mTOR promotes zebrafish larvae fin regeneration through regulating macrophages activation and CaM-mTOR-dnm1l axis

To investigate the functional and mechanistic roles of mTOR in zebrafish larvae fin regeneration, we firstly examined the spatiotemporal expression of mTOR in larvae fin and established a mTOR knockout (mTOR-KO) transgenic fish line using CRISPER / Cas9 gene editing technology. Moreover, mTOR was essential for the activation of macrophages, which is a key factor in maintaining the regenerative repair process. We also demonstrated that mTOR knockdown attenuated the proliferative capacity of bud embryo cell during the regenerative phase, while cell apoptosis was not affected. RNA-sequence analysis showed changes in mitochondrial function and dnm1l was identified as the main regulatory factor during the fin regeneration stage. We further suggested that mTOR may promote mitochondrial fission to support bud embryo cell regeneration via CaM-mTOR-dnm1l axis. Sequencing samples for the micro-transcriptome were obtained from tail fin tissues regenerated for 24 h. Fifty regenerated tail fins were extracted for each sample from the control and experimental groups treated with 1 uM Rapamycin. Each group had 3 biological replicates and samples were lysed using Trizol. Sequencing technology was provided by UNIKAWA Biotechnology Co.Ltd (Hangzhou, China).

为探究哺乳动物雷帕霉素靶蛋白（mTOR）在斑马鱼幼鱼鳍再生中的功能与作用机制，本研究首先检测了mTOR在幼鱼鳍中的时空表达模式，并利用CRISPR/Cas9基因编辑技术构建了mTOR敲除（mTOR-KO）转基因鱼株。此外，mTOR对巨噬细胞的激活至关重要，而巨噬细胞是维持再生修复进程的关键调控因素。本研究还证实，在再生阶段，mTOR敲低会减弱胚芽细胞的增殖能力，但对细胞凋亡无显著影响。RNA测序分析显示线粒体功能发生改变，且dnm1l被鉴定为鳍再生阶段的主要调控因子。本研究进一步提出，mTOR可能通过CaM-mTOR-dnm1l信号轴促进线粒体分裂，以支持胚芽细胞的再生。微转录组测序的样本取自再生培养24小时的尾鳍组织。对照组与经1μM雷帕霉素处理的实验组，每个样本均提取自50条再生尾鳍。每组设置3次生物学重复，样本采用Trizol法进行裂解。测序服务由中国杭州优卡沃生物技术有限公司（UNIKAWA Biotechnology Co.Ltd）提供。