ABSTRACT: PROJECT III, Systems Biology of Bone in Coronal Nonsyndromic Craniosynostosis The cranial bones are separated by sutures with mesenchymal cells that differentiate into osteoblasts at osteogenic fronts to produce bone. Abnormal bone development in craniosynostosis (CS) causes the premature fusion of calvarial bones. Mutations in more than 40 candidate genes are known to cause CS, usually involving the coronal suture. However, very little is known about normal osteogenesis or coronal nonsyndromic craniosynostosis (cNSC) that accounts for about a quarter of all CS cases. High-throughput technologies have generated data from genome-wide association and expression studies, but only a few regions of the genome and pathways involved in cNSC have been discovered. These large datasets make it feasible to apply systems biology approaches to increase our understanding of the molecular basis of normal osteogenesis and cNSC. Our objective is to study molecular networks involved in the development of normal bone and in cNSC using a novel systems biologic approach. We hypothesize that there will be key modules and networks that explain the development of normal osteogenesis and cNSC. Our specific aims are to: Specific Aim (SA) 1) Generate high-throughput datasets of RNA expression on human mesenchymal stem cells (MSCs), preosteoblasts, and osteoblasts derived from induced pluripotent stem cells (iPSCs) of 100 cNSC patients and 100 normals. For preliminary results, we have similar genomic and expression data on 50 human osteoblast cell cultures derived from surgically-removed cNSC sutures and 50 control calvarial samples of postnatal development. Additional molecular expression data will be generated for three developmental stages of normal bone and cNSC from MSCs, preosteoblasts, and osteoblasts derived from 200 iPSC cell lines. This information will give us insight into genetic regulation of gene expression and guide our network construction. SA2) Generate a predictive network framework for elucidating the pathogenesis of cNSC. We will use state-of-the-art and innovative top-down and bottom-up approaches to generate predictive models of the regulatory network of the mesenchymal-osteogenic lineage. SA3) Validate key driver genes and network predictions. We will knockdown or overexpress driver genes using shRNA and cDNA expression constructs transfected into iPSC-differentiated MSCs/osteoblast cell cultures and mouse calvarial cultures to validate novel and important causal relationships. Gene-editing by TALENs will be used to correct select lesions. This study will have a significant impact in identifying new candidate genes, pathways, and networks involved in normal osteogenesis and cNSC as well as other skeletal dysplasias, and will lead to novel therapeutic strategies to ameliorate these conditions. Synergy: The genomic and expression data for the MSCs, preosteoblasts, and osteoblasts will be generated by Molecular/Analytic Core B. Data from SA 1 and new candidate genes and pathways found in Projects I & II will contribute directly to the generation of a regulatory network for bone development and cNSC.