Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development

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.

A cleft lip and palate gene, Irf6, is involved in osteoblast differentiation of craniofacial bone

“…Previous studies had focused on the importance of IRF6 in epithelial cells and wound healing due to the obvious pathologies observed in VWS and PPS patients at birth (Ingraham et al, ; Vieira, ; Letra et al, ; Fakhouri et al, ; Joly et al, ), though this study concentrated on the effect of IRF6 on craniofacial bone formation due to aberrant skeletal phenotype observed in Irf6 ‐null mice (Ingraham et al, ) and dominant negative Irf6 in Zebrafish (Duncan et al, ). Although our previous study suggested an intercellular role of IRF6, which is highly expressed in epithelial cells on mandibular bone development (Fakhouri et al, ), no studies thus far have investigated whether IRF6 is involved in regulating bone and cartilage development in a cell‐autonomous manner. Interestingly, our finding of IHC staining extended the knowledge about the current perception of IRF6’s role in intramembranous bone formation by reporting the distinct expression in intramembranous‐differentiated osteogenic cells, as well as in hypertrophic chondrocytes of Meckel's and nasal cartilage.…”

“…Moreover, syngnathia and rib abnormalities have been reported in severe cases of PPS (Escobar and Weaver, ; Cardoso et al, ; Vandeweyer et al, ). Similarly, a complete loss of Irf6 in mice leads to skeletal abnormalities, including xiphoid and digit malformation (Ingraham et al, ; Fakhouri et al, ), while exogenous overexpression of IRF6 in basal epithelium partly rescued the craniofacial phenotype in Irf6 null embryos (Kousa et al, ). Despite all these findings, no studies have reported IRF6 expression in osteogenic cells during bone formation (Fakhouri et al, ; Manocha et al, ).…”

“…Similarly, a complete loss of Irf6 in mice leads to skeletal abnormalities, including xiphoid and digit malformation (Ingraham et al, ; Fakhouri et al, ), while exogenous overexpression of IRF6 in basal epithelium partly rescued the craniofacial phenotype in Irf6 null embryos (Kousa et al, ). Despite all these findings, no studies have reported IRF6 expression in osteogenic cells during bone formation (Fakhouri et al, ; Manocha et al, ). Our recently published study showed that IRF6 is not expressed in migratory cranial neural crest cells; however, it regulates the expression of endothelin 1 (Edn1) ligand in the mandibular epithelium, which binds at Edn1 receptors expressed in adjacent mesenchymal cells to stimulate the cell differentiation (Fakhouri et al, ).…”

Mandibular Explant Assay for Investigating Extrinsic Stimuli on Bone and Cartilage Development

“…In comparison to IRF6 , the TWIST1 gene regulates neural tube closure during embryonic development and cranial suture fusion during skull development. Mutations in TWIST1 can cause craniosynostosis, mandibular hypoplasia, and cleft palate (Fakhouri et al ., 2017). Inhibition or alteration of IRF6 and TWIST1 expression can be done similar to the methods performed by Miettinen et al .…”

“…However, difficulties arise in in vivo experiments when the study begins to incorporate genetic interactions and rescue experiments of two or more allelic mutations. In our recent study, the genetic interaction between Irf6 and Twist1 causes severe mandible abnormality and cleft of the secondary palate in the mouse model (Fakhouri et al ., 2017). …”