Significance of acquisition of retinoic acid signaling pathway during chordate evolution

视黄酸调节许多脊索动物特有的发育过程。这包括背侧中空神经索的前后形态形成、鳃裂的形成、神经嵴分化和肢体形态发生。由于维甲酸的受体（RAR）、合成酶（RALDH2）和降解酶（CYP26）仅在脊索动物中被发现，因此编码这些蛋白质的基因的获得被认为是脊索动物进化过程中的一个关键事件。在本研究中，我利用海鞘进行了以下项目。(1)鉴定了脊椎动物Raldh2、Cyp26和RAR基因的Ciona同源物。在正常和维甲酸处理的胚胎中检测了这些基因的表达模式。Raldh2 mRNA在3个最前部尾肌细胞中表达，而Cyp26 mRNA在一部分脑细胞中表达。它们的表达域之间存在着差距。这种表达模式与在脊椎动物中观察到的相似，这表明由这些酶产生的视黄酸的不均匀分布对乔纳胚胎的形态发生很重要。(2)从玉米基因组DNA中分离出RAR基因的一个增强子/启动子区。确定了中枢神经系统、表皮和肌肉细胞中负责激活的增强因子。(3)利用微阵列技术（cdna和寡核苷酸技术）鉴定黄颡鱼胚胎维甲酸靶基因。通过原位杂交检测了150多个候选靶基因在正常胚胎和维甲酸处理胚胎中的表达。在翻译抑制剂puromycin存在的情况下，对视黄酸有应答的基因被认为是主要靶基因，其转录可被RAR直接激活。这些基因包括Cyp26、Hox-1和一个功能未知的无名指蛋白编码基因。(4)这些数据发表为7篇原创研究论文和2篇综述文章。(5)还尝试用海鞘胚胎建立RNAi技术。从玉米基因组中分离到U6 snRNA基因启动子区。基因特异性短发夹rna编码基因与该启动子结合，通过电穿孔导入受精卵。用该方法对维甲酸靶基因进行了沉默。少

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