MECHANISMS OF RETINOID EFFECTS ON SKELETOGENESIS

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Despite the limb bud's scrutiny as a model of development for over half a century, limb malformations still afflict more than one out of every 2,000 live human births. The mechanisms underlying these often devastating defects are unclear, partly because we still have so much to learn about normal limb development. Perhaps the most fundamental aspect of limb development is outgrowth. The project's long term objective is to understand the regulation of vertebrate limb-bud outgrowth. The immediate goal is to test the hypothesis that the well- known, but poorly understood 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 the subjacent mesenchyme and thereby to limit the amount of free retinoic acid (RA) encountered by nuclear RA receptors, such as RA receptor-beta (RAR-beta), in these cells. A corollary hypothesis to be tested is that CRABP's effect involves promoting metabolic inactivation of free RA. Since RA's effects at physiologic concentrations can be largely generalized as promoting differentiation through interactions with nuclear receptors, limiting RA's availability could protect stem cells within the AER's zone of influence from precocious differentiation. Four specific aims are proposed to test this hypothesis. Aim 1 is to determine the AER's effect 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 in limbs to which extra AERs have been grafted. Unoperated contralateral limbs will serve as controls. Aim 2 is to determine the AER's effect on CRABP expression in serum-free cultures of mesenchyme from different wing-bud regions. CRABP expression will be examined in micromass cultures of wing mesenchyme overlain by isolated AERs. Controls will include cultures lacking an AER and cultures covered with other types of ectoderm. The ability of exogenous RA to thwart the ectodermal effect will also be tested. Aim 3 is to determine the AER's effect on RA metabolism in serum-free cultures of wing-bud mesenchyme from different limb regions. Radiolabeled RA will be added to selected culture types that exhibited different CRABP expression levels in Aim 2 and/or different levels of RAR-beta expression in Aim 4. Extracts of treated cultures will be analyzed to reveal any correlation between RA metabolism, CRABP expression and the activation of RA-driven genes. Aim 4 is to determine the AER's effect on the expression of RA-activated genes in vivo and in vitro. AER effects on the expression of RAR-beta will be examined taking the same approach used to analyze effects on CRABP expression in Aims 1 (in vivo) and 2 (in vitro). The methods will include in situ hybridization and immunohistochemistry to examine patterns of CRABP and RAR-beta expression and Northern and Western blotting to provide information about CRABP and RAR-beta isoforms present. Retinoid metabolism will be assessed using reverse phase HPLC of extracts from cultures treated with radiolabeled retinoids, scintillation counting of HPLC fractions, and comparison of peak retention times to standards. Aim 5 is to introduce minority biomedical science students to combined classic and molecular approaches to important problems in development biology.