Zebrafish (Danio rerio) are ray-finned fish of the teleost class, whose fins consist of an exoskeletal domain and an endoskeletal domain. The exoskeletal domain of the fins contains the fin rays and originate from embryonic fin folds that elongate as the fins are growing. The elongation of the fin fold is supported by two parallel sets of rigid fibrils oriented along the proximal-distal axis, called actinotrichia. Actinotrichia fibrils are composed of two primary components, a collagenous component and actinodin proteins. The actinodin proteins are encoded for by the actinodin (and) family of genes which are found in the genomes of finned fish while absent in limbed tetrapods. CRISPR/Cas9 was used to create loss-of-function deletions in the and1 and and2 genes, resulting in the absence of actinotrichia in the zebrafish double mutants. We hypothesised that the loss of actinotrichia during zebrafish development would result in developmental defects leading to fin ray defects in the adult zebrafish. The and1/2 mutants that lack actinotrichia presented with fin fold and cell migration defects during development that persisted into adulthood and resulted in shorter fins, disturbed fin ray patterning, and a decrease in ray number. In addition, an unexpected fusion between the hypurals of the caudal fin endoskeleton revealed an additional function of the actinotrichia fibrils in caudal fin endoskeletal patterning. During zebrafish development, actinotrichia fibrils play a vital role in ensuring normal fin development, normal patterning and formation of the fin rays, and the normal development of the caudal fin endoskeleton.

The exoskeleton of the fins comprises fin rays and actinotrichia; the latter are small unmineralized fibrils found at the distal margin of fin rays. Actinotrichia play a role in the growth and structure of the fins during fin development and regeneration. Our lab has previously identified the actinodin (and) gene family, which codes for structural proteins in actinotrichia. Interestingly, the loss of this gene family has been proposed to be involved in the loss of fin rays, an important step in the fin-to-limb transition during evolution. Furthermore, the and genes are expressed in the epithelial cells and in the migrating mesenchymal cells of the zebrafish embryonic pectoral and median fin fold. The presence of tissue-specific cis-acting regulatory elements were found within the 2 kilobase pair genomic region (2P) located upstream of and1’s first untranslated exon by performing analyses of the expression of a fluorescent reporter (EGFP) placed under the control of fragments of various lengths originating from the 2P genomic fragment in zebrafish transgenic lines. Using these various and1 reporter lines, tissue-specific and1 expression was previously characterized during the embryonic stage of zebrafish development. However, these transgenic reporter lines were not analyzed throughout important fin morphogenesis events occurring during fin development, such as the initial formation of lepidotrichia and the resorption of the median fin fold, and throughout fin regeneration as well. This study mainly enabled us to characterize in great details and1 expression throughout fin development and regeneration using the various tissue-specific and1 reporter lines by performing time course analyses. In doing so, we were able to demonstrate that these reporter lines recapitulate endogenous and1 expression through in iii situ hybridization and RT-PCR experiments. Furthermore, the distinct transgene expression patterns observed during lepidotrichia formation/regeneration in the various and1 reporter lines supports previous research that proposes and1-expressing cells may indirectly contribute to lepidotrichia formation not only during fin regeneration but during fin development as well. Furthermore, the characterization of the tissue-specific and1 reporter lines throughout development allowed us to characterize specific changes in the cis-acting regulation of and1 in the fins of adult fish when compared with the tissue-specific and1 reporter expression patterns characterized during the embryonic stage. All in all, this study provides further clues on the contribution of and1-expressing cells throughout fin development and regeneration.

The evolution of the tetrapod limb from paired fish fins involved drastic changes to the appendicular dermal and endochondral skeleton. Fish fin rays were lost, and the endochondral bone was modified and elaborated to form three distinct segments common to all tetrapod limbs: the stylopod, the zeugopod, and the autopod. Identifying the molecular mechanisms that contributed to these morphological changes presents a unique insight into our own evolutionary history. Chapter II of this thesis focuses on the actinodin gene family and how their disappearance from the tetrapod genome during the fin-to-limb transition may have contributed to the loss of dermal fin rays. The actinodin genes code for structural proteins in the actinotrichia, rigid fibers being the first exoskeletal elements formed during zebrafish fin development. We have identified tisse-specific cis-acting regulatory elements responsible for actinodin1 activation in the fin fold ectoderm and mesenchyme. These elements are only partially functional in transgenic reporter mouse limbs. We therefore propose that changes to actinodin gene regulation contributed to the loss of the actinodin genes during limb evolution. The actinotrichia also serve as a scaffold for the migration of cells from the distal fin mesenchyme, which has been shown to differentiate into fin ray osteoblasts. In fact, both actinotrichia and distal fin mesenchyme migration defects have been proposed as events that may lead to the loss of dermal bone during the fin-to-limb transition. Chapter III of this thesis tests the effects of distal fin mesenchyme ablation on larval and adult zebrafish fin development. Following the chemo/genetic ablation of these cells, zebrafish display actinotrichia, fin fold, and fin ray defects supporting the hypothesis the defects in distal fin mesenchyme may have contributed to the loss of dermal fin rays during tetrapod evolution. Previous research has shown that changes in the regulation of the 5’HoxA/D genes may have had consequences for both actinodin regulation and the migration of distal fin fold mesenchyme. Chapter IV of this thesis examines the contributions of Hoxa11 regulatory changes to the evolution of the pentadactyl, or five-digit state, in tetrapods. Through a novel tetrapod-specific enhancer, Hoxa11 is repressed from the presumptive limb autopod region in mice. In fish, hoxa11b is expressed distally and ectopic expression of Hoxa11 in the distal limb bud produces mice with polydactyly (extra digits), an ancestral tetrapod character state. In conclusion, we have provided evidence that actinotrichia defects (potentially though changes in actinodin regulation) and fin fold mesenchyme defects may have contributed to the loss of fin dermal bone during the fin-to-limb transition. Our data also shows these two events may have been linked as fin fold mesenchyme require actinotrichia to migrate correctly, while actinotrichia maintenance relies on Actinodin secretion from fin fold mesenchyme. Furthermore, we have also contributed to the growing body of evidence that proposes changes in 5’HoxA/D regulation during the fin-to-limb transition underlie changes in appendicular dermal and endochondral bone. Therefore, it is possible that modifications in shared gene regulatory networks underlie both dermal and endochondral bone evolution during the fin-to-limb transition.