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Lrp4 modulates extracellular integration of cell signaling pathways in development - PubMed

Lrp4 modulates extracellular integration of cell signaling pathways in development

Atsushi Ohazama et al. PLoS One. 2008.

Erratum in

  • PLoS ONE. 2009;4(1). doi: 10.1371/annotation/f19bff3b-227a-4159-b166-7dc3c19bec43. Choi, Hong J [corrected to Choi, Hong Y]

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.

Figure 2
Figure 2. The expression patterns of Lrp4 and Wise during early molar and incisor tooth development.

(A, B) Lrp4 was expressed in tooth epithelium whereas Wise expression was observed in tooth mesenchyme. (C, E) The expression of Lrp4 was restricted to the primary enamel knots. (D, F) Wise expression was found in both epithelium and mesenchyme but was absent from primary enamel knots. (G, H) Sections showing complementary expression of Lrp4 (G) and Wise (H) in incisor regions. Yellow arrow representing the region of Lrp4 expression (H). Tooth epithelium outlined in green (G, H). Radioactive in situ hybridisation on frontal sections showing Lrp4 expression (A, C, E, G) and Wise expression (B, D, F, H) in embryo heads at E12.5 (A B), E13.5 (C, D) and E14.5 (E–H).

Figure 3
Figure 3. Supernumerary teeth in incisor region.

Supernumerary teeth were observed in incisor region on both maxilla (arrows in B, G and arrowheads in C) and mandible (arrows in E, H and arrowheads in F) of both Lrp4 and Wise mutants. Sagittal view of maxillary incisor region (A and B), lingual view of mandibular incisors (D, E and H), occlusal view of maxillary incisors (G), sagittal sections (C) and frontal sections (F) of wild-type (A, D), Lrp4 mutants (B, C, E, F) and Wise mutants (G, H) at adult.

Figure 4
Figure 4. The molar tooth phenotypes of Lrp4 mutant mice and Wise mutant mice.

SEM images of maxillary molars (A–C), 3D reconstructions based of micro CT scans (D, M and N), horizontal micro CT sections (E–I, K and L) and dissected maxillary molars (O and P) of adult wild-type (A, E, F, M and O), Lrp4 mutants (B–D, G–I and N) adults, Wise mutants (J–L) and K14-Cre/Smoflox/flox mice (P) of adult (A–N) and P6 (O, P). One fused molar (B), fused molar with one relatively normal size tooth and lingual peg-shaped extra tooth (arrowhead in C), and fused molar with two reratively normal size teeth (D). Horizontal micro CT sections at the crown region (E and G) and root region (F, H and I). (F) In wild type, there are three groups of tooth roots in maxillary molars (red circle = three roots of the first molar; blue circle = three roots of the second molar; yellow circle = two roots of the third molar). (G–I) In Lrp4 mutants, the fused molar with one normal size tooth showed four groups of tooth roots, indicating that a supernumerary tooth was present in the quadrant. The fused molars (H) had three groups of roots (one root in green circle; three roots in red circle; three roots in blue circle in H), and one normal size molar had one root as one unit (yellow circle in I), suggesting that the fused molar includes the supernumerary tooth. At the horizontal section level showing the tooth roots of the fused molars, the roots of a normal size molar could not be seen (H). Fused molars were also found in the maxillae of Wise mutant mice (J). Wise mutant fused molars also showed the several groups of roots (K and L). Supernumerary teeth were observed anterior to the first maxillary molar tooth (sn in N) of Lrp4 mutant mice (N). Fused maxillary molar of K14-Cre/Smoflox/flox mice (P). b; buccal side, l: lingual side. In all images, left side is anterior side. Scale bar = 1 mm.

Figure 5
Figure 5. Gene expression in incisor region.

(A–D) Ectopic Shh expression in incisor region of Lrp4 mutant (arrowheads in B) and Wise mutants (arrowheads in C). (E, F) Wnt activity in enamel knot in wild-type mice (arrowhead in E). Ectopic Wnt activity in lingual side of endogeneous incisor tooth germ (arrowheads in F). Tooth epithelium outlined in green (E, F). Whole mount (A–D) and β-gal activity (E, F) showing Shh (A–D) expression and Wnt activity (E, F) at E14.5 in wild-type (A, C, E), Lrp4 mutants (B, F) and Wise mutants (D).

Figure 6
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).

Figure 7
Figure 7. Shh, Bmp and Wnt signalling in fused maxillary molars.

Sagittal section showing tooth bud epithelium of a supernumerary tooth in Lrp4 mutant at E14.5 (arrowhead in A') and E16.5 (Supernumerary tooth; blue arrowhead, the first molar tooth germ; yellow arrowhead, the second molar tooth germ; black arrowhead in G'). Transient epithelial swelling of vestigial remants of diastema tooth in wild-type at E14.5 (arrowhead in A) and no swelling at E16 (G). Weak expression of Lrp4 (B) and strong Wise (C) were observed at the posterior part of tooth germ at E14.5 (arrowheads in B, C). Shh expression domain was reduced in Lrp4 mutants (D'). Ptc1 (E) and Gli1 (F) expression are found at posterior part of tooth germ at E14.5 (arrowheads in E, F). The first molar epithelium is still continuous with the second molar epithelium at E16.5 in wild-type (G). The junction region between the first and second molar could be distinguished by the absence of inner enamel epithelium in wild-type whereas Lrp4 mutant showed continuouse inner enamel epithelium from first molar to second molar (red arrowhead in G–H'). H and H' are high magnification of the junction region in G and G'. Strong Wise expression was observed at the joint region between first and second molars but no Lrp4 expression in the region (arrowheads in I and J). Shh expression are observed in only inner enamel epithelium of the first molar and second molar (K). Gli1 is expressed in junctional epithelium as well as tooth germs (L). Gli2 and Ptc1 expression were not observed in the epithelium of the junction region in Lrp4 mutnats whereas they were expressed in the epithelium of the region (M–N'). Bmp7 were upregulated at the junction region in Lrp4 mutants whereas they were not expressed at the region in wild-type (O, O'). Phosphorylated-Smad1/5/8 (Pho-Smad) was not detected at the junction region in wild-type whereas it was found in region corresponding the junction region in Lrp4 mutant (arrowhead in P and P'). Wnt activity was observed indistinctly in molar region of wild type (Q) whereas it was obvious in molar region of Lrp4 mutant (Q'). Ectopic Wnt activity was also found where supernumerary molar develop (arrowhead in Q'). In wild types, sagittal sections showed Wnt activity were detected in inner enamel epithelium including enamel knot and stratum reticulum whereas it was not observed in the junction region (arrowhead in R). In Lrp4 mutants, Wnt was activated in the junction region (arrowhead in R'). Maxillary molar tooth at E14.5 (A–F) and E16.5 (G–R') of wild-type (A, B–D, E–G, H, I–M, N, O, P, Q, R) and Lrp4 mutant (A', D', G', H' M', N', O', P', Q' R'). Histology (A, A', G–H'), immunohistochemistry (P and P'), β-gal activity (Q–R') and radioactive in situ hybridisation (B–F, I–Q') on oral view (Q and Q') and sagittal sections (A–P', R, R').

Figure 8
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.

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References

    1. Nykjaer A, Willnow TE. The low-density lipoprotein receptor gene family: a cellular Swiss army knife? Trends Cell Biol. 2002;12:273–280. - PubMed
    1. Herz J, Bock HH. Lipoprotein receptors in the nervous system. Annu Rev Biochem. 2002;71:405–434. - PubMed
    1. Boucher P, Gotthardt M, Li WP, Anderson RG, Herz J. LRP: role in vascular wall integrity and protection from atherosclerosis. Science. 2003;300:329–332. - PubMed
    1. Herz J, Hui DY. Lipoprotein receptors in the vascular wall. Curr Opin Lipidol. 2004;15:175–181. - PubMed
    1. May P, Woldt E, Matz RL, Boucher P. The LDL receptor-related protein (LRP) family: an old family of proteins with new physiological functions. Ann Med. 2007;39:219–228. - PubMed

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