SPECC1L deficiency causes shortened primary cilia and genetically interacts with IFT‐A deficiency to affect palatogenesis

Abstract

Orofacial clefts are among the most common congenital malformations, affecting approximately 1 in 700 births. SPECC1L mutations in patients with syndromic cleft lip and/or palate cluster in the second coiled coil domain (CCD2), which facilitates interaction of SPECC1L with microtubules. We have shown that loss of SPECC1L in a null allele (Specc1l∆Ex4) resulted in perinatal lethality and delayed palatal shelf elevation, but no cleft palate (CP) or exencephaly at birth. However, mice with an in‐frame deletion of SPECC1L‐CCD2 exhibited CP and exencephaly, which are common manifestations of ciliopathies. Given that SPECC1L cytoplasmic expression includes localization around the cilia base and that SPECC1L regulates actin cytoskeleton, we investigated an association between SPECC1L and primary cilia. We observed that primary cilia were shortened relative to wild‐type in E13.5 Specc1l∆Ex4/∆Ex4 as well as Specc1l∆CCD2/∆CCD2 mutant palatal mesenchyme and epithelium. The cytoplasmic expression of SPECC1L around the cilia base was diminished in Specc1l∆CCD2/∆CCD2 cells. Thus, loss of SPECC1L association with microtubules appears sufficient to prevent its pericentriolar localization and to cause cilia shortening. Super‐resolution imaging for acetylated‐α‐tubulin and ciliary membrane protein, ARL13B, revealed bulbous structures in Specc1l mutant cilia, suggesting a defect in ciliary protein trafficking. Due to the overlapping phenotypes of shortened cilia, exencephaly and CP between the intraflagellar transport (IFT)‐A mouse mutant, Thm1aln/aln, and Specc1l∆CCD2/∆CCD2, we crossed these mutants to look for a genetic interaction. 18% of Specc1l∆CCD2/+, 0% of Thm1aln/+, and 33% of double heterozygous Specc1l∆CCD2/+;Thm1aln/+ embryos showed CP, confirming a genetic interaction. This was observed when the mother was Specc1l∆CCD2/+. However, remarkably, if the mother was Thm1aln/+, there was no CP incidence in Specc1l∆CCD2/+ and Specc1l∆CCD2/+;Thm1aln/+ embryos even though they had shortened cilia, suggesting Thm1aln/+ mothers provided a protective environment against CP phenotype. Consistent with our previous data, Specc1l∆Ex4;Thm1aln/+ mice resulted in no CP. These results suggest that shortened cilia inSpecc1l mutants do not cause but rather modulate CP phenotype. Overall, our studies indicate a novel role for SPECC1L in ciliogenesis, a novel genetic interaction between Specc1l andThm1, and a novel protective maternal genetic effect of Thm1 in palatogenesis.