Project 1 Kronenberg

项目概述（见说明书）：在骨发育早期，软骨细胞和成骨细胞谱系以一种受调控的方式增殖和分化。甲状旁腺激素相关蛋白（PTHrP）是这些过程的关键调节剂，通过G蛋白偶联受体PTH/PTHrP受体（PPR）发出信号，延缓胎儿期软骨细胞的肥厚分化。在初步研究中，我们发现，在出生后的生活中，PPR的移除会迅速导致生长板的消失（生长板融合）。在Aim 1中，我们将证实这些发现，使用遗传学工具确定特定G蛋白在介导PPR出生后行为中的作用，并确定该模型中生长板融合的机制。在Aim 2中，我们将继续确定调节软骨细胞肥大的方式。在第一个子目标中，我们将在体内和骨外植体中使用遗传工具来测试PTHrP通过阻止HDAC4与MEF2c的相互作用来调节肥大的假设。在第二个子目标中，我们将研究一种新的转录调节因子肢体芽和心脏（LBH）在调节软骨细胞肥大中的作用。我们对鸡胎儿肢体的研究表明，LBH通过抑制转录因子Runx2的表达来调节软骨细胞和成骨细胞的发育。在这里，我们将使用cre-lox方法将LBH从软骨细胞（胶原11启动子）和成骨细胞（成骨启动子）中敲除，以确定LBH在骨骼发育中的作用。我们将在小鼠敲除模型中验证LBH调节肥大，并探索LBH的作用与PTHrP-HDAC4-MEF2c轴之间可能的联系。在之前的资助周期中，我们使用了他莫昔芬调节的osterix-cre和胶原l-cre构建物来追踪早期骨发育中的成骨细胞谱系。在Aim 3中，我们将通过生成由Msx2和Runx2启动子驱动的tamixofen调控的cre系来关注成骨细胞谱系的早期步骤。这些小鼠将被用来评估成骨细胞早期细胞的作用和调控。评估成骨细胞谱系的非常早期细胞的作用和调节。

PROJECT SUMMARY (See instructions): Early in bone development, chondrocytes and cells of the osteoblast lineage proliferate and differentiate in a regulated fashion. Parathyroid hormone-related protein (PTHrP), a key modulator of these processes, signals through a G protein-coupled receptor, the PTH/PTHrP receptor (PPR) to delay hypertrophic differentiation of chondrocytes in fetal life. In preliminary studies we have found that in postnatal life, the removal of the PPR leads quickly to the disappearance of the growth plate (growth plate fusion). In Aim 1 we will confirm these findings, use genetic tools to determine the roles of specific G proteins in mediating the postnatal actions of the PPR, and determine the mechanism of growth plate fusion in this model. In Aim 2, we will continue to determine the ways that chondrocyte hypertrophy is regulated. In the first sub-aim we will use genetic tools both in vivo and in bone explants to test the hypothesis that PTHrP regulates hypertrophy by stopping the interaction of HDAC4 with MEF2c. In the second sub-aim, we will study the role of a novel transcriptional regulator, limb bud and heart (LBH) in regulating chondrocyte hypertrophy. Our studies in fetal chick limbs have demonstrated that LBH regulates both chondrocyte and osteoblast development by suppressing expression of the transcription factor, Runx2. Here we will use the cre-lox approach to knock LBH out of chondrocytes (collagen 11 promoter) and osteoblasts (osterix promoter) to determine to roles of LBH in bone development. We will verify in the mouse knockout model that LBH regulates hypertrophy and explore possible connections between the actions of LBH and the PTHrP-HDAC4-MEF2c axis. In the previous grant cycle, we used tamoxifen-regulated osterix-cre and collagen l-cre constructs to trace the osteoblast lineage in early bone development. In Aim 3, we will focus on earlier steps of the osteoblast lineage by generating further tamixofen-regulated cre lines driven by the Msx2 and Runx2 promoters. These mice will be used to assess the roles and regulation of very early cells of the osteoblast llneage.assess the roles and regulation of very early cells of the osteoblast lineage..