National Institute for Basic Biology

Developmental Biology

DIVISION OF EMBRYOLOGYProfessor

FUJIMORI, Toshihiko

Associate Professor:
NONOMURA, KeikoAssistant Professor:
KOYAMA, Hiroshi
Specially Appointed Assistant Professor:
ARATA, Masaki
Technical Staff:
OKA, Sanae
Postdoctoral Fellow:
KISHI, Kanae
SOKENDAI Graduate Student: SAKURAI, Jun
Visiting Graduate Student: MIKOSHIBA, Seiya
Visiting Scientist:
INOUE, Yuuki
Technical Assistant:
HIGUCHI, Yoko
KANIE, Akemi
NAKAGAWA, Mami
Admin Support Staff:
KATO, Azusa

The aim of our research is to understand the events underlying early mammalian development. One of the characteristics
of mammalian embryonic development is that embryogenesis proceeds in the oviducts and the uterus of the mother,and the interaction between the embryo and maternal tissue
is essential in this process. Another characteristic is thehighly regulative potential of embryos. Cells within embryos
interact with their environment and establish their own fatesand behaviors. We want to provide basic and fundamental
information about the specification of the differentiation ofcell lineages, embryonic axes, cell behaviors, body shape
regulation and tissue morphology in early mammalian devel-opment. We are also interested in the relationship between
embryos and their environment, the oviducts and the uterus.

I. Live observation of early mouse development

To understand the mechanisms underlying embryonicdevelopment and morphogenesis, it has become common toobserve the behavior of cells and gene expression in living
embryos in a variety of animal species. Progress in genetic
engineering and embryonic manipulation concomitantly withthe progress in optics and microscope technologies, including development of highly sensitive photo-detectors, hasallowed us to observe developing embryos, even mammalianones, in real time. We have established a series of transgenic
mouse lines for live imaging, which is part of a collaborative
project taking place in the Laboratory for Animal Resourcesand Genetic Engineering, Riken CLST. These mouse lines
have been used to visualize nuclei, cell shapes, the cytoskeleton and other organelles to observe cell behaviors in living
mouse embryos across many laboratories over the world. We
have also established mouse lines to monitor the cell cycle.
We have been studying the behavior of cells within embryos
by applying newly developed image analyses following theobservation of cell behaviors within embryos using thesemouse lines. We found that the proximal visceral endoderm
overlying the extra-embryonic ectoderm shows coherent cell
growth in a proximal-anterior to distal-posterior direction.
We also observed that directional cell migration is coupledwith cell elongation in the anterior region, suggesting that thebehaviors of visceral endoderm cells vary between regions
during peri-implantation stages.
We have also been establishing several reporter mouse lines
in the lab to study gene expression patterns during the periimplantation stage of mouse development. In these mice,
cDNA encoding fluorescent proteins are connected to themouse genomic DNA sequences of the enhancer/promoter
region of important genes encoding factors regulating celldifferentiation in these stages.
In addition to these reporter mouse lines, we generated transgenic (Tg) mice expressing photoactivatable-Cre(PA-Cre) proteins. Due to the limitation of high throughput
analyses in mouse embryos, we established embryonic stem
cells from the Tg mice for testing various conditions. We
then systematically investigated the conditions which enableefficient photoactivation for PA-Cre, in vitro and in silico. It
was suggested that repetitive pulsed illumination with shortpulse cycles efficiently induced DNA recombination withlow light energy. We also showed that repetitive short-pulsed
illumination induced DNA recombination in pre-implantationembryos expressing PA-Cre.

II. Histological observation of mouse embryos
developing in the uterus

A mouse fertilized egg and an embryo at 12 days after fertilization.
Embryonic body with axes of anterior-posterior, dorsal-ventral and leftright is formed from an egg with symmetrical shape. How is the information relating future body formed during early stages of development?

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Mammalian embryos develop in the uterus after implantation. Direct interaction between embryos and the cells of theuterus is important. In studies on the developmental biology
of mammalian embryos, embryos are usually removed fromthe uterus, and those that are isolated are analyzed. We havebeen analyzing the early embryonic development of themouse by comparing the changes in the uterine epithelium.

To observe the morphogenesis of embryos within the uterus,
serial sections of implanted uteruses were made, and imagesof the embryos within it were captured to make high resolution three-dimensional re-constructions. We are preparing
samples obtained from various developmental stages. Precise
positions of embryonic implantation and changes in theuterine epithelium correlating with embryonic development
have been examined using these images. We are identifying the molecules involved in the interaction between theembryo proper and uterine cells, which may play a majorrole in embryonic development.

Figure 1. Three-dimensional image reconstruction of the pregnant uterus.
A uterus obtained 5 days after fertilization was sectioned and images weredigitized by an image scanner, and reconstructed three-dimensionally toobserve the relationship between the embryo and the maternal tissue. The
right panel represents the pseudo 3D image observed from the lateral sideof the left image. Arrows indicate the embryo in the decidual tissue.

III. Polarity formation in the mouse oviductepithelium

The oviduct (Fallopian tube) is the organ connecting theperiovarian space and the uterine horn. The ova released fromthe ovary are transported through the oviduct, where the ova
are fertilized by spermatozoon entering from the uterus. The
epithelium of the mouse oviduct consists of multi-ciliatedcells and secretory cells. Ovulated oocytes are transported in
oviducts by unidirectional motility of multi-cilia. This cellularorientation in the oviduct is one example of planar cell polarity(PCP). PCP is seen in a variety of tissues, where cells sense
global axes of the tissue to which they belong and orient themselves to fulfill specialized functions.
Many evolutionally conserved genes are required for theformation of PCP, and several of those encode proteins thatare localized in a polarized manner within cells. We found that
Celsr1, a PCP core member, was strongly concentrated only at
the specific domains of cell boundaries which are perpendicular to the ovary-uterus axis, and that this polarized localization
appeared to precede the directional movement of cilia. In
Celsr1-deficient mutant oviducts, the beating direction of cilia
was not coordinated along the ovary-uterus axis throughout
the epithelia; some cells were oriented orthogonally to theaxis, while some others exhibited reverse polarity. Recently,
we found that Camsap3, a microtubule minus-end binding
protein, localized to the uterine side of the base of cilia and its
mutation disrupted cilia orientation in individual cells without
affecting Celsr1 localization. Thus, both Celsr1 and Camsap3
are responsible for the alignment of cilia along the ovary-uterus axis, but in distinct ways; Celsr1 orients cells, while
Camsap3 orients cilia within each cell.
In addition to the ciliary polarity phenotype, cellular shapepolarity was also impaired in the Celsr1-deficient mice. We
found a new feature of cellular polarity in the wild typeoviduct, e.g. that the apical surface of epithelial cells waselongated along the ovary-uterus axis. In Celsr1-deficient
mice, epithelial cells showed less elongation and a randomized
orientation.
Furthermore, we also found that the three-dimensionalarchitecture of oviductal epithelia was dramatically disrupted. In adult wild-type mice, the epithelial cell sheet is bent
multiple times, which forms around twenty longitudinal foldsparallel to the organ axis. However, in the mutant oviducts,
epithelial folds no longer run parallel to the organ axis. The
direction of the folds was randomized, and ectopic branchesof the folds were observed. This suggests that Celsr1 isimportant for the formation of multi-scale polarities in themouse oviduct ranging from the cellular to the tissue scale.
Epithelial folds are observed in various organs including
the oviducts, guts, and airways etc. Longitudinally aligned
folds are also observed in the oviducts of birds and frogs. To
investigate the mechanisms of the epithelial fold pattern formation, we utilized mathematical modeling and simulations.
By considering mechanical properties of the epithelial sheets,
we reproduced the longitudinally aligned and branched folds
which are observed in wild-type and Celsr1 mutant mice,
respectively (Figure 2). In addition, our model also successfully reproduced circumferentially aligned folds and zigzag
folds observed in other organs. We are also focusing onsome other PCP regulators, and are trying to understand how
oviduct epithelial cells establish and maintain their polarity.

IV. Analysis of the mechanical properties of cells
and tissues during embryonic development

Mechanics is one of the essential components of biological processes, including cell shape transformation and tissuemorphogenesis etc. To understand how mechanical forces
contribute to various patterns of morphogenesis, measuring cellular and tissue mechanical states is necessary. We
developed statistical techniques to infer mechanical statesusing fluorescent microscopic images during morphogenesis

Figure 2. Epithelial fold patterns in oviduct and the reproduction of thepatterns by computational simulations.

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National Institute for Basic Biology

Developmental Biology(Figure 3). By employing this method, we inferred mechanical forces in multi-cellular systems including cultured epithelial cells, and early embryogenesis in C. elegans and mice.
Further computational simulations based on the inferredmechanical information reproduced morphological featuresof the multi-cellular systems. Thus, the mechanical information will be useful to investigate physical mechanisms ofearly embryonic development and morphogenesis duringorganogenesis in late stages of development.

cluster and multiple cell clusters, etc. Difference in cellular
stiffness was critical for these pattern formations. The former
situation is actually observed during elongation of the notochord in mice. Together with active cell movements and thedifferential adhesion hypothesis which has been recognized
as a classical concept within biology, passive cell movements
can be effective for diverse pattern formation.

Figure 3. Theoretical inference of cellular/tissue mechanical states.
Schematic illustration of inference.

V. Mammalian tissue morphogenesis requiring
mechanosensor channel PIEZOs

Several examples have shown that mechanical stimuli
can work as key components for tissue or organ development. However, our knowledge about the involvementof mechanotransduction in biological phenomena or theirprecise mechanisms is still limited. It is partially because key
mechanosensors are not yet identified in many cell types.
PIEZOs are recently identified mechanically activated cation
channels functioning in mammalian cells (Figure 4). They
are activated when mechanical forces are applied to the cell
membrane. Series of data show that PIEZO2 serves as themain mechanosensor in sensory neurons for light touch sensation, proprioception and breathing. We recently found thatPIEZO1 in endothelial cells is required for lymphatic valve
formation (Figure 5). To further elucidate how PIEZO1mediated mechanotransduction is involved in lymphatic
valve formation, we have been developing systems to manipulate mechanical stimuli and monitor PIEZO activity in vitro
and in vivo. Analyses utilizing these systems and mouselines deficient in PIEZOs will clarify the relationship among
mechanical forces, PIEZO activation, cellular responses, andtissue morphogenesis.

Positive or
negative pressure,
shear stress

Ca2+, Na+,+ +Mg2+, K+++++Piezo+ ++ +non-selectivecation channel

How is Piezo-mediatedmechanotransduction
involved in tissuemorphogenesis?

Figure 4. Schematic illustration of Piezo mechanically activated nonselective cation channel and the key question of this study.

VI. Mechanics of cell population patterning
during development

During development, cells actively and/or passively move,resulting in various cell distribution patterns. We investigatedthe effect of passive cell movements provoked by frictional
forces from adjacent growing tissues. The passive movements generated various patterns, such as an elongated cell36

Figure 5. Reduced number of lymphatic valves in mice lacking PIEZO1
in endothelial cells.

Publication List:〔Original papers〕

T., Tohsato, Y., Itoga, H., Shioi, G., Kiyonari, H., Oka, S.,• Hirata,Fujimori, T., and Onami, S. (2021). NeuroGT: A brain atlas of••••neurogenic tagging CreER drivers for birthdate-based classification andmanipulation of mouse neurons. Cell Reports Methods 1, 100012. DOI:10.1016/j.crmeth.2021.100012
Kinoshita, N., Yamamoto, T.S., Yasue, N., Takagi, C., Fujimori, T., andUeno, N. (2022). Article Force-dependent remodeling of cytoplasmic
ZO-1 condensates contributes to cell-cell adhesion through enhancing
tight junctions. iScience 25, 103846. DOI: 10.1016/j.isci.2022.103846
Kishi, K., Koyama, H., Oka, S., Kato, A., Sato, M., and Fujimori,T. (2021). Repetitive short-pulsed illumination efficiently activatesphotoactivatable-Cre as continuous illumination in embryonic stem
cells and pre-implantation embryos of transgenic mouse. GENESIS 59,e23457. DOI: 10.1002/dvg.23457
Koyama, H., Suzuki, M., Yasue, N., Sasaki, H., Ueno, N., and Fujimori,T. (2022). Differential Cellular Stiffness Contributes to Tissue
Elongation on an Expanding Surface. Front. Cell Dev. Biol. 10, 864135.
DOI: 10.3389/fcell.2022.864135
Saito, H., Matsukawa-Usami, F., Fujimori, T., Kimura, T., Ide, T.,
Yamamoto, T., Shibata, T., Onoue, K., Okayama, S., Yonemura, S.,
Misaki, K., Soba, Y., Kakui, Y., Sato, M., Toya, M., and Takeichi,M. (2021). Tracheal motile cilia in mice require CAMSAP3 for theformation of central microtubule pair and coordinated beating. Mol.
Biol. Cell 32. ar12. DOI: 10.1091/mbc.E21-06-0303