MECHANISMS OF RETINOID EFFECTS ON SKELETOGENESIS

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Limb defects afflict more than one out of every 2,000 live human births. The mechanisms underlying these often devastating defects are unclear. A fundamental aspect of limb development is outgrowth. The project's long term objective is to understand the regulation of this process. The immediate goal is to test the hypothesis that the ability of the limb bud's apical ectodermal ridge (AER) to promote limb outgrowth involves an ability to maintain cellular retinoic acid-binding protein (CRABP) expression in sub-ridge limb mesenchyme and thereby to limit the retinoic acid (RA) encountered by nuclear RA receptors in these cells. Corollary hypothesis to be tested are 1) than an AER signal maintains CRABP expression, 2) that the AER signal is FGF-8, 3) that CRABP promotes RA degradation, 4) that RA degradation prevents stem-cell differentiation, 5) that preventing stem-cell differentiation prevents FGF-receptor (FGFR) isoform switching, and 6) that preventing FGF-R isoform switching maintains limb mesenchymal competence to respond to the AER. Six specific aims are proposed to test this hypothesis. Aim 1 is to document AER effects on CRABP expression in the subjacent limb-bud mesenchyme in vivo. Two approaches will be taken. CRABP expression will be examined in chick limbs whose AERs have been excised at different stages of limb outgrowth, and limbs into which extra AERs have been grafted. Unoperated contralateral limbs will serve as controls. Aim 2 is to determine the AER's effects on retinoic acid metabolism. Distal limb fragments, intact or with their AER's previously surgically removed, will be incubated in vitro with 2/H-RA. Incubated tissue and media extracts will be analyzed by HPLC to compare metabolite profiles produced by the different preparations. Aim 3 is to determine RA effects on CRABP expression. Serum- free micro-cultures of limb mesenchyme will be treated with all-trans-ERA, 9-cis-RA, or 3,4-didehydro-RA. CRABP protein levels will be assessed by in situ ELISA and Western blots of culture extracts. Aim 4 is to determine FGF effects on CRABP expression. Micro-cultures of mesenchyme from various limb regions grown in serum-free medium containing FGF-2, FGF-4, FGF-8, or FGF-10 will be subjected to whole-mount in situ hybridization using a digoxigenin-labeled CRABP riboprobe in the wells of the plate. Aim 5 is to determine CRABP's effect on responses to retinoic acid. Micro-cultures transiently transfected with a eukaryotic expression vector containing a 700 bp. CRABP cDNA construct, will be treated with all-trans, 9-cis-, or 3,4-didehydro-RA, and effects on growth, chondrogenesis, and FGFR expression will be measured. Aim 6 is to determine CRABP's effect on RA metabolism. Chick limb-bud mesenchymal cells will be transiently transfected with a eukaryotic expression vector containing a 700 bp CRABP cDNA. Transfected cells will be harvested, washed, suspended in serum-free medium, and incubated with RA radio-labeled to high specific activity.