Self-Organization in Limb Pattern Formation--Test of a Hypothesis

这个项目的目标是检验一种假说，即脊椎动物肢芽的细胞如何产生准周期排列的软骨棒和软骨节，这些软骨棒和软骨节在所有此类物种中都是肢体骨骼的原基。假说是，肢体间充质细胞在体外产生时空排列的软骨前凝集物和软骨结节的“自组织”特性也被用于在体内产生肢体骨骼模式。为了验证这一想法，将确定肢体芽间充质固有的关键基因调控过程，这为其以时空可控的方式产生软骨前凝聚提供了基础。由于细胞外基质蛋白纤维连接蛋白在体外和体内都是软骨前细胞凝聚的主要媒介，因此DNA构建物将被设计为包含鸡纤维连接蛋白基因启动子连接到一个易于可视化的报告分子-绿色荧光蛋白。将确定正常启动子序列的长度，该序列在体外指导真实的缩合相关基因表达，然后突变最小启动子，以确定哪些顺式作用位点对这种活性是必不可少的。这将允许描绘与发育相关的转录因子如Fast-1的作用的候选位点。这些结构对添加的生长和分化因子如转化生长因子-B2和成纤维细胞生长因子-2、-4和-8的反应将被用来帮助识别调节纤维连接蛋白表达的内源性因子。一旦在体外确定了相关决定因素的特征，它们将通过电穿孔或洗涤剂介导的转基因进入胚胎的四肢，以确定体内骨骼原基的纤维连接蛋白“预模式”的建立是否使用了与在体外产生这些模式的自组织过程中使用的相同的纤维连接蛋白调节通路。

0090499NewmanThe objective of this project is to test a hypothesis for how the cells of the vertebrate limb bud generate the quasi-periodic array of cartilage rods and nodules that serve as the primordia of the limb skeleton in all such species. The hypothesis is that the "self-organizing" properties of limb mesenchymal cells that lead them to generate spatiotemporally arranged precartilage condensations and cartilage nodules in vitro are also utilized in vivo in the generation of the limb skeletal pattern. To test this idea key gene regulatory processes will be identified that are intrinsic to limb bud mesenchyme which provide the basis for its ability to generate precartilage condensations in a spatiotemporally-controlled fashion. Because the extracellular matrix protein fibronectin is a major mediator of precartilage cell condensation both in vitro and in vivo, DNA constructs will be designed to contain the chicken fibronectin gene promoter linked to a readily visualized reporter molecule, Green Fluorescent Protein. The length of normal promoter sequence that directs authentic condensation-associated gene expression in vitro and then mutate the minimal promoter to determine which cis-acting sites are essential for this activity will be identified. This will permit the delineation of candidate sites for action of developmentally relevant transcription factors such as Fast-1. The response of these constructs to added growth and differentiation factors such as TGF-B2, and FGF-2, -4 and -8 will be used to help identify endogenous factors that regulate fibronectin expression. Once the relevant determinants have been characterized in vitro, they will be introduced into embryonic limbs by electroporation or detergent-mediated transfection to ascertain whether or not the establishment of fibronectin "prepatterns" for the skeletal primordia in vivo employ the same fibronectin regulatory pathways utilized in the self-organizing processes responsible for the generation of these patterns in vitro.