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

成骨细胞向骨细胞转变过程中 SP7 的综合研究

• 批准号: 10569846
• 负责人: Jialiang Wang
• 金额: $ 9.29万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569846
• 关键词: Acceleration; Address; Affect; Applications Grants; Award; Bioinformatics; Biology; Bone Development; Bone Diseases; Bone Resorption; Brain; Cell Culture Techniques; Cell Surface Extensions; Cells; Communication; Complex; Data; Dendrites; Development; Disease; Ensure; Etiology; Faculty; Future; Genes; Genetic; Genetic Transcription; Goals; Grant; Homeostasis; Human; Human Characteristics; In Vitro; Investigation; Knowledge; Lasers; Link; Mentorship; Metabolism; Methods; Microdissection; Modeling; Molecular; Mus; Mutation; Neurons; Neurosciences Research; Osteoblasts; Osteocytes; Osteogenesis; Osteogenesis Imperfecta; Osteoporosis; Pathologic; Pathway interactions; Patients; Pattern; Phase; Phenotype; Positioning Attribute; Process; Publications; Regulation; Regulatory Element; Research Personnel; Role; Signal Pathway; Training; Transgenic Mice; Variant; Work; bone; bone health; career; cell type; gene function; gene regulatory network; genome wide association study; in vitro testing; in vivo; innovation; interdisciplinary collaboration; mouse model; neuropeptide Y; novel; novel therapeutics; osteoblast differentiation; programs; single-cell RNA sequencing; skeletal; skeletal abnormality; skeletal disorder; targeted treatment; tenure track; transcription factor; transcriptome; transcriptomic profiling; transcriptomics

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.

项目摘要/摘要 现在，许多病理或疾病状况可以归因于破坏的骨细胞功能。但是，有限 在骨病中已经研究了富含骨细胞的基因的数量。我们以前的工作着重于 转录因子SP7及其在调节骨细胞树突形成中的作用。该提议的主要目标之一 是为了进一步阐明SP7的骨细胞功能，并解码SP7如何调节骨细胞发生。到 解决这个基本问题，我已经开发了一种基于体内和体外的全面方法 定义成骨的方法在骨细胞中引起骨质不完美的SP7 R316C突变的作用。结果 从这些方法中，结合单细胞转录组学和互补生物学 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，识别直接目标 受此突变选择性影响的基因以及R316C如何通过捕获来影响骨细胞的发生 该突变因成熟而阻止的骨细胞亚群。执行转录组 开发骨细胞的分析，我将开发新型的激光辅助微解剖方法来隔离可行 基质嵌入细胞用于单细胞RNA序列。像大脑中的神经元一样，骨骼中的骨细胞 通过广泛的树突连接网络相互通信。我会表演生物信息 分析以鉴定神经元和骨细胞中表达受限的基因以及功能性骨骼作用 候选共享基因的体外和体内将进行测试。总体而言，本提案中描述的目的 定义骨细胞特异性基因（例如SP7）在骨骼中的作用的强大潜力，并发现 骨细胞特异性基因在人骨骼疾病中的贡献。而且，这项工作可能导致身份证明 可以通过治疗剂来靶向的新途径，以确保成骨细胞向骨细胞的过渡。 我的长期职业目标是获得终身教师职位，并成功建立一个实验室 弥合骨细胞发育与骨骼健康之间的差距的最前沿。我希望这是K99阶段 提案，其中包括完成小鼠R316C突变的表征和鉴定 直接目标和受R316C突变影响的新途径需要1 - 2年，至少导致一个 高质量的出版物。在K99阶段提供的培训和指导将使我为我的强大做好准备 我继续学习的背景和起点，并作为独立研究者提供了授予申请。 然后 骨细胞生成，以及在体内共享基因的神经元骨细胞的骨骼影响。一起， 这些数据将用于证明我希望在R01赠款申请中提出的未来研究合理 独立阶段的第三年开始。