An Investigation of the Role of Actinotrichia During Zebrafish Caudal Fin Regeneration

Abstract

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.