Lrp4/Wise in Tooth Development Figure 7. Shh, Bmp and Wnt signalling in fused maxillary molars. Sagittal section showing tooth bud epithelium of a supernumerary tooth in Lrp4 mutant at E14.5 (arrowhead in A’) and E16.5 (Supernumerary tooth; blue arrowhead, the first molar tooth germ; yellow arrowhead, the second molar tooth germ; black arrowhead in G’). Transient epithelial swelling of vestigial remants of diastema tooth in wild-type at E14.5 (arrowhead in A) and no swelling at E16 (G). Weak expression of Lrp4 (B) and strong Wise (C) were observed at the posterior part of tooth germ at E14.5 (arrowheads in B, C). Shh expression domain was reduced in Lrp4 mutants (D’). Ptc1 (E) and Gli1 (F) expression are found at posterior part of tooth germ at E14.5 (arrowheads in E, F). The first molar epithelium is still continuous with the second molar epithelium at E16.5 in wild-type (G). The junction region between the first and second molar could be distinguished by the absence of inner enamel epithelium in wild-type whereas Lrp4 mutant showed continuouse inner enamel epithelium from first molar to second molar (red arrowhead in G–H’). H and H’ are high magnification of the junction region in G and G’. Strong Wise expression was observed at the joint region between first and second molars but no Lrp4 expression in the region (arrowheads in I and J). Shh expression are observed in only inner enamel epithelium of the first molar and second molar (K). Gli1 is expressed in junctional epithelium as well as tooth germs (L). Gli2 and Ptc1 expression were not observed in the epithelium of the junction region in Lrp4 mutnats whereas they were expressed in the epithelium of the region (M–N’). Bmp7 were upregulated at the junction region in Lrp4 mutants whereas they were not expressed at the region in wild-type (O, O’). Phosphorylated-Smad1/5/8 (Pho-Smad) was not detected at the junction region in wild-type whereas it was found in region corresponding the junction region in Lrp4 mutant (arrowhead in P and P’). Wnt activity was observed indistinctly in molar region of wild type (Q) whereas it was obvious in molar region of Lrp4 mutant (Q’). Ectopic Wnt activity was also found where supernumerary molar develop (arrowhead in Q’). In wild types, sagittal sections showed Wnt activity were detected in inner enamel epithelium including enamel knot and stratum reticulum whereas it was not observed in the junction region (arrowhead in R). In Lrp4 mutants, Wnt was activated in the junction region (arrowhead in R’). Maxillary molar tooth at E14.5 (A–F) and E16.5 (G–R’) of wild-type (A, B–D, E–G, H, I–M, N, O, P, Q, R) and Lrp4 mutant (A’, D’, G’, H’ M’, N’, O’, P’, Q’ R’). Histology (A, A’, G–H’), immunohistochemistry (P and P’), b-gal activity (Q–R’) and radioactive in situ hybridisation (B–F, I–Q’) on oral view (Q and Q’) and sagittal sections (A–P’, R, R’). doi:10.1371/journal.pone.0004092.g007 be hypomorphic [14–16]. An incisor tooth phenotype was originally reported in Lrp4 mutant mice which led us to investigate the tooth development in more detail in these mice. The most obvious tooth phenotype we observed in Lrp4 mutants are greatly enlarged molar teeth. These large molars arise from fusion of different molar tooth germs during development. The PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e4092