Hyaluronic acid synthesis is required for zebrafish tail fin regeneration

Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. has3 expression is induced in the wound epithelium within hours after tail amputation, and its onset and maintenance requires fibroblast growth factor, phosphoinositide 3-kinase, and transforming growth factor-β signaling. Inhibition of hyaluronic acid (HA) synthesis by the small molecule 4-methylumbelliferone (4-MU) impaired tail regeneration in zebrafish larvae by preventing injury-induced cell proliferation. In addition, 4-MU reduced the expression of genes associated with wound epithelium and blastema function. Treatment with the glycogen synthase 3-kinase inhibitors rescued 4-MU-induced defects in cell proliferation and tail regeneration, even though only a subset of wound epithelium and blastema markers was restored. Our findings uncover a role for HA biosynthesis in zebrafish tail regeneration and its epistatic relationships with other regenerative processes. Zebrafish embryos were raised until 2 days post-fertilization (dpf) and their caudal tail fins were amputated just posterior to the notochord. Uncut larvae and larvae amputated at 2 dpf were then raised for an additional day (1 day post-amputation, dpa) and posterior tissues were again amputated but then collected on ice. Total RNA was extracted from the posterior tissues and genomic DNA was removed. Approximately 1 ug of total RNA was isolated from 150 tails per each condition and two rounds of amplification was performed using the MessageAmp II aRNA Kit. cDNA synthesis and labeling, hybridization to the NimbleGen Zebrafish Gene Expression 385K array, microarray scanning, and data compilation were performed at the Stanford Functional Genomics Center, according to guidelines established by Roche NimbleGen. Subsequent analysis was performed using DNASTAR ArrayStar software.