Fu Yuanyuan, Liu Lei, Wang Chunlin, Zhu Fang, Liu Xiao
Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, Zhejiang, China; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, Zhejiang, China.
Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, Zhejiang, China; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, Zhejiang, China.
Comp Biochem Physiol B Biochem Mol Biol. 2019 Aug;234:41-49. doi: 10.1016/j.cbpb.2019.05.001. Epub 2019 May 8.
The functions of Wnt signalling in crab limb regeneration are poorly understood. Herein, we isolated and characterised the full-length cDNA of WNT4 from Portunus trituberculatus, designated PtWNT4. The 4831 bp cDNA encodes 323 amino acid polypeptide. Protein structure prediction showed that PtWNT4 has a conserved WNT domain. The PtWNT4 gene was expressed in all regenerative limb stages, and was upregulated from stage I, with highest expression in stage III, and expression then declined in stages IV and V. Immunohistochemistry revealed strong localisation at loose connective tissue during limb regeneration, but PtWNT4 protein levels decreased upon formation of muscle fibers. Injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels from 36 h to 5 days after injection, indicating successful gene silencing. RNA interference knockdown of PtWNT4 expression greatly retarded limb regeneration compared with the control group. Blastema emergence phenotype analysis revealed limb regeneration rates of 0, 31.5% and 40.5% for the dsWNT4 group at 36 h, 3 days and 5 days, respectively, compared with 29.95%, 83.0% and 92.5% for the saline-injected control groups (p <0.05). Expression analysis on the WNT4 using RNAi provide important information for understanding its functional mechanism during limb regeneration in P. trituberculatus. The results also contribute to our understanding of the role of Wnt signalling during limb regeneration in crustaceans.
Wnt信号通路在蟹肢体再生中的功能尚不清楚。在此，我们从三疣梭子蟹中分离并鉴定了WNT4的全长cDNA，命名为PtWNT4。该4831 bp的cDNA编码323个氨基酸的多肽。蛋白质结构预测表明PtWNT4具有保守的WNT结构域。PtWNT4基因在肢体再生的所有阶段均有表达，从第一阶段开始上调，在第三阶段表达最高，然后在第四和第五阶段表达下降。免疫组织化学显示在肢体再生过程中，PtWNT4在疏松结缔组织中有强烈定位，但在肌纤维形成后PtWNT4蛋白水平下降。向再生肢体注射WNT4双链RNA后，从注射后36小时到5天，PtWNT4 mRNA水平显著降低，表明基因沉默成功。与对照组相比，RNA干扰敲低PtWNT4表达极大地阻碍了肢体再生。芽基出现表型分析显示，dsWNT4组在36小时、3天和5天的肢体再生率分别为0、31.5%和40.5%，而注射生理盐水的对照组分别为29.95%、83.0%和92.5%（p<0.05）。利用RNAi对WNT4进行表达分析，为了解其在三疣梭子蟹肢体再生过程中的功能机制提供了重要信息。这些结果也有助于我们理解Wnt信号通路在甲壳类动物肢体再生中的作用。
