The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

Abstract

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.

Regional cell shape changes control form and function of Kupffer's vesicle in the zebrafish embryo

“…Antisense RNA probes were labeled with digoxygenenin (Roche DIG RNA labeling kit) to detect spaw expression via RNA in situ hybridization as described (Yu et al, 2011). …”

Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT–PIX complexes

“…Because Arf proteins have no intrinsic GTPase activity, active GTP-bound Arf proteins require ArfGAP proteins to deactivate them by converting bound GTP to GDP (Randazzo et al, 1994). Mammals express two GIT proteins, GIT1 and GIT2, whereas zebrafish -because they have two GIT2 genes (git2a and git2b) -express three GIT proteins (Yu et al, 2011). Caenorhabditis elegans and Drosophila melanogaster have each only a single GIT gene (git-1 and Git, respectively) (Lucanic and Cheng, 2008;Bahri et al, 2009).…”

“…In the β-PIX-deficient mouse, lethality results from defective cell migration during early embryogenesis (Omelchenko et al, 2014). Experiments with GIT2 in Xenopus (Köster et al, 2010) and GIT2a in zebrafish (Yu et al, 2011) demonstrated cell migration defects during gastrulation. GIT1-knockout lethality has been reported to result from poor angiogenesis in the lungs (Pang et al, 2009).…”

Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish

“…Our findings suggest that NMM-II activity is required for normal myoblast elongation and boundary capture primarily during the early and transitory phases of somite-to-myotome maturation. Previous work has implicated the Paxillin binding partner, Git2, in NMM-II activation and regulation of cell shape during zebrafish epiboly (Yu et al, 2011). Interestingly, activation of NMM-II can promote Integrin activation and ECM fibril formation in newly formed somites (Julich et al, 2015).…”