Zebrafish, danio rerio, are bony fish in the Teleost class. Zebrafish can regenerate most organs, including their fins. The caudal fin of zebrafish adults has bundles of rigid, uncalcified fibrils, termed actinotrichia, at the distal end of each lepidotrichia. During embryonic and early larval stages of fin development, actinotrichia are synthesized between the fin fold epidermis. Actinotrichia remain present in the fins of adult fish and during fin regeneration. They provide structural support and facilitate mesenchymal cell migration during fin development and regeneration. The importance of the role of actinotrichia for fin formation and regeneration can be studied through loss of function analysis of structural components of the actinotrichia. Our lab identified two genes, actinodin1 and actinodin2 (and1, and2), that are coding for structural proteins of the actinotrichia. CRISPR/Cas9 mediated knockout of and1 and and2 lead to loss of actinotrichia in the embryonic fins. Immunohistochemistry with anti-And1 and anti-ColII antibodies, in addition to controlled tissue degradation to observe actinotrichia fibers revealed the absence of actinotrichia in the caudal fins of adult and1/2 double homozygous mutants. We hypothesized that loss of actinotrichia will lead to the disruption of mesenchymal cell migration during development and regeneration, resulting in fin growth and morphology defects. The intact adult caudal fin of and1/2 double mutants have fewer rays (lepidotrichia) and these lepidotrichia are shorter and wavy when compared to WT. Along the lepidotrichia of actinodin mutants, there is an uneven distribution of joints revealing that there are defects in ray patterning. We carried out a regeneration time course analysis to compare caudal fin regeneration in WT and and1/2 double homozygous mutants. The and1/2 double homozygous mutants have a delay in bone re-formation and defects in joint patterning are observed in regenerating rays. From these results, and1 and and2 are shown to be required for actinotrichia formation. In addition, morphological analyses identified that actinotrichia is required for proper caudal fin growth and patterning during development and regeneration.

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.

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.