PROJECT SUMMARY/ABSTRACT – PROJECT 2 Osteogenesis imperfecta (OI) is usually caused by mutations in type I collagen genes and more rarely, by recessive mutations in genes involved in type I collagen processing (e.g., CRTAP). Because type I collagen is the most abundant matrix component secreted by bone forming osteoblasts, these cells have been considered the primary mediators of the disease presentation. Consequently, a possible role for osteocytes in the skeletal manifestations of OI has largely been overlooked. Thus, the overall goal of Project 2 is to elucidate the contribution of osteocytes to bone fragility in OI and identify changes in the osteocytic transcriptome that may lead to new therapeutic targets. Our preliminary data show that osteocytes express as much type I collagen as osteoblasts, suggesting that alterations in type I collagen, in addition to affecting osteoblasts, may also negatively impact osteocytes. In support of this, we found that the osteocyte transcriptomes from 2 mouse models of OI were significantly dysregulated compared to control mice. Moreover, osteocytes actively express all 19 genes associated with dominant and recessive forms of OI. However, it is unknown to what extent altered collagen in OI affects the function of osteocytes, and whether their transcriptional dysregulation is a direct consequence of the altered collagen expressed by osteocytes or due to their interaction with altered collagen produced by osteoblasts. Our central hypothesis is that osteocytes contribute to the bone fragility observed in OI and that expression of altered type I collagen in osteocytes disrupts osteocyte function and, in turn, alters the osteocytic expression of genes that regulate bone homeostasis. To test this hypothesis, we generated a novel knock-in mouse model in which Cre-mediated recombination can be used to express an OI glycine substitution (p.Gly1146Arg) mutation in the endogenous Col1a1 gene. We will breed this mouse with EIIa-Cre and Sost-Cre mice, to express the mutation globally or specifically in osteocytes, and then compare the skeletal phenotype of offspring using dual X-ray absorptiometry, micro-CT, bone biomechanics, bone histomorphometry, and Q-PCR to assess what aspects of the OI skeletal phenotype are reproduced in osteocyte-specific knock-in OIOCY mice (Aim 1). We will also generate a conditional allele for a collagen-modifying gene (Crtap) and determine the skeletal consequences of its deletion in osteocytes versus global inactivation using the methodologies described above (Aim 2). To determine if osteocyte function in OI is affected because they express abnormal collagen, because they interact with a defective bone matrix secreted by osteoblasts, or both, we will identify osteocyte transcriptome differences between osteocyte-specific Crtap KO, global Crtap KO, and WT mice using both bulk and single-cell RNA-seq (Aim 3). These studies will provide direct evidence of the extent to which altered collagen a...