An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors

Ophir D. Klein 0 1 2 David B. Lyons 2 Guive Balooch 5 Grayson W. Marshall 5 M. Albert Basson 4 Miroslav Peterka 3 Tomas Boran 3 Renata Peterkova 3 Gail R. Martin ) 2 0 Department of Orofacial Sciences 1 Department of Pediatrics, School of Medicine, University of California at San Francisco , San Francisco, CA 94143-2711 , USA 2 Department of Anatomy and Program in Developmental Biology 3 Department of Teratology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic , Prague , Czech Republic 4 Department of Craniofacial Development, King's College London , London, SE1 9RT , UK 5 Department of Preventive and Restorative Dental Sciences, School of Dentistry, University of California at San Francisco , San Francisco, CA 94143-0758 , USA Rodent incisors grow throughout adult life, but are prevented from becoming excessively long by constant abrasion, which is facilitated by the absence of enamel on one side of the incisor. Here we report that loss-of-function of sprouty genes, which encode antagonists of receptor tyrosine kinase signaling, leads to bilateral enamel deposition, thus impeding incisor abrasion and resulting in unchecked tooth elongation. We demonstrate that sprouty genes function to ensure that enamel-producing ameloblasts are generated on only one side of the tooth by inhibiting the formation of ectopic ameloblasts from self-renewing stem cells, and that they do so by preventing the establishment of an epithelial-mesenchymal FGF signaling loop. Interestingly, although inactivation of Spry4 alone initiates ectopic ameloblast formation in the embryo, the dosage of another sprouty gene must also be reduced to sustain it after birth. These data reveal that the generation of differentiated progeny from a particular stem cell population can be differently regulated in the embryo and adult. INTRODUCTION Rodent incisors are unusual among mammalian teeth in that they grow continuously throughout the life of the animal. This growth is fueled by stem cells in both the mesenchymal and epithelial compartments of the incisor, the progeny of which perpetually generate the various cell types in the tooth (Gronthos et al., 2002; Smith and Warshawsky, 1975). Incisor growth is counterbalanced by abrasion, without which the tooth would become excessively long and interfere with feeding. It would be difficult to abrade the incisors if enamel, the hardest component of the tooth, covered the entire surface of the incisor as it does in the molar. However, in rodent incisors enamel is normally present on the labial surface (facing the lip) and is absent on the lingual surface (facing the tongue) (see Fig. 1A). This asymmetry not only facilitates the abrasion that keeps incisor length relatively constant, but also ensures that the tooth will be filed down primarily on one side, thus generating a sharp tip (Addison and Appleton, 1915). Little is known about the stem cells that generate enamelproducing ameloblasts (ameloblast stem cells, or ASCs), because markers for them have not yet been identified. It has been proposed that ASCs reside in a niche located within a region called the cervical loop (CL) at the posterior end (base) of the incisor (Harada et al., 1999) (see Fig. 1A), but it is not known if ASCs give rise only to ameloblasts or also to the other epithelial cell types that must be continuously generated as the tooth grows. Based on models for the generation of differentiated progeny from stem cells in other tissues, such as the crypt of the intestinal villus (Fuchs et al., 2004), it has further been speculated that ameloblast formation begins when ASC progeny move out of the putative niche and develop into transitamplifying (T-A) cells that undergo a limited number of cell divisions (Harada et al., 1999; Wang et al., 2007). It is known that, once formed, ameloblasts move anteriorly along the length of the incisor (toward the tip) as they differentiate. After producing and depositing enamel, ameloblasts either undergo apoptosis or shrink in size (Smith and Warshawsky, 1975). Genetic analysis has shown that members of the fibroblast growth factor (FGF) family of secreted signaling molecules play a role in regulating ameloblast development or function, as Fgf3/ mice have defective enamel and Fgf3/;Fgf10+/ mice have very thin or no enamel (Wang et al., 2007). Furthermore, based on data from studies of incisors developing in vitro it has been suggested that Fgf10 regulates epithelial stem cell survival (Harada et al., 2002; Yokohama-Tamaki et al., 2006). In wild-type incisors, the lack of lingual enamel is due to the absence of ameloblasts on that side (Smith and Warshawsky, 1975). However, it is not yet known whether lingual ameloblasts are absent because their formation from ASCs is blocked or because there are no ASCs in the lingual CL. Interestingly, the lingual CL differs in morphology from the labial CL (see Fig. 1A), which might reflect a lack of either T-A cells or ASCs. These morphological differences are correlated with asymmetries in the expression patterns of genes encoding members of the FGF family. For example, Fgf3 is detected in the mesenchyme surrounding the labial but not the lingual CL, and Fgf10 is expressed at a higher level on the labial side than on the lingual side (Harada et al., 1999). Furthermore, ectopic Fgf3 expression in lingual mesenchyme is associated with the formation of lingual ameloblasts in embryos homozygous for a null allele of follistatin (Fst), which encodes an extracellular inhibitor of signaling by transforming growth factor (TGF ) superfamily members (Wang et al., 2007; Wang et al., 2004). Together, the available data suggest that suppression of FGF signaling on the lingual side is necessary to prevent ameloblast formation. Here we identify sprouty (Spry) genes, which encode intracellular antagonists of FGF and other receptor-tyrosine kinase signaling pathways (Mason et al., 2006), as essential for establishing and sustaining the asymmetry of enamel deposition necessary for normal incisor length and shape. We provide genetic evidence that sprouty genes prevent the generation of lingual ameloblasts by inhibiting an FGF-mediated epithelial-mesenchymal signaling loop on the lingual side. Furthermore, our data suggest that the earliest ameloblasts that form in the incisor do not arise from ASCs, but instead are derived from a transient embryonic ameloblast progenitor cell population that does not self-renew. MATERIALS AND METHODS Mouse lines Mouse lines carrying mutant alleles of Fgf9 (Colvin et al., 2001), Fgf10 (Min et al., 1998), Fst (Matzuk et al., 1995), Spry1 (Basson et al., 2005), Spry2 (Shim et al., 2005), Spry4 (Klein et al., 2006) and the K14-cre (Dassule et al., 2000) and Wnt1-cre (Danielian et al., 1998) transgenes were maintained and genotyped as reported, except that Fst wild-type and null alleles were genotyped using PCR rather than Southern blotting (primer sequences available on request). A (...truncated)