Genes Expressed in the Amphioxus Notochord Revealed by EST Analysis

Abstract

The notochord cell of the cephalochordate amphioxus adult is unique due to the occurrence of myofilaments in the cytoplasm. The present EST (expressed sequence tag) analysis targeted mRNAs of the amphioxus notochord to determine genes that are expressed there. Notochord cells were isolated from Branchiostoma belcheri adults, from which a cDNA library was constructed. Analysis of a set of 257 ESTs (both 5′ and 3′ ends) showed that about 11% of the cDNAs are related to muscle genes, while 9% of them are genes for extracellular matrix proteins associated with formation of the notochordal sheath. The muscle-related genes included actin, tropomyosin, troponin I, myosin regulatory light chain, myosin light chain kinase, myosin heavy chain, calmodulin, calponin, calcium vector protein, creatine kinase, muscle LIM protein, and SH3-binding glutamate-rich protein, suggesting that vertebrate skeletal and smooth muscle-type genes are simultaneously expressed in the amphioxus notochord. Nucleotide sequences of cDNAs for actin, tropomyosin, troponin I, and a few others were completely determined to substantiate the conclusions. The chordate muscle-type actin is distinguishable from the cytoplasmic-type actin by the usage of amino acid residues at 20 diagnostic positions. Interestingly, analysis of the usage of amino acid residues at these positions showed that the “amphioxus notochord actin” is a unique intermediate between muscle-type and cytoplasmic-type actins. These results strongly suggest that the notochord of adult amphioxus is a mechanical swimming organ and its role is quite different from the role of the vertebrate embryonic notochord, which functions as a source of signals required for body plan formation.