Peng Mou-Yun, Wen Hui-Ju, Shih Li-Jane, Kuo Ching-Ming, Hwang Sheng-Ping L
Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, ROC.
Mol Reprod Dev. 2002 Dec;63(4):422-9. doi: 10.1002/mrd.10201.
To investigate whether different myosin heavy chain (MHC) isoforms may constitute myofibrils in the trunk and tail musculature and if their respective expression may be regulated by spadetail (spt) and no tail (brachyury), we identified and characterized mRNA expression patterns of an embryonic- and tail muscle-specific MHC gene (named myhz2) during zebrafish development in wild type, spt, and ntl mutant embryos. The identified myhz2 MHC gene encodes a polypeptide containing 1,935 amino acids. Deduced amino acid comparisons showed that myhz2 MHC shared 92.6% sequence identity with that of carp fast skeletal MHC. Temporal and spatial myhz2 MHC mRNA expression patterns were analyzed by quantitative RT-PCR and whole-mount in situ hybridization using primer pairs and probes designed from the 3'-untranslated region (UTR). Temporally myhz2 MHC mRNA appears in pharyngula embryos and peaks in protruding-mouth larvae. The expression level decreased in 7-day-old hatching larvae, and mRNA expression was not detectable in adult fish. Spatially in pharyngula embryos, mRNA was localized only in the tail somite region, while in long-pec embryos, transcripts were also expressed in the two cranial muscle elements of the adductor mandibulae and medial rectus, as well as in pectoral fin muscles and the tail muscle region. Myhz2 MHC mRNA was expressed in most cranial muscle elements, pectoral fin muscles, and the tail muscle region of 3-day-old hatching larvae. In contrast, no expression of myhz2 MHC mRNA could be observed in spt prim-15 mutant embryos. In spt long-pec mutant embryos, transcripts were expressed in two cranial muscle elements and the tail muscle region, but not in pectoral fin muscles, while only trace amounts of myhz2 MHC mRNA were expressed in the remaining tail muscle region of 38 hpf and long-pec ntl mutant embryos.
为了研究不同的肌球蛋白重链（MHC）亚型是否可能构成躯干和尾部肌肉组织中的肌原纤维，以及它们各自的表达是否可能受spadetail（spt）和无尾（短尾）调控，我们在野生型、spt和ntl突变胚胎的斑马鱼发育过程中，鉴定并表征了一个胚胎和尾部肌肉特异性MHC基因（命名为myhz2）的mRNA表达模式。鉴定出的myhz2 MHC基因编码一个含有1935个氨基酸的多肽。推导的氨基酸比较表明，myhz2 MHC与鲤鱼快速骨骼肌MHC的序列同一性为92.6%。使用从3'-非翻译区（UTR）设计的引物对和探针，通过定量RT-PCR和整体原位杂交分析了myhz2 MHC mRNA的时空表达模式。在时间上，myhz2 MHC mRNA出现在咽胚中，并在突嘴幼虫中达到峰值。在7日龄孵化幼虫中表达水平下降，在成鱼中未检测到mRNA表达。在空间上，在咽胚中，mRNA仅定位于尾部体节区域，而在长胸鳍胚胎中，转录本也在内收下颌肌和内直肌的两个头部肌肉元件以及胸鳍肌肉和尾部肌肉区域中表达。Myhz2 MHC mRNA在3日龄孵化幼虫的大多数头部肌肉元件、胸鳍肌肉和尾部肌肉区域中表达。相比之下，在spt prim-15突变胚胎中未观察到myhz2 MHC mRNA的表达。在spt长胸鳍突变胚胎中，转录本在两个头部肌肉元件和尾部肌肉区域中表达，但在胸鳍肌肉中不表达，而在38 hpf和长胸鳍ntl突变胚胎的其余尾部肌肉区域中仅表达微量的myhz2 MHC mRNA。
