Irf6 and Jag2 are expressed normally in the oral epithelia of Jag2^ΔDSL/ΔDSL and Irf6^R84C/R84C homozygous mice, respectively. Section in situ hybridization of wild-type (A, C, E, G), Jag2^ΔDSL/ΔDSL (B, D) and Irf6^R84C/R84C (F, H) mice. At E12.5 (A, B) and E14.5 (C, D), Irf6 is expressed throughout the oral epithelia of both wild-type and Jag2^ΔDSL/ΔDSL littermate embryos. Similarly, at E12.5 (E, F) and E14.5 (G, H), Jag2 is expressed with a comparable pattern in wild-type and Irf6^R84C/R84C littermate embryos. p, palate; t, tongue. Scale bars: 300 μm.

Irf6^+/R84C;Jag2^+/ΔDSL and Irf6^+/R84C;Jag2^+/sm compound heterozygous embryos exhibit intra-oral epithelial adhesions and cleft palate. (A–L) Histological analysis of wild-type (A–D), Irf6^+/R84C;Jag2^+/ΔDSL (E–H) and Irf6^+/R84C;Jag2^+/sm (I–L) mice. (A–D) In wild-type mice, no adhesion between the oral epithelia is observed until the palatal shelves fuse to one another above the dorsum of the tongue (D). (E–H) In contrast, severe inter-epithelial adhesions are observed in Irf6^+/R84C;Jag2^+/ΔDSL mice which result in occlusion of the anterior region of the oral cavity (E). In addition, the palatal epithelia adhere to those covering the tongue and the mandible (arrows in F and G) such that the palatal shelves fail to elevate and remain cleft (H). Intra-oral adhesions are also observed between the maxilla and mandible in the regions of the molar tooth germs (arrows in H). (I–L) The oral adhesions observed in Irf6^+/R84C;Jag2^+/sm embryos are less severe than those observed in their Irf6^+/R84C;Jag2^+/ΔDSL counterparts at all developmental stages (arrows in I, J, K); nevertheless, cleft palate is observed in all Irf6^+/R84C;Jag2^+/sm embryos (L). p, palatal shelf; t, tongue. Scale bars: A, D, E, H, I, L, 500 μm; B, F, J, 300 μm; C, G, K, 100 μm.

Irf6^+/R84C;Jag2^+/ΔDSL and Irf6^R84C/R84C mice exhibit abnormal oral epithelial differentiation and altered Notch signalling. Immunofluorescence analysis of E13.5 wild-type (A, D, G, J), Irf6^R84C/R84C (B, E, H, K) and Irf6^+/R84C;Jag2^+/ΔDSL (C, F, I, L) embryos. (A–C) Immunofluorescence analysis reveals apparently normal levels of p63 expression throughout the oral epithelia of Irf6^R84C/R84C (B) and Irf6^+/R84C;Jag2^+/ΔDSL (C) embryos compared with wild-type littermates (A). (D–F) Higher magnification views indicate that p63 expression is restricted to the basal cells of the lingual and palatal epithelial in wild-type mice (D); in contrast, in Irf6^R84C/R84C and Irf6^+/R84C;Jag2^+/ΔDSL embryos, p63 staining is expanded into the superficial layers of the oral epithelia (arrows in E and F, respectively). (G–I) Immunofluorescence analysis of the activated form of Notch1 (Act N1) reveals expression predominantly in the periderm of the lingual epithelia of E13.5 wild-type mice (arrowheads in G). Similar analyses of Irf6^R84C/R84C (H) and Irf6^+/R84C;Jag2^+/ΔDSL (I) embryos reveals a complete loss of activated Notch1 expression; a similar staining pattern is observed for the Notch target, Hes1 (J–L). t, tongue; p, palate. Scale bars: A–C, 300 μm; D–L, 50 μm.

Irf6^+/R84C;Jag2^+/ΔDSL but not Irf6^R84C/R84C mice demonstrate MES degeneration. Analysis of wild-type (A, D, G, J, M, P), Irf6^R84C/R84C (B, E, H, K, N, Q) and Irf6^+/R84C;Jag2^+/ΔDSL (C, F, I, L, O, R) embryos. (A–C) At E15.5, the wild-type secondary palate has fused (A), whereas in Irf6^R84C/R84C (B) and Irf6^+/R84C;Jag2^+/ΔDSL embryos (C), it remains cleft as a consequence of inter-epithelial adhesions. While partial breakdown of the epithelia between the tongue and palate occurs in Irf6^+/R84C;Jag2^+/ΔDSL mice (arrow in C), the equivalent region remains intact in Irf6^R84C/R84C embryos (B). At E14.5, Tgfb3 is expressed throughout the wild-type MEE (D, G, J); in contrast, while Tgfb3 is detected in the anterior region of the secondary palate of Irf6^R84C/R84C mice where it fuses to the lateral aspect of the tongue (arrows in E), it is down-regulated in the mid- (H) and posterior (K) regions. (F, I, L) Tgfb3 is also expressed in the presumptive MEE of the secondary palate in Irf6^+/R84C;Jag2^+/ΔDSL mice; however, isolated areas of residual Tgfb3 expression are observed in the posterior region (L). Whereas Mmp13 expression is present in the MEE of E14.5 wild-type (M) and Irf6^+/R84C;Jag2^+/ΔDSL mice (O), no Mmp13 expression is observed in the secondary palate of Irf6^R84C/R84C mice (N). At E14.5, immunofluorescence reveals activated caspase 3-positive cells in the MES and oral/nasal epithelial triangles of wild-type embryos and in the epithelia between the tongue and the palate in Irf6^+/R84C;Jag2^+/ΔDSL (P and R, arrows); similar analyses in Irf6^R84C/R84C mice fail to reveal activated caspase 3-positive cells in the region of the MEE (Q). t, tongue; p, palate. Scale bars: A–O, 300 μm; P–R, 100 μm.

Irf6^R84C/R84C and Irf6^+/R84C;Jag2^+/ΔDSL embryos display abnormal oral periderm. (A–F) Histological analysis of E13.5 wild-type (A), Irf6^R84C/R84C (B) and Irf6^+/R84C;Jag2^+/ΔDSL mice (C) reveals abnormal lingual and palatal epithelia in mutant embryos (B, C). (D–F) Higher magnification views of the boxed areas in A, B and C. (D) In wild-type embryos, the epithelia consist of basal cuboidal cells and superficial, flattened periderm cells (D; arrowheads). (E) In contrast, Irf6^R84C/R84C embryos display thickened oral epithelia with disorganized basal cells (arrows) and absence of periderm. (F) Irf6^+/R84C;Jag2^+/ΔDSL mice also exhibit disorganized basal cells but a superficial layer of rounded cells lacking the flattened appearance of normal periderm is observed (arrowheads). (G) At E13.5, keratin 17 is expressed in the periderm and tooth germs of wild-type embryos. (H) In Irf6^R84C/R84C embryos, keratin 17 expression is reduced. (I) In Irf6^+/R84C;Jag2^+/ΔDSL mice, keratin 17 is expressed at similar levels to wild-type embryos, but has a patchy appearance especially in regions of epithelial adhesion (arrowed). (J–L) Deconvolved images of immunofluorescence for keratin 17 (green) and E-cadherin (red) at E13.5. (J) In wild-type mice, keratin 17-expressing periderm cells are distinguishable from the basal cells and, whereas dual labelling is evident on the basal surface of the periderm cells, E-cadherin expression is absent from their apical surface. (K) In contrast, Irf6^R84C/R84C mice lack keratin 17 expression on the lingual and palatal epithelia and multiple regions of E-cadherin expression are present on the apical surface of the exposed basal cells (arrowed). (L) While the oral epithelia of Irf6^+/R84C;Jag2^+/ΔDSL mice are bi-layered, superficial cells lack normal peridermal morphology and dual labelling for keratin 17 and E-cadherin is apparent (asterisks). p, palatal shelf; t, tongue. Scale bars: A–C, 100 μm; D–F, 50 μm; G–I, 300 μm; J–L, 20 μm.