EPITHELIAL/MESENCHYMAL INTERACTIONS IN LIMB PATTERNING

描述：拟议研究的长期目标是 更详细地了解顶端外胚层 Ridge(AER)促进和维护了 下伏的亚脊间充质。为此，申请人已 制定了四个具体目标。 第一个目的是确定AER对表达的影响 正常大鼠成纤维细胞生长因子-2、MSX-1和成纤维细胞生长因子受体(FGFRs)的表达 肢体发育。具体地说，这一目标是为了测量成纤维细胞生长因子-2 中胚层和外胚层的生物活性及成纤维细胞生长因子-2和MSX-1的表达 正常鸡肢芽和AER切除后的肢体中胚层。 成纤维细胞生长因子-2生物测定包括比较组织提取物与 用已知成纤维细胞生长因子的标准曲线抑制MM14细胞的分化 浓度。成纤维细胞生长因子-2和MSX-1的表达水平将通过 核糖核酸酶保护试验。成纤维细胞生长因子-2水平的相关性 生物活性和表达也将沿着 肢芽前后差值的前后轴 在成纤维细胞生长因子-2中，已经证实了反应性。 第二个目的是评价成纤维细胞生长因子-2对培养的肢芽的影响。 细胞。微团培养和改良四肢器官培养 远端肢体碎片将被用来评估任何区域依赖的或 成纤维细胞生长因子-2对异种肢芽细胞类型的依赖作用 间充质的我。外源性成纤维细胞生长因子-2对软骨分化的影响 将检测细胞增殖、MSX-1表达和FGFR表达， 以及不同的培养基组分和培养能力 底物来调节任何成纤维细胞生长因子-2的作用。 第三个目标是评估成纤维细胞生长因子-2的表达模式如何影响 它在促进肢芽生长和骨骼形成方面的功效。 成纤维细胞生长因子-2在不同部位的异位表达对血管内皮生长因子表达的影响 将对体内发育的肢体进行监测，以确定细胞是否 必须首先从远端接收到成纤维细胞生长因子-2信号 在缺乏AER的突变体中建立进展区的方向，以及 异位表达是否能诱导近端向外扩张 以增殖效应来衡量正常肢体的进展区， MSX-1的表达与分化 第四个目标是满足建立成纤维细胞生长因子-2作为 通过测定发育中的雏鸡肢体的AER信号 应用抗成纤维细胞生长因子-2抗体阻断体内成纤维细胞生长因子-2信号通路 阻断抗体和过表达显性负FGFR可以 消除AER的影响，并产生无肢者的表型 变种人。 这些目标旨在进一步确定成纤维细胞生长因子-2是否 事实上，AER衍生的生长促进因子看起来是 到目前为止。

DESCRIPTION: 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 is 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 is 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 is 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.