Components in hemidesmosome and Sox18 protein in <i>Ra</i> mice.

<p>TEM image of wild-type hemidesmosomes in the oral cavity (arrows in A). Schematic representation of molecular organization of basement membrane zone including hemidesmosomes (B). (C) Schematic representation of the Sox18 proteins in <i>Ra</i> mice. The numbering indicates the amino acid coordiates of the represented boxes.</p

mRNA level in <i>Ra^op</i> mice.

<p>Quantitative PCR analysis of Plectin (upper) and integrin β4 (lower) expression level. <i>Ra^opHE</i>; heterozygous <i>Ra^op</i> mice. <i>Ra^opHO</i>; homozygous <i>Ra^op</i> mice.</p

<i>Sox7</i> and <i>Sox17</i> expression in developing heads.

<p>Expression of <i>Sox7</i> (A, C) and <i>Sox17</i> (B, D) in heads at E11.5 (A, B) and E12.5 (C, D). Radioactive <i>in situ</i> hybridisation on wild type frontal sections. Tooth germ epithelium is outlined in red. E; RT-PCR analysis showed the presence of <i>Sox17</i> and <i>Sox7</i> expression and absence of <i>Msx1</i> or <i>Lhx7</i> in epithelium. Epi, total RNA extracted from only epithelium of mandible; Epi+Mesen, total RNA extracted from whole mandibles.</p

<i>Sox18</i> expression in head.

<p>Radioactive <i>in situ</i> hybridisation showed strong expression of <i>Sox18</i> in mesenchyme (m) at E9.5 (B), E10.5 (D), E11.5 (F) and E13.5 (G) of wild-type. Insets of A, C and E represent histological images of B, D and F, respectively. Arrows indicate mandible (B–F). A, B; Sagittal section, C–K; Frontal sections. B, D, F; Epithelia of jaws outlined by red dots. (G) <i>Sox18</i> expression is observed outside of condensed mesenchyme of tooth germs (arrowheads) and the buccal regions of maxillary jaws (arrows). (H, I) Blood cells were observed at the buccal side of maxillary mesenchyme (arrows in H) and outside the condensed mesenchyme of tooth germs (arrowheads in I). von-Willbrand factor proteins were detected in the same regions (arrowheads and arrows in J). The endothelial marker gene, <i>Claudin 5</i> showed a similar expression pattern to <i>Sox18</i> (arrowheads and arrows in K). (G, J, K) Tooth germ epithelium is outlined by red dots. L; RT-PCR analysis showed the presence of <i>Sox18</i> expression and absence of <i>Msx1</i> or <i>Lhx7</i> in epithelium. Epi, total RNA extracted from only epithelium of mandible; Epi+Mesen, total RNA extracted from whole mandibles. Scale bars: 100 µm (C–F); 125 µm (G, I–K).</p

The epithelial phenotype of <i>Ra^op</i> mice.

<p>Normal attached epithelium was observed in wild-type head (A, C, E, G). By contrast detached epithelium was observed in the skin of the snout (B), the mucosa of the incisor region (D), molar region (F), diastema (H) and the skin of trunk (L) of <i>Ra^op</i> mice. (D, F) Arrowheads indicate tooth germs. E12.5 (A–D, I, J) and E14.5 (E–H, K, L). Detachment of epithelium was found before histological processing (green arrowheads in J). Scale bars: 125 µm (C, D); 300 µm (E–H).</p

Ultrastructural analysis of <i>Ra^op</i> mice.

<p>Frontal sections of wild-type (A, C, G) and <i>Ra^op</i> mice (B, D, E, F, H) at E12.5. Epithelium consisted of two or three epithelial cell layers in unaffected regions of <i>Ra^op</i> mice (B). (C) Cytoplasmic plaques were seen as single plaque structures in wild-type (arrowheads). Cytoplasmic plaques were seen as single plaques in unaffected regions of <i>Ra^op</i> mice (D). (E, F, H) Detached epithelium of <i>Ra^op</i> mice. (F) High magnification images taken from boxed regions in panel E. Lamina densa and lamina lucida were retained on the mesenchymal side (arrowheads in F). (H) High magnification of region indicated by arrowhead in E. Cytoplasmic plaque were also seen and retained at basement membranes of affected region of <i>Ra^op</i> mice (arrowheads in H).</p

Haemorrhage in <i>Ra^op</i> mice.

<p>Mass of blood seen in the trunk (B), limb bud (D, H) and head (F, G) of <i>Ra^op</i> mice. (A, C, E) wild type mice. Detached epithelium could not be detected in the limb bud where mass of blood was observed (arrowheads in H). E12.5 (C–H) and E14.5 (A, B).</p

Hemidesmosome proteins of <i>Ra^op</i> mice.

<p>Immunohistochemistry showed that there was no significant changes in Collagen IV (green arrowheads in B), nidogen (red arrowheads in D), Keratin 5 (red arrowheads in F) and BPAG1 (red arrowheads in H) where epithelium was detached in <i>Ra^op</i> mice. Collagen IV, nidogen and BPAG1 were retained at mesenchyme, while Keratin 5 was observed in detached epithelium. (J) E12.5 mutant embryos showed both detached and undetached epithelium. (I, K, L) At E12.5 integrin ß4 level was decreased in intensity where epithelium was unaffected (green arrowheads), whereas complete lack of integrin ß4 protein was observed in detached epithelium (yellow arrowheads). J; adjacent section to I. K and L; High magnification of unaffected (K) and affected (L) region of I. At E14.5, integrin ß4 (green arrowheads in L), integrin α6 (red arrowheads in N) and plectin (red arrowheads in P) were not observed in <i>Ra^op</i> mice. WT; A, C, E, G, M, O, Q. <i>Ra^op</i> mice; B, D, F, H–L, N, P, R. Scale bars: 300 µm (A–H, M–R); 125 µm (I–L).</p

Apoptotic cells in <i>Ra^op</i> mice.

<p>Pearl-like organization of some groups of cells in the basal tooth epithelium of <i>Ra^op</i> (B) contrast with the normal organization observed in wild-type. Apoptotic cells were seen in epithelium of <i>Ra^op</i> mice (arrowheads in D) but were absent in wild-type embryos. (C, D) TUNEL assay. Scale bars: 30 µm (A, B).</p

Shh and Fgf signaling in <i>Lrp4</i> and <i>Wise</i> mutants during palatal rugae development.

(A, B, Q, R) Sagittal sections of developing palate between midline and molar region showing in situ hybridization of Gli1 (A, B) and Erm (Q, R) in wild-type (A, Q), Shh-K14 (B) mice and Lrp4 mutant mice (R) at E13.5. Arrowheads indicating Erm expression in developing palatal rugae. (A) Box showing large magnification images of developing rugae region. (C, D) SEM image of palatal rugae of wild-type (C) and Shh-K14 (D) mice at birth. Arrowheads indicating palatal rugae presumptive region. (E-P) Whole mount explants showing Shh (E-N) and Erm (O, P) transcription in palatal rugae of wild-type (E, G, I, K, M, O), Lrp4 (N) and Wise (F, H, J, L, P) mutant mice at E12.5 (G, H), 13.5 (I, J, O, P), E14.0 (K, L) and E14.5 (E, F, M, N). Scale bar; 200μm.</p