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Dev Biol. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2744848
NIHMSID: NIHMS139234

Fate of HERS during Tooth Root Development

Abstract

Tooth root development begins after the completion of crown formation in mammals. Previous studies have shown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fate of HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamic movement of HERS cells in vivo, we generated K14-Cre;R26R mice. HERS cells are detectable on the surface of 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 Malasez. HERS cells secrete extracellular matrix components onto the surface of the dentin before dental follicle cells penetrate the HERS network to contact dentin. HERS cells also participate in the cementum development and may differentiate into cementocytes. During root development, the HERS is not interrupted, and instead the HERS cells continue to communicate with each other through the network structure. Furthermore, HERS cells interact with cranial neural crest derived mesenchyme to guide root development. Taken together, the network of HERS cells is crucial for tooth root development.

Keywords: HERS, tooth root, Cre recombinase, LacZ, ROSA26 conditional reporter (R26R), K14 promoter, Wnt1 promoter

Introduction

A central issue in developmental biology is to understand pattern formation and its regulation. How do epithelial-mesenchymal interactions inform positional information and pattern formation during organogenesis and cell differentiation? The mammalian tooth organ is an excellent model for studies of heterotypic tissue interactions that inform morphogenesis and cytodifferentiation and the formation of unique extracellular matrices (enamel, dentine, cementum) associated with unique types of biomineralization (Vainio et al., 1993; Chen et al., 1996; Kratochwil et al., 1996; Vaahtokari et al., 1996; Neubüser et al., 1997; Jernvall et al., 1998; Thesleff et al., 1995; Maas and Bei, 1997; Thesleff and Sharpe, 1997, Cobourne et al 2004, Chai et al., 2003; Chai and Maxson 2006). Following crown formation a bilayered epithelial structure termed HERS (Hertwig's Epithelial Root Sheath) migrates apically and participates in root formations and the completion of the tooth organ. This bilayered structure is formed from ectodermally-derived outer and inner enamel epithelium. Morphologically, HERS is a structural boundary of two dental ectomesenchymal tissues: dental papilla and dental follicle, like a sandwich structure. During further root development, HERS breaks up into epithelial rests and cords, allowing other cells to come in contact with the outer dentin surface. The sandwich structure plays at least two important roles during root formation: bio-mineralization (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 possible outcomes of HERS have been proposed: (1) epithelial rests of Malassez (Wentz et al., 1950; Cerri et al., 2000; Cerri and Katchburian, 2005) (2) apoptosis (Kaneko et al., 1999; Cerri et al., 2000; Cerri and Katchburian, 2005) (3) incorporation into the advancing cementum front (Lester et al. 1969; Luan et al. 2006), (4) epithelial-mesenchymal transformation (Wentz et al., 1950; Thomas, 1995, Kaneko et al., 1999, Sonoyama et al., 2007), (5) migration toward the periodontal ligament (Andujar et al., 1985). (6) differentiation into cementoblasts (Zeichner-David et al., 2003; Yamamoto et al., 2004; Sonoyama et al., 2007).

Although dental epithelial cells can be detected along the developing root surface using an anti-Keratin, laminin, and heparanase antibody and Keratin-14 (K14) can be detected on the surface of the root in K14-LacZ transgenic mice (Alatli et al., 1996; Luan et al., 2006; Azumi and Hiroaki, 2006; Tummers et al., 2007), a comprehensive cell lineage analysis of the mammalian HERS cells has been limited. Using transgenic analysis of gene regulation we have analyzed the timing, patterns and duration of differential gene expression within HERS during mouse molar tooth organogenesis. K14-Cre;R26R mice provide an opportunity to study HERS cell fate determination in situ. In this Cre/loxp system, the ROSA26 conditional reporter (R26R) transgene exhibits constitutive β-galactosidase (β-gal) expression in cells activated by Cre (Soriano, 1999). This system is ideal for monitoring Cre mediated expression and cell lineage analysis in developmental time. By utilizing the K14 promoter, Cre expression is restricted to the precursors of the epithelial cells of tooth germ, and consequently the progeny of epithelial cells are marked indelibly during root development. Using this two-component genetic system, we have systematically followed the dynamic contribution of HERS cells during tooth root morphogenesis. We find that HERS cells may participate in the formation of acellular as well as cellular cementum that covers root surfaces. One fate of HERS cells is to become cementoblasts that synthesize and secrete alkaline phosphatase (ALPase) and bone sialoprotein (Bsp).

MATERIALS & METHODS

Generation of K14-Cre;R26R and Wnt1-Cre;R26R mice

Male mice carrying the K14-Cre allele (Andl et al., 2004) and Wnt1-Cre allele (Danielian et al., 1998) were crossed with females carrying the R26R conditional reporter allele (Soriano et al., 1999) to generate K14-Cre;R26R and Wnt1-Cre;R26R mice, respectively. Postnatal age was determined according to birth, with noon of the day of birth designated as postnatal day 0.5 (PN 0.5). Genomic DNA was isolated from tail biopsies of the mice at different ages (PN 0.5, PN 3.5, PN 7.5, PN 10.5, PN 13.5, PN 21.5, PN 30.5, and PN 60.5). Genotypes of the double transgenic animals were determined by PCR as previously described. (Chai et al., 2000; Soriano et al. 1999).

Detection of β-gal (LacZ) activities

Whole teeth (PN 13.5) were dissected from the mandible and stained for β-gal activity according to standard procedures as previously described (Chai et al., 2000). The teeth were fixed for 20 minutes at room temperature in 0.2% glutaraldehyde in phosphate buffered saline (PBS). Fixed samples were washed three times in rinse solution (0.005% Nonidet P-40 and 0.01% sodium deoxycholate in PBS). The teeth were stained overnight at room temperature using the standard staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.4% X-gal in PBS), rinsed twice in PBS and postfixed in 3.7% formaldehyde.

Cryostat section, X-gal staining and ALPase staining

Detection of β-gal activity in tissue sections was carried out as previously described (Chai et al., 2000). Samples from mice of different postnatal ages were frozen sectioned at 10 µm thickness (fixed in 0.2% glutaraldehyde and decalcified with 4.4% di-sodium EDTA) prior to X-gal staining. Detection of ALPase activity in tissue sections was carried out as previously described (Sasaki et al., 2006).

Mallorin-H staining

Samples were fixed in 10% buffered formalin and processed into serial paraffin wax-embedded sections using routine procedures. For general morphology, deparaffinized sections were stained with Mallory-Heidenhain solution using standard procedures.

Transmission electron microscopy (TEM) after LacZ staining

Samples from mice at PN 13.5 were fixed in 2.5% glutaraldehyde, frozen sectioned at 12 µm thickness, mounted on glass slides and stained by X-gal. Then the sections were post-fixed in osmic acid (1% in sodium cacodylate buffer for 2 hours at room temperature), dehydrated in graded ethanol and in propylene oxide, and embedded in epoxy resin. Thin slices (60 nm) were obtained, mounted on copper grids for TEM observation.

Immunohistochemistry

Tissues were fixed with 4% paraformaldehyde for immunohistochemistry. Paraffin blocks containing processed mouse tissue were sectioned (6 µm in thickness). The slides were heated in a 60°C oven for 30 min and subsequently hydrated through a series of decreasing concentrations of ethanol. The immunohistochemical staining was performed using the Zymed HistoStain SP kit, according to the manufacturer’s instructions. The specific anti-K14 antibody was obtained from Santa Cruz Biotechnology Inc.

In situ hybridization

Tissue was fixed with 4% paraformaldehyde in PBS, embedded in paraffin, serially sectioned, and mounted following standard procedures. DNA fragments of Bsp and type I Collagen were subcloned into vector plasmids. Digoxigenin (DIG)-labeled sense and antisense cRNA riboprobes were synthesized using the DIG RNA Labeling Mix (Roche Molecular Biochemicals). Paraffin-embedded sections were dewaxed and treated with proteinase K (20 µg/ml), 0.2 M HCl, acetylated, and hybridized overnight with Digoxigenin labeled probes as previously described (Xu et al., 2006).

Results

Fate of HERS during tooth root development

In order to track the HERS cells in vivo during toot development, we have analyzed the K14-Cre;R26R animal model. All epithelial cells of the molar were β-gal positive during crown formation (Xu et al., 2008) and at the newborn stage (Fig. 1A and H). Thus, K14-Cre;R26R mice are a good model to analyze HERS cell lineage throughout the entire process during root development. At PN 3.5, cells from the ameloblast layer and outer enamel epithelium were elongated and formed a bi-layer. These cells were β-gal-positive (Fig 1B and I). At PN 7.5, HERS formed a bi-layer of cells extending in an apical direction at the interface between the dental follicle and dental pulp (Fig 1C and J). At this stage, HERS showed strong LacZ expression, but we failed to detect β-gal in the periodontal ligament, dentin, odontoblasts and dental pulp. The β-gal-positive HERS was continuous with no interruptions. At PN 10.5, we detected a dissociation of the HERS (Fig 1D and K). Subsequently, the root continued development and the HERS dissociated further. These β-gal-positive cells remained on the surface of the root until PN 60.5 (Fig 1 E–G and L–O). In contrast, we detected no blue cells on the root surface in the wild type (Fig 1 P–S). As a control, we examined β-gal expression in Wnt1-Cre;R26R mice and found that, as expected, dental epithelial cells were β-gal negative and most cells derived from the cranial neural crest in periodontal ligament (PDL) and dental mesenchyme were β-gal positive (Fig 1 T–Y). Thus, β-gal-positive cells derived from dental epithelium remain on the surface of the root from the beginning of root formation to adulthood in K14-Cre;R26R mice.

Figure 1
The development of HERS

HERS cells contribute to acellular cementogenesis

In adults, acellular cementum covers the cervical two thirds or 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 (Fig 2A and B). At PN 13.5, cementum could be found on the outer surface of dentin, visualized with Mallorin-H staining (Fig 2D, indicated by arrows). Compared with the sections of Mallorin-H staining, many β-gal-positive cells could be detected on the surface of root in the K14-Cre;R26R mice. Moreover, we detected pre-cementum or cementum matrix between the β-gal-positive cells and the outer surface of dentin (Fig 2C and E). In Wnt1-Cre;R26R mice not all PDL cells derived from CNC contacted the dentin directly, but β-gal negative cells derived from dental epithelium were still located on the root at PN 13.5 (Fig 2 F–J). In PN13.5 K14-Cre;R26R mice, cementum matrix could be clearly detected between the epithelium derived cells and the dentin in the same section but different focal plane (Figure 2 K–M). We also used TEM to observe the ultrastructural morphology of the root surface in PN13.5 K14-Cre;R26R mice. The cytoplasma of dental epithelial cells (Fig 2 V–Y, indicated by asterisks) is identifiable by dark LacZ staining (Fig 2W, indicated by white arrowheads). Two layers of continuous HERS cells (Fig 2V and W, indicated by asterisks) attach to the dentin closely in the apical part of the root; whereas in the root surface of the acellular cementum, extracellular matrix (Fig 2Y, indicated by black arrows) is detectable between the dentin surface (Fig 2Y, indicated by white arrows in line) and HERS cells (Fig 2Y, indicated by asterisk). Ectomesenchymal cells from the dental follicle penetrated into the HERS, attached on the surface of the dentin (Fig 2C, E–M), and formed extracellular matrix. Some dental follicle cells were slender, similar to fibroblasts. Fibers were also formed at this stage (Fig 2E and J). Therefore, during cementogenesis, both HERS cells and dental follicle cells could form cementum-like tissue on the surface of the root. We also observed a network structure of HERS cells in the periphery of the tooth root surface at PN 13.5 using section and whole mount LacZ staining (Fig 2 P–T). Dental follicle cells could pass through this network structure to contact the surface of the root. We conclude that the geometrical intricacy of this relationship likely explains why the distribution of HERS did not appear the same in different sections of the samples at PN 13.5 (Fig 2N and O). In fact, the HERS was not interrupted and the HERS cells may still communicate with each other through the network structure (Fig 2P).

Figure 2
HERS cells contribute to acellular cementogenesis

HERS cells may participate in the formation of cellular cementum

Cellular cementum is often absent from single-rooted teeth and confined to the apical third and inter-radicular regions of molars. In this study, we found that β-gal-positive cells participated in cellular cementogenesis. In the apical area of the molar root, we detected β-gal-positive cells at PN 60.5 (Fig 3 A–H). Blue cells were embedded into the cellular cementum and may have differentiated into cementoblasts (Fig 3 B–H, indicated by arrows). Dental follicle cells could also be found as cementoblasts and produce cellular cementum in the apical part of root as well (Fig 3, indicated by arrowheads). In the inter-radicular regions of the molars, most β-gal-positive cells were on the surface of the root; a few of them were embedded into the cementum during cementogenesis (Fig 3 I–P). Most of the β-gal-positive cells were located on the surface of the furcation, and we detected no β-gal-positive cells on the surface of the cellular cementum in the apical area of the root edge. Therefore, the formation of cellular cementum in the apical root and furcation areas may be different. Our results suggest that there are three kinds of cementogenesis: acellular cementogenesis, apical cellular cementogenesis, and interradicular cementogenesis. During acellular cementum formation, the HERS cells were always on the surface of the root and few were embedded into the cementum. In the apical area of the root all the HERS cells were embedded into cellular cementum and no β-gal-positive cells were detected on the surface. In the inter-radicular regions, some HERS cells were embedded into the 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 differentiate into cementoblasts in vivo.

Figure 3
HERS cells participate in the formation of cellular cementum
Figure 4
Multiple forms of cementum

K14, Bsp and type I collagen expression in HERS

In order to confirm that HERS cells can differentiate into cementoblasts and cementocytes, we examined the expression of K14, β-gal and cementoblast markers, such as Bsp and type I collagen in K14-Cre;R26R mice. Keratin is expressed in dental epithelial cells during root development (Luan et al., 2006, Azumi and Hiroaki, 2006; Tummers et al., 2007). Comparing the expression of K14 and β-gal in K14-Cre;R26R mice, we found that the expression patterns of K14 and β-gal were indistinguishable before PN 7.5. After PN 7.5, the number of LacZ positive cells was greater than that of K14 positive cells, especially at PN 21.5 (Fig 5 A–H and Fig 6 A–H). At PN 21.5, we detected very little K14 expression on the surface of the root, and most cells adjacent to the surface of the root did not express K14 during root development (Fig 6 E–H). We only detected K14 positive cells in the epithelial rest of Malassez. In contrast, we detected many β-gal positive cells in the root at the same stage. Because the progeny of epithelial cells are traced by LacZ staining, our data suggests that HERS cells are derived from dental epithelium, but some of them do not express the epithelial marker K14 during later stage of root development.

Figure 5
K14, Bsp and type I collagen expression in HERS
Figure 6
Detailed comparison of K14, Bsp and β-gal expression in HERS at PN 21.5

Type I collagen and Bsp are considered markers for cementoblasts. Gene expression patterns of type I collagen and Bsp have been described in murine cementoblasts using in situ hybridization (MacNeil et al., 1996; Sommer et al., 1996; D’Errico et al., 1997). We examined the expression of type I collagen and Bsp in K14-Cre;R26R mice and compared them with the expression of K14 and β-gal. We detected expression of type I collagen in odontoblasts, PDL, and cementoblasts (Fig. 5 L–M). Bsp was expressed strongly in cementoblasts, pre-odontoblasts, and osteoblasts (Fig. 5 O–Q and Fig. 6 I–L). Comparing the percentage of β-gal positive cells and Bsp positive cells in different areas of the surface of the root at PN 21.5, we found that more than 37.62% of the cells on the furcation were β-gal positive and more than 87.67% of the cells expressed Bsp. This data suggests that at least 25% of the β-gal positive cells expressed Bsp, which indicates that some of the cells derived from dental epithelium express Bsp, the marker for mesenchymal cells and cementoblasts. Thus, these results lend support to the conclusion that HERS cells may participate in cementogenesis.

ALPase expression in HERS

Traditional methods such as ALPase detection for calcium deposits have been used to identify cells with osteogenic potential. Previous studies have reported high ALPase activity in alveolar bone and cementum (Groenevenld et al., 1995). The function of ALP is related to mineralization. In order to confirm that HERS cells can differentiate into cementoblasts and participate in mineralization, we performed double staining of ALPase and LacZ. ALPase could be detected in ameloblasts but not in HERS at PN 3.5, and 13.5 (Fig 7A, 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 stage, including PN 13.5, and 21.5, (Fig 7C, D, G, H, I, J, L). For the double staining process, we first documented the X-gal staining (Fig 7K), then performed the ALPase assay (Fig 7L). β-gal positive cells clearly also express ALPase. These results further support the conclusion that HERS cells may participate in mineralization and cementogenesis.

Figure 7
ALPase and β-gal expression in HERS

Discussion

Using the two-component Cre/loxp strategy (Chai et al., 2000), we have generated K14-Cre;R26R mice to follow HERS cells during tooth root development. The progeny of dental epithelial cells can be tracked using LacZ staining in these mice. Although Keratin has been 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 at PN 21.5. To date, the patterning and function of HERS has remained unknown (Diekwisch, 2001; Luan, et al 2006), although HERS is clearly important during root formation. The canonical theory of root development suggests that mesenchymal cells of the dental follicle become cementoblasts and secrete cementum after they transit through the barrier of the HERS (Paynter and Pudy, 1958; Lester, 1969; Chai et al, 2000; Diekwisch, 2001). Previously, the widely accepted theory of root development and cementogenesis suggests that cementum is a dental follicle-derived connective tissue that is formed subsequent to HERS disintegration (Diekwisch, 2001). This theory was developed from morphological observations that mesenchymal cells from the dental follicle penetrate the HERS bilayer and deposit an initial cementum matrix, although HERS cells are separated from the root surface by a basal lamina. In our current study, we found that HERS cells could be detected on the surface of the root from the beginning of root formation to PN 60.5. Using TEM, some pre-cementum or cementum matrix is present between the epithelial cells and the outer surface of dentin at PN 13.5 when acellular cementum is formed. Moreover, we determined that HERS cells might have differentiated into cementocytes in cellular cementum based on our finding that some of the cementocytes are β–gal positive and are embedded in the cellular cementum. In addition, HERS cells express type I collagen, Bsp, and ALPase, consistent with our hypothesis that dental epithelial cells may differentiate into cementocytes. CNC-derived-mesenchymal cells from the dental follicle are critical for root formation. They penetrate into the HERS, attach to the surface of the dentin, and form the extracellular matrix. Interestingly, HERS is partially dissociated, but still maintains a network of connections during root development. The HERS epithelial network provides space for penetration by the dental follicle cells, which are then able to contact the surface of the root. Some of the cells of the dental follicle differentiate into slender fibroblasts, which secrete collagen and other proteins to form fibers. Our data suggests that both HERS cells and dental follicle cells form cementum-like tissue. The fibroblasts derived from dental follicle cells form the fibers (also known as Sharpey’s fibers) that are embedded in the developing cementum. In cellular cementum, which can be found in the apical and interradicular regions of the tooth, the majority of the cementocytes in the apical area of the root is derived from the dental follicle. We detected no HERS cells on the surface of the apical cellular cementum.

Our results suggest that the two regions of cellular cementum (the apical area of the root and the furcation) are different. The HERS in the interradicular region of the first molar is disassociated after PN 10.5. However, unlike the HERS cells on the surface of the acellular cementum, the epithelial cells in the interradicular region are still active until PN 60.5. Some of the HERS cells in the interradicular region are embedded in the cellular cementum and may become cementoblasts, but others remained on the root surface. The layer of the cellular cementum in the molar furcation is not as thick as in the apical region. Taken together, these observations indicate that the mechanism of cellular cementogenesis is likely different in the apical root region versus the furcation. The mechanism of cementogenesis in the furcation is similar to acellular cementogenesis. Most HERS cells are active in the furcation and some of them are embedded in cementum to become cementocytes. However, cellular cementogenesis in the apical region of the root is similar to osteogenesis. Most cementocytes and almost all of the cementoblasts on the surface of the cellular cementum are β-gal-negative cells. This indicates that these cementocytes and cementoblasts in the apical root are not primarily derived from HERS, but from dental follicle cells.

HERS provides a structural boundary between two mesenchymal structures, the dental follicle and the dental papilla, suggesting that it may play a critical function in tissue-tissue interactions. Previous studies suggested that HERS cells expressed not only epithelial molecules such as cytokeratin, E-cadherin, ameloblastin, but also mesenchymal molecules such as Bsp, vimentin, and N-cadherin (Fong et al., 1996; Fong and Hammarström, 2000; Zeichner-David et al., 2003; Yamamoto et al., 2004; Sonoyama et al., 2007). Previous gene expression studies during mouse molar root development have suggested that some growth factors, including bone morphogenetic proteins (Liu et al., 2005, Andl et al., 2004), epidermal growth factors (Vaahtokari et al., 1996), Shh (Khan et al,. 2007; Nakatomi et al., 2006), insulin-like growth factor-1 (Fujiwara et al,. 2005), Fgf10 (Yokohama et al,. 2006) and transcriptional factors such as Gli, Msx1, Msx2 and Runx2 are involved in the growth and differentiation of odontoblasts and/or cementoblasts and in the mineralization of dentin and/or cementum (Nakatomi et al., 2006, Yamashiro et al., 2002, Yamashiro et al., 2003). The transcription factor, Nfic, is essential for root development, because the root fails to form in Nfic−/− mice (Steele-Perkins et al., 2003). Nevertheless, the mechanism of interaction of HERS and dental mesenchyme and their signaling pathways during root development has yet to be determined (Thesleff and Sharpe, 1997; Miletich and Sharpe, 2003). There are many intriguing questions that remain to be answered: (1) How is the HERS formed? (2) How are odontoblasts induced to differentiate during root dentin formation? Is HERS the inducer? (3) Do infiltrating dental follicle cells receive reciprocal signal from the dentin or the surrounding HERS cells? (4) How do HERS cells differentiate directly into cementoblasts? (5) What is the function of the epithelial cell rests of Malassez? (6) How are fibroblasts induced to secrete Sharpy’s fiber? (7) What is the difference between acellular and cellular cementum? (8) What determines the number of roots formed? In this study, we have found that HERS cells remain on the surface of the root throughout root formation and can differentiate into cementoblasts and cementocytes to participate in cementogenesis. We also detected a network structure of HERS that suggests the HERS cells may be a signal center of interaction between the dental epithelium and mesenchyme during root elongation, odontogenesis, and cementogenesis. Future studies will help to elucidate the process of root development, the interaction between epithelial and mesenchymal cells, and the signaling pathway in this process. This knowledge may eventually lead to our ability to promote tooth or root regeneration with obvious clinical applications.

Acknowledgements

We thank Sarah Millar for K14-Cre mice and Julie Mayo for critical reading of the manuscript. This study was supported by grants from the NIDCR, NIH (DE012711 and DE014078) to Yang Chai and the Beijing New Star Program (2007B54) to Xiaofeng Huang.

Footnotes

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