Understanding Connexin43 dependent Skeletal Morphogenesis - Progress made and Promises ahead

Skeletal morphogenesis is a complex process through which bones grow to their correct size and shape and find their location in the body. A number of signaling pathways are involved in proper regulation of this process. Among them, Gap Junctional Intercellular Communication (GJIC) is important for proper bone growth and patterning. In our lab, we use regenerating caudal fin of a zebrafish to study skeletal morphogenesis. Our examination of a fin length mutant sofb123 has identified a gap junction protein called Connexin43 (Cx43). Cx43 regulates growth and patterning during regeneration by promoting cell proliferation and suppressing joint formation, respectively. The function of Cx43 is conserved in vertebrates. In humans, mutations in Cx43 cause a malformation of craniofacial and limb skeleton, known as Occulodentodigital dysplasia (ODDD). In mice and chicks as well, similar defects in bone development are observed following mutations in the gene Cx43. What we do not understand, however, is how does communication through a gap junctional channel give rise to such tangible phenotypes? To answer this question, we need to identify how cx43 regulates other genes during regeneration in zebrafish. We compared two fin length mutants with opposite phenotypes through a microarray. The mutant sofb123, which shows reduced fin length, reduced segment length and reduced level of cell proliferation, demonstrates a lower level of cx43 mRNA and protein. The other mutant alfdty86, on the other hand, shows an elevated level of cx43 expression and demonstrates increased fin length, longer segments due to stochastic joint placement and increased level of cell proliferation. Identification of genes that are upregulated in alfdty86 and downregulated in sofb123 are hypothesized to be part of the Cx43 dependent growth and patterning pathway during regeneration.One of the genes identified and validated through this microarray is a gene called serpinh1b, which codes for a protein called Hsp47. The first part of my thesis demonstrates that serpinh1b is functionally downstream of cx43. Knockdown of Hsp47, recapitulates the sof phenotype. Furthermore, knockdown of Hsp47 results in disruption of collagen II dependent actinotrichia leading to defective growth and patterning events during regeneration.Another gene previously validated from the microarray is semaphorin3d (sema3d). Sema3d mediates the downstream effect of Cx43 through interactions with its two putative receptors, Neuropilin2a (Nrp2a) and PlexinA3 (PlxnA3). The second part of my thesis characterizes the significance of Sema3d binding to its receptors. Our data suggest that Sema3d binding reduces homomeric interaction between Nrp2a receptors, and that PlxnA3 can act as a competitor for Nrp2a homo-oligomerization. We also hypothesize that the juxtamembrane MAM domain of Nrp2a receptor can act as an alternate Sema3d binding domain, besides its usual role as a facilitator of receptor oligomerization. The third part of my thesis characterizes the AB9 fibroblast cell line, derived from regenerating caudal fin of an adult zebrafish as a complementary tool for pilot studies on gene regulation. Our data suggest that AB9 cells express major players of the Cx43 dependent growth and patterning pathway. We believe that this cell line can be a used as a simpler tool to do gene manipulation studies, as well as to complement our findings in vivo.In conclusion, I believe that my thesis encompasses three different aspects of studying skeletal patterning events during regeneration. It characterizes one more member of the Cx43 dependent growth and patterning pathway during regeneration and identifies its significance in formation of an important fin structure, the actinotrichia. The findings suggest that actinotrichia co-ordinates between the dividing cells of the blastema and the differentiating cells in the lateral patterning compartment. My thesis also validates the functional significance of ligand-receptor interactions and suggests possibilities on how it might affect cell division and differentiation events. My thesis also introduces a new cell culture tool to be used in the future to perform pilot studies on genes involved in skeletal regeneration in zebrafish.