Significance of acquisition of retinoic acid signaling pathway during chordate evolution

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Retinoic acid regulates many chordate-specific developmental processes. These include anterior posterior pattern formation in the dorsal hollow nerve cord, formation of branchial gill slits, neural crest differentiation and limb morphogenesis. Since the receptor (RAR), synthesizing enzyme (RALDH2), and degrading enzyme (CYP26) for retinoic acid have been identified exclusively in chordates, acquisition of genes encoding these proteins was thought to be a crucial event during chordate evolution. In this study, I carried out the following projects, using the ascidian Ciona intestinalis.(1)Ciona homologs of vertebrate Raldh2, Cyp26, and RAR genes were identified. Expression pattern of these genes were examined in normal and retinoic acid-treated embryos. Raldh2 mRNA was expressed in three anterior-most tail muscle cells, while Cyp26 mRNA was expressed in a subset of brain cells. There is a gap between their expression domains. This expression pattern is similar to that observed in vertebr … More ates, suggesting that uneven distribution of retinoic acid, created by these enzymes, is important for the morphogenesis in the Ciona embryo.(2)I isolated an enhancer/promoter region of the RAR gene from the Ciona intestinalis genomic DNA. The enhancer elements responsible for activation in the central nervous system, epidermis and muscle cells were identified.(3)Microarray analyses (both cDNA-based and oligonucleotide-based) were performed to identify retinoic acid target genes in the Ciona intestinalis embryo. Expression of more than 150 candidate target genes was examined by in situ hybridization in normal and retinoic acid-treated embryos. Genes that respond to retinoic acid in the presence of a translation inhibitor, puromycin, were suggested to be primary target genes, of which transcription was directly activated by RAR. These included Cyp26, Hox-1 and a ring-finger protein-encoding gene whose function is unknown.(4)These data was published as 7 original research papers and 2 review articles.(5)Establishment of RNAi technique was also attempted using ascidian embryos. The promoter region of U6 snRNA gene was isolated from the Ciona intestinalis genome. Gene-specific short hairpin RNA-encoding gene was combined with this promoter and introduced into fertilized eggs by electroporation. Retinoic acid target genes were silenced using this method. Less