments more quickly.
As in normal development, regenerative proliferation of segments involved the addition of rows of cells provided by theanterior segment, characterized by expression of PCNA,
with one new segment forming for each five new rows ofcells.
To determine the molecular mechanisms underlying thisprocess, Niwa looked at signaling pathways common tosegmentation in arthropods and vertebrates, suspectingthat these might be conserved in polychaetes as well. The
polychaete homolog of the Drosophila gene wingless (wg)is expressed in a stripe at the posterior edge of each segment during proliferation, and faces a similar band of thepolychaete hedgehog (hh) homolog in the cell at the anterior margin of the newly forming segment. A closer observation of the expression of these genes suggested that wg,
hh, and PCNA are sequentially expressed in an anterior toposterior direction during segment formation, suggestive ofa central instructive role for wg in the process. This sequence plays out repeatedly during segment proliferation,such that the program reinitiates for every fifth new cell,ensuring that individual segments are consistent in length.
The picture that emerges from these findings is that P. nuntia differs from other taxa in its approach to forming new
segments—rather than generate signals from the edge ofthe newly forming segment, the signaling in polychaete appears to move from pre-existing cells at the border of theproliferation zone, a mechanism that bears resemblance tothe theory of homeogenetic induction first proposed by
Spemann and Mangold in 1927.
“We were surprised to find that segment formation in thepolychaete differs so significantly from that in insects andvertebrates, specifically in its unique ability to regeneratesegments,” says Hayashi. “I never cease to be amazed at
the surprises the rich diversity of the animal world holds instore.”

Electron micrograph of Perinereis nuntia, showing tail and segments (left). Expression of hedgehog (hh) and wingless (wg) in a
newly forming segment (top right); expression of PCNA in the last row of cells in the terminal segment shows active proliferation(bottom right).

41Organizer signaling scales to embryo size

While body sizes may differ significantly between individuals
within the same species, body shape tends to remain constant. Even in closely related species, such as mouse andrat, while the overall size of the individual animals is quite
different, the shape of their bodies are very similar. In a more
extreme demonstration of this phenomenon, it has beenshown that, after being cut in two, the dorsal half of a frog
embryo can develop into a smaller, but morphologically ordinary tadpole, rather than one featuring only dorsal structures. The reason behind this conservation of pattern in
differently sized embryos, however, has remained a mystery.
A new study by Hidehiko Inomata and colleagues in the
Lab for Organogenesis and Neurogenesis (Yoshiki Sasai,Group Director), in collaboration with the Lab for Physical
Biology (Tatsuo Shibata, Research Unit Leader), now points
to a role for gradients of signaling factors from the embryonic organizer region of the African clawed frog, Xenopus
laevis, in maintaining proportional patterning. Published inCell, these new findings stand to answer a longstanding
question of how embryonic shape can be scaled to embryosize.
In early amphibian development, BMP signals from the ventral side of the embryo are countered by BMP antagonists
secreted from the organizer regions, first described byHans Spemann in the 1920s. The interaction between
these two streams generate a dorsal–ventral signaling gradient, which in turn sets the stage for further developmentalroutines: neural lineages form in dorsal regions of higherorganizer signaling activity, while lower activity at the ventral
side of the embryo triggers differentiation into blood andother lineages. What has remained an enigma is that thatsome mechanism must enable the maintenance of this gradient, even when the size of the embryo is halved, as suggested by the bisection studies described above.

Tatsuo SHIBATA, Hidehiko INOMATA

42

To dig deeper into this question, Inomata first developed a‘hyperventralized’ embryo to allow him to zero in on andanalyze the functions of discretely expressed genes in reconstructing the dorsal–ventral (D–V) axis. When he misexpressed the organizer signal Chordin, he found that a gradient akin to that seen in wildtype embryos was established,leading to the formation of a normal D–V axis. Gradients aredefined by distribution, so Inomata was curious about what©2013 Elsevier B.V.
Inomata H, et al. Scaling of Dorsal-Ventral Patterning by Embryo Size-Dependent
Degradation of Spemann’s Organizer Signals. Cell 153.1296-1311 (2013)

Scaling of Dorsal-Ventral Patterning
by Embryo Size-Dependent Degradationof Spemann’s Organizer Signals
Hidehiko Inomata,1,3,* Tatsuo Shibata,2 Tomoko Haraguchi,1 and Yoshiki Sasai1,*
1Organogenesis and Neurogenesis Group
2Laboratory for Physical Biology
RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
3Precursory Research for Embryonic Science and Technology (PRESTO) Program, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi, Saitama, 332-0012, Japan*Correspondence: hideino@cdb.riken.jp (H.I.), yoshikisasai@cdb.riken.jp (Y.S.)
http://dx.doi.org/10.1016/j.cell.2013.05.004

SUMMARY

Spemann’s organizer plays a key role in dorsalventral (DV) patterning in the amphibian embryo by
secreting diffusible proteins such as Chordin, anantagonist to ventralizing bone morphogenetic proteins (BMPs). The DV patterning is so robust that anamphibian embryo with its ventral half surgicallyremoved can develop into a smaller but proportionally patterned larva. Here, we show that this robust
patterning depends on facilitated Chordin degradation and requires the expression of the Chordinproteinase inhibitor Sizzled on the opposite side.
Sizzled, which is stable and diffuses widely along
the DV axis, stabilizes Chordin and expands its distribution in the ventral direction. This expanded Chordin distribution, in turn, limits BMP-dependent
Sizzled production, forming an axis-wide feedback
loop for shaping Chordin’s activity. Using bisection
assays, we demonstrate that Chordin degradation
is dynamically controlled by embryo-size-coupled
Sizzled accumulation. We propose a scaling modelthat enables the DV pattern to adjust proportionallyto embryonic axis size.
INTRODUCTION
In Xenopus, sperm entry triggers the dorsal-ventral (DV) polarity
formation, causing cortical rotation and activating canonical Wnt
signaling in a dorsally predominant manner (Heasman, 2006).
This Wnt activation, which depends on maternal b-catenin, induces the gene network that determines the side of Spemann’sorganizer after the midblastula transition (Spemann and Mangold, 1924), including genes that encode organizer-specifying
transcription factors, such as Goosecoid, and secreted organizer factors (De Robertis, 2009; De Robertis and Kuroda, 2004).
In Xenopus, positional information along the DV axis is primarily given by local bone morphogenetic protein (BMP) signals,

which instruct ventralization. The organizer provides dorsal positional values by secreting BMP antagonists such as Chordin and
Noggin (Piccolo et al., 1996; Sasai et al., 1994; Smith and Harland, 1992), which generate a reverse BMP signal gradient along
the DV axis (dorsal-low/ventral-high). If the organizer factors are
depleted, the whole embryo is hyperventralized because ofincreased activity of BMP4, which is autonomously produced
from zygotic transcripts (Khokha et al., 2005). The gastrula of
Xenopus laevis has a diameter of 1.1 mm, which is fairly large
compared to the mammalian gastrula. Given the substantial perturbations that can affect an amphibian embryo developing in thewild, it is surprising that a single signaling center, the organizer, isable to shape a robust pattern across this relatively long distanceduring the short period of frog gastrulation.
Closely related amphibian species that are thought to havepatterning molecules with similar properties can vary widely inembryo sizes. In fact, eggs and gastrulae of the same species(e.g., Xenopus laevis) occasionally differ in size; yet despite thesize differences, they still give rise to embryos with indistinguishable patterns (Figures S1A–S1C available online). An even more
remarkable example of the robust shape similarity in Xenopus is
the self-regulatory phenotype after bisection (Kimelman andPyati, 2005; Reversade and De Robertis, 2005). When a blastulaembryo is physically bisected into dorsal and ventral halves, thedorsal piece, which autonomously forms the organizer, develops
into a proportionally patterned smaller embryo along the DV
axis—instead of having disproportionately large dorsal tissues,
as would be expected from its origin. Thus, the dynamic adjustment of the axial pattern to the embryonic size, termed scaling(Ben-Zvi et al., 2011), is an important characteristic of DV
patterning by the organizer, one that cannot be easily explainedby simple diffusion of the same set of molecules. In our present
study, we address this scaling issue by elucidating an axis-wide
feedback control system for Chordin distribution, which iscontrolled by the secreted Chordin proteinase inhibitor Sizzled(Lee et al., 2006; Muraoka et al., 2006).
Xenopus DV patterning involves multiplex feedback mechanisms with strong resilience and robustness. For instance,even if a half of the organizer tissue is removed by resection,
the embryo can develop normally by regaining proper organizer

1296 Cell 153, 1296–1311, June 6, 2013 ª2013 Elsevier Inc.

factors that control how Chordin is distributed in the embryo, and discovered that the action of Sizzled, which inhibits its degradation, plays a key role in stabilizing the Chordin
molecule along a grade. Specifically, when he changed thelevel of Sizzled expression, he found that the Chordin gradient was altered dramatically, tilting from a gradual slope toa steep cliff. Inomata determined that the interaction between these two factors involved the following feedback
loop: stabilization by Sizzled enables the broader diffusionof Chordin, which Chordin itself limits by inhibiting further
Sizzled expression.
What then of dorsalized embryos? Knowing their ability togrow into small but proportional larvae, Inomata examinedgene expression in bisected embryos, and found that theexpression levels of dorsal markers in dorsal halves also
dropped by around 50%, even as overall shape was maintained. But in loss-of-Sizzled-function embryos, bisection
triggered no such reduction in dorsal gene expression, andthe embryos failed to develop normally, suggesting that
Sizzled is responsible for maintaining the Chordin gradientin a manner scaled to embryo size.
The group’s conclusion, that stabilization of Chordin by
Sizzled and inhibition of Sizzled by Chordin maintain the
Chordin gradient in a size-dependent manner, gained further support from the results of simulations of various starting values and perturbations for these factors and embryosize.
“It will be interesting to discover whether a similarly balanced feedback mechanism is at work in the embryos ofother species,” says Sasai, “as well as to determine whether the scaling mechanism changes over time as the embryo
grows.”

Sizzled concentration scales to embryo size, which proportionally affects
Chordin stability (degradation), resulting in the formation of a Chordin gradientscaled to the size of the embryo and maintenance of homology.

43

Mesoderm induction: As in frog, so in bird?

Yuping WU, Cantas ALEV

British developmental biologist Lewis Wolpert famously remarked that the most important events in one’s life takeplace during gastrulation. This crucial stage of embryogenesis involves dynamic morphological rearrangements thatgive rise to the three germ layers of the body: ectoderm,
mesoderm, and endoderm. While gastrulation as a general
phenomenon is highly conserved, the details differ between
major phylogenetic groups. In fish and amphibians (anamniotes), for example, the induction of mesoderm is centeredon a site known as the blastopore, while in amniotes suchas birds and mammals, mesoderm forms at the primitive
streak. Despite the central importance of this process,however, the evolutionary roots for this diversity in its developmental program have remained elusive.

44

Cantas Alev and colleagues in the Lab for Early Embryogenesis (Guojun Sheng, Team Leader) have now showed
that, with a little manipulation, chicken embryos can bemade to undergo an anamniote-like form of mesoderm induction. Published in Development, these results shed newlight into the evolution of gastrulation strategies.
In frog and other anamniote embryos, gastrulation proceeds radially outward from the blastopore, inducing a ringof cells that migrate to the embryo interior, forming the mesodermal layer. In amniotes such as chicken, however, mesoderm cells are formed solely from a region known as theprimitive streak, which evolved as an alternative to the ‘circumblastoporal’ routine. Despite these differences, bothforms of gastrulation involve the expression of conserved

RESEARCH REPORT 2691

©2013 The Company of Biologists
Alev C, et al. Decoupling of amniote gastrulation and streak formation reveals a morphogenetic
unity in vertebrate mesoderm induction. Development 140.2691-6 (2013)

Development 140, 2691-2696 (2013) doi:10.1242/dev.094318© 2013. Published by The Company of Biologists Ltd

Decoupling of amniote gastrulation and streak formation
reveals a morphogenetic unity in vertebrate mesoderm
induction
Cantas Alev, Yuping Wu, Yukiko Nakaya and Guojun Sheng*
SUMMARY
Mesoderm is formed during gastrulation. This process takes place at the blastopore in lower vertebrates and in the primitive streak(streak) in amniotes. The evolutionary relationship between the blastopore and the streak is unresolved, and the morphogenetic andmolecular changes leading to this shift in mesoderm formation during early amniote evolution are not well understood. Using thechick model, we present evidence that the streak is dispensable for mesoderm formation in amniotes. An anamniote-likecircumblastoporal mode of gastrulation can be induced in chick and three other amniote species. The induction requires cooperative
activation of the FGF and Wnt pathways, and the induced mesoderm field retains anamniote-like dorsoventral patterning. We proposethat the amniote streak is homologous to the blastopore in lower vertebrates and evolved from the latter in two distinct steps: an
initial pan-amniote posterior restriction of mesoderm-inducing signals; and a subsequent lineage-specific morphogenetic modificationof the pre-ingression epiblast.
KEY WORDS: Amniotes, Avian, Blastopore, Gastrulation, Mesoderm, Primitive streak

Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology,
2-2-3 Minatojima-minamimachi, Chuo-Ku, Kobe, Hyogo 650-0047, Japan.
*Author for correspondence (sheng@cdb.riken.jp)
Accepted 10 April 2013

signaling factors, such as FGF, Wnt, and TGFβ. What Alev
sought to determine in the present study was whether hecould recapitulate the more ancient, anamniote approachto gastrulation in an amniote embryo by tweaking these signaling pathways.
He first developed a new method for injecting molecularsignals into the subgerminal cavity of the developing embryo, and used it to test the effects of widespread ectopic
presence of prominent signaling factors. He found that injection of FGF4 induced a ring of mesoderm (as labeled byBrachyury) in the marginal zone, irrespective of whether theprimitive streak formed or not (the streak formed in fewer
than half of the injected embryos). The mesoderm thus
formed expressed epithelial–mesenchymal transition (EMT)
markers, as in normal chick gastrulation, and dorsal–ventral
axis formation was unperturbed, suggesting that these bird
embryos could be led to undergo a very frog-like form ofgastrulation, independent of primitive streak formation.
In these manipulated embryos, FGF was not able to induce
mesoderm in the central epiblast, raising the likelihood that

In chicken embryos mesoderm is normally formedin the primitive streak. (blue: mesoderm marker
Brachyury)

mesoderm mimics that in anamniote blastopore. Using additional
avian and reptilian species, we demonstrated that the ability to
uncouple mesoderm and streak formation, and to generate
mesoderm in an ancestral ‘circumblastoporal’ mode, is conserved
among amniotes. Finally, we presented a model to explain theevolutionary shift from a radial to a dorsally restricted mode of
mesoderm formation.
MATERIALS AND METHODS
Subgerminal cavity injection

Fertilized chicken, quail, emu and turtle eggs were purchased from local
farms. The following growth factors were used: FGF4 (#235-F4, R&D
Systems; final concentration 50 ng/l), FGF2 (#03-0002, Stemgent; 50 ng/l),
FGF8 (#F6926, Sigma; 50 ng/l), activin A (#338-AC, R&D Systems; 50
ng/l) and Wnt3a (#1324-WN, R&D Systems; 10 ng/l). SB431542 was from
Sigma (#S4317-5MG; 25 M). Growth factors/chemical inhibitors werediluted in Pannett-Compton to their final concentrations, together with FastGreen for visualization (#061-00031, Wako; final 0.1%). After brief warm-up(30 minutes at 38.5°C), chicken eggs were opened and their content
transferred to pre-cut foster shells. For quail, emu and turtle eggs, host shells
were cut with a drill or small scissors to leave the cut-edges smooth. A volumeof 1-2 l was injected by mouth-pipetting using a pulled glass capillary (#2000-050, Drummond). The subgerminal cavity can hold up to 5 l (Petitte etal., 1990; Sang, 2004; van de Lavoir et al., 2006) and control-injected embryos
developed normally. Afterwards, shells were filled up with thin albumen,covered with thin cling-film (from Nippon Paper-Pak), and assembled using
plastic rings (Showa Furanki Company) and rubber bands (supplementarymaterial Fig. S1). Eggs were incubated horizontally at 38.5°C in a humidified
incubator. Turtle eggs were incubated at 32°C vertically after covering
injected embryos with cling-film.
Embryology and imaging

In situ hybridization for chicken, quail, emu and turtle embryos followed
standard chicken protocol (Nagai et al., 2011; Stern, 1998), as with paraffinembedded sectioning (10 m, Microm-HM325 microtome), whole-mountembryo imaging (Olympus-SZX12; Olympus-DP70) and section imaging(Olympus-BX51; Olympus-DP70). For immunofluorescence (Fig. 1;
supplementary material Fig. S1), unincubated embryos were fixed in ovo(4% paraformaldehyde), collected and stained with phalloidin (AlexaFluor-

DEVELOPMENTINTRODUCTION
The mesoderm is one of the three principle germ layers and isformed during gastrulation. Mesoderm induction is regulated byevolutionarily conserved molecular mechanisms (Harvey et al.,2010; Kimelman, 2006), yet morphogenetic events during its
formation vary in different vertebrate clades (Arendt and NüblerJung, 1999; Meinhardt, 2006; Shook and Keller, 2008; SolnicaKrezel, 2005). In lower vertebrates (anamniotes), mesoderm cells
are induced radially at the blastopore. This circumblastoporal modeof mesoderm formation was lost during early amniote evolution. In
birds and mammals, mesoderm cells are generated exclusively fromthe streak, a transitory structure unique to the amniotes. The
evolutionary origin of the streak has long been debated and its
relationship to the blastopore remains unresolved. The reptiles, thestem amniote lineage from which both the mammals and birds
evolved, do not have a streak (Bertocchini et al., 2013).
Furthermore, mammals and birds have been hypothesized to
employ different cellular mechanisms to generate a streak (Arnold
and Robertson, 2009; Chuai and Weijer, 2008; Stern, 2004;
Voiculescu et al., 2007; Williams et al., 2012).
In this work, we used the chick model and examined therelationship between streak formation and mesoderm induction andthat between the streak and blastopore. We found that irrespectiveof whether a streak forms or not, mesoderm cells can be induced
radially (‘circumblastoporally’) in the marginal zone. These
ectopically formed mesoderm cells undergo migration and initiate
terminal differentiation. This capacity to generate mesoderm
radially was revealed by manipulating the availability of mesoderm
inducer FGF and requires cooperative activation of the FGF and
Wnt signaling pathways. Dorsoventral patterning in the induced

other factors important for mesoderm formation were also
involved. When Alev co-electroporated Wnt and FGF signaling factors into the medial epiblast of chick embryos , hefound that the combined activity resulted in induction of
mesoderm even in this central region. Additional experiments modulating a third signaling molecule, TGFβ, revealed that this pathway plays roles in the dorsalization of
the embryo as well as the ingression of mesoderm cells.
Suspecting that the effects of FGF activation might be conserved in amniotes other than chick, Alev repeated the experiment in quail, emu, and turtle embryos, and observed
similar reversion to an anamniote-like circumblastoporalmode of gastrulation.
“Seeing dramatic differences in the morphogenetic processes taking place during gastrulation between anamniotes and amniotes, one would expect that this would alsoentail a significant change in the underlying molecularmechanisms” says Sheng. “But the results from these newexperiments clearly show that, whether in frog or in bird, theessential process is fundamentally similar.”Subgerminal cavity injection of FGF, however, induces a ring of
mesoderm not associated with the primitive streak. (blue: mesoderm marker Brachyury)45

Evolutionary Morphology
By studying the evolutionary designs of diverse animal species, I hope to gain a deeper insight
into the secrets behind the fabrication of morphological designs. Integrating the fields ofevolutionary morphology and molecular genetics, our lab seeks to expand the understanding of the relationship between genome and morphology (or body plan) through investigating the evolutionary changes in developmental processes, and also the process of evolutionin which phenotypic selection shapes developmental programs. Our recent studies have focused on novel traits found in the vertebrates, such as the jaw, the turtle shell, and the mammalian middle ear. By analyzing the history of developmental patterns, I seek to open new avenues toward answering as-yet unresolved questions about phenotypic evolution in vertebrates
at the level of body plans.

Shigeru KURATANI Ph.D.
http://www.cdb.riken.go.jp/en/kuratani
Shigeru Kuratani received his masters and Ph.D.
from the Kyoto University Department of Zoology.
He spent the period from 1985 to 1988 studying
comparative embryology in the Department of
Anatomy, University of the Ryukyus, and 1988 to
1991 working in experimental embryology in the
Department of Anatomy at the Medical College ofGeorgia before moving to the Biochemistry Department at the Baylor College of Medicine, where he
was engaged in molecular embryological research.
He returned to Japan in 1994 to take a position as
associate professor at the Institute of Medical Embryology and Genetics in the Kumamoto University
School of Medicine. He moved to Okayama University to assume a professorship in the Department of
Biology in 1997, where he remained until he wasappointed team leader at the CDB. He was appointed group director in 2005.

Through the study of evolutionarily novel structures, our lab has identified changes in developmental mechanisms that have obliterated the structural homologies between organisms
as evidenced in such novel structures as the jaw in gnathostomes (jawed vertebrates) andthe turtle shell. Developmental studies of the cranial region of the lamprey are intended toshed light on the true origins of the vertebrate head and neck, as lampreys lack a number ofimportant features, including jaws, true tongues, and trapezius muscles, that are possessed
only by gnathostomes. We aim to resolve the question of what primary factors have changed
during evolution by comparing the developmental patterns that yield such innovations, and by
the experimental construction of phenocopies in one animal that mimic structures in another.
The turtle’s shelled body pattern appears at the end of a graded series of changes in thefossil record. Our lab’s research into turtle carapace development addresses the developmental changes that resulted in this abrupt and dramatic morphological change, and is aimed at
identifying genes that function differently in turtle and other amniotes, which it is hoped will
provide a key to discovering the true targets of natural selection in the acquisition of a shell.

Staff
Group Director

Student Trainee

Shigeru KURATANI

Hiroki HIGASHIYAMA
Mana NAMETA

Research Scientist

Noritaka ADACHI
Tatsuya HIRASAWA
Naoki IRIE
Christian MITGUTSCH
Takayuki ONAI
Juan PASCUAL ANAYA
Fumiaki SUGAHARATechnical Staff

Satoko FUJIMOTO
Tamami HIRAI
Yasuhiro OISI
Iori SATO

InternJessica PLANADE
Part-Time Staff

Shigemi SHIBUYAKazuko YAMAMOTO
Assistant

Naeko MINAMI
Tomoko OYANAGI

Recent Publications
Hirasawa T, et al. The endoskeletal origin of the turtle carapace. Nat Commun 4.2107 (2013)
Wang Z, et al. The draft genomes of soft-shell turtle andgreen sea turtle yield insights into the development and evolution of the turtle-specific body plan. Nat Genet 45.701-6(2013)
Oisi Y, et al. Craniofacial development of hagfishes and theevolution of vertebrates. Nature 493.175-80 (2013)
Ota K. G, et al. Identification of vertebra-like elements andtheir possible differentiation from sclerotomes in the hagfish.
Nat Commun 2.373 (2011)
Nagashima H, et al. Evolution of the turtle body plan by thefolding and creation of new muscle connections. Science325.193-6 (2009)
Ota K G, et al. Hagfish embryology with reference to the evolution of the neural crest. Nature 446.672-5 (2007)

CT-scanned skeleton of Pelodiscus sinensis juvenile48

Sensory Development
Organogenesis is a monumental undertaking. From siting a given organ to its correct location, to confirming that it is correctly engineered and constructed, to ensuring the necessaryfunctional connections with other organs, the embryo has a daunting task before it; indeed,
perhaps we should be surprised that the result is so frequently successful. Taking sensory
organogenesis as our model system, we hope to be able to follow the route an organ takes
in its development, and seek to understand the steps that allow it to reach maturity. One aim
is to be able to recapitulate these steps in vitro, with the ultimate goal of providing potentially clinically relevant applications.
The ear provides a particularly rich system for study. The inner ear, which converts mechanical stimulation to electrical impulses, is formed from a piece of tissue originally destined
to become skin; a fate that is altered by instructive signals in a process known as induction.
My lab has been focusing on the role that extrinsic signals play in its development. In particular, the roles that they play in cell fate, in balancing proliferation and differentiation and in
directing morphogenesis. For this we have predominantly focused on the fibroblast growth
factor (FGF) family. Our research aims is to bridge developmental events with the cellularmechanisms that drive development, either controlling quiescence, proliferation or imparting
the distinctive phenotypic characteristics of particular cell types.
Our investigations have elucidated a number of mechanisms involved in the development of
the inner ear. We have been instrumental in describing the earliest events of inner ear induction providing the clearest view of the developmental decisions that are made during induction. We have also discovered a role for extrinsic signals in regulating the cytoskeletal changes
involved in tissue remodelling during the invagination of the inner ear. More recently, we havebeen investigating later signalling events in the generation of hair cells, the sensors of theinner ear, and how then their morphology is specified and maintained.
Environmental effects, genetic disorders or just the passage of time can damage hair cells,
and the damage impairs our ability to hear. Our hope is that understanding inner ear formation allows us insights into potentially repairing or, at least mitigating the damage, elicited onhair cells.

Raj LADHER Ph.D.
http://www.cdb.riken.go.jp/en/ladher
Raj Ladher received his B. Sc. in biochemistry from
Imperial College, London in 1992 and his Ph. D. indevelopmental biology from the National Institutefor Medical Research in 1996, for his thesis on thecloning of the Xenopus homeobox gene, Xom. He
worked as a research associate at King's College,
London from 1996 to 2000 in the lab of Pip FrancisWest, and first came to Japan during that period as
a visiting scientist at the Tokyo Medical and Dental
University. In 2000, he moved to the University of
Utah as a postdoctoral fellow in Gary Schoenwolf'slaboratory. He was appointed team leader at the
CDB in 2002.

Staff
Team Leader

Research Associate

Rajesh LADHER

Kazuya ONO

Research Scientist

Part-Time Staff

Akira HONDA
Siu Shan MAKPaul O'NEILL
Xiao Rei SAI

Yoshiko KONDO
Anna WRABELAssistant

Noriko HIROIVisiting Researcher

Stephen FREEMAN
Tomoko KITA
Yuko MUTA

Recent Publications
Ono K, et al. FGFR1-Frs2/3 signalling maintains sensory progenitors during inner ear hair cell formation. PLoS Genet
10(1): e1004118 (2014)
Paschaki M, et al. Retinoic Acid maintains olfactory progentior cells during neurogenesis and regeneration. Neural Development 5: 13 (2013)
Honda A, et al. From placode to labyrinth: Culture of thechicken inner ear. Methods (2013)
Freter S, et al. Pax2 modulates proliferation during the specification of the otic and epibranchial placodes. Dev Dyn
241.1716-1728 (2012)
O’Neill P, et al. The amniote paratympanic organ develops froma previously undiscovered sensory placode. Nat Commun
3.1041 (2012)

Ciliated mechanoreceptors, or inner ear hair cells, transduce sound information in the organ of corti,
part of the mammalian cochlea. Shown are the four rows of inner ear hair cells that make up the organof corti, stained with alpha-tubulin (to show kinocilia in green) and phalloidin to show the actin-basedstereocilia (in red).

Ladher R. K., et al. From shared lineage to distinct functions:the development of the inner ear and epibranchial placodes.
Development 137.1777-85 (2010)49

Cell Asymmetry
Our group explores the mechanisms underlying the organization of cells into highly orderedstructures in the developing brain. During brain development, neural stem cells generate alarge number of neurons and glia of different fates at appropriate points in time; the framework and size of the brain depend on the spatiotemporal behavior of neural stem cells,
which are highly dynamic in their modes of division and gene expression. We focus ourstudy on the programs by which behaviors of neural stem cells are controlled, using invertebrate (Drosophila) and vertebrate (mouse) model systems.

Fumio MATSUZAKI Ph.D.
http://www.cdb.riken.go.jp/en/matsuzaki
Fumio Matsuzaki is engaged in the study of the genetic programs underlying neural development,with a focus on the asymmetric division of neuralstem cells. His laboratory has discovered several
key mechanisms controlling asymmetric divisions inneural progenitor cells using Drosophila and mouse
as model systems. He has also recently found anovel type of self-renewing progenitor in the developing cerebral cortex in rodents, providing new insights into the enormous increase in brain size during mammalian evolution.

Staff
Group Director

Fumio MATSUZAKI
Research Scientist

Ikumi FUJITA
Hiroshi ISHIMOTO
Daijiro KONNO
Keiko NAKANISHI
Atsunori SHITAMUKAI
Shigeki YOSHIURA
Research Associate

Noriko YUTSUDO
Visiting Scientist

Ayano KAWAGUCHI
Yoichi KOSODO
Technical Staff

Chiaki KISHIDA
Raymond TerhuneKUNIKANE
Taeko SUETSUGU
Nobuko TANIYAMA

Drosophila neural stem cells, called neuroblasts, provide an excellent model system for theinvestigation of fundamental aspects of asymmetric division, a process essential to the generation of cells of divergent type during proliferation. We have been investigating mechanisms controlling asymmetric divisions, including the cell polarity and spindle orientation. We
also extend our research scope to understand how neurogenesis is controlled in tissue
space depending on the environments that surround the nervous system. We recently identified an extrinsic mechanism that controls the orientation of division (cell polarity) in neuroblasts relative to the overriding ectoderm (Yoshiura et al., 2012), which determines the orientation of neural tissue growth.
The vertebrate brain evolved rapidly, resulting in an expansion of the size of the brain, which
comprises a larger number of neurons arranged in a vastly more complex functional network
than that in invertebrate. Neural stem cells typically adopt three states-proliferative (symmetrically dividing), neurogenic (asymmetrically dividing), and resting-and undergo transitions among the states, on which the basic organization of the brain depend. We are investigating mechanisms that determine the individual states of neural stem cells, and control
transitions between states in mouse as well as mechanisms for generating neural progenitor
cell diversity (see figure). We recently discovered a novel transition in the division mode in thedeveloping mouse cortex from radial glia (typical neural stem cells with the epithelial structure) to translocating neural stem cells, basal radial glia (Shitamukai et al., 2011), whichbecome a major population of neural stem cells in mammals with gyrencephalic brains, suchas primates and ferrets. We are investigating the mechanisms that underlie the formation,
maintenance, and expansion of these neural stem cells, by using model mice that produce
large numbers of basal radial glia as well as ferrets.

Student Trainee

Shun MASE
Kaga SAKAI
Part-Time Staff

Hiromi ISHIMOTO
Erina MATSUMOTO
Yoko OTSUKA
Megumi SHIBATA
AssistantJunko ISHIGAI

Tomoko AOKI

Recent Publications
Pilz GA, Shitamukai A, Reillo I, Pacary E, Schwausch J, Stahl
R, Ninkovic J, Snippert HJ, Clevers H, Godinho L, Guillemot
F, Borrell V, Matsuzaki F, Götz M. Amplification of progenitors
in the mammalian telencephalon includes a new radial glial
cell type. Nat Commun. 4:2125. doi: 10.1038/ncomms3125(2013)
Konno D, Iwashita M, Satoh Y, Momiyama A, Abe T, Kiyonari
H, and Matsuzaki F. The mammalian DM domain transcription factor Dmrta2 is required for early embryonic development of the cerebral cortex. PLoS One. 7(10):e46577. doi:
10.1371 (2012).
Matsuzaki F, and Shitamukai A. Cell division modes andcleavage planes of neural progenitors during mammaliancortical development. Cold Spring Harb Perspect Biol. Mammalian Develop. doi 10.1101/cshperspect.a015719
Iwano T, et al. Prox1 postmitotically defines dentate gyrus
cells by specifying granule cell identity over CA3 pyramidal
cell fate in the hippocampus. Development 139.3051-62(2012)
Yoshiura S, et al. Tre1 GPCR signaling orients stem cell divisions in the Drosophila central nervous system. Dev Cell
22.1-13 (2012)
Kosodo Y, et al. Regulation of interkinetic nuclear migration
by cell cycle-coupled active and passive mechanisms in thedeveloping brain. EMBO J 30.1690-704 (2011)50

During brain development, the ganglionic eminence in the ventral telencephalon generates a large
number of diverse types of neurons including GABAergic interneurons that migrate tangentially into
the cerebral cortex. Compared to the dorsal cortex, which has a well-organized pseudo-stratified
structure, the ganglionic eminence is organized differently with a thicker germinal zone composed bya large number of intermediate progenitor cells. In collaboration with the Goetz laboratory in Germany,
we have revealed that the ganglionic eminence produces novel types of self-renewing progenitor cells
as well as inter mediate progenitor cells that extensively proliferate. This results in rapid expansion of
the population of descendant cells, and produces a range of different cell lineages. RG, radial glia;
SAP, subapical progenitor; BP, basal progenitor.
Ref: Pilz GA, Shitamukai A, Reillo I, Pacary E, Schwausch J, Stahl R, Ninkovic J, Snippert HJ, Clevers
H, Godinho L, Guillemot F, Borrell V, Matsuzaki F, Götz M. Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type. Nat Commun. 4:2125. doi: 10.1038/ncomms3125 (2013)

Lung Development
The organs of the body all play critical functional roles, which are made possible by the arrangement of differentiated cells into the structures specific to that organ. Such structures
are formed throughout development, with the late embryonic and immediate postnatal periods being particularly important for the functional maturation of organ systems. Defects
that arise during these organogenetic processes are closely linked to a wide range of diseases, while after birth the body is constantly exposed to potentially damaging environmental stresses. The adult body does manifest a certain degree of regenerative ability, although
this is by no means complete. To study organ formation, repair and regeneration, we havefocused our research on the respiratory system in mouse.
Respiratory organs in higher mammals are characterized by their efficient gas exchange,
enabled by the functions of specialized cells. The development of such organs relies on thecoordinated activities of both epithelial and mesenchymal tissue types, which arise fromtissue-specific populations of stem cells in the developing embryo. The epithelial tissues of
the conducting airways serve as the channel for the intake and exhalation of gases in the
respiratory cycle, and are composed mainly of Clara, ciliated, and neuroendocrine cells. In
our work to date, we have shown how these various cells interact and exchange information
to maintain the appropriate balance in their respective cell numbers and distributions.
Despite its location in the body’s interior, the respiratory tract is constantly exposed to environmental factors, such as infection by viruses and bacteria, smoke, and chemical toxins, thatmay damage the airway epithelium. This damage is rapidly repaired by regenerating epithelial cells supplied by somatic stem cells in the adult tissue, and as in development, thenumbers and distribution of cells in the epithelium must be maintained at levels appropriate
to each region of the airway.
We focus on issues of how tissue morphology influences the formation of stem cell niches
in the development, repair and regeneration of respiratory organs, as well as mechanisms
regulating cell proliferation and differentiation in developmental and regenerative processes.

Mitsuru MORIMOTO Ph.D.
http://www.cdb.riken.go.jp/en/morimoto
Mitsuru Morimoto received his Ph.D. in life sciencesin 2003 from Tokyo University of Pharmacy and Life
Sciences. From 2003 to 2006, he studied the molecular mechanisms of somitogenesis using mouse
genetics at the National Institute of Genetics. He
then moved to the Washington University School of
Medicine in St. Louis to work with Dr. Raphael Kopan, where he extended his research to lung organogenesis. He returned to the National Instituteof Genetics in 2010, and was appointed Team
Leader at the RIKEN CDB in 2012.

Staff
Team Leader

Technical Staff

Mitsuru MORIMOTO

Chisa MATSUOKA

Research Scientist

Part-Time Staff

Keishi KISHIMOTO

Junko SAKAI

Research Scientist

Assistant

Masafumi NOGUCHI

Yuka NODA

Recent Publications
Morimoto M, et al. Different assemblies of Notch receptors
coordinate the distribution of the major bronchial Clara, ciliated and neuroendocrine cells. Development 139.4365-73(2012)
Morimoto M, et al. Canonical Notch signaling in the developing lung is required for determination of arterial smoothmuscle cells and selection of Clara versus ciliated cell fate.
J Cell Sci 123.213-24 (2010)
Demehri S, et al. Skin-derived TSLP triggers progression
from epidermal-barrier defects to asthma. PLoS Biol 7.
e1000067 (2009)
Morimoto M. and Kopan R. rtTA toxicity limits the usefulnessof the SP-C-rtTA transgenic mouse. Dev Biol 325.171-8(2009)
Morimoto M, et al. The negative regulation of Mesp2 bymouse Ripply2 is required to establish the rostro-caudal patterning within a somite. Development 134.1561-9 (2007)

Airway epithelium is composed of various specialized cell types that benefit functional airways. Immunofluorescence staining of E18.5 lung with anti-CC10 (green), anti-CGRP (red) and anti-SSEA-1(blue) shows localizations of Clara, neuroendocrine and SPNC cells.

Morimoto M, et al. The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity. Nature
435.354-9 (2005)

51

Developmental Morphogeometry
Developmental phenomena comprise a multiscalar system extending across a range ofspatial scales, from molecular to cellular to histological. Such phenomena are also multiphysical, in that they involve the transmission and reception of positional information throughdiffusion and reactions of chemicals, and the generation of forces within tissues and concomitant geometrical deformation through the proliferation and migration of cells. The Laboratory for Developmental Morphogeometry takes theoretical and experimental approaches tothe study of phenomena comprising multiple scales and properties, and the quantitative
measurement of dynamic and coordinated interactions between such phenomena.

Yoshihiro MORISHITA, Ph.D.
http://www.cdb.riken.go.jp/en/morishita
Yoshihiro Morishita received his M.Sc. and Ph.D.
from the University of Tokyo Graduate School of
Frontier Sciences. From 2005 to 2007, he served
as Project Assistant Professor in the Kyushu University Department of Biology. From 2007 to 2011,
he served both as assistant professor in the Theoretical Biological Laboratory at Kyushu University
and as a researcher funded under several JST programs. He was named Research Unit Leader at the
RIKEN CDB in 2012.

Staff
Research Unit Leader

Technical Staff

Yoshihiro MORISHITA

Kaori TSUJINO
Yu WATAOKA

Research Scientist

Yukitaka ISHIMOTO
Daisuke OHTSUKA
Student Trainee

Part-Time Staff

Yuko FUJIYAMA
Megumi MORIOKA

An example of this is seen in organogenesis, a process in which the various tissue regionsthat give rise to the organ exhibit changes in volume at different rates, or anisoptropic expansion and contraction. Such deformations can be quantified as tensor quantities (geometricalcharacteristics). The morphological differences between various organs, or between homologous organs in different species can thus be explained as spatio-temporal patterns in thetensor quantities of each object. We are now able to extract such patterns using organlevel quantitative imaging and statistical analysis. By combining and comparing such data withthe accumulated body of molecular and cellular evidence, we hope to develop clearer insights into the relationships between macro-scale organ morphogenesis and micro-scale
phenomena.
The ability of individual cells to recognize and respond to (for example, through proliferationand differentiation) their positions within a tissue is also essential to tissue growth and patterning. This necessitates accurate “spatial recognition” on the part of cells, which receive
environmental cues (such as gradients of growth factors or interactions with neighboring
cells), but this is complicated by uncertainty arising from perturbations within the organism(such as inter-individual variations in morphogen expression levels). Questions of how to
maximize the accuracy of the transmission and reading of information against a backgroundof uncertainty is formalized as problems in information coding. Analysis of such problems
has revealed optimal sites of the expression of information sources (morphogens) and optimal designs for the form and parameter values of response functions implemented by intracellular biochemical reactions. By comparing the results of these theoretical analyses withexperimental observations, we can begin to assess the extent to which actual developmental systems are designed to optimal criteria.
Our lab will seek to use 1) analysis of measured data and 2) study system designs throughtheoretical formulation and computer simulations with an eye to developing a better understanding of these phenomena.

Ken-ichi HIRONAKA
Sangwoo LEE

Recent Publications
Hironaka K. and Morishita Y. Cellular sensory mechanisms
for detecting specific fold-changes in extracellular cues. Biophys J (2014)
Hironaka K. I. and Morishita Y. Encoding and decoding of
positional information in morphogen-dependent patterning.
Curr Opin Genet Dev 22.553-61 (2012)
Morishita Y. and Iwasa Y. Coding design of positional information for robust morphogenesis. Biophys J 101.2324-35(2011)
Morishita Y. and Iwasa Y. Estimating the spatiotemporal pattern of volumetric growth rate from fate maps in chick limb
development. Dev Dyn 238.415-22 (2009)
Morishita Y. and Iwasa Y. Growth based morphogenesis ofvertebrate limb bud. Bull Math Biol 70.1957-78 (2008)
Morishita Y. and Iwasa Y. Optimal placement of multiple morphogen sources. Phys Rev E Stat Nonlin Soft Matter Phys
77.041909 (2008)

Optimal arrangement of morphogen source (Shh expression region) to maximize the precision of positional information in vertebrate limb bud.

52

Germline Development
Germ cells are the only cell types capable of transmitting genetic information across generations, and their formation is characterized by unique developmental processes as well. In
many types of animals, including the Drosophila fruit fly, the formation and differentiation ofgerm cells is controlled by mRNAs and proteins localized in a specific cytoplasmic region
within eggs, called germ plasm. Germ plasm mRNAs are translated in a spatiotemporally
regulated manner, but the means by which the germ plasm is formed and regulates germ
cell development remain largely unknown. Our research team studies the establishment of
the Drosophila germ line as a model of the processes of germ plasm assembly and germ
cell differentiation. We expect that our study will also provide insights into the general mechanisms of mRNA localization and translation, which are important to a number of other developmental processes including asymmetric cell division, cell migration and axis formation,and postnatal synaptic plasticity as well.
One area of interest is the mechanism of translational repression in germline development.
In one example of this critically important form of regulation, the translation of the RNA for thematernal gene oskar, which functions in embryonic patterning and the formation of germline
cells in Drosophila, is repressed during its transport to the posterior pole of the oocyte. We
are now studying the function of proteins, such as the recently identified factor, Cup, that regulates the repression of oskar translation during its localization to the oocyte posterior. In
another concurrent project, we are focusing on the roles of wunen2 and polar granule component (pgc), which are known to function in lipid signaling and global transcriptional regulation in germline cells during embryogenesis.
In addition to the study of fruit fly germline development, we are also beginning to undertake
investigations using the ascidian, Ciona intestinalis. Our team will explore the genetic regulation of ascidian germline development by characterizing promoter regions of germline specific genes and trans-acting factors that regulate germline specific gene expression.

Akira NAKAMURA Ph.D.
http://www.cdb.riken.go.jp/en/nakamura
Akira Nakamura received both his baccalaureateand his Ph.D. from the University of Tsukuba. He
spent a year as a post-doctoral fellow at the same
institution before moving to the Department of Biology at McGill University in Montreal in 1995 to work
as a postdoc under Paul Lasko. He returned to Japan in 1997 as a research associate at the University of Tsukuba. He was appointed assistant professor in the university's Gene Research Centerand Institute of Biological Sciences in 2000, andbegan a three-year term as a PRESTO researcher
in the Japan Science and Technology Corporation(JST) in December 2001. He was appointed CDB
team leader in March 2002. From March 2013, Dr.
Nakamura has been a professor in the Institute of
Molecular Embryology and Genetics, Kumamoto
University.

Staff
Team Leader

Akira NAKAMURA
Research Scientist

Kazuko HANYU-NAKAMURA
Yasuko KATO
Yuko SUKEGAWA
Assistant

Yoko UMEGAKI

Recent Publications
Shirae-Kurabayashi M, et al. Ci-Pem-1 localizes to the nucleus and represses somatic gene transcription in the germlineof Ciona intestinalis embryos. Development 138.2871-81(2011)
Tanaka T, et al. Drosophila Mon2 couples Oskar-induced
endocytosis with actin remodeling for cortical anchorage ofthe germ plasm. Development 138.2523-32 (2011)
Tanaka T and Nakamura A. The endocytic pathway acts
downstream of Oskar in Drosophila germ plasm assembly.
Development 135.1107-17 (2008)
Hanyu-Nakamura K, et al. Drosophila Pgc protein inhibits PTEFb recruitment to chromatin in primordial germ cells. Nature 451.730-3 (2008)
Shirae-Kurabayashi M, et al. Dynamic redistribution of vasa
homolog and exclusion of somatic cell determinants during
germ cell specification in Ciona intestinalis. Development
133.2683-93 (2006)
Nakamura A, et al. Drosophila Cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNAtranslation in oogenesis. Dev Cell 6.69-78 (2004).

Migrating germ cells (blue) in stage 10 Drosophila embryo

53

Chromatin Dynamics
Multicellular organisms are made up of diverse populations of many different types of cells,
each of which contains an identical set of genetic information coded in its DNA. Cell differentiation and the process of development itself depend on the ability of individual cells tomaintain the expression of different genes, and for their progeny to do so through multiple
cycles of cell division. In recent years, we have begun to understand that the maintenanceof specific patterns of gene expression does not rely on direct modifications to the DNA
sequence encoding the organism’s genome, but rather takes place in a heritable, “epigenetic” manner. DNA methylation, chromatin modifications, and RNA silencing are some of thebest known epigenetic phenomena. Recent studies have begun to show that these different
mechanisms are closely inter-related, but a detailed understanding of these systems has yet
to be developed.

Jun-ichi NAKAYAMA Ph.D.
http://www.cdb.riken.go.jp/en/nakayama
Jun-ichi Nakayama received his bachelor's, master's and Ph.D. degrees in bioscience from the Tokyo Institute of Technology, the last in 1999 for his
work on the cloning and characterization of mammalian telomerase components. He spent the period from 1999 to 2001 as a postdoctoral researcher at Cold Spring Harbor Laboratory in Shiv Grewal'slab, before returning to Japan in December 2001 as
a PRESTO researcher in the Japan Science andTechnology Corporation (JST). He was appointed
team leader of the Laboratory for Chromatin Dynamics in 2002.

Our team investigates how modifications to the structure and configuration of chromatin(complexes of nuclear DNA and proteins that provide the structural basis of chromosomes)
contribute to epigenetic gene regulation and how such modifications are transmitted overgenerations of cellular division by studying events at the molecular scale in the excellent
model organism, fission yeast (Schizosaccharomyces pombe), and in cultured mammaliancells.
Histones are a set of DNA packing proteins present in nucleosomes, the fundamental building blocks of chromatins. In our current studies, we are particularly interested in determining
the specific histone modifications and the molecular recognition processes that enablemodified histones to work together to establish and maintain higher-order chromatin structures. We also seek to clarify the picture of how dynamic rearrangements of chromatin
structure are triggered by examining the structure and function of protein complexes thatbind to and modify histones. Through these approaches we aim to understand the molecularmechanisms that underlie complex epigenetic phenomena in developmental processes.

Staff
Team Leader

Technical Staff

Jun-ichi NAKAYAMA

Yasuko OHTANI

Research Scientist

Assistant

Aki HAYASHI

Shinobu SHIMAHARA

Research Associate

Mayumi ISHIDA

Recent Publications
Mano Y, et al. Single cell visualization of yeast gene expression shows correlation of epigenetic switching between multiple heterochromatic regions through multiple generations.
PLoS Biol 11. e1001601 (2013)
Kato H, et al. Spt6 prevents transcription-coupled loss of
posttranslationally modified histone H3. Sci Rep 3. 2186
(2013)
Ishida M, et al. Intrinsic nucleic acid-binding activity of Chp1
chromodomain is required for heterochromatic gene silencing. Mol Cell 47.228-41 (2012)
Kawakami K, et al. A novel RNAi protein, Dsh1, assembles
RNAi machinery on chromatin to amplify heterochromatic
siRNA. Genes Dev 26.1811-24 (2012)
Hayashi A, et al. Heterochromatin protein 1 homologue Swi6
acts in concert with Ers1 to regulate RNAi-directed heterochromatin assembly. Proc Natl Acad Sci USA 109.6159-64(2012)
Hiragami-Hamada K, et al. N-terminal phosphorylation of
HP1α promotes its chromatin binding. Mol Cell Biol 31.1186200 (2011)54

Intrinsic nucleic acid-binding activity of Chp1 chromodomain (CD) is required for heterochromatin assembly. Electrophoretic-mobility shift assay(EMSA) of four CDs in fission yeast (upper, left). The 3D NMR structure of
Chp1-CD in complex with H3K9me3 peptide (upper, right). Model for therole of CD-protein’s intrinsic RNA binding in heterochromatin assembly(lower).

Stem Cell Biology
The stem cell system can be thought of as a means by which organisms maintain cell renewal. All forms of life require the endless recycling of both materials and energy, making it
impossible for either organisms or individual cells to live independent of their environments.
In bodies made up of many cells working in cooperation, such as our own, the need to replace and renew is not limited to simple materials and energy supply; the constant generation of new cells is also essential to the maintenance of the individual, a process that hasdeveloped in the evolution of multicellular life. In this sense, the process of cell renewal inmaintaining the body’s integrity and function provides a meaningful example of the relationship of the cell to the organism, the individual to the whole, which we hope to explore more
fully using the biology of stem cells as our model.
We are especially interested in the question of how cells maintain the never-ending self-renewal that sustains the organism as a whole. This will require, at the very least, solving theriddles of two essential processes. The first of these is the need for old cells destined for
replacement to be able to disengage or otherwise be dislocated from their environmental
milieux. The second is the requirement for preparing new cells in replacement. To investigate
the first of these mechanisms, our group uses a system for labeling cells with special dyes,
which allows us to monitor their location and behavior. We have also developed a system fordifferentiating embryonic stem (ES) cells in culture to study the second question of new cell
production. Currently, we are focusing on developing different stem cell systems, particularly hematopoietic stem cells. This ongoing research project in our lab will allow us to explore how the self-renewing stem cell system is formed and will lead to the development ofmethods to generate bona fide hematopoietic stem cells from ES cells.

Shin-Ichi NISHIKAWA M.D., Ph.D.
http://www.cdb.riken.go.jp/en/nishikawa
Shin-Ichi Nishikawa received his M.D. from the
Kyoto University School of Medicine in 1973. He
performed his internship and residency in the Department of Internal Medicine at the Kyoto University Chest Disease Research Institute, before taking
an assistant professorship in the same department
in 1979. He spent the period from 1980-82 at the
University of Cologne Institute for Genetics before
returning to the Chest Disease Research Institute,
where he was appointed associate professor in the
Department of Microbiology in 1983. He moved to
the Kumamoto University Faculty of Medicine in
1987 to take a professorship in the Department of
Immunopathology, and returned to Kyoto in 1993,
as professor in the Department of Molecular Genetics at the Kyoto University Faculty of Medicine. He
was appointed CDB group director in 2000.

Staff
Group Director

Visiting Researcher

Shin-Ichi NISHIKAWA

Takao YAMADA

Research Scientist

Technical Staff

Guo DING
Yosuke TANAKA
Yan-Fung WONG
Research Specialist

Kumiko KOBAYASHI
Satoko MORIWAKI
Satomi NISHIKAWA
Iori SATO

Lars Martin JACT

Part-Time Staff

Collaborative Scientist

Shin KAWAMATA
Visiting Scientist

Teruo AKUTA
Keitaro IMAIZUMIHiroshi KATAOKA
Herve Le MOUELLIC
Taro MUTO

Miho HOSHIJIMA
Yoko OTSUKA
Noriaki SATO
Kazumi YANAIAssistantJunko KOYA
Naeko MINAMI

Recent Publications
Jakt L. M, et al. A continuum of transcriptional identities visualized by combinatorial fluorescent in situ hybridization.
Development 140.216-25 (2013)
Kataoka H, et al. Region-specific Etv2 ablation revealed thecritical origin of hemogenic capacity from Hox6-positive
caudal-lateral primitive mesoderm. Exp Hematol 41.567-81
e9 (2013)
Nakagawa R, et al. Peyer’s patch inducer cells play a leadingrole in the formation of B and T cell zone architecture.
J Immunol 190.3309-18 (2013)
Kobayashi K, et al. Role of Etv2-positive cells in the remodeling morphogenesis during vascular development. Genes
Cells 18.704-21 (2013)
Wong Y. F, et al. Prolonged treatment with DNMT inhibitors
induces distinct effects in promoters and gene-bodies. PLoS
One 8.e71099 (2013)

Group Directors assemble for Dr.Nishikawa's farewell party

55

Growth Control Signaling
The processes of animal development, including organ size and body size, are geneticallypredetermined, but these processes are also influenced by environmental factors such as nutrition and temperature. The close link between cell and tissue growth control and environmental cues ensures that developmental transitions occur at the appropriate time during
animal development.

Takashi NISHIMURA Ph.D.
http://www.cdb.riken.go.jp/en/nishimura
Takashi Nishimura obtained his Ph.D. in Kozo Kaibuchi’s lab at the Nagoya University Graduate
School of Medicine for his work on the regulation ofneuronal polarization. He did postdoctoral researchin the same laboratory before moving to the Institute of Molecular Biotechnology in Vienna, Austria
in 2006 to pursue further postdoctoral work in Jürgen Knoblich’s group. He returned to Japan in
2009, working briefly as a visiting scientist at the
RIKEN Center for Developmental Biology prior to
his appointment as team leader in July of the same
year.

Cell proliferation and differentiation in each tissue and organ are kept under strict regulation
both spatially and temporally. Research has revealed the nature of spatial signals, such as
growth factors and morphogens, but the way in which these signals direct cell and tissue
growth over time remains understood. In addition, growth and developmental timing arealso governed by nutrient availability. Most species have a standard body size, but developing organisms are also capable of adapting their growth to fluctuating nutritional states
through metabolic regulation. Therefore, linking the nutrient sensing system to an endocrine
signaling network allows organisms to control the timing of cell proliferation and differentiation.
Our team’s research aims to shed light on the molecular basis for growth control and developmental timing at the cellular and tissue/organ level using Drosophila as a model system.
In particular, we are interested in addressing the following questions: 1) how do organisms
adapt their growth program to changes in energy needs and states; 2) what are the molecular mechanisms that sense nutrient availability and regulate body size; and 3) how do
endocrine signals interact with metabolic and growth regulators?
To better understand the interface between nutrient availability and growth regulation, we
are focusing on how nutrition controls systemic growth through Drosophila insulin-like peptides (Dilps). Members of the insulin family of peptides have conserved roles in the regulationof growth and metabolism in a wide variety of metazoans. We are now analyzing the molecular mechanism underlying the nutrient-dependent expression of Dilp genes. We have also
conducted in vivo RNAi screening to identify new players regulating growth and developmental timing at the organismal level. We described the first demonstration of the glia-derived endocrine factor regulating systemic body growth. The identification of SDR protein in
Drosophila provides a new concept for the regulation of insulin/IGF signaling.

Staff
Team Leader

Student Trainee

Takashi NISHIMURA

Miki YOSHIDASpecial Postdoctoral
Researcher

Part-Time Staff

Naoki OKAMOTO
Research Associate

Hiroko MATSUDA
Eizo NAKAGAWA

Okiko HABARA
Kanako HIGUCHI
Noriko KAWASAKI
Tomomi OKUGOUCHI
Junko SHINNOTechnical Staff

Tomoyuki TAKAHASHI
Takayuki YAMADA
Yuki YAMAUCHI

Recent Publications
Okamoto N, et al. A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila. Genes
Dev 27.87-97 (2013)
Okamoto N, et al. Conserved role for the Dachshund proteinwith Drosophila Pax6 homolog Eyeless in insulin expression.
PNAS 109.2406-11 (2012)
Wirtz-Peitz F, et al. Linking cell cycle to asymmetric division:
Aurora-A phosphorylates the Par complex to regulate Numb
localization. Cell 135.161-73 (2008)
Nishimura T and Kaibuchi K. Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3.
Dev Cell 13.15-28 (2007)

Wild-type female fly (right) and insulin-like receptor mutant female (left)

56

Pluripotent Stem Cell Studies
Pluripotency is a term used to describe the ability of certain types of stem cells that are capable of giving rise to at least one type of cell from all three germ layers-endoderm, mesoderm and ectoderm. Stem cells are also characterized by their ability to produce copies ofthemselves with similar differentiative potential, as well as more specialized cells with different properties through differentiation. Embryonic stem (ES) cells are the best known example of a type of stem cell possessing these properties of pluripotency and self-renewal. In
our lab, we use ES cells as a model system for studying the molecular mechanisms that
determine and enable the mysterious properties of pluripotent cells.
Pluripotency can be primarily determined by a particular set of transcription factors, for as
we now know, pluripotency can be induced by four transcription factors. These transcription
factors should form a self-organizing network able to stabilize their expression and maintainpluripotency. At the same time, the network should have the sensitivity to signals that induce
differentiation. How can the transcription factor network satisfy these contradictory requirements? What is the principle behind the structure of the transcription factor networks governing developmental processes? We are now trying to answer these questions by studying
ES cells and the changes they undergo in their differentiation toward trophoblast stem cells,
extraembryonic endoderm cells, and primitive ectoderm cells.

Hitoshi NIWA M.D., Ph.D.
http://www.cdb.riken.go.jp/en/niwa
Hitoshi Niwa received his M.D. from Nara Medical
University in 1989, and his Ph.D. in medical physiology in 1993 from the Kumamoto University Graduate School of Medicine for his work in gene trap
methods. From 1993 to 1994, he worked as a research associate in the Department of Developmental Genetics at the same university, before taking a postdoctoral fellowship with Austin Smith at
the University of Edinburgh Centre for Genome
Research. He returned to Japan in 1996 as a research associate in the Department of Nutrition andPhysiological Chemistry at the Osaka University
Graduate School of Medicine, where he remained
until taking his current position as team leader at
the RIKEN CDB. He was appointed project leaderof the Laboratory for Pluripotent Stem Cell Studies
in October 2009.

Staff
Project Leader

Technical Staff

Hitoshi NIWA

Setsuko FUJII

Research Scientist

Junior Research
Associate

Yoko NAKAI-FUTATSUGISatomi NISHIKAWA-TORIKAI
Satoshi OHTSUKA
Masaki SHIGETA
Robert Odell STEPHENSON
Toshimi SUGIMOTO
Tomoyuki TSUKIYAMA
Research Associate

Mariko YAMANE
Visiting Scientist

Shunsuke ITOStudent Trainee

Marius KLANGWART
Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI
AssistantMiho SAKURAI

Yasuhide OHINATA
Hiroki URA

Recent Publications
Adachi K, et al. Context-Dependent Wiring of Sox2 Regulatory Networks for Self-Renewal of Embryonic and Trophoblast Stem Cells. Mol Cell 52.380-92 (2013)
Ohtsuka S, et al. E-cadherin promotes incorporation ofmouse epiblast stem cells into normal development. PLoS
One 7.e45220 (2012)
Martello G, et al. Esrrb is a pivotal target of the gsk3/tcf3 axis
regulating embryonic stem cell self-renewal. Cell Stem Cell
11.491-504 (2012)

Chimeric embryos generated by injection ofGata6GR ES cells into blastocysts. These ES
cells carry the constitutively-active Egfp transgene and contribute to primitive ectoderm (upper panels), whereas they contribute to parietal
endoderm after induction of Gata6 activity withdexamethasone (lower panels).

Schematic model of transcription factor networks for pluripotent stem cells, trophectoderm and primitive endoderm.

Niwa H, et al. A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460.11822 (2009)
Niwa H. How is pluripotency determined and maintained?
Development 134.635-46 (2007)57

Seeing neuronal circuitry with SeeDB

Takeshi IMAI, Satoshi FUJIMOTO, Meng-Tsen KE

Developmental biology is one of the most visually stunningfields of in the life sciences, and the ability to ‘see’ within theembryo is a fundamentally important approach to its study.
The advent of fluorescent marker proteins, such as GFP,
has made it possible to visualize the localization of the expression of labeled proteins, but the scattering of fluorescent signals in opaque biological samples means that such
imaging can only peer to within a few hundreds of micrometers of the surface of the tissue, even when using advanced technologies such as two-photon microscopy. This
creates significant problems for the study of complex threedimensional tissues, such as neuronal circuits; the traditional approach of slicing the tissue to obtain two-dimensional data to be used in attempts to reconstruct 3D

58interconnected structures presents herculean difficulties. A
number of alternative strategies involving the ‘clearing’ oftissue with solvents to render it more or less transparent
have been developed, but these have also been complicated, time-consuming, and may affect fluorescent signals
from labeled proteins.
Meng-Tsen Ke and colleagues in the Lab for Sensory Circuit formation (Takeshi Imai, Team Leader) now look to improve the toolkit with a report of a new technique for optically clearing embryonic tissues, which they have named
SeeDB. This new method, published in Nature Neuroscience, uses a water-based agent that preserves fluorescence and tissue structure, and enables the deep-layer visualization of neuronal circuits.

©2013 Nature Publishing Group
Ke M. T, et al. SeeDB: a simple and morphology-preserving optical clearing agent
for neuronal circuit reconstruction. Nat Neurosci 16.1154-61 (2013)

In seeking a better optical clearing agent, Ke and colleagues resolved to identify aqueous solutions with refractive properties similar to those of the tissues they wished to
study. Other groups had reported some success using sucrose solutions, but the Imai team decided to test fructose
as an alternative. After long experimentation, they refined
the recipe to what they named SeeDB (for See Deep Brain),a solution that made it possible to render an embryo optically transparent in just three days, a major advance overprevious techniques requiring 2–3 weeks. SeeDB also excels in preserving the size and morphology of embryonicstructures, and avoids the quenching of fluorescence frommolecular labels such as GFP. Additional information for
scientists interested in this new optical clearing solution areavailable at SeeDB Resources.
Ke et al. next sought to determine the tissue depths at
which SeeDB imaging could be used, using samples fromadult mouse brain. Working with a microscope manufacturer, they customized a two-photon microscope with alonger working distance lens, to overcome depth limitationsof the commercially available version. Fluorescence imaging of 6 mm-thick samples cleared by SeeDB yielded beautiful, high-resolution images of even the deepest layers,demonstrating the utility of this technique at a scale suitablefor whole-brain studies in mouse.

Embryo prior to (left) and after (right) clearing with SeeDB agent

In a further test of the new clearing agent’s utility, the teamlabeled axons of the corpus callosum, which link the left
and right hemispheres of the brain, and traced their projections in a mouse brain made transparent with SeeDB. They
were able to trace individual axon fibers over long distancesfrom one side of the brain to counterparts projecting fromthe opposite hemisphere, and found that such callosalaxon projection relies not only on positional guidance fromtarget tissue, but also sorting at the pre-target site.
The team last moved to the lab’s core interest of studying
the circuits formed in the olfactory bulb of the brain. In themouse, olfactory sensory neurons of like type converge inglomeruli within the olfactory bulb, which is the site of odor
information processing. Odor inputs to a glomerulus arethen relayed to mitral cells through dendritic connections,but the wiring of these mitral connections has remained unresolved. Using SeeDB, Ke et al. were able for the first timeto get an unobstructed view of the circuit layout for the fullset of mitral cells associated with a single glomerulus, revealing an unexpected architectural complexity.
“I am hopeful that this new technology will help usher in a
new era of ‘3D biology’ aimed at a better understanding ofneural circuit formation and other complex developmental
processes,” says Imai. “However, we still need to fine-tune
the microscopy and image data processing technqiues tomake this even more suitable for a wide range of studies.”

3D imaging of mouse brain treated with SeeDB

59

Role for RA in olfactory neurogenesis

Marie PASCHAKI, Yuko MUTA

Uniquely among sensory tissues, olfactory neurons havethe capacity to regenerate throughout life, a necessity given
their direct exposure to the external environment. The olfactory neuroepithelium itself derives from a sensory placodeincludes a variety of supporting cell types in addition toneuronal cells, which are generated by the activity of progenitors also present in the tissue. Despite our increasingly
detailed understanding of olfactory neural circuitry andfunctions, however, we still have much to learn about the
ontogeny and regeneration of olfactory neurons.
Work by Marie Paschaki and colleagues in the Lab for Sensory Development (Raj Ladher, Team Leader) published in
Neural Development has now begun to provide some insights into those processes. By studying the function of60

retinoic acid in olfactory tissues from mouse and chicken,
Paschaki et al. show that this signaling molecule plays arole in olfactory neurogenesis.
Retinoic acid (RA) is involved in a broad range of developmental processes, and had been implicated as a possible
signal involved in olfactory epithelial patterning. But the results of this previous work had left open the question ofwhether it was involved only in induction of the tissue, or inprompting neuronal differentiation as well. Paschaki set out
to pinpoint this role, as well as to determine the extent (ifany) to which retinoic acid affects olfactory neurogenesis,by studying RA and an enzyme responsible for its synthesis, RALDH, in chicken and mouse.

©2013 BioMed Central Ltd
Paschaki M., et al. Retinoic acid regulates olfactory progenitor cell fate anddifferentiation. Neural Dev 8.13 (2013)

Retinoic acid regulates olfactory progenitor cellfate and differentiation
Paschaki et al.
Paschaki et al. Neural Development 2013, 8:13
http://www.neuraldevelopment.com/content/8/1/13

She first examined whether inhibition of RA signaling would
perturb the induction of the olfactory placode in chicken
embryos, but found that interference with RA synthesis had
no perceptible effect. In mouse mutants lacking Raldh function as well, showed no abnormal placode phenotype, indicating that retinoic acid is dispensable in olfactory placodeinduction.
That picture changed dramatically however when she tested RA function later in development. When she implanted
beads soaked in an inhibitor of RALDH activity into chicken
embryos, she found that this led to a failure in the differentiation of olfactory stem cells, suggesting that RA is necessary for inducing or maintaining the olfactory progenitor
population. In tissue explants, culture medium containing
vitamin A, from which retinoic acid is metabolized, revealed
an inhibitory effect on neurogenesis, pointing to the possibility that RA maintains stem cells by suppressing differentiation.
These observations raised the issue of where in the olfactory lineage RA plays its part. Loss of RA synthesis had noaffect on apoptosis, but did increase the number of prolifer-ating cells, prompting Paschaki to look at the olfactory progenitor phenotype in a mouse Raldh mutant. She foundthat a specific population of quiescent Pax6-expressing
progenitors was depleted in the mutant embryos. Turning
again to explanted tissue, she examined the effects of vitamin A and found that its addition to the culture medium
supported greater numbers of progenitors than did medium without this RA precursor. More strikingly when she
grew Raldh null mutant explants in the same conditions, theprogenitor population collapsed irrespective of whether vitamin A was included in the medium, suggesting that massive differentiation had occurred at the expense of progenitor self-renewal. This hypothesis was borne out in the
mutant embryos, which showed fewer Pax6-expressing
progenitors than in wildtype, and consequent failure to
maintain olfactory neurogenesis.
“The olfactory epithelium is the only neuronal population in
mammals that can repair itself,” says Ladher. “If we canunderstand how it uses its developmental history to achievethis, it may give us some ideas on how we can help otherneuronal populations repair themselves.”

Mouse olfactory epithelium at day 18.5 of development. Neurons are stained green, andcan be seen through the epithelium, sending out axons, arranged as bundles, in the cephalic mesenchyme.

61Endoskeletal origins of the turtle carapace

Exoskeletons are widely associated with invertebrates, typically envisioned as the chitinous armor of a beetle or crustacean. But vertebrates have exoskeletal elements too. Our
own cranium, which lacks an overlying layer of muscle, isthe exo- exception to our predominantly interior skeletal
system. A number of mammals have also developed more
extensive protective outer coats, but these vary dramatically in their composition – an armadillo’s scutes are completely different, for example, from a turtle’s carapace. While
the armadillo’s scales are exoskeletal in origin, and the animal maintains internal ribs as in other vertebrates, only inthe turtle does the endoskeleton make its way to the bodyexterior. Students of turtle evolution and development, however, have long debated whether there might not be some
exoskeletal contribution to the formation of the carapace.
Tatsuya Hirasawa and colleagues from the Laboratory for
Evolutionary Morphology (Shigeru Kuratani, Group Director)
have now found new evidence that may help put that argument to rest, through analyses of anatomical and paleontological data that squarely trace the origins of the carapace
to exteriorized ribs of endoskeletal origin. Published in Nature Communications, this work adds compelling new evidence to support the theory that the turtle’s shell grows out
from the inside.
Our skeletons evolved through a process of territorial giveand take, with some elements advancing while other gave
ground. By studying how such movements differ in embryos of various species, biologists have gained insights into
the mutability of fundamentally conserved body plans. For
the past two centuries, however, the turtle has remained
something of a conundrum. While some hold that the carapace represents a fusion of endo- and exoskeletal elements(as the result of signaling by endoskeletal elements, i.e.,ribs, that have shifted into the dermis, triggering the formation of exoskeletal tissue), others view it as a purely endoskeletal structure that nonetheless finds its way outside.

Shigeru KURATANI, Tatsuya HIRASAWA

62

To find an answer to this age-old question, Hirasawa analyzed the dorsal carapace of the Chinese soft-shelled turtle
in close detail. The formation of this carapace begins withdorsal positioning of ribs and intercostal muscle primordia,
after which the muscle element is gradually lost, leaving the©2013 Nature Publishing GroupARTICLE
Received 7 Dec 2012 | Accepted 3 Jun 2013 | Published 9 Jul 2013

Hirasawa T, et al. The endoskeletal origin of the turtle carapace.
Nat Commun 4.2107 (2013)

DOI: 10.1038/ncomms3107

OPEN

The endoskeletal origin of the turtle carapace
Tatsuya Hirasawa1, Hiroshi Nagashima2 & Shigeru Kuratani1

The turtle body plan, with its solid shell, deviates radically from those of other tetrapods. The
dorsal part of the turtle shell, or the carapace, consists mainly of costal and neural bonyplates, which are continuous with the underlying thoracic ribs and vertebrae, respectively.
Because of their superficial position, the evolutionary origins of these costo-neural elements
have long remained elusive. Here we show, through comparative morphological andembryological analyses, that the major part of the carapace is derived purely from endoskeletal ribs. We examine turtle embryos and find that the costal and neural plates develop notwithin the dermis, but within deeper connective tissue where the rib and intercostal muscle
anlagen develop. We also examine the fossils of an outgroup of turtles to confirm that thestructure equivalent to the turtle carapace developed independently of the true osteoderm.
Our results highlight the hitherto unravelled evolutionary course of the turtle shell.

1 Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan. 2 Division of Gross Anatomy andMorphogenesis, Department of Regenerative and Transplant Medicine, Niigata University, Niigata 951-8510, Japan. Correspondence and requests formaterials should be addressed to T.H. (email: hirasawa@cdb.riken.jp).
NATURE COMMUNICATIONS | 4:2107 | DOI: 10.1038/ncomms3107 | www.nature.com/naturecommunications

1& 2013 Macmillan Publishers Limited. All rights reserved.

periosteum of rib to expand laterally and serve as a scaffold
that is subsequently filled in by bony trabecula. These bonyelements later invade the intercostal spaces to form a complete carapace entirely as the endoskeleton.
The group next compared the formation of the turtle carapace with that of ribs in a bird (chicken) and the bony dermal plates (osteoderm) of the alligator. While the carapace
and avian ribs followed broadly comparable developmental
sequences, the formation of the osteoderm differed dramatically, as it apparently involves epithelial-mesenchymal
interactions within the dermis, further cementing the argument for an endoskeletal origin of the carapace.
The paleontological record also holds clues to carapace
evolution. Odontochelys, an ancient ancestoral turtle, hadbony plate-like outgrowth derived from ribs, although it
lacked a fully-closed carapace. Most vertebrates have
movable joints between ribs and vertebrae, which facilitate
respiration, so the rigidity of these bones in both Odonto-

chelys and modern turtles is a remarkable feature. Morerecently unearthed intermediate fossils also underscore theevolution from axial endoskeleton to rigid shell. Examining
the collection at the Institute of Vertebrate Paleontology andPaleoanthropology (IVPP) in the Chinese Academy of Sciences (CAS), Hirasawa identified a marine reptile, Sinosaurosphargis yunguiensis, which shows anatomical similarities with both Odontochelys and turtles that suggest thegrowth of an immovable shell within the subdermal tissue
otherwise typically occupied by intercostal muscle.
“We usually think of the turtle’s carapace as a form of protection, but it may have first emerged as an aid to swimming – a kind of surfboard if you like – that only latterly became hardened,” says Kuratani. “The study of unusual
adaptations such as the turtle shell can teach us not only
about the great diversity of the animal world, but the evolutionary programs that led to our own body plans as well.”Development of turtle carapace (left) and chicken rib (right). Subdermal expansion of periosteum and bony trabeculae are seen in bothprocesses.

63

Microtubules have a firm CLASP on the epiblast basement
membrane

Yukiko NAKAYA, Erike Widyasari SUKOWATIIn higher animals, gastrulation lays the groundwork for subsequent development by establishing the three germ layersthat give rise to all the tissue in the body. In chicken andother birds, cells of the epiblast at the body surface migrate
to a site called the primitive streak, and subsequently moveinward to the embryonic interior, forming the endoderm andmesoderm. Epiblast cells are epithelial in nature, form adhesive bonds and attach to a basement membrane. But
during their ingression from the primitive streak, these cells
shed their connections with other cells and the basement
membrane, transforming into mobile mesenchymal cells in
the process. Such epithelial–mesenchymal transitions(EMT) play roles in numerous developmental processes,
but the molecular machinery that controls EMT is still poorly understood.

64

Yukiko Nakaya and colleagues in the Lab for Early Embryogenesis (Guojun Sheng, Team Leader) have now identified
once such mechanism, finding that the microtubule-binding protein CLASP is required for maintaining cells in anepithelial state, and that downregulation of CLASP induces
the degradation of basement membrane, a necessary
event in gastrulation. Published in Journal of Cell Biology,
this work was featured on the cover of the August issue.
Previous studies of CLASP had shown that it associateswith microtubule plus-ends and maintain their density, but
the physiological and developmental role of the protein has
been a mystery. Nakaya had observed that EMT in gastrulating chick embryos appeared to involve microtubule-mediated anchoring of epiblast cells to the basement mem-Published August 12, 2013This article has original data in the JCB Data Viewer

JCB: Article

http://jcb-dataviewer.rupress.org/jcb/browse/6202

Epiblast integrity requires CLASP and Dystroglycanmediated microtubule anchoring to the basal cortex©2013 The Rockefeller University Press

Yukiko Nakaya, Erike W. Sukowati, and Guojun Sheng
Laboratory for Early Embryogenesis, Institute of Physical and Chemical Research (RIKEN) Center for Developmental Biology, Chuo-Ku Kobe, Hyogo 650-0047, JapanTHE JOURNAL OF CELL BIOLOGY

A

mniote epiblast cells differentiate into mesoderm
and endoderm lineages during gastrulation
through a process called epithelial-to-mesenchymaltransition (EMT). Molecular regulation of gastrulation EMT
is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules
to the basal cortex via CLIP-associated protein (CLASP), a
microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm
formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation

epiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediatedthrough the CLASP-binding partner LL5. Live-imaging
using EB1–enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilizedbasal microtubules. We further show that Dystroglycan
is localized to basolateral membrane in epiblast cells.
Basal but not lateral localization of Dystroglycan was
regulated by CLASP. We propose that epiblast–BM interaction requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which
initiates gastrulation EMT.

Introduction
Epithelial-to-mesenchymal transition (EMT) is a phenomenon
in which cells change their shape and intercellular organizationfrom an epithelial type to a mesenchymal one. Correct executionof EMT and its reverse process MET is vital for tissue morphogenesis during animal development, and their abnormal reinitiation leads to organ fibrosis and tumor metastasis (Moreno-Bueno
et al., 2008; Kalluri and Weinberg, 2009; Thiery et al., 2009;
Kerosuo and Bronner-Fraser, 2012; Lim and Thiery, 2012). Cellbiologically, EMT has been well characterized as a multistep
process that includes dissolution of local basement membrane(BM), loss of the epithelial polarity and tight junctions (TJs),
switch of the adherens junction (AJ) subtypes, and mesenchymal cell migration. Molecularly, transcriptional factors suchas Snail, Twist, Zeb1, and Zeb2 are viewed as key mediators
between signaling input (e.g., TGFB, FGF, EGF, and HGF) on
one hand and cell biological output on the other, measured mainlyby E-cadherin down-regulation and loss of epithelial polarity
markers (Moreno-Bueno et al., 2008; Kalluri and Weinberg,
2009; Thiery et al., 2009; Kerosuo and Bronner-Fraser, 2012;Lim and Thiery, 2012). Recently, control of epithelial cell–BMinteraction has been recognized as another crucial componentof EMT regulation (Levayer and Lecuit, 2008; Nakaya et al.,2008; Rowe and Weiss, 2009; Hagedorn and Sherwood, 2011;
Williams et al., 2012). However, it is unclear how this regulationis achieved molecularly and whether it utilizes the same signaling and transcriptional mediators as those involved in E-cadherin
and apicobasal polarity regulations.
Gastrulation, one of the best-known examples of EMT,
is a conserved developmental process during which the threeprincipal germ layers (ectoderm, mesoderm, and endoderm)
are generated (Nakaya and Sheng, 2008, 2013; Acloque et al.,2009; Lim and Thiery, 2012; Solnica-Krezel and Sepich, 2012).
Gastrulation EMT in amniotes (birds and mammals) involveschanges of epiblast cells from a proper epithelium with the fullarray of epithelial characteristics to mesenchymal-shaped mesoderm cells (Nakaya and Sheng, 2008). The primitive streak,a transient embryonic structure where gastrulation EMT takes
place, is composed of cells that have initiated EMT, but are stillconnected to the rest of the epiblast as a continuous sheet. Thesecells are considered to be metastable, partial-EMT cells, having

Correspondence to Guojun Sheng: sheng@cdb.riken.jp© 2013 Nakaya et al. This article is distributed under the terms of an Attribution–Noncommercial–
Share Alike–No Mirror Sites license for the first six months after the publication date (see http://
www.rupress.org/terms). After six months it is available under a Creative Commons License(Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Abbreviations used in this paper: BM, basement membrane; CLASP, CLIP-associatedprotein; DG, Dystroglycan; eGFP, enhanced GFP; EMT, epithelial-to-mesenchymaltransition; MO, morpholino; MT, microtubule; PH, pleckstrin homology.

The Rockefeller University Press $30.00
J. Cell Biol. Vol. 202 No. 4 637–651
www.jcb.org/cgi/doi/10.1083/jcb.201302075

Downloaded from jcb.rupress.org on September 18, 2013

Nakaya Y, et al. Epiblast integrity requires CLASP and Dystroglycan-mediatedmicrotubule anchoring to the basal cortex. J Cell Biol 202. 637-51 (2013)

Supplemental Material can be found at:
http://jcb.rupress.org/content/suppl/2013/08/08/jcb.201302075.DC1.html
Original image data can be found at:
http://jcb-dataviewer.rupress.org/jcb/browse/6202
JCB

637

brane, presumably linked to its maintenance and degradation.
Looking first at the expression of CLASP during this period,
she observed that while it was expressed in the epiblast, it
was downregulated in cells at the primitive streak. Overexpression of CLASP caused stabilization of the basement
membrane, which is ordinarily destined to decay during
EMT, while conversely, knocked down CLASP outside thestreak led to abnormal degradation of basement membrane components.

membrane and other intracellular factors. Overexpression
or inhibition of Dystroglycan in the epiblast yielded similar
results to those she observed in her experiments with
CLASP, inappropriate stabilization or degradation of thebasement membrane. Co-immunoprecipitation revealed
colocalization of the two proteins in the basal cell cortex,
indicating a physical interaction. And when she looked at
Dystroglycan expression in the primitive streak, she found it
decreased during EMT, just as with CLASP.

Investigating CLASP function, Nakaya found that it works
with its binding partner LL5 to tether microtubules to thebasal cortex in cells, suggesting that its expression maintains epiblast cells in an epithelial state, while its downregulation is a necessary step in EMT. But how does a cytoplasmic molecule such as CLASP have this effect on theextracellular basement membrane? For a promising candidate, Nakaya next turned to Dystroglycan, a transmembrane protein known to interact both with the basement

This series of experiments makes a solid case for the combined roles of CLASP and Dystroglycan in anchoring microtubules to the basal cell cortex and stabilization of thebasement membrane in epiblast cells. “But we are still curious about why it is that loss of CLASP causes the basement membrane to decay,” says Sheng. “We suspect thismay involve vesicle transport by microtubules, but we will
have to study that possibility more closely in a future project.”

Anterior

Primitive streak

Epiblast(Epithelial)

EMT
Mesoderm(Mesenchyme)BMdegradation

Basement membrane(BM)Endoderm(Top) Cartoon scheme of epithelial-mesenchymal transition, showing degradation of basement
membrane beneath the primitive streak and ingression of cells. (Bottom) Forced expression of
CLASP (green) in the streak results in the stabilization of the basement membrane (yellow arrows).

65Baseline receptor activity steers olfactory axon targetingalong the A-P axis

Takeshi IMAI

All the many odors we recognize and remember are firstdetected as molecular signals by G-protein coupled receptors (GPCRs) on specialized neurons in the olfactory system. The mouse has around 1000 such odorant receptors,
which, when bound to an odorant molecule, convey signals
to the olfactory bulb in the brain. An equal number of structures, known as glomeruli, serve as convergence points forneurons bearing the same odorant receptor, patterns of thecollective activity of which are perceived as olfactory sensations by the brain. Establishing the right connections fromreceptor to glomerulus requires the precise wiring of olfactory neurons into elaborate circuits, by processes that remain imperfectly understood.

66

Takeshi Imai, Team Leader of the Lab for Sensory Circuit
Formation, and colleagues have now discovered an essential role for the activation of olfactory receptors unprovokedby ligand binding in the control of axon targeting from olfactory neuron to glomerulus. Reporting in Cell, they also describe how the co-action of the protein Gs supports the efficient transduction of such weak, unstable signals, making
guided axon projection in the fetal mouse possible even in
the absence of odor stimuli.
Odorant receptors are expressed on neurons lining the olfactory epithelium, each of which typically expresses only asingle receptor type. Axons from these neurons of the same
type converge to glomeruli specific to that odorant receptor
profile, following a general targeting gradient along the an-©2013 Elsevier B.V.
Nakashima A, et al. Agonist-Independent GPCR Activity Regulates AnteriorPosterior Targeting of Olfactory Sensory Neurons. Cell 154.1314-25 (2013)

Agonist-Independent GPCR Activity
Regulates Anterior-Posterior Targetingof Olfactory Sensory Neurons
Ai Nakashima,1,2,9 Haruki Takeuchi,1,2,9 Takeshi Imai,1,3,4,9 Harumi Saito,1 Hiroshi Kiyonari,5 Takaya Abe,5 Min Chen,6
Lee S. Weinstein,6 C. Ron Yu,7 Daniel R. Storm,8 Hirofumi Nishizumi,1 and Hitoshi Sakano1,2,*
1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
2Department of Brain Function, School of Medical Science, University of Fukui, Fukui 910-1193, Japan
3Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
4PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan
5Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
6Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
7Stowers Institute for Medical Research, Kansas City, MO 64110, USA
8Department of Pharmacology, University of Washington, Seattle, WA 98195, USA
9These authors contributed equally to this work
*Correspondence: sakano@mail.ecc.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.cell.2013.08.033

SUMMARY

G-protein-coupled receptors (GPCRs) are known topossess two different conformations, active andinactive, and they spontaneously alternate between
the two in the absence of ligands. Here, we analyzed
the agonist-independent GPCR activity for its
possible role in receptor-instructed axonal projection. We generated transgenic mice expressing
activity mutants of the b2-adrenergic receptor, awell-characterized GPCR with the highest homology
to odorant receptors (ORs). We found that mutants
with altered agonist-independent activity changed
the transcription levels of axon-targeting molecules—e.g., Neuropilin-1 and Plexin-A1—but not ofglomerular segregation molecules—e.g., Kirrel2 andKirrel3—thus causing shifts in glomerular locations
along the anterior-posterior (A-P) axis. Knockout
and in vitro experiments demonstrated that Gs, but
not Golf, is responsible for mediating the agonistindependent GPCR activity. We conclude that theequilibrium of conformational transitions set byeach OR is the major determinant of expression
levels of A-P-targeting molecules.
INTRODUCTION
In the mouse olfactory system, various odorants are detected by1,000 types of odorant receptors (ORs) expressed in the olfactory epithelium (OE) (Buck and Axel, 1991). It is well establishedthat each olfactory sensory neuron (OSN) expresses only one
functional OR gene in a monoallelic manner (Chess et al.,
1994; Serizawa et al., 2000, 2003). Furthermore, OSNs expressing the same type of OR converge their axons to a specific set ofglomeruli at stereotyped locations in the olfactory bulb (OB)(Feinstein et al., 2004; Mombaerts et al., 1996; Wang et al.,1998). Based on these two principles, ‘‘one neuron-one OR’’and ‘‘one glomerulus-one OR,’’ odor information received in
the OE is represented as a topographic map of activatedglomeruli in the OB (Mori and Sakano, 2011). During development, the glomerular map is formed by the combination of ORindependent and -dependent processes. The dorsal-ventral(D-V) arrangement of glomeruli is OR independent and is regulated by the anatomical location of OSNs within the OE (Vassar
et al., 1994; Ressler et al., 1994; Miyamichi et al., 2005; Takeuchi
et al., 2010). In contrast, anterior-posterior (A-P) targeting is instructed by expressed OR proteins using cyclic AMP (cAMP)
as a second messenger (Chesler et al., 2007; Imai et al., 2006,2009).
We have previously reported that both global targeting along
the A-P axis and local sorting of OSN axons for glomerular segregation are regulated by OR-derived cAMP (Imai and Sakano,2008). In the OB, A-P-targeting molecules such as Neuropilin-1(Nrp1) and Plexin-A1 (PlxnA1) are detected on axon termini ofOSNs, forming a complementary gradient in a glomerular map(Imai et al., 2006; Imai and Sakano, 2008). OR-derived cAMPsignals also regulate the expression of glomerular segregation
molecules (e.g., Kirrel2 and Kirrel3) for olfactory map refinement(Serizawa et al., 2006). Unlike A-P-targeting molecules,
glomerular segregation molecules are regulated in a neuronal
activity-dependent manner and show mosaic distribution in theglomerular map. How is it, then, that A-P targeting and glomerular segregation are differentially regulated by the expressed
ORs although both are under the control of OR-derived cAMP?
What is the source of cAMP signals, and how are the signals
generated? These important questions had never been formallyaddressed.
G-protein-coupled receptors (GPCRs), including ORs, are
known to possess two different conformation states, active
and inactive. Agonists stabilize the receptor in an active form,

1314 Cell 154, 1314–1325, September 12, 2013 ª2013 Elsevier Inc.

terior-posterior (A-P) axis, followed by more precise local
sorting. Both of these have been thought to rely on signals
arising from odorant receptor activation, but the details ofthese mechanisms have eluded explication.

role of Gs and how it works with Golf had never been determined. Undertaking a detailed biochemical analysis, Imaifound that while Gs effectively responded to baseline OR
activity, Golf did not.

Binding of a GPCR by an odorant molecule triggers a shift
from inactive to active state that entails structural changes
in the conformation of the receptor molecule. Past studies
have shown, however, that activation by such an agonist is
not necessary; GPCRs show a certain baseline level of activity even in the absence of ligand binding, although thishas often been considered little more than noise.

The team next generated knockout Golf and Gs mice; the Gs
KO was necessarily conditional, as the mutant is embryonic lethal. Imai found that A-P axon guidance was dramatically affected in the Gs mutant, and glomerular sortingin the Golf KO, and that these defects were specific to therespective mutations. Gs mutants additionally showed a highfrequency of axonal wiring errors, indicating that its role is toregulate A-P axon targeting in response to baseline signaling by still-developing olfactory neurons.

Imai and colleagues set out to determine whether this agonist-independent receptor activation plays a role in olfactory
neuron axon guidance along the A-P axis by developing
mutant mice with altered baseline activity. A-P axon guidance is governed by factors during mid-development, while
those responsible for local sorting are expressed later. In an
extensive screening study, Imai was able to identify Gs as
the A-P factor and Golf as the glomerular segregation molecule. Both proteins are structurally similar, and mediate
odorant receptor cAMP signals; Golf is known to cooperatewith odorant receptors in detection of odor stimuli but the“The ability odorant receptors to achieve two highly distinct
functions simply by the use of differential responses by Gs
and Golf to baseline signals is truly amazing,” says Imai.
“Many have suggested that stimulus-independent signaling
might play a role in neural circuit formation, and it appears
that is just what we are observing here. We next want tostudy the mechanism behind how connection specificity
during embryonic neural circuit formation depends on neuronal activity.”

Axon projection from olfactory neurons expressing a specific receptor (OR-I7). While the Golf knockout mouse shows noabnormalities, the GS conditional knockout shows disturbance of axonal targeting. Top row shows stained sections ofolfactory bulb (OR-I7 in green), bottom row shows wholemount olfactory bulb preparations.

67

New roles for microtubules in actin regulation

Shigenori NAGAE

Microtubules are components of the cytoskeleton known
for their roles in maintaining cellular structure and cytoplasmic transport. These elongated proteins form two distinct
populations, one anchored to the centrosome by its minusend and a second that grows from other sites within thecell, through interactions with other molecules. It has previously been shown that the cytoplasmic protein CAMSAP3(also known as Nezha) stabilizes non-centrosomal microtubules, but it remains unclear whether the roles of non-centrosomal in cellular architecture and other functions differ
from those of the centrosomal type.
Now, a study by Shigenori Nagae of the Laboratory for Cell
Adhesion and Tissue Patterning (Masatoshi Takeichi,
Group Director) and the Kyoto University Graduate School

68

of Biostudies, working in collaboration with the Institute of
Genetics and Developmental Biology, Chinese Academy of
Sciences (Beijing, China), reveals that non-centrosomal microtubules mediated by CAMSAP3 regulate the organization of a second cytoskeletal protein, F-actin, which plays
roles in cellular morphology and behavior. Published in
Genes to Cells, these findings shed new light on the molecular machinery underlying microtubule function.
CAMSAP, which serves as the nucleus of non-centrosomal
microtubule formation, was recently identified by the Takeichigroup, who showed that CAMSAP stabilizes microtubules
through binding their minus-ends. In this latest study,
Nagae et al. used the HeLa line of immortalized human cells
to study the function of microtubules bound to CAMSAP.

©2013 John Wiley & Sons, Inc.

Non-centrosomal microtubules regulate F-actinorganization through the suppression of GEF-H1 activity
Shigenori Nagae1,2, Wenxiang Meng1,3 and Masatoshi Takeichi1*
1

RIKEN Center for Developmental Biology, 2-2-3 Chuo-ku, Kobe 650-0047, Japan
Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of
Sciences, Beijing 100101, China

2

Nagae S, et al. Non-centrosomal microtubules regulate F-actin organization
through the suppression of GEF-H1 activity. Genes Cells 18.387-96 (2013)

3

Animal cells contain two populations of microtubules: one radiating from the centrosome andthe other growing from non-centrosomal sites. Whether or not they have differing roles incellular architecture and function remains not fully understood. The cytoplasmic protein
Nezha (also known as CAMSAP3) stabilizes non-centrosomal microtubules by attaching totheir minus ends. Here, we found that depletion of CAMSAP3 in HeLa cells resulted in a relative increase in centrosomal microtubules, and this change was accompanied by accelerated
actin stress fiber formation. In these cells, RhoA activity was upregulated, and the soluble fraction of GEF-H1, a RhoGEF whose activity is inhibited by binding to microtubules, increased,
explaining why stress fiber formation was promoted. We further found that CAMSAP3 depletion led to an increase in detyrosinated microtubules, and these microtubules did not interact
with GEF-H1. These findings suggest that CAMSAP3-anchored non-centrosomal microtubules
capture GEF-H1 more efficiently than other microtubules do and that a balance between thesemicrotubules is important to maintain proper actin organization.

Introduction
Microtubules play a number of important roles in cellstructure and function during interphase of the cell
cycle. It is well established that microtubule polymerization is nucleated by the c-tubulin ring complex
located in the centrosome, resulting in the formationof radial microtubule arrays that converge on thissubcellular structure (Kollman et al. 2011). In manycell types, however, microtubules are not always
anchored to the centrosome, instead connected to
non-centrosomal sites via their minus ends. The proportions of the centrosomal and non-centrosomal
microtubules differ among cell types. For example,
fibroblasts generally exhibit radial microtubules,
whereas epithelial cells have microtubules with theirminus ends oriented toward the apical cell cortex,
but not converging on the centrosome (Bacallao et al.
1989; Bartolini & Gundersen 2006). Neurons andCommunicated by: Kozo Kaibuchi*Correspondence: takeichi@cdb.riken.jp

myotubes are also enriched with non-centrosomal
microtubules (Bartolini & Gundersen 2006).
Non-centrosomal microtubules are tethered tovarious subcellular sites, such as cell junctions (Lechler & Fuchs 2007; Meng et al. 2008; Bellett et al.
2009), cell cortex (Mogensen et al. 2000), the nucleus(Hagan & Petersen 2000), and the Golgi apparatus(Efimov et al. 2007; Rivero et al. 2009), via known
or unknown mechanisms. Microtubules polymerizing
from different sites are expected to play distinct roles
in structural organization of cells or their behavior. It
has been indeed demonstrated that Golgi-derived andcentrosomal microtubules control the distribution of
Golgi membranes via dissimilar ways (Miller et al.
2009; Hurtado et al. 2011; Vinogradova et al. 2012).
However, when the functions of microtubules in acell are discussed, centrosomal and non-centrosomal
microtubules are not always distinguished, despite thepossibility that these two populations of microtubules
are functionally not equivalent.
Recent studies identified a small family of proteinsthat regulate microtubule minus-end stability: Nezha(also termed CAMSAP3) (Meng et al. 2008) andDOI: 10.1111/gtc.12044© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

The group focused specifically on CAMSAP3, which theyfound to be expressed in a punctate fashion throughout thecells, binding with microtubule minus ends, with the plus
ends growing outward from these CAMSAP loci.
Using RNAi to inhibit CAMSAP expression, the group foundthat the ordinarily smooth-edged cells became spiky anddistorted, taking on a distinct ‘thorny’ morphology, with aconcomitant increase in the number of centrosomal microtubules. Curious about the cause of the morphologicalchanges, they next examined the activity of a second cytoskeletal protein, actin, in CAMSAP-inhibited cells, andfound that actin stress fiber was upregulated on loss of
CAMSAP function.
But how then do changes in non-centrosomal microtubuleformation affect actin fibers? In looking for the underlying
mechanism, they next focused on RhoA, a protein known
to induce actin stress fiber formation. Intriguingly, theyfound that depletion of CAMSAP3 led to an increase in

Genes to Cells (2013) 18, 387–396

387

detyrosinated microtubules (which were found to be at
higher levels in centrosomal microtubules than in non-centrosomal microtubules). Due to this, the Rho activator GEFH1 became unbound from microtubules, leaving it free toactivate RhoA, resulting in the promotion of actin stress fiber formation and, ultimately, changes in cell shape. Thus,
it appears that under normal conditions binding of microtubules by CAMSAP3 inhibits RhoA activity maintaining actin
fiber formation at a fixed level. These new insights help toclarify the mechanistic and functional differences between
centrosomal and non-centrosomal microtubules.
“We are now finding that a balance between these two
types of microtubules serves to regulate actin fibers, which
may contribute to cell type-dependent morphological processes,” says Takeichi. “By clearly distinguishing between
these different microtubule populations, we hope to contribute even more to our understanding of how these molecules work in the cell.”In HeLa cells, inhibition of CAMSAP3 (right panel) results in an increase in actin fibers (green) and changes in cell morphology. Control cells shown to left.

69

AwardeePositionLaboratory

Award

Organization

The Young
Scientists’ Prize,
The Commendationfor Science andTechnology

The Minister of Education,
Culture, Sports, Scienceand Technology

Miki Ebisuya

Unit Leader

Reconstitutive
Developmental
Biology

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Systems of Biology
Award

The Society of Photo Optical
Instrumentation Engineers

Tatsuya Hirasawa

Research Scientist

Evolutionary
Morphology

Best Presentation
Award for Young
Researchers(1st place winner)

The Society of Evolutionary
Studies

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Hans Sigrist Prize

Hans Sigrist Foundation of
University Bern

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Uehara Award

The Uehara Memorial
Foundation

71

Cellular Reprogramming
All organisms possess instincts to survive exposure to external stresses by adapting to theirenvironment and, to some degree, regenerating injured tissues or organs. It is thus unsurprising to observe dramatic cellular plasticity after exposure to significant external stresses,
such as an injury. In both vertebrates and plants, cellular reprogramming can be induced byexposure to external stimuli, which suggests that organisms that possess significant capacity for regeneration show great plasticity when exposed to external stresses and generate“stem cells” from somatic cells. Given these observations, true ‘stemness’ appears to belinked to cellular plasticity. The main goal of our research is to elucidate the relationship between stemness and cellular plasticity.

Haruko OBOKATA Ph.D.
http://www.cdb.riken.go.jp/en/obokata

ControlStress-treated cells

Bright Field

Haruko Obokata graduated from Waseda University with a B.Sc. in applied chemistry (2006), and a
M.Sc. (2008) and Ph.D. (2011) in engineering. Herforay into regenerative medicine and tissue engineering began during her master’s, doing her bench
work as a student trainee at the Tokyo Women’s
Medical University. During her doctoral program,she studied for almost two years under Charles Vacanti at the Brigham and Women’s Hospital, Harvard University Medical School looking at the relationship between stem cells and regeneration. She
took a postdoctoral fellowship in Vacanti’s lab after
completing her Ph.D. in 2011 to continue the research she began in his lab, and also began collaborations with Teruhiko Wakayama (now at Yamanashi University) at the CDB as a visiting scientist. In March 2013, she was appointed to her current position as a unit leader at the CDB.

Haruko OBOKATA
Technical Staff

Kumi MATSUURA
Azusa NOMA
Rieko YAKURA-NISHIZAWA

Oct4-GFP

Staff
Research Unit Leader

Student Trainee

Yukari TERASHITA

Recent Publications
Obokata H, et al. Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 505.641-647 (2014)

Oct4 expressing cell generation from CD45 positive somatic cells.
Oct4-GFP expression of stress treated cells. Stress-treated cells express Oct4-GFP, while
untreated controls did not. Magnification of Oct4-expressing colony is shown in the upper
right in the stress-treated group. Scale bar indicates 100 μm.

Obokata H, et al. Bidirectional developmental potential in
reprogrammed cells with acquired pluripotency. Nature 505.
676-680 (2014)
Obokata H, et al. Reproducible subcutaneous transplantation of cell sheets into recipient mice. Nat Protoc 6.1053-9(2011)

4N embryos at E9.5 generated with stress altered somatic cells.
Stress altered somatic cells contributed not only to fetus but also to placenta and amniotic membrane.

72

Mammalian Epigenetic Studies
The stable inheritance of cellular traits and gene regulatory programs through multiple
rounds of cell division is a fundamental process in animal development and homeostasis.
Chemical modifications and remodeling of chromatin play critical roles in such epigenetic
processes. Research in our laboratory has focused on cytosine DNA methylation, a heritablegenome-marking mechanism. In mammals, DNA methylation is catalyzed by three DNA
methyltransferases, Dnmt1, Dnmt3a and Dnmt3b, and plays important roles in the regulation of gene expression and development. During early embryogenesis, genome-wide reprogramming of DNA methylation establishes new DNA methylation profiles of the embryos.
Once established, these cell-specific DNA methylation profiles are stably maintained in a
lineage-dependent manner. Using mouse embryos and embryonic stem (ES) cells as model systems, we investigate the regulation and functions of DNA methylation in mammaliandevelopment.
During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on cell type and developmental stage. Using a drug-inducible
Gata4 system and an ES cell model of mesoderm differentiation, we found that DNA methylation modulates transcription factor output in the context of cell differentiation. The activation of inducible Gata4 in mouse ES cells is known to drive their differentiation into endoderm. We found that the differentiation of wild-type ES cells into mesoderm blocks their
Gata4-induced endoderm differentiation, while mesoderm cells derived from Dnmt3a-/-Dnmt3b-/- ES cells can retain their response to Gata4, allowing lineage conversion from mesoderm to endoderm. Our study revealed groups of endoderm and mesoderm developmental
genes whose expression were induced by Gata4 only when DNA methylation was lost,
suggesting that DNA methylation restricts the ability of these genes to respond to Gata4,
rather than controlling their transcription per se. Our data indicate that epigenetic regulation
by DNA methylation functions as a heritable safeguard to prevent transcription factors from
activating inappropriate downstream genes, thereby restricting the differentiation potentialof somatic cells.

Masaki OKANO Ph.D.
http://www.cdb.riken.go.jp/en/okano
Masaki Okano received his baccalaureate andmaster's degrees from Kyoto University, and his
doctorate from the Research Division in Agriculture
at the same institution in 1994. He spent the period
from 1994 to 1995 as a research fellow in cell biology at the Nagoya University BioScience Center
before moving to Massachusetts, where he worked
as a research fellow in medicine at Harvard Medical
School and Massachusetts General Hospital from1995 to 2002. He was appointed team leader at
the RIKEN Center for Developmental Biology in
2001, and returned to work in Japan full-time in
2002. In 2012, he served as a senior investigator at
the CDB.

Staff
Senior Investigator

Masaki OKANO
Research Scientist

Takashi TAMURA
Technical Staff

Mesoderm ES

Chisa MATSUOKA

Recent Publications
Oda M, et al. DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem
cell differentiation. PLoS Genet 9.e1003574 (2013)
Kubiura M, et al. Chromosome-wide regulation of euchromatin-specific 5mC to 5hmC conversion in mouse ES cells
and female human somatic cells. Chromosome Res 20.83748 (2012)
Ehara T, et al. Role of DNA methylation in the regulation of
lipogenic glycerol-3-phosphate acyltransferase 1 gene expression in the mouse neonatal liver. Diabetes 61.2442-50(2012)
Sakaue M, et al. DNA methylation is dispensable for thegrowth and survival of the extraembryonic lineages. Curr Biol
20.1452-7 (2010)

Gene expression changes (blue, low; yellow, high) in wild-type or Dnmt3a-/-Dnmt3b-/- cells
in response to stimulation with transcription factor Gata4. The mesoderm cells deficientfor DNA methylation are hypersensitive to the transcription factor-stimulation.

73Organogenesis and Neurogenesis
The complexity of the fully formed brain defies description, yet this organ arises from a nondescript clump of cells in the embryo. The specification of the dorsal-ventral axis is significant in neural development in that the central nervous system forms on the dorsal side of
the body in all vertebrate orders. This process is dictated by the effects of a number of signaling factors that diffuse from organizing centers and direct dorsal ectoderm to maintain aneural fate. These molecules, which include Noggin, Chordin, Follistatin and their homologs,
participate in elaborate signaling networks in which factors collaborate and compete to initiate the embryonic nervous system.

Yoshiki SASAI M.D., Ph.D.
http://www.cdb.riken.go.jp/en/sasai
Yoshiki Sasai received his M.D. from the Kyoto University School of Medicine in 1986, subsequently
performing internships in general practice andemergency medicine. He completed the Ph.D.
course at the same institution in 1992, for work onneural specific transcriptional regulators. In 1993,
he took a postdoctoral fellowship in the De Robertis
lab at the UCLA School of Medicine, remaining
there until 1996 when he was appointed associate
professor at the Kyoto University School of Medicine. He assumed a professorship at the Kyoto
University Institute for Frontier Medical Sciences in
1998, and was appointed group director at the
CDB in 2000. He serves on the editorial boards ofCell, Neuron, Developmental Cell, Genesis, andDevelopmental Dynamics.

Using the African clawed frog, Xenopus laevis, as a model in molecular embryological studies, our group is engaged in clarifying the structure and extent of the signaling networks involved in setting up the dorsal-ventral axis and determining neural fate in the ectoderm.
These studies focus on molecules that operate at early stages in embryonic development tolay down a complex of permissive, instructive and inhibitory patterns that determines thegrowth and differentiation of the brain in its earliest stages of development.
Our group is now also actively developing effective methods of inducing neuralization in
mammals, work which it is hoped will contribute to basic research by providing in vitro experimental systems for use in the analysis of mammalian neurogenesis. In addition, thisapproach has potential for application in the treatment of neurodegenerative disorders, suchas Parkinson’s disease. Using a system developed in our lab, we have succeeded in inducing
mouse and human embryonic stem (ES) cells to differentiate into a range of specialized neuronal
types, including those of the cerebral cortex, cerebellar cortex and retina.
By studying very early neurogenesis, and the mechanisms of neuronal differentiation, our lab
aims to understand the molecular basis underpinning the formation of so intricate a system
as the mammalian neural network. In addition, we seek to understand the principles underlying the shapes of organs, and are currently studying self-organization of cortex, retina, andadenohypophysis using three-dimensional ES cell culture.

Staff
Group Director

Technical Staff

Yoshiki SASAI

Hidehiko INOMATA

Masayo FUJIWARA
Tomoko HARAGUCHI
Masako KAWADA
Ayaka NISHIYAMA
Mika SOEN
Rieko YAKURA-NISHIZAWA

Research Scientist

Junior Research Associate

Research Specialist

Keiko MUGURUMA
Senior Scientist

Satoru OKUDA
Nozomu TAKATA
Momoko WATANABE
Foreign Postdoctoral
Researcher

Nicholas Robert LOVE
Visiting Scientist

Satoshi ANDO
Atsushi KUWAHARA
Makoto NASU
Morio UENO

Hideya SAKAGUCHI
Student Trainee

Taisuke KADOSHIMA
Atsushi SHIRAISHI
Kazuhiro TAKASHIMA
Part-Time Staff

Masako SUZUKI
Assistant

Mako MIYAGI-INOMATA
Masayo OKUGAWA
Ayumi TANAKA-IKEYA
Fumi WAGAI

Recent Publications
Inomata H, et al. Scaling of Dorsal-Ventral Patterning by Embryo Size-Dependent Degradation of Spemann’s Organizer
Signals. Cell 153.1296-1311 (2013)
Suga H, et al. Self-formation of functional adeno-hypophysis
in three-dimensional culture Nature 480.57–62 (2011)
Eiraku M, et al. Self-organizing optic-cup morphogenesis inthree-dimensional culture. Nature 472.51-56 (2011)
Kamiya D, et al. Intrinsic transition of ES cell differentiation
into neural progenitors. Nature 470.503-509 (2011)
Muguruma K, et al. Ontogeny-recapitulating generation andtissue integration of ES cell-derived Purkinje cells. Nat Neurosci 13.1171-80 (2010)

Self-organized formation of optic cup from human ES cells

74Early Embryogenesis
The lab studies how mesoderm cells form and differentiate during early vertebrate development, using the chick as the main model organism. Our research currently focuses on two
aspects: 1) the formation and early regionalization of mesoderm cells during gastrulation; 2)differentiation of the ventral-most mesoderm cell types.
Mesoderm cells are derived from the epiblast during gastrulation through a process called
epithelial to mesenchymal transition (EMT). During EMT, mesoderm cells leave epithelialshaped epiblast sheet and become migratory. We are interested in understanding the molecular and cellular mechanisms governing this EMT. We are also interested in understanding how
this process is linked to mesoderm fate specification in the epiblast and to mesoderm regionalization along the dorsal-ventral axis during gastrulation.
After their formation, mesoderm cells give rise to several well-defined lineages: axial, paraxial, intermediate, lateral plate and extraembryonic. The extraembryonic lineage contains
three cell types: blood, endothelial and smooth muscle. We are investigating how thesethree cell types are specified and how they are organized to form functional hematopoietic
and vascular systems.

Guojun SHENG Ph.D.
http://www.cdb.riken.go.jp/en/sheng
Guojun Sheng received his B.S. in microbiology
from Fudan University in Shanghai, China in 1990.
He entered the Rockefeller University the next year,
attending the summer course in embryology at the
Woods Hole Marine Biological Laboratory in 1996
and receiving his Ph.D. under Claude Desplan in
1997. He spent the period from 1998 to 2000 in
Claudio Stern's laboratory in Columbia University,
moving with Dr. Stern to University College London
in 2001. He was appointed team leader at the
RIKEN CDB in 2004.

Staff
Team Leader

Technical Staff

Guojun SHENG

Hiroki NAGAI
Kanako OTA
Erike Widyasari SUKOWATIYuPing WU

Research Scientist

Cantas ALEV
Yukiko NAKAYA
Wei WENG

Recent Publications
Weng W and Sheng G. Five transcription factors and FGF
pathway inhibition efficiently induce erythroid differentiation in
the epiblast. Stem Cell Reports in press (2014)
Nakaya Y, et al. Epiblast integrity requires CLASP and Dystroglycan-mediated microtubule anchoring to the basal cortex. J Cell Biol 202.637-51 (2013).
Alev C, et al. Decoupling of amniote gastrulation and streak
formation reveals a morphogenetic unity in vertebrate mesoderm induction. Development 140.2691-6 (2013)

An anamniote-like, circumblastoporal ring of mesoderm precursors(Blue: Brachyury) is induced by subgerminal cavity injection of FGFs,
suggesting that the primitive streak is not an essential component of
mesoderm formation in amniotes.

Alev C, et al. Transcriptomic landscape of the primitive streak.
Development 137.2863-74 (2010)
Shin M, et al. Notch mediates Wnt and BMP signals in theearly separation of smooth muscle progenitors and blood/
endothelial common progenitors. Development 136.595603 (2009)
Nakaya Y, et al. RhoA and microtubule dynamics control
cell-basement membrane interaction in EMT during gastrulation. Nature Cell Biol 10.765-775 (2008)

This is a cross-section of the primitive streak. Epiblast cells in the middle of the streak are undergoing
EMT. Red: beta-Dystroglycan (basolaterally localized in lateral epiblast cells and downregulated inmedial epiblast cells); Green: nuclei.

75Physical Biology
Advances in measurement technologies have afforded us glimpses into dynamic functional
processes, such as morphogenesis and information processing, in cells and tissues involved in development and regeneration. The truly organic dynamism of the biological phenomena exhibited by living cells, individually or in groups, emerges from the coordinatedinteraction of numerous molecular and genetic factors, and the need for integrated, systems-based approaches to the study of design and operating principles in such “living”
phenomena is becoming increasingly clear. This will require not only technologies for the measurement of such elements but the development of applicable mathematical methods aswell. In Laboratory for Physical Biology, we will seek to use concepts and methodology frommathematical sciences such as physics in the study and elucidation of these emerging
questions in biology.

Tatsuo SHIBATA Ph.D.
http://www.cdb.riken.go.jp/en/shibata
Tatsuo Shibata received his B.Sc. in Physics from
Kyoto University, and Ph.D. from the Graduate
School of Arts and Sciences, University of Tokyo in
1999, after which he worked as a postdoctoral research fellow at the Kyoto University Research Institute for Mathematical Sciences for two years. In
2001, he moved to Germany to do a second postdoc at the Fritz-Haber-Institut. He returned to Japan in 2002, taking an associate professorship at
Hiroshima University. In 2007, he was additionallyappointed as a researcher under the PRESTO program coordinated by the Japan Science and Technology Agency (JST). In October of 2010, he took
his current position of research unit leader at the
RIKEN CDB.

Staff
Research Unit Leader

Technical Staff

Tatsuo SHIBATA

Satoru IWATANI
Yuko WADA

Research Scientist

Akinori BABA
Naotoshi NAKAMURA
Katsuhiko SATO

Junior Research Associate

Foreign Postdoctoral
Researcher

Toshinori NAMBA

Marcel HOERNING

One example of such a phenomenon is seen in cellular chemotaxis, in which cells recognize
concentration gradients of attractant molecules and respond by directional movements necessary for functions such as the exploration of the environment by single-celled organisms,
and morphogenesis in metazoa. Chemotactically responsive cells are able to detect differentials in the concentration of an attractant molecule of only a few percent, which, given cell
sizes of ranging in the tens of micrometers, translates to a real difference of just a few dozen
molecules. Cells are capable of interpreting this minute difference as a gradient that guides
the direction of its movement, raising the question of how cells are able to detect and follow
such weak and noisy signals. We now know that within their tiny intracellular spaces, cells
comprise many interacting molecules that work in a highly orchestrated fashion, and thus
give rise to emergent order enabling their orientation. Using quantitative fluorescence imaging data and the analysis of mathematical models, we seek to gain a better understandingof such mechanisms.
Mathematical modeling of the essential aspects of observed phenomena of interest is a
useful approach to evaluating whether we have sufficient knowledge of associated molecules, reactions, and cellular interactions to explain them. The abstracted mathematical idea
of particular phenomena may further reveal general principles that underlie the living systems
more broadly across diverse taxa. We seek to contribute to the thorough exploration of these
fascinating problems in biology through concepts and methods adapted from the mathematical sciences.

Prabhat SHANKAR
Part-Time Staff
Assistant

Satoko KINOSHITA

Visiting Researcher

Seirin LEE

Recent Publications
Shibata T, et al. Intracellular encoding of spatiotemporal
guidance cues in a self-organizing signaling system for chemotaxis in dictyostelium cells. Biophys J 105.2199-209(2013)
Hiraiwa T, et al. Theoretical model for cell migration with gradient sensing and shape deformation. Eur Phys J E Soft Matter 36.9846 (2013)
Baba A, et al. Directional sensing of deformed cells underfaint gradients, Phys. Rev. E 86.060901(R) (2012)
Shibata T, et al. Modeling the self-organized phosphatidylinositol lipids signaling system in chemotactic cells using
quantitative image analysis. J Cell Sci 125.5138-50 (2012)
Namba T, et al. The relation of signal transduction to thesensitivity and dynamic range of bacterial chemotaxis.
Biophys J 103.1390-9 (2012)
Arai Y, et al. Self-organization of the phosphatidylinositol lipids signaling system for random cell migration.
Proc Natl Acad Sci U S A 107.12399-404 (2010)(A) Spontaneous asymmetry formation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3,
green) and PTEN (red), and an explanatory theoretical model.(B, C) Spatiotemporal diagrams of themembrane distributions.(D-F) Stochastic numerical simulations of the theoretical model. (G-H) Experiment of gradient response in PtdIns(3,4,5)P3 (green) (Shibata, et al. 2012, 2013).

76

Retinal Regeneration
The retina has been called the “approachable part of the brain,” owing to its relatively simple
structure and its location near the body surface, and for these reasons it serves as a usefuland experimentally amenable model of the central nervous system. Until very recently, it wasthought that in adult mammals the retina was entirely incapable of regenerating, but we now
know that at least new retinal neurons can be generated after being damaged. This has
opened up new hope that the ability to regenerate neurons and even to reconstitute theneural network may be retained in the adult retina. We are now exploring the exciting prospect that, by transplanting cells from outside of the retina or by regeneration from intrinsic
progenitor cells, it may one day be possible to restore lost function to damaged retinas.
Our research into retinal regeneration seeks to achieve clinical applications by developing
methods for inducing stem cells or embryonic stem cells to differentiate into retinal neurons
and pigment epithelial cells in sufficient quantities for use in the treatment of patients suffering from conditions in which such cells have been damaged or lost. We must also ensurethat such cells establish viable grafts upon transplantation and induce the reconstitution offunctional neural networks. We also hope to develop means of promoting true regenerationby activating endogenous stem cells to replace cells lost to trauma or disease and thus repair damaged tissues. Access to a broad spectrum of developmental biological research
information will be key to the achievement of these goals, and we appreciate the opportunities for exchange that working in the environment provided by the RIKEN CDB.
Therapeutic applications cannot be developed from basic research alone; the clinical approach – a thorough understanding of the medical condition to be treated is equally important. For conditions such as retinitis pigmentosa, even the successful transplantation of cells
in animal models may not necessarily be translatable to a human clinical therapy without anunderstanding of the underlying genetics and possible immunological involvement. Our goal
is to study retinal regeneration based on both a strong foundation in basic research andsolid clinical evidence.

Masayo TAKAHASHI M.D., Ph.D.
http://www.cdb.riken.go.jp/en/takahashi
Masayo Takahashi received her M.D. from Kyoto
University in 1986, and her Ph.D. from the same
institution in 1992. After serving as assistant professor in the Department of Ophthalmology, Kyoto
University Hospital, she moved to the Salk Institutein 1995, where she discovered the potential ofstem cells as a tool for retinal therapy. She returned
to Kyoto University Hospital in 1997, and since2001 served as an associate professor at theTranslational Research Center in the Kyoto University Hospital. She joined the CDB as a team leaderof the retinal regeneration research team in 2006.
Her clinical specialty is retinal disease – maculardiseases and retinal hereditary diseases in particular. Her aim is to understand these diseases at afundamental level and develop retinal regeneration
therapies.

Staff
Project Leader

Technical Staff

Masayo TAKAHASHI

Momo FUJII
Kanako KAWAI
Tomoyo HASHIGUCHI
Naoko HAYASHI
Shino HIRAHARA
Yukako HIRAO
Ayumi HONO
Kyoko ISEKI
Hiromi ITO
Miwa OKADA
Noriko SAKAI
Yumiko SHIBATA
Hitomi TAKAKUBO
Motoki TERADA
Chikako YAMADARyo YOSHIDA

Deputy Project Leader

Michiko MANDAI
Sunao SUGITA
Research Scientist

Hiroshi AKIMARU
Jun KANEKONaoshi KOIDE
Chikako MORINAGA
Akishi ONISHI
Akiko SUGA
Research Associate

Chie ISHIGAMI
Akihiro TACHIBANA

Part-Time Staff

Tomoko KATAYAMA
Ryoko TAKEDA
Tomoko YOKOTA

1
Human iPS cells

2
Recent Publications

Retinal pigment epithelial cells
differentiated from iPS cells

Kamao H, et al. Characterization of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell
Sheets Aiming for Clinical Application. Stem Cell Reports
2.1–14 (2014)
Jin ZB., et al. Modeling retinal degeneration using patientspecific induced pluripotent stem cells. PLoS One 6. e17084(2011)

3
Purified retinal pigment epithelial cells

4
Photoreceptor cells differentiatedfrom iPS cells

Osakada F, et al. Toward the generation of rod and cone
photoreceptors from mouse, monkey and human embryonicstem cells. Nat Biotechnol 26. 215-24 (2008)
Osakada F, et al. Wnt signaling promotes regeneration in theretina of adult mammals. J Neurosci 27. 4210-9 (2007)
Ooto S, et al. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina. Proc Natl Acad Sci
USA 101. 13654-9 (2004)
Haruta M, et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonicstem cells. Invest Ophthalmol Vis Sci 45. 1020-5 (2004)

77

Cell Adhesion and Tissue Patterning
Animal cells organize into tissues with complex architecture. Our lab is exploring the molecular mechanisms by which individual cells assemble into a tissue-specific multicellular
structure, such as epithelial sheets and neural networks. We are also interested in the molecular basis of how tissue architecture is disrupted during carcinogenesis, a process that isthought to accelerate the metastasis of cancer cells. For these studies, we are focusing on
the roles played by cell-cell adhesion and recognition molecules, the cadherin family of adhesion molecules in particular, as these are known to be indispensable for tissue construction. Our current studies are divided into three categories:

Masatoshi TAKEICHI Ph.D.
http://www.cdb.riken.go.jp/en/takeichi
Masatoshi Takeichi is director of the RIKEN Center
for Developmental Biology as well as director of the
Cell Adhesion and Tissue Patterning researchgroup. He completed the B. Sc. and M. S. programs in biology at Nagoya University before receiving a doctorate in biophysics from Kyoto University in 1973. After attaining his Ph.D., he took aresearch fellowship at the Carnegie Institution Department of Embryology under Richard Pagano. He
then returned to Kyoto University, attaining a professorship in the Department of Biophysics (19861999), before becoming professor in the Department of Cell and Developmental Biology in the
Graduate School of Biostudies at the same university. He assumed the directorship of the CDB in
2000.

1) Cell-cell adhesion is a dynamic process, and this nature of cell-cell adhesion is implicatedin various cell behaviors, such as contact-dependent regulation of cell movement and cancer
metastasis. A growing body of evidence suggests that cadherins cooperate with cytoskeletal and/or motility machineries, such as actin regulators, non-muscle myosins, and Rho GTPases, in modulating cell assembly. We are therefore studying the molecular mechanisms underlying the crosstalk between cadherins and such cytoskeletal systems.
2) A second area of interest to our lab is to gain a better understanding of how the cell-celladhesion machinery contributes to animal morphogenesis. Using mouse and chicken embryos, we are analyzing the roles of cadherins and associated proteins in various morphogenetic processes, including neural tube closure and neural crest migration. We are also
investigating the roles of members of the cadherin superfamily known as protocadherins,
deficiencies of which have been implicated in brain disorders. Through these studies, we
expect to gain deeper mechanistic insights into the ways by which cells build the elaborate
structures of the animal body.
3) In addition, we have recently begun analyzing the functions of microtubule minus endassociated proteins, Nezha/CAMSAPs. These proteins regulate microtubule assembly patterns, centrosomal function, and organelle positioning. We are exploring molecular mechanisms
underlying such regulatory activity, as well as the roles of these molecules in cellular morphogenesis, such as polarized epithelial formation and axon growth, with the aim of uncoveringnovel functions of non-centrosomal microtubules.

Staff
Group Director

Technical Staff

Masatoshi TAKEICHI

Sylvain HIVER
Yoko INOUE
Saeko KOBAYASHI
Miwako NOMURA
Hiroko SAITO

Research Scientist

Shuichi HAYASHI
Shoko ITO
Shigenori NAGAE
Anna PLATEKNobutoshi TANAKA
Mika TOYA
Vassil VASSILEV

Part-Time Staff

Haruka MORISHITA
AssistantMutsuko AISO-WATANABE

Collaborative Scientist

Wenxiang MENG
Shoko NAKAMURA
Tamako NISHIMURA

Recent Publications
Tanaka N, et al. Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of non-centrosomal
microtubules. Proc. Natl. Acad. Sci. USA 109.20029-34(2012)
Nishimura T, et al. Planar cell polarity links axes of spatial
dynamics in neural-tube closure. Cell 149.1084-97 (2012)
Ishiuchi T. and Takeichi M. Willin and Par3 cooperatively
regulate epithelial apical constriction through aPKC-mediated ROCK phosphorylation. Nat Cell Biol 13.860-6 (2011)
Taguchi K, et al. Mechanosensitive EPLIN-dependent remodeling of adherens junctions regulates epithelial reshaping. J Cell Biol 194.643-56 (2011)
Ishiuchi T, et al. Mammalian Fat and Dachsous cadherins
regulate apical membrane organization in the embryonic cerebral cortex. J Cell Biol 185.959-67 (2009)
Meng W, et al. Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts.
Cell 135.948-59 (2008)78

Double-immunostaining for F-actin (green) and Kusabira Orange-tagged E-cadherin (red) introduced into A431D cells. In these cells, E-cadherin dynamicallymoves along cortical actin filaments, resulting in the unique distributions shown
here.

Systems Biology
Recent large-scale efforts in genome-sequencing, expression profiling and functional
screening have produced an embarrassment of riches for life science researchers and biological data can now be accessed in quantities that are orders of magnitude greater thanwere available even a few years ago. The growing need for interpretation of data sets, aswell as the accelerating demand for their integration to a higher level understanding of life,
has set the stage for the advent of systems biology, in which biological processes and phenomena are approached as complex and dynamic systems. Systems Biology is a naturalextension of molecular biology, and can be defined as "biology after the identification of key
genes." We see systems-biological research as a multi-stage process, beginning with thecomprehensive identification and quantitative analysis of individual system components andtheir networked interactions, and leading to the ability to drive existing systems toward adesired state and design new ones based on an understanding of structure and underlying
principles.
Over the last several years, the Laboratory for Systems Biology (LSB) has worked to establish experimental systems biology at the molecular-to-cellular level and apply them to system-level questions of complex and dynamic biological systems, such as the mammalian
circadian clock. In October 2009, our laboratory was re-designated as a Project Lab in theCenter Director’s Strategic Program for Systems Biology research to promote challenging
research endeavors. Based on the achievements over the past years, we strongly feel that
it is now the time for us to take the next step forward toward experimental systems biology
at the cellular-to-organism level.
Over the next several years, we intend to develop an efficient experimental platform to identify, monitor, and perturb cellular networks within organism. To this aim, we will attempt toinvent and combine several key technologies ranging from (i) rapid engineering of the genome of ES cells, (ii) generation of “100% chimera” animals for F0 phenotyping, and (iii)
phenotype analysis of a small number of the generated animals (ideally with a single animal).
Full utilization of these technologies will formulate cellular-to-organism-level systems biology, which will provide new strategies and concepts for the diagnosis, treatment, and prevention of biological-time-related disorders, including rhythm disorder, seasonal affective
disorder, and sleep disorder.

Hiroki R. UEDA M.D., Ph.D.
http://www.cdb.riken.go.jp/en/ueda
Hiroki R. Ueda received his M.D. from the Faculty of
Medicine of the University of Tokyo in 2000, andreceived his Ph.D. from the University of Tokyo
Graduate School of Medicine in March 2004. Whilean undergraduate, he worked as a research assistant on a biological simulation system project at
Sony Computer Science Laboratories. As a graduate student he next went on to work, first as a researcher (2000) and then as a group leader (2002),
at Yamanouchi Pharmaceuticals, on a project studying biological clock mechanisms in fly and mouse.
He was appointed team leader of Laboratory for
Systems Biology in 2003, and Leader of the Functional Genomics Unit at the CDB in 2004. He wasalso appointed as Professor (Invited) of Osaka University in 2006. In 2009, he was promoted project
leader of the Laboratory for System Biology under
CDB’s Center Director’s Strategic Program.

Staff
Project Leader

Visiting Researcher

Hiroki R. UEDA

Arthur MILLIUS

Research Scientist

Technical Staff

Yohei M. KOYAMA
Yasutaka NIWA
Genshiro SUNAGAWA
Hideki UKAI
Rikuhiro G. YAMADA

Hiroshi FUJISHIMA
Junko HARA
Naoshi KOIDE
Kaori TSUJINO
Maki UKAI-TADENUMA
Junko YOSHIDASpecial Postdoctoral
Researcher

Hiroki DANNO
Etsuo A. SUSAKI
Foreign Postdoctoral
Researcher

Craig C. JOLLEY
Dimitri PERRIN

Student Trainee

Genki KANDA
Hiroyuki TAMIYA
Visiting Scientist

Kohei MASUMOTO
Atsushi WADA
Assistant

Noriko OKAMOTO

Recent Publications
Tsujino K, et al. Establishment of TSH β real-time monitoring
system in mammalian photoperiodism. Genes Cells 18.57588 (2013)
Sunagawa G. A, et al. FASTER: an unsupervised fully automated sleep staging method for mice. Genes Cells 18. 50218 (2013)
Jolley C. C, et al. A design principle for a posttranslational
biochemical oscillator. Cell Rep 2. 938-50 (2012)

Post-translational processes such as protein phosphorylation are vital for circadian rhythms in manyorganisms. In cyanobacteria, circadian proteins can be incubated with ATP to form an in vitro posttranslational oscillator (PTO) that operates in the absence of transcription and translation. It is still
unknown whether components of the mammalian clock may also be able to function as a PTO. In a
recent paper, Jolley, Ode, and Ueda developed a mathematical model to examine the possibility ofoscillations in a simple system with only three components. They found that two essential design
motifs are necessary for sustained post-translational oscillation: a preferred ordering of phosphorylation states ("single-molecule oscillators") and synchronization of these autonomous oscillators by
enzyme sequestration.

Ukai-Tadenuma M, et al. Delay in feedback repression by
cryptochrome 1 is required for circadian clock function.
Cell 144. 268-81 (2011)
Masumoto K. H, et al. Acute induction of Eya3 by late-night
light stimulation triggers TSHβ expression in photoperiodism.
Curr Biol 20. 2199-206 (2010)
Isojima Y, et al. CKIepsilon/delta-dependent phosphorylation
is a temperature-insensitive, period-determining process inthe mammalian circadian clock. Proc Natl Acad Sci USA
106. 15744-9 (2009)79

Genomic Reprogramming
A theoretically limitless number of clones of an animal can be generated from its somatic
cells. Only a few years ago, such a statement would have belonged to the realm of science
fiction, but now thanks to advances in the technology known as micromanipulation, whichallows researchers to work with individual cells and their nuclei, that fiction has become reality. The efficiency of the cloning procedure (measured as the ratio of live offspring to cloning
attempts), however, remains quite low, at only a few percent, and even in those attempts
that do lead to live births, the cloned animals consistently exhibit a range of severe developmental abnormalities and pathologies.

Teruhiko WAKAYAMA Ph.D.
http://www.cdb.riken.go.jp/en/wakayama
Teruhiko Wakayama received his B.Sc. and M.Sc.
from Ibaraki University, and was awarded a Ph.D. inreproductive biology from the University of Tokyo
Department of Veterinary Anatomy in 1996. He received a postdoctoral fellowship from the JapaneseSociety for the Promotion of Science in 1996 andspent the next two years at the Yanagimachi lab in
the University of Hawaii Medical School, where hesucceeded in creating the world's first cloned mouse.
He was appointed to an assistant professorship at
the same institution in 1998, and moved to theRockefeller University as a research assistant professor in 1999. He spent a year as a researcher at
Advanced Cell Technology before returning to Japan to take his current position at the RIKEN CDB.

The cause of these defects is thought to be due to imperfections in the nuclear transfer
technique used to remove the nucleus from a host cell and replace it with another from adonor; imperfections which presumably lead to total or partial failure of reprogramming, theprocess in which a cell’s developmental potential is reset to a totipotent state (such as is
exhibited by a naturally fertilized egg). However, the specific details of the technical flaws, theways in which they lead to reprogramming failure, and possible solutions to the problem all
remain completely unknown. Despite these challenges, cloning continues to be a biotechnology of great promise, given its potential applications in the field of regenerative medicine,such as the generation of embryonic stem (ES) cells by nuclear transfer, which may one day
allow us to grow replacement cells that perfectly match a patient’s own genetic and immune
profile, potentially eliminating the risk of rejection.
We use the mouse as a model system to study cloning under a range of experimental conditions with the goals of achieving improvements in the efficiency of the cloning procedure,
gaining a better understanding of reprogramming mechanisms and analyzing the properties of
ES cells derived via somatic nuclear transfer. We also use nuclear transfer technology to develop
methods for preserving embryonic lethal and infertile strains of laboratory mice, and continue toexplore the development of new and better techniques for sperm and oocyte preservation andother reproductive biological research technologies.

Staff
Team Leader

Student Trainee

Teruhiko WAKAYAMA
Research Scientist

Hirohisa KYOGOKU
Yukari TERASHITA

Sayaka WAKAYAMATechnical Staff

Visiting Scientist

Satoshi KISHIGAMI
Koji KOJIMAHaruko OBOKATA
Mikiko TOKORO
Kazuo YAMAGATA

Yuko SAKAIDE
Kaori YAMANAKA
Assistant

Sakura HIRAUCHI

Recent Publications
Wakayama S, et al. Successful Serial Recloning in the Mouseover Multiple Generations. Cell Stem Cell 12:293-297 (2013)(Cover article of issue)
Terashita Y. et al. Latrunculin A treatment prevents abnormal
chromosome segregation for successful development ofcloned embryos PLoS One 8. e78380 (2013)
Terashita Y, et al. Latrunculin A Can Improve the Birth Rateof Cloned Mice and Simplify the Nuclear Transfer Protocol by
Gently Inhibiting Actin Polymerization. Biol Reprod.86:180
1-6 (2012)
Yamagata K, et al. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy. PLoS
One 7.e31638 (2012)
Mizutani E, et al. Abnormal chromosome segregation at
early cleavage is a major cause of the full-term developmental failure of mouse clones. Dev Biol 364.56-65 (2012)

Transfer of a somatic nucleus into an enucleated egg

80Epithelial Morphogenesis
The central question in developmental biology is how cells, tissues and organs acquire their
specific functions and shapes. A large body of work over the past several decades has
yielded a broad understanding of how functional specialization is achieved through differential
gene expression. In contrast, far less is known about how cell shapes and tissue structures arecontrolled and remodeled. Although a general theme has emerged whereby cytoskeletal elements control the cell shapes, while alteration of individual cell shapes collectively organizes
the tissue architecture, the underlying molecular and mechanical mechanisms remain poorly understood. My lab aims at identifying novel mechanisms that orchestrate the formationof three-dimensional epithelial structures. Our long-term goal is to comprehensively understand the mechanistic principles of tissue morphogenesis in order to conceptualize the originof morphological diversity both within an organism and among evolutionary lineages.
We are currently focusing on how modifications of epithelial cell polarity control cell shapes
using gastrulating Drosophila embryos as the model system. Our previous work identified anovel mechanism for cell shape changes whereby cell shortening is induced upon a basal
repositioning of the apical-basal polarity and cell-cell adhesive apparatus adherens junctions. The cell shortening occurs in two narrow strips of cells, producing heterogeneities in cellheight within the tissue, thereby allowing it to bend. The polarity-based mechanism represents
the first instance wherein the initiation of epithelial folding does not involve the canonical myosindependent apical constriction. Since cell-cell adhesion and apical-basal polarization are two
fundamental features of epithelial tissues, our work potentially heralds a general mechanism forcell shape changes and epithelial folding. In addition, we found that after initiation, thedepths of epithelial folds differ depending on the degrees of neighboring cell invagination.
Genetic evidence suggests that the strength of mechanical coupling between adherens junctions
and their underlying actin cytoskeleton determines the extent of cell invagination. Our ongoingwork promises to identify genes and forces that sculpt distinct morphological features.
We employ an integrated approach that combines genetic manipulation, two-photon deep
tissue live imaging and computational cell shape reconstruction. We are also in the processof designing novel imaging strategies that could be used to visualize mechanical forces andcomputational algorithms that reconstruct and quantify 4D cell shapes. Furthermore, we will
launch a multidisciplinary, international collaboration that combines genetics, computationaland evolutionary approaches to analyze the history and function of transiently formed epithelial structures that do not eventually contribute to a body part or organ.

Yu-Chiun WANG Ph.D.
http://www.cdb.riken.go.jp/en/wang
Yu-Chiun Wang completed his B.Sc. (1996) and
M.S. (1998) from the National Taiwan University,
Taiwan, and received his doctorate in 2006 fromthe University of Chicago, U.S.A. under the supervision of Prof. Edwin Ferguson for his work on theregulation of BMP signaling during dorsal-ventral
patterning in the Drosophila for which he wasawarded the 2007 Larry Sandler Memorial Awardfor best dissertation of Drosophila research. In
2007, he moved to Princeton University to work as
a postdoctoral fellow in the laboratory of Eric Wieschaus and was also awarded a postdoctoral research fellowship from the Helen Hay Whitney
Foundation for his work on understanding themechanisms of the formation of the cephalic furrow
and dorsal transverse folds during Drosophila gastrulation. He was appointed team leader of theLaboratory for Epithelial Morphogenesis at the CDB
in October 2013.

Staff
Team Leader

Assistant

Yu-Chiun WANG

Yuko FUJIYAMA

Research Scientist

Niraj K. NIRALA
Technical Staff

Mustafa SAMI
Michiko TAKEDA

Recent Publications
Wang Y. C, et al. Distinct Rap1 activity states control theextent of epithelial invagination via alpha-catenin. Dev Cell
25.299-309 (2013)
Wang Y. C, et al. Differential positioning of adherens junctions is associated with initiation of epithelial folding. Nature
484.390-3 (2012)
Wang Y. C. and Ferguson E. L. Spatial bistability of Dpp-receptor interactions during Drosophila dorsal-ventral patterning. Nature 434.229-34 (2005)
Podos S. D, et al. The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev Cell 1.567-78 (2001)

The formation of dorsal folds during Drosophila
gastrulation.

Reconstruction of 3D cell shapeduring dorsal fold formation.

The dorsal epithelium of the gastrulating Drosophila embryo forms two epithelial (anterior and posterior) folds.
These pictures show mid-sagittal optical sections of anearly (A) and a late (B) embryos that display the initiation(A) and final morphology (B) of these dorsal folds. The
highlighted areas are shown on the right panels. Adherens
junctions are labeled in green; plasma membrane in magenta.

EDGE4D software was developed incollaboration with Dr. Zia Khan (University of Maryland) to perform 3D reconstruction of cell shape based oncell membrane labeling. Immunolabeling of adherens junction (orange in A
and B) defines the boundaries between the apical and basal-lateralcompartments (A’ and B’).

81

Sox2 sits atop different transcriptional networks in ESC and
TSC self-renewal

The first unmistakable differentiation events in mammaliandevelopment take place just as the blastocyst is forming,with the outer layer of cells committing to form the extraembryonic trophectoderm, and the inner cell mass giving
rise to the earliest embryonic lineages. When grown in culture, these inner cell mass cells are known as embryonicstem cells (ESCs), and show the ability, known as pluripotency, to give rise to every embryonic lineage, as well as theability to proliferate indefinitely by self-renewal in vitro.
Trophectoderm likewise arises from trophectoderm stem
cells (TSCs), which form the embryonic component of theplacenta. The genetic regulation of such ‘stemness’ in
ESC, TSC and neural stem cells relies in part on the function of the transcription factor Sox2, but how the function ofthis protein functions differs across these cell types has
never been resolved.
A new in-depth study by Kenjiro Adachi and colleagues inthe Lab for Pluripotent Stem Cell Studies (Hitoshi Niwa,
Project Leader) reveals that Sox2 sits atop two different
networks of stemness genes in mouse ESCs and TSCs.
Conducted in collaboration with the CDB labs for Genomic
Reprogramming, Functional Genomics, Systems Biology
and Genome Resources and Analysis, this work was published in Molecular Cell.
Previous work by the Niwa lab had shown that deletion of
Oct4, a known pluripotency gene, caused ESCs to transform into TSCs, which are known to rely on the activity of
FGF4. In the present study, Adachi and colleagues examined changes in gene expression during this transition, anddetermined that in TSCs, FGF4 activates Sox2 and a second gene, Esrrb. They found that by misexpressing thesegenes in TSCs, they could maintain their stemness even in
the absence of FGF4. In contrast, in mouse ESCs, Sox2 isunder the control of LIF, indicating to the team that Sox2
might interact with different sets of genes to maintain stemness and inhibit differentiation in trophoblast and embryonic stem cells.

Kenjiro ADACHI

82

What then is responsible for the switch in Sox2 targets?
Adachi et al. next looked at Sox2 DNA and protein binding,and discovered something interesting: Sox2 does not act
alone, but functions by binding with other transcription fac-

Molecular Cell

Article©2013 Cell Press

Context-Dependent Wiring of Sox2 Regulatory
Networks for Self-Renewalof Embryonic and Trophoblast Stem Cells

Adachi K, et al. Context-Dependent Wiring of Sox2 Regulatory Networks for SelfRenewal of Embryonic and Trophoblast Stem Cells. Mol Cell 52.380-92 (2013)

Kenjiro Adachi,1,9,10,* Itoshi Nikaido,2,9,11 Hiroshi Ohta,3,12 Satoshi Ohtsuka,1 Hiroki Ura,1 Mitsutaka Kadota,5Teruhiko Wakayama,3,6 Hiroki R. Ueda,2,4 and Hitoshi Niwa1,7,8,*
1Laboratory for Pluripotent Stem Cell Studies
2Functional Genomics Unit
3Laboratory for Genome Reprogramming
4Laboratory for Systems Biology
5Genome Resource and Analysis Unit
RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 6500047, Japan
6Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 4008510, Japan
7Laboratory for Development and Regenerative Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo-ku,
Kobe 6500017, Japan
8JST, CREST, Sanbancho, Chiyoda-ku, Tokyo, 1020075, Japan
9These authors contributed equally to this work
10Present address: Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
11Present address: Bioinformatics Research Unit, Advanced Center for Computing and Communication, RIKEN, Wako,
Saitama 3510198, Japan
12Present address: Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku,
Kyoto 6068501, Japan*Correspondence: kenjiro.adachi@mpi-muenster.mpg.de (K.A.), niwa@cdb.riken.jp (H.N.)
http://dx.doi.org/10.1016/j.molcel.2013.09.002

SUMMARY

Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential rolein different types of multipotent stem cells, raising
the possibility that Sox2 governs the common
stemness phenotype. Here we show that Sox2 isa critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal oftrophoblast stem cells (TSCs). Sustained expressionof Sox2 together with Esrrb or Tfap2c can replace
FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and
TSCs combined with inducible knockout systems,
we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct
sets of genes with unique functions in these twodifferent yet developmentally related types ofstem cells. Our findings provide insights into thefunctional versatility of transcription factors during
embryogenesis, during which they can be recursively utilized in a variable manner within discrete
network structures.

INTRODUCTION
The transcriptional output of a given cell type is controlled byunique combinations of transcription factors under the control

of extrinsic signals that can modulate the expression and activityof transcription factors, forming a gene regulatory network that
dictates a specific cellular phenotype. Tissue-specific transcription factors play deterministic roles in cell-type specification,
which is manifested as lineage reprogramming by forced expression of such transcription factors (Graf and Enver, 2009; Zhou
and Melton, 2008). Sox2 is one such transcription factor required
for the maintenance of pluripotent stem cells in vivo (Avilion et al.,2003) and in vitro (Masui et al., 2007) and for the induction ofpluripotency (Takahashi and Yamanaka, 2006). However, it isalso preferentially expressed in neural, retinal, and trophoblaststem cells (TSCs) (Avilion et al., 2003; Pevny and Nicolis,2010), suggesting a possible role for Sox2 in governing a common stemness phenotype.
In embryonic stem cells (ESCs), Sox2 forms a heterodimer
with Oct3/4 (also known as Pou5f1) on DNA with the OCT-SOX
composite motifs, and these factors cooperatively activate
pluripotency-related target genes such as Nanog, Fgf4, Utf1,
Lefty1, and Fbxo15, as well as their own expression (Nakatake
et al., 2006, and references therein). Oct3/4-knockout ESCs
are differentiated along the trophoblast lineage in a highly homogeneous manner (Niwa et al., 2000). In contrast, the loss of Sox2
causes differentiation of ESCs accompanied by upregulation ofmarkers for trophoblast and embryonic germ layers, although
artificial maintenance of Oct3/4 from the transgene can sustain
self-renewal and pluripotency of Sox2-null ESCs (Masui et al.,2007), suggesting that the unique function of Sox2 may be
to maintain Oct3/4 expression. These two core transcription
factors, along with Nanog, form an interconnected and hierarchical network downstream of the leukemia inhibitory factor (LIF)Stat3 and LIF-phosphatidylinositol 3-kinase (PI3K) signaling

380 Molecular Cell 52, 380–392, November 7, 2013 ª2013 Elsevier Inc.

tors in a cell context-dependent fashion. While it binds Oct4
in ESCs, in TSCs it partners with Tfap2c; these changes inbinding partner trigger conformational changes that enableinteractions with independent networks of target genes inthese stem cell types.
“The idea that combinations of the activity of various signaling pathways helps to give rise to cellular diversity is widely
accepted, but it is generally thought that the factors involved and the order of activation within a pathway are
fairly uniform,” says Niwa. “But what we have shown here isthat changes in upstream and downstream factors can playsignificant roles in driving cell diversity, and we suspect perhaps evolutionary processes as well.”A
Oct3/4 KO
Day 0-1 kb 0 +1 kb

Sox2
Day 1

Day 2

Day 3

Day 4

A schematic of genetic networks regulating stemness in early embryoniccells. In mouse ESCs, Sox2 is controlled by LIF, and in TSCs by FGF4. The
switch of Sox2’s binding partner from Oct4 to Tfap2c changes the set ofdownstream genetic interactions, inducing a transition from ESC to TSC
state. In this scheme, Oct4 and Tfap2c are mutually inhibitory.

Oct3/4 KO
Day 0

B

Esrrb
Day 1

Day 2

Day 3

Day 4

-1 kb 0 +1 kb

Heatmaps of Sox2 and Esrrb ChIP-seq signals around peak regions. Sox2 binding sites changed
dynamically after elimination of Oct3/4; most of the peaks were found specifically in ESCs or
TSCs. In contrast, Esrrb binding showed less dynamic behavior, with only persistent or ESCspecific peaks apparent.

83

GDNF levels control neuronal migration and differentiation in
the ENS

Toshihiro UESAKAThe neurons of the enteric nervous system undergo among
one of the longest journeys in development, as precursors
migrate downward and proliferate to innervate the digestive
tract from end to end. This process involves two main stages: primary caudal migration by neural crest-derived precursors, and a second round of radial migration of a subset
of precursors that radiate to the submucosal layer. While
precursors in the myenteric ganglia (MG) typically leave mitosis by the time of birth, many submucosal ganglia (SMG)
precursors remain active for two weeks thereafter, suggesting that these cells at least in part remain in an incompletely differentiated state. The molecular controls over this exquisitely coordinated activity, however, have remained elusive.
Toshihiro Uesaka and colleagues in the Lab for Neuronal
Differentiation and Regeneration (Hideki Enomoto, Team84

Leader) now reveal that GDNF signaling is an essential
regulator of both the primary and secondary migration of
ENS precursors in mouse. Published in The Journal of Neuroscience, these new findings also show that intracellularsignaling levels controlled by GDNF regulate differentiation
states in these precursors as well.
At embryonic day 9.5 in mouse embryonic development,
neural crest-derived cells begin to enter the foregut and migrate down the enteric tract toward the colon, completing
this primary migration by embryonic day 14. Most of thisfirst wave of migrants differentiates to form myenteric ganglia, but a subpopulation secondarily migrates radially into
the submucosal layer, giving rise to submucosal ganglia.
GDNF is expressed in the gut mesenchyme, and activates
the RET tyrosine kinase through binding with the receptor

16372 • The Journal of Neuroscience, October 9, 2013 • 33(41):16372–16382©2013 Society for Neuroscience
Uesaka T, et al. GDNF signaling levels control migration and neuronaldifferentiation of enteric ganglion precursors. J Neurosci 33.16372-82 (2013)

Development/Plasticity/Repair

GDNF Signaling Levels Control Migration and Neuronal
Differentiation of Enteric Ganglion Precursors
Toshihiro Uesaka,1,3 Mayumi Nagashimada,1,2 and Hideki Enomoto1,2,3
1Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan, 2Department of Development
and Regeneration, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan, and 3Division of Neural Differentiation and Regeneration,
Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan

Pleiotropic growth factors play a number of critical roles in continuous processes of embryonic development; however, the mechanisms
by which a single regulatory factor is able to orchestrate diverse developmental events remain imperfectly understood. In the development of the enteric nervous system (ENS), myenteric ganglia (MGs) form initially, after which the submucosal ganglia (SMGs) develop byradial inward migration of immature ENS precursors from the myenteric layer. Here, we demonstrate that glial cell line-derived neurotrophic factor (GDNF) is essential for the formation not only of the MGs, but the SMGs as well, establishing GDNF as a long-term acting
neurotrophic factor for ENS development in a mouse model. GDNF promotes radial migration of SMG precursors. Interestingly, premigratory SMG precursors in the myenteric layer were distinguished from the surrounding neuronally differentiating cells by their lower
activation of the GDNF-mediated MAPK pathway, suggesting that low activation of GDNF downstream pathways is required for the
maintenance of the immature state. ENS precursors devoid of GDNF signaling during midgestation halt their migration, survive, andremain in an undifferentiated state over the long-term in vivo. Reactivation of GDNF signaling in these dormant precursors restores theirmigration and neuronal differentiation in gut organ culture. These findings suggest that pleiotropic function of GDNF is at least in partgoverned by modulating levels of intracellular activation of GDNF downstream pathways; high activation triggers neuronal differentiation, whereas low activation is crucial for the maintenance of progenitor state.

Introduction
Embryonic development is a highly complex, dynamic processthat requires exquisite orchestration of cellular proliferation, migration, differentiation, and maintenance of progenitor cells.
Cells are exposed to a number of growth factors during development, some of which are expressed for long periods, inducing
various cellular responses in tissue progenitors and their progeny.
Because impaired regulation of progenitors causes many developmental disorders, it is crucial to understand how a single
growth factor governs the behavior of progenitors, including migration, proliferation, and differentiation, and regulates themaintenance of in-tissue progenitors in an undifferentiated state.
The enteric nervous system (ENS) serves as an interestingmodel for the study of long-term developmental processes in
Received May 16, 2013; revised Sept. 4, 2013; accepted Sept. 6, 2013.
Author contributions: T.U. and H.E. designed research; T.U. and M.N. performed research; T.U. analyzed data; T.U.
and H.E. wrote the paper.
This work was supported by RIKEN and Ministry of Education, Culture, Sports, Science and Technology of Japan(Grant-in-Aid for Scientific Research on Innovative Areas, “Cellular and Molecular Basis for Neuro-Vascular Wiring”).
We thank the Laboratory for Animal Resources and Genetic Engineering for their excellent technical assistance. We
also are grateful to D. Sipp and H.S. Hiraga for editing this manuscript.
The authors declare no competing financial interests.
Correspondence should be addressed to Hideki Enomoto, Division of Neural Differentiation and Regeneration,
Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, 7-5-1, Kusunokicho,
Chuo-ku, Kobe 650-0017, Hyogo, Japan. E-mail: enomotoh@med.kobe-u.ac.jp.
M. Nagashimada’s present address: Department of Cell Metabolism and Nutrition, Brain/Liver Interface Medicine
Research Center, Kanazawa University, Kanazawa, Japan.
DOI:10.1523/JNEUROSCI.2079-13.2013
Copyright © 2013 the authors 0270-6474/13/3316372-11$15.00/0

GFRα1. All of these factors are essential for proper ENS
development, as evidenced by the loss of enteric neurons
in mice lacking any of these genes. Previous work by the
Enomoto lab has shown that enteric precursors are receptive to GDNF signaling into the postnatal period, suggestingthat this factor plays a long-term central role in controlling
multiple events during ENS development.
Uesaka used a number of conditional knockout mouselines to gain a more detailed understanding of GDNF’s
function. When he examined GFRα1 expression, he foundthat in both myenteric and submucosal ganglia, GFRα1+cells (i.e., those carrying receptors for GDNF) also expressed Sox10, a marker of undifferentiated cells. Knocking out GFRα1 at day 10.5, he found that mutant precursor
cells lost most of the migratory activity seen in control ENS
precursors. Knockout of the same gene at day 15.5, duringsecondary migration, caused a near-complete failure of
submucosal ganglia formation. These results indicated that
not only does GDNF signaling play important roles throughout ENS development, but that its activity changes overtime.
Looking at temporal changes in GDNF expression, theteam found that while GDNF was highly expressed in boththe longitudinal and circular myenteric layers, by day 18.5,
when MG formation was complete, its expression was con-response to a single growth factor. In mice, vagal neural crest(NC) cells enter the foregut at embryonic day (E) 9.5 (Durbec etal., 1996) and migrate caudally until they colonize the entire
length of the gut, at �E14. After colonization, the majority of
ENS precursors form the myenteric ganglia (MGs), while a subset
of ENS precursors undergoes radial migration from the myenteric to the submucosal layer to form the submucosal ganglia(SMGs) (Gershon et al., 1993). Most MG precursors become
postmitotic by birth, but some SMG precursors proliferate until
postnatal day 15 (P15; Pham et al., 1991). Thus, ENS precursors
forming the SMGs remain in an immature state for a long periodof time.
Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gut mesenchyme (Young et al., 2001; Natarajan etal., 2002), binds preferentially to GDNF family receptor �1(GFR�1), and activates the RET tyrosine kinase (Jing et al., 1996;
Baloh et al., 2000), which is expressed in ENS precursor cells(Pachnis et al., 1993; Durbec et al., 1996; Young et al., 2003). As
GDNF signaling is required for ENS precursor proliferation andmigration (Chalazonitis et al., 1998; Hearn et al., 1998; Heuckeroth et al., 1998; Taraviras et al., 1999; Gianino et al., 2003; Uesaka
et al., 2007), null mutations of Gdnf, Gfr�1, or Ret cause intestinal
aganglionosis in mice (Schuchardt et al., 1994; Moore et al., 1996;
Pichel et al., 1996; Sánchez et al., 1996; Cacalano et al., 1998;
Enomoto et al., 1998). Interestingly, ENS precursors retain responsiveness to GDNF signaling even after birth (Wang et al.,2010), suggesting a role for GDNF in regulating precursor behaviors during both embryonic and postnatal ENS development.

fined mainly to the circular muscle layer, highlighting thepossibility that this shift in the site of GDNF expression
might be responsible for inducing secondary migration.
The formation of submucosal ganglia subsequent to MG
formation indicates that even during their differentiation, theMG population must retain some undifferentiated cells.
Here again, the team found that differential GDNF expression plays a role, as shown by the higher levels of a GDNF
downstream factor in differentiated enteric neurons, andlower levels in undifferentiated precursors at day 12.5. Uesaka tested this hypothetical role for GDNF signaling incontrolling cell state by knocking out RET, which interrupts
GDNF signaling. Whereas most precursors would have differentiated into neurons within a few days, he found that in
the RET knockout many remained undifferentiated. Recovery of GDNF signaling rescued the defects in migration anddifferentiation, sealing the case for its broad functionality.
“This work presents a very reasonable system for the longterm maintenance of precursors in an undifferentiatedstate, and show why even seemingly minor changes in
GDNF signaling receptors can lead to major developmental
defects,” says Enomoto. “At the same time, we know thatmany other factors are also involved in regulating ENS development, so we will be curious to learn more about theseare orchestrated in the embryonic gut.”(Left) ENS precursors migrate down digestive tract at embryonic days 9.5–14 (primary migration) and then radiate inward from the myenteric layer to thesubmucosa (secondary migration). (Right) In a condition knockout in which GDNF signaling is blocked during secondary migration (E15.5), submucosal
ganglia (green) nearly entirely fail to form. (Pink shows neuronal marker PGP9.5)

85

Spontaneous fluctuations in cells support chemotaxis

Chemotaxis refers to movement by cells to or away fromchemical stimuli in their environments. In the multicellularbody, this process plays a central role in directing immune
responses by white blood cells and the formation of nervous system tissues. For more primitive eukaryotes, suchas the slime mold Dictyostelium, chemotaxis plays what isperhaps an even more fascinating role, guiding individual
cells in food-poor environments to assemble into a multicellular aggregate, or ‘slug,’ capable of reproduction. The
mechanism underlying this chemotaxis resembles thatseen in mammalian immune cells, and allows tiny cells justa few micrometers in diameter to respond to minute differences of a few percent in the concentration of a chemical
stimulus. However, cells do not need an excuse to move;even in the absence of a chemotactic signal, cells move in
random direction under the control of internal fluctuations.
The question then is, how do cells override these random
internal signals so as to be able to detect, amplify, and respond to gradients of external stimuli?
In a report published in Journal of Cell Science, Research
Unit Leader Tatsuo Shibata and colleagues in the Lab for
Physical Biology used fluorescence imaging to reveal amechanism for controlling the spontaneous polarization of
Dictyostelium cells, from which they developed a quantitative model able to reconstruct the phenomenon. A second
paper by the same lab, published in Biophysical Journal,
reports a model of how this self-organizing mechanism robustly facilitates the exquisite sensitive of these cells tochemical stimuli. Both studies were conducted in collaboration with researchers from the RIKEN Quantitative Biology
Center.
Dictyostelium cells respond chemotactically to gradients ofcAMP by initiating inositol phospholipid metabolism at thecell surface. Cell surface regions presented with a highcAMP concentration begin to produce the phosphatidylinositol lipid PIP3 from PIP2, while regions facing lower concentrations elicit the opposite reaction, catalyzed by the
enzyme PTEN. Regions of high PIP3 mobilize actin fiber
formation to extend a pseudopod in the direction of thegradient, driving chemotaxis.
Tatsuo SHIBATA

86

Shibata wondered, however, how would cells behave in environments lacking cAMP, and simultaneously prevented
from generating cAMP themselves. He found that PIP3 domains appeared to move at random on the surface of such

Biophysical Journal Volume 105 November 2013 2199–2209©2013 Cell Press
Shibata T, et al. Intracellular encoding of spatiotemporal guidance cues in a selforganizing signaling system for chemotaxis in dictyostelium cells. Biophys J
105.2199-209 (2013)

2199

Intracellular Encoding of Spatiotemporal Guidance Cues in a SelfOrganizing Signaling System for Chemotaxis in Dictyostelium Cells
Tatsuo Shibata,†‡§* Masatoshi Nishikawa,†§ Satomi Matsuoka,§{ and Masahiro Ueda§{jj†
Laboratories for Physical Biology, RIKEN Center for Developmental Biology, Kobe, Japan; ‡PRESTO, Japan Science and Technology
Agency (JST), Saitama, Japan; §Japan Science and Technology Agency (JST), CREST, Osaka, Japan; {Laboratory for Cell Signaling
Dynamics, RIKEN Quantitative Biology Center, Osaka, Japan; and jjLaboratory of Single Molecule Biology, Graduate School of Science,
Osaka University, Osaka, Japan

ABSTRACT Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to movedirectionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases.
It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activatesthe motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network havebeen reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues.
Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision ofpolarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency
modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for thesensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-timewaves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.

INTRODUCTION
The ability of cells to sense an external chemical gradient isessential to cellular functions such as chemotaxis and otherdiverse processes (1). Eukaryotic cells such as leukocytes,
neurons, and amoebae detect differentials in the concentration of chemicals along their periphery, without rapid motile
translocation. A concentration differential of a few percent
across a cell body of a few tens of micrometers can be
sufficient to induce directional migration. In addition, thegradient information must be maintained in the presenceof noise in the signaling network and amplified sufficiently
for downstream processes to produce directional migration(2,3). What type of gradient sensing mechanism can afford
such astonishing sensitivity in noisy cellular systems?
In Dictyostelium discoideum, several signaling pathways
have been identified to mediate chemotaxis in parallel (4–6).
Among them, the phosphatidylinositol (PtdIns) lipids reaction is the key signaling event responsible for chemotaxis
in a shallow chemoattractant gradient (7). The PtdIns
signaling pathway also works for chemotaxis in othereukaryotic cells (8). Along a chemoattractant gradient,phosphoinositide-3-kinase (PI3K) catalyzes the production

Submitted April 13, 2013, and accepted for publication September 23, 2013.
*Correspondence: tatsuoshibata@cdb.riken.jp
M. Nishikawa’s present address is Max Planck Institute of Molecular Cell
Biology and Genetics, Dresden, Germany.
This is an Open Access article distributed under the terms of the Creative
Commons-Attribution Noncommercial License (http://creativecommons.
org/licenses/by-nc/2.0/), which permits unrestricted noncommercial use,
distribution, and reproduction in any medium, provided the original work
is properly cited.

of PtdIns(3,4,5)P3 from PtdIns(4,5)P2 at the membraneregion facing the higher chemoattractant concentration,
whereas the phosphatase and tensin homolog (PTEN) catalyzes the reverse reaction in the membrane region facing
the lower concentration. As a result, accumulation of
PtdIns(3,4,5)P3 induces actin polymerization to form a
pseudopod in the region of higher chemoattractant, leading
to chemotaxis. However, accumulation of PtdIns(3,4,5)P3
can be induced even in the absence of a gradient. Dictyostelium cells exhibit random cell migration using the same
motility apparatus (9–12). We hypothesized that some
type of intracellular signaling must be spontaneously
produced at random positions to polarize and activate themotility apparatus.
Spontaneous activity of chemotaxis systems in theabsence of a chemical gradient have been reported previously, including cell motility (13,14), actin waves (15–17),
and formation of PtdIns(3,4,5)P3–localized domains (18–22). We reported recently that the PtdIns lipids system is
responsible for generating spontaneous signals that promote random cell migration (21). In the absence of an
external gradient, the PtdIns lipids system can self-organize to produce a domain of high PtdIns(3,4,5)P3 concentration on the membrane (21,23), as shown in Fig. 1 A and
B. The domain exhibits a variety of spatiotemporal dynamics, such as persistent domain formation, that lasts
for more than 30 min (Fig. 1 A, left), and transient domain
formation lasts 10 s to 1 min (Fig. 1 B, left). We haveproposed a model to explain the mechanism of theseself-organization activity, which was developed based on
spatiotemporal statistical analysis of fluorescence image

Editor: Edda Klipp.
 2013 The Authors
0006-3495/13/11/2199/11 $2.00

cells, indicating an internal mechanism for establishing polarity, ensuring that PIP3 and PTEN localized in a mutually
exclusive fashion. Statistical analysis revealed that this activity could not be explained simply as the result of interactions between enzymes and substrates, but that rather
PTEN seemed to be ‘attracted’ to areas in which PIP3 levels were low. Using a reaction–diffusion model to simulate
this phenomenon, Shibata et al. were able to show how
domains of persistent PIP3 expression, in agreement with
their experimental observations. Adjusting the simulatedparameters, he was able to evoke cycles of PIP3 oscillation, resembling the behavior of cells in which cAMP was
not blocked. These validations of this quantitative model
point to possible broader applications in the analysis of spontaneous polarization, such as seen in neurons, which can
exhibit robust responses to even seemingly minor stimuli.

http://dx.doi.org/10.1016/j.bpj.2013.09.024“In these studies, we were able to construct quantitativemodels that closely recapitulate the results of experiments
using known molecules observed by fluorescence imaging,” says Shibata. “These models have led to new insights
into molecular mechanisms and gradient sensing, which
we next hope to validate using live cells. We are also hopeful that the self-organizing activity we have described willplay a role in the explication of other biological functions.”

In the second study, the team looked at cells confronted
with chemotactic stimuli. Using ordinary Dictyostelium
cells, they observed that while some would manifest stable
domains in response to cAMP, others showed transient domain patterns oriented toward higher cAMP concentrations. They next made a stochastic reaction–diffusion model of these two patterns, along with that of an inactive state
absent the self-organized polarity formation, and tested
how they would respond to stepwise increases in cAMP
gradient steepness. The persistent and transient domain
models responded much more strongly than did that of theinactive state, indicating that the self-organizing system
plays a role in promoting the response to cAMP. They next
turned to quantitatively analyzing PIP3 domain formation,and found that such domains formed more frequently in thepresence of a stimulus and became distributed with increasing precision in the direction of the gradient as thechemotactic signal rose.
In following cAMP gradients, Dictyostelium cells need tomove toward increases in concentration, and avoid pursuing lower levels of cAMP. Shibata suspected that this might
be possible through the combined effect of the extracellular
gradient, which introduces a small bias in the PIP3 domain
induction on the side of the cell facing the higher concentration, and self-organizing activity which establishes front–back orientation. In line with his predictions, the model showedthat the self-organizing system lies behind the robust, sensitive response by these cells to even shallow gradients.

(Top) Self-organization of polarity formation in Dictyostelium cells; PIP3
shown in green, PTEN in red. (Center left) Kymograph of PIP3 and PTEN
along cell surface. (Center right) Alternating cell surface concentrationsof PIP3 and PTEN. (Bottom) Simulation of mathematical model in threecases: No self-organization activity, persistent domain formation, ortransient domain formation.

87

The case of the missing nasopalatine: The maxillary nerve injaw evolution

As its name implies, the trigeminal nerve has three major
branches in vertebrates: a first division into the ophthalmic
nerve and the maxillomandibular, which further bifurcates
into the maxillary and mandibular nerves. The trigeminal
governs sensation and muscle control in the face and jaw.
In humans, it develops from a primordium just above thepharyngeal arches, from which the ophthalmic nerve diverges and extends toward the eye, and the maxillary andmandibular nerves toward their respective prominences.
The structure of the jaw is conserved from fish to mammals,so the how its innervation patterns were acquired during
evolution are a question of fundamental importance.
In a comparative study of trigeminal development in diverse
gnathostome taxa published in Journal of Morphology, Hiroki Higashiyama and Group Director Shigeru Kuratani of
the Lab for Evolutionary Morphology have discovered thata branch of the trigeminal (called the nasopalatine nerve inhuman) innervates derivatives of the medial nasal prominence. Only diapsids, such as gecko and chicken, lacked ahomolog of this nerve, suggesting it had been secondarily
lost in this lineage.
Textbook accounts of maxillary nerve development typicallystate that it is localized to the maxillary prominence andgoes on to innervate the entire upper jaw. But recent embryological studies have shown that the upper jaw arises
not only from the maxillary prominence, but rather forms
from the fusion of this primordial structure with the medial
nasal domain, suggesting an apparent discrepancy between neural morphology and developmental pattern. The
medial nasal prominence is the site of origin for the upper
lip, anterior palate, and front teeth, and gives rise to the upper jaw (maxilla) by merging with the maxillary prominence.
But despite the region’s origins in a fusion with the medial
nasal prominence, to which the maxillary nerve does not
localize, the maxillary nerve appears to control the entire
maxillary region.

Shigeru KURATANI, Hiroki HIGASHIYAMA

88

Interested in this conundrum, Higashiyama examined the
distribution of trigeminal nerve branches in mouse. At day
E10.5, he noted the clear projection of the maxillary nerve
to the maxillary prominence, but by day E11.5 he saw that
this nerve had begun to ramify rostrally. This branch, cor-

JOURNAL OF MORPHOLOGY 00:00–00 (2013)©2013 John Wiley & Sons, Inc.

On the Maxillary Nerve
Hiroki Higashiyama1,2 and Shigeru Kuratani2*

Higashiyama H. and Kuratani S. On the maxillary nerve.
J Morphol (2013)

1
2

Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan
Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

ABSTRACT The trigeminal, the fifth cranial nerve ofvertebrates, represents the rostralmost component of
the nerves assigned to pharyngeal arches. It consists of
the ophthalmic and maxillomandibular nerves, and in
jawed vertebrates, the latter is further divided into two
major branches dorsoventrally. Of these, the dorsal one
is called the maxillary nerve because it predominantly
innervates the upper jaw, as seen in the human anatomy. However, developmentally, the upper jaw isderived not only from the dorsal part of the mandibular
arch, but also from the premandibular primordium: themedial nasal prominence rostral to the mandibular
arch domain. The latter component forms the premaxillary region of the upper jaw in mammals. Thus, there
is an apparent discrepancy between the morphologicaltrigeminal innervation pattern and the developmental
derivation of the gnathostome upper jaw. To reconcile
this, we compared the embryonic developmental patterns of the trigeminal nerve in a variety of gnathostome species. With the exception of the diapsid species
studied, we found that the maxillary nerve issues abranch (nasopalatine nerve in human) that innervatesthe medial nasal prominence derivatives. Because thetrigeminal nerve in cyclostomes also possesses a similar branch, we conclude that the vertebrate maxillomandibular nerve primarily has had a premandibular
branch as its dorsal element. The presence of thisbranch would thus represent the plesiomorphic condition for the gnathostomes, implying its secondary loss
within some lineages. The branch for the maxillary
process, more appropriately called the palatoquadratecomponent of the maxillary nerve (V2), represents theapomorphic gnathostome trait that has evolved in association with the acquisition of an upper jaw. J. Morphol.
000:000–000, 2013. VC 2013 Wiley Periodicals, Inc.

in the trunk part (Gans and Northcutt, 1983; Shimada et al., 2013). The migrating cephalic neuralcrest cells predominantly localize to the ventral
part of the head, forming three massive ectomesenchymal cell populations. These cell populations
are called, from anterior to posterior: the trigeminal crest cells that fill the mandibular arch andmore rostral part of the head; hyoid crest cells inthe second arch; and circumpharyngeal crest cells
filling the postotic pharyngeal arches at the interface between the head and trunk portions of theembryonic body (Kuratani, 1997; Matsuoka et al.,2005). Of these, the trigeminal crest cells are socalled because the distribution of these cells
largely corresponds to the sensory innervation
domains of the trigeminal nerves; this ectomesenchymal region is divided into mandibular and premandibular components (Johnston, 1966; Kuratani
and Tanaka, 1990; Kuratani, 1997; Kuratani andHorigome, 2000). This domain is partly covered by
the surface ectodermal region of trigeminal placodes. Therefore, the trigeminal ganglionic primordia also differentiate in this neural crest cellstream (Batten, 1957a; Noden, 1988; O’Neill et al.,2007; McCabe and Bronner-Fraser, 2008), similar
to other sensory ganglia for branchiomeric nerves(VII–X) that develop both from cephalic crest cells
and dorsal/epibranchial placodes in a segmental
pattern, as seen in the pharyngeal arches (Fig.
1A). Thus, the peripheral nerve morphology istightly associated with the craniofacial developmental programs of vertebrates.

KEY WORDS: vertebrates; cranial nerves; pharyngeal
arches; trigeminal nerve
Additional Supporting Information may be found in the online version of this article.

INTRODUCTION
A major component of craniofacial primordia invertebrates is the cephalic neural crest-derived
ectomesenchyme (Le Douarin, 1982; Noden, 1983,
1988; Kuratani, 2012). Specified between the surface ectoderm and the neural plate, the neuralcrest produces delaminated, migratory cells withpluripotency, being able to differentiate into various cell types (Le Douarin, 1982; Gans and Northcutt, 1983; Noden, 1988). Of the neural crest, thecephalic component is characterized by its skeletogenic and mesenchymal differentiation, not foundContract grant sponsor: JSPS Research Fellowship; Contract
grant number: 23.0778 (H.H.).
*Correspondence to: Shigeru Kuratani; Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, 2-2-3
Minatojima-minami, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
E-mail: saizo@cdb.riken.jp
Received 3 June 2013; Revised 6 August 2013;
Accepted 8 August 2013.
Published online 00 Month 2013 inWiley Online Library (wileyonlinelibrary.com).
DOI 10.1002/jmor.20193

C 2013 WILEY PERIODICALS, INC.
V

responding to the human nasopalatine nerve, follows thenasal septum to innervate the front teeth and soft palate,indicating that it is the nasopalatine that controls the medial
nasal region in mammals. But what of other taxa? When
Higashiyama looked at relatively closely related diapsids,
such as reptiles and birds, he was surprised to find that thenasaopalatine was missing, with its function assumed byposterior facial nerves.
This raised the question of which state was ancestral, andwhich derived. Higashiyama broadened his search to anamniotes, such as cartilaginous and bony fishes, and were
happy to see that the maxillary and medial nasal prominences did not fuse, considerably simplifying the investigation. In these embryos, the maxillary nerve formed two
branches, extending into both the maxillary and medial nasal territories, suggesting that the arrangement seen in

mammals is common to all gnathostomes, and only secondarily absent in diapsids. When he looked at the lamprey,a jawless fish, Higashiyama found something even moreinteresting – the nerve projecting to its round mouth corresponds to the nasopalatine, suggesting that it, not the
maxillary, is the more ancient structure.
“Studies of vertebrate morphology often focus on hard
structures, such as skeletal elements, but our bodies aremostly made up of softer bits, including the nerves welooked at in this study,” says Kuratani. “Similar to the trigeminal, the vagal nerve plays an important role in controlling enteric functions, but the relationship between thisnerve and embryonic muscle development remain mysterious. By observing embryos to learn more about the makingof the body, we sometimes find it teaches about its evolution as well.”Jaw structures and innervation patterns during development. Jaw skeletal mesenchyme (prospective
maxillomandibular prominence) is innervated by the maxillomandibular nerve (v23). In most gnathostomes, the medial nasal prominence is innervated by the nasopalatine nerve or a homologous branch,
but this appears to have been lost secondarily during diapsid evolution.

89

Self-organized neocortex from human ESCs

The characteristic ‘wrinkled’ surface of the human neocortex is one the keys to the brain’s capacity for rational andanalytic thought and language, and evolved as a means ofincreasing surface area to a remarkable extent. Studies of
cerebral development, however, have largely been confined
to model organisms, such as mouse, leaving gaps in our
understanding of just this sort of human-specific feature.
What is needed is a better way to study the same processes in vitro.
Taisuke Kadoshima and colleagues in the Lab for Organogenesis and Neurogenesis (Yoshiki Sasai, Group Director)
have now succeeded in recapitulating the human neocortical development up to the equivalent of second trimesterdevelopment in an in vitro environment, building on previous work by the same lab in inducing self-organized tissue
differentiation from embryonic stem cells (ESCs). This work
was featured on the cover of The Proceedings of the National Academy of Sciences, in which it was published.
The Sasai group had previously shown that a 3D culture
technique known as SFEBq could be used to induce steer
the differentiation of human ESCs into a four-layered cortical-like structure, akin to that of a first-trimester fetus. Taking that study as their starting point, the group revisited theculture media and growth factors used in that protocol, andidentified a number of opportunities for optimization, enabling even higher efficiency cortical induction.
Using this modified SFEBq system, they found that nearly
all ESC aggregates became positive for the telencephalicmarker gene foxg1 on day 26, and rapidly began to differentiate into neuroepithelium by day 34. Following progressby a combination of live imaging and marker gene expression, the group observed the spontaneous onset of dorsocaudal–ventrororsal polarity in the neuroepithelium. The
dorsocaudal epithelium further began to exhibit a rolling
form of morphogenesis, resulting in curvature of the inward
tissue surface, evaginating to form a hemispheric, enclosed
cavity, closing resembling the process of brain formation in
vivo.

Taisuke KADOSHIMA, Hideya SAKAGUCHI, Mika SOEN90

©2013 National Academy of Sciences
Kadoshima T, et al. Self-organization of axial polarity, inside-out layer pattern, andspecies-specific progenitor dynamics in human ES cell-derived neocortex.
Proc Natl Acad Sci U S A 110.20284-9 (2013)

Self-organization of axial polarity, inside-out layer
pattern, and species-speciﬁc progenitor dynamics
in human ES cell–derived neocortex
Taisuke Kadoshimaa,b,1, Hideya Sakaguchia,b, Tokushige Nakanoa,2, Mika Soena, Satoshi Andoa,2, Mototsugu Eirakuc,and Yoshiki Sasaia,b,3
aLaboratory of Organogenesis and Neurogenesis and cFour-Dimensional Tissue Analysis Unit, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan;
and bDepartment of Medical Embryology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan

Edited by Chen-Ming Fan, Carnegie Institution of Washington, Baltimore, MD, and accepted by the Editorial Board October 17, 2013 (received for review
August 21, 2013)

Here, using further optimized 3D culture that allows highly selective
induction and long-term growth of human ES cell (hESC)-derived
cortical neuroepithelium, we demonstrate unique aspects of selforganization in human neocorticogenesis. Self-organized corticaltissue spontaneously forms a polarity along the dorsocaudalventrorostral axis and undergoes region-speciﬁc rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronalzones (subplate, cortical plate, and Cajal-Retzius cell zones) andthree progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate,late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitorsthat share characteristics with outer radial glia abundantly foundin the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence ofcomplex neocortical features.
corticogenesis| stratiﬁcationThe mammalian neocortex has a multilayered structure (layers
I–VI) (1). The neocortex arises from the neuroepithelium(NE) of the dorsal telencephalon, which evaginates to form
a semispherical brain vesicle (Fig. S1A) (2). The dorsocaudal
side of the neocortex is ﬂanked by the cortical hem, whereas its
ventrorostral side is neighbored by the lateral ganglionic eminence (LGE; striatum anlage) and septum. Layer I [its fetal
primordium is called the marginal zone (MZ); Fig. S1B] isqualitatively different from other layers, as this superﬁcial-mostlayer is mainly composed of Reelin+ Cajal-Retzius (CR) cells,
which are largely derived from neighboring tissues such as thecortical hem and septum (3) (in the case of human cortex, some
Reelin+ cells also arise directly from neocortical NE) (4). The
rest of the cortical layers are generated with the “inside-out”
pattern: the deeper the layer, the earlier the neurons are bornfrom cortical progenitors (Fig. S1B) (5, 6).
A detailed understanding of early human corticogenesis
remains elusive because of the limited access to fetal brain tissues. We previously established a 3D culture of mouse andhuman ES cell (hESC) aggregates that recapitulates early steps ofcorticogenesis [or serum-free ﬂoating culture of embryoid bodylike aggregates with quick reaggregation (SFEBq)] (7–9). Thismethod has been also applied to human induced pluripotent stem
(iPS) cell culture (10). In this self-organization culture, large
domains of cortical NE self-form within a ﬂoating hESC aggregate and spontaneously develop ventricular zone (VZ), corticalplate (CP) (mostly deep-layer neurons), and MZ by culture day
40–45. This cortical NE was still immature, mimicking humancorticogenesis during the ﬁrst trimester (Fig. S1C) (7).
Here, using an optimized culture, we revealed unique selforganizing aspects of human corticogenesis. Moreover, the20284–20289 | PNAS | December 10, 2013 | vol. 110 | no. 50

The previous SFEBq method had made culture possible forup to around 45 days, but tweaks to the modified approach
resulted in a dramatic lengthening of this time span, making
it possible for the hESC-derived brain-like tissue to matureeven more in vitro. By around day 70 of culture, the neuroepithelium had thickened to more than 200 μm, andshowed a six-layer laminar structure corresponding to thatseen in the fetal neocortex. By day 91, the tissue had deepened to 350 μm, and each layer had grown morphologically similar to that seen in a second-trimester fetus.
Tracing of the movements of labeled cells over time revealed another distinctive feature of neocortical development, in which later born superficial neurons localized more

optimized culture generates species-speciﬁc progenitors in theouter subventricular zone (oSVZ), called outer radial glia(oRG), which are abundantly present in the human neocortex(11, 12) but rare in the mouse cortex (13, 14). Thus, an unexpectedly wide range of self-organizing events is internallyprogrammed within the cortical NE.
Results
Intracortical Polarity in Self-Organized Cortical NE. For the improved
SFEBq culture (Fig. S2 A and A′), we formed aggregates byplating 9 × 103 dissociated hESCs into each well of V-bottomed
96-well plates (15) and culture them in medium supplemented
with the Rho kinase inhibitor (16) (Fig. S2A). Then, the aggregates were transferred to petri dishes and cultured under 40% O2
conditions. The addition of chemically deﬁned lipid concentrate,
FBS, heparin, and a low concentration of Matrigel improved
long-term maintenance of VZ progenitors, whereas the additionof a TGFβ inhibitor and a Wnt inhibitor for the ﬁrst 18 d moderately promoted telencephalic differentiation.
Under these conditions, all hESC aggregates were positive forfoxg1::Venus (marking telencephalic tissue) (2) on days 26 (Fig.
1A and Fig. S2B), and >75% of total cells (day 34) expressed
foxg1::Venus, in contrast to the previous culture (Fig. 1B and Fig.
S2C). The foxg1::Venus+ NE contained epithelial domes with aventricle-like cavity inside (Fig. 1C; day 42). These thick epithelia had a cell-dense VZ positive for Pax6 and Sox2 on theluminal side (Fig. 1 D and E), whereas pH3+ progenitors undermitosis were found exclusively in its innermost part (Fig. 1F), as
seen in vivo. TuJ1+ neurons occupied the zone outside of the VZ(CP; Fig. 1G), and expressed markers of early CP neurons such
Signiﬁcance
Using 3D culture of human ES cells, we show new self-organizing
aspects of human corticogenesis: spontaneous development of
intracortical polarity, curving morphology, and complex zone
separations. Moreover, this culture generates species-speciﬁcprogenitors, outer radial glia, which are abundantly present in
the human, but not mouse, neocortex. Our study suggests an
unexpectedly wide range of self-organizing events that are
driven by internal programs in human neocortex development.
Author contributions: T.K. and Y.S. designed research; T.K., H.S., T.N., M.S., S.A., and M.E.
performed research; T.K. and Y.S. analyzed data; and T.K. and Y.S. wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission. C.-M.F. is a guest editor invited by the
Editorial Board.
1

Present address: Faculty of Exploratory Pharmacology, Asubio Pharma Co., Ltd., Kobe
650-0047, Japan.

2

Present address: Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd.,
Osaka 554-8558, Japan.

3

To whom correspondence should be addressed. E-mail: yoshikisasai@cdb.riken.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1315710110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1315710110

superficially than older, deeper layer neurons, resembling asimilar inside-out pattern seen in fetal corticogenesis. Another intriguing finding was that region of the in vitro tissue
correspondig to the outer subventricular zone developed
large numbers of basal progenitors akin to outer radial glia
found abundantly in human.
“These new refinements to the SFEBq culture system willallow us to begin to more closely study important questions, such as the mechanisms underlying the spontaneous
acquisition of polarity by the neuroepithelium, or the processes involved in its curvature and rolling morphogenesis,”says Sasai. “These are exciting times for the study of human neurodevelopment.”

Neuroepithelium derived from human ESCs shows rolling morphogenesis (day 35 of culture)91

Animal Resources and Genetic Engineering
Genetically engineered mice are one essential tool in modern biomedical research. The quality and efficacy of research are greatly
dependent on how efficiently mutant mice can be generated, propagated, and housed. The major role of the Laboratory for Animal
Resources and Genetic Engineering (LARGE) is to provide comprehensive services to develop mutant mouse models that are usefulfor research in animal development and regeneration, and to maintain and enhance the experimental rodent resources at the CDB.
LARGE comprises two complementary units that work to accomplish these missions.

Yasuhide FURUTA Ph.D.

Genetic Engineering Unit
Yasuhide FURUTA Ph.D.

Staff
Unit Leader

Technical Staff

Yasuhide FURUTA

Kana BANDO
Naoko HATAMOTO
Michiko HIGASHIKAWA
Yoshiko MUKUMOTO
Megumi WATASEYui YAMASHITA

Senior Advisor

Shinichi AIZAWA
Research Specialist

Takaya ABE
Go SHIOI
Visiting Scientist

Toshihiko FUJIMORIThe Genetic Engineering Unit works with researchlabs within the CDB, as well as others in Japan andthroughout the Asia-Pacific region to develop genetically engineered mice useful for the study of development and regeneration. In these joint development projects, we receive sequence information of
the subject genes from our collaborators, and perform all subsequent stages of mutant mouse production from constructing the targeting vectors togenerating chimeras, making about one hundred
new mutant mouse lines every year. In addition, wedevelop new tools and technologies for bioimaging
to aid in the visualization of mouse development at
tissue, cell, and organelle levels.

An E9.5 embryo expressing theR26p-Fucci2 cell cycle probes

Animal Resource Unit
Hiroshi KIYONARI Ph.D.

Staff
Unit Leader

Hiroshi KIYONARI
Attending Veterinarian

Naoko KAGIYAMA
Research Consultant

Kazuki NAKAO
Technical Staff

Karin AKIYAMA
Kenichi INOUE
Mari KANEKO

Yuki KANEKO
Takuya KAWADA
Masako NODA
Miho SATO
Mayo SHIGETA
Aki SHIRAISHI
Tomoko TOKUNAGA
Sachi YAKAWA
Assistant

Yuki TSUJI

Recent Publications
Abe T, et al. Visualization of cell cycle in mouse embryos with
Fucci2 reporter directed by Rosa26 promoter. Development
140.237-46 (2013)
Hoshina N, et al. Protocadherin 17 regulates presynaptic assembly in topographic corticobasal Ganglia circuits. Neuron
78.839-54 (2013)

The Animal Resource Unit maintains and cares for
the CDB’s laboratory mouse and rat resources in aSpecific Pathogen Free (SPF) environment. We also
handle transfer of mutant mice both within the CDB
and to/from other domestic and overseas institutions. In addition, we provide pregnant females, fertilized mouse eggs, and services for colony expansion and strain cryopreservation. We also carry out
research projects aimed at developing new technologies in reproductive biology. The unit has recently established colonies of a metatherian species,
the gray short-tail opossum (Monodelphis domestica),
and a reptilian species, the gecko (Paroedura picta).
We are currently in the process of expanding thesecolonies, and determining their optimal environmentand breeding conditions, with the aim to establish a
distribution service of these new model animals.

Injection of C57BL/6 ES(HK3i) cells into
8 cell stage embryo for chimeric mouseproduction

Gecko

Imuta Y, et al. Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T
loci. Genesis 51.210-8 (2013)
Lee J, et al. Angiopoietin-1 guides directional angiogenesis
through integrin alphavbeta5 signaling for recovery of ischemic retinopathy. Sci Transl Med 5.203ra127 (2013)
Nakashima A, et al. Agonist-independent GPCR activity
regulates anterior-posterior targeting of olfactory sensory
neurons. Cell 154.1314-25 (2013)
Sakaguchi M, et al. The phosphatase Dullard negatively
regulates BMP signalling and is essential for nephron maintenance after birth. Nat Commun 4.1398-1407 (2013)
Takao D, et al. Asymmetric distribution of dynamic calcium
signals in the node of mouse embryo during left-right axis
formation. Dev Biol 376.23-30 (2013)

92

OpossumElectron Microscope
Detailed knowledge of the shapes of individual cells and fine subcellular structures is vital to the understanding of biological structuresand organization. In developmental processes in particular, changes in cell morphology and behavior play fundamental roles. The
Electron Microscope Laboratory provides technical assistance and expert guidance in the use of transmission and scanning electron
microscopy (TEM and SEM) and related technologies in morphological analyses.

Shigenobu YONEMURA Ph.D.

Electron Microscope
Shigenobu YONEMURA Ph.D.
Our technical support activities aim to assist scientists with all procedures related to conventional transmission and scanning electron microscopy, including the preparation of specimensof any type, assistance in the use of equipment, and the recording and printing of images. We
also provide instructions equipment use, specimen preparation, and interpretation of images.
In all cases, we seek to provide researchers with specific advice as to the appropriate electron
micrograph analysis before and during the observation, in order to facilitate the efficient use ofelectron- microscopy in the CDB’s research activities.
Our lab additionally conducts research into cytoskeletal elements, and the biophysical aspects of morphological rearrangements in epithelial cells.

Staff
Laboratory Head

Research Fellow

Shigenobu YONEMURA
Research Scientist

Akira ONODERA
Ayuko SAKANE

Hanako HAYASHI

Student Trainee

Technical Staff

Kisa KAKIGUCHI
Kazuyo MISAKIYu AMANO
Assistant

Mai SHIBATA

Visiting Scientist

Masatsune TSUJIOKA

Recent Publications
Ikenouchi J, et al. Sphingomyelin clustering is essential forthe formation of microvilli. J Cell Sci 126.3585-92 (2013)
Ebrahim S, et al. NMII forms a contractile transcellular sarcomeric network to regulate apical cell junctions and tissue
geometry. Curr Biol 23.731-6 (2013)
Tsujioka M, et al. Talin couples the actomyosin cortex to theplasma membrane during rear retraction and cytokinesis.
Proc Natl Acad Sci USA 109.12992-7 (2012)

Cells organizing the notochord of zebrafish embryo

α-catenin molecules stretched by forces produced
by Myosin Ⅱ(green) are selectively labeled with α18
antibody(red).

Yonemura S. A mechanism of mechanotransduction at thecell-cell interface: emergence of alpha-catenin as the centerof a force-balancing mechanism for morphogenesis in multicellular organisms. Bioessays 33.732-6 (2011)
Yonemura S. Cadherin-actin interactions at adherens junctions. Curr Opin Cell Biol 23.515-22 (2011)
Yonemura S, et al. alpha-Catenin as a tension transducer
that induces adherens junction development. Nat Cell Biol
12.533-42 (2010)93

Bio-Imaging
The role of the Bioimaging Laboratory is to assist in the operation and maintenance of the Center’s core imaging facility and supportscientists through the provision of superior optical imaging technologies, analysis and presentation of results. The lab will manage
central optical microscopy systems, image analysis software and the associated IT environment, and promote a secure environment
for the effective use of these resources.

Shigeo HAYASHI Ph.D.

Optical Image Analysis Unit
Yuko KIYOSUE Ph.D.
This unit runs the CDB’s common-use Imaging facility.

Staff
Unit Leader

Part-Time Staff

Yuko KIYOSUE

Emiko MAEKAWA

Technical Staff

Agency Staff

Tomoko HAMAJI

Shinobu SHIMAHARA

Recent Publications

Bio-imaging is an interdisciplinary process that integrates molecular cell biology and biochemistry, as well as technology from optics, engineering, and computer sciences. It has enabledscientists to visualize biological processes at the cellular and molecular levels and today, has
become an indispensable field for research on biological systems. We aim to design an imaging environment that can adapt to changing scientific demands and can contribute to theprogress of biology. We hope to accelerate biological research by matching research concepts with the latest technologies from the different specialized fields.
In addition, we aim to shed light on the molecular mechanisms controlling the microtubule
cytoskeleton in the cell/tissue morphogenesis by making full use of the available technology.

van der Vaart B, et al. CFEOM1-Associated Kinesin KIF21A
Is a Cortical Microtubule Growth Inhibitor. Dev Cell 27.14560 (2013)
Shimozawa T, et al. Improving spinning disc confocal microscopy by preventing pinhole cross-talk for intravital imaging. Proc Natl Acad Sci USA 110.3399-404 (2013)
Nakamura S, et al. Dissecting the Nanoscale Distributions
and Functions of Microtubule-End-Binding Proteins EB1 andch-TOG in Interphase HeLa Cells. PLoS One 7.e51442(2012)
Mimori-Kiyosue Y. Shaping microtubules into diverse patterns: molecular connections for setting up both ends. Cytoskeleton (Hoboken) 68.603-18 (2011)
Kosodo Y, et al. Regulation of interkinetic nuclear migration
by cell cycle-coupled active and passive mechanisms in thedeveloping brain. EMBO J 30.1690-704 (2011)
Hotta A, et al. Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex throughLL5alpha/beta. J Cell Biol 189.901-17 (2010)

Inverted microscope maintained by the Optical Image Analysis Unit

94

Genomics
The Genomics Laboratory works to support a wide range of genomic and epigenomic research and functional genomics research,
providing genome-scale, high-throughput services in sequencing gene expression analysis to all CDB labs. All projects can be initiated and followed using an internal website designed to ensure a smooth workflow and timely reporting of results.

Fumio MATSUZAKI Ph.D.

Genome Resource and Analysis Unit
Shigehiro KURAKU Ph.D.
The Genome Resource and Analysis Unit (GRAS) provides a broad range of biologist-oriented
support for DNA sequencing, sequence informatics and gene expression profiling. In parallel,
we conduct our original research projects on vertebrate comparative genomics, focusing on
evolution of developmental programs. Overall, GRAS aims to create an integrative researchsupport station with active communication between laboratory staffs and bioinformaticians
and take full advantage of evolving massively parallel sequencing technologies for transcriptomic, epigenetic and genomic projects arising in the whole institute.

Staff
Unit Leader

Technical Staff

Shigehiro KURAKU

Tetsutaro HAYASHI
Kazu ITOMI
Kana MIYAGISHI
Osamu NISHIMURA
Chiharu TANEGASHIMA
Kaori TATSUMI
Kenichiro UNO

Research Specialist

Mitsutaka KADOTA
Research Scientist

Yuichiro HARA
Munazah ANDRABI
Miyuki NORO

Assistant

Sayo SAITO

Recent Publications
Kuraku S, et al. aLeaves facilitates on-demand exploration ofmetazoan gene family trees on MAFFT sequence alignment
server with enhanced interactivity. Nucleic Acids Res 41.
W22-8 (2013)
Smith J. J, et al. Sequencing of the sea lamprey (Petromyzon
marinus) genome provides insights into vertebrate evolution.
Nat Genet 45.415-21, 21e1-2 (2013)

Our main sequencing platform, illumina HiSeq 1500, in
operation since spring 2012

Microfluidics-based equipment for
single cell gene expression profiling newly installed

Adachi K, et al. Context-dependent wiring of sox2 regulatory
networks for self-renewal of embryonic and trophoblast stem
cells. Mol Cell 52.380-92 (2013)
Oisi Y, et al. Craniofacial development of hagfishes and theevolution of vertebrates. Nature 493.175-80 (2013)

Functional Genomics Unit
Hiroki R. UEDA M.D., Ph.D.
The Functional Genomics Unit (FGU) has two missions: to provide functional genomics services to the laboratories within the CDB, and develop and introduce cutting-edge technologies related to functional genomics in order to accelerate the Center’s research. We are striving to implement two types of technologies: 1) expression analysis, and 2) high-throughput
measurement and perturbation. For expression analysis, we first introduced GeneChip technology, which is mainly used to measure expression profiles of genes in cells or tissues. For
high-throughput measurement and perturbation, we introduced cell-based screening technology, which examines gene functions in cells. We will integrate technologies for expression
analysis and high-throughput measurement and perturbation to develop new functional genomics methods. In particular, we will focus on strengthening single-cell expression analysis,
and developing three-dimensional expression analysis in organs.

Staff
Unit Leader

Technical Staff

Hiroki R. UEDA

Kenichiro UNO

Special Postdoctoral
Researcher

Assistant

Yohei SASAGAWA

Yuka TOKUMINE

Research Specialist

Takeya KASUKAWA
Itoshi NIKAIDO

Recent Publications
Sasagawa Y, et al. Quartz-Seq: a highly reproducible andsensitive single-cell RNA sequencing method, reveals nongenetic gene-expression heterogeneity. Genome Biol 14.
R31 (2013)
Kasukawa T, et al. Human blood metabolite timetable indicates internal body time. Proc Natl Acad Sci USA 109.
15036-41 (2012)
Kasukawa T, et al. Quantitative expression profile of distinct
functional regions in the adult mouse brain. PLoS One 6.
e23228 (2011)
Masumoto K. H, et al. Acute induction of Eya3 by late-night
light stimulation triggers TSHβ expression in photoperiodism.
Curr Biol 20. 2199-206 (2010)95

Proteomics
The identification of proteins from trace amounts of biologically important protein complexes help researchers to discover novel molecular pathways and has become an indispensable approach to the study of development and regeneration. A better understandingof the protein components of cells and tissues may yield new insights into the molecular structure and function that underlies the
spectrum of biological phenomena and improve our ability to manipulate and recapitulate them.

Shigeo HAYASHI Ph.D.

Mass Spectrometry Analysis Unit
Shigeo HAYASHI Ph.D.

Staff
Lab Head

Shigeo HAYASHI
Research Specialist

Reiko NAKAGAWA

The Mass Spectrometry Analysis Unit uses LC-MASS spectrometry, a technology that enables the high-speed analysis of protein components from even minute biological samples. Its
support activity takes the forms of protein identification services and analysis of protein modifications such as phosphorylation, ubiquitination, and proteolysis. The unit supports laboratories within the CDB, and collaborations with a number of laboratories at other institutions. The
lab receives gel slices containing proteins from researchers and conducts all subsequent
steps, including de-staining of gels, in-gel digestion of proteins, peptide extraction, and operating the LC-MS/MS.

Recent Publications
Tsutsui T, et al. Mediator complex recruits epigenetic regulators via its two cyclin-dependent kinase subunits to repress
transcription of immune response genes. J Biol Chem
288.20955-65 (2013)
Kamata K, et al. C-terminus of the Sgf73 subunit of SAGA
and SLIK is important for retention in the larger complex andfor heterochromatin boundary function. Genes Cells 18.82337 (2013)
Kuroha K, et al. The Upf factor complex interacts with aberrant products derived from mRNAs containing a prematuretermination codon and facilitates their proteasomal degradation. J Biol Chem 288.28630-40 (2013)
McCloskey A, et al. hnRNP C tetramer measures RNA length
to classify RNA polymerase II transcripts for export. Science335.1643-6 (2012)
Shiomi Y, et al. Two different replication factor C proteins,
Ctf18 and RFC1, separately control PCNA-CRL4Cdt2-mediated Cdt1 proteolysis during S phase and following UV irradiation. Mol Cell Biol 32.2279-88 (2012)
Terabayashi T, et al. Phosphorylation of Kif26b promotes its
polyubiquitination and subsequent proteasomal degradation
during kidney development. PLoS One 7.e39714 (2012)

96

LC-MASS spectrometry system used in the Mass Spectrometry
Analysis Unit

Human Stem Cell Technology
The Division of Human Stem Cell Technology (DHSCT) was established to provide support services to any lab with an interest in using
human embryonic stem cells (hESCs) and their derivatives in their research. The DHSCT provides technological expertise, training andsupport in hESC culture, maintenance, distribution and management, as well as monitoring and analysis of global trends in stem cellresearch and regulation.

Yoshiki SASAI M.D., Ph.D.(Deputy Chief Hitoshi NIWA M.D., Ph.D.)

Human Stem Cell Technology Unit
Yoshiki SASAI M.D., Ph.D.
Stem cells – both embryonic and somatic-can be challenging to study and manipulate in vitro.
The Division of Human Stem Cell Technology Unit was established to provide a full spectrumof support services to labs within the CDB and throughout Japan with an interest in using human embryonic stem cells (hESCs) and their derivatives in their research. The unit provides
expertise, training, and support in hESC culture, maintenance, distribution and management,
for users in the life sciences community. We seek to contribute to achieving the goals of translational research, in line with the CDB mission to establish a solid scientific foundation for regenerative medicine.

Staff
Unit Leader

Technical Staff

Yoshiki SASAI
Deputy Unit Leader

Michiru MATSUMURA-IMOTO
Maki MINAGUCHI

Masatoshi OHGUSHI

Junior Research Associate

Research Specialist

Chikafumi OZONE

Hiroyuki KITAJIMA

Part-Time Staff

Visiting Scientist

Yoshinori NAKAI

Tokushige NAKANO
Daiki NUKAYA

Recent Publications
Nakano T, et al. Self-formation of optic cups and storable
stratified neural retina from human ESCs. Cell Stem Cell
10.771-85 (2012)
Ohgushi M, et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7.225-39 (2010)

Four-Dimensional Tissue Analysis Unit
Mototsugu EIRAKU, Ph.D.
Recent advances in stem cell technology have enabled the generation of various potentially
medically useful cell types from ES and iPS cells, but the extent to which such cells mimic theirin vivo function when plated on culture dishes is limited. The Four-dimensional Tissue Analysis
Unit seeks to develop new approaches to cell culture that will allow for more realistic in vitro
recapitulation through the formation of three-dimensional tissue from stem cells. We will establish efficient 3D culture of ES cell-derived brain and retinal tissues, and develop cuttingedge live imaging technologies and optic devices for the 4D analysis of large tissues. We also
support and work with users of these technologies within and outside the CDB.

Staff
Unit Leader

Technical Staff

Mototsugu EIRAKU

Eriko SAKAKURA

Visiting Scientist

Junior Research Associate

Hidetaka SUGA

Yuiko HASEGAWA

Recent Publications
Eiraku M, et al. Self-organizing optic-cup morphogenesis inthree-dimensional culture. Nature 472.51-56 (2011)
Eiraku, M. and Sasai, Y. Mouse embryonic stem cell culture
for generation of three-dimensional retinal and cortical tissues. Nature Protocols 7.69-79 (2011)

Science Policy and Ethics Studies Unit
Douglas SIPPThe field of stem cell research has been subject to legal, social, and ethical tensions across a
broad range of issues, from the research use of human embryos to the optimization of pathways for the translation of basic research into clinical applications. We seek to compare different science policy approaches to these issues and identify regulatory frameworks best suited
to the development and promulgation of stem cell applications. We will further explore approaches to the clinical translation of human stem cell research.

Staff
Unit Leader

Douglas SIPP

Recent Publications
Sipp D. Direct-to-consumer stem cell marketing and regulatory responses. Stem Cells Transl Med 2.638-40 (2013)
Sipp D. and Turner L. U.S. Regulation of Stem Cells as
Medical Products. Science 338.1296-1297 (2012)97107