Wang Sixiang, Fu Yuanyuan, Zheng Xia, Zhou Jie, Liu Lei, Wang Chunlin
School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, China.
Ningbo Institute of Oceanography, Ningbo, Zhejiang, China.
Int J Biol Macromol. 2025 Aug;319(Pt 1):145219. doi: 10.1016/j.ijbiomac.2025.145219. Epub 2025 Jun 16.
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.
在许多甲壳类动物中都观察到了再生这一生物学现象，尤其是在常见的物种中，如虾和蟹。然而，甲壳类动物再生的分子机制仍知之甚少。三疣梭子蟹是一种具有重要经济意义且具有显著再生能力的物种。本研究从形态学和组织学角度报道了三疣梭子蟹肢体再生的过程。同时，我们基于先前的转录组数据鉴定出Wnt基因家族的一个成员PtWnt7b，它在动物发育和细胞增殖中起着关键作用。从三疣梭子蟹中克隆了PtWnt7b的全长cDNA，其包含一个1053 bp的开放阅读框，编码一个具有保守Wnt1结构域的350个氨基酸的蛋白质。与其他组织相比，PtWnt7b在再生组织中的表达显著升高，在整个肢体再生过程中呈上升趋势。原位杂交和免疫荧光分析显示PtWnt7b及其蛋白在再生肢体中广泛表达，主要集中在上皮细胞中。此外，靶向PtWnt7b的RNA干扰导致再生效率显著降低，表现为再生肢芽生长延迟、蜕皮后再生肢体畸形以及恢复率降低。此外，通过荧光素酶报告基因检测，我们证实PtWnt7b的表达激活了Wnt/β-连环蛋白信号通路。这些发现表明，PtWnt7b在三疣梭子蟹肢体再生过程中促进细胞增殖和分化方面起着关键作用，而这对于再生肢体的正常形成至关重要。
