Characterization of SPECC1L Function in Palatogenesis
Hall, Everett Gordon
University of Kansas
Anatomy & Cell Biology
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Orofacial clefts are among the most common congenital birth defects, occurring in as many as 1 in 800 births worldwide. Genetic and environmental factors contribute to the complex etiology of these anomalies. SPECC1L encodes a cytoskeletal protein with roles in adhesion, migration, and cytoskeletal organization. SPECC1L mutations have been identified in patients with atypical clefts, Opitz G/BBB syndrome, and Teebi hypertelorism syndrome. Our lab has previously shown that knockout of Specc1l in mice with gene trap alleles results in early embryonic lethality with defects in neural tube closure and neural crest cell delamination, as well as reduced PI3K-AKT signaling. However, the early lethality phenotype rendered these models incapable of recapitulating the human anomalies. To validate a role for SPECC1L in palatogenesis, we generated additional gene trap and truncation mutant Specc1l alleles. Specc1lgenetrap/truncation compound heterozygote embryos survive to the perinatal period, allowing analysis at later developmental stages. Examination of compound mutant palates revealed a delay in palate elevation, abnormal oral adhesions, and ectopic expression of adherens junction protein β-catenin at the apical surface of oral epithelial periderm cells, as well as a global increase in actin filament staining. We also establish that transcription factor IRF6, which has a causative role in many cases of orofacial clefting, is required for SPECC1L expression in the palate. After establishing a role for SPECC1L in the palatal epithelium, we asked if it also acted in the migration of palatal mesenchyme. Indeed, time-lapse imaging analysis of primary mouse embryonic palatal mesenchyme (MEPM) cells from Specc1lgenetrap/truncation mutant embryos show defects in migration. We also demonstrate that activation of the PI3K-AKT pathway with a small molecule activator improves mutant wound closure speed. Furthermore, random 2-D motility cultures revealed that MEPM cells have attributes of collective migration that are reduced in mutant cells. Lastly, we generated a mouse allele with the T397P point mutation, located in the second coiled-coil domain (CCD2) of SPECC1L, that was previously identified in patients with Opitz G/BBB syndrome. Embryos with this allele displayed omphalocele and completely penetrant cleft palate defects that recapitulated the phenotype of patients with SPECC1L mutations. Together, these data identify roles for SPECC1L in palate epithelial integrity and mesenchymal remodeling, and thus firmly establish a requirement for SPECC1L in palatogenesis.
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