TRANSGENIC MODULATION OF GAP JUNCTION INTERACTIONS

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Various studies suggest that the connexin 43 (Cx43) gap junction gene plays in important role in conotruncal heart development. This likely involves modulating the development of cardiac neural crest (NC) cells. This is indicated by the finding that the conotruncal heart defects and neonatal lethality of the Cx43 knockout (KO) can be rescued [partially, at least] by restoring Cx43 expression to subpopulations of NC cells via a CMV43 transgene. Furthermore, in transgenic mice expressing Cx43 constructs that up or down regulate gap junctional communication in NC cells, conotruncal heart defects also arise. In light of these findings, the proposed research seeks to understand the role of Cx43 in neural crest development. Dr. Lo will use these existing transgenic mouse lines (and others to be made) to carry out studies focused on three main objectives. First, determine how changes in Cx43 function affect the emergence and migratory behavior of cardiac NC cells. Second, determine whether changes in Cx43 function may perturb NC differentiation. Third, determine whether defects in NC cells alone account for the Cx43 KO lethality. For these studies, a combination of in vitro (Aims 1-3) and in vivo (Aims 4,5) approaches will be utilized. Aim 1 is to characterize the timing of the onset of neural crest migration, and determine whether cell-cell adhesion and cell signaling pathways important in regulating the onset of NC migration may be altered. Aim 2 is to quantitate the rate and directionality of neural crest migration, and characterize the locomotory responses of NC cells to different matrix environments and various chemotropic agents. Aim 3 is to characterize NC differentiation by quantitating the expression of differentiation markers for smooth muscle cells, cartilage, and melanocytes. Aim 4 is to confirm the in vitro results by examining NC migration in vivo using a green fluorescent protein tag driven by a 6.5 kb Cx43 promoter sequence known to specify transgene expression in neural crest cell lineages (Lo et al. 1997). Am 5 is to determine whether long term survival of the Cx43 KO mouse may be achieved when Cx43 expression is restored to all neural crest lineages via a Cx43 expression vector driven by the Cx43 promoter.