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Epithelial Fibroblast Growth Factor Receptor 1 Regulates Enamel Formation
1 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA; and Correspondence: * corresponding author, ychai{at}usc.edu
The interaction between epithelial and mesenchymal tissues plays a critical role in the development of organs such as teeth, lungs, and hair. During tooth development, fibroblast growth factor (FGF) signaling is critical for regulating reciprocal epithelial and mesenchymal interactions. FGF signaling requires FGF ligands and their receptors (FGFRs). In this study, we investigated the role of epithelial FGF signaling in tooth development, using the Cre-loxp system to create tissue-specific inactivation of Fgfr1 in mice. In K14-Cre;Fgfr1fl/fl mice, the apical sides of enamel-secreting ameloblasts failed to adhere properly to each other, although ameloblast differentiation was unaffected at early stages. Prior to eruption, enamel structure was compromised in the K14-Cre;Fgfr1fl/fl mice and displayed severe enamel defects that mimic amelogenesis imperfecta (AI), with a rough, irregular enamel surface. These results suggest that there is a cell-autonomous requirement for FGF signaling in the dental epithelium during enamel formation. Loss of Fgfr1 affects ameloblast organization at the enamel-secretory stage and, hence, the formation of enamel.
Key Words: Fgfr1 • K14-cre • amelogenesis imperfecta • conditional knockout • mice
The interaction between epithelial and mesenchymal tissues plays an important role in organogenesis. Many factors—including growth factors, their receptors, transcription factors, and extracellular matrix proteins—are involved in regulating tissue-tissue interactions. Fibroblast growth factor (FGF) signaling is involved in a reciprocal relationship between epithelial and mesenchymal tissue in limb and lung development, such that FGF ligands secreted from epithelial cells interact with FGF receptors in mesenchymal cells (Xu et al., 1999; Warburton et al., 2005). FGF signaling controls cell growth, differentiation, and motility during skeletal and neural development (Ornitz and Itoh, 2001). During craniofacial development, FGF signaling is involved not only in craniofacial bone formation, but also in the formation of the palate, salivary glands, teeth, craniofacial muscles, and tongue muscles (Nie et al., 2006). The importance of FGFR2 in tooth development has been well-documented. Mice with soluble dominant-negative Fgfr2 lack tooth buds (Celli et al., 1998). Fgfr2IIIb–/– mice possess defects in tooth and salivary gland development. Specifically, tooth development fails to progress beyond the bud stage (De Moerlooze et al., 2000). Additionally, FgfR2-IIIb–/lacZ mice show agenesis of the tooth bud (Revest et al., 2001). However, the role of Fgfr1 in tooth morphogenesis is still unknown. Unfortunately, Fgfr1 null mice show growth retardation and defects of mesodermal patterning, and die at E7.5-9.5 (Yamaguchi et al., 1994). To bypass the gastrulation defect of Fgfr1 null mice, hypomorphic (partial loss of function) and conditional knockout mice have been generated (Partanen et al., 1998; Trokovic et al., 2003). These transgenic mice reveal that Fgfr1 is required in a spatially and temporally specific manner. For instance, Fgfr1 plays a critical role in the ectoderm for neural crest migration during the development of the second branchial arch. In contrast, Fgfr1 is required by cranial neural crest cells during palatogenesis (Trokovic et al., 2003). To date, the function of Fgfr1 in epithelial tissue is still unknown, although Fgfr1 is expressed in the dental epithelium and mesenchyme throughout tooth development (Kettunen et al., 1998). To investigate the biological function of Fgfr1 in tooth epithelial tissue, we have generated epithelial-specific Fgfr1 conditional knockout mice.
Generation of K14-Cre;Fgfr1fl/fl Mutant Mice All animal studies were performed according to IACUC guidelines. A K14-Cre transgene was used as previously reported (Andl et al., 2004). The Fgfr1 floxed allele contains 2 loxP sites inserted into introns flanking exons 8–15 of the Fgfr1 gene. In the presence of Cre, exons 8 to 15 recombine, and the Fgfr1 gene is removed, resulting in a null allele, as previously described (Trokovic et al., 2003). Mating K14-Cre;Fgfr1fl/+ with Fgfr1fl/fl mice generated K14-Cre;Fgfr1fl/fl. Null alleles were genotyped with PCR primers as previously described (Yu et al., 2003).
Two-component Genetic System for Marking the Progeny of Ectoderm-derived Cells
Histological Procedure
Scanning Electron Microscopy
Immunohistochemistry
In situ Hybridization
Abnormal Ameloblast Structure in the Incisors of K14-Cre;Fgfr1fl/fl Mice To investigate the biological function of Fgfr1 in the epithelial cells, we generated an epithelial-specific gene inactivation in mice using the K14 promoter. The K14 promoter is active in surface ectoderm and basal cells from embryonic day E9.5 in developing hair follicles and tooth epithelia (Byrne et al., 1994; Dassule et al., 2000). We confirmed that Cre recombination driven by the K14 promoter was detectable in the dental epithelium, implying that the ameloblasts had lost the Fgfr1 gene (Fig. 1A  ). Next, we analyzed hematoxylin-eosin-stained sections of lower incisors from control (K14-Cre;Fgfr1fl/+) and K14-Cre;Fgfr1fl/fl newborn mice (Figs. 1B-1K). During enamel formation, dental epithelial cells proceed through proliferating, post-mitotic, polarizing, and secretory stages (Gritli-Linde et al., 2002). Dental epithelial cells in the cervical loop, which contains the ameloblast stem cell lineage (Harada et al., 2002), were indistinguishable in K14-Cre;Fgfr1fl/fl mice from those of control mice (Figs. 1D, 1H). The ameloblasts of K14-Cre;Fgfr1fl/fl mice in the proliferating stage were also unchanged as compared with those in control mice (Figs. 1E, 1I). We observed a basal localization of nuclei that were well-organized at the enamel-secretory stage in control ameloblasts (Fig. 1F). In contrast, the ameloblasts of K14-Cre;Fgfr1fl/fl mice appeared to have a wavy structure, although we did see basal localization of the nuclei (Fig. 1J). The dentin-enamel junction (DEJ) of control mice was easily detached through the decalcification process (Fig. 1G). In K14-Cre;Fgfr1fl/fl mice, the enamel component was easily broken internally (Fig. 1K). The surface of the broken enamel structure from K14-Cre;Fgfr1fl/fl mice was rough, with a lava-like shape (Fig. 1K). Throughout tooth development, odontoblast and dentin formation in K14-Cre;Fgfr1fl/fl mice appeared normal (Fig. 1). These observations imply that loss of Fgfr1 in the dental epithelium resulted in abnormal ameloblast structure from the enamel-secretory stage, although ameloblast differentiation appeared to be unaffected at early stages.
Compromised Ameloblast Organization in K14-Cre;Fgfr1fl/fl Mice To confirm the specificity of Fgfr1 gene inactivation in the dental epithelia, we performed Fgfr1 in situ hybridization. In the control mice, Fgfr1 was expressed in ameloblasts as well as in odontoblasts (Fig. 2A , arrows). In K14-Cre;Fgfr1fl/fl mice, Fgfr1 was detectable in odontoblasts and the surrounding bone (Fig. 2B, black arrows), but not in ameloblasts (*), consistent with the deletion of Fgfr1 in ameloblast cells. Amelogenin is a marker for ameloblast differentiation (Snead et al., 1988). To assess ameloblast differentiation status in K14-Cre;Fgfr1fl/fl mice, we analyzed Amelogenin expression using in situ hybridization. We found that Amelogenin expression in ameloblasts of K14-Cre;Fgfr1fl/fl mice was indistinguishable from that of the control (Figs. 2C, 2D). Next, we investigated the pattern of Amelogenin protein expression (Figs. 2E, 2F). Control mice expressed Amelogenin at the tip of ameloblasts (Fig. 2E, black arrowheads). In contrast, K14-Cre;Fgfr1fl/fl mice lost this localization pattern and displayed broad expression of Amelogenin (Fig. 2F, white arrowheads). Previous studies have reported that ameloblasts express E-cadherin, which is a cell-cell adhesion molecule, in a strongly polarized distribution pattern in ameloblasts at the secretory stage (Terling et al., 1998; Gritli-Linde et al., 2002). We examined the expression pattern of E-cadherin using immunohistochemistry. In control mice, E-cadherin was located not only at the base of ameloblasts, next to the basement membrane, but also at the tips of ameloblasts (Fig. 2G). In contrast, we found that E-cadherin expression was detectable only at the base of ameloblasts in K14-Cre;Fgfr1fl/fl mice (Fig. 2H). Taken together, these expression patterns suggest that the organization of ameloblasts is compromised in K14-Cre;Fgfr1fl/fl mice; in particular, Tomes’ processes may be absent or defective.
Incisor Enamel Defects in K14-Cre;Fgfr1fl/fl Mice K14-Cre;Fgfr1fl/fl mice were indistinguishable from K14-Cre;Fgfr1fl/+ (control) mice with respect to viability, size, and weight (data not shown). However, we observed defects in enamel formation upon oral examination of K14-Cre;Fgfr1fl/fl mice at 8 wks after birth (Figs. 3A, 3B ). The incisor enamel surface was dimpled irregularly, and the tips of the enamel region were more worn than those of control mice (Figs. 3A, 3B). By scanning electron microscopy (SEM), the surface of tooth enamel from control mice appeared smooth and covered with well-formed enamel prisms at 8 wks after birth (Fig. 3C). In contrast, K14-Cre;Fgfr1fl/fl incisor enamel appeared rough, like lava boulders (Fig. 3D). Internally, the enamel structure of K14-Cre;Fgfr1fl/fl incisors appeared amorphous when compared with that of the control (Figs. 3E, 3F).
Molar Enamel Defects in K14-Cre;Fgfr1fl/fl Mice The molars of K14-Cre;Fgfr1fl/fl mice were also affected. By SEM, we found that the surfaces of molars were smooth in control mice, but rough in K14-Cre;Fgfr1fl/fl mice (Fig. 4 ). Although the defect in molars was milder than that of incisors (Figs. 3D , 4D), our results demonstrate that all tooth enamel was defective in K14-Cre;Fgfr1fl/fl mice. This phenotype is consistent with a diagnosis of amelogenesis imperfecta.
In this study, we investigated the functional significance of FGFR1 signaling in regulating tooth development. In K14-Cre;Fgfr1fl/fl mice, ameloblasts differentiate normally at early stages, but the function of enamel-secreting ameloblasts is compromised at the newborn stage. Furthermore, we found that the enamel structure of all teeth is defective in adult K14-Cre;Fgfr1fl/fl mice, resulting in a phenotype similar to the human disorder, pitted hypoplastic amelogenesis imperfecta (AI). AI is a condition of abnormal tooth formation. Although it has been classified with 14 subtypes (Witkop, 1988), clinically it is divided into hypocalcifed, hypoplastic, and hypomature phenotypes. Inheritance patterns for AI can be autosomal-dominant, autosomal-recessive, or X-linked. Four gene mutations have been identified for this disorder: amelogenin (AMELX), enamelin (ENAM), kallikrein (KLK4), and metalloproteinase 20 (MMP20) (PS Hart et al., 2002, 2004; TC Hart et al., 2004; Kim et al., 2005a,b; Ozdemir et al., 2005). However, some human AI cases cannot be attributed to any of these gene mutations. Thus, numerous efforts with the use of mouse models to investigate genes related to enamel formation have been carried out to identify additional candidate genes. The K14 promoter has been used as a powerful tool to investigate ameloblast differentiation. For instance, K14 promoter-directed loss of Shh signaling results in compromised ameloblast cytodifferentiation in mice (Dassule et al., 2000; Gritli-Linde et al., 2002). The overexpression of Follistatin, a BMP inhibitor, driven by the K14 promoter, results in compromised enamel formation, implying that BMP4 is critical for ameloblast differentiation (Wang et al., 2004). Wnt3 overexpression, driven by the K14 promoter, also results in the loss of ameloblasts after birth (Millar et al., 2003). Furthermore, Fgf10 null mice display not only the loss of the ameloblast stem cell lineage, but also defects in secretory ameloblast differentiation (Harada et al., 2002; Yokohama-Tamaki et al., 2006). Previous studies have suggested that FGFR1b binds to ligands FGF1, 2, 3, and 10, and FGFR1c binds FGF1, 2, 4, 5, and 6 (Powers et al., 2000). Mesenchymal FGFs, especially FGF3 and FGF10, are important regulators of epithelial cell proliferation during tooth morphogenesis (Kettunen et al., 2000). In incisors, expression of Fgf3 and Fgf10 is restricted to the mesenchyme underlying the rapidly proliferating inner enamel epithelium. FGF10, expressed in dental mesenchyme, stimulates proliferation of both stem cell and transit-amplifying cells (inner enamel epithelial cells), and FGF3 stimulates proliferation of transit-amplifying cells (Harada et al., 1999). Incisor development is arrested in FGF10 null mice, because the stem cell lineage of ameloblasts is lost. We have found that the cervical loop of K14-Cre;Fgfr1fl/fl mice appears to be unaffected, although ameloblast organization is altered at the enamel-secretory stage. Interestingly, Kallman syndrome, which is associated with FGFR1 mutation, includes craniofacial defects, such as cleft palate, and dentitional anomalies, including enamel defects (Molsted et al., 1997; Dode et al., 2003). Future studies will help to elucidate the ligands and down-stream targets important in this FGFR1 signaling pathway during tooth development.
We thank Dr. Julie Mayo for critical reading of the manuscript, and Dr. David Ornitz and Sarah Millar for Fgfr1 floxed and K14-Cre mice, respectively. This study was supported by grants from the National Institute of Dental and Craniofacial Research, NIH (DE012711, DE014078, and DE017007) to Y.C. Received for publication April 30, 2007. Revision received November 21, 2007. Accepted for publication December 2, 2007.
Journal of Dental Research, Vol. 87, No. 3,
238-243 (2008) This article has been cited by other articles:
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