Examination of the role of Cx43 gap junctional communication in murine embryogenesis using a dominant negative disruption strategy

Gap Junctions are membrane channels that allow direct intracellular transfer of ions and small molecules and thus mediate cell-cell signaling and metabolic cooperation. We are interested in the role(s) of gap junctions in murine embryogenesis. In order to examine this we focused our studies on the gap junction gene connexin 43 (Cx43). The expression pattern of Cx43 suggests that it may mediate key processes during mouse development. As a first step in this analysis, we cloned and characterized the mouse Cx43 gene. The murine Cx43 gene consists of two small exons separated by a 10.5 kb intron. This gene structure is conserved among all the connexin genes described, suggesting that regulatory functions may be subserved by this genomic structure. In order to engineer perturbations in Cx43, we sought to develop a mutant form of the protein that could disrupt endogenous Cx43 function. To this end, we constructed a Cx43/$\beta$-galactosidase fusion protein using the coding region of the Cx43 gene. Because gap junction channels are made of hexameric arrays of connexin polypeptides, we hypothesized that such a fusion protein would retain the ability to associate with endogenous Cx43, but would disrupt the trafficking, assembly, or permeability of Cx43 encoded gap junctions. We examined gap junctional communication in tissue culture cells expressing this fusion protein and found that it was inhibited. Our analysis suggest that this may be due to disruption of Cx43 trafficking to the cell surface. We went on to generate transgenic animals expressing the Cx43/$\beta$-gal fusion protein. We observed various developmental perturbations in these mice. The defects included cardiac and cranial neural tube abnormalities, phenotypes that are strikingly similar to those described in the Cx43 null mutant mice. Our observations suggest that these developmental deficiencies may arise from the perturbation of neural crest cells. Thus, our data raise the possibility that Cx43 gap junctions may play an important role in neural crest cell development. These studies further show that the Cx43/$\beta$-gal fusion protein is a valuable approach for targeting Cx43 perturbations in transgenic mice