Project 3 - Tabin

项目摘要(见说明)：分泌的蛋白质指导发育中的骨骼元素的启动、生长和构图。印度刺猬(IHH)就是这样一个因素的重要例子。像许多诱导信号一样，IHH除了与靶细胞上的受体相互作用外，还与细胞外基质成分发生物理作用。这种细胞外相互作用可以隔离信号，影响形态原在组织中的移动和/或介导配体-受体相互作用。刺猬蛋白先前已被证明与硫酸肝素蛋白多糖(HSPGs)特异性相互作用。虽然还没有直接研究IHH，但参与HSPG链延长的两个基因ext1和ext2的突变会导致骨骼发育不良，称为外移症，IHH信号在ext1突变体中受到损害。为了直接阐明HSPG相互作用对IHH功能的重要性，我们将构建一个IHH的等位基因，该等位基因缺少HSPG结合的Cardin-Wetraub(C-W)结构域。IHH对骨骼发育的作用是通过直接作用于增殖的软骨细胞，也通过间接作用于软骨膜-包裹骨骼间质的扁平细胞鞘来实现的。事实上，已知的是，软骨在调节下伏骨骼元素分化的许多方面发挥着重要作用。我们最近发现了一系列标记物，表明这种组织的组织复杂程度令人惊讶。我们将构建cre重组酶处于启动子转录控制下的转基因小鼠，这些启动子驱动不同软骨膜区域的表达。这些将被用来绘制软骨膜不同层中的细胞的命运图，并测试骨骼调节因子在该组织中的作用，包括IHH和HOX基因。

PROJECT SUMMARY (See instructions): Secreted proteins direct the initiation, growth and patterning of the developing skeletal elements. Indian hedgehog (Ihh) is an important example of such a factor. Like many inductive signals, Ihh physically interacts with extracellular matrix components in addition to receptors on it's target cells. Such extracellular interactions can sequester the signals, influence morphogen movement across tissues and/or mediate ligand-receptor interactions. Hedgehog proteins have previously been shown to specifically interact with Heparin Sulfate Proteoglycans (HSPGs). While this has not been directly explored for Ihh in particular, mutations in two genes involved in HSPG chain elongation, Ext1 and Ext2, cause skeletal dysplasias known as exotoses and Ihh signaling is compromised in Ext1 mutants. To directly address the importance of HSPG interacting for Ihh function, we will construct an allelle of Ihh that lacks the puttaive HSPG-binding Cardin- Weintraub (C-W) domain. Ihh acts on skeletal development through direct effects on prolioferating chondrocytes, and also through indirect effects via the perchondrium a membrous sheath of flattened cells encapsulating the skeletal aniagen. Indeed, the perchondrium is known to play important roles in a number of aspects of regulating the differentiation of subjacent skeletal elements. We recently identified a series of markers indicating a surprising level of complexity in the organization of this tissue. We will construct transgenic mice in which cre-recombinase is placed under the transcriptional control of promoters driving expression in different perichondrial domains. These will be used to fate-map the cells in different layers of the perichondrium and to test the roles of skeletal regulators in this tissue, including Ihh and Hox genes.