X-gal staining of tooth root sections from K14-Cre;R26R mice. A: Cellular cementum in the apical region of the root at PN 30.0. All the HERS cells are embedded into the cellular cementum and no β-gal-positive cell are detectable on the surface. B: Cellular cementum of the furcation at PN 21.5. In the interradicular regions, some HERS cells are embedded into the cellular cementum and others remain on the surface of the root. C: Acellular cementum at PN 13.5. The HERS cells are detectable on the surface of the root and few are embedded into the cementum during acellular cementum formation.
AB: alveolar bone, AC: acellular cementum, CC: cellular cementum, D: dentin, DP: dental pulp, PDL: periodontal ligament

Double staining of ALPase and X-gal staining. ALPase could be found in ameloblasts but not in HERS at PN 3.5 and 13.5 (A, B, E, F). ALPase and LacZ could both be detected in the cells on the lateral surface, furcation and apical area of the root at the different stages, including PN 13.5 and 21.5 (C, D, G, H, I, J, L, indicated by arrows). During the double staining, we first documented the sample following X-gal staining (K), then ALPase essay was performed (L). The β-gal positive cells express ALPase (as indicated by red arrows).
D: dentin, DP: dental pulp, PDL: periodontal ligament, CC: cellular cementum. Scale bars (A, B, E, and F) = 200µm, Scale bars (C, D, G– L) = 20µm.

X-gal staining (A–D), immunohistochemical staining of K14 (E–H), and in situ hybridization for Bsp (I–L) of PN 21 in K14-Cre;R26R mice. Many β-gal positive cells are detectable on the root surface, in the apical area and furcation (A–D). K14 is scarcely detectable on the root, although it is clearly detectable in the epithelial rest of Malasis (E–H). Bsp is detectable on the surface of the root and alveolar bone (I–L).
More than 40% of the cells on the surface of the furcation are β-gal positive and more than 80% cells express Bsp.
B: alveolar bone, D: dentin, DP: dental pulp, PDL: periodontal ligament. Scale bars (A, E, and I) = 200µm, Scale bars (B, F, J) = 80µm, Scale bars (C, D, G, H, K, and L) = 40µm.

X-gal staining of apical (A–H) and interradicular (I–P) tooth root sections from post-natal K14-Cre;R26R mice. A–H: In the apical region of the molar root, β-gal-positive cells are detectable until at least PN 60.5. HERS cells appear to be embedded into the cellular cementum and may have differentiated into cementocytes (B–H, indicated by arrows). β-gal-negative mensenchymal cells are detectable as cementoblasts and cementocytes (C–H, indicated by arrowheads). I–P: In the interradicular regions of the molars, most β-gal positive cells are detectable on the surface of the root; a few are embedded into the cementum during cementogenesis (N, indicated by arrow). Dental mesenchymal cells are detectable that penetrate into the HERS and have attached onto the surface of the dentin (N, indicated by arrowhead).
AB: alveolar bone, CC: cellular cementum, D: dentin, DP: dental pulp, PDL: periodontal ligament. Scale bars (A–H, and M–P) = 20µm, Scale bars (I–L) = 40µm.

A–H: Immunohistochemical staining for K14 (A–D) and X-gal staining (E–H) of tooth root sections from different stages of post-natal K14-Cre;R26R mice. The expression of K14 and β-gal overlap significantly at PN 3.5 (A, E). After PN 7.5, the number of LacZ positive cells is greater than that of K14 positive cells (B–D, F–K). I–Q: X-gal staining (I–K), in situ hybridization of type I collagen (L–N), and in situ hybridization of Bsp of PN13.5 K14-Cre;R26R mice. β-gal, Bsp and type I collagen are all detectable on the surface of the root at PN 13.5 (I–Q).
AB: alveolar bone, AC: acellular cementum, CC: cellular cementum, D: dentin, DP: dental pulp, PDL: periodontal ligament. Scale bars (A– H) = 40µm, Scale bars (I, L, and O) = 80µm, Scale bars (J, K, M, N, P, and Q) = 20µm.

LacZ staining of sections showing upper molars of K14-Cre;R26R mice (A–G) and lateral surface of the root in K14-Cre;R26R mice (H–O), wild type (Wt: P–S), and Wnt1-Cre;R26R (R–W) mice. A– O: At PN 0.5, all the epithelial cells in K14-Cre;R26R teeth are β-gal-positive (A and H). At PN 3.5, cells from the ameloblast layer and the outer enamel epithelium are elongated and begin to form a bi-layer. These cells are β-gal-positive (B and I). At PN 7.5, the HERS forms a bi-layer of flat cells and extends in an apical direction at the interface between dental follicle and dental pulp (C and J). Dissociation of HERS is observed at PN 10.5 (D and K). The root developed and the HERS are dissociated further after PN 10.5, and β-gal-positive cells remain detectable on the surface of the root until PN 60.5 (E–G and L–O, β-gal-positive cells are indicated by arrows in O). P–S: No blue cells are detectable on the root surface in wild type mice. T–Y: In Wnt1-Cre;R26R mice, dental epithelial cells are β-gal negative, and most of the cells derived from the cranial neural crest in the PDL and dental mesenchyme are β-gal positive.
AB: alveolar bone, D: dentin, DP: dental pulp, PDL: periodontal ligament. Scale bars (A–G) = 200µm, Scale bars (H–Y) = 40µm.

X-gal (A, C, E, and F–Y) and Mollary–H (B, D) staining of tooth root sections from K14-Cre;R26R (A–E, K–Y) and Wnt1-Cre;R26R (F–J) mice; X-gal staining of dissected teeth from PN 13.5 K14-Cre;R26R mice (Q–T) and TEM of PN 13.5 K14-Cre;R26R mice (V–Y). A, B: At PN 10.5, the HERS (blue in X-gal staining) appears to be interrupted. C–P: At PN 13.5, cementum is detectable on the outside of the dentin in K14-Cre;R26R samples (D, indicated by arrows), and many β-gal-positive cells are detected on the surface of the root (C, E, K–P). Pre-cementum, or cementum matrix is detectable between the β-gal-positive cells and the outer surface of dentin (C, E, K–P). K–M show different focal planes of the same section highlighting the extracellular matrix (arrows) between the β-gal-positive cells and the dentin. The distribution of the HERS is variable in different sections at PN 13.5 (N and O). Also at PN 13.5, ectomesenchymal cells from the dental follicle penetrate into the HERS and attach on the surface of dentin in Wnt1-Cre;R26R samples (F–J). P–T: The HERS cells appear to form a network structure in the periphery of the tooth root surface. R and T are enlarged from the boxes in Q and S, respectively. U–Y: Dental epithelial cells are first labeled by LacZ staining (U), then analyzed by TEM (V–Y), in which HERS cells (asterisks) are stained by X-gal. In the apical part of the root, the HERS is continuous and attaches the dentin closely (W). In the root surface where HERS cells are interrupted, extracellular matrix (Y, indicated by black arrows) is detected between HERS cells (Y, indicated by asterisk) and dentin surface ( Y, indicated by white arrows in line).
D: dentin, PDL: periodontal ligament; OD: odontoblast, DP: dental pulp. Scale bars (A, B and P) = 40µm, Scale bars (C– O) = 20µm, Scale bars (Q and S) = 200µm, Scale bars (R and T) = 80µm, Scale bars (V and X) = 10µm, Scale bars (W and Y) = 2µm.