(A) Schematic diagram of an adult incisor. Enamel and dentin, the calcified tissues of the tooth, are produced by ameloblasts and odontoblasts, respectively. Note that in the normal incisor, ameloblasts and enamel are present only on the labial surface. (B,F) Side views of mandibles from wild-type and Spry4^-/-;Spry2^+/- adult mice with soft tissue removed. Note the abnormal length and thickness (red bar) of the mutant incisor, as well as the absence of a sharp tip (asterisk). (C-E) Coronal sections from an XTM analysis of wild-type, Spry4^-/- (4^-/-), and Spry4^-/-;Spry2^+/- (4^-/-;2^+/-) incisors. The colors in the bar on the left indicate mineral content. In the XTM sections, enamel is false-colored blue and purple, and dentin and bone are false-colored green and yellow. The white arrow points to ectopic enamel. (G,H) Sagittal sections of the mandibular incisor from postnatal day 14 wild-type and Spry4^-/-;Spry2^+/- animals. Here, and in all other panels in this and subsequent figures, anterior is to the left and posterior to the right. Yellow arrowheads point to a layer of enamel. Black arrows point to the cervical loops (CLs) at the posterior end of the lingual and labial sides of the incisor. (I-L) Higher magnification views of regions boxed in G and H. Note that in the wild-type and mutant incisors, dentin (stained pink and/or blue) and odontoblasts are present on both lingual and labial sides, whereas enamel and ameloblasts are found only on the labial side in wild-type and on both labial and lingual sides in the Spry4^-/-;Spry2^+/- incisor. (M-P) High magnification views showing lingual and labial CL morphology in wild-type and Spry4^-/-;Spry2^+/- incisors. Note that the mutant lingual CL is more similar to the labial CL than to the wild-type lingual CL. Abbreviations: A, anterior; Am, ameloblasts; CL, cervical loop; De, Dentin; En, enamel; Ep, epithelium; Mes, mesenchyme; M1 and M2, first and second molar; Od, odontoblasts; P, posterior; wt, wild-type. Scale bars: H, 500 μm; L, 100 μm; P, 50 μm.

(A-I) Gene expression analyzed by RNA in situ hybridization using the indicated probes on paraffin sections of wild-type and mutant embryonic incisors. The stage at which the embryos were collected is indicated. In this and subsequent figures, a dotted line outlines the incisor epithelium. In panel D, the Shh expression domain appears to extend slightly into the lingual epithelium, but as this was not reproducibly observed it is likely an artifact of the plane of section. The red arrowheads in panels E and H point to a domain in which Shh is ectopically expressed on the lingual side of Spry4^-/-;Spry2^+/- and Spry4^-/- incisors. A similar domain is detected in Fst null incisors (panel I). The open arrowheads in panel I point to an additional anterior domain in which Shh is ectopically expressed on the lingual side of Fst null incisors. Abbreviations as in the legend to Fig. 1. Scale bars = 100 μm.

(A-K) Gene expression was analyzed by RNA in situ hybridization using the probes indicated on paraffin sections of E16.5 incisors of the genotypes indicated. Yellow asterisks indicate gene expression domains in the mesenchyme, and arrows point to the anterior and posterior ends of gene expression domains in the epithelium. All samples are shown at the same magnification.

(A-C) Sagittal section of postnatal day (P) 5 Spry4^-/- incisor. The areas boxed in panel A are shown at higher magnification in panels B and C. Note that unlike on the labial side, the lingual ameloblast layer does not extend to the posterior end of the incisor. (D-G) RNA in situ hybridization assays for Shh and Fgf3 expression in paraffin sections of P2 incisors of the genotypes indicated. Yellow asterisks indicate mesenchymal expression, and black arrows demarcate the extent of epithelial expression. Note that no ectopic expression of either gene is detected in the Spry4^-/- incisors at this stage (H) Frequency of the tusk phenotype (presence of lingual enamel) in Spry4^-/-;Spry2^+/- adult mice, and the effects of reducing the dosage of Fgf9 or Fgf10. The Spry4^-/-;Spry2^+/- animals evaluated were pooled from both crosses used to generate animals with reduced FGF gene dosage. p values were calculated using the Fisher exact test. Abbreviations as in the legend to Fig. 1.

Gene expression was analyzed by RNA in situ hybridization using the probes indicated on paraffin sections of embryonic incisors of the genotypes indicated at E16.5 or E17.5. (A-F) A comparison of the expression domains of Sprouty gene family members in the incisor at the stages indicated. Yellow asterisks indicate mesenchymal expression. (G-K) Tissue-specific inactivation of Spry4. The absence of Spry4 expression is indicated by red circles in the epithelium of an incisor carrying K14-cre, one Spry4^fl and one Spry4^- allele (panel G) and by red asterisks in the mesenchyme of an incisor carrying Wnt1-cre and two Spry4^fl alleles (panel H). For each genotype shown in panels I-K, the diagram illustrates the tissue in which Cre-mediated recombination occurred (green fill), the Sprouty alleles that were inactivated by Cre (white lettering), or that were inherited as nulls (black lettering). The photograph shows Shh expression, which marks cells that are differentiating along the ameloblast lineage. The red arrowheads point to the ectopic lingual Shh expression domain.

(A) Functions of Sprouty genes in inhibiting the establishment of a lingual FGF epithelial-mesenchymal signaling loop. Arrows indicate a stimulatory effect and the symbol ⊥ indicates an inhibitory effect of one signaling molecule on the expression of another. In wild-type, Sprouty genes are expressed on the labial side, but they do not prevent (dashed ⊥ symbol) reciprocal signaling between FGF9 in epithelium and FGF3/FGF10 in mesenchyme. On the lingual side, Sprouty genes inhibit signaling to adjacent tissues by the low levels of FGF9 in the epithelium and of FGF10 in the mesenchyme, and consequently there is no upregulation of FGF gene expression in either tissue. However, in Spry4 null incisors a reciprocal signaling loop between epithelium and mesenchyme is established because, in the absence of SPRY4, these tissues are hypersensitive to the low level of FGF signaling. In turn, the increase in FGF signaling on the lingual side results in the generation of ameloblasts from self-renewing stem cells in the CL.
(B) A proposal for how ameloblasts develop in the embryonic incisor. At E15.5, wild-type incisor epithelium contains “early ameloblast progenitor” (EAP) cells capable of limited proliferation. On the labial side, their descendants (in the domain colored pink) differentiate “in situ” into enamel-producing cells. Similar cells are present on the lingual side (in the domain colored lighter pink), but their differentiation is inhibited by Follistatin. Between E15.5 and E16.5, an ameloblast stem cell (ASC) population is established in the labial cervical loop (CL). Unlike EAP cells, ASCs have the capacity to self-renew (circular arrow), as well as give rise to ameloblasts. ASC descendants that will develop into enamel-producing cells may first form transit-amplifying (T-A) cells in the anterior CL. After several divisions, their descendants move out of the CL, and begin differentiating. In the E16.5 incisor, these ASC-derived pre-ameloblasts are found in a domain in the labial epithelium (colored dark brown), between the EAP domain and the CL. The diagram illustrates the possibility that ASCs are normally also present on the lingual side. However, no pre-ameloblasts derived from these ASCs are present in the lingual epithelium anterior to the CL (in the domain colored tan), because the generation of ameloblasts from lingual ASCs is blocked due to the inhibitory effects of Sprouty as well as FST function on lingual FGF gene expression.