肌间刺缺失突变对斑马鱼胚胎发育过程中肌肉发育的影响
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摘要
为阐明肌间刺缺失对斑马鱼(Danio rerio)胚胎发育时期肌肉发育的影响,采用qRT-PCR的方法,检测分析了斑马鱼不同肌间刺表型间5个肌肉特异性基因(mef2、myf5、myod、myog和sox6)在囊胚期(3hpf,hourspost fertilization)、原肠胚期(6 hpf)、体节期(12 hpf)、咽囊期(24 hpf)和孵化期(72 hpf)5个发育时期的表达情况,对比分析了肌间刺缺失对斑马鱼胚胎发育过程中肌肉发育特异性基因的时序表达差异和肌肉发育的影响,以及对受精率、孵化率和畸形率的影响。结果显示, 5个特异性基因在肌间刺缺失突变型与野生型斑马鱼各发育时期的表达量无显著性差异(P>0.05)。其中,myf5、myod基因在原肠胚期表达量升高,在体节期和咽囊期开始逐步下降;mef2、myog和sox6基因在囊胚期和原肠胚期表达量很低,在体节期和咽囊期表达量迅速升高,并在孵化期开始下降。此外,受精率、孵化率和畸形率的统计结果显示,肌间刺突变型与野生型间亦无显著性差异(P>0.05)。综上,推测肌间刺的缺失对斑马鱼胚胎发育及胚胎发育过程中肌肉的发育没有影响。
In order to explore the difference in embryonic muscle development between an intermuscular bone(Imb) deficiency mutant and wildtype zebrafish, we used a qRT-PCR method to analyze the expression of 5 muscle-specific genes, including mef2, myf5, myod, myog, and sox6, in the development stages blastula(3 hpf, hours post fertilization), gastrula(6 hpf), segmentation(12 hpf), pharyngula(24 hpf), and hatching period(72 hpf), and discussed the impact of Imb's deficiency on muscle development during embryonic development. Furthermore, the differences in fertilization rate, hatching, and abnormal rate of larvae were assessed. The results showed that the expression of genes myf5 and myod were elevated in the gastrula stage, and downregulated in the segmentation and pharyngula stages; the expression levels of mef2, myog, and sox6 in the blastula and gastrula stages were extremely low, and were upregulated significantly in the segmentation and pharyngula stages before being downregulated in the hatching period. The results also showed that there were no significant differences in 5 muscle-specific genes' expression between the wildtype zebrafish and its Imb deficiency mutant. Additionally, no significant difference in fertilization rate, hatching, and abnormal rate occurred between the two Imb phenotypes. In summary, the deletion of Imb do not impact muscle development or the survival of embryos during embryonic development of zebrafish.
In order to explore the difference in embryonic muscle development between an intermuscular bone(Imb) deficiency mutant and wildtype zebrafish, we used a qRT-PCR method to analyze the expression of 5 muscle-specific genes, including mef2, myf5, myod, myog, and sox6, in the development stages blastula(3 hpf, hours post fertilization), gastrula(6 hpf), segmentation(12 hpf), pharyngula(24 hpf), and hatching period(72 hpf), and discussed the impact of Imb's deficiency on muscle development during embryonic development. Furthermore, the differences in fertilization rate, hatching, and abnormal rate of larvae were assessed. The results showed that the expression of genes myf5 and myod were elevated in the gastrula stage, and downregulated in the segmentation and pharyngula stages; the expression levels of mef2, myog, and sox6 in the blastula and gastrula stages were extremely low, and were upregulated significantly in the segmentation and pharyngula stages before being downregulated in the hatching period. The results also showed that there were no significant differences in 5 muscle-specific genes' expression between the wildtype zebrafish and its Imb deficiency mutant. Additionally, no significant difference in fertilization rate, hatching, and abnormal rate occurred between the two Imb phenotypes. In summary, the deletion of Imb do not impact muscle development or the survival of embryos during embryonic development of zebrafish.
引文
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[26]Nie C H,Wan S M,Tomljanovic T,et al.Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development[J].BMC Genomics,2017,18:147.
[2]Dong Z J,Huang D Z,Li L J,et al.Preliminary study on intermuscular bones of several cultured cyprinids[J].Journal of Shanghai Fisheries University,2006,15(4):425-429.[董在杰,黄代中,李丽娟,等.几种常见鲤科养殖鱼类肌间刺的初步研究[J].上海水产大学学报,2006,15(4):425-429.]
[3]Bing Z.On the myoseptal spines of the carp(Cyprinus carpio L.)[J].Acta Zoologica Sinica,1962,14(2):175-178.[秉志.幼鲤大侧肌隔骨针的观察[J].动物学报,1962,14(2):175-178.]
[4]Gao W J.A study on the morphological and distribution of Os.intermuscular of Ctenopharyngodon idellus[J].Journal of Agricultural University of Hebei,1984,7(4):178-184.[高维竞.草鱼的肌间骨(Os.intermuscu Iar)形态及其分布的研究[J].河北农业大学学报,1984,7(4):178-184.]
[5]Ke Z H,Zhang W,Jiang Y,et al.Developmental morphology of the intermuscular bone in Hypophthalmichthys molitrix[J].Chinese Journal of Zoology,2008,43(6):88-96.[柯中和,张炜,蒋燕,等.鲢肌间小骨发育的形态学观察[J].动物学杂志,2008,43(6):88-96.]
[6]Ma L X,Dong Z J,Su S Y,et al.Research progress of fishes intermuscular bones[J].Jiangsu Agricultural Sciences,2012,40(4):234-235,240.[马良骁,董在杰,苏胜彦,等.鱼类肌间刺的研究进展[J].江苏农业科学,2012,40(4):234-235,240.]
[7]ChargéS B P,Rudnicki M A.Cellular and molecular regulation of muscle regeneration[J].Physiological Reviews,2004,84(1):209-238.
[8]Gros J,Scaal M,Marcelle C.A two-step mechanism for myotome formation in chick[J].Developmental Cell,2004,6(6):875-882.
[9]He X,Yan Y L,De Laurier A,et al.Observation of miRNAgene expression in zebrafish embryos by in situ hybridization to micro RNA primary transcripts[J].Zebrafish,2011,8(1):1-8.
[10]Choi J,Costa M L,Mermelstein C,et al.Myo D converts primary dermal fibroblasts,chondroblasts,smooth muscle,and retinal pigment epithelial cells into striated,mononucleated myoblasts and multinucleated myotubes[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(20):7988-7992.
[11]Weintraub H,Dwarki V J,Verma I,et al.Muscle-specific transcriptional activation by Myo D[J].Genes&Development,1991,5:1377-1386.
[12]Rudnicki M A,Schnegelsberg P N,Stead R H,et al.Myo Dor Myf5 is required for the formation of skeletal muscle[J].Cell,1993,75:1351-1359.
[13]Coutelle O,Blagden C S,Hampson R,et al.Hedgehog signaling is required for maintenance of myf5 and myo D expression and timely terminal differentiation in zebrafish adaxial myogenesis[J].Developmental Biology,2001,236(1):136-150.
[14]Weinberg E S,Allende M L,Kelly C S,et al.Developmental regulation of zebrafish Myo D in wild-type,no tail and spadetail embryos[J].Development,1996,122:271-280.
[15]Sun W.Cloning,expression and functional analysis of genes regulating muscle development from zebrafish[D].Qingdao:Institute of Oceanology,Chinese Academy of Sciences,2009:20.[孙威.斑马鱼肌肉发育相关基因的克隆、表达与功能分析[D].青岛:中国科学院海洋研究所,2009:20.]
[16]Molkentin J D,Black B L,Martin J F,et al.Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins[J].Cell,1995,83:1125-1136.
[17]Yun K,Wold B.Skeletal muscle determination and differentiation:story of a core regulatory network and its context[J].Current Opinions in Cell Biology,1996,8:877-889.
[18]Jackson H E,Ono Y,Wang X G,et al.The role of Sox6 in zebrafish muscle fiber type specification[J].Skeletal Muscle,2015,5(1):2.
[19]Charlrs B K,William W B,Seth R K,et al.Stages of embryonic development of the zebrafish[J].Developmental Dynamics,1995,203:253-310.
[20]Kenneth J L,Thomas D S.Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the2-(35)(35)Ct Method[J].Methods,2001,25:402-408.
[21]Kubota T,Zhang Q,Wrana J L,et al.Multiple forms of SppI(secreted phosphoprotein,osteopontin)synthesized by normal and transformed rat bone cell populations:Regulation by TGF-β[J].Biochemical and Biophysical Research Communications,1989,162(3):1453-1459.
[22]Tan X G.Muscle development in fish and evolution of myogenic factors[D].Qingdao:Institute of Oceanology,Chinese Academy of Sciences,2003:20.[谭训刚.鱼类肌肉发育及成肌因子的进化[D].青岛:中国科学院海洋研究所,2003:30]
[23]Yao W J,Lv Y P,Gong X L,et al.Different ossification patterns of intermuscular bones in fish with different swimming modes[J].Biology Open,2015,4(12):1727-1732.
[24]Lv Y P,Yao W J,Chen J,et al.Newly identified gene muscle segment homeobox C may play a role in intermuscular bone development of Hemibarbus labeo[J].Genetics and Molecular Research,2015,14:11324-11334.
[25]Wan S M,Yi S K,Zhong J,et al.Identification of micro RNA for intermuscular bone development in blunt snout bream(Megalobrama amblycephala)[J].International Journal of Molecular Sciences,2015,16:10686-10703.
[26]Nie C H,Wan S M,Tomljanovic T,et al.Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development[J].BMC Genomics,2017,18:147.