Regulation of the yolk microtubule and actin cytoskeleton by Dachsous cadherins during zebrafish epiboly.

Epiboly is a crucial morphogenetic process during early animal embryogenesis that expands surface area of embryonic tissues while thinning them. During zebrafish development, epiboly spreads the superficial enveloping layer (EVL), germ layers, and yolk syncytial layer (YSL) to cover the yolk cell. Here we investigated functions of the three zebrafish dchs genes, dchs1a, dchs1b, and dchs2 that encode large atypical cadherins and report that they have partially overlapping functions in epiboly progression. By inserting GFP or mNeongreen fluorescent proteins at the C-terminal Dchs1b intracellular domain of the endogenous dchs1b locus using homologous recombination, we observed the resulting Dchs1b fluorescent fusion proteins localized in both the cell membrane and the cytoplasm of EVL, embryonic cells and YSL during gastrulation. The dynamic microtubule and actin cytoskeleton of the yolk cell are essential for epiboly. Our studies demonstrate that the yolk cell microtubules are more bundled and show faster polymerization during epiboly in dchs triple loss-of-function mutant embryos than in wild-type embryos, indicating that dchs genes are required for limiting microtubule polymerization and promoting dynamics during epiboly. The epiboly progression defects of dchs1b-deficient mutants were suppressed by mutations in the tetratricopeptide repeat protein 28 (ttc28) gene encoding a cytoplasmic protein previously shown to bind to Dchs1b intracellular domain and alter microtubule dynamics during early cleavages. We further demonstrate that MZdchs1b mutants exhibit abnormal organization and dynamics of yolk cell actin cytoskeleton during epiboly. Together, these lines of evidence as well as our transcriptomic analyses support the notion that, like during early embryonic cleavages, Dchs1b plays a major role, while Dchs1a and Dchs2 proteins have supporting roles regulating microtubule dynamics and organization of both microtubule and actin cytoskeleton to ensure normal epiboly.