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 脊（AER）促进和保持特殊的功能特性， 下面的脊下间充质。 为此，申请人已 提出了四个具体目标。 第一个目的是确定AER对表达的影响 FGF-2，msx-1和FGF受体（FGFR）的模式在正常 肢体发育 具体而言，这一目标是测量FGF-2 生物活性以及中胚层和外胚层中的FGF- 2和msx-1表达 正常鸡肢芽和切除AER后的肢中胚层中。 FGF-2生物测定涉及比较组织提取物与 用已知FGF标准曲线抑制MM 14细胞的分化 浓度的 FGF-2和msx-1表达水平将通过以下方法测量： RNA酶保护测定。 FGF-2水平之间的相关性 生物活性和表达也将沿着 由于前后差异，肢体芽的前后轴 在FGF-2反应性中的作用已经在以前得到证实。 第二个目的是评价FGF-2对体外培养肢芽的影响 细胞 全肢微团和改良肢器官培养， 远端肢体碎片将用于评估任何区域依赖性或 成纤维细胞生长因子-2对异种肢芽的细胞类型依赖性作用 间充质 外源性FGF-2对软骨分化的影响， 将检查增殖、msx-1表达和FGFR表达， 以及不同培养基成分和培养物的能力 基质来调节任何FGF-2作用。 第三个目的是评估FGF-2的表达模式如何影响 具有促进肢芽生长和骨骼生成的功效。 FGF-2在不同部位异位表达对人乳腺癌细胞的影响 将监测体内发育肢体以确定细胞是否 在肢体领域必须首先从远端接收FGF-2信号， 指导在缺乏AER的突变体中建立进展区，以及 异位表达是否可以诱导近端向扩展的 通过对增殖的影响测量的正常肢体的进展区， msx-1表达和分化。 第四个目标是满足将FGF-2确定为 发育中的鸡肢中的AER信号， 使用抗FGF-2在体内阻断FGF-2信号传导途径 阻断抗体和通过显性阴性FGFR的过表达， 消除AER的影响，并产生一个无肢的表型 变种人 这些目标旨在进一步确定FGF-2是否是 事实上，AER衍生的生长促进因子， 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.