The research described in this proposal will further define the role of retinoic acid (RA) and related retinoids (vitamin A derivatives) in chick limb morphogenesis, and is important for a proper understanding of the physiological mechanisms underlying patterns formation in vertebrate limbs. Previous work suggests that RA most likely is a morphogen that plays a role in specifying the limb pattern along its anteroposterior axis. The main evidence is that when RA is exogenously applied to the developing limb bud, it triggers the formation of additional digits in a very specific way, and that RA is present endogenously in the limb anlage in the form of an anteroposterior concentration gradient. Proposed research: (1) We will continue the search for endogenous retinoids in early chick limb buds and embryos, and chemically identify these yet unknown retinoids by a combination of high performance liquid chromatography, derivatization and spectroscopy. We will examine whether any biologically active retinoid in addition to RA is present in buds in the form of a gradient. To provide information about their time of action, we will determine the concentration of endogenous retinoids during limb outgrowth. (2) We will remove the posterior half from wing buds, the domain that contains the zone of polarizing activity (ZPA). Such """"""""anterior only"""""""" buds, when left to develop, give rise to extremities lacking a hand. We have discovered that targeting RA to such truncated limb buds results in the formation of intact limbs. Using this system, allows us to determine the mode of RA action free from interference by the ZPA. (3) To determine how the gradient of RA is generated, we will target 3H-retinol and retinal into wing buds and study their in situ conversion to RA. (4) We will localize the site(s) of retinoid metabolism in limb buds and embryos. Our strategy is to isolate the metabolic enzymes involved and prepare antibodies against them. These agents will then be used to localize the anitgen in situ by immunohistochemical procedures. (5) We will test a series of aldehyde oxidase inhibitors for their ability to inhibit the conversion of retinal to RA in the limb bud. We expect that such antagonists block the formation of a normal limb pattern, and therefore provide a stringent test for the hypothesis that RA is a morphogen in the chick limb system. These studies are basic to an undestanding of pattern formation in vertebrate limbs and are a contribution towards rationalizing drug-induced and congential malformations not only in limbs but in the embryo as a whole.
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