Orofacial clefts are among the most common birth defects in the U.S., occurring in 1/800 live-births. The lifetime cost for medical treatment, educational services and lost productivity averages more than $100,000 per affected person. While a number of contributory genes have been identified, there is a continued need to understand the underlying pathogenetic mechanisms. We identified the first de novo mutations in a novel cytoskeletal SPECC1L gene in patients with oblique facial clefts. Recent identification of SPECC1L mutations in patients with syndromic and nonsyndromic cleft lip and palate suggests that insights into the cellular and molecular mechanism of SPECC1L function will be directly relevant to both severe and common facial malformations. Our data show that Specc1l is expressed in the neural folds at E8.5, including in pre-migratory cranial neural crest cells (CNCCs). Later at E9.5, it is expressed in post-migratory CNCCs, and in the branchial arches at E10.5. To study SPECC1L function, we have generated severe, hypomorphic and conditional mouse alleles of Specc1l deficiency. The severe mouse allele is embryonic lethal at E9.5 with defective neural tube (NT) closure and incomplete delamination of post-migratory CNCCs. The hypomorphic allele shows incompletely penetrant perinatal exencephaly phenotype. Moderate mutants with one hypomorphic and one severe allele show highly penetrant palate closure delay. In addition, SPECC1L-deficient cells show altered adherens junctions (AJs) and reduced PI3K-AKT signaling, both in vitro in cultured cells and in vivo in Specc1l mutant tissue. Modulation of cell-cell contacts is important not only for CNCC delamination from the neuroectoderm, but also for ?collective? migration of CNCCs to their defined destinations. The central hypothesis of this project is that SPECC1L modulates cell adhesion and collective migration by regulating the density of epithelial and mesenchymal cell-cell contacts through PI3K-AKT signaling.
In Aim 1, we propose to investigate the effect of Specc1l dosage on craniofacial development using our hypomorphic, severe, and conditional mouse alleles.
In Aim 2, we will investigate SPECC1L mediation of AJ stability and PI3K-AKT signaling using small molecule modulators of PI3K-AKT pathway. We will also explore the mechanism underlying SPECC1L mediation of AKT stability.
In Aim 3, we will use live-imaging to quantitatively assess changes in collective cell behavior. Analyses will be conducted in ex vivo cultured E8.5 neural plate explants, with CNCCs marked with Wnt1-Cre or Sox10-Cre. Successful completion of these studies will establish novel SPECC1L-based links between AJs, cell polarity and PI3K-AKT signaling during facial morphogenesis and palate closure. Understanding the correlation between SPECC1L dosage or functional deficiency and collective cell migration will provide targets for future therapeutic or preventative strategies against orofacial clefting.
Orofacial clefts are a common birth defect, yet mechanisms underlying their pathogenesis are incompletely understood. We show that level of deficiency of a novel cytoskeletal protein, SPECC1L, may determine the severity of orofacial clefts in humans and mice. SPECC1L regulates cell adhesion and migration by modulating cell-cell communication. Knowledge of this cell-cell communication at the molecular level will help identify potential therapeutic strategies either early in embryonic development or later during the perinatal period to improve postnatal surgical outcome in patients with orofacial clefts.
