Expression of Lrp4 and Wise in early tooth development In order to determine the temporal-spatial relationships between Wise and Lrp4, expression of Lrp4 and Wise were analyzed in the developing heads of mouse embryos between days 12.5 and 14.5 of gestation (E12.5–E14.5) using radioactive in situ hybridisation. Thickening of the oral epithelium to form dental placodes takes place from E12.5. At this stage, weak Wise expression was observed in molar tooth mesenchyme whereas Lrp4 was exclusively expressed in tooth epithelium (Figures 2A and 2B). At E13.5, the bud stage, the expression of Lrp4 became restricted to the epithelial cells at tips of molar tooth buds. Wise expression was observed in epithelium and mesenchyme but it was absent from the tip of tooth bud epithelium (Figures 2C and 2D). At E14.5, the cap stage, Lrp4 showed restricted expression in the primary enamel knots (Figure 2E). Wise expression was observed in tooth mesenchyme and thus Lrp4 and Wise are expressed in a complementary manner in bud and cap stage molar tooth germs (Figure 2F). Similar complementary pattern of expression of Lrp4 and Wise were observed in incisor tooth germs (Figures 2G and 2H). Incisor teeth in Lrp4 and Wise deficient mice Mice have only one incisor in each jaw quadrant. Supernu- merary incisors were observed in both the maxilla and mandible in Lrp4 mutants. In the maxillary incisor region, supernumerary teeth were located on the lingual sides of each endogenuous incisor, although the locations of the supernumerary teeth were slightly variable (Figures 3A–3C). In the mandible incisor region, two supernumerary incisors were found midline between the endog- enous incisors in Lrp4 mutants (Figures 3D–3F). The supernu- merary teeth had single roots and lance-like tips. All supernumer- ary teeth however were reduced in size and had abnormal shapes. Supernumerary incisors were also observed in both the maxilla and mandible of Wise mutant mice (Figures 3G and 3H; [29]). The size, shape and location of these supernumerary incisors appeared identical to those in Lrp4 mutant mice. Molar teeth in Lrp4 and Wise deficient mice Mice have only one incisor and three molars in each jaw quadrant that are divided by a tooth-less region, the diastema. In a quadrant, the first molar is the most anterior and largest molar followed progressively by the second and third molars (Figure 4A). We examined the molars of eleven Lrp4 mutants (44 quadrants) and found none to have a normal phenotype in the maxilla. In the maxillae, 18 quadrants (out of 22 quadrants) had abnormally large teeth in the first molar position (Figures 4B–4D and S1). The occurence of second and third molars and also the presence of supernumerary teeth, anterior (mesial) to the first molars were also observed with varying degrees of penetrance (Figure S1). The remaining 4 quadrants that did not show the large teeth and a supernumerary tooth mesial to the first molar (Figure 4N and S1). In the mandible, the molar tooth phenotype penetrance was low, with only three quadrants showing a large molar and three quadrants showing supernumerary teeth (Figure S1). To examine whether the abnormally large molars developed from a single tooth germ or were created by fusion of several molar tooth germs, micro CT analysis was performed. In wild-type jaws, each maxillary molar has several roots; three roots in the first molar; three roots in the second molar; one or two in the third molar (Figures 4E and 4F). The large maxillary molars in Lrp4 mutants typically had seven roots that could be distinguished as being organized as three or four separate groups. In most cases, the most anterior aspect of the tooth had one root, followed by two groups of three roots each (Figures 4G–4I). Micro CT analysis of the supernumerary teeth found in the quadrants without the large Lrp4/Wise in Tooth Development Figure 2. The expression patterns of Lrp4 and Wise during early molar and incisor tooth development. (A, B) Lrp4 was expressed in tooth epithelium whereas Wise expression was observed in tooth mesenchyme. (C, E) The expression of Lrp4 was restricted to the primary enamel knots. (D, F) Wise expression was found in both epithelium and mesenchyme but was absent from primary enamel knots. (G, H) Sections showing complementary expression of Lrp4 (G) and Wise (H) in incisor regions. Yellow arrow representing the region of Lrp4 expression (H). Tooth epithelium outlined in green (G, H). Radioactive in situ hybridisation on frontal sections showing Lrp4 expression (A, C, E, G) and Wise expression (B, D, F, H) in embryo heads at E12.5 (A B), E13.5 (C, D) and E14.5 (E–H). doi:10.1371/journal.pone.0004092.g002 molars showed these all had a single root. This suggests therefore that the large molars have formed from a fusion of first and second molars with a anterior supernumerary tooth. Other examples where quadrants had a large molar and a separate anterior supernumerary tooth indicated that this large molar root pattern was derived from a fusion of first and second molars or first, second and third molars (Figure S1). Having shown the binding of Wise to Lrp4 and their complementary expression patterns during tooth development, we next compared the molar tooth phenotype of Lrp4 mutants with that of Wise mutants. Wise mutant maxillary molars were also very large and had similar root patterns to Lrp4 mutants, suggesting that their large molars were formed by a similar fusion process (Figures 4J–4L). The incidence of large molars and supernumerary teeth on the mandible of Wise mutants was higher PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e4092