| Cortactin ,a novel member of filament actin binding protein family and the main substrate of non-receptor Src protein kinase ,plays an important role in the dynamic organization of cell cortex cytoskeleton. Recently,great progress has been made in understanding of the mechanism of cell movement in vitro,and probably,cortactin acts directly on the molecular machines of cell motility. However,in vivo experimental evidence is still in great need because of tissue untransparancy and deficiency of normal moving cells in adult tissues. On the other hand,cell movement is one of the most attractive subjects of developmental biology research for centuries. With the efforts of generations of developmental biologists,detailed cell migration pathways in different kind of vertebrate embryos have been clarified,but unfortunately,mechanism underlying has been poorly known by now partially because of the uncertainty of the cellular events happening.Here,we tried to bridge up theses seemingly separate areas byanalyzing the expressing pattern of cortactin in zebrafish early embryos using Western Blotting,whole-mount antibody staining and laser confocal scanning microscope techniques. Taking advantage of microinjection of anti-cortactin antibodies,we also investigated the function of cortactin in zebrafish embryogenesis.The results shew that no cortactin was found in unfertilized eggs and the 1-cell stage embryo;cortactin appeared after the first cleavage and the blastomeres were highly stained. Asymmetrical distribution of cortactin appeared when gastrulation starts,with higher staining in the dorsal part of the embryo,while much lower in the ventral part. Embryonic shield and its neighboring areas were especially highly stained and cortactin marked the cells undertaking active convergencing movement. The stained areas were belt -like along the anterior and posterior axis,firstly short and wide,with the progression of gastrulating,the stained belt was extending interiorly and narrowed,but the anterior and posterior terminals were sustainedly highly stained. In the early neurula,cortactin staining signals were detected in the developing primodium of forebrain as early as 10.5 hpf (hours post fertilization),at the same time the tailbud areas were highly stained too. Later,the staining was concentrating in the developing neural rod while paraxial mesoderm derivatives were not stained. As for the sub-cellular locations of cortactin,laser confocal scanning microscope pictures shew that in cleavage stage,cortactin wasdistributed ubiquitously in the cytoplasm with the exception that in cells undertaking cytokinesis,positive stained signals were detected in the cleavage furrow. From middle blast transition ,cortatin location dramatically changed to the cell cortex and there were no signals detected in cytoplasm anymore. And it was found that in the embryonic shield area where convergence movements happening most extensively,cells' cortex were most highly stained.We also stained the embryos with anti-Arp3 antibody and found that these two kinds of antigen are generally co-localized but Arp3 were not detected in cleavage stage embryos. It was deduced that cortactin functions in the cytokinesis independent of Arp3. And microinjection of anti-cortactin antibodies into the I cell stage embryos leaded to fatal developmental defects because of the unequal cytokinesis.In this paper we reported the engagement of cortactin in cell movements in vivo for the first time. We also found that ,besides the traditional idea about the role it plays in cell motility,cortactin was also involved in cytokinesis and played undispensensable role in nleava stage independent of Arp2 3 complex. And also,it was firstly reported here that cortactin functions in the developing primordium of forebrain as early as 10.5 hpf ,much more earlier than what is known by now about the forebrain primordium molecular marker such as HNK-1(12hpf)(46). The expression pattern of cortactin in the developing neural system ofzebrafish embryo also suggest...
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