Proect 2 - McMahon

项目SuIVIMARY(请参阅说明)： 一个矿化的内骨骼支撑着哺乳动物的身体。骨骼也是重要的矿物质储存库，并培养非骨骼细胞类型，特别是造血系统的干细胞。骨骼异常是最常见的出生缺陷之一，骨折修复问题出现在不愈合的骨折中，骨骼疾病在老龄化人口中越来越普遍。两种关键的生物合成细胞类型产生哺乳动物骨骼的特定细胞外基质：分泌软骨的软骨细胞和分泌骨骼的成骨细胞。这些细胞有不同的来源(神经脊、体细胞和侧板中胚层)，并通过不同的过程生成骨：直接来自间充质干细胞(膜骨)或通过软骨模板(软骨内骨)生成骨。小鼠和人类的遗传学已经确定了一些关键的信号通路，以及三个关键的转录因子，它们是软骨细胞(Sox9)和成骨细胞(Runx2和OSx)程序的主要调节因子。Sox9和Runx2的单蛋白充足与人类骨骼缺陷有关。虽然这些因素的重要性得到了很好的记录，但他们的监管行动却没有。为此，我们将确定支持软骨细胞和成骨细胞发育中每一个关键骨骼决定因素的调控作用的基因调控网络。具体目标1：我们将在小鼠胚胎干细胞中使用基因打靶技术来创造内源性Sox9、Runx2和OSX蛋白被修饰的小鼠，方法是在它们的C末端添加一个小肽。这一表位将使随后通过共同的方法分析每个因子的表达和活性成为可能。具体目标2：将对每个标记蛋白进行ChlP-seq分析，以直接确定它们在原代软骨细胞或成骨细胞中的DNA靶标。具体目标3：来自正常软骨细胞和成骨细胞或Sox9、Runx2和OSX基因敲除后的相同群体的基于微阵列的转录图谱数据将与ChlP-seq数据相交，以预测每个调控因子作用的靶点。选定的调控模型将在转基因小鼠研究中进行检验。这些数据将为驱动哺乳动物骨骼形成的基因调控网络提供第一个基因组规模的洞察。

PROJECT SUIVIMARY (See instructions): A mineralized endoskeleton supports the mammalian body. Bone also serves as a vital mineral repository and nurtures non-skeletal cell types, notably stem cells of the hematopoietic system. Skeletal anomalies are amongst the most common birth defects, problems in fracture repair are observed in non-union fractures and bone disorders are increasingly prevalent in an ageing population. Two key biosynthetic cell types generate the specific extracellular matrix of the mammalian skeleton: cartilage-secreting chondrocytes and bone secreting osteoblasts. These cells have diverse origins (neural crest, somitic and lateral plate mesoderm), and generate bone by distinct processes: directly from a mesenchymal stem cell (membranous bone) or via a cartilage template (endochondral bone). Mouse and human genetics have identified a number of critical signaling pathways, and three key transcription factors that are master regulators of chondrocyte (Sox9) and osteoblast (Runx2 and Osx) programs. Haploin sufficiency for Sox9 and Runx2 associates with human skeletal deficiencies. While the importance of these factors is well documented, their regulatory actions are not. To this end, we will determine the gene regulatory networks that underpin the regulatory actions of each of these critical skeletal determinants in chondrocyte and osteoblast development. Specific Aim 1: We will use gene-targeting in mouse embryo stem cells to create mice whose endogenous Sox9, Runx2 and Osx proteins are modified by appending a small peptide at their C-terminus. This epitope will enable subsequent analysis of the expression and activity of each factor through common approaches. Specific Aim 2: ChlP-seq analysis will be performed with each tagged protein to directly identify their DNA targets in primary chondrocytes or osteoblasts. Specific Aim 3: Data from microarray-based transcriptional profiling of normal chondrocytes and osteoblasts, or these same populations following knock-down of Sox9, Runx2 and Osx, will be intersected with ChlP-seq data to predict the targets of each regulatory factors actions. Selected regulatory models will be examined in transgenic mouse studies. These data will provide the first genome scale insight into the gene regulatory networks driving mammalian skeletogenesis.