Identification of RAR Target Genes

这项研究的长期目标是了解类视黄醛信号在脊椎动物发育过程中的作用。这些研究将集中在从受精到神经发育结束的这段时间，这段时间发生了关键的发育决定，但在高等脊椎动物中大多无法操纵。我们的研究重点将是通过爪蟾视黄酸受体（xRARs）确定真正的体内信号靶点。非洲爪蟾的早期胚胎特别适合实验操作，是分子生物学研究发育方法的丰富材料来源。布隆伯格博士将利用非洲爪蟾胚胎的这些独特特征来表征这些新发现的RAR靶基因，并确定它们在早期胚胎的发育等级中所处的位置。富含xRAR靶基因的胚胎将通过RAR拮抗剂TTNPB处理产生，而缺乏xRAR靶基因的胚胎将使用高度特异性的RAR拮抗剂AGN193109产生。来自这两个胚胎群体的cdna之间的减法杂交将用于鉴定不受xRAR信号调节或下调的cdna。这些新发现的靶基因的转录本将通过原位杂交定位，并利用反向遗传方法确定其在发育过程中的功能。受体亚型选择性激动剂和拮抗剂将使我们能够确定三种RARs中哪一种，α， β或γ调节每个靶基因。在最近类维生素a药理学的改进之前，这样的研究是不可行的。这是一个重要的问题，因为我们的初步数据表明rarα信号在前模式中更重要，而作为另一种RAR， RARgamma可能负责后模式。综上所述，类维生素a一直被认为对发育过程中的各种过程都很重要。提出的研究采用新的工具来阐明胚胎模式过程中类视黄醇信号的分子要求。研究结果可广泛应用于其他脊椎动物模型系统的发育研究，并为理解RARs在发育过程中的作用提供重要证据。

The long-term goal of the proposed research is to understand the role of retinoid signaling during vertebrate development. These investigations will be focused from fertilization through the end of neurulation - a time when critical developmental decisions occur but that is mostly inaccessible to manipulation in higher vertebrates. The focus of our research will be identify bona fide in vivo targets for signaling through Xenopus retinoic acid receptors (xRARs). The early Xenopus laevis embryo is uniquely suited for experimental manipulation and is a rich source of materials for molecular biological approaches to development. Dr. Blumberg will exploit these unique characteristics of the Xenopus embryo to characterize these newly identified RAR target genes and determine where they fit into the developmental hierarchies that pattern the early embryo.Embryos enriched for xRAR target genes will be generated by treatment with the RAR panagonist TTNPB while embryos depleted for xRAR targets will be produced using the highly specific RAR antagonist, AGN193109. Subtractive hybridization between cDNAs derived from these two populations of embryos will be employed to identify cDNAs that are unregulated or downregulated by xRAR signaling. Transcripts from these newly identified target genes will be localized by in situ hybridization and reverse genetic approaches will be utilized to determine their functions during development. Receptor subtype-selective agonists and antagonists will enable us to determine which of the three RARs, alpha, beta or gamma regulates each target gene. Such a study was not feasible before recent improvement in retinoid pharmacology. This is an important issue because our preliminary data suggest that RARalpha signaling is more important in anterior patterning where as another RAR, likely RARgamma is responsible for posterior patterning.In summary, retinoids have long been known to be important for a variety of processes during development. The proposed research employs new tools to elucidate molecular requirements for retinoid signaling in embryonic patterning processes. The results obtained should be widely applicable to the study of development in other vertebrate model systems and provide critical evidence toward understanding the role of RARs during development.