mutations cause two allelic autosomal dominant mixed clefting type syndromes: VWS and PPS, in which clefts of both the primary and secondary palates may be observed even within families. Recently, two Irf6
mutant alleles were reported that partially model the VWS and PPS phenotypes and identified a role for Irf6
as a key determinant of the keratinocyte proliferation-differentiation switch. Embryos homozygous for a gene trap null allele (Ingraham et al., 2006
) have defects in stratified epidermis formation, skeletal defects secondary to the skin defect, cleft secondary palate and epidermal adhesions in the oral cavity. These oral adhesions are found at much reduced frequency in heterozygous embryos. Interestingly, the cleft palate in the homozygous embryos seems to be a defect in elevation of the palatal shelves. A second knock-in allele has been made with a mutation identical to that found in PPS patients (Richardson et al., 2006
). These have a highly penetrant heterozygous phenotype of mild intraoral adhesions between the ventral surface of the tongue and the mandible, but no cleft lip or palate. In the homozygous state, the mutation causes similar defects in skin development and cleft secondary palate due to abnormal adhesion between the palate and the tongue.
The Irf6clft1mutant allele appears to be a milder allele than either previously reported for Irf6. We observe no gross defects in skin development (as determined by the permeability assay), and only rare expression of more severe phenotypes, including a loop tail and forelimb defects (). We observe no defects in heterozygous mice, and a significant fraction of clft1 mutants with cleft palate show anterior secondary palate shelf elevation and fusion, which is never seen in more severe Irf6 mutant alleles. This finding is contradictory to our expectation that palate shelf fusion would not occur if palate shelf elevation could be uncoupled from the oral adhesions, based upon the fact that human VWS and PPS cases exhibit palate and lip fusion defects in the absence of severe oral adhesions. This observation is similar to that found for the PPS-like Irf6R84C heterozygous mice, in which multiple intraoral adhesions occur but palate fusion is complete.
The difference in allelic expressivity may be due to differences in genetic backgrounds. Previously published Irf6
alleles were on a mixed 129, C57BL/6 background (Ingraham et al., 2006
; Richardson et al., 2006
), and our allele is on a mixed A/J, FVB background. As such, we cannot formally exclude strain-specific modifier effects contributing to these phenotypic differences.
The coding mutation in the Irf6clft1
allele is in the Proline-39 amino acid that is mutated in a family with VWS. Biochemical studies have shown that the majority of VWS and PPS mutations that alter amino acids within the IRF6 DNA-binding domain abrogate binding to the IRF6 target DNA binding sequence motif, regardless of whether they make contact with DNA, (Little et al., 2009
). The Proline-39 residue is not predicted to make direct contact with DNA (Kondo et al., 2002
), but alteration of a proline within the IRF6 DNA-binding domain, which contains three α-helices, is likely to perturb its conformation. However, while the Irf6clft1
carries the same mutation as found in an affected human family, it does not appear to have cleft palate as a primary defect. Nonetheless, the Irf6clft1
hypomorphic allele is the first to contain a specific human VWS-like point mutation and will facilitate the molecular and biochemical study of its etiology in mice.