Dorsal convergence of gastrula cells requires Vangl2 and an adhesion protein-dependent change in protrusive activity.

Lateral zebrafish hypoblast cells initiate dorsal convergence near mid-gastrulation and exhibit non-polarized morphologies, limited cell-cell contact and indirect migration trajectories. By late gastrulation, mesodermal cells become packed as they engage in planar cell polarity (PCP)-dependent movement. Here, we aimed to understand this transition in cell behavior by examining the relationship between protrusion dynamics and establishment of PCP and directed migration. We found that wild-type cells undergo a reduction in bleb protrusions near late gastrulation accompanied by a VANGL planar cell polarity protein 2 (Vangl2)-regulated increase in filopodia number and polarization. Manipulation of blebs is sufficient to interfere with PCP and directed migration. We show that Vangl2, fibronectin and cadherin 2 function to suppress blebbing. Vangl2 maintains ezrin b (Ezrb) protein levels and higher Ezrb activation rescues defective mediolateral cell alignment and migration paths in vangl2 mutant embryos. Transplantation experiments show that loss of vangl2 disrupts protrusion formation cell-autonomously while fibronectin acts non-autonomously. We propose that dorsal convergence requires the coordinated action of Vangl2, Ezrb and cell-adhesion proteins to inhibit blebs and promote polarized actin-rich protrusive activity and PCP.

Vangl2-dependent regulation of membrane protrusions and directed migration requires a fibronectin extracellular matrix.

During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells.