Comprehensive investigation of SP7 during the osteoblast-to-osteocyte transition

项目摘要/摘要 许多病理或疾病状况现在可以归因于骨细胞功能障碍。然而，一个有限的 许多富含骨细胞的基因已经在骨骼疾病中进行了研究。我们之前的工作主要集中在 转录因子SP7及其在骨细胞树突形成中的调控作用这项提议的一个主要目标 目的是进一步阐明SP7的成骨功能，破译SP7如何调节成骨细胞的生成。至 为了解决这个根本问题，我开发了一种基于体内和体外的全面方法 方法确定一种成骨缺陷导致的骨细胞SP7R316C突变的影响。结果 从这些方法中，结合单细胞转录组学和互补生物信息学 分析，将阐明人类R316C突变在骨细胞发育中的性质；这包括 确定该突变是否选择性地影响SP7的骨细胞功能，确定直接靶点 受此突变选择性影响的基因，以及R316C如何通过捕获 被该突变阻止成熟的骨细胞亚群。进行转录切分 对发育中的骨细胞进行分析，我将开发新的激光辅助显微切割方法来分离活的 用于单细胞RNA测序的基质包埋细胞。就像大脑中的神经元，骨骼中的骨细胞 通过树枝状连接的广泛网络相互通信。我将执行生物信息学 分析鉴定神经元和骨细胞中表达受限的基因及其在功能骨骼中的作用 的候选共享基因将在体外和体内进行测试。总体而言，这项提案中描述的目标有 很有可能确定骨细胞特异性基因(例如，SP7)在骨骼中的作用，以及揭示 骨细胞特异性基因在人类骨骼疾病中的作用。此外，这项工作可能会导致识别 为确保成骨细胞向骨细胞的转变，治疗学家可以针对新的途径进行研究。 我的长期职业目标是获得一个终身教职的职位，并成功地建立一个实验室，它是 在骨细胞发育和骨骼健康之间架起桥梁的前沿。我预计这一阶段的K99 建议，包括完成R316C突变在小鼠身上的表征和鉴定 受R316C突变影响的直接靶点和新途径，需要1-2年时间，并导致至少一个 高质量的出版物。在K99阶段提供的培训和指导将为我提供强大的 作为一名独立研究员，我继续学习和申请助学金的背景和起点。 接下来的R00阶段将允许我进一步探讨SP7在 成骨细胞生成，以及检测体内神经元-骨细胞共享基因对骨骼的影响。一起， 这些数据将被用来证明R01拨款申请中提出的未来研究的合理性，我预计将在 独立阶段第三年的开始。

Project Summary/Abstract Many pathologic or disease conditions can now be ascribed to disrupted osteocyte functions. However, a limited number of osteocyte-enriched genes have been studied in bone disease. Our previous work focused on the transcription factor SP7 and its role in regulating osteocyte dendrite formation. One major goal of this proposal is to further elucidate the osteocytic function of SP7 and decode how SP7 regulates osteocytogenesis. To address this fundamental question, I have developed a comprehensive approach based on in vivo and in vitro methods to define the effect of an osteogenesis imperfecta-causing SP7 R316C mutation in osteocytes. Results from these approaches, in combination with single-cell transcriptomics and complementary bioinformatic analysis, will illuminate the nature of the human R316C mutation in osteocyte development; this includes determining whether this mutation selectively affects the osteocytic function of SP7, identifying direct target genes that are selectively affected by this mutation, and how R316C influences osteocytogenesis by capturing the osteocyte subpopulations that are blocked by this mutation from maturation. To perform transcriptomic profiling of developing osteocytes, I will develop novel laser-assisted microdissection methods to isolate viable matrix embedded cells for single cell RNA-sequencing. Like neurons in the brain, osteocytes in bone communicate with one another through an extensive network of dendritic connections. I will perform bioinformatic analyses to identify genes with restricted expression in neurons and osteocytes, and the functional skeletal roles of candidate shared genes will be tested in vitro and in vivo. Overall, the aims described in this proposal have strong potential to define the role of osteocyte-specific genes (e.g., SP7) in bone, as well as uncover the contribution of osteocyte-specific genes in human skeletal disease. Moreover, this work may lead to identification of new pathways that can be targeted by therapeutics to ensure the osteoblast-to-osteocyte transition. My long-term career goal is to obtain a tenure-track faculty position and successfully establish a lab that is at the forefront of bridging the gap between osteocyte development and bone health. I expect the K99 phase of this proposal, which includes completing the characterization of R316C mutation in mice and the identification of direct targets and novel pathways affected by the R316C mutation, to take 1-2 years and result in at least one high quality publication. The training and mentorship provided during the K99 phase will prepare me with strong background and starting point for my continuing studies and grant applications as an independent investigator. The following R00 phase of the award will then permit me to further explore the regulation of SP7 during osteocytogenesis, as well as to examine the skeletal impact of neuron-osteocyte shared genes in vivo. Together, these data will be used to justify future studies proposed in an R01 grant application that I expect to submit at the beginning of the third year of the independent phase.