fusions and molecular changes that include reduction in Shh signalling [15]. In Lrp4 mutants the differentiation of epithelium into inner enamel epithelium is normal, except for ectopic differentiation of epithelium at the junctions between developing molars. The formation of second and third molars in mice occurs by a process that is similar to that involved in the development of permanent tooth germs from deciduous tooth germs during human embryogenesis. The essential role of Lrp4 in this process implies that this protein may play an important but as yet unidentified role in the development of the permanent teeth in humans. Supernumerary teeth The formation of supernumerary teeth (mesial) anterior to the first molars, in the position of a premolar, has been described in several different mice with mutations that affect Fgf, Eda, Bmp and Shh signalling ([28,39,40]; Sharpe lab unpublished). The supernumerary teeth in Lrp4 mutants closely resemble those found in Wise mutants and their development can be first visualised by an ectopic patch of Shh expression in the diastema at E14.5. Interestingly at this same stage, the expression of Shh in the developing molars, located a few microns more proximally, is significantly reduced in Lrp4 mutant embryos. This suggests parallels with the context-dependent role of Wise in Wnt signalling described in Xenopus where it can either activate or antagonise Wnt signalling [18]. The possible primary role of Fgf signalling in supernumerary tooth formation in Lrp4 mutants can be excluded since no changes in Sprouty expression or Fgf signalling were observed (data not shown), suggesting that the Bmp/Shh interaction lies downstream of Fgf signalling. The formation of supernumerary incisors Supernumerary incisors were observed in both the maxilla and mandible of Lrp4 mutants that phenocopy the Wise mutants [41]. In wild-type mice, vestigial tooth germs are found in the incisor region that degenerate by apoptosis during development [42]. The supernumerary incisors in Wise mutants are thought to form as a result of the successive development of the rudimentary tooth germs, since apoptosis is reduced in the incisor region [41]. Ectopic Shh expression in developing incisor regions of Lrp4 and Wise mutants is indicative of the survival of rudimentary tooth germs. Bmp and Wnt signaling were also found to be upregulated in incisor regions of Wise mutants and which we also observed in Lrp4 mutants [41]. Presence of supernumerary teeth in both incisor and molar regions suggests common pathways regulating molar and incisor tooth number where Lrp4 is required for the correct modulation and integration of multiple pathways. Lrp4-Wise interaction – a role in extracellular signaling integration The accepted view of cell signaling by secreted proteins is that cell-cell communication is mediated by ligands binding to specific cell surface receptors that transmit an intracellular response. Secreted antagonists may also bind to the ligands to prevent pathway activation and presumably, but not well understood, the ligands must be removed from the extracellular environment, possibly by endocytosis. This removal of ligands is of critical importance in signaling in epithelial-mesenchymal interactions in development. In tooth development for example, the same ligands are repeatedly used as epithelial signals and mesenchymal signals at different times and this can only work if the ligands are removed very rapidly and effectively. Lrp4/Wise in Tooth Development The status of Bmp proteins in the extrcellular environment is communicated to epithelial cells expressing Lrp4, which in turn modulate intracellular Wnt activitys. Materials and Methods Immunoprecipitation HEK293A cells were transfected with pCDNA3-AP, pCDNA3- RAP-AP, pCDNA3- Wise-AP, pCDNA3-RSpondin2-AP, or pCDNA3-DKK1-AP constructs using FuGENE 6 (Roche) in DMEM plus 0.2% BSA medium to produce media containing AP or AP-tagged proteins. After 48 hr transfection, media were collected and the production levels of AP and AP-tagged proteins in the media were determined by AP activity assay using p- Nitrophenyl phosphate (Calbiochem) as a substrate and western blotting with anti-AP antibody (Sigma). To test binding of AP and AP-tagged proteins to LRP4 in cell- free system, medium containing ectodomain of LRP4 was produced by transfection of HEK293A cells with pCDNA3.1- LRP4ecto-Fc constructs in DMEM plus 0.2% BSA medium. The LRP4ecto-Fc medium was incubated with Protein A-Agarose (Sigma) at 4 C for 2 hrs to make LRP4ecto-Fc-Agarose conjugates and equal volumes of media containing AP or AP-tagged proteins were precleared with Protein A-Agarose at 4 C for 2 hrs prior to incubation with the conjugates at 4 C overnight. The agarose conjugates were washed three times with PBS, resuspended in 40 ul Leammli sample buffer, and anti-AP western blotting was performed. To test binding of AP and AP-tagged proteins in cell system, HEK293A cells were transfected with pCDNA3.1-LRP4 con- structs for 48 hrs, washed once with PBS plus 0.1%BSA, and incubated in equal volumes of media containing AP or AP-tagged proteins at 37 C for 1 hr. The cells were washed once with PBS, incubated with a cross-linker, dithiobis[succinimidylpropionate] (250 mM, Pierce), at room temperature for 30 min, harvested, washed three times with PBS, and lysed in 50 mM Tris-HCl buffer, pH 7.5 containing 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2,1% Triton X-100, and protease inhibitors (Roche). After determination of protein concentration by Lowry assay, equal protein amounts of the cell lysates were subjected to immunopre- cipitation using anti-AP antibody and Protein A-Agarose. The immunoprecipitates were reduced with 5% b-mercaptoethanol and anti-LRP4 western blotting was performed. The polyclonal rabbit anti-LRP4 antibody was generated in our laboratory against C-terminal peptide CWKHERKLSSESQV. Production and analysis of transgenic mice Lrp4 mutant mice were produced as described by Johnson et al. [15]. Wise mutant mice were produced as described by Kassai et al. [28]. Mice with a K14-Cre/Smoflox/flox mice were produced as described by Gritli-Linde et al. [33]. BAT-gal mice were produced as described by Maretto et al. [33]. Day E0 was taken to be midnight prior to finding a vaginal plug. To accurately assess the age of embryos, somite pairs were counted and the stage confirmed using morphological criteria e.g. relative sizes of maxillary and mandibular primordia, extent of nasal placode invagination, and the size of limb buds. Embryos were harvested at the appropriate time and genotyped using PCR and Southern blot analysis of genomic DNA extracted from unused embryonic or extraembryonic tissue. PCR assays and Southern blot hybridization were carried out. Lrp4 mutant mice and wild- type mice heads from E10 to newborn were fixed in 4% paraformaldehyde (PFA), wax embedded and serially sectioned at 7 mm. Sections were split over 5–10 slides and prepared for PLoS ONE | www.plosone.org 9 December 2008 | Volume 3 | Issue 12 | e4092