DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

Assistant Professor:

YABE, Taijiro
MII, Yusuke
Specially Appointed Assistant Professor:
SHINOZUKA, Takuma
Technical Staff:
UTSUMI, Hideko
Postdoctoral Fellow:
TAKADA, Ritsuko
SOKENDAI Graduate Student: HATAKEYMA, Yudai
TRAN, Thi Hong Nguyen
SUZUKI, Minako
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
SAKAI, Kimiko
Admin Support Staff:
NOBATA, Ryuko

The morphology of the body and tissues is established ina spatio-temporarily regulated manner. A number of genes
involved in the process of morphogenesis have been identified, but it is still uncertain how either spatial and temporal
information are established and converted to morphology
during embryogenesis. Therefore, our aim is to understandthe mechanism by which this spatial information is established, and how temporal and periodical information is converted into morphology through the application of several
different approaches.

Secreted signal molecules are important in forming spatial
information during the development of many tissues. Thesemolecules are secreted from the cells that produce them andtransported to surrounding cells, thus resulting in the formation of concentration gradients. Given that their concentration decreases in accordance with their distance from thesource, their specific signal gradient defines the relative positions of receiving cells in developing tissues. Many genetic
studies have revealed that a limited number of secreted signal
proteins, including Wnt, BMP, and Hedgehog, function
during tissue and embryo morphogenesis. However, in spiteof the accumulation of genetic evidence, the molecularmechanism that regulates their distribution in certain developing tissues is yet to be elucidated. To this end, we havevisualized signal proteins and monitored their movement intissues. Furthermore, we are also examining the biochemical characteristics and functions of these molecules, whichappear to affect how they are spread.
In contrast to secreted signal proteins, the segmental subregions of several specific tissues, like somites, appear not
to be simply directed by the gradient of the signal molecules,
but by a unique mechanism that functions periodically.
Somites are sequentially generated in an anterior-to-posteriororder via the conversion of temporal periodicity, createdby a molecular clock, into periodical structures. However,
the molecular mechanism underlying this conversion andmorphological segmentation is not yet fully understood.
Therefore, another goal of our current research is to reveal
the molecular mechanism of this differing and unique modeof patterning that underlies the periodical and sequential subdivision in the development of somites.

Figure 1. Model of Wnt protein diffusion: Wnt trimers are the smallest
unit of the HMW complex. Both the trimer and the HMW complex
appear to exist in the extracellular milieu. The HMW complex isprobably less mobile when interacting with the plasma membrane, resulting in the restriction of Wnt diffusion range. Some Wnt molecules canbe dissociated by local interaction with Frizzled receptor (Fzd), resulting
in a short-range signal (local action). In contrast, the HMW complex,
probably as well as the trimer itself, can also be dissociated by interaction with soluble Wnt binding proteins (partner proteins), including sFRP.
Due to this dissociation, Wnt turns out to be more mobile and its diffusion range is expanded (diffusible action).

I. Regulation of spatial distribution and functionof Wnt proteins in vertebrates

Visual overview of this lab’s work.

By combining biochemical and structural analyses, wehave already shown that Wnt3a proteins are not secreted ina monomeric form, rather in homo-trimer and larger HMWcomplexes. Secreted Wnt3a proteins were able to be dissociated via interaction with their receptor Frizzled8 and witha secreted Wnt binding protein, sFRP2, in vitro. Similarly,
this dissociation was detected in vivo by Fluorescence31National Institute for Basic BiologyDevelopmental Biology

Correlation Spectroscopy (FCS). Several lines of evidenceshow that large assemblies of Wnt3a are less mobile, andWnt/sFRP2 heterodimer, which is generated through thebinding of dissociated Wnt with sFRP2, diffuse more freely.
Based on these results, we have proposed a model which
contends that the assembly and dissociation of dissociableoligomers modulate Wnt signaling range (Figure 1).

suggesting a dynamic exchange of a bound form of Wnt
proteins. Based on these results and our previous findings,
we have proposed a basic mathematical model to explain distribution and dispersion of secreted proteins (Figure 3). Thismodel, which is based on the dynamic exchange of the boundform of Wnt proteins, can recapitulate a graded distributionof the bound, not free, state of Wnt proteins.
In an attempt to investigate the physiological role ofWnt signaling, we collaborated with Dr. Fujimori and Dr.
Takahama at the University of Tokushima and NIH, respectively, to examine gain-of-function (GOF) and loss-of-function (LOF) of β-catenin highly specific in mouse thymic
epithelial cells (TECs). GOF of β-catenin in TECs results
in severe thymic dysplasia and T-cell deficiency beginningfrom the embryonic period. By contrast, loss-of-functionof β-catenin in TECs reduces the number of cortical TECs
and thymocytes modestly and only postnatally. These results
indicate that fine-tuning of β-catenin expression within apermissive range is required for TECs to generate an optimal
microenvironment to support postnatal T-cell development.

Figure 2. Fluorescence decay after photoconversion (FDAP) at cell coundaries. A Photoconvertable fluorescent protein, mKikGR can be switch its fluorescence from green to red. Decay of red (photoconverted) fluorescence canbe fitted with a model of dissociation from cell surface scaffolds.

II. T he molecular mechanism of metamericstructures in vertebrate development

To increase our insight into the intercellular transmissionof Wnt proteins in embryonic tissue, we precisely examinedthe extracellular dynamics of Wnt, comparing with sFRP
in Xenopus embryos. Here, we focused on Wnt8 and amember of sFRPs, Frzb, both of which are involved in theanteroposterior patterning of the vertebrate embryo. While
Venus-tagged Wnt8 was found on the surfaces of cells close
to Wnt-producing cells, we also detected its dispersal overa long range from the source cells. We further examined
their dynamics by FCS and fluorescence decay after photoconversion (FDAP)-based measurements in the embryonictissue. In particular , we refined FDAP-based analysis byfocusing on a limited area across the cell surface, whichenabled us to obtain dynamics comparable to those measured
by FCS (Figure 2). Combination of fluorescence correlation
spectroscopy and quantitative imaging revealed that only asmall proportion of Wnt8 proteins diffuse freely, whereas
most of them are bound to the cell surface. FDAP analysis,that we refined by focusing on a limited area across thecell surface, showed that Wnt8 proteins that were bound tothe cell surface were rapidly and exponentially decreasing,

The segmental sub-regions of several specific tissues,
including the paraxial mesoderm (or somites) and the pharyngeal pouches is not likely to be simply directed by thegradient of the signal molecules, but by a unique mechanism
proceeding periodically. For instance, somites are sequentially generated in an anterior-to-posterior order by converting
the temporal periodicity created by a molecular clock into
periodical structures. The molecular mechanism underlying
this conversion and morphological segmentation, however, is
not yet fully understood.
Somites are segmental epithelial blocks located symmetrically on either side of the neural tube. Somitogenesis periodically proceeds in an anterior-to-posterior manner from theirprecursor, the presomitic mesoderm (PSM), which is locatedat the posterior of newly formed somites. This periodic generation is achieved by a complex and dynamic mechanism
within the PSM. The molecular clock, the so-called segmentation clock, essentially creates oscillatory expression ofparticular genes, hairy and some notch-related genes, in theposterior PSM.
As the oscillatory waves of the segmentation clock movefrom posterior to anterior PSM, the temporal dynamics of thesegmentation clock are transformed into the spatial patternFigure 3. Mathematical model considering free diffusion and binding to heparan sulfate clusters. The free population (u) does not virtually contribute tospatial distribution, whereas the bound population (v) directly contributes to the graded distribution of a morphogen. This model suggests both populations
contribute to rapid formation of a stable gradient, which gives insight into a perceived dilemma between speed and stability of gradient formation.

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of somites. One of the key processes shaping the spatial
pattern of somites is the generation of inter-somite boundaries. Evidences indicate that inter-somite boundary is definedby the anterior edge of the Tbx6 protein expression region.
It has been reported by us and other groups that this anteriorborder is newly generated in each segmentation cycle, byperiodic degradation of Tbx6 protein via physical interaction
with Ripply1 and Ripply2.
We are currently using a genetic approach in zebrafish to
elucidate the mechanisms of the interaction between thesegmental clock and the Tbx6/Ripply system. Based on
the results obtained from the genetic approach, we provide
a mathematical model showing the minimal network that
forms the metameric pattern of somites and the periodic cessation of the segmentation clock.

Publication List:〔Original papers〕

D., Wang, J., Yang, W., Li, J., Lin, X., Zha, F., Wang, X., Ma, L.,• Feng,Choi, N.T., Mii, Y., Takada, S., Huen, M.S.Y., Guo, Y., Zhang, L., and•••••Gao, B. (2021). Regulation of Wnt/PCP signaling through p97/VCPKBTBD7-mediated Vangl ubiquitination and endoplasmic reticulumassociated degradation. Sci. Adv. 7, eabg2099. DOI: 10.1126/sciadv.
abg2099
Fujimori, S., Ohigashi, I., Abe, H., Matsushita, Y., Katagiri, T., Taketo,M.M., Takahama, Y., and Takada, S. (2022). Fine-tuning of betacatenin in mouse thymic epithelial cells is required for postnatal T-celldevelopment. eLife 11, e69088. DOI: 10.7554/eLife.69088
Matsuda, S., Schaefer V, J., Mii, Y., Hori, Y., Bieli, D., Taira, M.,
Plueckthun, A., and Affolter, M. (2021). Asymmetric requirementof Dpp/BMP morphogen dispersal in the Drosophila wing disc. Nat.
Commun. 12, 6435. DOI: 10.1038/s41467-021-26726-6
Mii, Y., Nakazato, K., Pack, C.-G., Ikeda, T., Sako, Y., Mochizuki,A., Taira, M., and Takada, S. (2021). Quantitative analyses reveal
extracellular dynamics of Wnt ligands in Xenopus embryos. eLife 10,e55108. DOI: 10.7554/eLife.55108
Okubo, T., Hara, K., Azuma, S., and Takada, S. (2021). Effect ofretinoic acid signaling on Ripply3 expression and pharyngeal archmorphogenesis in mouse embryos. Dev. Dyn. 250, 1036–1050. DOI:
10.1002/dvdy.301
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada,S., Ueno, N., Sawai, S., Kondo, Y., and Aoki, K. (2021). Optogeneticrelaxation of actomyosin contractility uncovers mechanistic roles ofcortical tension during cytokinesis. Nat. Commun. 12, 7145. DOI:
10.1038/s41467-021-27458-3〔Review article〕

T., and Takada, S. (2021). Morphological and functional• Shinozuka,
changes of roof plate cells in spinal cord development. J. Dev. Biol. 9,30. DOI: 10.3390/jdb9030030

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