Cilp基因对斑马鱼肌间骨和脊椎发育的影响

Effect of cilp on the development of the intermuscular bones and vertebra in zebrafish

为阐明cilp基因在斑马鱼椎骨和肌间骨发育中的作用,利用CRISPR/Cas9建立斑马鱼cilp基因敲除纯合突变系(cilp^(-/-)),对其进行骨骼表型的观察,并进一步采用qRT-PCR分析14个骨骼发育相关基因在突变体胚胎发育阶段和成鱼骨骼中的表达水平变化。结果显示:与野生型斑马鱼相比,90 dph(Days post hatching)cilp^(-/-)斑马鱼的肌间骨数量显著减少了10.27%,而肌间骨的长度无明显变化;同时突变体斑马鱼中椎骨发生异常融合及髓棘缺失。qRT-PCR结果显示:与野生型相比,col1a1a、sp7、smad4a和smad5基因在胚胎发育的整个时期都存在显著的差异,在突变体成鱼尾部骨骼组织中bmp2a、bmp2b、smad5、sp7、runx2a、runx2b和bglap基因的表达量均显著低于野生型,表明cilp基因敲除导致了BMPs和SMADs家族基因的表达水平下降,并下调了下游的成骨细胞发育相关基因的表达量,推测cilp功能缺失可能通过抑制BMPs信号通路影响成骨细胞分化和骨形成,从而导致了肌间骨数量的减少和脊椎骨异常融合。

To investigate the molecular mechanism of cilp in the development of vertebrae and intermuscular bones in zebrafish,we established a zebrafish cilp^(-/-)homozygous mutant strain by CRISPR/Cas9.The bones staining of wild type zebrafish and cilp^(-/-)strain showed that the loss function of cilp caused abnormal fusion of vertebrae and deficiency of neural spine in mutants,and the number of intermuscular bones in cilp^(-/-)strain was significantly reduced by 10.27%,while the length of intermuscular bones did not change significantly.Furthermore,we analyzed the expression levels of 14 bone development-related genes in five embryonic development stages and 90 dph(days post hatching)adult fish skeletal tissue.The results showed that the col1a1a,sp7,smad4a,and smad5 genes were significantly differentially expression throughout the embryonic development stages compared to the wild type,the expression levels of bmp2a,bmp2b,smad5,sp7,runx2a,runx2b,and bglap in the skeleton of the 90 dph mutant adult fish were significantly lower than those of the wild type zebrafish,which indicated that the cilp gene knockout down-regulated the expression levels of genes in BMPs and SMADs families which consequentially repressed the expression of genes related to osteoblast development.In summary,dysfunctions of the cilp gene might affect the development of intermuscular bones and skeleton in zebrafish by suppressing the BMPs signaling pathway to affect the development of osteoblasts and bones formation.