SPECC1L-deficient palate mesenchyme cells show speed and directionality defect

Abstract

Clefts of the lip and/or palate (CL/P) are common anomalies that occur in 1/800 live births. Pathogenic SPECC1L variants identified in patients with rare atypical clefts and syndromic CL/P suggest the gene plays a primary role in face and palate development. We have generated Specc1l gene-trap (Specc1lcGT) and truncation (Specc1l{Delta}C510) alleles that cause embryonic or perinatal lethality, respectively. Specc1lcGT/{Delta}C510 compound mutants show delayed and abnormal palatal shelf elevation at E14.5. By E15.5, the mutant shelves do elevate and fuse, however, the palatal rugae form abnormally. Palatogenesis requires extensive mesenchymal remodeling, especially during palatal shelf elevation. We posit that this remodeling involves collective movement of neural crest-derived palatal mesenchyme cells. Live time-lapse microscopy was performed to visualize in vitro wound-repair assays with wildtype and SPECC1L-deficient primary mouse embryonic palatal mesenchyme (MEPM) cells. SPECC1L-deficient MEPM cells consistently showed delayed closure in wound-repair assays. To evaluate which features of cellular movement were responsible, we performed automated particle image velocimetry (PIV) and manual cell tracking. The analyses revealed that both cell speed and directionality are disrupted in SPECC1L-deficient cells compared to controls. To determine if primary MEPM cells can move collectively, we assayed stream formation, which is a hallmark of collective movement. Indeed, MEPM cultures displayed correlated movement of neighboring cells. Importantly, correlation length was reduced in SPECC1L-deficient cultures, consistent with a role for SPECC1L in collective migration. Furthermore, we demonstrated that activation of the PI3K-AKT pathway with the 740Y-P small molecule can rescue the wound-closure delay in SPECC1L-deficient MEPM cells. Cell tracking analyses showed that this rescue was due to both increased speed and improved directionality. Altogether, our data showed a novel role for SPECC1L in guided movement through modulation of PI3K-AKT signaling.