Project 1 Kronenberg

项目总结（见说明）：在骨发育的早期，软骨细胞和成骨细胞谱系的细胞以受调节的方式增殖和分化。甲状旁腺激素相关蛋白（PTHrP）是这些过程的关键调节因子，通过G蛋白偶联受体（PTH/PTHrP受体（PPR））发出信号，以延迟胎儿期软骨细胞的肥大分化。在初步研究中，我们发现，在出生后的生活中，PPR的去除迅速导致生长板的消失（生长板融合）。在目的1中，我们将证实这些发现，使用遗传工具来确定特定G蛋白在介导PPR的出生后作用中的作用，并确定该模型中生长板融合的机制。在目标2中，我们将继续确定软骨细胞肥大的调节方式。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通过停止HDAC 4与MEF 2c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，肢芽和心脏（LBH）在调节软骨细胞肥大中的作用。我们在鸡胚胎肢体中的研究表明，LBH通过抑制转录因子Runx 2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方法敲除软骨细胞（胶原11启动子）和成骨细胞（osterix启动子）中的LBH，以确定LBH在骨发育中的作用。我们将在小鼠基因敲除模型中验证LBH调节肥大，并探索LBH和PTHrP-HDAC 4-MEF 2c轴的作用之间可能的联系。在前一个研究周期中，我们使用了他莫昔芬调节的osterix-cre和胶原l-cre构建体来追踪早期骨发育中的成骨细胞谱系。在目标3中，我们将通过产生由Msx 2和Runx 2启动子驱动的进一步的他莫昔芬调节的cre系来关注成骨细胞谱系的早期步骤。这些小鼠将被用于评估成骨细胞谱系的极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..