Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling

Abstract

Much of our knowledge about mammalian evolution comes from examination of dental fossils, because the highly calcified enamel that covers teeth causes them to be among the best-preserved organs. As mammals entered new ecological niches, many changes in tooth number occurred, presumably as adaptations to new diets. For example, in contrast to humans, who have two incisors in each dental quadrant, rodents only have one incisor per quadrant. The rodent incisor, because of its unusual morphogenesis and remarkable stem cell-based continuous growth, presents a quandary for evolutionary biologists, as its origin in the fossil record is difficult to trace, and the genetic regulation of incisor number remains a largely open question. Here, we studied a series of mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor-tyrosine kinase signaling. In sprouty loss-of-function mutants, splitting of gene expression domains and reduced apoptosis was associated with subdivision of the incisor primordium and a multiplication of its stem cell-containing regions. Interestingly, changes in sprouty gene dosage led to a graded change in incisor number, with progressive decreases in sprouty dosage leading to increasing numbers of teeth. Moreover, the independent development of two incisors in mutants with large decreases in sprouty dosage mimicked the likely condition of rodent ancestors. Together, our findings indicate that altering genetic dosage of an antagonist can recapitulate ancestral dental characters, and that tooth number can be progressively regulated by changing levels of activity of a single signal transduction pathway.

Fig. 1.

Incisor duplication in Spry2^+/–;Spry4^–/– mice. (A) Schematic of incisor in a perinatal mouse; dotted vertical black line indicates plane of section for D and E. (B) Schematic of incisor in an adult mouse. (C) Adult mouse skull showing the incisor, toothless diastema and molars. (D) Section of wild-type (WT) incisor at E18.5. (E) Section of Spry2^+/–;Spry4^–/–incisor at E18.5. (F) Adult wild-type incisors in frontal view. (G) Incisor duplication in adult Spry2^+/–;Spry4^–/– mutant mice. CL, cervical loop; DP, dental papilla. In D and E, white asterisks indicate labial ameloblasts and blue asterisks indicate ectopic ameloblasts. Arrows in F and G point to the incisors. Scale bars: 500 μm.

Fig. 2.

Upper incisor development in wild-type and Spry2^+/–;Spry4^–/– embryos. (A) Schematic of the epithelium-mesenchyme dissociation method. (B-G) Photographs of dissociated incisor epithelia in wild-type embryos from E12.5 to E17.5. (B′-G′) Schematic of upper incisor development in wild-type embryos. (H-M) Photographs of dissociated incisor epithelia in Spry2^+/–;Spry4^–/– embryos from E12.5 to E17.5. (H′-M′) Schematic representation of upper incisor development in Spry2^+/–;Spry4^–/– embryos. Yellow dashed lines outline the incisor epithelium; red line indicates position of septum. EK, enamel knot; Epi, epithelium; Mes, mesenchyme; la, labial; li, lingual; laCL, labial cervical loop; liCL, lingual cervical loop.

Fig. 3.

Septum formation during incisor development in Spry2^+/–;Spry4^–/– embryos. (A) Schematic of epithelium re-orientation. (B-D) Photographs of epithelia after re-orientation of wild-type embryos at E12.5, E13.5 and E14.5. (E-G) Photographs of epithelia of Spry2^+/–;Spry4^–/– embryos at E12.5, E13.5 and E14.5. (B′-G′) Schematics of epithelial development. Yellow dashed lines outline the incisor; red arrowheads in F point to notch; red line in G indicates septum. la, labial; li, lingual; EK, enamel knot; laCL, labial cervical loop; liCL, lingual cervical loop.

Fig. 4.

Cell death and proliferation in incisors of control and Spry2^+/–;Spry4^–/– embryos. (A-F) Three-dimensional reconstructions viewed from mesenchymal aspect and location of apoptotic elements (red dots) in epithelia of control (A-C) and Spry2^+/–;Spry4^–/– embryos (D-F) at E12.5 (A,D), E13.5 (B,E) and E14.5 (C,F). Scale bar: 100 μm. (G-I) Apoptotic rate (G), mitotic index (H) and epithelial volume (I) in control and Spry2^+/–;Spry4^–/– embryos. Mean±s.e.m. are plotted. ^*P<0.05, ^**P<0.01.

Fig. 5.

In situ hybridization in incisors of wild-type and Spry2^+/–;Spry4^–/– embryos. (A-F) Whole-mount RNA in situ hybridization for Shh in wild-type (A,C,E) and Spry2^+/–;Spry4^–/– (B,D,F) embryos at E11.5, E12.5 and E13.0. Red arrowheads point to the left incisor expression domain. (G,H) Whole-mount RNA in situ hybridization for Fgf4 in dissected incisor epithelia viewed from mesenchymal side of wild-type (G) and Spry2^+/–;Spry4^–/– (H) embryos at E14.5. EK, enamel knot; laCL, labial cervical loop; liCL, lingual cervical loop.

Fig. 6.

Incisor phenotypes in a Sprouty gene dosage series. (A-E) Frontal histological sections of embryonic head at E15.5 (A) and E18.5 (B-E) showing the phenotype of upper and lower incisors in K14-Spry4 (Spry4^GOF) (A), wild-type (B), Spry2^+/–;Spry4^–/– (C), Spry1^–/–;Spry4^–/– (D) and Spry2^–/–;Spry4^–/– (E) embryos. (A′-E′) High magnification of upper incisor. (A′-E′) High magnification of lower incisor. (F) Schematic with indication of the number of incisors and enamel organs for each genotype. EO, enamel organ(s); In, incisor(s). Dotted line in A indicates that this composite image is composed of upper and lower jaw images from different sections.

Fig. 7.

Upper incisor evolution in Euarchontoglires. (A) Phylogenetic tree of Euarchontoglires and schematic of the general upper incisor pattern of each taxon. Three patterns are indicated by color coding: one pair of incisors in Simplicidentata (green), two pairs of incisors (labial and lingual, each pair in tandem) in Duplicidentata (blue) and two pairs of incisors (frontals, each pair side by side) in Euarchonta (red). 1, Euarchontoglires; 2, Glires; 3, Simplicidentata; 4, Duplicidentata; 5. Euarchonta. (A′) Upper incisor phenotypes of mutants discussed in the text. (B) Schematic of incisor evolution in early Euarchontoglires showing the appearance of the three main incisor patterns.