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

Nathan R Wilson,Andras Czirok,Eric C Liao,Adam J Olm-Shipman,Bryan C Bjork,Kanagaraj Palaniyandi,Guerin J Smith,Kelly M Stumpff,Diana S Acevedo,Lenore Pitstick,Everett G Hall,Edina Kosa,Irfan Saadi

doi:10.1038/srep17735

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.

Highlights

Delamination of Cranial neural crest cells (CNCCs) from the neural epithelium requires dissolution of adherens junctions (AJs), composed of a protein complex, containing E-cadherin, β -catenin, α -E-catenin and α -actinin among others, tethered to actin filaments[2]
Given the association of SPECC1L with actin cytoskeleton[18,23], we hypothesized that SPECC1L interacts with actin-based adherens junctions (AJs)
We previously reported that SPECC1L expression in the first pharyngeal arch at E10.5 is present in both the epithelium and the underlying mesenchyme[18], consistent with CNCC lineage

Summary

Introduction

Delamination of CNCCs from the neural epithelium requires dissolution of adherens junctions (AJs), composed of a protein complex, containing E-cadherin, β -catenin, α -E-catenin and α -actinin among others, tethered to actin filaments[2]. Recent studies show that loss of murine PDGFα based PI3K-AKT signaling leads to craniofacial malformations including cleft palate and neural tube defects[12]. A link between the PI3K-AKT pathway and AJ stability in CNCC delamination is not clear. More than half of Opitz G/BBB syndrome cases are X-linked (OMIM #300000), caused by mutations in MID1 gene[21], which encodes a microtubule-associated cytoskeletal protein[22]. We proposed that SPECC1L, a microtubule- and actin cytoskeleton-associated protein, may mediate transduction of signals required to remodel the actin cytoskeleton during cell adhesion and migration[18]. Using in vitro and in vivo studies, we describe SPECC1L as a novel regulator of AJ stability through PI3K-AKT signaling. SPECC1L functions in the highly regulated cell adhesion based signaling required for proper CNCC function during facial morphogenesis

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Results

Conclusion