Dynamics of actinotrichia regeneration in the adult zebrafish fin

Abstract

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFβ/Activin-βA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

Regeneration of caudal fin inPoecilia latipinna: Insights into the progressive tissue morphogenesis

“…The differentiation of the proliferating blastemal cells into lepidotrichia and actinotrichia can be observed for the first time in the tail tissues of 5 dpa stained with alcian bluealizarin red. Similar observations were made by Konig et al 20 for adult zebrafish fin regeneration at 5 dpa.…”

Zebrafish fin regeneration involves transient serotonin synthesis

“…Immunofluorescence stainings were performed as previously described. 22 Briefly, the slides were blocked for 1 hour in blocking solution (5% goat serum (Jackson Immunoresearch, West Grove, Pennsylvania, United-States) in 0.3% Triton-X in PBS (PBST)), incubated overnight in primary antibodies diluted in blocking solution, washed on the next day in PBST, incubated in secondary antibody for 2 hours, washed and mounted in 90% glycerol in 20 mM Tris pH 8 with 0.5% N-propyl gallate (Sigma-Aldrich, Saint-Louis, Missouri, United-States). For BrdU visualization, the sections were incubated for 40 minutes in a solution of 2 N HCl before immunofluorescence staining.…”

“…In situ hybridization on sections was performed as previously described. 22 Primers for the generation of the template for the probes were the following:…”

Inhibition of mmp13a during zebrafish fin regeneration disrupts fin growth, osteoblasts differentiation, and Laminin organization

“…Since mmp13a is essentially expressed in the blastema, we speculate that mmp13a knockdown is primarily affecting the blastema and defects in actinotrichia may be a secondary effect of impaired blastema. Indeed, at least three of the four actinodin genes are strongly expressed in the blastema 2,43 and we show that mmp13a knockdown strongly reduces and1 expression which, as a consequence, disrupts actinotrichia formation and compromises Collagen II incorporation into actinotrichia. It is also important to note that the Collagen II antibody is detecting collagen present in actinotrichia only; it does not detect collagen elsewhere (eg, within cell cytoplasm).…”

“…42 How this turnover is regulated remains unclear although involvements of osteoblasts, growth rate, and signaling network have been proposed. 43 It is possible that Mmp13 may be involved in the turnover of actinotrichia as Collagen II is a known substrate of Mmp13. 29 However it is unexpected that reduced Mmp13a level resulted in diminished Collagen II and actinotrichia, as one would expect an excess of Collagen II in the fin in such case.…”