Holland L Z, Pace D A, Blink M L, Kene M, Holland N D
Scripps Institution of Oceanography, University of California San Diego, La Jolla 92093-0202, USA.
Dev Biol. 1995 Oct;171(2):665-76. doi: 10.1006/dbio.1995.1313.
The lower chordate amphioxus, widely considered the closest living invertebrate relative of the vertebrates, is a key organism for understanding the relationship between gene duplications and evolution of the complex vertebrate body plan. In tetrapod vertebrates, the alkali myosin light chain genes (MLC-alk), which code for proteins associated with the globular head of the myosin heavy chain, constitute a large family with stage-, tissue-, and fiber-type-specific expression of different isoforms thought to have arisen by duplication of a single ancestral gene. In protostome invertebrates, e.g., arthropods, molluscs, and nematodes, only one MLC-alk gene has been found, but the number of such genes in deuterostome invertebrates and lower vertebrates is unknown. The present report, describing the sequence and expression throughout development of the amphioxus gene for alkali myosin light chain (AmphiMLC-alk), thus fills a major gap in understanding the relation between gene duplication and increasing diversity of muscle-cell types. A full-length clone (1 kb) of AmphiMLC-alk was isolated from a larval amphioxus cDNA library. It coded for a 149-amino-acid protein most closely related to the vertebrate embryonic form of MLC-alk. Southern blot analysis revealed only one copy of AmphiMLC-alk and suggested that it is the only MLC-alk gene in amphioxus. Northern blot analysis indicated that this gene produces only one transcript, which is expressed at all stages of development and in adults. In situ hybridizations showed expression initially in the myotomes of somites 2-5 of neurula embryos and soon thereafter in the myotomes of somite 1 and of newly forming somites progressively added posteriorly. Myotomal expression continues throughout larval development and into the adult stage as the myotomal cells differentiate into striated, mononucleate muscle cells--unlike vertebrate striated muscle cells, those of amphioxus never become multinucleate. In late larvae and adults myotomal expression of AmphiMLC-alk is localized along the medial edge of the myotome and at the ends of the cells. This is the first demonstration of intracellular localization of MLC transcripts in muscle cells of any animal. Expression of AmphiMLC-alk was also detected in smooth muscles as well as in striated muscles not derived from the myotome. These expression data are consistent with the Southern blot analysis in suggesting that there is only one MLC-alk gene in amphioxus. Thus, duplication of an ancestral vertebrate MLC-alk gene probably occurred after the vertebrate and amphioxus lineages split.(ABSTRACT TRUNCATED AT 400 WORDS)
文昌鱼作为一种低等脊索动物，被广泛认为是现存与脊椎动物亲缘关系最近的无脊椎动物，是理解基因复制与复杂脊椎动物身体结构进化之间关系的关键生物。在四足脊椎动物中，碱性肌球蛋白轻链基因（MLC-alk）编码与肌球蛋白重链球状头部相关的蛋白质，构成了一个大家族，不同亚型具有阶段、组织和纤维类型特异性表达，这些亚型被认为是由单一祖先基因复制产生的。在原口无脊椎动物，如节肢动物、软体动物和线虫中，只发现了一个MLC-alk基因，但在后口无脊椎动物和低等脊椎动物中此类基因的数量尚不清楚。本报告描述了文昌鱼碱性肌球蛋白轻链基因（AmphiMLC-alk）的序列及其在整个发育过程中的表达情况，填补了在理解基因复制与肌肉细胞类型多样性增加之间关系方面的一个主要空白。从文昌鱼幼虫cDNA文库中分离出了一个全长为1 kb的AmphiMLC-alk克隆。它编码一种149个氨基酸的蛋白质，与脊椎动物MLC-alk的胚胎形式关系最为密切。Southern印迹分析显示文昌鱼中只有一个AmphiMLC-alk拷贝，表明它是文昌鱼中唯一的MLC-alk基因。Northern印迹分析表明该基因只产生一种转录本，在发育的各个阶段以及成体中均有表达。原位杂交显示，该基因最初在神经胚2-5体节的肌节中表达，此后不久在第1体节以及新形成的、逐渐向后添加的体节的肌节中表达。随着肌节细胞分化为有横纹的单核肌肉细胞，肌节表达在整个幼虫发育过程中持续存在并进入成体阶段——与脊椎动物有横纹的肌肉细胞不同，文昌鱼的肌肉细胞从不形成多核。在晚期幼虫和成体中，AmphiMLC-alk的肌节表达定位于肌节的内侧边缘和细胞末端。这是首次在任何动物的肌肉细胞中证明MLC转录本的细胞内定位。在平滑肌以及非源自肌节的有横纹肌肉中也检测到了AmphiMLC-alk的表达。这些表达数据与Southern印迹分析一致，表明文昌鱼中只有一个MLC-alk基因。因此，脊椎动物祖先MLC-alk基因的复制可能发生在脊椎动物和文昌鱼谱系分化之后。（摘要截选至400字）
