Spatiotemporal Expression and Functional Analysis of PtWnt7b During Limb Regeneration in Portunus trituberculatus

The biological phenomenon of regeneration is observed in many crustaceans, particularly in commonly encountered species, such as shrimp and crabs. However, the molecular mechanisms underlying crustacean regeneration remain poorly understood. The swimming crab, Portunus trituberculatus, is an economically significant species that possesses a remarkable regenerative capacity. This study reports the process of limb regeneration in P. trituberculatus from both morphological and histological perspectives. Concurrently, we identified a member of the Wnt gene family, PtWnt7b, based on prior transcriptomic data, which is known for its crucial role in animal development and cell proliferation. The full-length cDNA of PtWnt7b was cloned from P. trituberculatus, which contains a 1053 bp open reading frame, encoding a 350 amino acid protein with a conserved Wnt1 domain. PtWnt7b expression was significantly elevated in regenerating tissues compared with that in other tissues, showing an increasing trend throughout the limb regeneration process. In situ hybridization and immunofluorescence analyses revealed the widespread expression of PtWnt7b and its protein in regenerating limbs, with a predominant focus in epithelial cells. Furthermore, RNA interference targeting PtWnt7b resulted in a marked inhibition of regeneration efficiency, characterized by delayed growth of regenerating limb buds, malformed regenerated limbs after molting, and reduced recovery rates. Additionally, using a luciferase reporter assay, we confirmed that PtWnt7b expression activates the Wnt/β-catenin signaling pathway. These findings indicated that PtWnt7b plays a critical role in promoting cell proliferation and differentiation during limb regeneration in P. trituberculatus, which are essential for the proper establishment of the regenerated limb.