Inhibition of Wnt signaling by Wise (Sostdc1) and negative feedback from Shh controls tooth number and patterning

Abstract

Mice carrying mutations in Wise (Sostdc1) display defects in many aspects of tooth development, including tooth number, size and cusp pattern. To understand the basis of these defects, we have investigated the pathways modulated by Wise in tooth development. We present evidence that, in tooth development, Wise suppresses survival of the diastema or incisor vestigial buds by serving as an inhibitor of Lrp5- and Lrp6-dependent Wnt signaling. Reducing the dosage of the Wnt co-receptor genes Lrp5 and Lrp6 rescues the Wise-null tooth phenotypes. Inactivation of Wise leads to elevated Wnt signaling and, as a consequence, vestigial tooth buds in the normally toothless diastema region display increased proliferation and continuous development to form supernumerary teeth. Conversely, gain-of-function studies show that ectopic Wise reduces Wnt signaling and tooth number. Our analyses demonstrate that the Fgf and Shh pathways are major downstream targets of Wise-regulated Wnt signaling. Furthermore, our experiments revealed that Shh acts as a negative-feedback regulator of Wnt signaling and thus determines the fate of the vestigial buds and later tooth patterning. These data provide insight into the mechanisms that control Wnt signaling in tooth development and into how crosstalk among signaling pathways controls tooth number and morphogenesis.

Fig. 1.

Dosage-dependent rescue of Wise tooth phenotypes in Lrp5 and Lrp6 mutants. (A-G) Tooth phenotypes in Wise-null mutants carrying varying doses of the Lrp5 and Lrp6 Wnt-coreceptor genes. The genotypes are listed on the left and the respective incisor or molar regions noted at the top. M, molar; Mn, mandibular; Mx, maxillary; T, tooth. Asterisks mark the supernumerary incisor and the black arrowhead marks the supernumerary lateral molar. (H) Summary of the genetic interaction with the number of scored jaw quadrants shown as n. All phenotypes were scored with littermates in a mixed background of C57/BL6 and 129Sv/Ev. SN, supernumerary.

Fig. 2.

Elevated Wnt signaling and continuous development of the R2 vestigial bud. (A-H) Wholemount Top-Gal expression in tooth germs of E12.5-E15.5 mandibles. (A′-H′) Parasagittal sections (anterior to the left) of the tooth germs from panels A-H show Top-Gal expression in the epithelial signaling center of the vestigial buds MS and R2, and in M₁. (I-J′) Tamoxifen (Tmx)-inducible mutation of β-catenin leads to ectopic Wnt activation in Shh-expressing cells of R2 and M₁ and continued development of R2. Tmx was injected at E13.5. (K-L′) Wise-LacZ BAC reporter expression in E14.5 tooth germs. Parasagittal (K,L) and frontal (K′,L′) sections showing reporter expression in mesenchymal cells. The dotted lines indicate the boundary between the dental epithelium and mesenchyme.

Fig. 3.

Abnormal partitioning of the tooth field in Wise-null mice. (A) Schematic diagrams summarizing the fate of tooth buds in control and Wise-null mice. (B-D) The root pattern indicates that the tooth field was repartitioned in Wise-null mice. Mandibular molars (top) and corresponding coronal sections (bottom).

Fig. 4.

Overexpression of Wise disrupted tooth development. (A) K14-Wise transgene. (B-E) Molars of transgenic mice were smaller with an abnormal cusp pattern and M₃ was frequently missing in the maxilla (E). (F-I) In the K14-Wise;Top-Gal embryo, tooth germs were growth-retarded with reduced Top-Gal expression, as shown in wholemount lower jaws (F,G) and frontal sections (H,I). Mn, mandibular; Mx, maxillary.

Fig. 5.

Proliferation and fate-mapping of the vestigial buds. (A-D) BrdU incorporation assays measuring cell proliferation in tooth germs. Asterisks mark mesenchymal cells underlying R2. (E-H) Immunostaining for cleaved caspase 3 to detect apoptotic cells. (I-J′) Shh was ectopically expressed in R2 of Wise-null tooth germs at E15.5, as shown in the whole mandibles (I,J) and on parasagittal sections (I′,J′). (K-L′) Shh-expressing cells and their descendants were marked by β-gal staining of the Shh^+/GFPcre;R26R embryos at E15.5. (M-N′) Tmx-induced labeling of the descendants of Shh-expressing cells indicates that MS contributes to the anterior outer enamel epithelium of M₁.

Fig. 6.

Genetic interaction between Wise, Shh, Ptch1 and Lrp6. (A-C) Adult molars in the mandible. The asterisk marks a supernumerary tooth. (D-L) Gfp expression from the GfpCre knock-in Shh allele in wholemount tooth germs shown as inverted gray scale. (M) Exacerbation of Wise-null tooth phenotypes by reducing Shh dosage. (N) Rescue of Wise^+/−;Shh^+/− supernumerary tooth by reducing dosage of Ptch1 or Lrp6. The Shh^GFPcre allele in a C57/BL6 background was used for analyses. n, number of mandibular jaw quadrants.

Fig. 7.

Shh negatively regulates Wnt signaling in tooth germs. (A-H) Wnt signaling was elevated in R2 of Wise^+/−;Shh^+/− mice, as shown by Top-Gal expression in whole mandibles and parasagittal sections. (I-L) Top-Gal expression was elevated in Shh-deficient (K14-cre;Shh^neo/fx) tooth germs. Increased Top-Gal expression was also observed in fungiform taste papillae (I″,J″). (M-R) Top-Gal expression in the maxillary tooth germs of Wise (M,N), K14-cre;Shh^neo/fx (O,P) and Shh^fx/CreERT (Q,R; Tmx injection at E13.5) mice. (S) qPCR analysis of selected genes in Wise^+/− and Wise^+/−;Shh^+/− tooth germs at E13.5.

Fig. 8.

Model for signaling network in diastema tooth development. Schematic diagrams of the signaling network regulating development of the diastema R2 bud at E13.5-E14.5 in wild-type and genetic mutant backgrounds. In wild-type teeth, Wnt signaling is required for the bud-to-cap transition of R2 by inducing Fgfs. Wise and Spry2/4 antagonize Wnt and Fgf signaling, respectively, to suppress the transition. Shh is a downstream target of Wnt signaling and acts as a negative-feedback regulator of Wnt signaling via Dkk1 and other targets. Elevated Wnt signaling feeds back to stimulate Wise expression. The thickness of the lines represents relative levels of activity. Red indicates repressive input and black indicates positive input to activity.