Delta/notch signaling regulates blastema formation during limb regeneration in Eriocheir sinensis

Limb autotomy and regeneration represent specialized adaptive responses employed by crustaceans to cope with threats from other animals or environmental stress. However, the molecular regulatory mechanisms underlying these adaptations remain poorly understood. In this study, we established a limb autotomy and regeneration model in Eriocheir sinensis, characterized in detail the morphological and structural features of regenerated limbs post-amputation, analyzed expression patterns of key components of the Notch signaling pathway genes, and evaluated their functional roles during limb regeneration utilizing long-term RNA interference (RNAi) technology. Specifically, the regenerative blastema appeared approximately 14 days post- amputation, with both the receptor EsNotch and the ligand EsDelta expressed across distinct stages of limb regeneration and exhibiting distinct expression patterns. Notably, both genes were significantly up-regulated during the blastema growth stage. Subsequently, we observed that long-term knockdown of either EsNotch or EsDelta significantly suppressed the formation of blastema during limb regeneration and increased crab mortality. Furthermore, we found that their silencing triggered the down-regulation of genes involved in cell proliferation, differentiation, and other regeneration-related processes. Overall, our findings underscore the critical functional role of Notch signaling in crab limb regeneration via modulation of regeneration-related gene expression, significantly advancing understanding of molecular mechanisms underlying crustacean limb regeneration and paving the way for future regenerative biology research.