Skeletal morphogenesis requires the integration of multiple signals for coordinated growth and patterning. For example, cell proliferation must be coordinated with the differentiation of osteoblasts and joint-forming cells. However, the mechanisms responsible for coordinating multiple cellular events are largely unknown. Our analyses using the zebrafish regenerating fin as a model system have identified two fin length mutants that provide insight into the coordination of fin growth and skeletal patterning. The short fin (sof b123) mutant has short fins due to the development of short fin ray segments (i.e. or premature joint formation). In contrast, the another long fin (alf dty86) mutant exhibits fin overgrowth and overlong segments due to stochastic joint failure. Interestingly, sof b123and alf dty86 also exhibit important and opposing differences in the expression of the gap junction gene connexin43 (cx43). The sof b123phenotypes are caused by hypomorphic mutations in cx43, while the joint failure phenotype in alf dty86 is the result of cx43 over-expression. Indeed, cx43 knockdown in alf dty86regenerating fins rescues joint formation. Together, these and other data suggest that Cx43 function both promotes fin growth and suppresses joint formation. Moreover, mutations in human CX43 cause oculodentodigital dysplasia, a syndrome characterized by morphological defects of the craniofacial and distal limb skeletons. Thus, Cx43 exhibits conserved, but unknown, functions in skeletal morphogenesis. In order to understand molecularly how Cx43 mediates its effects on growth and patterning of the fin skeleton, we identified genes expressed downstream of cx43. Interestingly, the gene for a secreted semaphorin, sema3d, is expressed in the regenerating fin in a cx43-dependent manner. Strikingly, knockdown of sema3d leads to reduced fin length, reduced cell proliferation, reduced segment length, and rescue of joint formation in alf dty86, similar to the cx43-knockdown phenotypes. Together, our findings suggest that sema3d functions in a common pathway with cx43 to regulate fin growth and joint formation. Semaphorins are members of a large evolutionarily conserved class of signaling molecules, best understood as providing guidance cues for axons and blood vessels. More recent studies revealed that semaphorins are expressed broadly during development and initiate a wide diversity of cellular outcomes. Loss of semaphorin function leads to human diseases including cancer progression, neurodegenerative diseases, multiple sclerosis, and rheumatoid arthritis. Thus, semaphorins contribute broadly to normal and pathological development.
The aims of this proposal are to identify the cx43-dependent cellular outcomes affected by manipulating Sema3d signaling and to identify the components of the signaling pathway downstream of Sema3d. Results obtained from this proposal will reveal the underlying mechanism of Cx43-Sema3d action during fin regeneration, and will serve as preliminary data for a more comprehensive proposal on defining the functional relationship between Cx43 and Sema3d, and the molecular pathway(s) required for the coordination of growth and patterning of the vertebrate skeleton.
Mutations in the CX43 gene cause defects in skeletal morphology that lead to human disease. Our analyses reveal that zebrafish Cx43 coordinates bone growth and cellular differentiation, and that the signaling molecule Sema3d mediates Cx43-dependent functions. The underlying goal of this proposal is to reveal the Sema3d-dependent cellular outcomes contributing to normal bone growth and patterning, providing tangible insights into the molecular mechanisms regulating size and shape of the vertebrate skeleton.
|Barton, Rachael; Palacio, Danica; Iovine, M Kathryn et al. (2015) A cytosolic juxtamembrane interface modulates plexin A3 oligomerization and signal transduction. PLoS One 10:e0116368|
|Ton, Quynh V; Kathryn Iovine, M (2012) Semaphorin3d mediates Cx43-dependent phenotypes during fin regeneration. Dev Biol 366:195-203|