SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination

Abstract

Cranial neural crest cells (CNCCs) delaminate from embryonic neural folds and migrate to pharyngeal arches, which give rise to most mid-facial structures. CNCC dysfunction plays a prominent role in the etiology of orofacial clefts, a frequent birth malformation. Heterozygous mutations in SPECC1L have been identified in patients with atypical and syndromic clefts. Here, we report that in SPECC1L-knockdown cultured cells, staining of canonical adherens junction (AJ) components, β-catenin and E-cadherin, was increased, and electron micrographs revealed an apico-basal diffusion of AJs. To understand the role of SPECC1L in craniofacial morphogenesis, we generated a mouse model of Specc1l deficiency. Homozygous mutants were embryonic lethal and showed impaired neural tube closure and CNCC delamination. Staining of AJ proteins was increased in the mutant neural folds. This AJ defect is consistent with impaired CNCC delamination, which requires AJ dissolution. Further, PI3K-AKT signaling was reduced and apoptosis was increased in Specc1l mutants. In vitro, moderate inhibition of PI3K-AKT signaling in wildtype cells was sufficient to cause AJ alterations. Importantly, AJ changes induced by SPECC1L-knockdown were rescued by activating the PI3K-AKT pathway. Together, these data indicate SPECC1L as a novel modulator of PI3K-AKT signaling and AJ biology, required for neural tube closure and CNCC delamination.

SPECC1L-deficient palate mesenchyme cells show speed and directionality defect

“…Control and SPECC1L-kd U2OS cells were generated previously from U2OS osteosarcoma cells (ATCC HTB-96) as reported (Saadi et al 2011). U2OS cells were cultured in DMEM (HyClone, SH30243.01) containing 10% FBS (Corning, 35-010-CV) as shown previously (Wilson et al 2016).…”

“…We previously reported that loss of SPECC1L results in decreased PI3K-AKT signaling along with defects in cell-adhesion and cell-shape (Wilson et al 2016).…”

“…Mutations in SPECC1L have been identified in multiple patients with craniofacial malformations, including cleft palate (Bhoj et al 2018;Bhoj et al 2015;Kruszka et al 2015;Saadi et al 2011). Mouse embryos homozygous for a null Specc1l gene-trap allele die between E9.5-E10.5 with open neural-folds and defects in cranial neural crest cell delamination (Wilson et al 2016). Loss of SPECC1L also results in poor cell migration, increased filamentous actin staining, and abnormal staining of adherens junction markers β-catenin and E-cadherin (Saadi et al 2011;Wilson et al 2016).…”

“…Mouse embryos homozygous for a null Specc1l gene-trap allele die between E9.5-E10.5 with open neural-folds and defects in cranial neural crest cell delamination (Wilson et al 2016). Loss of SPECC1L also results in poor cell migration, increased filamentous actin staining, and abnormal staining of adherens junction markers β-catenin and E-cadherin (Saadi et al 2011;Wilson et al 2016). We have recently reported generation of a new gene-trap allele (Specc1l cGT ), and a Specc1l-truncation allele lacking 510 C-terminal amino acids (Specc1l DC510 ).…”

Zebrafish models of orofacial clefts

“…In 2011, mutations in SPECC1L were identified in two patients with oblique facial clefts (Saadi et al, 2011). They were later also found in patients with Opitz G/BBB syndrome, the letters referring to the initials of patients first diagnosed with it, which is associated with cleft lip and/or palate (Kruszka et al, 2015), and in a family with Teebi hypertelorism syndrome, which is characterized by the presence of frontonasal dysplasia (Bhoj et al, 2015;Wilson et al, 2016). SPECC1L is a cytoskeletal protein that interacts with both microtubules and the actin cytoskeleton.…”