Figure 6. Gene expression in diastema and molar region. (A) Lrp4 expression in incisor teeth (blue arrowhead), in molar teeth (red arrowhead) and in the diastema (green arrowhead). (B) Sections of the diastema region showed Lrp4 expression in epithelium (arrowhead). (C, D) Wise expression in mesenchyme of diastema region (green arrowheads in C and D). Tooth epithelium was comfirmed by Shh expression in adjacent specimen (yellow domain pointed by arrow in C). (E–H) Ectopic Shh expression in diastema (green arrowhead in F and H). Reduced intensity of Shh expression in molar tooth germ (red arrowhead in F and H). Whole mount (A, E–H) and radioactive in situ hybridization (B–D) showing Lrp4 (A, B), Wise (C, D) and Shh (E–H) expression at E12.5 (A–D) and E14.5 (E–H) in wild-type (A–E and G), Lrp4 mutants (F) and Wise mutants (H). Sagittal section (C) and frontal sections (B and D). doi:10.1371/journal.pone.0004092.g006 tooth development in Lrp4 mutants, we performed immunohisto- chemistry using anti-phosphorylated Smad1/Smad5/Smad8 (phospho-Smad1/5/8) antibody. Smad1, Smad5 and Smad8 are phosphorylated by Bmp receptors following binding [34]. In wild- type molar tooth germs, phospho-Smad1/5/8 positive cells were restricted to the inner enamel epithelium and were absent from the junction regions between the first and second molars (Figure 7P). At E16.5 Bmp4 and Bmp7 expression could not be detected in either epithelium or mesenchyme of the junctional region but they Lrp4/Wise in Tooth Development were expressed in mesenchyme facing the inner enamel epithelium in wild-type (Figures 7O and S2E). In Lrp4 muatnts, Bmp4 and Bmp7 expression was expanded into the region corresponding to the junctional region between first and second molars (Figures 7O’ and S2F). Similality, a Lrp4 mutant mice, continuous phospho- Smad1/5/8 positive cells were also found from anterior to posterior of the large fused tooth (Figure 7P’), suggesting that Bmp signalling was ectopically activated in the region correspond- ing to the junction between the first and second molars where fusion occurs in the mutants. Ectopic phospho-Smad1/5/8 positive cells were also found in the junctional region in Wise mutants whereas that could not be detected in the junctional region in K14-Cre/Smoflox/flox mice (Figures S3C, S3D, S4C and S4D). In order to identify changes in canonical Wnt signalling in molar region of Lrp4 mutant embryos, Wnt activity was detected by crossing the Lrp4 mutant mice with BAT-gal reporter mice. In wild-type embryos, Wnt activity was observed in tooth epithelium but was absent from the junction region of wild type at E16 (Figure 7R). In Lrp4 mutants, ectopic Wnt activity was seen in the developing supernumerary (diastema) teeth at E14.5 (Figure 7Q’) and also in the region corresponding the junctional epithelium of the fused molars at E16.5 (Figure 7R’). Axin2 expression was upregulated at the junctional region in Wise mutants whereas significant differences in Axin2 expression could not be detected in the junctional region between wild-type and K14-Cre/Smoflox/flox mice (Figures S3E, S3F, S4E and S4F). Downregulation of Shh and upregulation Bmp and Wnt activity were thus present in the tooth epithelium of the junction region between the first and second molars in Lrp4 and Wise mutants. These changes in Bmp and Wnt activity can be ascribed to the direct role of Lrp4 in binding Wnt proteins and the Bmp antagonist Wise. In order to determine the likely signaling hierarchy we examined Wnt and Bmp activity in the inner enamel epithelium between the developing first and second molars in K14-Cre/Smoflox/flox mice. No obvious upregulation of either of these pathways was evident, suggesting that Shh lies downstream of Wnt and Bmp activity (Figure 8). The consequence of these signaling changes in the mutants is the inappropriate (ectopic) differentiation of the epithelium into inner enamel epithelium which links (fuses) the adjacent tooth germs. Discussion LDL receptor-related proteins control a wide range of cellular functions including cell migration, pericellular proteolysis, signal transduction, antigen presentation, Ca influx, transcytosis and synaptic plasticity [1,2,35]. Originally identified through functions as endocytic receptors in lipoprotein metabolism, a fundamental role in the control of cell signalling pathways was first revealed when the LDL receptor family members Apoer2 (Lrp8) and its partner Vldlr were found to mediate the positional signals that are conveyed by the signalling protein Reelin to migrating neurons during embryonic brain development [6]. This highly conserved signalling pathway involves the clustering-induced activation of Src family tyrosine kinases and is essential for the lamination of neuronal cortical layers. Subsequently, Lrp5 and Lrp6 (Arrow in Drosophila) were found to bind Wnt proteins and act as essential modulators of Wnt signalling. Lrp4 shares overlapping structural elements within its extracellular domain with Lrp5 and Lrp6, particularly with the region that bind Wnts and Wise. Introduction of a stop codon just upstream of the transmembrane segment of Lrp4 results in mutant animals that survive but have polysyndac- tyly and fusion of digits [15]. This allele of Lrp4 is thus assumed to PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e4092