The role of primary cilia and C2cd3 in craniofacial skeletogenesis

Primary cilia are ubiquitous, microtubule-based extensions that transduce molecular signals within a cell. Defects in primary cilia result in ciliopathies, a pleiotropic group of debilitating, and sometimes life-threatening disorders. One third of ciliopathies are defined by severe craniofacial anomalies. Currently there are no recognized treatments for these patients, mostly because we have an incomplete understanding of how the cilium integrates cellular/molecular signals and influences cell behaviors. Oral-facial-digital syndrome 14 (OFD14) is a human ciliopathy that is caused by mutations in the centriolar protein, C2 Calcium-Dependent Domain Containing 3 (C2CD3) and is characterized by multiple craniofacial skeletal anomalies including cleft palate and micrognathia. Our previous work revealed hypoplastic cranial neural crest cell (NCC) derived skeletal elements in OFD14 models; however, the mechanism of how C2cd3 functions in this context is unknown. Based on the known role of C2cd3 in ciliogenesis, its localization to the centriole, and the presence of numerous C2 protein binding domains we hypothesize that the craniofacial phenotypes present in OFD14 are molecularly due to impaired to Hh/Gli-driven NCC skeletal differentiation and cellularly due to impaired association between the centriole and actin cytoskeleton. To test these hypotheses, we will utilize knockdown and conditional knockout transgenic murine lines (C2cd3Ex2; C2cd3Exon4-5fl/fl; C2cd3Exon9fl/fl) as well as human induced pluripotent stem cells (hIPSCs) that either lack C2CD3 (C2CD3-/-) or carry OFD14 causing C2CD3 variants (C2CD3Trp65Cys, C2CD3Ile477*). Experiments in this proposal will (Aim 1) determine if skeletal differentiation is impaired in hIPSC-derived NCCs carrying OFD14 causing C2CD3 variants, (Aim 2) determine if C2CD3 functions to anchor the centriole to the actin cytoskeleton and if OFD14 causing C2CD3 variants impair this association, and (Aim 3) test if C2cd3 mutations and OFD14 causing C2CD3 variants impair Gli3/DNA and Gli3/co-factor binding, and subsequent Gli3 transcription. Together these studies will address both the cellular and molecular etiology of the human ciliopathy OFD14 and identify possible areas of therapeutic intervention for craniofacial phenotypes that arise in all ciliopathies. Project Number: 1R01DE034357-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Samantha Brugmann | Institution: CINCINNATI CHILDRENS HOSP MED CTR, CINCINNATI, OH | Award Amount: $646,212 | Activity Code: R01 | Study Section: Skeletal Biology Development and Disease Study Section[SBDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11244048