Synovial joints are critical for skeletal form and function, but are susceptible to debilitating diseases such as osteoarthritis and to injury. While much is known regarding the composition and mechanics of functioning joints, the molecular mechanisms defining how joint-forming cells are specified in a particular location remain largely unknown. We use the zebrafish regenerating fin to address fundamental questions regarding skeletal development, including the specification and commitment of fin ray joints. Indeed, there are important similarities between fin ray joints and synovial joints during the initial stages of joint formation, suggesting that findings in the fin ray joints will be applicable to synovial joins. For example, the synovial joint is initiated in uninterrupted cartilaginous elements by the formation of the interzone. The interzone is a discrete band of chondrocytes that condense at the site of the future joint and guides later stages of joint formation. The fin ray joint is similrly initiated in a region of uninterrupted matrix by a group of cells that condense at the location of the future joint. These cells represent the fin ray joint interzone, and similarly appear to guide joint morphogenesis. How the cells of the interzone decide to initiate joint formation, in any presumptive joint, remains a gap in our knowledge. Evaluation of two fin length mutants, short fin (sof b123) and another long fin (alf dty86), suggest that the gap junction protein Connexin43 (Cx43) determines where a fin ray joint will be produced. For example, the sof b123 mutant exhibits short fin ray segments caused by premature joint formation. In contrast, alf dty86 exhibits long segments on average due to stochastic joint failure. Notably, cx43- knockdown rescues joint formation in alf dty86. These findings suggest that cx43 suppresses joint formation. The underlying hypothesis of this proposal is that transient abrogation of Cx43 function is required to permit the commitment of joint-forming cells, and thereby permit joint formation. The two aims of this proposal are to provide tangible evidence in support of this hypothesis.
In Aim 1 we propose to demonstrate that preventing the abrogation of Cx43 causes joint failure. This will be accomplished by generating a transgenic line that overexpresses Cx43 in a heat-inducible manner, and to prevent Cx43 turnover using pharmacological treatments.
In Aim 2 we propose to reveal the molecular requirements for the commitment of joint-forming cells. This will be accomplished by manipulating the expression of genes required in either joint- forming cells (i.e. evx1) or osteoblast-forming cells (i.e. osx) to reveal the functional requirements for joint- forming cell commitment. Similarly to Aim 1, it will be determined if abrogation of Cx43 is required for the commitment of joint-forming cells. In both Aims, we will further test the hypothesis that gap junctional intercellular communication is required for Cx43 function during both joint commitment and joint formation. Together, completion of these aims will reveal key mechanistic details critical for our understanding of the initial events of joint formation and patterning in the vertebrate skeleton.
Synovial joints are subject to injuries and diseases such as osteoarthritis and rheumatoid arthritis. Despite the intense research focus on synovial joints, relatively little is known about their early development, including how specific regions of the skeleton are specified and committed to making a joint. My research in the zebrafish regenerating fin skeleton has provided new insights into how joints may be specified. The goal of this proposal is to elucidate early signaling events required for the commitment of joint-forming cells in the vertebrate skeleton.
| Bhattacharya, Shashwati; Gargiulo, Domenic; Iovine, M Kathryn (2018) Simplet-dependent regulation of ?-catenin signaling influences skeletal patterning downstream of Cx43. Development 145: |
| Dardis, Gabrielle; Tryon, Robert; Ton, Quynh et al. (2017) Cx43 suppresses evx1 expression to regulate joint initiation in the regenerating fin. Dev Dyn 246:691-699 |
