Connexins and early zebrafish development (gap junctions, notochord, Danio rerio)

Until recently, evidence for the role(s) of gap junction proteins (i.e., "connexins") in embryonic development and organogenesis has been correlative, based upon connexin expression in developmentally significant domains and sites of inductive interactions. The transparent zebrafish (Danio rerio) embryo, with its external development, provides an excellent model system to elucidate the role(s) of gap junctional communication throughout development. To this end, the first objective of this thesis was to isolate zebrafish connexins. A 2.8 kb cDNA encoding a 381 amino acid polypeptide was isolated in a moderate stringency screen of a zebrafish cDNA library. Based on sequence comparison and predicted molecular mass (43,455 Da), this cDNA clone was named zebrafish connexin43 (zfCx43). RNase protection, in situ hybridization and immunofluorescence studies demonstrated that expression of zfCx43 is first readily detected after the completion of gastrulation. Its spatial expression is similar to that in the mouse, with particularly striking conservation of very discrete expression patterns within several developing tissues including heart, lens, and fin buds. Previous characterization of another connexin, zfCx43.4 (homologue of mammalian Cx45), suggested a role in notochord and tail morphogenesis with the possibility that its expression is regulated by the No tail (Ntl) transcription factor. The second objective of this thesis was to determine if Ntl activates zfCx43.4 transcription. RNase protection analysis demonstrates an increase in zfCx43.4 expression with overexpression of Ntl. The third objective of this thesis was to determine if zfCx43.4 expression is necessary for normal notochord and tail morphogenesis. Results of antisense zfCx43.4 experiments in wild type zebrafish embryos and zfCx43.4 overexpression in ntl mutant zebrafish embryos suggest that expression of zfCx43.4 is necessary for the coalescence of notochord precursor cells into the notochord primordium and for proper somite development. In summary, I show that the Cx43 expression pattern is highly conserved, suggesting conservation of function among vertebrates. Additionally, I demonstrate that expression of Cx43.4 is important for the proper morphogenesis of the notochord and patterning of adjacent tissue and further that its expression is a downstream consequence of Ntl activation.