Functions of PTH/PHTrP Receptor, PTHrP and PTH in vivo

甲状旁腺激素相关蛋白（PTHrP）是软骨细胞和成骨细胞增殖和分化的重要调节因子。本项目将探讨PTHrP在骨发育过程中作用的分子机制。它还将表征与软骨细胞分化相关的基因，并将表征体内成骨细胞谱系细胞的分化。PTHrP抑制软骨细胞中Runx 2的表达。目的分析这种抑制在介导甲状旁腺激素受体（PTHrP）延迟软骨细胞分化中的作用。Indian hedgehog（Ihh）刺激软骨细胞中PTHrP的合成。为了确定Ihh刺激PTHrP表达的作用是否是对产生PTHrP的软骨细胞的直接作用，将在Aim IB中的嵌合生长板中分析缺失smoothened的软骨细胞中PTHrP的表达。为了鉴定介导PTHrP作用的基因，在短暂的PTH治疗后对E14.5小鼠跖骨进行微阵列分析。 将进行，并在目标1C中分析选择基因的作用。为了表征与软骨细胞分化相关的遗传变化，将在Aim ID中对从离散软骨细胞组扩增的RNA进行微阵列分析，并分析选择基因的作用。成骨细胞系细胞分化的早期阶段和这些阶段细胞的增殖能力在体内知之甚少。在目标II中，将使用位点特异性重组，在成骨细胞发育的早期阶段具有活性的启动子，以研究这些细胞的特性。连接活性重组酶将用于标记离散的细胞群，并确定这些细胞在胎儿期和成年期的自我更新能力。微阵列将用于表征参与早期 成骨细胞谱系的步骤。PTH/PTHrP受体在胎儿成骨细胞发育过程中的作用将使用PTH/PTHrP受体的突变等位基因进行评估。

Parathyroid hormone-related protein (PTHrP) is an important regulator of the proliferation and differentiation of both chondrocytes and osteoblasts. This project will explore the molecular mechanisms of PTHrP action during bone development. It will also characterize the genes associated with differentiation of chondrocytes and will characterize the differentiation of cells of the osteoblast lineage in vivo. PTHrP suppresses the expression of Runx2 in chondrocytes. Aim IA will analyze the role of this suppression in mediating the action of PTHrP to delay the differentiation of chondrocytes. Indian hedgehog (Ihh) stimulates the synthesis of PTHrP in chondrocytes. To determine whether the action of Ihh to stimulate PTHrP expression is a direct action on PTHrP-producing chondrocytes, the expression of PTHrP in chondrocytes missing smoothened will be analyzed in chimeric growth plates in Aim IB. To identify genes mediating the action of PTHrP, microarray analysis of E14.5 mouse metatarsals after brief PTH treatment will be performed, and the roles of select genes will be analyzed in Aim 1C. To characterize the genetic changes associated with differentiation of chondrocytes, microarray analysis of RNA amplified from discrete groups of chondrocytes will be conducted in Aim ID, and the roles of select genes will be analyzed. The early stages of differentiation of osteoblast lineage cells and the proliferative capacity of cells at these stages are poorly understood in vivo. In Aim II, site-specific recombination will be used, with promoters active in early stages of osteoblast development, to study the properties of such cells. Conditionally active recombinases will be used to mark discrete cell populations and determine the self-renewing capacity of these cells both in fetal life and adulthood. Microarrays will be used to characterize genes involved in early steps of the osteoblast lineage. The role of the PTH/PTHrP receptor during fetal osteoblast development will be assessed using a floxxed allele of the PTH/PTHrP receptor.