Identification of RAR Target Genes

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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.