Zebrafish fins are composed of multiple bony rays each of which is comprised of multiple bony segments separated by joints. As in all vertebrates these joints must be formed at precise positions to ensure the flexibility of the skeletal system. During regeneration (as well as during growth), there is a sequential addition of new bony segments and joints in a proximal to distal direction. Therefore, the youngest tissue is located most distally. Not much is known about the genes involved in joint morphogenesis in this context. Now a paper from the laboratory of M. Kathryn Iovine (http://www.ncbi.nlm.nih.gov/pubmed/24278401) has characterized a pathway involved in joint regeneration during zebrafish fin regeneration.
Previous reports have suggested that the transcription factor even-skipped 1 (evx1), an eve-related homeobox gene, is required for joint formation during regeneration. Genes that are expressed during joint formation are expressed in discrete groups of cells located within the lateral population of skeletal precursor cells. Based on the expression pattern the authors found that three genes dlx5a (distal-less homeobox-5a), mmp9 (matrix metalloproteinase 9) and col10a1b showed a similar expression than evx1 in this lateral mesenchymal compartment of the ray. In order to characterize the relationship between these three genes and evx1, they first checked how their expression was affected in evx1 mutants. Whereas the expression of col10a10b was not affected in evx1 mutants, dlx5 and mmp9 were significantly reduced, although not completely abolished. These results suggested that dlx5 and mmp9 were expressed downstream of evx1. Next, the authors characterized the function of dlx5 and mmp9 on fin regeneration. Morpholino-mediated knockdowns of these genes resulted in increased ray segment length suggesting that dlx5 and mmp9 are necessary for correct joint placement.
During fin ray regeneration genes required at early stages of differentiation are expressed in more distal regions, whereas proximal regions include late differentiation genes. Evx1 was expressed in the most distal domain of the skeletal precursor cells, consistent with this gene acting earlier. On the other side, dlx5a was expressed more proximally than evx1 and mmp9 was expressed even more proximally than evx1 and dlx5a. These results suggested a linear pathway initiated by evx1 followed by dlx5a and then followed by mmp9. This was further supported by the observation that mmp9 expression was reduced in dlx5a knockdowns. In contrast dlx5a expression was not affected after mmp9 knockdown. Similarly evx1 expression was not affected in dlx5a or mmp9 knockdowns.
This same laboratory had previously reported that the activity of Cx43 (a gap junction protein) suppresses joint formation. Cx43 is expressed in the medial compartment adjacent to the lateral population of skeletal precursor cells. Mutants for this gene are characterized by short segments because of premature joints. Accordingly, and as predicted, the expression of evx1, dlx5 and mmp9 was initiated more distally (meaning sooner) in regenerating fins of Cx34 mutants. This premature expression of these genes would account for the short segments observed in Cx34 mutants.
In summary, the authors propose a model in which Cx43 would inhibit evx1 which is itself required to sequentially activate dlx5a and mmp9 for proper joint formation.