In-frame deletion of SPECC1L microtubule association domain results in gain-of-function phenotypes affecting embryonic tissue movement and fusion events.

Patients with autosomal dominant SPECC1L variants show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L variants largely cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1lΔEx4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or omphalocele. Given the clustering of human variants in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1lΔCCD2) resulted in exencephaly, cleft palate and ventral body wall closure defects (omphalocele). Interestingly, exencephaly and cleft palate were never observed in the same embryo. Further examination revealed a narrower oral cavity in exencephalic embryos, which allowed palatal shelves to elevate and fuse despite their defect. In the cell, wild-type SPECC1L was evenly distributed throughout the cytoplasm and colocalized with both microtubules and filamentous actin. In contrast, mutant SPECC1L-ΔCCD2 protein showed abnormal perinuclear accumulation with diminished overlap with microtubules, indicating that SPECC1L used microtubule association for trafficking in the cell. The perinuclear accumulation in the mutant also resulted in abnormally increased actin and non-muscle myosin II bundles dislocated to the cell periphery. Disrupted actomyosin cytoskeletal organization in SPECC1L CCD2 mutants would affect cell alignment and coordinated movement during neural tube, palate and ventral body wall closure. Thus, we show that perturbation of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 variants are gain-of-function.

SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects.

Cleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.