Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development

Walid D Fakhouri,Brian C Schutte,Angela Quispe-Salcedo,Sarah Bakhiet,Larissa Nitschke,Ali Naji,Youssef A Kousa,Kareem Metwalli

doi:10.1038/s41598-017-06310-z

Abstract

Interferon Regulatory Factor 6 (IRF6) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos (Irf6+/−; Twist1+/−) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel’s cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53. Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.

Highlights

Mandibular disorders are common congenital malformations that can occur as part of genetic syndromes or as an isolated form that can lead to a sequence of anomalies associated with tongue, palate and pharynx[11, 12]
We identified a genetic interaction between Irf[6] and Twist[1], two genes that are essential for craniofacial development
While single heterozygous mice for Irf[6] and Twist[1] are grossly normal, our data suggests a genetic interaction in Irf[6], Twist[1] compound heterozygous mice that can lead to severe micrognathia and cleft palate

Summary

Introduction

Mandibular disorders are common congenital malformations that can occur as part of genetic syndromes or as an isolated form that can lead to a sequence of anomalies associated with tongue, palate and pharynx[11, 12]. Conditional knockout of Twist[1] in cranial neural crest cells results in severe mandibular hypoplasia, cleft palate and loss of frontal and maxillary bones[18]. These data show that Twist[1] is critical in tissues derived from the first pharyngeal arch. Individual heterozygous embryos for Irf[6] or Twist[1] show no phenotype at birth, the double heterozygous pups had a severe mandible abnormality and cleft of the secondary palate. We conducted in vivo and ex vivo studies to further characterize the phenotype and signaling pathway in murine embryos to better understand the nature of this novel interaction and to rescue the phenotype genetically and pharmacologically

Methods

Results

Conclusion