Modeling Campomelic Dysplasia Using Induced Pluripotent Stem Cells Identifies a SOX9-WNT Signaling Axis Involved in Epithelial-to-Mesenchymal Transition

Campomelic dysplasia (CD) is a congenital disease causing skeletal defects, such as shortened and angulated long bones, hypoplastic scapulae, hypomineralized thoracic pedicles, and craniofacial defects. Neonatal death often occurs due to respiratory complications caused by underdeveloped tracheobronchial cartilage. Non-skeletal malformations also occur such as sex reversal in XY individuals and cardiac, pancreatic, renal, and sensory organ defects. This doctoral thesis investigates the molecular pathogenesis of CD using induced pluripotent stem cells (iPSCs) as the model. CD is caused by heterozygous mutations in SOX9, the master regulator of chondrogenesis. The pleiotropic effects of these mutations are due to the developmental role of SOX9 in various lineages, which have been uncovered using mouse models and reductionist approaches. In addition, SOX9 regulates certain adult stem cell populations and epithelial-to-mesenchymal transition (EMT) in neural crest development. In order to understand the pathogenesis of CD in humans, iPSCs were generated from two CD patients with distinct SOX9 coding mutations. In preliminary experiments, differentiation towards mesendoderm-derived cartilage or neural crest demonstrated that EMT and differentiation were severely impaired in one of the CD patients. Using a systems level approach, comparative transcriptome analysis of CD iPSCs versus those from healthy individuals undergoing differentiation was combined with published SOX9 chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) data to generate a SOX9 gene regulatory network (GRN). Analysis of the GRN uncovered a SOX9-WNT axis that is imbalanced in this particular CD patient. In testing this network we found that SOX9 overexpression at high levels exacerbated the phenotype but exogenous WNT activation rescued the phenotype. Thus, using CD iPSCs, a critical role for SOX9 in the regulation of the WNT signaling pathway was identified and restoring WNT signaling partially rescues the CD phenotype.Ph.D