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.

项目总结/摘要 现在许多病理或疾病状况可归因于骨细胞功能的破坏。然而，有限的 在骨疾病中已经研究了许多骨细胞富集基因。我们以前的工作集中在 转录因子SP7及其在调节骨细胞树突形成中的作用。这项提案的一个主要目标是 目的是进一步阐明SP7的骨细胞功能，并阐明SP7是如何调节骨细胞发生的。到 为了解决这个基本问题，我开发了一种基于体内和体外的综合方法， 方法来确定骨细胞中导致骨生成障碍的SP7 R316C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，将阐明骨细胞发育中人类R316C突变的性质;这包括 确定该突变是否选择性地影响SP7的骨细胞功能， 基因选择性地受到这种突变的影响，以及R316C如何通过捕获 被这种突变阻止成熟的骨细胞亚群。进行转录组学 分析发育中的骨细胞，我将开发新的激光辅助显微切割方法，以分离可行的 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 通过广泛的树突状连接网络相互交流。我会进行生物信息学 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 将在体外和体内测试候选共享基因。总的来说，本提案所述的目标 确定骨细胞特异性基因的作用的强大潜力（例如，SP7）在骨骼中，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 新的途径，可以有针对性的治疗，以确保成骨细胞到骨细胞的过渡。 我的长期职业目标是获得终身教职，并成功建立一个实验室， 弥合骨细胞发育和骨骼健康之间差距的前沿。我希望K99阶段 该提案包括完成小鼠R316C突变的表征和 直接靶点和新途径受R316C突变影响，需要1 - 2年，并导致至少一个 高质量出版物。在K99阶段提供的培训和指导将使我具备强大的 作为一名独立调查员，我继续学习和申请补助金的背景和起点。 接下来的R00阶段的奖励将允许我进一步探索SP7的监管， 骨细胞生成，以及检查体内神经元-骨细胞共享基因对骨骼的影响。在一起， 这些数据将被用于证明未来的研究建议在R01赠款申请，我预计提交 独立第三年的开始。