Galpha(12/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(12) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(12/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Galpha(12/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Galpha(12) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4-labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Galpha(12/13) signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Galpha(12/13) regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Galpha(12/13) modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Galpha(12/13) regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.