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Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation

Periodic Reporting for period 2 - MECSPEC (Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation)

Reporting period: 2019-01-01 to 2020-06-30

In the past funding period, we have been working on the question of how cell/tissue morphogenesis and mechanics interact with cell fate specification and differentiation during early zebrafish embryogenesis. In developmental biology, enormous progress has been made on deciphering the gene regulatory pathways controlling cell fate specification and embryo patterning. Likewise, research at the interface between developmental biology and physics has provided novel insight into the key mechanisms by which mechanical forces function in cell and tissue morphogenesis during development. However, remarkably little is yet known about how cell/tissue mechanics influence the gene regulatory pathways involved in cell fate specification and vice versa. To address this important question, we have chosen to study early development of zebrafish, a vertebrate model organism amendable to a wide range of experimental techniques from different disciplines, ranging from genetics to biophysics.

Specifically, we have focussed on the interaction between mesendoderm specification and blastoderm epiboly movements (objective 1) and the self-organization of germ layer progenitor cells ex vivo (objective 3). We have also begun addressing the interplay between blastoderm morphogenesis and enveloping layer (EVL) specification (objective 2). However, while addressing the role of YAP/TAZ signalling as a mechanosensitive effector pathway in this process, we also discovered an earlier important function of this pathway in cell specification during oogenesis, on which - in addition to the EVL - we have focussed during the past funding period.
For objective 1, we have analysed how mesendoderm specification affects tissue material properties, such as tissue viscosity/fluidity, and how changes in tissue viscosity/fluidity feed back on tissue morphogenesis during zebrafish gastrulation (Petridou et al., 2018). We found that at the onset of zebrafish morphogenesis, the blastoderm undergoes rapid, pronounced and spatially patterned tissue fluidization, which is important for blastoderm spreading. This fluidization is temporally controlled by mitotic cell rounding-dependent cell-cell contact disassembly during the last rounds of cell cleavages. Finally, we found that local activation of non-canonical Wnt-signalling within the blastoderm margin downstream of mesendoderm cell fate specification increases cell contractility and cell cohesion, which counteracts the effect of mitotic rounding on contact disassembly. As part of objective 1, we have also investigated how interstitial fluid (IF) accumulations within the blastoderm affect mesendoderm specification and internalization, and, vice versa, how mesendoderm specification affects IF accumulation within the blastoderm. We found that the IF, previously localized under the surface cells, gradually sieves through the tissue toward the yolk interface, following the migration of underlying mesendoderm cells. In mutants lacking mesendoderm specification, IF does not exhibit gradual sieving and overall IF re-localization is inefficient, despite normal epiboly progression. Finally, we found that pharmacological treatments causing IF depletion resulted in defective mesendoderm internalization movements in respect to cellular crowding and internalization speed.

For objective 2, we have analysed how EVL morphogenesis/spreading and EVL cell fate specification are related to each other. Specifically, we have analysed how pulling forces, acting at the margin of the EVL, trigger maturation and growth of tight junctions (TJ) connecting the EVL margin with the yolk cell, where the pulling forces are generated through actomyosin contraction. By analysing TJ formation at the interface between the EVL and Yolk Syncytial Layer (YSL) within the gastrulating zebrafish embryo, we found that ZO-1 accumulation at TJ directly scales with actomyosin tension within the YSL. Actomyosin tension triggers ZO-1 junctional accumulation by driving retrograde actomyosin flows within the YSL that transport non-junctional ZO-1 aggregates towards TJ by advection. Finally, we found that non-junctional ZO-1 aggregates form by phase separation and their accumulation is dependent on the intrinsically disordered region (IDR) of ZO-1, previously implicated in ZO-1 binding to Actin. When the IDR of ZO-1 is removed, the non-junctional ZO-1 pool disappears, TJ lose their mechanosensitivity, and, consequently, EVL-YSL movements are reduced.
In addition to analysing the interplay between mechanical forces and EVL maturation, we have also investigated how in zebrafish oogenesis the future the future micropyle precursor cell (MPC) is specified (Peng et al., 2019). We found that a group of cells within the granulosa cell layer at the oocyte animal pole acquires elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, thereby mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz-/- follicles.

For objective 3, we have analysed how the interplay between mechanical and biochemical signals determines the self-organizing capability of germ layer progenitor cells during gastrulation. By analysing self-organization of germ layer progenitors in blastoderm explants ex vivo, we found that explants can give rise to a broad range of mesendodermal cell types. An initial analysis of the distribution of key signaling molecules of mesendoderm patterning further revealed several differences in the establishment of their signaling domain between explants and intact embryos. In contrast to mesendoderm patterning, the morphogenesis of blastoderm explants can only partially recapitulate normal development. Blastoderm explants form an “exogastrula” with mesendodermal progenitors arranging in an extension instead of internalizing.

To understand how cell-cell contact formation functions in germ layer self-organisation, we have also studied the influence of actomyosin cell cortex tension on the formation of adhesive contacts between zebrafish progenitor cells. We found that when cortex tension was massively up-regulated, cell-cell contacts did not grow big, as expected from previous models, but, instead, remained very small. This unexpected effect, by which the cell-cell contacts size limits rather than promotes contact expansion, is due to cortical tension reducing turnover of cadherin adhesion molecules at the contact, required for fast contact growth.
Our findings so far have unravelled a number of novel and unexpected feedback mechanisms determining the interplay between cell/tissue mechanics and cell fate specification. In particular, we discovered an important function of mesendoderm specification in modulating tissue fluidity, and, conversely, tissue fluidization in directing proper tissue morphogenesis leading to mesendoderm cell fate specification (Petridou et al., 2018). We also identified how differential cell growth can trigger lateral inhibition in cell fate specification by modulating TAZ-dependent gene transcription through mechanical compression (Xia et al., 2019). These findings provide novel insight of the role of cell/tissue mechanics in tissue rheology and cell fate specification that go far beyond the state of the art. In addition, we made several still preliminary findings on the role of interstitial fluid in mesendoderm specification and internalization, tight junction mechanosensitivity and germ layer tissue self-organization during gastrulation. Together, we expect that these projects will decisively advance our understanding of the interplay and feedback between physical mechanisms driving cell/tissue morphogenesis and the gene regulatory pathways determining cell fate specification in development.