Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development Atsushi Ohazama1, Eric B. Johnson 2 , Masato S. Ota3, Hong J. Choi2, Thantrira Porntaveetus1, Shelly Oommen1, Nobuyuki Itoh 4 , Kazuhiro Eto3, Amel Gritli-Linde5, Joachim Herz2, Paul T. Sharpe1* 1 Department of Craniofacial Development, Dental Institute, King’s College London, Guy’s Hospital, London, United Kingdom, 2 Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, Texas, United States of America, 3 Section of Molecular Craniofacial Embryology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, 4 Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto, Japan, 5 Department of Oral Biochemistry, Sahlgrenska Academy at Goteborg University, Goteborg, Sweden Abstract The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP’s and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway. Citation: Ohazama A, Johnson EB, Ota MS, Choi HJ, Porntaveetus T, et al. (2008) Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development. PLoS ONE 3(12): e4092. doi:10.1371/journal.pone.0004092 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received October 3, 2008; Accepted November 21, 2008; Published December 31, 2008 Copyright: 2008 Ohazama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Wellcome Trust and MRC. A.O. is an RCUK Fellow. J.H. is supported by grants from the NIH and is the recipient of a Wolfgang Paul Award from the Alexander-von-Humboldt Foundation (HL20948 and HL63762). A.G.L. is supported by the Swedish Research Council-Medicine (grants 2789, 14100 and 15181) and Faculty of Odontology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: paul.sharpe@kcl.ac.uk Introduction The integration of different cell signaling pathways is increas- ingly recognized as being of fundamental importance in development. Most attention has necessarily focused on the intracellular links between pathways since ligand-receptor-antag- onist interactions that occur outside the cell are pathway specific. However the concurrent secretion of ligands in developmental processes suggests that pathways of extracellular integration must exist. Here we describe an integration between a secreted BMP antagonist, Wise (also known as USAG-1, Sosdc1 and Ectodin), and a negative Wnt co-receptor, Lrp4, that provides a novel method of extracellular communication between mesenchymal and epithelial cells based on the integration of Wnt and Bmp pathways. This integration occurs in the context of epithelial- mesenchymal signaling controlling processes that regulate tooth number. The low-density lipoprotein (LDL) receptor family is a large evolutionarily conserved group of transmembrane proteins (for reviews, see [1,2]). The LDL receptor was first identified as an endocytic receptor that transports the lipoprotein LDL into cells by receptor-mediated endocytosis. In this process, specific ligands are internalized after binding to their receptors on the cell surface from where they are moved to an intracellular vesicle (endosome) and then discharged to other compartments inside the cell. The LDL receptor mainly regulates the concentration of lipoproteins in the extracellular fluids and delivers them to cells (i.e. for uptake of cholesterol). More recent findings have shown that LDL receptor family members can also function as direct signal transducers or modulators for a broad range of cellular signalling pathways. For example, LDL receptor-related protein 1 (Lrp1) is involved in the modulation and integration of PDGF and TGFb signals in smooth muscle cells of the vascular wall [3–5], Apoer2 (Lrp8) and its partner Vldlr controls brain development [6] and synaptic transmission [7,8] through their common signalling ligand Reelin (reviewed in [2]), and Lrp5 and Lrp6 function as co-receptors in the Wnt signalling cascade [9–11]. Canonical Wnt/b-catenin signalling mediated by Lrp5 and Lrp6 plays a central role in mammalian bone density regulation [12]. Loss of Lrp5 function results in osteoporosis pseudoglioma syndrome that is character- ized by a juvenile onset of decreased bone mass [13]. Lrp4 (also called Megf7) belongs to the LDL receptor family and ENU- induced Lrp4 mutants die at birth with defects in formation of multiple embryonic tissues [14]. However, several other allelic mutations at the Lrp4 locus have been reported that survive [15– 17]. A retroviral-derived allele appears to be hypomorphic, because wild-type transcripts are present in these mutants [16]. A second allele was generated by targeted mutation by introducing a stop codon just upstream of the transmembrane domain. This allele is also assumed to be hypomorphic, since it has an identical phenotype to the retrovirally-derived alleles [15,16]. Lrp5/6 have been shown to be able to modulate both Wnt and Bmp signalling by the direct binding of Bmp antagonists such as Wise, replacing binding of Wnts [18–21]. Similarly, Lrp4 was PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e4092