Lrp4/Wise in Tooth Development Figure 8. Schematic representation of Lrp4 in tooth development. Signalling of Lrp4 and Wise regulating tooth development. BMPs bind to the Ectodin/Wise, which in turn binds to Lrp4 inhibiting Wnt signalling. In the absence of Lrp4 or Wise, the excess BMPs bind to their receptor and activate both Wnt and Bmp signalling, which can result in downregulation of signalling. doi:10.1371/journal.pone.0004092.g008 incidence of the different molar fusions varies between individuals but is consistently more penetrant on the maxilla than the mandible. When we compared the molar teeth from Lrp4 mutants with those in mutants of the Bmp/Wnt antagonist Wise we observed an almost identical range of molar fusions and supernumerary teeth. In addition to the tooth phenotype, disorganised palatal rugae were observed in both Wise and Lrp4 and mutants (data not shown). The only significant difference phenotypically between the two mutants was the higher pene- trance of abnormal teeth on the mandible in Wise mutants, which we attribute to the fact that the Lrp4 mutation is hypomorphic, whereas Wise is believed to be a null. This remarkable phenocopy implies that a common genetic pathway through which Lrp4 and Wise control tooth development. Since Wise had previously been shown to bind to a specific region in the extracellular domain of Lrp5/6, we aligned this region in Lrp5/6 with Lrp4 revealing a remarkable degree of conservation. Using standard biochemical protein-protein inter- action assays, we found that Wise can also bind to Lrp4. Wise can modulate both the Bmp and Wnt pathways by acting as a high affinity BMP ligand antagonist and by competing for binding to Lrp’s with Wnts [18–21]. The complementary expression patterns of Bmp4 and Wise during early stages of tooth development have been interpreted as an example of a classic ‘‘activator-inhibitor morphogenetic interactions [28]. The Bmp inhibitory action of Wise was established in kidney cells and to a lesser extent in MC3T3 cells, whereas the inhibitory activity in vivo in tooth development has largely been inferred [19,20,28,29]. Wise was originally identified as a context-dependent activator or inhibitor of Wnt signalling in Xenopus animal cap assays [18]. In this contexts however, Wise did not appear to have any effect on Bmp signalling. We detected altered Bmp and Wnt signalling in Lrp4 mutants using phosphorylated Smads and BAT-Gal reporter mice as indicators of active Bmp and canonical Wnt pathways, respectively. Wnt and Bmp signalling was increased in those areas that are involved with molar tooth fusion, indicating that Lrp4 is essential for normal Bmp and Wnt signalling during the physical separation of molar tooth primordia. The fact that an identical phenotype was observed in Wise mutants is consistent with Lrp4 modulating Wnt and Bmp signaling by binding and thereby sequestering, presenting or endocytosing Wise in a context- dependent manner. This also implies that Wise inhibition of Bmp’s may require the presence of Lrp4. However, no obvious differences in Lrp4 or Wise expression could be detected in tooth germs of Wise or Lrp4 mutants, respectively (data not shown). Fused molars Increased Wnt and Bmp signalling and reduced Shh signalling, as a result of loss of either Wise or Lrp4, results in fusion of molar teeth. This fusion occurs when the epithelial cells in junctional regions differentiate into inner enamel epithelial cells. The reduction in Shh signalling that accompanies the increase in Bmp/Wnt activity is functionally important since conditional loss of Shh in dental epithelium also produces similar molar tooth fusions [33,36]. K14-Cre mediated Smo deletion produced normal inner enamel epithelium differentiation in molar teeth whereas K14-Cre mediated Shh deletion resulted in a slight disruption of inner enamel epithelium [33,36]. Another member of the LDL receptor family, Megalin, has been reported to bind Shh in addition to Bmps [37]. Loss of Megalin results in increased Bmp signalling and reduction of Shh expression in ventral forebrain development [38]. This common morphogenetic pathway may also include limb development since Lrp4 mutants exhibit polysyndactyly with digit PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e4092