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. 2012 Aug 14;23(2):317-28.
doi: 10.1016/j.devcel.2012.05.012. Epub 2012 Jul 19.

Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors

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Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors

Emma Juuri et al. Dev Cell. .
Free PMC article


The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal.


Figure 1
Figure 1. Sox2 Expression in the Oral Epithelium Is Progressively Restricted to the Labial CL during Incisor Development
(A) Schematic illustrations of the mouse incisor. Left: Lower jaw and higher magnifications of the incisor in a frontal section, and a sagittal section from the proximal part illustrating labial and lingual CLs. Right: 3D reconstruction from histological sections of the proximal part of the incisor. (B) In situ hybridization in the mouse lower incisor from E12 to P2 reveals gradual restriction of Sox2 mRNA expression to a subset of SR cells and adjacent enamel epithelium in the labial CL. The arrow at E15 indicates disappearance of Sox2 expression in the lingual CL. The dotted line marks the border between epithelium and mesenchyme. All sections are in the sagittal plane unless indicated otherwise. Am, ameloblasts; CL, cervical loop; ERM, epithelial cell rests of Malassez; IEE, inner enamel epithelium; Lab, labial; Lat, lateral; Lin, lingual; Med, medial; OEE, outer enamel epithelium; SC, stem cell; SR, stellate reticulum; TA, transient amplifying cells. Scale bar, 100 μm. See also Figure S1.
Figure 2
Figure 2. Culture of Sox2-GFP Mouse Incisor Demonstrates Restriction of Sox2+ Cells to Labial CL and Their Migration toward Enamel Epithelium
(A) Expression of GFP in the Sox2-GFP reporter mouse incisor in tissue culture initiated at E14.5. The white arrow marks the disapperance of GFP signal from lingual CL. (B) Confocal images of a living tissue slice of labial CL of Fucci-red;Sox2-GFP mouse at P2 with GFP (green) and mKO2 (red) expression on the left and only GFP expression on the right. GFP expression is intense in a subset of cells within the labial CL (yellow dotted line), whereas GFP gradually disappears from IEE when the cells reach the level of TA cells. (C) Sox2 antibody staining in labial CL of P2 wild-type incisor. The arrow points to the border in the IEE where the Sox2 protein level decreases. (Da–Dc) (Da) Living tissue slice of labial CL of P2 Fucci-red;Sox2-GFP mouse at P2 showing the imaged area and orientation for cell movement analysis. (Db and Dc) Still images of time-lapse movies show tracking (Movies S1 and S2) and polar plot analysis the orientation of the movement (small arrows point to the direction of overall movement of individual cells) of Sox2+ (Db) and Sox2-negative (Dc) cells (yellow circles). Scale bars: 100 μm (A), 60 μm (B and C), 15 μm (D). See also Figure S2.
Figure 3
Figure 3. Genetic Inducible Fate Mapping Demonstrates that Sox2+ Cells in the Adult Incisor Are SCs
(A) Timing of tamoxifen induction and histological analysis in Sox2Cre-ER;R26R mice. (B) Timing of tamoxifen double induction and histological analysis. (Ca–Ck) LacZ expression 48 hr (Ca), one week (Cb, Cj, and Ck), and one month (Cc–Cf) after single tamoxifen induction. Frontal sections (Cd–Cf) show the distinct lacZ+ epithelial cell types derived from Sox2+ cells: SR (Cd), IEE and OEE (Ce), ameloblasts and SI cells (Cf), ERMs (Ch and Ci), and IEE/OEE ridge (Cj and Ck). (Cg–Ci) LacZ expression one month after double induction. The region marked in (Ch) is shown in higher magnification in (Ci). The arrowhead in (Ci) points to ERM cells on the lateral side of the incisor. CL, cervical loop; ERM, epithelial cell rests of Malassez; IEE, inner enamel epithelium; OEE, outer enamel epithelium; SR, stellate reticulum; TA, transient amplifying cells; Am, ameloblasts. Scale bar: 100 μm, except in (Ci), where it is 50 μm. See also Figure S3.
Figure 4
Figure 4. The Early Progeny of Sox2+ SCs Express Sfrp5
(Aa–Ae) In situ hybridization of Sfrp5 (Aa–Ac) and Shh (Ad and Ae) in frontal and sagittal (Ac) sections of P2 incisor. Notice that Sfrp5 is absent from part of the lingual CL, as indicated by the arrow in (Aa). The planes of the frontal and sagittal sections are shown in the sagittal and coronal views of the proximal part of the incisor in (B). (B) 3D reconstruction of Sfrp5 (red) and Sox2 (green) expression patterns from in situ hybridization of serial sections. Notice that in the coronal view, Sfrp5 expression forms a circle marking the cells of the IEE/OEE ridge in the proximal opening of the incisor. Sox2 expression is restricted to the tip of the labial CL and does not overlap with Sfrp5 expression in either coronal or sagittal views. Arrows in coronal and sagittal views point out Sfrp5-negative cells. (C) Schematic 3D representation of the LRCs of rat incisor redrawn from Smith (1980). 3H-Thymidine incorporation in rat incisor was monitored 2 and 4 days after injection. After 2 days, LRCs were localized in both the labial CL and IEE/OEE ridge. After 4 days, only the cell population within the labial CL exhibited labeling. (D) 3D illustration of the proximal area of the P2 incisor shows the coronal section plane of thick slices from the Sox2-GFP incisor in (E) and (F). (E) GFP expression in a thick coronal slice with BodipyTR and Draq5 counterstaining (left), and alone (right). The arrow shows the proposed direction of cell movement. (F) DiI-labeled labial CL cells are detected in the IEE/OEE ridge 24 hr after injection. The arrow shows the direction of cell migration. Lat, lateral; Med, medial. Scale bars: 60 μm in (E), 100 μm in (A) and (F). See also Figure S4.
Figure 5
Figure 5. FGF8, but Not FGF10, Is Required for Sox2 Expression in the CL
(A) Sox2-driven GFP expression in a cultured labial CL of P2 incisor under global inhibition of FGFs by SU-5402 (bottom) and in control with DMSO (top). Note the suppression of GFP expression after 24 hr with SU-5402 (yellow arrow) and the reappearance of expression within 24 hr after removal of inhibition. (B) Sox2-driven GFP expression (green) and apoptotic cells (red) in a cultured labial CL of P2 incisor in control (top row) and under inhibition of FGF10 and FGF8 (middle and bottom rows, respectively) by blocking antibodies. Note the suppression of GFP expression and unaffected apoptosis by FGF8 inhibition (yellow arrows) and increased apoptosis in the periphery of the explants under FGF10 inhibition. Scale bars, 100 μm. See also Figure S5A.
Figure 6
Figure 6. Sox2 Expression Is Induced by FGF8, and Expression of Both Sox2 and Fgf8 Is Regulated by miRNAs
(Aa–Af) In situ hybridization of Fgf8 and FgfrIc in sagittal (Aa and Ad) and frontal (Ab, Ac, Ae, and Af) sections of the P2 incisor. Fgf8 is expressed in the labial CL in a restricted cell population in the distal part of the SR, as indicated by black arrows (Aa and Ac), and in preodontoblasts, as indicated by white arrows (Aa and Ab). Fgfr1c expression is restricted to the CL epithelium and localizes to the same area as Fgf8 in the distal part of the CL seen in frontal section (Af). Dashed lines in (Aa) show the positions of frontal sections depicted in (Ab) and (Ac). (B) Effects of FGF8, FGF9, and FGF10 proteins on Sox2 expression in E14 incisor as measured by qRT-PCR. (C) Effects of miRNAs on Fgf8 and Sox2 expression in a Luciferase assay. (Da–Dc) Sox2 expression in sagittal sections from P5 Shh-Cre;Dicerfl/fl (Da and Db) and Shh-Cre;Dicerfl/wt incisors (Dc). (Da) Note ectopic Sox2 expression in the lingual CL (arrow) and in the preameloblasts (asterisks). Expression seems to be more intense in the IEE and OEE of labial CL (arrowheads) (Da) compared to Sox2 expression in the control (Dc). In the mutant, Sox2 expression is found also in the tips of the extra foldings of epithelium (arrow) (Db). (E) Expression of miR-200b and miR-720 in sagittal sections of P2 wild-type incisor. MiR-200b expression is located in the SR of the labial CL, whereas in the lingual CL, expression is seen in all epithelial cells. MiR-720 is located in the enamel epithelium and in the mesenchyme next to the TA cells and in the preodontoblasts (arrows). No expression is detected in the SR. Bars in (B) and (C) correspond to average ± SD, n = 3. Asterisk indicates p < 0.01. Lab, labial; Lat, lateral; Lin, lingual; Med, medial. Scale bars: 100 mm. See also Figure S5B.
Figure 7
Figure 7. Schematic Presentation of the Movement of Sox2+ SC Progeny and the Generation of Distinct Epithelial Cell Types
(A) Sox2+ SCs (green) in the distal tip of the labial CL give rise to progeny that move in four main directions (blue arrows), all going through the Sfrp5+ area (red): the ameloblast lineage along the labial surface of the incisor, the IEE/OEE ridge on both sides, and the OEE in the posterior part of the labial CL. (B) The progeny from Sox2+ SCs (blue) form ameloblasts and SI, SR, and OEE cells and take part in the renewal of the ERM (yellow). CL, cervical loop; ERM, epithelial rests of Malassez.

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