SEQUENCE AND EXPRESSION OF AMPHIOXUS ALKALI MYOSIN LIGHT-CHAIN (AMPHIMLC-ALK) THROUGHOUT DEVELOPMENT - IMPLICATIONS FOR VERTEBRATE MYOGENESIS

The lower chordate amphioxus, widely considered the closest living inv ertebrate relative of the vertebrates, is a key organism for understan ding the relationship between gene duplications and evolution of the c omplex vertebrate body plan. In tetrapod vertebrates, the alkali myosi n light chain genes (MLC-alk), which code for proteins associated with the globular head of the myosin heavy chain, constitute a large famil y with stage-, tissue-, and fiber-type-specific expression of differen t isoforms thought to have arisen by duplication of a single ancestral gene. In protostome invertebrates, e.g., arthropods, molluscs, and ne matodes, 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 (Amphi MLC-alk), thus fills a major gap in understanding the relation between gene duplication and increasing diversity of muscle-cell types. A ful l-length clone (1 kb) of AmphiMLC-alk was isolated from a larval amphi oxus cDNA Library. It coded for a 149-amino-acid protein most closely related to the vertebrate embryonic form of MLC-alk. Southern blot ana lysis revealed only one copy of AmphiMLC-alk and suggested that it is the only MLC-alk gene in amphioxus. Northern blot analysis indicated t hat this gene produces only one transcript, which is expressed at all stages of development and in adults. In situ hybridizations showed exp ression initially in the myotomes of somites 2-5 of neurula embryos an d soon thereafter in the myotomes of somite 1 and of newly forming som ites progressively added posteriorly. Myotomal expression continues th roughout larval development and into the adult stage as the myotomal c ells differentiate into striated, mononucleate muscle cells-unlike ver tebrate striated muscle cells, those of amphioxus never become multinu cleate. In late larvae and adults myotomal expression of AmphiMLC-alk is localized along the medial edge of the myotome and at the ends of t he cells. This is the first demonstration of intracellular localizatio n of MLC transcripts in muscle cells of any animal. Expression of Amph iMLC-alk was also detected in smooth muscles as well as in striated mu scles not derived from the myotome. These expression data are consiste nt with the Southern blot analysis in suggesting that there is only on e MLC-alk gene in amphioxus. Thus, duplication of an ancestral vertebr ate MLC-alk gene probably occurred after the vertebrate and amphioxus lineages split. We conclude that development of a segmented axial musc ulature preceded the evolution of multiple MLC-alk isoforms, which evi dently arose about the time of multinucleation. Since myogenesis in am phioxus is similar to but far simpler than myogenesis in vertebrates a t both the structural and gene levels, an understanding of myogenesis in amphioxus can give insights into both the evolutionary history and the detailed mechanisms of vertebrate myogenesis. (C) 1995 Academic Pr ess, Inc.