Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors

Abstract

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

Fig. 1.

Attenuation of FGFR2b signaling during mid- and late embryonic development impairs ameloblast formation and cervical loop morphology. (A) Schematic of a sagittal section through the proximal region of the mouse incisor. The lingual and labial cervical loops (liCL and laCL, respectively) are believed to contain epithelial stem cell niches. Only stem cells in the laCL give rise to enamel (En)-producing ameloblasts (Am), which abut the mesenchymally derived dentin (De)-secreting odontoblasts (Od). Stem cells in the laCL, which are thought to reside in the stellate reticulum (SR) core or the outer enamel epithelium (OEE), produce transit-amplifying (T-A) cells in the inner enamel epithelium (IEE), which, in turn, give rise to pre-ameloblasts (pre-Am) that develop into mature ameloblasts. The IEE (black and white dotted bracket) includes the T-A cells, the pre-ameloblasts and the ameloblasts. (B) Timecourse for doxycycline (DOX) treatment of females carrying rtTA;tet(O)sFgfr2b (DTG) and single-transgenic control embryos. (C-D′) Hematoxylin and Eosin staining of sagittal sections through the proximal incisor of control (C-C′) and DTG embryos (D-D′) at E18.5. C′, C′, D′ and D′ are magnifications of the areas indicated in C and D. Asterisk in D′ represents missing columnar cells in DTG embryos. Blue dotted lines outline the epithelium. Di, distal; Pr, proximal.

Fig. 2.

Decreased FGFR2b signaling during late incisor development leads to reduction in T-A cells, impaired amelogenesis and cervical loop expansion. (A-N) Hematoxylin and Eosin staining (A,B,D,E), analysis of cell proliferation (C,F), immunohistochemistry (G-L) and Shh in situ hybridization (M,N) of E18.5 control (A-C,G-I,M) and DTG embryos (D-F,J-L,N) after 48 hours exposure to doxycyline (treatment timecourse shown at the top). Red arrowheads in C and F delimit the approximate T-A cell regions in control and DTG embryos, respectively, and black arrowheads indicate mesenchymal proliferation. Asterisk in E represents abnormally short, less organized ameloblasts. Note that FGFR2 is most highly expressed in the T-A cells (region between blue arrowheads in I and L). Dotted lines outline the epithelium. Am, ameloblasts; Od, odontoblasts.

Fig. 3.

Long-term attenuation of FGFR2b signaling in adult mice causes loss of maxillary incisors. (A) Timecourse for postnatal doxycycline treatment. (B-I′) Whole-mount images of control (B-E′) and DTG (F-I′) mice after 14 (B,F), 28 (C,G), 56 (D,H) and 76 (E,E′,I,I′) days of doxycyline treatment. (J) Diagram of the region analyzed by microCT. Blue represents mesenchyme and dark brown represents epithelium. (K-L′) MicroCT analysis showing enamel and bone (K,L) or enamel only (K′,L′). Green line and pink line indicate enamel in the maxillary and mandibular incisor, respectively. Dotted yellow lines indicate the absence of enamel in the proximal regions of the DTG incisors. Di, distal; laCL, labial cervical loop; liCL, lingual cervical loop; M, molars; P, postnatal day; Pr, proximal.

Fig. 4.

Long-term postnatal reduction of FGFR2b signaling leads to defective amelogenesis in maxillary incisors. (A) Timecourse for doxycycline treatment. (B) Dotted lines in schematic indicate areas shown in sagittal sections in C-D′ and planes of coronal sections in E-N. Blue represents mesenchyme and dark brown represents epithelium. (C-D′,E,J) Hematoxylin and Eosin staining of maxillary incisor sections from control (C,C′,E) and DTG (D,D′,J) mice. C′ and D′ are magnifications of the areas indicated in C and D, respectively. Note defects in the dentin and enamel matrices (blue and green asterisks, respectively), ectopic blood vessels (small black asterisks), and connective tissue (CT) in the proximal incisor region (J) where ameloblasts are normally found in controls (E). (F-I,K-N) Immunostaining of coronal sections with antibodies against FGFR1 (F,K), FGFR2 (G,L), amelogenin (H,M) and K14 (L,N). Black arrowheads point to ameloblasts.

Fig. 5.

Ameloblast formation and ameloblast progenitor proliferation are reduced after 4 weeks of postnatal FGFR2b signaling attenuation in mandibular incisors. (A-B′) Hematoxylin and Eosin staining of sagittal sections of mandibular incisors of control (A-A′) and DTG mice (B-B′) treated with doxycyline for 28 days (treatment timecourse shown at top). Dotted boxes in A and B indicate regions magnified in A′, A′, B′ and B′. Note the presence of ectopic blood vessels (BV) near the labial CL of DTG mice (B′). (C,D) BrdU staining on sagittal sections of control (C) and DTG (D) incisors. Dotted lines indicate the outline of the epithelium. (E) Number of BrdU-positive cells (mean ± s.e.m.) in the cervical loop of control and DTG mice. ^*P<0.05. Am, ameloblasts; Od, odontoblasts.

Fig. 6.

Postnatal expression of dominant-negative FGFR2b causes downregulation of FGFR signaling target genes and changes in expression of dental epithelial markers. (A-L) In situ hybridization of sagittal sections of the proximal incisor of untreated controls (A,C,E,G,I,K) and doxycycline-treated DTG mice (B,D,F,H,J,L). Expression of Shh (A,B), Etv4 (C,D), Etv5 (E,F), Spry2 (G,H) and Notch1 (I,J) is markedly decreased in treated DTG mice relative to controls, whereas Notch2 expression (K,L) appears to be maintained. Treatment timecourse is shown at the top.

Fig. 7.

Effects of 4-week attenuation of FGFR2b signaling in postnatal incisors are reversible. (A) Timecourse for postnatal doxycycline treatment followed by different lengths of doxycyline-free chases. (B-O) Whole-mount images of incisors of control (B-H) and DTG (I-O) mice after 4 weeks of doxycyline exposure and 0 (P42; B,I), 28 (P70; C,J), 49 (P91; D,K), 56 (P99; E,L), 66 (P109; F,M), 70 (P113; G,N) and 77 (P120; H,O) days doxycyline-free period before sacrifice. Red and green arrowheads point to abnormal maxillary and mandibular incisors, respectively. (P-R′) Hematoxylin and Eosin staining of proximal incisor sections of a control animal (P,P′) and DTG animals 28 days (P70; Q,Q′) and 77 days (P120; R,R′) post-doxycyline treatment. Boxes in P, Q and R indicate areas magnified in P′, Q′ and R′, respectively. Am, ameloblasts; Od, odontoblasts.