Differential actinodin1 regulation in embryonic development and adult fin regeneration in Danio rerio.

Actinotrichia are the first exoskeletal elements formed during zebrafish fin development. These rigid fibrils serve as skeletal support for the fin fold and as substrates for mesenchymal cell migration. In the adult intact fins, actinotrichia are restricted to the distal domain of the fin. Following fin amputation, actinotrichia also reform during regeneration. The actinodin gene family codes for structural proteins of actinotrichia. We have previously identified cis-acting regulatory elements in a 2kb genomic region upstream of the first exon of actinodin1, termed 2P, required for tissue-specific expression in the fin fold ectoderm and mesenchyme during embryonic development. Indeed, 2P contains an ectodermal enhancer in a 150bp region named epi. Deletion of epi from 2P results in loss of ectodermal-specific activity. In the present study, we sought to further characterize the activity of these regulatory sequences throughout fin development and during adult fin regeneration. Using a reporter transgenic approach, we show that a site within the epi region, termed epi3, contains an early mesenchymal-specific repressor. We also show that the larval fin fold ectodermal enhancer within epi3 remains functional in the basal epithelial layer during fin regeneration. We show that the first non-coding exon and first intron of actinodin1 contains a transcriptional enhancer and an alternative promoter that are necessary for the persistence of reporter expression reminiscent of actinodin1 expression during adulthood. Altogether, we have identified cis-acting regulatory elements that are required for tissue-specific expression as well as full recapitulation of actinodin1 expression during adulthood. Furthermore, the characterization of these elements provides us with useful molecular tools for the enhancement of transgene expression in adulthood.

A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.