Determining How Defects in Connexin 43 Cause Skeletal Diseases

The zebrafish fin is composed of multiple bony fin rays. Each fin ray is comprised of multiple segments separated by joints. Regulatory mechanisms that control joint morphogenesis and ray segment length in zebrafish fins are not fully understood. We utilize the fin length mutants short fin (sof b123) and another long fin (alf dty86) to provide insight into these processes. The sof b123 mutant has short fins and short segments due to a mutation in the gap junction protein gene connexin43 (cx43). In contrast, the alf dty86 mutant has long fins, long segments, and over-expression of cx43. Thus, the two mutants exhibit two opposing phenotypes. For example, the sof b123 mutant exhibits reduced cx43 mRNA and the alf dty86 mutant exhibits increased cx43 mRNA. Cx43 knockdown in alf dty86 rescues the segment length phenotype suggesting that Cx43 activity regulates joint formation. These data suggest that Cx43 is involved in two independent pathways: promoting cell division and suppressing joint formation. This thesis dissertation is a collection of my entire graduate work mainly focused on Cx43-dependent events that coordinate cell proliferation and joint formation. Identification of genes acting downstream of cx43 revealed semaphorin3d (sema3d). Sema3d is a secreted ligand and is known to play various roles including axon guidance, vasculature patterning and cell proliferation. Here I found sema3d is functionally downstream of cx43 and mediates cx43-dependent pathways. Moreover, independent receptors of Sema3d were identified that may regulate cell proliferation and joint formation in zebrafish fin regeneration. I provide evidence that through Sema3d, Cx43 regulates an evx1-dependent joint pathway to suppress joint differentiation. Additional data show that to permit the joint pathway, cx43 mRNA is transiently reduced. Continued studies on joint morphology and gene expression in the sof and alf mutants as well as characterization of the Cx43 protein will provide evidence whether Cx43 indeed regulates joint development. These future findings will ultimately provide us with keen insights into roles of Cx43 that are reflected via a specific time line of gene expression.