Zebrafish are capable of fully regenerating organs and tissue such as their caudal fin, which is similar to a human regrowing an arm or a leg. In contrast, most mammals including humans have a greatly reduced capacity for wound healing. The ability of zebrafish to undergo this regenerative process, called epimorphic regeneration, hinges on the capacity to form a blastema at the wound site. The blastema quickly recapitulates the developmental processes involved in complex tissue formation to restore lost or damaged tissue. One key mechanism for inducing blastema formation is global repression of genes involved in tissue differentiation and maintenance.
Induction of repressive factors, such as microRNAs (miRNAs), are involved in reprogramming cells during epimorphic regeneration. The upstream mechanism by which zebrafish undergo epimorphic regeneration remains elusive. Furthermore, while focus is shifting toward regulatory RNAs such as miRNAs, the full complement of their repressive activities is unknown. We took a transcriptomics approach to investigating epimorphic regeneration and fin development. Parallel sequencing of total RNA and small RNA samples was performed on regenerating fin tissue at 1 day post-amputation (dpa). Most miRNAs had increased expression, consistent with global repression of genes involved in cell specialization during de-differentiation. We identified predicted interactions between miRNAs and genes involved in transcriptional regulation, chromatin modification, and developmental signaling. miR-146a and miR-146b are anti-inflammatory miRNAs that were predicted to target eya4, which is involved in chromatin remodeling and innate immunity. miR-132-3p and miR-21 were predicted to cooperate in repression Bone morphogenic protein (BMP) signaling antagonists. Depletion of miR-489 and miR-92b-3p were predicted to be involved in expression of cell cycle regulators mycn and foxm1, as well as other transcription factors associated with regeneration. We also identified Hedgehog and Scatter factor signaling as possibly targets of miRNAs regulation during regeneration.
Next, we performed microarray analysis on tissue exposed to the glucocorticoid beclomethasone diproprionate (BDP). We identified cripto-1 as a highly upregulated gene flowing BDP exposure. Morpholino oligo knockdown of cripto-1 permitted regeneration during BDP exposure. We were able to phenocopy the effects of BDP exposure by exposing zebrafish to an Activin signaling inhibitor, SB431542. Promoter analysis identified binding sites for genes that were differentially expressed in our data. BDP exposure of murine embryonic stem cells confirmed that glucocorticoid-mediated induction of cripto-1 is conserved in mammals.
Finally, we investigated a unique caudal fin duplication, called x-fin, resulting from embryonic exposure an exogenous ligand of the aryl hydrocarbon receptor (AHR), benzo[k]fluoranthene (BkF). We determined that the phenotype is Ahr2-dependent. We also determined that it is independent of hepatic metabolism using a hepatocyte ablation model. RNA seq analysis of trunk tissue at four time points of revealed that BMP and Fibroblast growth factor (FGF) ligands were robustly induced. Enrichment analysis revealed similarities to fin regeneration processes as well as inflammation. Altogether, we leveraged transcriptomics as a useful tool for identifying mechanisms associated with tissue regeneration and development.