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

0090499 Newman本项目的目的是检验脊椎动物肢芽细胞如何产生软骨棒和结节的准周期阵列的假设，这些软骨棒和结节在所有这些物种中充当肢骨骼的原基。假设是肢体间充质细胞的“自组织”特性导致它们在体外产生时空排列的前软骨致密物和软骨结节，在体内也用于肢体骨骼模式的产生。为了测试这一想法的关键基因调控过程将被确定，是内在的肢芽间充质提供了基础，其能力，以时空控制的方式产生precarcinoma凝聚。因为细胞外基质蛋白纤连蛋白是体外和体内前软骨细胞凝聚的主要介质，所以DNA构建体将被设计为含有与易于可视化的报道分子绿色荧光蛋白连接的鸡纤连蛋白基因启动子。将鉴定正常启动子序列的长度，所述正常启动子序列指导真实的体外缩合相关基因表达，然后突变最小启动子以确定哪些顺式作用位点对于该活性是必需的。 这将允许描绘发育相关转录因子如Fast-1的作用的候选位点。 这些构建体对添加的生长和分化因子如TGF-β 2和FGF-2、FGF-4和FGF-8的反应将用于帮助鉴定调节纤连蛋白表达的内源性因子。 一旦相关的决定因素已在体外的特点，他们将被引入胚胎肢体电穿孔或去污剂介导的转染，以确定是否建立纤连蛋白的骨骼原基在体内的“prepatterns”采用相同的纤连蛋白的调控途径，在自组织过程中负责这些模式的产生在体外。

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.