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

Abstract

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, wildtype 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.

Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos

“…We found that almost 39% of C57BL/6J female embryos had not completed PS elevation by E14.25, whereas 100% of C57BL/6J male embryos showed completed PS elevation. We also recently reported a mouse model ( Specc1l ΔCCD2 ) on a mostly C57BL/6J background (N3 generation) mixed with some FVB/NJ that the cleft palate phenotype in heterozygotes was more prevalent in female (17%) vs. male (5%) embryos (Goering et al 2021b). In fact, we saw a drastic decrease in Specc1l ΔCCD2/+ heterozygous females as we further backcrossed to C57BL/6J background, with almost no Specc1l CCD2/+ females at weaning in the N7 generation.…”

“…We recently reported that deficiency of Specc1l resulted in PS elevation delay, but the PS shelves still eventually elevated and fused (Goering et al 2021b;Hall et al 2020).…”

“…We recently reported that deficiency of Specc1l resulted in PS elevation delay, but the PS shelves still eventually elevated and fused (Goering et al 2021b; Hall et al 2020). The PS elevation delay provides a model for nonsyndromic cleft palate where a delay in elevation can be considered a sensitized background that combined with additional negative genetic or environmental factors, may put the affected individual above the threshold for isolated cleft palate.…”

To Stick or Not to Stick: Adhesions in Orofacial Clefts

“…However, mice homozygous for a truncation allele showed an altered palatal rugae phenotype with oral adhesions and a reduction in interferon regulatory factor 6 (IRF6) in these structures, but no cleft palate [ 127 ]. Homozygotes for in-frame deletions in the second coiled-coil domain resulted in exencephaly, cleft palate, and ventral body wall closure defects [ 128 ], demonstrating the essential functional role of the coiled-coil domain. In vitro, functional studies demonstrated that SPECC1L colocalized with microtubules and filamentous actin in palatal mesenchymal cells [ 126 ].…”

“…Deletions or mutations in the coiled-coil domain severely affected its ability to associate with microtubules, reducing its ability to traffic in the cells and resulting in a perinuclear accumulation. This uneven distribution of SPECC1L in the cells resulted in increased actin and non-muscle myosin II bundles at the cell periphery [ 128 ]. Ultimately, the disrupted actomyosin cytoskeleton would affect cell alignment and coordinated movement required for elevation and fusion of the palatal shelves.…”