Mild cryoinjury in zebrafish fin induces regenerative response without blastema formation

Previous studies have shown that tissue regeneration induces expression of genes that play important roles in regeneration. Recently, several studies have identified regeneration-response enhancers (RREs) that activate gene expression by tissue injury. Particularly, we showed that RREs contain two transcription factor-binding motifs: a bHLH transcription factor-binding motif, an E-box, and an AP-1/bZIP transcription factor-binding motif, a 12-O-Tetradecanoylphorbol 13-acetate response element (TRE). However, the triggers and subsequent signals generated by injury are still unclear. In this study, we analyzed RRE activation using various injury models. Although inter-ray incisions and skin exfoliation injuries did not activate RREs or regeneration genes, the fin puncture injury activated RREs and several regeneration-response genes. After fin puncture injury, msxc was activated only on the proximal side of the hole where blastema-like tissue was formed, whereas RREs, junbb, and fibronectin 1b (fn1b) were activated on both the proximal and distal sides, implying that activation of RREs, junbb, and fn1b is independent of blastema formation. Here, we also established a mild cryoinjury method. After this injury, transient vascular destruction, an increase in cell death, and an accumulation of myeloid cells were observed; however, no major morphological damage was observed. Importantly, msxc was not induced by cryoinjury, whereas fn1b, junbb, and 1.8 k RRE (-1.8 kb promoter of fn1b) were activated, suggesting that cryoinjury induces the responses of fn1b, junbb, and 1.8 k RRE without forming the blastema. Thus, our study shows that the cryoinjury model and the RRE transgenic (Tg) zebrafish may provide a useful platform for exploring injury signals.