National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

place in the sea in which to live. We also investigate the evolution of the notochord using ancestral chordates.

I. ZO-1 condensates in mouse hatching embryos
Professor

Associate Professor

Assistant Professor:
Technical Staff:
Technical Assistant:

TAKAHASHI, Hiroki
TAKAGI, Chiyo
YAMAMOTO, Takamasa
YASUE, Naoko
MIYAKE, Satoko
TSUGE, Toyoko

UENO, Naoto

Admin Support Staff:

KINOSHITA, Noriyuki

In addition to genes and proteins that are widely known
to govern biological phenomena and have been extensively studied over the past several decades, a growing bodyof evidence now suggests that physical environments that
include light, temperature, and internal and external forces
influence both cellular and organismal behaviors. In particular, we are currently investigating the contribution ofphysical forces using mouse embryos and light-dependent
behaviors using coral larvae to understand the impact ofphysical environments on organisms. As reported below, theinner pressure of the blastocyst cavity generates tensile force
upon trophectodermal cells in mouse embryos, and enhances
their cell junction. During this process, a physical phenomenon, liquid-liquid phase separation (LLPS) of the tightjunction protein ZO-1, plays a pivotal role. The coral planula
larvae change their swimming activities by sensing types oflight of specific wavelengths to settle upon an appropriate

A graphical overview of our laboratory’s works.

28

Cells sense and respond to not only molecular factors but
also mechanical forces, which in turn play various importantroles in biological events, such as tissue homeostasis, differentiation, and cell migration. During early embryogenesis,
various dynamic morphogenetic movements occur, including
the convergent extension of the axial mesoderm and epibolyof the ectoderm. These movements generate physical forcesat both the cellular and tissue levels. However, it is still not
fully understood how these forces influence morphogenetic
processes.
To investigate how embryonic cells respond to mechanicalstimuli, we artificially applied mechanical forces to Xenopus
embryos. We found that tensile force applied to ectodermal
tissue induces a mesenchymal-epithelial transition (MET)like phenotype, enhancing the junctional structure andincreasing ectodermal tissue stiffness. Among these changes,
we focused on the behavior of the tight junction protein,
ZO-1. Before the force application, ZO-1 forms cytoplasmicgranules, but the mechanical force induces its accumulation
at the tight junction. Significantly, mechanical forces generated by gastrulation movements also induce similar changes
in the developing ectodermal tissue.
To examine whether the behavior of ZO-1 protein is conserved across species, in mammals in particular, we firstobserved the localization of ZO-1 in the mouse embryo.
We focused on E3.5 and E4.5 embryos since they hatch outof the zona pellucida (ZP) and expand their shape (Figure1A). We immunostained E3.5 and E4.5 embryos with aZO-1 antibody and found that the cells of E3.5 embryos
showed a significantly higher number of ZO-1 puncta inthe cytoplasm relative to E4.5 embryos. As development
proceeded, the surface area of the trophectoderm (TE) cells
expanded and became thinner. The number of cytoplasmic
ZO-1 puncta was reduced, and ZO-1 signal intensity at theFigure 1. Change of ZO-1 localization in mouse hatching embryos.
A. At E3.5, the embryo is covered with the zona pellucida (ZP). During
hatching, the embryo is enlarged and emerges from the ZP. Embryos
were stained with an anti-ZO-1 antibody and Alexa Fluor 546 Phalloidin.
Scale bar, 20 µm. The inset in the E3.5 phalloidin image was acquiredwith higher laser power, demonstrating that the structure of corticalF-actin is formed at E3.5 even though the signal intensity was weaker
than that in the E4.5 embryo. B. Schematic diagram of ZO-1 behavior
shuttling between the tight junction and cytoplasmic droplets.

plasma membrane in E4.5 embryos became much higher
than those of E3.5 embryos at the expense of their cytoplasmic pool. Importantly, this change coincides well with theaccumulation of F-actin in the cell cortex in E4.5 embryos.
This result suggests that the shuttling of ZO-1 protein fromthe cytoplasmic puncta to cell junctions occurred as development progressed.
To confirm that the tensile force induces this ZO-1 behaviorin TE cells, we inhibited the expansion of said cells using
two methods: one was the Na+/K+ ATPase inhibitor, ouabain
treatment, and the other was piercing the embryo with aglass needle. Both methods reduced the inner pressure of thecavity and inhibited ZO-1 accumulation in the cell junction.
These results suggested that tensile force being applied to theTE cells. Furthermore, we assumed that the ZO-1 puncta inE3.5 embryos were formed by phase separation and treatedembryos with 1,6-hexanediol, which is known to dissolve
LLPS assembly. As expected, 5% 1,6-hexanediol treatment
reduced the number of particles within a few minutes, thus
suggesting that these puncta are liquid droplets generated byphase separation, and that mechanical force regulates ZO-1
phase separation (Figure 1B).

In contrast, migrating cells around the wound site, ones
that were forming ZO-1 droplets, reduced cortical actin anddeveloped more stress fibers. This correlation between ZO-1
and F-actin is consistent with our previous finding that themouse E3.5 embryo with ZO-1 droplets had less F-actin thanthe E4.5 embryo with junctional ZO-1. Thus, ZO-1’s phaseseparation is regulated by cell-cell interaction and F-actin
remodeling.
We also analyzed LLPS using bacterially-produced ZO-1
in vitro. We found that ZO-1 IDR4 was sufficient to form
liquid droplets in vitro (Figure 2F), and the full-length ZO-1
can bind to F-actin (Figure 2G). This system will be usefulin analyzing detailed molecular mechanisms of ZO-1 phaseseparation.

II. I nteraction with F-actin regulates ZO-1
phase separation

To analyze the nature of ZO-1 liquid droplets in various
cell environments, we first expressed GFP-ZO1 in A6 cells.
GFP-ZO-1 is mainly localized in the cell periphery andcolocalized with F-actin bundles. We thus treated cells withlatrunculin B, which disrupted F-actin (Figure 2B). We
found that the formation and extinction of cytoplasmic ZO-1
granules depended on the destruction and development of theF-actin network. Furthermore, we confirmed that latrunculin
B-induced ZO-1 granules rapidly recovered in the FRAPassay. These observations indicate that the efficiency andgrowth of ZO-1 condensation are negatively regulated by theinteraction with F-actin in A6 cells.
To inhibit the interaction between ZO-1 and F-actin ina different way, we constructed GFP-ZO-1ΔABD, whichlacks the actin-binding domain (ABD) (Figure 2A), andexpressed it in A6 cells. GFP-ZO-1ΔABD subsequently
formed droplets with a smooth surface (Figure 2C). Thissuggests that cytoplasmic droplet formation is restricted by
the binding of ZO-1 to F-actin and that GFP-ZO-1ΔABD
lost its capacity for the interaction. It is known that when
proteins form liquid droplets, intrinsically disordered regions(IDRs) play an important role in phase separation. In human
ZO-1, four IDRs are predicted (Figure 2A). Among these, we
found that the C-terminal IDR4, is essential for phase separation. Since IDR4 includes ABD, binding to F-actin may
affect the LLPS-inducing activity of the IDR4.
We observed ZO-1 behavior in the wound healing assay
using canine MDCK cells. Initially, most of the cells hadfew condensates in a confluent cell sheet. However, afterscratching, cells started active migration toward the woundsite, forming a significant number of droplets (Figure 2D).
In these cells, F-actin distribution and intensity also changed(Figure 2E). Initially, cortical F-actin was well-developedin the non-migrating polygonal cells with junctional ZO-1.

Figure 2. Interaction with F-actin regulates ZO-1 phase separation.
A. The domain structure and the intrinsically disordered regions (IDRs)of ZO-1. B. A6 cells were treated with latrunculin B (Lat) to disrupt
F-actin. Bar = 20 µm. C. GFP-ZO-1 lacking the actin binding domain(ABD) was expressed in A6 cells. Bar = 20 µm. D. A wound healing
assay using MDCK cells. Bar = 50 µm. E. 10 hours after the woundhealing assay, cells close to the wound site ("edge") and in the inner
region ("inner") were stained with phalloidin. Bar = 20 µm. F. Purified
GFP-ZO1-IDR4 formed liquid droplets in vitro. Bar = 5 µm. G. Purified
GFP-ZO-1 interacts with F-actin. Bar = 10 µm.

29National Institute for Basic Biology

Developmental Biology

III. C ephalochordate Brachyury enhancers
involved in the evolution of the chordate
notochord and somites

The notochord and somite are the most central organs inchordates, and Brachyury (Bra) plays a pivotal role in theirformation. The question of how Bra gained its enhancer
activity in the notochord-specific expression is critical to ourunderstanding of chordate evolution. In cephalochordates,
Bra is duplicated into two genes (Figure 3). We examined theenhancer activity of Branchiostoma floridae Bra (BfBra1 andBfBra2) by lacZ reporter assay using a Ciona-embryo hostsystem. In the 5’, 3’ regions and introns, the signal intensity was higher and broader in BfBra2 than BfBra1 (Figure3). In some cases, lacZ expression is expanded to posterior
muscle cells in the former. The intron enhancers displayed
another difference; primary expression of BfBra1 occurredin the notochord, whereas both muscle and the notochordindicated BfBra2 expression. These results, along with otherdata, predicted an EvoDevo scenario in which BfBra2 ismore ancestral and mother-like, while BfBra1 is daughterlike and duplicated by BfBra1. Originally, ancestral BfBra2
was likely to gain enhancer machinery for gene expression inmuscle and notochord, as has been deduced from its spatial
expression profile: the 5’ region for somite expression, the3’ region for notochord expression, and introns for somite/
notochord expression. In contrast, sister BfBra1 became
more specialized for notochord expression using the intron
enhancers.

test whether the observed light response resulted in aggregation or dispersal under specific light fields.
First, we precisely observed the larval swimming activity
under fluctuating light conditions and found that larvae temporarily stopped swimming ~30 s after rapid light intensity
reduction, thus exhibiting a step-down photophobic response.
This behavior was also observed when we rapidly changed
the spectral composition of light. Further experiments using
the Okazaki Large Spectrograph revealed that the loss of
short wavelengths of light (blue/green light) induced thistype of behavior.
The analysis of mathematical simulations of this step-down
photophobic response indicates that larvae will aggregate
in the lighter areas of two-dimensional large rectangularfields. These results suggest that the step-down photophobic
response of coral larvae may play an important role in determining where larvae settle on the reef.

Figure 4. A wild colony of Acropora tenuis (photo taken by Masayuki Hatta
at Ochanomizu University). The inset shows a planula larvae of A. tenuis.

Figure 3. A summary of results of the present reporter assay and their
possible interpretation.
Enhancer activity of 5’ upstream sequences in muscle is shown in brown
and that of the 3’ downstream in notochord is in blue. The activity of
BfBra2 introns in muscle and notochord is shown in purple.

IV. A step-down photophobic response inlarvae of the common reef coral, Acropora
tenuisMany reef-building corals form a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium.
Corals mostly depend on photosynthetic products fromthese symbionts as their energy source, and thus light conditions in habitats can influence post-settlement survival.
Previous studies reported that light environments play anessential role in larval habitat selection. However, due to alack of basic photobiological studies in corals, how corallarvae perceive and respond to the light in their environment
remains largely unknown. To answer these questions, weanalyzed the swimming behavior of larvae of the common
reef coral Acropora tenuis (Figure 4) under various light conditions. In addition, we developed a mathematical model to30

Publication List:〔Original papers〕

N., Yamamoto, T.S., Yasue, N., Takagi, C., Fujimori, T., and• Kinoshita,Ueno, N. (2022). Force-dependent remodeling of cytoplasmic ZO-1•

condensates contributes to cell-cell adhesion through enhancing tightjunctions. iSCIENCE 25, 103846. DOI: 10.1016/j.isci.2022.103846
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada,S., Ueno, N., Sawai, S., Kondo, Y., and Aoki, K. (2021). Optogenetic
relaxation of actomyosin contractility uncovers mechanistic roles ofcortical tension during cytokinesis. Nat. Commun. 12, 7145. DOI:
10.1038/s41467-021-27458-3