Periodic Reporting for period 4 - MECSPEC (Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation)
Reporting period: 2022-01-01 to 2022-06-30
In this project we have analyzed the mechanistic underpinning of various mechanochemical feedback loops in early zebrafish embryogenesis. We have been able to show that doming, the spreading of the blastoderm over the yolk cell at the onset of zebrafish embryogenesis, depends on both the specification of mesoderm and endoderm (mesendoderm) progenitor cells at the blastoderm margin and the regulation of tissue fluidity at the blastoderm centre. These two processes are interconnected by mesendoderm cell fate specification locally suppressing tissue fluidity by activating the Wnt/planar cell polarity pathway and cell compaction promoting mesendoderm specification by promoting paracrine Nodal signalling. We also found that specification of the enveloping cell layer (EVL) and junction formation to the adjacent yolk cell in the early zebrafish embryo is required for EVL spreading and that EVL spreading is required for EVL specification and junction formation. The crosstalk between these two processes is achieved by EVL specification and junction formation promoting actomyosin contraction within the yolk cell, required for EVL spreading, and that proper force generation within the yolk cell promotes EVL specification and junction formation by enhancing the transport and accumulation of critical molecular components needed for EVL specification and junction formation. Finally, we showed in ex vivo preparations how cell-cell contact formation depends on mesoderm specification and actomyosin-mediated mechanical force generation. Collectively, these findings have considerably advanced our understanding of the molecular, cellular and biophysical mechanisms by which the gene regulatory pathways controlling fate specification and the mechanical processes underlying cell and tissue morphogenesis function in concert during early zebrafish embryogenesis. more generally, our findings obtained in this project shed light on the mechanistic basis of early embryonic development and, consequently, how malfunction of these mechanisms led to defects observed in human congenital disorders.
For objective 1, we have followed up on our previous observations that at the onset of zebrafish morphogenesis, the blastoderm undergoes rapid, pronounced and spatially patterned tissue fluidization, which is important for blastoderm spreading, and that this fluidization is temporally controlled by mitotic cell rounding-dependent cell-cell contact disassembly during the last rounds of cell cleavages (Petridou et al., 2019, Nature Cell Biology). To understand how the blastoderm tissue undergoes fluidization, we have used – in collaboration with Prof. Hannezo at IST Austria – percolation theory to theoretically describe how changes in blastoderm cell-cell contacts can lead to an abrupt and strong tissue fluidization, as observed within the blastoderm at the onset of gastrulation. We found that there is a critical point in cell connectivity within the blastoderm, at which the blastoderm undergoes a rigidity transition leading to its fluidization (Petridou et al, 2021, Cell). This provides a conceptual framework for explaining how cell connectivity within tissues relate to tissue material properties, and what role such relationship might play in embryonic development.
For objective 2, we have analysed how enveloping layer (EVL) morphogenesis/spreading and EVL cell fate specification/differentiation are related to each other. Specifically, we have analysed the interplay between intermediate filament expression, hallmark of EVL specification, and EVL spreading during zebrafish gastrulation. We found that during the spreading process the EVL tissue becomes increasingly stiffer, and that this tissue stiffening process relies on keratin intermediate filament expression and organization into a filamentous network within EVL cells. We also found that interfering with keratin expression within the EVL leads to reduced EVL stiffness and impaired epiboly movements. Finally, we observed that tension within the plane of the EVL tissue, as a result of the yolk syncytial layer (YSL) pulling on the margin of the EVL, triggers keratin expression within the EVL, pointing at the possibility of keratin expression being regulated by mechanical signals. Collectively, these findings suggest that EVL cell specification and differentiation, as evident by their expression of keratin intermediate filaments, is controlled by tissue tension and leads to tissue stiffening, thereby revealing a mechanism by which mechanical tension control cell fate specification and tissue stiffening.
For objective 3, we have focussed on understanding how cell-cell contact formation functions in germ layer self-organisation by studying 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 (Slovakova et al., 2022, PNAS).
For objective 2, we have analysed how enveloping layer (EVL) morphogenesis/spreading and EVL cell fate specification/differentiation are related to each other. Specifically, we have analysed the interplay between intermediate filament expression, hallmark of EVL specification, and EVL spreading during zebrafish gastrulation. We found that during the spreading process the EVL tissue becomes increasingly stiffer, and that this tissue stiffening process relies on keratin intermediate filament expression and organization into a filamentous network within EVL cells. We also found that interfering with keratin expression within the EVL leads to reduced EVL stiffness and impaired epiboly movements. Finally, we observed that tension within the plane of the EVL tissue, as a result of the yolk syncytial layer (YSL) pulling on the margin of the EVL, triggers keratin expression within the EVL, pointing at the possibility of keratin expression being regulated by mechanical signals. Collectively, these findings suggest that EVL cell specification and differentiation, as evident by their expression of keratin intermediate filaments, is controlled by tissue tension and leads to tissue stiffening, thereby revealing a mechanism by which mechanical tension control cell fate specification and tissue stiffening.
For objective 3, we have focussed on understanding how cell-cell contact formation functions in germ layer self-organisation by studying 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 (Slovakova et al., 2022, PNAS).
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 Nature Cell Biology & 2021, Cell). 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, Cell). 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 tissue tension and EVL cell fate specification and, vice versa, on the role of EVL cell differentiation, and the expression of intermediate filaments, on EVL tissue stiffness. 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.