Role of Indian hedgehog in endochondral Bone Formation

描述（由申请人提供）：印度刺猬（Ihh）在骨骼发生过程中具有多种功能。缺乏Ihh基因的小鼠表现出严重的骨骼异常，包括软骨细胞增殖明显减少和异常成熟，缺乏成熟的成骨细胞。由于Ihh及其受体smoothened（smo）在软骨细胞和成骨细胞中表达，目前的动物模型不能提供足够的信息，无论是Ihh对成骨细胞有直接影响，还是在软骨内骨化过程中对骨的影响是间接介导的。在该授权申请中，我们提出在体内选择性地消融来自出生前和出生后的II型胶原表达细胞以及来自X型胶原表达细胞的子集的Ihh基因。此外，我们将选择性地从软骨细胞中删除smo并比较表型。这将使我们能够确定软骨细胞衍生的Ihh对软骨内骨形成的具体作用。我们计划使用我们产生的动物，并与从我们的合作者那里获得的col 2alpha 1-cre、col 2alpha 1-creER* 和col 10alpha 1-cre小鼠杂交。在具体目标1中，我们建议在出生前选择性地从软骨细胞中删除Ihh或smo基因，并分析和比较突变表型以确定Ihh在软骨内骨形成过程中的作用。在具体目标2中，我们将分析出生后从软骨细胞中选择性删除Ihh基因的小鼠。我们将利用他莫昔芬诱导的ere，这是在II型胶原启动子的控制下，以确定急性影响IHH对已建立的软骨内骨。在特定目标3中，将生成亚型小鼠模型，其中仅删除生长板中的软骨细胞亚群以表达Ihh。为此目的，将X型胶原蛋白cre基因敲入小鼠与Ihh动物杂交。使用该模型，我们将能够检测软骨细胞对Ihh表达降低的反应。我们预计这些小鼠的表型不太严重，并将在II型胶原和X型胶原表达细胞中缺乏Ihh基因的动物之间进行比较分析。在这项资助申请中提出的创新小鼠模型将产生一种新的体内系统，这将有助于我们阐明Ihh的作用，不仅在软骨内骨形成过程中，而且在维持软骨内骨中。这些新的信息将为我们理解Ihh如何调节软骨细胞和成骨细胞的发育和功能提供一个更安全的框架。因此，这种体内分子研究可能导致发现其他涉及Ihh基因的人类骨骼疾病，并发现旨在调节骨骼异常的新治疗策略。

DESCRIPTION (provided by applicant): Indian hedgehog (Ihh) has multiple functions during skeletogenesis. Mice lacking the Ihh gene exhibit severe skeletal abnormalities, including markedly reduced chondrocyte proliferation and abnormal maturation, with absence of mature osteoblasts. Since Ihh and its receptor, smoothened (smo), are expressed in chondrocytes as well as in osteoblasts, current animal models do not provide sufficient information whether Ihh has a direct effect on osteoblasts, or whether the effects on bone are indirectly mediated through chondrocytes during endochondral ossification. In this grant application we propose to selectively ablate, in vivo, the Ihh gene from collagen type II expressing cells, pre- and postnatally, and from a subset of collagen type X expressing cells. In addition, we will delete smo selectively from chondrocytes and compare the phenotypes. This will allow us to determine the specific role of chondrocyte-derived Ihh on endochondral bone formation. We plan to use animals that we generated and cross bred with col2alpha1-cre, col2alpha1-creER*, and col10alpha1-cre mice, obtained from our collaborators. In Specific Aim 1 we propose to selectively delete either the Ihh or the smo gene from chondrocytes, before birth, and to analyze and compare the mutant phenotypes to define the role of Ihh during the process of endochondral bone formation. In Specific Aim 2 we will analyze mice in which the Ihh gene will be selectively deleted from chondrocytes after birth. We will take advantage of a tamoxifen-inducible ere that is under the control of the collagen type II promoter to determine the acute effects of Ihh on established endochondral bone. In Specific Aim 3 a hypomorph mouse model will be generated, in which only a subset of chondrocytes in the growth plate will be deleted for the expression of Ihh. For this purpose, collagen type X cre knock-in mice will be bred with floxed Ihh animals. Using this model we will be able to detect the response of chondrocytes to reduced Ihh expression. We expect a less severe phenotype in these mice, and will perform a comparative analysis between animals lacking the Ihh gene in collagen type II and collagen type X expressing cells. The innovative mouse models proposed in this grant application will generate a novel in vivo system that will help us to clarify the role of Ihh, not only during endochondral bone formation, but also in maintaining endochondral bones. This new information will give us a more secure framework for understanding how Ihh regulates chondrocyte and osteoblast development and function. Such in vivo molecular studies, could, therefore, lead to the discovery of other human skeletal diseases involving the Ihh gene and to novel therapeutic strategies aimed at modulating skeletal anomalies.