Analyzing the Role of Wnt Signaling in Bone Development

描述（由申请人提供）：Wnt信号通路是成骨细胞分化和功能的关键通路，WLS/GPR177是我们十多年来研究的重点。具体来说，WLS的缺失使任何细胞都不能分泌任何Wnt蛋白。这种竞争性更新将测试WLS在成骨细胞谱系中的作用是否影响骨量。这建立在我们研究Wnt受体Lrp5和Lrp6在成骨细胞谱系中建立和维持正常骨量的重要性的基础上。位于人类染色体1p31.3上GPR177/Wntless基因（WLS）内含子内的两个单核苷酸多态性（snp）与腰椎和股骨颈骨矿物质密度（BMD）的显著降低有关。虽然这些研究确定WLS是建立和维持骨密度的候选基因，但这些结果需要得到验证，并确定分子机制以将这些信息纳入生物学背景。关键的一步是确定基因产物影响骨密度的细胞类型。我们将通过创建基因工程小鼠模型来做到这一点，其中Wls缺陷仅限于特定的细胞类型。两个具体目标将验证WLS/GPR177在成骨细胞内控制骨形成和/或体内平衡的总体假设。特异性目的1将集中于研究Wls在分化成骨细胞中的功能。亚目标将包括表征与骨钙素介导的成骨细胞中Wls条件缺失相关的表型，通过使用他莫昔芬诱导的cre （Col1a1-cre）评估骨骼成熟小鼠成骨细胞中Wls的功能，并将由于Lrp5突变导致的高骨量小鼠与缺乏Wls的小鼠杂交以检查所产生的表型。目的2将通过使用Dermo1-cre介导的Wls缺失来研究Wls在骨软骨祖细胞中的作用。此外，我们将比较Wls缺失和控制的初代颅骨成骨细胞的分化，以评估我们在对成骨细胞谱系中Wls靶向缺失的小鼠进行的初步研究中看到的骨量缺失的机制。

DESCRIPTION (provided by applicant): WLS/GPR177 is specifically required for Wnt signaling, a key pathway in osteoblast differentiation and function and that has been the focus of our research for more than a decade. Specifically, deletion of WLS makes any cell incapable of secreting any Wnt protein. This competitive renewal will test whether the actions of WLS within cells of the osteoblast lineage affect bone mass. This builds on our work examining the importance of the Wnt receptors, Lrp5 and Lrp6, in establishing and maintaining normal bone mass within the osteoblast lineage. Two single nucleotide polymorphisms (SNPs) located within an intron of the GPR177/Wntless gene (WLS) on human chromosome 1p31.3 are associated with significant reductions in bone mineral density (BMD) in the lumbar spine and femoral neck. While these studies identify WLS as a candidate gene in establishing and maintaining BMD, the results need to be validated and a molecular mechanism determined to put the information into biological context. A key step is to identify the cell type(s) in which the gene product functions o impact BMD. We will do this by creating genetically engineered mouse models in which Wls deficiency is restricted to specific cell types. Two specific aims will test the overarching hypothesis that WLS/GPR177 functions within osteoblasts to control bone formation and/or homeostasis. Specific Aim 1 will focus on examining Wls function in differentiated osteoblasts. Subaims will include characterizing the phenotypes associated with Osteocalcin-cre- mediated conditional deletion of Wls in osteoblasts, assessing the function in Wls in osteoblasts of skeletally mature mice by using a tamoxifen-inducible cre (Col1a1-cre), and crossing mice with high bone mass due to an Lrp5 mutation to those lacking Wls to examine the resulting phenotype. Aim 2 will examine the role of Wls within osteochondral progenitors by using Dermo1-cre mediate Wls deletion. In addition, differentiation of Wls- deficient and control primary calvarial osteoblasts will be compared to assess mechanisms which underlie the deficiencies in bone mass we have seen in our preliminary studies examining mice with targeted deletions of Wls in the osteoblast lineage.