Analyzing the Role of Wnt Signaling in Bone Development

描述(申请人提供)：WLS/GPR177是Wnt信号通路所特需的，Wnt信号通路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成骨细胞系细胞内的WLS的活动是否影响骨量。这项工作建立在我们研究Wnt受体LRP5和LRP6在建立和维持成骨细胞谱系中正常骨量方面的重要性的基础上。位于人类染色体1p31.3上GPR177/Wnless基因内含子内的两个单核苷酸多态(SNPs)与腰椎和股骨颈骨密度(BMD)显著降低相关。虽然这些研究确认WLS是建立和维持BMD的候选基因，但结果还需要验证，并需要确定将信息纳入生物学背景的分子机制。关键的一步是确定基因产物在其中发挥作用的细胞类型(S)，以影响骨密度。我们将通过创造基因工程小鼠模型来做到这一点，在该模型中，WLS缺陷仅限于特定细胞类型。两个特定的目标将检验WLS/GPR177在成骨细胞中发挥作用以控制骨形成和/或内稳态的首要假设。具体目标1将重点研究WLS在分化的成骨细胞中的功能。Subaims将包括表征成骨细胞中与骨钙素cre介导的条件删除wls相关的表型，通过使用他莫昔芬诱导的cre(Col1a1-cre)来评估骨骼成熟小鼠成骨细胞中wls的功能，以及将由于LRP5突变而具有高骨量的小鼠与那些缺乏wls的小鼠杂交，以检查所产生的表型。目的2通过使用Dermo1-cre介导WLS缺失，研究WLS在骨软骨前体细胞中的作用。此外，我们还将比较WLS缺陷和对照原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不足的机制。

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.