A Wnt-bmp feedback circuit controls intertissue signaling dynamics in tooth organogenesis

Abstract

Many vertebrate organs form through the sequential and reciprocal exchange of signaling molecules between juxtaposed epithelial and mesenchymal tissues. We undertook a systems biology approach that combined the generation and analysis of large-scale spatiotemporal gene expression data with mouse genetic experiments to gain insight into the mechanisms that control epithelial-mesenchymal signaling interactions in the developing mouse molar tooth. We showed that the shift in instructive signaling potential from dental epithelium to dental mesenchyme was accompanied by temporally coordinated genome-wide changes in gene expression in both compartments. To identify the mechanism responsible, we developed a probabilistic technique that integrates regulatory evidence from gene expression data and from the literature to reconstruct a gene regulatory network for the epithelial and mesenchymal compartments in early tooth development. By integrating these epithelial and mesenchymal gene regulatory networks through the action of diffusible extracellular signaling molecules, we identified a key epithelial-mesenchymal intertissue Wnt-Bmp (bone morphogenetic protein) feedback circuit. We then validated this circuit in vivo with compound genetic mutations in mice that disrupted this circuit. Moreover, mathematical modeling demonstrated that the structure of the circuit accounted for the observed reciprocal signaling dynamics. Thus, we have identified a critical signaling circuit that controls the coordinated genome-wide expression changes and reciprocal signaling molecule dynamics that occur in interacting epithelial and mesenchymal compartments during organogenesis.

Fig. 1.

Bioinformatic analyses reveal concordant genome-wide transcriptional dynamics in dental epithelium and mesenchyme and the primacy of Wnt and Bmp signaling. (A) Principal components (PC) analysis of microarray gene expression profiles from LCM and manually dissected first lower molar epithelial (Epi) and mesenchymal (Mes) tissues from the E10.0 (initiation) to E14.5 (enamel knot, EK) stages. n = 2 to 3 biological microarray replicates. (B) Contingency table reveals significant concordance in differentially regulated genes (DRG) between epithelium and mesenchyme. Five hundred fifty and 900 genes were concordantly regulated, compared to 91 and 44 that were discordantly regulated (P < 10⁻¹⁵, χ² test; concordance score = 0.83). Bud, bud stage; init, initiation stage. (C) Average scaled expression of extracellular signaling molecules, with decreasing epithelial expression and increasing mesenchymal expression between E11.0 and E12.5, which recapitulates the dynamic shift in tooth instructive potential from epithelium to mesenchyme at E12.5 (9). (D and E) Molecular concept maps showing significant overlap (FDR <0.05 and odds ratio >2) between differentially regulated genes caused by endogenous signaling or by addition of Wnt, Bmp, Shh, and Fgf agonists to mesenchyme (initiation stage) or epithelium (bud stage). n = 3 biological microarray replicates.

Fig. 2.

The epithelial-mesenchymal intertissue gene regulatory network for early odontogenesis encodes a Wnt-Bmp feedback circuit. (A and B) Epithelial-mesenchymal gene regulatory networks describing ligand regulation by Wnt and Bmp pathways. Each signaling pathway node (that is, Wnt pathway, Bmp pathway) represents a gene set encoding components of the respective signal transduction pathway; each extracellular signal node (for example, Shh, Bmp4, and so on) represents gene expression for the respective signaling molecule at E13.5. Color represents scaled expression. Evidence for each edge (black arrows) was inferred from a combination of gene expression data (this study) and from genetic evidence from the literature and is available at http://compbio.med.harvard.edu/ToothCODE. (C) Epithelial and mesenchymal gene regulatory networks in (A) and (B) are coupled through the action of Wnt and Bmp4 extracellular signaling molecules to generate a single integrated intertissue gene regulatory network. Other signaling molecules shown in (A) and (B) are omitted for clarity. Nodes for epithelial- and mesenchymal-derived Wnt and Bmp4 are considered functionally equivalent and are indicated by the orange rectangle. Orange arrows represent signal transduction through ligand-receptor interaction. (D and E) Gene expression of Bmp4 and average expression of canonical Wnt genes based on time course microarray data.

Fig. 3.

Constitutive epithelial Wnt signaling produces a short circuit and bypasses the requirement for mesenchymal Bmp4 expression. (A) Epithelial Apc loss of function (constitutive canonical Wnt pathway activity, outlined in red) is predicted to short-circuit the intraepithelial Wnt-Bmp feedback circuit by uncoupling dependence on mesenchymal Bmp4 expression, which is decreased in Pax9- and in Msx1-null mutants (X); this enables supernumerary tooth formation. (B) Amelogenin (Amel) and Dentin sialophosphoprotein (Dspp) expression confirms ameloblast and odontoblast differentiation, respectively, in E17.5 sagittal sections of compound epithelial Apc loss-of-function; Pax9-null mutants (Apc^f/f; Pax9^−/−). The epithelium is denoted by the dashed red lines. n = 3 nonadjacent sections. (C) Coronal sections at E14.0 showing increased epithelial Wnt6 expression in Apc^f/f; Pax9^−/− mutants compared to control (Apc^+/f; Pax9^+/−). Expression of epithelial Bmp4 is increased and that of mesenchymal Bmp4 expression is decreased in Apc^f/f; Pax9^−/− mutants compared to control. n = 3 nonadjacent sections. Scale bars, 200 μm (B) and 100 μm (C). See also figs. S11 to S14.

Fig. 4.

Loss of Bmpr1a signaling breaks the Wnt-Bmp circuit and prevents tooth formation induced by constitutive epithelial Wnt signaling. (A) Epithelial Bmpr1a loss of function (X) breaks the odontogenic circuit and results in decreased expression of genes encoding canonical Wnt ligands. The Wnt pathway node outlined in red represents constitutive Wnt pathway activity. (B) Sagittal sections at E17.5 in compound epithelial Apc loss-of-function; Bmpr1a loss-of-function (Apc^f/f; Bmpr1a^f/f) mutants reveal undetectable Amel and Dspp expression and failure of tooth differentiation. The epithelium is denoted by the dashed red lines. n = 3 nonadjacent sections. (C) At E14.5, qRT-PCR reveals decreased Wnt3a, Wnt6, and Wnt10a expression in Apc^f/f; Bmpr1a^f/f compared to epithelial Apc loss-of-function (Apc^f/f; Bmpr1a^+/f) mutants. Expression of Wnt10b was similar in Apc^f/f; Bmpr1a^f/f and Apc^f/f; Bmpr1a^+/f. Data are means ± SD (n = 6; two biological replicates run in technical triplicate) normalized to Hprt. (D) Decreased epithelial Wnt6 expression in Apc^f/f; Bmpr1a^f/f compared to Apc^f/f; Bmpr1a^+/f mutants in E14.0 coronal sections. Similar epithelial expression of Bmp4 in Apc^f/f; Bmpr1a^f/f compared to Apc^f/f; Bmpr1a^+/f mutants. n = 3 nonadjacent sections. Scale bars, 200 μm (B) and 100 μm (D). See also figs. S15 to S17.