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Nebulin is a 770 kDa protein that is localized along the thin filaments of skeletal muscles in vertebrates. It is also present in the striated muscles of Amphioxus , an invertebrate cephalochordate that is phylogenetically close to vertebrates. However, the nebulin of urochordate ascidians or its expression in invertebrate hearts has not been investigated. In this study, we investigated the structure and cardiac expression of the nebulin gene in Ciona intestinalis , a urochordate whose phylogeny lies between cephalochordates and vertebrates. As a result of the gene structure analysis, we found that the Ciona nebulin gene predicted to be 62 kb and consists of 143 exons. The nebulin was expected to consist of a unique N-terminal region, followed by 155 nebulin repeats, another unique region, a Ser-rich region and a C-terminal SH3 domain. Whole-mount in situ hybridization experiments showed that the Ciona nebulin gene was expressed in a variety of muscles, including hearts. However, Western blot analysis using antibody to Ciona nebulin did not detect the presence of full-length nebulin. Alternatively, RT-PCR experiments on samples of Ciona heart detected the expression of nebulette-like and nrap-like isoforms from the Ciona nebulin gene. These results indicate that, similarly to vertebrate hearts, Ciona hearts do not express nebulin, but rather nrap- and nebulette-like isoforms. These results also imply that the nebulin, nebulette and nrap genes in vertebrates were separated from an ancestral invertebrate nebulin gene during vertebrate evolution. • Nebulin of urochordate or its expression in invertebrate hearts had not been revealed. • Structure and cardiac expression of the Ciona nebulin gene were investigated. • Nebulin was absent but nebulette and nrap were present in urochordate hearts. • Expression of nebulin family protein is conserved among chordate hearts. • Neb, Nebl and Nrap genes of vertebrates were separated from an invertebrate Neb gene. [ABSTRACT FROM AUTHOR]