Ye Weidong, Shi Mijuan, Cheng Yingyin, Liu Yuhang, Ren Keyi, Fang Yutong, Younas Waqar, Zhang Wanting, Wang Yaping, Xia Xiao-Qin
State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Hubei Hongshan Laboratory, Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture and Rural Affairs, The Innovation Academy of Seed Design, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; Department of Vascular Surgery, Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou 324000, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; The Joint Innovation Center for Engineering in Medicine, Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou 324000, China.
State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Hubei Hongshan Laboratory, Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture and Rural Affairs, The Innovation Academy of Seed Design, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
Int J Biol Macromol. 2025 May;308(Pt 1):142397. doi: 10.1016/j.ijbiomac.2025.142397. Epub 2025 Mar 22.
The evolutionary process of intermuscular bones (IBs) is complex, the molecular regulatory mechanisms of their development are not clear, and even the genes involved in the evolution and development of IBs are poorly understood. In this study, comparative genomic analysis of four fish species with IBs and eleven fish species without IBs identified 106 genes that are more conservatively evolved in fish species with IBs, but highly variable in fish species without IBs. These genes are mainly involved in swimming behavior and BMP signaling pathways. We performed single-cell transcriptome sequencing of IBs origin tissues in zebrafish before and after IBs formation and found that osteoblasts and mesenchymal stem cells (MSCs) increased significantly after IBs formation. RNA velocity analysis showed that osteoblasts in IBs differentiate from MSCs, and the differentiation trajectory of MSCs into osteoblasts was successfully constructed by pseudo-time analysis. Combined with the results of multi-omics analysis, seven candidate genes associated with IBs development were screened and knocked out in zebrafish. It was found that foxn3 mutation resulted in a delay in IB development, whereas bmp6 mutation resulted in a total loss of IB. By comparing the transcriptome of IBs tissues between bmp6 zebrafish and bmp6 zebrafish, we found that bmp6 deletion may inhibit the differentiation of MSCs into osteoblasts while promoting the formation of osteoclasts and ultimately inhibiting the formation of IBs. This study provides new insights into the molecular regulatory mechanisms and evolutionary processes of IB development.
肌间骨（IBs）的进化过程复杂，其发育的分子调控机制尚不清楚，甚至参与IBs进化和发育的基因也知之甚少。在本研究中，对四种有肌间骨的鱼类和十一种无肌间骨的鱼类进行比较基因组分析，鉴定出106个基因，这些基因在有肌间骨的鱼类中进化更为保守，但在无肌间骨的鱼类中高度可变。这些基因主要参与游泳行为和骨形态发生蛋白（BMP）信号通路。我们对斑马鱼肌间骨形成前后的肌间骨起源组织进行了单细胞转录组测序，发现肌间骨形成后成骨细胞和间充质干细胞（MSCs）显著增加。RNA速度分析表明，肌间骨中的成骨细胞由间充质干细胞分化而来，并通过伪时间分析成功构建了间充质干细胞向 成骨细胞的分化轨迹。结合多组学分析结果，筛选出七个与肌间骨发育相关的候选基因，并在斑马鱼中进行敲除。发现foxn3突变导致肌间骨发育延迟，而bmp6突变导致肌间骨完全缺失。通过比较bmp6斑马鱼和野生型斑马鱼肌间骨组织的转录组，我们发现bmp6缺失可能抑制间充质干细胞向成骨细胞的分化，同时促进破骨细胞的形成，最终抑制肌间骨的形成。本研究为肌间骨发育的分子调控机制和进化过程提供了新的见解。
