histology or radioactive in situ hybridisation. Decalcification using 0.5 M EDTA (pH 7.6) was performed after fixation of E16 and newborn mice. In situ hybridisation In situ hybridisation with [35S]UTP-labeled riboprobes was carried out as described previously by Wilkinson [43], with modifications. Embryonic heads were sectioned at 8 mm and floated onto TESPA(3-aminopropyltriethoxysilane)-coated slides. The slides were pretreated with 5 mg/ml proteinase K and 0.25% (vol/vol) acetic anhydride to reduce background. Hybridisation was carried out overnight in a humidified chamber at 55uC. The slides were then washed twice at high stringency in 26 standard saline citrate (SSC), 50% formamide, 10 mM dithiothreitol (DTT) at 65uC for 20 min and treated with 40 mg/ml RNAse A for 30 min at 37uC to remove any nonspecifically bound probe. The high stringency washes (at 65uC in 26 SSC, 50% formamide, 10 mM DTT) were repeated, followed by a further wash at 65uC in 0.16 SSC, 10 mM DTT. The sections were then washed in 0.16 SSC at room temperature and dehydrated through 300 mM ammonium acetate in 70% ethanol, 95% ethanol, and absolute ethanol. The slides were air-dried and dipped in Ilford K.5 photographic emulsion. Autoradiography was performed by exposing the sections in a light-tight box at 4uC for 10–14 days. Slides were developed using Kodak D19, fixed in Kodak UNIFIX, counter- stained with malachite green or hematoxylin, and mounted with DePex (BDG). For photography, in some of sections, the darkfield images were inverted, artificially stained red, and combined with the brightfield image by using Adobe Photoshop. Whole-mount in situ hybridisation was carried out as described by Pownall et al. [44] and Dietrich et al. [45]. Briefly, explants were pretreated with proteinase K at 37uC, refixed in fresh 4% PFA and then prehybridised for 5 hours at 60uC in a hybridisation buffer including 50% formamide, 50 mg/ml heparin and 50 mg/ ml yeast tRNA. The proteinase K concentration was 10 mg/ml, and the length of the proteinase K treatment was modified according to the size of the tissue. The probe was added at concentration of approximately 1 mg/ml of hybridization mix. After hybridisation, tissues were washed in high-stringency conditions and preblocked in antibody blocking solution, then incubated with preabsorbed antibody. DIG-labelled antisense and sense riboprobes were detected with alkaline phosphatase-coupled anti-DIG antibodies using NBT and BCIP as the color substrates in NMT solution. FITC-labelled antisense and sense riboprobes were detected with alkaline phosphatase-coupled anti-FITC antibodies using Fast Red (Sigma). Following visualisation of the stain, the tissues were postfixed and cleared in 50% glycerol before photography. The radioactive or DIG antisense probes or fluorescent antisense probes were generated from mouse cDNA clones that were gifts from several laboratories: Axin2 (W. Birchmeier), Bmp4 (B. Hogan), and Shh (A. McMahon). Micro CT analysis Heads of Lrp4 mutant, Wise mutant and wild-type mice were scanned with Explore Locus SP (GE Pre-clinical imaging) high resolution Micro-CT with a voxel dimension of 8 mm. Three- dimension reconstruction was performed by three structure analysis software, Microview (GE Pre-clinical imaging). Immunohistochemistry analysis After deparaffinization of sections, sections were treated by proteinase K and then incubated with antibody to Phosphorylat- Lrp4/Wise in Tooth Development ed-Smad 1/5/8 (Cell signaling Technology). As a negative control, normal rabbit serum or normal goat serum were used instead of primary antibody. Tyramide signal amplification system was performed (Parkin Elmer Life Science) for detecting Phosphory- lated-Smad 1/5/8 or active-caspase-3. Slides were mounted with Aquamount. Pictures were taken with same exposure between control, wild-type Wise and Lrp4 mutant mice. Wnt activity detection Tissues were fixed in 2% paraformaldehyde and 0.2% Glutaraldehyde with 1% Na deoxycholic Acid and 10% noni P40 for 30 min at 4uC. Explants were then assayed for b-gal activity by staining with XGal staining solution overnight at 37uC. Scanning Electron Microscope (SEM) analysis Both jaws were coated with gold and photographed using scanning electron microscopy. Supporting Information Figure S1 Frequency of molar tooth phenotypes in Lrp4 and Wise mutant mice. Red circles, blue circles and green circles represent fused tooth, relatively normal sized molar and lingual peg-shaped extra teeth, respectively. Found at: doi:10.1371/journal.pone.0004092.s001 (0.38 MB TIF) Figure S2 Shh and Bmp signalling in molar tooth development. Ptc1 (A), Smo (arrowhead in B) and Gli3 (arrowhead in C) expression was found at the posterior part of tooth epithelium at E14.5. (A) High magnification of posterior part of tooth germ of Figure 7E. Smo was expressed in junction region between first and second molars at E16.5 (arrowhead in D). Bmp4 were upregulated at the junction region in Lrp4 mutants whereas they were not expressed at the region in wild-type (E, F). Radioactive in situ hybridisation on sagittal sections in tooth germs of embryo heads at E14.5 (A–C) and E16.5 (D–F) of wild-type (A–E) and Lrp4 mutants (F). Found at: doi:10.1371/journal.pone.0004092.s002 (2.83 MB TIF) Figure S3 Bmp and Wnt signalling in Wise mutants mice. Differentiated inner enamel epithelium were found at junction region in Wise mutants (A, B). Phosphorylated-Smad1/5/8 (Pho- Smad) positive cells were found in region corresponding the junction region between the first molar (1st) and the second molar (2nd) in Wise mutant (C, D). sn; supernumerary tooth. Axin2 expression were upregulated at the junctional region in Wise mutants (F). B and D are high magnification of the junction region in A and C, respectively. Histology (A, B), immunohistochemistly (C, D) and radioactive in situ hybridisation (E, F) on sagittal sections in upper molar at E16.5 of wild-type (E) and Wise mutants (A–D, F). Found at: doi:10.1371/journal.pone.0004092.s003 (3.12 MB TIF) Figure S4 Bmp and Wnt signalling in K14-Cre/Smoflox/flox mice. Differentiated inner enamel epithelium were found at junction region in K14-Cre/Smoflox/flox mice (A, B). Phosphor- ylated-Smad1/5/8 (Pho-Smad) positive cells could not be detected in region corresponding the junction region between the first molar (1st) and the second molar (2nd) in K14-Cre/Smoflox/flox mice (arrow in C, D). Significant differences of Axin2 expression were not found at the junctional region between wild-type (E) and K14-Cre/Smoflox/flox mice (F). B and D are high magnification of the junction region in A and C, respectively. Histology (A, B), immunohistochemistly (C, D) and radioactive in situ hybridisation (E, F) on sagittal sections in upper molar at E15.5 (E, F), E16.5 (A– D) of wild-type (E) and K14-Cre/Smoflox/flox mice (A–D, F). Found at: doi:10.1371/journal.pone.0004092.s004 (3.23 MB TIF) PLoS ONE | www.plosone.org 10 December 2008 | Volume 3 | Issue 12 | e4092