Understanding how Connexin43 regulates joint formation in the regenerating zebrafish fin

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Summary Synovial joints are critical for skeletal form and function, but are susceptible to debilitating diseases such as osteoarthritis and injury. While much is known about the composition and mechanics of functioning joints, relatively little is known about how joint-forming cells (JFCs) are specified in a particular location, thereby permitting the development of joints that provide flexibility. Addressing this gap in knowledge could facilitate the development of novel therapies for joint disease by targeting molecular pathways influencing cell fate decisions. The zebrafish regenerating fin is an important model system for addressing fundamental questions of skeletal development, including the specification and commitment of JFCs. The fin skeleton is made of bony fin rays, and each fin ray is made of bony segments separated by joints. Thus, the fin is a rich source of joints, which are produced regularly during typical outgrowth and during regeneration. The osteoblasts and JFCs that build the fin skeleton are derived from a common skeletal precursor cell (SPC) located in the lateral fin ray mesenchyme. Recent studies from the Iovine lab provide strong evidence that Connexin43 (Cx43), via gap junctional intercellular communication (GJIC), influences JFC specification by suppressing expression of evx1. Evx1 is a transcription factor required for joint formation. The overall objectives for this proposal are to reveal the nature of Cx43-GJIC, and to reveal the subsequent mechanism of evx1 suppression. The central hypothesis of this proposal is that Cx43-GJIC influences joint formation by transducing changes in membrane potential to the SPCs; changes in membrane potential in turn trigger changes in gene expression that influence evx1 expression, JFC specification, and joint formation. This hypothesis was formulated on the basis of prior and preliminary data. For example, Cx43-GJIC promotes simplet (smp) expression in SPCs. Smp is required to bring -catenin to the nucleus. In concert with Lef1 or TCF7 transcription factors, -catenin (directly or indirectly) suppresses evx1 transcription. The Aims of this proposal are designed to test the central hypothesis. Aim 1 is to test if changes in membrane resting potential are sufficient to influence gene expression in SPCs. This will be accomplished by driving membrane hyperpolarization or depolarization in Cx43-expressing cells, and evaluating smp and evx1 expression in SPCs. Aim 2 is to identify cis-regulatory elements (CREs) that influence evx1 transcription. Putative CREs will be deleted from a BAC reporter for evx1, and impacts on reporter expression will be assessed to reveal likely enhancers or silencers acting on evx1. Completion of these Aims will significantly advance our understanding of signals mediated by GJIC influencing cell specification, and will further provide novel insights into the spatiotemporal regulation of JFC specification.