I propose to investigate the cellular and biochemical mechanisms underlying pattern formation in the well-defined developmental field of the embryonic chick limb bud. In particular, the normal digit pattern of a chick wing can be systematically altered by the local release of several carboxylated retinoids (vitamin A and its congeners) from a small ion-exchange bead implanted into the anterior margin of a 3 1/2 day old wing bud. In all aspects, retinoic acid-like molecules behave like a morphogenetic signaling molecule. Such a morphogen was postulated from previous studies to be released from a small group of cells (known as polarizing region or zone of polarizing activity) located at the posterior margin of the early limb bud. I believe that having a known compound that exhibits morphogenetic activity provides a powerful tool for sutdying pattern formation mechanisms. The following questions will be addressed. How do retinoids that are locally released in the limb become distributed in the wing bud - for example, do they form a gradient? Do retinoids that are supplied systemically to vitamin A deficient embryos become concentrated in particular areas of the bud? How are the applied retinoids metabolized by limb bud cells? To experimentally study these questions, I will use radiolabeled retinoids and follow their fate in the bud by both high-performance liquid chromatography and autoradiography. Using very sensitive chemical methods, I will also study the retinoids that occur naturally in the limb. I hope to be able to demonstrate that the polarizing region contains cells that release retinoic acid or a related active species. Finally, the limb bud will be examined for the presence of proteins that specifically bind biologically active retinoids. Such """"""""receptor proteins"""""""" are likely to be involved in any retinoid mediated patterning process. Using a combination of autoradiography, photocrosslinking techniques and filter binding assays I will attempt to localize such receptors and investigate their developmental history. If my studies are successful, they should both help in uncovering pattern formation mechanisms and increase our understanding of the as yet largely unexplored role that retinoids play in developing organisms. Work on such a central aspect of vertebrate limb development is likely to provide a mechanistic rationale for many congenital and drug-induced limb malformation.
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