Lrp4/Wise in Tooth Development Figure 3. Supernumerary teeth in incisor region. Supernumerary teeth were observed in incisor region on both maxilla (arrows in B, G and arrowheads in C) and mandible (arrows in E, H and arrowheads in F) of both Lrp4 and Wise mutants. Sagittal view of maxillary incisor region (A and B), lingual view of mandibular incisors (D, E and H), occlusal view of maxillary incisors (G), sagittal sections (C) and frontal sections (F) of wild-type (A, D), Lrp4 mutants (B, C, E, F) and Wise mutants (G, H) at adult. doi:10.1371/journal.pone.0004092.g003 than in Lrp4 (Figure S1). In addition to fused and supernumerary molars, small lingual peg-shaped extra teeth were also evident in some Lrp4 mutant quadrants (Figurs 4C and S1). These have also been reported to occur with low frequency in Wise mutants (Figure S1; [28]). Molecular analysis of supernumerary incisor development Ectopic Shh expression was observed at incisor region of both Lrp4 mutant at E14.5 (Figure 5B). Ectopic Shh expression was also observed in Wise mutant mice at E14.5 and its location and size were identical to it in Lrp4 mutant (Figure 5D). It is established that both Lrp4 and Wise are involved in Wnt signaling [15,18]. In order to identify changes in canonical Wnt signalling in Lrp4 mutant embryos, Wnt activity was detected by crossing the Lrp4 mutant mice with BAT-gal reporter mice that express the LacZ under b- catenin/Tcf responsive elements [32]. In wild-type embryos, Wnt activity was observed in the enamel knots of endogenous incisors (Figure 5E). In Lrp4 mutants, ectopic Wnt activity was found in lingual mesenchyme as well as the enamel knots (Figure 5F). Molecular analysis of supernumerary molar development Both Lrp4 and Wise mutants showed supernumerary teeth in the diastema mesial to the first molars. To explore the role of Lrp4 and Wise in the diastema, we examined Lrp4 and Wise expression by in situ hybridization in this area. In the diastema region of E12.5 embryos, Lrp4 was expressed in the epithelium, whereas Wise expression was observed in the mesenchyme (Figures 6A–6D). Vestigial remants of diastemal teeth can be seen in early mouse embryos as transient epithelial swellings at E13 that express Shh and which are rapidly eliminated at E14 by apoptosis (Figure 7A; [30,31]). Supernumerary tooth buds that develop anterior to the first molar were visible at E14.5 in Lrp4 mutants that were continuous with the first molar epithelium (Figure 7A’). Ectopic Shh expression was observed in the maxillary diastema of Lrp4 deficient mice (Figure 6E and 6F), that was associated with retention of a vestigial swelling (blue arrowheads in Figures 7A’ and 7G’). Significantly, although Shh was ectopically expressed, the level of expression in the developing molars was reduced (Figures 6F and 7D’). Ectopic Shh expression in the diastema region and reduced expresseion level of Shh were also observed in Wise mutants (Figure 6G and 6H; [28]). The retention of diastema buds mesial to the first molar in Lrp4 and Wise deficient embryos thus correlates with ectopic expression of Shh. Mechanisms of molar fusion The fusion phenotype (between the supernumerary tooth and first molar, and first molar and second molar) observed in Lrp4 and Wise mutants suggests these molecules interact to regulate the separation of individual teeth. In order to examine the role of Lrp4 and Wise in molar development, we analyzed gene expression in anterior or posterior parts of the first molar tooth germ and the anterior parts of second molar tooth germs. At E14.5, unlike in primary enamel knots, only weak expression of Lrp4 was found in both anterior and posterior parts of the first molar epithelium (Figure 7A and 7B). Wise was expressed throughout the mesenchyme of these regions (Figure 7C). The expression of Shh was considerably reduced in the first molar epithelium of Lrp4 and Wise mutants at this stage (Figure 6F, 6H and 7D’) suggesting that loss of Shh signaling is linked to the molar fusion process. The Shh receptor, Ptc1, was expressed weakly in posterior regions of first molar epithelium whereas strong expression was found in the mesenchyme at E14.5 (Figure 7E and S2A). The Shh signalling activator, Smoothened (Smo) and transcriptional effector Gli1 and Gli3 were expressed in posterior cells of first molar epithelium at E14.5 (Figure 7F, S2B and S2C). This implies that the Shh pathway is active in cells in posterior regions of the first molar epithelium at E14 although Shh is not transcribed in these cells. In order to investigate if loss of Shh is sufficient to cause molar fusion, we examined the molars of mice with conditional mutation of Smo under keratin (K) 14 promotor (K14-Cre/Smoflox/flox ; [33]). We observed fusion between first and second molars in K14- Cre/Smoflox/flox mice (Figure 4P). In wild-type embryos, second molars develop from posterior regions of first molars and then start to separate from first molar after cervical loops form which takes place at around E16.5. Micro CT scanning revealed that the fused molars contain a single, continuous large pulp chamber indicating that fusion took place before the formation of cervical loops (Figure 4G). The junction between the developing first and second molars is distinguished by the absence of a differentiated inner enamel epithelium at E16.5 (Figures 7G and 7H). In the developing molars of Lrp4 mutants a differentiated inner enamel epithelium was present at the junction between the first and second molars (Figures 7G’ and 7H’). Similar differentiated inner enamel epithelium was also observed in the junctional region in Wise and K14-Cre/Smoflox/flox mice (Figures S3A, S3B, S4A and S4B). Lrp4 expression could not be detected in this junctional region in wild type embryos, but Wise was expressed in both the epithelium and surrounding mesenchyme (Figures 7I PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e4092