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AbstractSeveral homeobox-containing genes related to Distal-less (Dlx/Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. In Drosophila only one Distal-less gene has been identified so far, and in vertebrates many components of the Distal-less family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Distal-less gene via gene duplication. Three Dll homeoboxes have been isolated from the protochordate Ciona intestinalis and their clustered arrangement has been described. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution. The goal of this project was to clone coding sequences from the DllA/DllB cluster of Ciona intestinalis in order to observe the gene expression using in situ hybridization. During my study of DllA and DllB genes in Ciona, I used various techniques including PCR, restriction digest, sticky-end ligation, and transformation, to obtain cDNA from each gene. These cDNAs, DNA copies of the mRNA sequences, were used to create digoxigenin-labeled antisense RNA probes by in vitro transcription with T7 RNA polymerase. The in-situ hybridization documented gene expression in the epithelial cells and neural tissue, which is consistent with the expression patterns found in other species. The DllA probe was expressed in the anterior epithelial tissue in mid-tail stage embryos, on the adhesive papillae of late-tail stage embryos, and the bilateral primordia of the atrial siphon of larvae. DllB probe in blastula through early tail stages showed expression patterns in epidermal and neuroectodermal cells.