斑马鱼ngs基因突变对骨骼发育的影响

Effects of ngs Mutation on Skeletal Development in Zebrafish

脊索颗粒表面(notochord granular surface,ngs)基因是维持脊椎动物脊索正常发育和稳定的必需基因,然而却在镜鲤(Cyprinus carpio)肌间刺和椎骨组织中显著表达,暗示其可能与肌间刺和椎骨的发育有关。为探索ngs基因对肌间刺和主轴骨骼发育的影响,本研究以斑马鱼(Danio rerio)为实验对象,首先采用RNA荧光原位杂交(RNA fluorescence in situ hybridization,RNA-FISH)证实了ngs基因与成骨细胞标志性基因sp7在肌间刺和椎骨组织中共表达。其次利用CRISPR/Cas9构建了ngs基因突变体,胚胎发育观察显示ngs基因突变导致斑马鱼脊索发育异常,72 hpf(hours post fertilization,受精后小时数)的ngs^(-/-)胚胎体长显著低于野生型胚胎的体长(P<2.22×10^(-16));90 dph(days post hatching,孵化后天数)的突变体骨骼染色表明ngs^(-/-)椎骨异常融合,肌间刺数量显著少于野生型的(P<0.05)。进一步采用RT-qPCR分析了12个骨骼发育相关基因在5个胚胎时期(12 hpf体节期、24 hpf咽囊期、36 hpf原基-25期、48 hpf孵化期和72 hpf早幼期)和90 dph成鱼组织(骨骼和肌肉)中的表达。结果表明,突变体中BMP和SMAD家族相关基因和成骨细胞标志基因的表达量在胚胎发育后期显著下调(P<0.05),但12个基因在突变体和野生型成鱼骨骼组织中的表达量无明显差异(P>0.05)。在90 dph成鱼肌肉组织中,除bmp2b、smad5和runx2b在ngs^(-/-)中的表达量显著低于野生型的(P<0.05)外,其他基因的表达量无显著差异。综上,ngs基因突变导致了脊索发育异常,进而致使椎骨发育畸形,生长发育减缓,同时造成了肌间刺数量的减少;基因表达分析结果表明,ngs基因突变可能在胚胎期通过影响BMP信号通路相关基因的表达使胚胎成骨作用减弱,椎骨发育异常,而在胚后发育阶段可能通过影响肌肉中bmp2b、smad5和runx2b基因的表达从而影响肌间刺的数量。

The notochord granular surface(ngs)is essential to maintain the normal development and stability of notochord in vertebrates,however,the ngs gene was significantly expressed in the vertebra and intermuscular bones of mirror carp(Cyprinus carpio),which suggested that it might play a role in the development of intermuscular bones and vertebra in mirror carp.To explore the effect of ngs on the development of intermuscular bones and skeleton,in this study,firstly,RNA fluorescence in situ hybridization(RNA-FISH)was used to confirm that the ngs gene was co-expressed with the osteoblast signature gene sp7 in intermuscular bones.Secondly,a ngs mutant strain in zebrafish(Danio rerion)was constructed by CRISPR/Cas9 genome editing technology,and was used to compare the embryonic development with wild-type zebrafish.The results showed that the mutation of ngs caused abnormal development of the notochord in ngs^(-/-),and the body length of mutants at 72 hpf(hours post fertilized)was significantly lower than that of the wild-type zebrafish(P<2.22×10^(-16)).Skeletal staining showed that abnormal fusion was observed in the vertebrae of ngs^(-/-)mutants,moreover,the number of intermuscular bones in the ngs^(-/-)strain was significantly less than that in the wild-type zebrafish(P<0.05).Furthermore,the expressed levels of 12 skeletal development-related genes in embryos at five developmental stages(12 hpf Segmentation period,24 hpf Pharyngula,36 hpf Pharyngula period Prim-25,48 hpf Hatching and 72 hpf Early larva)and in skeleton and muscle of 90 dph adult fish were analyzed by RT-qPCR.The results showed that the expression of BMP and SMAD families and osteoblast signature genes were significantly down regulated in the ngs^(-/-)during late embryonic developmen(P<0.05),whereas the expressed levels of 12 genes in skeletal tissues of 90 dph wild-type and mutant adult fish were not significant(P>0.05).In the muscle tissue of 90 dph adult fish,the expressed levels of bmp2 b,smad5 and runx2 b in ngs^(-/-)were significantly lower than those in the wild-type zebrafish(P<0.05).In conclusion,ngs deficiency caused abnormal development of the embryonic notochord,which in turn resulted in malformed vertebrae development and slowed growth,as well as a decrease in the number of intermuscular bones;the results of gene expression analyses indicated that ngs deficiency might weaken the bone formation and cause abnormal vertebrae development by suppressing the expression of genes in BMP pathway during the embryonic stage,while the number of intermuscular bone might be affected by the expression of bmp2 b,smad5 and runx2 b genes in muscle during the postembryonic developmental stage.