Hippo Signaling Regulates Blastema Formation During Limb Regeneration in Chinese Mitten Crab (Eriocheir sinensis).

Limb autotomy and regeneration are specific adaptations of crustaceans in response to external stress and attacks, which make them a suitable model to investigate the mechanism of organ regeneration in invertebrates. In this study, the Hippo gene of Eriocheir sinensis (EsHPO) was identified, and the effects of Hippo signaling on limb regeneration were evaluated. The expression of EsHPO and other key components of Hippo signaling was down-regulated during the basal growth phase in response to limb autotomy stress and then up-regulated during the proecdysial growth phase. The descending expression patterns of Hippo signal components were correlated with transcriptional activation of YKI and downstream target genes during the blastema formation stage, which suggested that Hippo signaling plays a key role during limb regeneration in E. sinensis. To further test the hypothesis, the transcription factor YKI was blocked via verteporfin injection after autotomy, which disrupted limb regeneration by repressing wound healing and preventing blastema emergence. Furthermore, our experiments revealed that the proliferation of blastema cells was blocked by verteporfin. In addition, the expression of genes related to ECM remodeling, cell cycle progression, and apoptosis resistance was down-regulated following the injection of verteporfin. Our findings therefore indicate that Hippo signaling is essential for successful wound healing and limb regeneration in E. sinensis by inducing ECM remodeling, as well as promoting the proliferation and repressing the apoptosis of blastema cells.