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

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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.