Significance of acquisition of retinoic acid signaling pathway during chordate evolution

视黄酸调节许多脊索动物特定的发育过程。这些包括背侧中空神经索的前后模式形成、鳃裂的形成、神经嵴的分化和肢体形态发生。由于视黄酸的受体（RAR）、合成酶（RALDH 2）和降解酶（CYP 26）仅在脊索动物中被鉴定，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关键事件。在这项研究中，我进行了以下项目，使用海鞘玻璃海鞘。（1）鉴定了脊椎动物Raldh 2、Cyp 26和RAR基因的玻璃海鞘同源物。这些基因的表达模式进行了检查，在正常和视黄酸处理的胚胎。Raldh 2 mRNA在三个最前面的尾肌细胞中表达，而Cyp 26 mRNA在一部分脑细胞中表达。它们的表达域之间存在间隙。这种表达模式与在脊椎动物中观察到的相似， ...更多信息 这表明，由这些酶产生的视黄酸的不均匀分布对玻璃海鞘胚胎的形态发生很重要。(2)I从玻璃海鞘基因组DNA中分离RAR基因的增强子/启动子区域。鉴定了负责中枢神经系统、表皮和肌肉细胞激活的增强子元件。（3）利用基因芯片技术（包括基于cDNA和基于荧光素酶的基因芯片技术）对玻璃海鞘胚胎中的视黄酸靶基因进行鉴定。在正常和维甲酸处理的胚胎中，通过原位杂交检测了150多个候选靶基因的表达。在翻译抑制剂嘌呤霉素存在下对视黄酸有反应的基因被认为是主要的靶基因，其转录被RAR直接激活。其中包括Cyp 26、Hox-1和一个功能未知的环指蛋白编码基因。（4）这些数据以7篇原创研究论文和2篇综述文章的形式发表。（5）以海鞘胚胎为实验材料，尝试建立RNAi技术。从玻璃海鞘基因组中分离U6 snRNA基因的启动子区。将基因特异性短发夹RNA编码基因与该启动子结合，通过电穿孔法导入受精卵。使用该方法沉默视黄酸靶基因。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