shown to suppress Wnt signalling, likely by competing for LRP5/6 in the Wnt/Fz complex [15]. We have identified a domain in Lrp4 that contains the highly conserved region where Wnts and Wise bind in Lrp5/6 and provide biochemical evidence that Wise can bind to Lrp4. The tooth is an organ that develops as a result of sequential and reciprocal interactions between the oral epithelium and neural crest-derived mesenchyme. The first morphological sign of tooth development is thickening of the oral epithelium. The thickened epithelium progressively takes the form of ‘‘bud’’, ‘‘cap’’ and ‘‘bell’’ configurations as differentiation and morphogenesis pro- ceeds [22]. Epithelial cells and mesenchymal cells (dental papilla) differentiate into enamel-secreting ameloblasts and dentin-secret- ing odontoblasts, respectively. It has been established that many different signalling pathways such as Bmp, Fgf, Wnt, Shh and Tnf are involved at multiple stages of tooth development (for reviews, see [23–25]). A role for Lrps in any of these signalling pathways in tooth development has however not been established. We report here that Lrp4 is expressed in spatially restricted patterns in epithelial cells during tooth development. Changes in Bmp and Wnt signalling were observed during tooth development in both Lrp4 and Wise mutants. Lrp4 mutants display a range of tooth number abnormalities that are identical to those seen in Wise mutants and include fused molars and supernumerary incisors and molars. We observed upregulation of both Wnt and Bmp activities in Lrp4 and Wise mutants that were accompanied by a downstream loss of Shh activity. The antagonism of BMP signaling by Wise thus does not occur in the absence of Lrp4. We propose that the ability of Wise to bind BMP’s and to Lrp4 allows it to act as an extracelluar, mesenchyme to epithelial signaling protein that is capable of BMP with Wnt signaling. Results Interaction between WISE and LRP4 Wise acts to modulate both BMP and Wnt signalling during development. The action of Wise on BMP signaling is as a secreted antagonist that binds BMP ligands [19,20]. The Wnt modulation by Wise is mediated by its binding to the extracellular domain of Lrp6. [18]. The extracellular domain of Lrp6 contains four EGF repeats and Wise (a cysteine knot protein) shares repeats 1–2 of the domain of Lrp6 essential for interaction with Wnts. Alignment of the amino acid sequences of EGF-like repeats 1 and 2 of Lrp5 and Lrp6 showed this region to be highly conserved in Lrp4 (Figure 1A), raising the possibility that Lrp4 might also interact with Wise and thereby mediate the integration of Wnt and Bmp signals during morphogenesis. A similar mechanism in which Lrp1 was shown to integrate PDGF and TGFb signals in the vascular wall has been proposed by Boucher et al. [3]. In order to test for a physical interaction between Wise and Lrp4, we performed two types of binding assays in cultured cells and in vitro. In the first assay, HEK293A cells expressing Lrp4 were incubated with media containing the alkaline phosphatase (AP)-tagged known ligand, (RAP), and the putative ligands for Lrp4, Wise and R-spondin2. AP-fusion proteins were then isolated with an antibody against AP and immunoblotted to detect co-precipitated Lrp4 (Figure 1C). In another assay, the converse experiment was performed were Fc- Lrp4 fusion protein was immobilized on ProteinA beads and incubated with the AP-tagged putative ligands. Bound proteins were detected by immunoblotting (Figure 1D). Both assays gave equivalent results, revealing interaction of Lrp4 with Wise, but not with R-spondin2, another modulator of the Wnt signalling pathway [26,27]. AP and AP-RAP served as negative and positive controls, respectively. Lrp4/Wise in Tooth Development Figure 1. RAP and Wise, but not AP and RSpondin2 (RS2), bind to LRP4. (A) Sequence alignment of Lrp4, Lrp5 and Lrp6. Alignment of amino acid sequence of EGF-like repeats 1 and 2 of mouse in the extracellular domains of Lrp5/6 and Lrp4. (B) Media containing AP or AP-tagged proteins were produced by transfection of HEK293A cells with indicated constructs for 48 hrs. AP activity measured in media shows various expression levels and no presence of AP in control medium. (C) HEK293A cells expressing LRP4 were incubated in equal volumes of media containing indicated proteins, treated with a cross- linker dithiobis[succinimidylpropionate], and lysed prior to analysis of LRP4-binding proteins by immunoprecipitation with anti-AP antibody followed by immunoblotting with anti-LRP4 antibody. (D) LRP4 ectodomain fused with human Fc was produced as a secreted protein, conjugated to Protein A-Agarose, and incubated in equal volumes of media containing indicated proteins prior to analysis of LRP4 binding proteins by immunoblotting with anti-AP antibody. doi:10.1371/journal.pone.0004092.g001 PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e4092