Regulation of Avian Gastrulation by Fibroblast Growth Factor, Non-Canonical Wnt, and Eph-Ephrin Signaling Pathways

Abstract

Gastrulation is a key early developmental event that generates the three primary germ layers (ectoderm, mesoderm, and endoderm) from which organ systems subsequently develop. The physical mechanisms of germ layer formation differ significantly in amniotes (reptiles, birds, and mammals) and anamniotes (e.g. frog and fish), as amniote gastrulation includes an epithelial-mesenchymal transition (EMT) that is absent from anamniote gastrulation. Despite this striking difference, much of our knowledge regarding the mechanisms underlying gastrulation is derived from frog and fish studies. To better understand amniote gastrulation, the work herein investigates three signaling pathways that regulate amniote gastrulation with distinct and overlapping functions. The central hypothesis is that multiple signaling pathways function cooperatively to precisely modulate cell migration through the primitive streak during avian gastrulation.First, I describe a novel function of Fibroblast Growth Factor (FGF) signaling in the preingression epiblast adjacent to the avian primitive streak, where it governs the expression of molecules from diverse signaling pathways and transcription factor families, and which is mediated largely through the Ras/MAPK pathway. Importantly, FGF signaling also regulates cell migration during avian gastrulation.Next, I report the isolation of a novel chicken non-canonical Wnt ligand (Wnt11b) that is specifically expressed in the primitive streak and adjacent preingression epiblast during gastrula stages. In gain and loss of function studies, Wnt11b and Wnt5a/b participate in regulating cell migration through the streak in a largely redundant fashion. Signaling specifically targets the non-canonical pathway, as similar cell migration defects are observed with a non-canonical mutant of Dishevelled, and activating the canonical pathway has no effect on cell migration.Finally, I investigate the function of A-class Eph-ephrin signaling during avian gastrulation, and describe that Eph receptor forward signaling negatively regulates the migration of cells through the primitive streak. This modulation of cell migration occurs independently of the EMT that accompanies avian gastrulation, as cells are able to undergo the normal cadherin transition and the basal lamina is unaffected.Altogether, the work presented herein provides a significant contribution to our understanding of signaling pathways that modulate gene expression and ongoing cell migration during germ layer formation in amniote gastrulation.