The long-range goal of the proposed studies is to understand, in more detail, the mechanism whereby the apical ectodermal ridge (AER) promotes and maintains the special functional properties of the underlying subridge mesenchyme. To this end, the applicant has devised four specific aims.
The first aim seeks to determine the effect of the AER on the expression of FGF-2, msx-1, and the pattern of FGF receptors (FGFRs) during normal limb development. Specifically, this aim is to measure FGF-2 bioactivity, and FGF- 2 and msx-1 expression in the mesoderm and ectoderm of normal chick limb buds and in the limb mesoderm after AER excision. The FGF-2 bioassay involves comparing the ability of tissue extracts to inhibit differentiation in MM14 cells with a standard curve of known FGF concentrations. FGF-2 and msx-1 expression levels will be measured by RNAse protection assays. Correlations between levels of FGF-2 bioactivity and expression will also be assessed along the anteroposterior axis of the limb bud since an anteroposterior difference in FGF-2 responsiveness has been previously demonstrated.
The second aim i s to evaluate the effects of FGF-2 on cultured limb bud cells. Micromass and modified limb-organ cultures of whole limbs and distal limb fragments will be used to assess any region-dependent or cell-type dependent effects of FGF-2 on the heterogeneous limb-bud mesenchyme. Effects of exogenous FGF-2 on cartilage differentiation, proliferation, msx-1 expression and FGFR expression will be examined, together with the ability of different medium components and culture substrata to modulate any FGF-2 effect.
The third aim i s to evaluate how the pattern of FGF-2 expression affects its efficacy in promoting limb-bud outgrowth and skeletogenesis. Effects of ectopic expression of FGF-2 at various sites in the developing limb in vivo will be monitored to determine whether the cells in the limb field must initially receive the FGF-2 signal from a distal direction to establish a progress zone in mutants lacking an AER, and whether ectopic expression can induce proximal ward expansion of the progress zone of normal limbs as measured by effects on proliferation, msx-1 expression and differentiation.
The fourth aim i s to satisfy a final criterion for establishing FGF-2 as the AER signal in the developing chick limb by determining whether blocking the FGF-2 signalling pathways in vivo using an anti-FGF-2 blocking antibody and by overexpression of a dominant negative FGFR can obliterate the effect of the AER and produce a phenocopy of the limbless mutant. Together these aims are intended to further establish whether FGF-2 is indeed the AER-derived outgrowth promoting factor as it appears to be thus far.
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