Ectodysplasin regulates activator-inhibitor balance in murine tooth development through Fgf20 signaling

Abstract

Uncovering the origin and nature of phenotypic variation within species is the first step in understanding variation between species. Mouse models with altered activities of crucial signal pathways have highlighted many important genes and signal networks regulating the morphogenesis of complex structures, such as teeth. The detailed analyses of these models have indicated that the balanced actions of a few pathways regulating cell behavior modulate the shape and number of teeth. Currently, however, most mouse models studied have had gross alteration of morphology, whereas analyses of more subtle modification of morphology are required to link developmental studies to evolutionary change. Here, we have analyzed a signaling network involving ectodysplasin (Eda) and fibroblast growth factor 20 (Fgf20) that subtly affects tooth morphogenesis. We found that Fgf20 is a major downstream effector of Eda and affects Eda-regulated characteristics of tooth morphogenesis, including the number, size and shape of teeth. Fgf20 function is compensated for by other Fgfs, in particular Fgf9 and Fgf4, and is part of an Fgf signaling loop between epithelium and mesenchyme. We showed that removal of Fgf20 in an Eda gain-of-function mouse model results in an Eda loss-of-function phenotype in terms of reduced tooth complexity and third molar appearance. However, the extra anterior molar, a structure lost during rodent evolution 50 million years ago, was stabilized in these mice.

Fig. 1.

Fgf20 expression in mouse molar development. (A-D) Fgf20 is expressed in the dental epithelium: at initiation (A,B), placode (C) and bud (D). (E,F) At the cap stage, expression is concentrated in the primary enamel knot (E) and at the bell stage in the secondary enamel knots (F).

Fig. 2.

Fgf20 expression is regulated by Eda. (A) Treatment of E13 Eda^–/– mouse molars with Eda protein upregulates Fgf20 transcription after 4 hours of culture (mean ± s.d.). ^*P<0.05. (B-J) Expression of Fgf20 at E12 (B-D), E13 (E-G) and E14 (H-J) in developing teeth of Eda^–/–, WT and K14-Eda mice. i, incisor; m, molar. Asterisk indicates extra molar.

Fig. 3.

Adult Fgf20^βGal/βGal molars are small and show defects in anteroconid cusp patterning. (A-C) Molars in Fgf20^βGal/βGal mice are smaller compared with WT, resembling Eda^–/– mice. The cusp pattern is affected in the anterior part of the first molar (dashed line). (D-G) All molars (m1, m2, m3) are shorter (D) and narrower (E), and thus smaller in two-dimensional size (F) compared with WT teeth. The width of the whole jaw is unaffected (G). (H) The distance (normalized to molar diameter) between the cusps in the first cusp pair (1) is shorter, whereas the distances in other cusp pairs (2-7) show no differences compared with the WT molars (cusp pairs indicated in A). Mean ± s.d. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Fig. 4.

Fgf20^βGal/βGal molars are reduced in size and cusp number during development. (A,B) In tissue culture conditions, WT mouse molars (A) typically form five cusps, lacking the most posterior cusp, the hypoconulid. Eda^–/– molars (B) typically form only three cusps, lacking the most anterior cusp, the anteroconid (white arrow). The two most posterior cusps, the talonid cusps (black arrows), are represented by only one cusp. (C-E) Fgf20^βGal/βGal molars show a variety of phenotypes ranging from WT-like five cusps (C) to Eda-null-like four cusps (D). Most commonly, the anteroconid is abnormally small (E) or missing completely. (F) Measurement of tooth area at E13+7 days. Mean ± s.d. ^**P<0.01. NS, non-significant.

Fig. 5.

Fgf20 has similar effects on embryonic dental mesenchyme as Fgf4 and Fgf9 and acts redundantly with Fgf9. (A-D) Beads supplemented with Fgf4, Fgf9 and Fgf20, but not BSA, induce the expression of Fgf3 in WT molar mesenchyme after 24 hours. (E,F) Fgf20 induces Runx2 expression around the bead. Asterisk indicates endogenous expression of Runx2 in an island of osteogenic mesenchyme. (G-J) Fgf20 protein stimulates cell proliferation in isolated dental mesenchyme (EdU-positive cells; red) after 24 hours of culture, whereas BSA has no effect. The position of the protein-releasing bead is indicated (dashed line). (K-N) Fgf9^–/–;Fgf20^βGal/βGal molars have developed into cap stage at E14.5, similar to WT control and Fgf9 and Fgf20 single mutants, and express Shh in the enamel knot (red). (O) However, the enamel knot is significantly shorter in Fgf9^–/–;Fgf20^βGal/βGal compared with single mutants and controls. (P) Expression of Fgf4 and Fgf9 is not induced after 4 hours Eda treatment in E13 (Fgf9) or E14 (Fgf4) Eda^–/– molars. Mean ± s.d. ^*P<0.05, ^**P<0.01. NS, non-significant.

Fig. 6.

The number, size and shape of K14-Eda molars are affected by deletion of Fgf20. (A,B) K14-Eda mice have an extra molar (EM) anterior to m1 in ∼50% of tooth rows (A with an EM; B without an EM, black arrow). (C,G) Inactivation of one Fgf20 allele (K14-Eda;Fgf20^+/βGal) results in a slightly smaller m2, without otherwise affecting the K14-Eda phenotype (mean ± s.d.). (D-F) In complete knockout of Fgf20 (K14-Eda;Fgf20^βGal/βGal), m1-m3 are smaller and the cusp pattern is abnormal, with reduced cusp number and complexity. (H) Tooth size in K14-Eda;Fgf20^βGal/βGal mice shows a trend towards increased reduction of more posterior molars compared with K14-Eda. (I) Also, the relative size of molars within a tooth row is changed: m2/m1 and m3/m1 are altered in K14-Eda;Fgf20^βGal/βGal mice compared with K14-Eda (linear regression of means). Upper table: mean molar proportions (m2/m1 and m3/m1) ± s.d. Lower table: Frequency of the EM is increased in K14-Eda;Fgf20^+/βGal and, more clearly, in K14-Eda;Fgf20^βGal/βGal tooth rows compared with K14-Eda mice. Also, m3 is lost in 31% of the K14-Eda;Fgf20^βGal/βGal tooth rows. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Fig. 7.

Eda upregulation and Fgf20 inhibition independently stabilize the foci of Shh signaling in the anterior end of m1. (A-L) Shh localization in E13.5 and E14.5 mandibles by whole-mount in situ hybridization. In WT mice, a small dot of Shh expression (blue, black arrow) is detected in the anterior end (AE) of m1 in one third of the E13.5 jaws (A). This Shh expression focus has disappeared by E14.5 (G). However, partial (Fgf20^+/βGal, B,H) or complete (Fgf20^βGal/βGal, C,I) loss of Fgf20 stabilize the Shh focus at E13-14.5. Eda upregulation alone (K14-Eda, D,J) or combined to partial (K14-Eda;Fgf20^+/βGal, E,K) or complete (K14-Eda;Fgf20^βGal/βGal, F,L) loss of Fgf20 also stabilize the Shh expressing foci. (M) Percentage of Shh-expressing foci present or absent in E13.5 half mandibles and the number of samples. (N) In K14-Eda;Fgf20^βGal/βGal, the Shh foci are larger compared with K14-Eda;Fgf20^+/βGal at E14.5 ^*P<0.05, ^**P<0.01. Error bars represent mean ± s.d.

Fig. 8.

Fgf20 regulates the expression of Spry2 and Spry4. (A-D) E14.5 molar (m1) epithelium (A,B) or mesenchyme (C,D) cultured with heparin beads soaked in Fgf20 recombinant protein or BSA. Fgf20 induces Spry2 (blue; B) in the epithelium and Spry4 in the mesenchyme (D), whereas BSA does not (A,C). (E-L) Sections of E14.5 molar (m1) from the anterior end (AE) and the largest area of the cap (as illustrated in the schematic). Spry2 is expressed in the epithelium in the AE and cap of K14-Eda molar (E,G) and is reduced in K14-Eda;Fgf20^βGal/βGal especially in AE (F,H). Spry4 expression is confined to mesenchyme (I,K), and is reduced in AE in K14-Eda;Fgf20^βGal/βGal (J,L) compared with K14-Eda. (M,N) Etv5 expression is high in the AE of K14-Eda;Fgf20^βGal/βGal in the epithelium but reduced in the mesenchyme (N) compared with the AE of K14-Eda (M). Dashed line indicates the margin of the epithelium.

Fig. 9.

Integration of Eda/Edar/NF-κB signaling in the enamel knot with an Fgf signal loop regulates tooth crown development. Schematic based on our findings and reported data. Activation of Edar receptor by Eda in the enamel knot (EK) leads to activation of NF-κB and Fgf20 transcription. Fgf20, together with Fgf4 and Fgf9, moves to mesenchyme, induces the transcription of Runx2 and Fgf3 and stimulates cell proliferation. Fgf3 and Fgf10 signal back to the epithelium and stimulate proliferation. Fgf20 induces Spry4 expression in the mesenchyme and Spry2 expression in the epithelium. Spry4 and Spry2 inhibit Fgf signaling in the mesenchyme and epithelium, respectively.