Regulatory Mechanisms Underlying Regeneration of the Adult Zebrafish Exoskeleton
Creators: name: Phan, Hue-Eileen
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2018
Subjects: fin regeneration
joint regeneration

description: The fin exoskeleton of the zebrafish is comprised of lepidotrichia (or fin rays) and actinotrichia. In uncut fins, the fin rays span the entire length of the fin and the actinotrichia are found at the distal tips of each of the fin rays. Both of these fin exoskeletal components are capable of regenerating following amputation or injury. The regulation of the regeneration of these exoskeletal components is the central topic of this thesis and is explored in two different projects. The first project focuses on zebrafish fin ray regeneration during which bone segments are periodically added at the distal tips of each fin ray, each segment being separated by a joint. Joint formation involves the expression of a unique set of genes: hoxa13a, evx1, and pthlha. The alternation between bone segment formation and joint formation during fin ray regeneration seems to closely correlate with positional outgrowth during regeneration. We investigated whether or not the calcineurin and retinoic acid (RA) signalling pathways, both of which may be potential regulators of positional outgrowth, are involved in regulating joint formation. FK506-induced calcineurin inhibition and RA treatments each resulted in the suppression of joint marker expression. In RA-treated fins, bone deposition occurs in the joints as a result of joint cells being induced to differentiate into osteoblasts. These results suggest that the calcineurin and RA pathways may provide the positional information that regulates joint and bone segment formation. The second project focuses on the regulation of actinotrichia formation during adult fin regeneration. Throughout the early to intermediate stages of fin ray regeneration, actinotrichia fibers are found deep to the regenerating hemirays. As regeneration progresses, these actinotrichia fibers become gradually restricted to the distal domain of the fin regenerate. Actinotrichia contain structural proteins known as actinodin. There are four actinodin genes in zebrafish, actinodin1-4. We studied the comparative activity of the cis-acting regulatory elements of actinodin1 during fin regeneration. We have previously identified tissue-specific cis-acting regulatory elements in a 2kb genomic region upstream of the first exon, termed 2P, that drive reporter expression in the fin fold ectoderm and mesenchyme during embryonic development. Within 2P is a 150bp region, named epi, which contains an ectodermal/epithelial enhancer. Using in silico analysis, we have identified four main clusters of transcription factor binding sites within epi, termed epi1-4. Using a reporter transgenic approach, we have identified epi3 as a site containing an early mesenchymal-specific repressor and an epithelial-specific enhancer. We have also shown that the first exon and intron of actinodin1 contains a general transcriptional enhancer in adulthood and an alternative promoter. Overall, these results suggest that there is a difference between the regulation of actinodin1 during embryonic development and that of adult fin regeneration.