Identification of RAR Target Genes

这项研究的长期目标是了解类维生素A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胚形成结束的过程中--这是一个关键的发育决定发生的时间，但在高等脊椎动物中，这一时间大多无法操纵。 我们的研究重点将是通过非洲爪蟾视黄酸受体（xRARs）识别真正的体内信号转导靶点。 早期非洲爪蟾胚胎是唯一适合实验操作，是一个丰富的材料来源的分子生物学方法的发展。Blumberg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发育层次中的位置。xRAR靶基因富集的胚胎将通过RAR泛激动剂TTNPB处理产生，而xRAR靶基因耗尽的胚胎将使用高度特异性RAR拮抗剂AGN 193109产生。 将采用来源于这两个胚胎群体的cDNA之间的消减杂交来鉴定未被xRAR信号转导调节或下调的cDNA。 这些新发现的靶基因的转录本将通过原位杂交和反向遗传方法进行定位，以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RAR α、β或γ中的哪一种调节每个靶基因。 在类维生素A药理学最近得到改进之前，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，RAR α信号是更重要的是在前面的图案，而作为另一个RAR，可能RAR γ是负责后patterning.In总结，类维生素A长期以来一直被认为是重要的各种过程中的发展。 拟议的研究采用新的工具来阐明胚胎图案化过程中维甲酸信号传导的分子要求。 所获得的结果应广泛适用于其他脊椎动物模型系统的发展研究，并提供关键的证据，了解RAR在发展过程中的作用。

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.