Connexin43 (Cx43) encodes a gap junction protein. Cx43 is specifically expressed in blood vessel endothelial cells in the zebrafish embryo as demonstrated by in situ hybridization. When Cx43 is knocked down with antisense morpholinos, the zebrafish embryos have defects in the intersegmental vessel branching and vascular perfusion. These phenotypes reflect problems in endothelial cell migration. Further analysis with microangiography revealed that the endothelial tubes or lumens fail to form normally. The co-injection of Cx43 morpholinos and p53 morpholinos rescued the vascular defects to limited degrees. The co-injection of Cx43 morpholinos with VE-Cadherin morpholino enhanced the effects observed with the injection of Cx43 morpholino alone. The transgenic zebrafish expressing the fusion protein of Cx43 and egfp under control of flk1 promoter (Tg[flk1: Cx43-egfp]) has been generated for studying the dynamic regulation of Cx43 protein in zebrafish endothelium. From live imaging of this transgenic line, green fluorescent puncta from Cx43-gfp can be found in the endothelial cells. Those puncta could be patches of gap junctional channels or undocked hemichannels on the cell-to-cell borders. The existence of hemichannels could support a role of Cx43 in cue sensing for migration or in the endothelial lumen formation. Immunohistochemistry of VE-Cadherin in Tg[flk1: Cx43-egfp] line shows that not all the Cx43-egfp puncta are co-localized with the VE-Cadherin. The endogenous Cx43 appears on the putative surface of the endothelial cells of the sprouting intersegmental vessels at 22 hours post fertilization (hpf), as determined by staining with an antibody against Cx43 in transgenic fish with gfp-highlighted endothelial cells. This suggests a potential role of Cx43 in endothelial migration. It is possible that Cx43 forms gap junction channels with other cell types or functions as hemichannels. To test the function of Cx43 in vascular formation, Cx43 dominant negative mutants (DN) are expressed in an endothelium-specific manner. In order to control the expression of those DNs during the generation of the transgenic zebrafish, a Loxp flanked fluorescent protein expression cassette is inserted between the promoter and DN expression cassette, which will be excised when Cre is introduced.
|Advisor:||Essner, Jeffrey J.|
|Commitee:||Coffman, Clark R., Johansen, Kristen M.|
|School:||Iowa State University|
|Department:||Genetics, Development and Cell Biology|
|School Location:||United States -- Iowa|
|Source:||MAI 47/05M, Masters Abstracts International|
|Keywords:||Angiogenesis, Connexin43, Dominant negative mutant, Endothelial lumen formation, Endothelial migration|
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