Tent5c基因对斑马鱼肌间刺发育的影响

TENT5C ON THE DEVELOPMENT OF INTERMUSCULAR BONES IN ZEBRAFISH

为探究tent5c/fam46c基因(Family with sequence similarity 46c,家族序列相似性46 c)在斑马鱼(Danio rerio)肌间刺发育中的作用,选取11种鱼类分析tent5c系统演化特征,采用原位杂交技术和qRT-PCR分析tent5c基因在斑马鱼发育中的时空表达特征,并利用CRISPR/Cas9建立了斑马鱼tent5c基因敲除纯合突变系(tent5c^(-/-)),对其骨骼表型和肌肉转录组进行分析。结果显示,tent5c编码TENT5C氨基酸在不同鱼类中具有高度的保守性,但在无肌间刺鱼类里发生了相同的氨基酸替换;tent5c基因在48hpf(Hours post fertilization)斑马鱼的眼睛和肌隔表达,在60dpf(Days post fertilization)斑马鱼的脊椎和肌间刺特异高表达。与野生型斑马鱼相比,tent5c^(-/-)斑马鱼肌间刺表现为生长减缓,矿化程度降低以及数量减少。比较转录组结果表明,在tent5c基因敲除之后,斑马鱼肌肉组织有112个基因表达下调,118个基因表达上调。差异基因主要富集于肌肉收缩和骨骼发育等相关通路,由此推测tent5c可能是通过调控这些肌肉和骨骼相关通路来对肌间刺发育产生影响。研究初步表明了tent5c基因在斑马鱼肌间刺发育中的作用,为进一步研究鱼类肌间刺等骨骼发育的分子调控机制提供了科学依据。

In order to investigate the role of the tent5c/fam46c gene in the development of intermuclar bones in zebrafish(Danio rerio),we analyzed the evolutionary characteristics of the tent5c gene across 11 fish species.Spatiotemporal expression patterns of tent5c gene during the zebrafish development were analyzed using in situ hybridization(ISH)and qRT-PCR.Homozygous tent5c^(-/-)mutant lines were established via CRISPR/Cas9 to observe skeletal phenotype and analyze gene expression changes.Analysis of the amino sequence revealed high conservation of the tent5c gene among 11 different fish species,all sharing a common structural domain.The ISH results showed that tent5c exhibited RNA level expression in the eyes and vertebrae of zebrafish at 48hpf(hours post fertilization),with particularly high expression in intermusclar bones and vertebrae at 60dpf(days post fertilization).Bone staining of tent5c^(+/+)wild type zebrafish and tent5c^(-/-)strain showed that loss of the tent5c gene resulted in a reduced growth of intermuscular bones,decreased mineralization,and a slight decline in numbers.Transcriptome analysis revealed that tent5c knockout down-regulated 112 genes and up-regulated 118 genes.KEGG enrichment analysis indicated enrichment of differently expressed genes(DEGs)mainly in motor proteins,ECM-receptor interaction,VEGF signaling pathway,and p53 signaling pathway.These findings indicated that tent5c may influence the development of intermuscular bones through pathways related to muscle and bone.Overall,this study provides preliminary evidence of the role of the tent5c gene in zebrafish intermuscular bone development,laying a scientific foundation for further exploration of the molecular regulatory mechanisms underlying fish intermusclar bone and skeletal development.