Analyzing the Role of Wnt Signaling in Bone Development

描述（由申请人提供）：WLS/GPR 177是Wnt信号传导所必需的，Wnt信号传导是成骨细胞分化和功能的关键途径，十多年来一直是我们研究的重点。具体地，WLS的缺失使得任何细胞不能分泌任何Wnt蛋白。这种竞争性更新将测试成骨细胞谱系细胞内WLS的作用是否影响骨量。这建立在我们研究Wnt受体Lrp 5和Lrp 6在成骨细胞谱系中建立和维持正常骨量的重要性的工作基础上。位于人类染色体1p31.3上GPR 177/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）显著降低相关。虽然这些研究将WLS确定为建立和维持BMD的候选基因，但需要验证结果并确定分子机制以将信息纳入生物学背景。关键步骤是鉴定基因产物在其中发挥作用以影响BMD的细胞类型。我们将通过创建基因工程小鼠模型来实现这一点，其中Wls缺陷仅限于特定的细胞类型。两个具体目标将测试总体假设，即WLS/GPR 177在成骨细胞中起作用以控制骨形成和/或稳态。具体目标1将集中于检查Wls在分化成骨细胞中的功能。子项目将包括表征与成骨细胞中骨钙素cre介导的Wls条件性缺失相关的表型，通过使用他莫昔芬诱导的cre（Col 1a 1-cre）评估骨骼成熟小鼠成骨细胞中Wls的功能，以及将由于Lrp 5突变而具有高骨量的小鼠与缺乏Wls的小鼠杂交，以检查产生的表型。目的2通过Dermo 1-cre介导的Wls缺失，研究Wls在骨软骨前体细胞中的作用。此外，将比较Wls缺陷型和对照原代颅骨成骨细胞的分化，以评估我们在检查成骨细胞谱系中Wls靶向缺失的小鼠的初步研究中所见的骨量缺陷的基础机制.

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.