Molecular Mechanisms Of Cell Movements During Embryogenesis-Cortactin Mediates Gastrulating Cell Migration Both In Zebrafish (Danio Rerio) And Amphioxus (Branchiostoma Belcheri Tsingtaoense)

Cell migration is essential to direct embryonic cells to specific sites where their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. In the present research, the structure, function and evolution conservation of cortactin are systematically analysized. Using zebrafish and amphioxus gastrula as experimental modles, the role cortactin plays in morphogenic cell movements was investigated, in addition, the evolution of morphogenetic movements was studied by comparing zebrafish gastrulation with that in amphioxus. Zebrafish cortactin gene was cloned and analyzed. Protein sequence alignments show that zebrafish cortactin is highly conserved to known higher vertebrate cortactin, all of which contain the typical four functional domains: the NTA domain at the N terminus, the SH3 domain at the C terminus and between them, the cortactin repeat domain and the α-helix domain. The zebrafish cortactin gene is located to the chromosome 19 in zebrafish genome. The whole genomic sequence of zebrafish cortactin gene covers a range of about 7kb including 15 exons and 14 introns. A synteny gene linkage cluster CCND1-ORAOV1-FGF19-FGF4-FGF3-CORTACTIN- SHANK2 was identified to be highly conserved from zebrafish to mouse and human. By genome comparing, three novel zebrafish genes, ORAOV1, SHANK2 and HS1, were identified related to this cluster in zebrafish genome. By Western Blotting analysis, an expression peak of cortactin was detected at the gastrula stage. Correspondingly, immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the germ shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Overexpression of cortactin in zebrafish embryos enhances cell migration and leads to the acceleration of gastrulation while does not interfere with cell fate determination. Therefore, these data suggest that cortactin mediated actin polymerization is implicated in the cell movement during zebrafish gastrulation and perhaps the development of the central neural system as well. The cephalochordate amphioxus shares a similar basic body plan with vertebrates. During gastrulation, it undergoes a primitive and relatively simple morphogetic procedure to lay down this body plan. However, cellular mechanisms underlying amphioxus gastrulation movements remain unknown. This study shows that amphioxus gastrula exhibit a marked cortical activity increase of cortactin in the invaginating vegetal cells. Similar expression pattern was also observed with Arp2/3 complex. Distribution of cortactin in the cell cortex of these cells was impaired by treatment of the initial gastrula with an FGF receptor inhibitor, SU5402. This drug also suppressed lammellipodia formation and cause dramatical changes on cell surface morphology and cell motility in amphioxus gastrula. The vegetal mesendoderm cells could not invaginate and gastrulation was greatly retarded. These findings reveal an FGF signaling mechanism whereby the invagination movements during amphioxus gastrulation are mediated by cortactin-regulated actin polymerization. Together with the evidence from zebrafish gastrula, it thus appears clear that the molecular mechanisms of morphogenetic cell movements which are mediated by cortactin is conserved through chordate evolution although morphogenetic processes of gastrulation have been known to be highly diversified in the phylum of chordate.