Abstract
Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.
Highlights
Grants ES10820, ES00210, and ES03850 from the NIEHS, National Science Foundation Grant 0641409, a Oregon Medical Research Foundation grant, and a pre-doctoral fellowship from the American Heart Association
Adult zebrafish caudal fin regeneration occurs by epimorphic regeneration, which involves reprogramming and differentiation of blastema cells to different cell types to restore the tissue to its original form (2, 4 – 6)
RNA isolated from adult zebrafish caudal fins and the adult heart regeneration list and we identified 189 common gene hearts [18, 19]
Summary
Zebrafish Lines and Care—For the larval fin regeneration studies, fertilized eggs were obtained from AB strain zebrafish (University of Oregon, Eugene, OR). For analysis of larval transcript abundance, 100 ng of total RNA from 0, 1, 2, and 3 dpa larval fin tissue were used to generate biotinylated complementary RNA (cRNA) using the Two-Cycle Target Labeling kit (Affymetrix, Santa Clara, CA). For the adult fin regeneration study, 2.5 g of total RNA was used to generate biotinylated cRNA for each treatment group using the One-Cycle Target Labeling kit (Affymetrix, Santa Clara, CA). From the double-stranded cDNA, biotinylated cRNA was synthesized using T7 RNA polymerase and a biotin-conjugated pseudouridine containing nucleotide mixture provided in the IVT Labeling Kit (Affymetrix, Santa Clara, CA) For both larval and adult fin regeneration experiments, 10 g of purified and fragmented cRNA from each experimental sample was hybridized to zebrafish genome arrays (Zebrafish430_2) according to the Affymetrix GeneChip Expression Analysis Technical Manual (701021 Rev. 5). The BrdUrd-labeled fluorescent cells were quantified with the acquired images using ImagePro Plus software program (Media Cybernetics, Inc., Silver Spring, MD)
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