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Modeling and experimental results suggest a role for Planar Cell Polarity-dependent multi-cellular rosette structures in ensuring correct epithelial cell migration in the mouse visceral endoderm., The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner., Author Summary The mouse visceral endoderm (VE) is a simple epithelium in the egg cylinder stage mouse embryo. Many functions associated with epithelia require them to undergo extensive remodelling through changes in the shape and relative positions of constituent cells, a process about which we understand relatively little. The anterior visceral endoderm (AVE) is a specialized group of cells in the simple epithelium of the VE, and their stereotypic migratory behaviour is essential for establishing the orientation of the anterior-posterior axis in the early mouse embryo. We show that AVE migration is linked to changes in cell packing in the VE and an increase in “rosettes,” which are striking collections of five or more cells meeting at a central point. To probe the role of rosettes during AVE migration, we have developed a mathematical model of cell movement in the VE. Simulations suggest that rosettes are not essential for AVE migration, but are crucial for the orderliness of this migration. We also explored the role of Planar Cell Polarity (PCP) signalling, which is known to coordinate cell polarization and rearrangement in many different tissues. We find that mutants in which PCP signalling is disrupted have fewer rosettes, altered epithelial packing, and abnormal AVE migration. We suggest that rosettes in the mouse VE are a PCP-dependent arrangement of cells that act to buffer the disturbances in cell packing generated by AVE migration, thereby enabling AVE cells to migrate in an orderly manner.

The anterior visceral endoderm (AVE), a signalling centre within the simple epithelium of the visceral endoderm (VE), is required for anterior-posterior axis specification in the mouse embryo. AVE cells migrate directionally within the VE, thereby properly positioning the future anterior of the embryo and orientating the primary body axis. AVE cells consistently come to an abrupt stop at the border between the anterior epiblast and extra-embryonic ectoderm, which represents an end-point to their proximal migration. Little is known about the underlying basis for this barrier and how surrounding cells in the VE respond to or influence AVE migration. We use high-resolution 3D reconstructions of protein localisation patterns and time-lapse microscopy to show that AVE cells move by exchanging neighbours within an intact epithelium. Cell movement and mixing is restricted to the VE overlying the epiblast, characterised by the enrichment of Dishevelled-2 (Dvl2) to the lateral plasma membrane, a hallmark of Planar Cell Polarity (PCP) signalling. AVE cells halt upon reaching the adjoining region of VE overlying the extra-embryonic ectoderm, which displays reduced neighbour exchange and in which Dvl2 is excluded specifically from the plasma membrane. Though a single continuous sheet, these two regions of VE show distinct patterns of F-actin localisation, in cortical rings and an apical shroud, respectively. We genetically perturb PCP signalling and show that this disrupts the localisation pattern of Dvl2 and F-actin and the normal migration of AVE cells. In Nodal null embryos, membrane localisation of Dvl2 is reduced, while in mutants for the Nodal inhibitor Lefty1, Dvl2 is ectopically membrane localised, establishing a role for Nodal in modulating PCP signalling. These results show that the limits of AVE migration are determined by regional differences in cell behaviour and protein localisation within an otherwise apparently uniform VE. In addition to coordinating global cell movements across epithelia (such as during convergence extension), PCP signalling in interplay with TGFβ signalling can demarcate regions of differing behaviour within epithelia, thereby modulating the movement of cells within them., Author Summary The orientation of the head-tail axis is determined during embryogenesis by the movements of a subset of cells called the AVE (anterior visceral endoderm). These cells migrate from their initial position within the simple epithelium of the visceral endoderm (VE) to a location from which they eventually induce anterior pattern in the adjacent epiblast. Little is understood about how AVE cells migrate within the VE, why they stop migrating where they do, and how surrounding cells in the VE respond to or influence AVE migration. In this study, we use time-lapse microscopy and high-resolution 3D reconstructions of protein localisation patterns to address these issues. Our results show that AVE cells move by exchanging neighbours within an intact epithelium. The limits of AVE migration are determined by regional differences in cell behaviour and protein localisation within an otherwise apparently uniform VE. Finally, we examine the role of planar cell polarity (PCP) signalling, which is responsible for coordinating morphogenetic events across different epithelia. We show that in addition to this traditional role in coordinating global cell movements, PCP signalling along with TGFβ signalling can demarcate regions of differing behaviour within epithelia, thereby modulating the movement of cells within them.