Nihei Yoshiaki, Kobiyama Atsushi, Ikeda Daisuke, Ono Yosuke, Ohara Satoshi, Cole Nicholas J, Johnston Ian A, Watabe Shugo
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
J Exp Biol. 2006 Jan;209(Pt 1):188-98. doi: 10.1242/jeb.01978.
Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYHemb1, MYHemb2 and MYHemb3. MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10 degrees C (MYHS10) and 30 degrees C (MYHS30). Phylogenetic analysis demonstrated that MYHemb1 and MYHemb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYHemb3 was similar to the adult slow muscle isoforms, MYHS10 and MYHS30. MYHemb1 and MYHemb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYHemb1 continued to be expressed at low levels in 7-month-old juveniles when MYHemb2 was not detectable. MYHemb3 transcripts appeared at almost the same stage as MYHemb1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYHemb1 and MYHemb2 were expressed in the inner part of myotome, whereas MYHemb3 was expressed in the superficial compartment. MYHS10 and MYHS30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYHS10 mRNA was expressed at an acclimation temperature of 10 and 20 degrees C, but not at 30 degrees C. In contrast, MYHS30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10 degrees C-acclimated adult fish (MYHcard). MYHcard mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.
从鲤鱼（Cyprinus carpio L.）中克隆出了三种胚胎II类肌球蛋白重链（MYHs），即MYHemb1、MYHemb2和MYHemb3。还从适应10摄氏度（MYHS10）和30摄氏度（MYHS30）的成年鲤鱼慢肌中分离出了MYH DNA克隆。系统发育分析表明，MYHemb1和MYHemb2属于快速骨骼肌MYH进化枝。相比之下，MYHemb3的序列与成年慢肌同工型MYHS10和MYHS30相似。通过Northern印迹分析，在受精后61小时（h.p.f.）心跳阶段的胚胎中首次检测到MYHemb1和MYHemb2转录本，在1月龄幼鱼中表达达到峰值。当MYHemb2无法检测到时，MYHemb1在7月龄幼鱼中仍以低水平持续表达。MYHemb3转录本几乎与MYHemb1转录本在同一阶段（61 h.p.f.）出现，并且这些基因显示出相似的表达模式。整体原位杂交分析表明MYHemb1和MYHemb2的转录本在肌节内部表达，而MYHemb3在表层区域表达。MYHS10和MYHS30 mRNA在孵化时首次被检测到。在成年阶段，慢肌MYH mRNA的表达取决于适应温度。MYHS10 mRNA在10和20摄氏度的适应温度下表达，但在30摄氏度时不表达。相比之下，MYHS30 mRNA在所有适应温度下都强烈表达。在成年慢肌和孵化时的胚胎中发现的主要MYH转录本，在某些适应温度下在成年快肌中表达，但在其他温度下不表达。从适应10摄氏度的成年鱼心肌中分离出一个MYH DNA克隆（MYHcard）。MYHcard mRNA在61 h.p.f.时首次被检测到，但仅在成年心肌中观察到强信号。因此，本研究揭示了MYH基因在发育阶段、肌肉类型和适应温度方面的复杂表达模式。到目前为止，在晚期幼鱼阶段尚未发现已鉴定的骨骼肌MYHs中有强烈表达的，这表明MYH II基因家族中仍有待发现更多受发育调控的成员。
