Lrp4/Wise in Tooth Development Figure 4. The molar tooth phenotypes of Lrp4 mutant mice and Wise mutant mice. SEM images of maxillary molars (A–C), 3D reconstructions based of micro CT scans (D, M and N), horizontal micro CT sections (E–I, K and L) and dissected maxillary molars (O and P) of adult wild-type (A, E, F, M and O), Lrp4 mutants (B–D, G–I and N) adults, Wise mutants (J–L) and K14-Cre/Smoflox/flox mice (P) of adult (A–N) and P6 (O, P). One fused molar (B), fused molar with one relatively normal size tooth and lingual peg-shaped extra tooth (arrowhead in C), and fused molar with two reratively normal size teeth (D). Horizontal micro CT sections at the crown region (E and G) and root region (F, H and I). (F) In wild type, there are three groups of tooth roots in maxillary molars (red circle = three roots of the first molar; blue circle = three roots of the second molar; yellow circle = two roots of the third molar). (G–I) In Lrp4 mutants, the fused molar with one normal size tooth showed four groups of tooth roots, indicating that a supernumerary tooth was present in the quadrant. The fused molars (H) had three groups of roots (one root in green circle; three roots in red circle; three roots in blue circle in H), and one normal size molar had one root as one unit (yellow circle in I), suggesting that the fused molar includes the supernumerary tooth. At the horizontal section level showing the tooth roots of the fused molars, the roots of a normal size molar could not be seen (H). Fused molars were also found in the maxillae of Wise mutant mice (J). Wise mutant fused molars also showed the several groups of roots (K and L). Supernumerary teeth were observed anterior to the first maxillary molar tooth (sn in N) of Lrp4 mutant mice (N). Fused maxillary molar of K14-Cre/Smoflox/flox mice (P). b; buccal side, l: lingual side. In all images, left side is anterior side. Scale bar = 1 mm. doi:10.1371/journal.pone.0004092.g004 and 7J). At E16.5, Shh was expressed only in the inner enamel epithelium whereas Ptc1, Gli1, Gli2 and Smo were expressed in the epithelium of the junctional region between first and second molars (Figure 7K–7M, 7N and S2D). Thus, Shh signaling is active in epithelial cells in the junctional region at E16.5 with Shh being produced from the adjacent epithelium. In Lrp4 mutants, Ptc1 and Gli2 were found to be slightly downregulated in the epithelium of junctional region (Figures 7M’ and 7N’), suggesting that Shh signalling is reduced specifically in this epithelium. Changes in Bmp expression have previously been reported in the limb buds of Lrp4 mutant mice identifying a possible role of Lrp4 in the control of Bmp signalling [15]. The binding of the Bmp antagonist Wise to Lrp4 provides a mechanism to explain changes in Bmp signaling in Lrp4 muatnts [19,20]. Our identification of apparently identical tooth phenotypes in Lrp4 and Wise mutants supports the premise that Lrp4 has a role in the modulation of Bmp signalling. To examine whether Bmp signalling was altered during Figure 5. Gene expression in incisor region. (A–D) Ectopic Shh expression in incisor region of Lrp4 mutant (arrowheads in B) and Wise mutants (arrowheads in C). (E, F) Wnt activity in enamel knot in wild- type mice (arrowhead in E). Ectopic Wnt activity in lingual side of endogeneous incisor tooth germ (arrowheads in F). Tooth epithelium outlined in green (E, F). Whole mount (A–D) and b-gal activity (E, F) showing Shh (A–D) expression and Wnt activity (E, F) at E14.5 in wild- type (A, C, E), Lrp4 mutants (B, F) and Wise mutants (D). doi:10.1371/journal.pone.0004092.g005 PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e4092