Founctional Analysis Of PI3K-Rac1 Signalling During Epiboly Movement In Zebrafish

The establishment of the original body plan occurs during gastrulation and depends on massive cell differentiation and orchestrating morphogenetic movements after the cleavage stage of zebrafish early embryo.The morphogenetic movements in gastrulation involves epiboly,involution,cell migration and convergence&extention,in which epiboly provides the driving force for expansion of germ layers and the engulfment of the yolk cell.The dynamic assembly of microfilament cytoskeleton not only provides direct force for epiboly,but also is essential for cell cohension,another critical factor for this process.Previous work showed that PI3K-Racl axis regulates F-actin dynamics and promotes the migration of macrophages and neutrophils in zebrafish.However,it still remains elusive whether and how this pathway functions during epiboly.In this study,we used LY294002,NSC23766 and dnRac1 to supress PI3K-Racl signaling in a series of developmental stages and found that this pathway can promote epiboly movement by differentially regulating several F-actin structures,such as marginal actomyosin ring,cortical F-actin bundles of both enveloping layer cells and the vegetal yolk cell.We further showed that the formation of marginal actomyosin ring have the highest sensitivity to the treatment of inhibitors.We also showed that these actin structures could be compromised in different treatment windows.In particular,inhibition of PI3K-Racl activity at 50%-epiboly stage can specifically disrupt the marginal actomyosin ring,which leads to displacement of nuclei in the external yolk syncytial layer(E-YSN)away from the marginal zone.These data suggest a novel function for the marginal actomysin ring in keeping the E-YSN within the yolk syncytial layer for coordinated movement with the leading edge of the enveloping layer.They also indicate that the vegetal cortex F-actin associates with and may exert a pulling force on the E-YSN.In summary,this study uncovers an essential role of PI3K-Racl signaling in the epiboly movement,and reveals novel functions of different F-actin structures during gastrulation of zebrafish embryos.