The research described in this application explores the role of retinoic acid (RA) and other biologically active retinoids in vertebrate development and is important for a proper understanding of the physiologic role of RA in vertebrate limb morphogenesis. The dose-dependent effect of RA on the limb digit pattern, the similarity between the effect of RA and of the ZPA (an endogenous signaling region), the presence in the limb bud of RA, of retinoid receptors, of RA biosynthetic enzymes and of retinoid binding proteins, collectively lend credence to the hypothesis that RA is or closely mimics a hypothetical morphogen released by the ZPA. However, tissue isolated from an RA-exposed bud can induce pattern duplications, hence RA may convert non-ZPA tissue into ZPA and not be a morphogen. To test these two possibilities and to further examine the physiologic role of RA in limb development, we propose the following studies: (1) We now have evidence that only pharmacologic but not physiologic doses of RA induce ZPA. To pursue this important finding, we will exploit the fact that grafts of authentic ZPA induce in the host HoxD11, 12, and 13 genes. Hence, we will treat quail wing buds with different doses of RA, transplant the tissue into a chick host and measure the induction of HoxD genes in the host tissue in dependence of the dose of RA to which the graft was exposed. (2) To determine whether the effect of two naturally occurring retinoids, RA and 9-cis-RA is mediated either by homodimers of retinoid-X-receptors (RXRs) or RA-receptors (RARs) or by RXR-RAR heterodimers, we will treat wing buds with RAR- or RXR-specific synthetic retinoids. (3) In vivo experiments have revealed local difference in retinoid metabolism along the anterioposterior axis of the limb bud. To quantify these differences, anterior and posterior limb bud tissue will be incubated separately in media containing either retinol, retinal, RA, or 9-cis-RA, and the relative rates of enzymatic conversion will be determined. This will reveal whether or not the ZPA is a high point of RA and/or 9-cis-RA production. (4) Retinol dehydrogenase (RDH) and retinal dehydrogenase (RalDH) are two key enzymatic activities catalyzing RA synthesis, and are both present in chick limb buds and embryos. Using sensitive enzymatic assays for RDH and RalDH, we will purify both enzymes to homogeneity from embryos. Partial protein sequence will be used to generate oligonucleotide probes to clone the corresponding genes. To reveal where and when in the limb bud and in the embryo RDH and RalDH are expressed and hence which sites serve as local retinoid sources, embryos will be subjected to in situ hybridization using RDH- and RalDH-specific probes. Although this proposal centers on the role of retinoid in vertebrate limbs; the resulting information will increase our general understanding of this important class of signaling molecules in normal development as well as in embryos exposed to high, teratogenic doses of retinoids.
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