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Developmental Biology 334 (2009) 22–30

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Developmental Biology

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Fate of HERS during tooth root development

Xiaofeng Huang a,b, Pablo Bringas Jr. a, Harold C. Slavkin a, Yang Chai a,⁎

a Center for Craniofacial Molecular Biology (CCMB), School of Dentistry, University of Southern California, 2250 Alcazar St. CSA 103, Los Angeles, CA 90033, USA b Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China

article info abstract

Article history: Tooth root development begins after the completion of crown formation in mammals. Previous studies have Received for publication 11 November 2008 shown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fate Revised 20 June 2009 of HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamic Accepted 22 June 2009 movement of HERS cells in vivo, we generated K14-Cre;R26R mice. HERS cells are detectable on the surface of Available online 1 July 2009 the root throughout root formation and do not disappear. Most of the HERS cells are attached to the surface of the cementum, and others separate to become the epithelial rest of Malassez. HERS cells secrete extracellular Keywords: HERS matrix components onto the surface of the dentin before dental follicle cells penetrate the HERS network to Tooth root contact dentin. HERS cells also participate in the cementum development and may differentiate into Tooth root development cementocytes. During root development, the HERS is not interrupted, and instead the HERS cells continue to Cre recombinase communicate with each other through the network structure. Furthermore, HERS cells interact with cranial LacZ neural crest derived mesenchyme to guide root development. Taken together, the network of HERS cells is ROSA26 conditional reporter (R26R) crucial for tooth root development. K14 promoter © 2009 Elsevier Inc. All rights reserved. Wnt1 promoter Cementum

Introduction alization (cementogenesis and dentin formation) and induction of root organization (Owens, 1978; Diekwisch, 2001). To date, the fate of HERS and its function is not clear. At least six A central issue in developmental biology is to understand pattern possible outcomes of HERS have been proposed: (1) epithelial rests of formation and its regulation. How do epithelial–mesenchymal Malassez (Wentz et al., 1950; Cerri et al., 2000; Cerri and Katchburian, interactions inform positional information and pattern formation 2005) (2) apoptosis (Kaneko et al., 1999; Cerri et al., 2000; Cerri and during organogenesis and cell differentiation? The mammalian tooth Katchburian, 2005) (3) incorporation into the advancing cementum organ is an excellent model for studies of heterotypic tissue front (Luan et al. 2006), (4) epithelial–mesenchymal transformation interactions that inform morphogenesis and cytodifferentiation and (Wentz et al., 1950; Thomas, 1995, Kaneko et al., 1999, Sonoyama et al., the formation of unique extracellular matrices (enamel, dentine, 2007), (5) migration toward the periodontal ligament (Andujar et al., cementum) associated with unique types of biomineralization (Vainio 1985). (6) differentiation into cementoblasts (Zeichner-David et al., et al., 1993; Chen et al., 1996; Kratochwil et al., 1996; Vaahtokari et al., 2003; Yamamoto et al., 2004; Sonoyama et al., 2007). 1996; Neubüser et al., 1997; Jernvall et al., 1998; Thesleff et al., 1995; Although dental epithelial cells can be detected along the Maas and Bei, 1997; Thesleff and Sharpe, 1997, Cobourne et al., 2004, developing root surface using an anti-Keratin, laminin, and hepara- Chai and Slavkin, 2003; Chai and Maxson, 2006). Following crown nase antibody and Keratin-14 (K14) can be detected on the surface of formation a bi-layered epithelial structure termed HERS (Hertwig's the root in K14-LacZ transgenic mice (Alatli et al., 1996; Luan et al., Epithelial Root Sheath) migrates apically and participates in root 2006; Azumi and Hiroaki, 2006; Tummers et al., 2007), a compre- formations and the completion of the tooth organ. This bi-layered hensive cell lineage analysis of the mammalian HERS cells has been structure is formed from ectodermally-derived outer and inner limited. Using transgenic analysis of gene regulation we have analyzed enamel epithelium. Morphologically, HERS is a structural boundary the timing, patterns and duration of differential gene expression of two dental ectomesenchymal tissues: dental papilla and dental within HERS during mouse molar tooth organogenesis. K14-Cre;R26R follicle, like a sandwich structure. During further root development, mice provide an opportunity to study HERS cell fate determination in HERS breaks up into epithelial rests and cords, allowing other cells to situ. In this Cre/loxp system, the ROSA26 conditional reporter (R26R) come in contact with the outer dentin surface. The sandwich structure transgene exhibits constitutive β-galactosidase (β-gal) expression in plays at least two important roles during root formation: biominer- cells activated by Cre (Soriano, 1999). This system is ideal for monitoring Cre mediated expression and cell lineage analysis in ⁎ Corresponding author. developmental time. By utilizing the K14 promoter, Cre expression is E-mail address: [email protected] (Y. Chai). restricted to the precursors of the epithelial cells of tooth germ, and

0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.06.034 X. Huang et al. / Developmental Biology 334 (2009) 22–30 23 consequently the progeny of epithelial cells are marked indelibly Immunohistochemistry during root development. Using this two-component genetic system, we have systematically followed the dynamic contribution of HERS Tissues were fixed with 4% paraformaldehyde for immunohisto- cells during tooth root morphogenesis. We find that HERS cells may chemistry. Paraffin blocks containing processed mouse tissue were participate in the formation of acellular as well as cellular cementum sectioned (6 μm in thickness). The slides were heated in a 60 °C oven that covers root surfaces. One fate of HERS cells is to become for 30 min and subsequently hydrated through a series of decreasing cementoblasts that synthesize and secrete alkaline phosphatase concentrations of ethanol. The immunohistochemical staining was (ALPase) and bone sialoprotein (Bsp). performed using the Zymed HistoStain SP kit, according to the manufacturer's instructions. The specificanti-K14 antibody was Materials and methods obtained from Santa Cruz Biotechnology Inc.

In situ hybridization Generation of K14-Cre;R26R and Wnt1-Cre;R26R mice Tissue was fixed with 4% paraformaldehyde in PBS, embedded in fi Male mice carrying the K14-Cre allele (Andl et al., 2004) and paraf n, serially sectioned, and mounted following standard proce- Wnt1-Cre allele (Danielian et al., 1998) were crossed with females dures. DNA fragments of Bsp and type I collagen were subcloned into carrying the R26R conditional reporter allele (Soriano, 1999)to vector plasmids. Digoxigenin (DIG)-labeled sense and antisense cRNA generate K14-Cre;R26R and Wnt1-Cre;R26R mice, respectively. Post- riboprobes were synthesized using the DIG RNA Labeling Mix (Roche fi natal age was determined according to birth, with noon of the day of Molecular Biochemicals). Paraf n-embedded sections were dewaxed μ birth designated as post-natal day 0.5 (PN 0.5). Genomic DNA was and treated with proteinase K (20 g/ml), 0.2 M HCl, acetylated, and isolated from tail biopsies of the mice at different ages (PN 0.5, PN 3.5, hybridized overnight with Digoxigenin labeled probes as previously PN 7.5, PN 10.5, PN 13.5, PN 21.5, PN 30.5, and PN 60.5). Genotypes of described (Xu et al., 2006). the double transgenic animals were determined by PCR as previously described. (Chai et al., 2000; Soriano, 1999). Results

Detection of β-gal (LacZ) activities Fate of HERS during tooth root development

In order to track the HERS cells in vivo during root development, Whole teeth (PN 13.5) were dissected from the mandible and we have analyzed the K14-Cre;R26R animal model. All epithelial cells stained for β-gal activity according to standard procedures as of the molar were β-gal-positive during crown formation (Xu et al., previously described (Chai et al., 2000). The teeth were fixed for 2008) and at the newborn stage (Figs. 1A and H). Thus, K14-Cre;R26R 20 min at room temperature in 0.2% glutaraldehyde in phosphate mice are a good model to analyze HERS cell lineage throughout the buffered saline (PBS). Fixed samples were washed three times in rinse entire process during root development. At PN 3.5, cells from the solution (0.005% Nonidet P-40 and 0.01% sodium deoxycholate in ameloblast layer and outer enamel epithelium were elongated and PBS). The teeth were stained overnight at room temperature using the formed a bi-layer. These cells were β-gal-positive (Figs. 1B and I). At standard staining solution (5 mM potassium ferricyanide, 5 mM PN 7.5, HERS formed a bi-layer of cells extending in an apical direction potassium ferrocyanide, 2 mM MgCl2, 0.4% X-gal in PBS), rinsed twice at the interface between the dental follicle and dental pulp (Figs. 1C in PBS and post-fixed in 3.7% formaldehyde. and J). At this stage, HERS showed strong LacZ expression, but we failed to detect β-gal in the periodontal ligament, dentin, odontoblasts Cryostat section, X-gal staining and ALPase staining and dental pulp. The β-gal-positive HERS was continuous with no interruptions. At PN 10.5, we detected a dissociation of the HERS (Figs. 1D and K). Subsequently, the root continued development and the β Detection of -gal activity in tissue sections was carried out as HERS dissociated further. These β-gal-positive cells remained on the previously described (Chai et al., 2000). Samples from mice of surface of the root until PN 60.5 (Figs. 1E–G and L–O). In contrast, we μ different post-natal ages were frozen sectioned at 10 m thickness detected no blue cells on the root surface in the wild type (Figs. 1P–S). fi fi ( xed in 0.2% glutaraldehyde and decalci ed with 4.4% di-sodium As a control, we examined β-gal expression in Wnt1-Cre;R26R mice EDTA) prior to X-gal staining. Detection of ALPase activity in tissue and found that, as expected, dental epithelial cells were β-gal- sections was carried out as previously described (Sasaki et al., 2006). negative and most cells derived from the cranial neural crest in periodontal ligament (PDL) and dental mesenchyme were β-gal- Mallorin-H staining positive (Figs. 1T–Y). Thus, β-gal-positive cells derived from dental epithelium remain on the surface of the root from the beginning of Samples were fixed in 10% buffered formalin and processed into root formation to adulthood in K14-Cre;R26R mice. serial paraffin wax-embedded sections using routine procedures. For general morphology, deparaffinized sections were stained with HERS cells contribute to acellular cementogenesis Mallory-Heidenhain solution using standard procedures. In adults, acellular cementum covers the cervical two thirds or Transmission electron microscopy (TEM) after LacZ staining more of the root. During root development and cementogenesis, we found that β-gal-positive cells remained on the surface of the root, although the HERS was dissociated after PN10.5 (Figs. 2A and B). At PN Samples from mice at PN 13.5 were fixed in 2.5% glutaraldehyde, 13.5, cementum could be found on the outer surface of dentin, frozen sectioned at 12 μm thickness, mounted on glass slides and visualized with Mallorin-H staining (Fig. 2D, indicated by arrows). stained by X-gal. Then the sections were post-fixed in osmic acid (1% Compared with the sections of Mallorin-H staining, many β-gal- in sodium cacodylate buffer for 2 h at room temperature), dehydrated positive cells could be detected on the surface of root in the K14-Cre; in graded ethanol and in propylene oxide, and embedded in epoxy R26R mice. Moreover, we detected pre-cementum or cementum resin. Thin slices (60 nm) were obtained, mounted on copper grids for matrix between the β-gal-positive cells and the outer surface of TEM observation. dentin (Figs. 2C and E). In Wnt1-Cre;R26R mice not all PDL cells 24 X. Huang et al. / Developmental Biology 334 (2009) 22–30

Fig. 1. The development of HERS. 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 panel 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)=200 μm, Scale bars (H–Y)=40 μm. X. Huang et al. / Developmental Biology 334 (2009) 22–30 25

Fig. 2. HERS cells contribute to acellular cementogenesis. 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 panels 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)=40 μm, scale bars (C–O)=20 μm, scale bars (Q and S)=200 μm, scale bars (R and T)=80 μm, scale bars (V and X)=10 μm, scale bars (W and Y)=2 μm. derived from CNC contacted the dentin directly, but β-gal-negative asterisks) attach to the dentin closely in the apical part of the root; cells derived from dental epithelium were still located on the root at whereas in the root surface of the acellular cementum, extracellular PN 13.5 (Figs. 2F–J). In PN13.5 K14-Cre;R26R mice, cementum matrix matrix (Fig. 2Y, indicated by black arrows) is detectable between the could be clearly detected between the epithelium derived cells and dentin surface (Fig. 2Y, indicated by white arrows in line) and HERS the dentin in the same section but different focal plane (Figs. 2K–M). cells (Fig. 2Y, indicated by asterisk). Ectomesenchymal cells from the We also used TEM to observe the ultrastructural morphology of the dental follicle penetrated into the HERS, attached on the surface of the root surface in PN13.5 K14-Cre;R26R mice. The cytoplasma of dental dentin (Figs. 2C, E–M), and formed extracellular matrix. Some dental epithelial cells (Figs. 2V–Y, indicated by asterisks) is identifiable by follicle cells were slender, similar to fibroblasts. Fibers were also dark LacZ staining (Fig. 2W, indicated by white arrowheads). Two formed at this stage (Figs. 2E and J). Therefore, during cementogen- layers of continuous HERS cells (Figs. 2V and W, indicated by esis, both HERS cells and dental follicle cells could form cementum- 26 X. Huang et al. / Developmental Biology 334 (2009) 22–30 like tissue on the surface of the root. We also observed a network detected no β-gal-positive cells on the surface of the cellular structure of HERS cells in the periphery of the tooth root surface at PN cementum in the apical area of the root edge. Therefore, the formation 13.5 using section and whole mount LacZ staining (Figs. 2P–T). Dental of cellular cementum in the apical root and furcation areas may be follicle cells could pass through this network structure to contact the different. Our results suggest that there are three kinds of cemento- surface of the root. We conclude that the geometrical intricacy of this genesis: acellular cementogenesis, apical cellular cementogenesis, and relationship likely explains why the distribution of HERS did not inter-radicular cementogenesis. During acellular cementum forma- appear the same in different sections of the samples at PN 13.5 (Figs. tion, the HERS cells were always on the surface of the root and few 2N and O). In fact, the HERS was not interrupted and the HERS cells were embedded into the cementum. In the apical area of the root all may still communicate with each other through the network structure the HERS cells were embedded into cellular cementum and no β-gal- (Fig. 2P). positive cells were detected on the surface. In the inter-radicular regions, some HERS cells were embedded into the cellular cementum HERS cells may participate in the formation of cellular cementum and others remained on the surface of the root (Fig. 4). Thus, HERS cells participate in the formation of cellular cementum and may Cellular cementum is often absent from single-rooted teeth and differentiate into cementoblasts in vivo. confined to the apical third and inter-radicular regions of molars. In this study, we found that β-gal-positive cells participated in cellular K14, Bsp and type I collagen expression in HERS cementogenesis. In the apical area of the molar root, we detected β- gal-positive cells at PN 60.5 (Figs. 3A–H). Blue cells were embedded In order to confirm that HERS cells can differentiate into into the cellular cementum and may have differentiated into cementoblasts and cementocytes, we examined the expression of cementoblasts (Figs. 3B–H, indicated by arrows). Dental follicle cells K14, β-gal and cementoblast markers, such as Bsp and type I collagen could also be found as cementoblasts and produce cellular cementum in K14-Cre;R26R mice. Keratin is expressed in dental epithelial cells in the apical part of root as well (Fig. 3, indicated by arrowheads). In during root development (Luan et al., 2006, Azumi and Hiroaki, 2006; the inter-radicular regions of the molars, most β-gal-positive cells Tummers et al., 2007). Comparing the expression of K14 and β-gal in were on the surface of the root; a few of them were embedded into the K14-Cre;R26R mice, we found that the expression patterns of K14 and cementum during cementogenesis (Figs. 3I–P). Most of the β-gal- β-gal were indistinguishable before PN 7.5. After PN 7.5, the number of positive cells were located on the surface of the furcation, and we LacZ positive cells was greater than that of K14 positive cells, especially

Fig. 3. HERS cells participate in the formation of cellular cementum. X-gal staining of apical (A–H) and inter-radicular (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 mesenchymal cells are detectable as cementoblasts and cementocytes (C–H, indicated by arrowheads). (I–P) In the inter-radicular 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)=20 μm, scale bars (I–L)=40 μm. X. Huang et al. / Developmental Biology 334 (2009) 22–30 27

Fig. 4. Multiple forms of cementum. 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 inter-radicular 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. at PN 21.5 (Figs. 5A–H and Figs. 6A–H). At PN 21.5, we detected very described in murine cementoblasts using in situ hybridization little K14 expression on the surface of the root, and most cells adjacent (MacNeil et al., 1996; Sommer et al., 1996; D'Errico et al., 1997). We to the surface of the root did not express K14 during root development examined the expression of type I collagen and Bsp in K14-Cre;R26R (Figs. 6E–H). We only detected K14 positive cells in the epithelial rest mice and compared them with the expression of K14 and β-gal. We of Malassez. In contrast, we detected many β-gal-positive cells in the detected expression of type I collagen in odontoblasts, PDL, and root at the same stage. Because the progeny of epithelial cells are cementoblasts (Figs. 5L–M). Bsp was expressed strongly in cemento- traced by LacZ staining, our data suggests that HERS cells are derived blasts, pre-odontoblasts, and osteoblasts (Figs. 5O–Q and Figs. 6I–L). from dental epithelium, but some of them do not express the Comparing the percentage of β-gal-positive cells and Bsp positive cells epithelial marker K14 during later stage of root development. in different areas of the surface of the root at PN 21.5, we found that Type I collagen and Bsp are considered markers for cementoblasts. more than 37.62% of the cells on the furcation were β-gal-positive and Gene expression patterns of type I collagen and Bsp have been more than 87.67% of the cells expressed Bsp. This data suggests that at

Fig. 5. K14, Bsp and type I collagen expression in HERS. (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)=40 μm, scale bars (I, L, and O)=80 μm, scale bars (J, K, M, N, P, and Q)=20 μm. 28 X. Huang et al. / Developmental Biology 334 (2009) 22–30

Fig. 6. Detailed comparison of K14, Bsp and β-gal expression in HERS at PN 21.5. 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 Malassez (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)=200 μm, scale bars (B, F, J)=80 μm, scale bars (C, D, G, H, K, and L)=40 μm. least 25% of the β-gal-positive cells expressed Bsp, which indicates Discussion that some of the cells derived from dental epithelium express Bsp, the marker for mesenchymal cells and cementoblasts. Thus, these results Using the two-component Cre/loxp strategy (Chai et al., 2000), we lend support to the conclusion that HERS cells may participate in have generated K14-Cre;R26R mice to follow HERS cells during tooth cementogenesis. root development. The progeny of dental epithelial cells can be tracked using LacZ staining in these mice. Although Keratin has been ALPase expression in HERS used as a marker for dental epithelium, we have found that most of the cells adjacent to the root did not express K14 during root development Traditional methods such as ALPase detection for calcium deposits at PN 21.5. To date, the patterning and function of HERS has remained have been used to identify cells with osteogenic potential. Previous unknown (Diekwisch, 2001; Luan et al., 2006), although HERS is studies have reported high ALPase activity in alveolar bone and clearly important during root formation. The canonical theory of root cementum (Groeneveld et al., 1995). The function of ALP is related to development suggests that mesenchymal cells of the dental follicle mineralization. In order to confirm that HERS cells can differentiate become cementoblasts and secrete cementum after they transit into cementoblasts and participate in mineralization, we performed through the barrier of the HERS (Paynter and Pudy, 1958; Lester, double staining of ALPase and LacZ. ALPase could be detected in 1969; Chai et al., 2000; Diekwisch, 2001). Previously, the widely ameloblasts but not in HERS at PN 3.5, and 13.5 (Figs. 7A, B, E, F). accepted theory of root development and cementogenesis suggests ALPase and LacZ could both be detected in the cells on the lateral that cementum is a dental follicle-derived connective tissue that is surface, furcation and apical area of the root at the different stages, formed subsequent to HERS disintegration (Diekwisch, 2001). This including PN 13.5, and 21.5, (Figs. 7C, D, G–J, L). For the double staining theory was developed from morphological observations that process, we first documented the X-gal staining (Fig. 7K), then mesenchymal cells from the dental follicle penetrate the HERS bi- performed the ALPase assay (Fig. 7L). β-gal-positive cells clearly also layer and deposit an initial cementum matrix, although HERS cells are express ALPase. These results further support the conclusion that separated from the root surface by a basal lamina. In our current study, HERS cells may participate in mineralization and cementogenesis. we found that HERS cells could be detected on the surface of the root X. Huang et al. / Developmental Biology 334 (2009) 22–30 29

Fig. 7. ALPase and β-gal expression in HERS. 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–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)=200 μm, scale bars (C, D, G–L)=20 μm. from the beginning of root formation to PN 60.5. Using TEM, some pre- the cellular cementum in the molar furcation is not as thick as in the cementum or cementum matrix is present between the epithelial cells apical region. Taken together, these observations indicate that the and the outer surface of dentin at PN 13.5 when acellular cementum is mechanism of cellular cementogenesis is likely different in the apical formed. Moreover, we determined that HERS cells might have root region versus the furcation. The mechanism of cementogenesis in differentiated into cementocytes in cellular cementum based on our the furcation is similar to acellular cementogenesis. Most HERS cells finding that some of the cementocytes are β-gal-positive and are are active in the furcation and some of them are embedded in embedded in the cellular cementum. In addition, HERS cells express cementum to become cementocytes. However, cellular cementogen- type I collagen, Bsp, and ALPase, consistent with our hypothesis that esis in the apical region of the root is similar to osteogenesis. Most dental epithelial cells may differentiate into cementocytes. CNC- cementocytes and almost all of the cementoblasts on the surface of derived-mesenchymal cells from the dental follicle are critical for root the cellular cementum are β-gal-negative cells. This indicates that formation. They penetrate into the HERS, attach to the surface of the these cementocytes and cementoblasts in the apical root are not dentin, and form the extracellular matrix. Interestingly, HERS is primarily derived from HERS, but from dental follicle cells. partially dissociated, but still maintains a network of connections HERS provides a structural boundary between two mesenchymal during root development. The HERS epithelial network provides space structures, the dental follicle and the dental papilla, suggesting that it for penetration by the dental follicle cells, which are then able to may play a critical function in tissue–tissue interactions. Previous contact the surface of the root. Some of the cells of the dental follicle studies suggested that HERS cells expressed not only epithelial differentiate into slender fibroblasts, which secrete collagen and other molecules such as cytokeratin, E-cadherin, ameloblastin, but also proteins to form fibers. Our data suggests that both HERS cells and mesenchymal molecules such as Bsp, vimentin, and N-cadherin (Fong dental follicle cells form cementum-like tissue. The fibroblasts derived et al., 1996; Fong and Hammarström, 2000; Zeichner-David et al., from dental follicle cells form the fibers (also known as Sharpey's 2003; Yamamoto et al., 2004; Sonoyama et al., 2007). Previous gene fibers) that are embedded in the developing cementum. In cellular expression studies during mouse molar root development have cementum, which can be found in the apical and inter-radicular suggested that some growth factors, including bone morphogenetic regions of the tooth, the majority of the cementocytes in the apical proteins (Liu et al., 2005, Andl et al., 2004), epidermal growth factors area of the root is derived from the dental follicle. We detected no (Vaahtokari et al., 1996), Shh (Khan et al., 2007; Nakatomi et al., HERS cells on the surface of the apical cellular cementum. 2006), insulin-like growth factor-1 (Fujiwara et al., 2005), Fgf10 Our results suggest that the two regions of cellular cementum (the (Yokohama et al., 2006) and transcriptional factors such as Gli, Msx1, apical area of the root and the furcation) are different. The HERS in the Msx2 and Runx2 are involved in the growth and differentiation of inter-radicular region of the first molar is disassociated after PN 10.5. odontoblasts and/or cementoblasts and in the mineralization of However, unlike the HERS cells on the surface of the acellular dentin and/or cementum (Nakatomi et al., 2006, Yamashiro et al., cementum, the epithelial cells in the inter-radicular region are still 2002, Yamashiro et al., 2003). The transcription factor, Nfic, is essential active until PN 60.5. Some of the HERS cells in the inter-radicular for root development, because the root fails to form in Nfic−/− mice region are embedded in the cellular cementum and may become (Steele-Perkins et al., 2003). Nevertheless, the mechanism of interac- cementoblasts, but others remained on the root surface. The layer of tion of HERS and dental mesenchyme and their signaling pathways 30 X. Huang et al. / Developmental Biology 334 (2009) 22–30 during root development has yet to be determined (Thesleff and Groeneveld, M.C., Everts, V., Beertsen, W., 1995. Alkaline phosphatase activity in the periodontal ligament and gingiva of the rat molar: its relation to cementum Sharpe, 1997; Miletich and Sharpe, 2003). There are many intriguing formation. J. Dent. Res. 74, 1374–1381. questions that remain to be answered: (1) How is the HERS formed? Jernvall, J., Aberg, T., Kettunen, S., Keranen, S., Thesleff, I., 1998. The life history of an (2) How are odontoblasts induced to differentiate during root dentin embryonic signalling center: BMP-4 induces p21 and is associated with apoptosis in – formation? Is HERS the inducer? (3) Do infiltrating dental follicle cells the mouse tooth enamel knot. Development 125, 161 169. Kaneko, H., Hashimoto, S., Enokiya, Y., Ogiuchi, H., Shimono, M., 1999. Cell proliferation receive reciprocal signal from the dentin or the surrounding HERS and death of Hertwig's epithelial root sheath in the rat. Cell Tissue Res. 298, 95–103. cells? (4) How do HERS cells differentiate directly into cementoblasts? Khan, M., Seppala, M., Zoupa, M., Cobourne, M.T., 2007. Hedgehog pathway gene (5) What is the function of the epithelial cell rests of Malassez? (6) expression during early development of the molar tooth root in the mouse. Gene Expr. Patterns 7, 239–243. How are fibroblasts induced to secrete Sharpy's fiber? (7) What is the Kratochwil, K., Dull, M., Farinas, I., Galceran, J., Grosschedl, R., 1996. Lef1 expression is difference between acellular and cellular cementum? (8) What activated by BMP-4 and regulates inductive tissue interactions in tooth and hair – determines the number of roots formed? development. Genes Dev. 10, 1382 1394. Lester, K.S., 1969. The incorporation of epithelial cells by cementum. J. Ultrastruct. Res. 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