DORSAL CONVERGENCE OF GASTRULA CELLS REQUIRES A VANGL2 AND ADHESION PROTEIN-DEPENDENT CHANGE IN PROTRUSIVE ACTIVITY

Abstract

During zebrafish gastrulation, planar cell polarity (PCP) describes the elongation and mediolateral alignment of cells parallel to their path of dorsal migration. The PCP pathway consists of a core group of proteins that includes VANGL planar cell polarity protein 2 (Vangl2) and a non-canonical Wnt/Glypican4 signaling pathway. Loss of function of a core PCP protein produces an embryo with a severe convergence and extension (C&E) phenotype characterized by a shortened and broadened dorsal axis. From mid- to late gastrulation mesodermal cells undergo a key transitional period. At 80% epiboly (mid-gastrulation) mesendodermal cells exhibit rounder cell shapes, limited cell contacts, and indirect migration trajectories. By late gastrulation mesodermal cells elongate, pack together, and align into files as they engage in directed migration. The goal of this study was to gain an understanding of this transition to polarized cell behaviors by examining the relationship between protrusion formation and the establishment of PCP and directed migration towards the dorsal axis. We found wild-type cells exhibit a reduction in the number of bleb protrusions formed between mid- and late gastrulation accompanied by a Vangl2 regulated increase in filopodia. Increased bleb protrusions by Ezrinb knockdown disrupts PCP and directed migration. Additionally, we show that Vangl2, fibronectin, and N-cadherin are required for the suppression of blebbing at late gastrulation. Vangl2 likely regulates blebbing by regulating Ezrinb protein levels, and increased Ezrinb activation in vangl2 mutant embryos rescues the mediolateral cell alignment and directness defects of mesodermal cells. Lastly, transplantation experiments vangl2 functions cell-autonomously to regulate protrusion formation while fibronectin is required non-autonomously.