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, USAb Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China

⁎ Corresponding author.E-mail address: ychai@usc.edu (Y. Chai).

0012-1606/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.ydbio.2009.06.034

a b s t r a c t

a r t i c l e i n f o

Article history:Received for publication 11 November 2008Revised 20 June 2009Accepted 22 June 2009Available online 1 July 2009

Keywords:HERSTooth rootTooth root developmentCre recombinaseLacZROSA26 conditional reporter (R26R)K14 promoterWnt1 promoterCementum

Tooth root development begins after the completion of crown formation in mammals. Previous studies haveshown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fateof HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamicmovement of HERS cells in vivo, we generated K14-Cre;R26Rmice. HERS cells are detectable on the surface ofthe root throughout root formation and do not disappear. Most of the HERS cells are attached to the surface ofthe cementum, and others separate to become the epithelial rest of Malassez. HERS cells secrete extracellularmatrix components onto the surface of the dentin before dental follicle cells penetrate the HERS network tocontact dentin. HERS cells also participate in the cementum development and may differentiate intocementocytes. During root development, the HERS is not interrupted, and instead the HERS cells continue tocommunicate with each other through the network structure. Furthermore, HERS cells interact with cranialneural crest derived mesenchyme to guide root development. Taken together, the network of HERS cells iscrucial for tooth root development.

© 2009 Elsevier Inc. All rights reserved.

Introduction

A central issue in developmental biology is to understand patternformation and its regulation. How do epithelial–mesenchymalinteractions inform positional information and pattern formationduring organogenesis and cell differentiation? The mammalian toothorgan is an excellent model for studies of heterotypic tissueinteractions that inform morphogenesis and cytodifferentiation andthe formation of unique extracellular matrices (enamel, dentine,cementum) associated with unique types of biomineralization (Vainioet 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 and Slavkin, 2003; Chai and Maxson, 2006). Following crownformation a bi-layered epithelial structure termed HERS (Hertwig'sEpithelial Root Sheath) migrates apically and participates in rootformations and the completion of the tooth organ. This bi-layeredstructure is formed from ectodermally-derived outer and innerenamel epithelium. Morphologically, HERS is a structural boundaryof two dental ectomesenchymal tissues: dental papilla and dentalfollicle, like a sandwich structure. During further root development,HERS breaks up into epithelial rests and cords, allowing other cells tocome in contact with the outer dentin surface. The sandwich structureplays at least two important roles during root formation: biominer-

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alization (cementogenesis and dentin formation) and induction ofroot organization (Owens, 1978; Diekwisch, 2001).

To date, the fate of HERS and its function is not clear. At least sixpossible outcomes of HERS have been proposed: (1) epithelial rests ofMalassez (Wentz et al., 1950; Cerri et al., 2000; Cerri and Katchburian,2005) (2) apoptosis (Kaneko et al., 1999; Cerri et al., 2000; Cerri andKatchburian, 2005) (3) incorporation into the advancing cementumfront (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 thedeveloping root surface using an anti-Keratin, laminin, and hepara-nase antibody and Keratin-14 (K14) can be detected on the surface ofthe root in K14-LacZ transgenic mice (Alatli et al., 1996; Luan et al.,2006; Azumi and Hiroaki, 2006; Tummers et al., 2007), a compre-hensive cell lineage analysis of the mammalian HERS cells has beenlimited. Using transgenic analysis of gene regulationwe have analyzedthe timing, patterns and duration of differential gene expressionwithin HERS during mouse molar tooth organogenesis. K14-Cre;R26Rmice provide an opportunity to study HERS cell fate determination insitu. In this Cre/loxp system, the ROSA26 conditional reporter (R26R)transgene exhibits constitutive β-galactosidase (β-gal) expression incells activated by Cre (Soriano, 1999). This system is ideal formonitoring Cre mediated expression and cell lineage analysis indevelopmental time. By utilizing the K14 promoter, Cre expression isrestricted to the precursors of the epithelial cells of tooth germ, and

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consequently the progeny of epithelial cells are marked indeliblyduring root development. Using this two-component genetic system,we have systematically followed the dynamic contribution of HERScells during tooth root morphogenesis. We find that HERS cells mayparticipate in the formation of acellular as well as cellular cementumthat covers root surfaces. One fate of HERS cells is to becomecementoblasts that synthesize and secrete alkaline phosphatase(ALPase) and bone sialoprotein (Bsp).

Materials and methods

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

Male mice carrying the K14-Cre allele (Andl et al., 2004) andWnt1-Cre allele (Danielian et al., 1998) were crossed with femalescarrying the R26R conditional reporter allele (Soriano, 1999) togenerate K14-Cre;R26R and Wnt1-Cre;R26R mice, respectively. Post-natal age was determined according to birth, with noon of the day ofbirth designated as post-natal day 0.5 (PN 0.5). Genomic DNA wasisolated 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 ofthe double transgenic animals were determined by PCR as previouslydescribed. (Chai et al., 2000; Soriano, 1999).

Detection of β-gal (LacZ) activities

Whole teeth (PN 13.5) were dissected from the mandible andstained for β-gal activity according to standard procedures aspreviously described (Chai et al., 2000). The teeth were fixed for20 min at room temperature in 0.2% glutaraldehyde in phosphatebuffered saline (PBS). Fixed samples were washed three times in rinsesolution (0.005% Nonidet P-40 and 0.01% sodium deoxycholate inPBS). The teeth were stained overnight at room temperature using thestandard staining solution (5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide, 2 mMMgCl2, 0.4% X-gal in PBS), rinsed twicein PBS and post-fixed in 3.7% formaldehyde.

Cryostat section, X-gal staining and ALPase staining

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

Mallorin-H staining

Samples were fixed in 10% buffered formalin and processed intoserial paraffin wax-embedded sections using routine procedures. Forgeneral morphology, deparaffinized sections were stained withMallory-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 andstained by X-gal. Then the sections were post-fixed in osmic acid (1%in sodium cacodylate buffer for 2 h at room temperature), dehydratedin graded ethanol and in propylene oxide, and embedded in epoxyresin. Thin slices (60 nm) were obtained, mounted on copper grids forTEM observation.

Immunohistochemistry

Tissues were fixed with 4% paraformaldehyde for immunohisto-chemistry. Paraffin blocks containing processed mouse tissue weresectioned (6 μm in thickness). The slides were heated in a 60 °C ovenfor 30 min and subsequently hydrated through a series of decreasingconcentrations of ethanol. The immunohistochemical staining wasperformed using the Zymed HistoStain SP kit, according to themanufacturer's instructions. The specific anti-K14 antibody wasobtained from Santa Cruz Biotechnology Inc.

In situ hybridization

Tissue was fixed with 4% paraformaldehyde in PBS, embedded inparaffin, serially sectioned, and mounted following standard proce-dures. DNA fragments of Bsp and type I collagen were subcloned intovector plasmids. Digoxigenin (DIG)-labeled sense and antisense cRNAriboprobes were synthesized using the DIG RNA Labeling Mix (RocheMolecular Biochemicals). Paraffin-embedded sections were dewaxedand treated with proteinase K (20 μg/ml), 0.2 M HCl, acetylated, andhybridized overnight with Digoxigenin labeled probes as previouslydescribed (Xu et al., 2006).

Results

Fate of HERS during tooth root development

In order to track the HERS cells in vivo during root development,we have analyzed the K14-Cre;R26R animal model. All epithelial cellsof the molar were β-gal-positive during crown formation (Xu et al.,2008) and at the newborn stage (Figs. 1A and H). Thus, K14-Cre;R26Rmice are a good model to analyze HERS cell lineage throughout theentire process during root development. At PN 3.5, cells from theameloblast layer and outer enamel epithelium were elongated andformed a bi-layer. These cells were β-gal-positive (Figs. 1B and I). AtPN 7.5, HERS formed a bi-layer of cells extending in an apical directionat the interface between the dental follicle and dental pulp (Figs. 1Cand J). At this stage, HERS showed strong LacZ expression, but wefailed to detect β-gal in the periodontal ligament, dentin, odontoblastsand dental pulp. The β-gal-positive HERS was continuous with nointerruptions. At PN 10.5, we detected a dissociation of the HERS (Figs.1D and K). Subsequently, the root continued development and theHERS dissociated further. These β-gal-positive cells remained on thesurface of the root until PN 60.5 (Figs. 1E–G and L–O). In contrast, wedetected no blue cells on the root surface in the wild type (Figs. 1P–S).As a control, we examined β-gal expression in Wnt1-Cre;R26R miceand found that, as expected, dental epithelial cells were β-gal-negative and most cells derived from the cranial neural crest inperiodontal ligament (PDL) and dental mesenchyme were β-gal-positive (Figs. 1T–Y). Thus, β-gal-positive cells derived from dentalepithelium remain on the surface of the root from the beginning ofroot formation to adulthood in K14-Cre;R26R mice.

HERS cells contribute to acellular cementogenesis

In adults, acellular cementum covers the cervical two thirds ormore of the root. During root development and cementogenesis, wefound that β-gal-positive cells remained on the surface of the root,although the HERSwas dissociated after PN10.5 (Figs. 2A and B). At PN13.5, 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 cementummatrix between the β-gal-positive cells and the outer surface ofdentin (Figs. 2C and E). In Wnt1-Cre;R26R mice not all PDL cells

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), andWnt1-Cre;R26R (R–W)mice. (A–O) At PN 0.5, all the epithelial cells in K14-Cre;R26R teeth areβ-gal-positive (A andH). At PN 3.5, cells from the ameloblast layer and theouter 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 apicaldirection 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 dissociatedfurther 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 thecranial 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, Scalebars (H–Y)=40 μm.

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Fig. 2.HERS cells contribute to acellular cementogenesis. X-gal (A, C, E, and F–Y) andMollary-H (B, D) staining of tooth root sections fromK14-Cre;R26R (A–E, K–Y) andWnt1-Cre;R26R(F–J) mice; X-gal staining of dissected teeth from PN 13.5 K14-Cre;R26Rmice (Q–T) and TEM of PN 13.5 K14-Cre;R26Rmice (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 aredetected on the surface of the root (C, E, K–P). Pre-cementum, or cementummatrix is detectable between the β-gal-positive cells and the outer surface of dentin (C, E, K–P). K–Mshowdifferent 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 indifferent 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 inWnt1-Cre;R26Rsamples (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, theHERS 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 betweenHERS 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, Band 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.

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derived from CNC contacted the dentin directly, but β-gal-negativecells derived from dental epithelium were still located on the root atPN 13.5 (Figs. 2F–J). In PN13.5 K14-Cre;R26R mice, cementum matrixcould be clearly detected between the epithelium derived cells andthe dentin in the same section but different focal plane (Figs. 2K–M).We also used TEM to observe the ultrastructural morphology of theroot surface in PN13.5 K14-Cre;R26R mice. The cytoplasma of dentalepithelial cells (Figs. 2V–Y, indicated by asterisks) is identifiable bydark LacZ staining (Fig. 2W, indicated by white arrowheads). Twolayers of continuous HERS cells (Figs. 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, extracellularmatrix (Fig. 2Y, indicated by black arrows) is detectable between thedentin surface (Fig. 2Y, indicated by white arrows in line) and HERScells (Fig. 2Y, indicated by asterisk). Ectomesenchymal cells from thedental follicle penetrated into the HERS, attached on the surface of thedentin (Figs. 2C, E–M), and formed extracellular matrix. Some dentalfollicle cells were slender, similar to fibroblasts. Fibers were alsoformed at this stage (Figs. 2E and J). Therefore, during cementogen-esis, both HERS cells and dental follicle cells could form cementum-

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like tissue on the surface of the root. We also observed a networkstructure of HERS cells in the periphery of the tooth root surface at PN13.5 using section and whole mount LacZ staining (Figs. 2P–T). Dentalfollicle cells could pass through this network structure to contact thesurface of the root. We conclude that the geometrical intricacy of thisrelationship likely explains why the distribution of HERS did notappear the same in different sections of the samples at PN 13.5 (Figs.2N and O). In fact, the HERS was not interrupted and the HERS cellsmay still communicatewith each other through the network structure(Fig. 2P).

HERS cells may participate in the formation of cellular cementum

Cellular cementum is often absent from single-rooted teeth andconfined to the apical third and inter-radicular regions of molars. Inthis study, we found that β-gal-positive cells participated in cellularcementogenesis. In the apical area of the molar root, we detected β-gal-positive cells at PN 60.5 (Figs. 3A–H). Blue cells were embeddedinto the cellular cementum and may have differentiated intocementoblasts (Figs. 3B–H, indicated by arrows). Dental follicle cellscould also be found as cementoblasts and produce cellular cementumin the apical part of root as well (Fig. 3, indicated by arrowheads). Inthe inter-radicular regions of the molars, most β-gal-positive cellswere on the surface of the root; a few of themwere embedded into thecementum during cementogenesis (Figs. 3I–P). Most of the β-gal-positive cells were located on the surface of the furcation, and we

Fig. 3. HERS cells participate in the formation of cellular cementum. X-gal staining of apical(A–H) In the apical region of the molar root, β-gal-positive cells are detectable until at leastdifferentiated into cementocytes (B–H, indicated by arrows). β-gal-negative mesenchymal c(I–P) In the inter-radicular regions of the molars, most β-gal-positive cells are detectable on(N, indicated by arrow). Dental mesenchymal cells are detectable that penetrate into the Halveolar bone, CC: cellular cementum, D: dentin, DP: dental pulp, PDL: periodontal ligamen

detected no β-gal-positive cells on the surface of the cellularcementum in the apical area of the root edge. Therefore, the formationof cellular cementum in the apical root and furcation areas may bedifferent. Our results suggest that there are three kinds of cemento-genesis: acellular cementogenesis, apical cellular cementogenesis, andinter-radicular cementogenesis. During acellular cementum forma-tion, the HERS cells were always on the surface of the root and fewwere embedded into the cementum. In the apical area of the root allthe HERS cells were embedded into cellular cementum and no β-gal-positive cells were detected on the surface. In the inter-radicularregions, some HERS cells were embedded into the cellular cementumand others remained on the surface of the root (Fig. 4). Thus, HERScells participate in the formation of cellular cementum and maydifferentiate into cementoblasts in vivo.

K14, Bsp and type I collagen expression in HERS

In order to confirm that HERS cells can differentiate intocementoblasts and cementocytes, we examined the expression ofK14, β-gal and cementoblast markers, such as Bsp and type I collagenin K14-Cre;R26R mice. Keratin is expressed in dental epithelial cellsduring root development (Luan et al., 2006, Azumi and Hiroaki, 2006;Tummers et al., 2007). Comparing the expression of K14 and β-gal inK14-Cre;R26R mice, we found that the expression patterns of K14 andβ-gal were indistinguishable before PN 7.5. After PN 7.5, the number ofLacZ positive cells was greater than that of K14 positive cells, especially

(A–H) and inter-radicular (I–P) tooth root sections from post-natal K14-Cre;R26R mice.PN 60.5. HERS cells appear to be embedded into the cellular cementum and may haveells are detectable as cementoblasts and cementocytes (C–H, indicated by arrowheads).the surface of the root; a few are embedded into the cementum during cementogenesisERS and have attached onto the surface of the dentin (N, indicated by arrowhead). AB:t. Scale bars (A–H, and M–P)=20 μm, scale bars (I–L)=40 μm.

Fig. 4.Multiple forms of cementum. X-gal staining of tooth root sections from K14-Cre;R26Rmice. (A) Cellular cementum in the apical region of the root at PN 30.0. All the HERS cellsare 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 thesurface of the root and feware 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.

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at PN 21.5 (Figs. 5A–H and Figs. 6A–H). At PN 21.5, we detected verylittle K14 expression on the surface of the root, andmost cells adjacentto the surface of the root did not express K14 during root development(Figs. 6E–H). We only detected K14 positive cells in the epithelial restof Malassez. In contrast, we detected many β-gal-positive cells in theroot at the same stage. Because the progeny of epithelial cells aretraced by LacZ staining, our data suggests that HERS cells are derivedfrom dental epithelium, but some of them do not express theepithelial marker K14 during later stage of root development.

Type I collagen and Bsp are considered markers for cementoblasts.Gene expression patterns of type I collagen and Bsp have been

Fig. 5. K14, Bsp and type I collagen expression in HERS. (A–H) Immunohistochemical staininpost-natal K14-Cre;R26Rmice. The expression of K14 and β-gal overlap significantly at PN 3.5cells (B–D, F–K). (I–Q) X-gal staining (I–K), in situ hybridization of type I collagen (L–N), andare all detectable on the surface of the root at PN 13.5 (I–Q). AB: alveolar bone, AC: acellular ceScale bars (A–H)=40 μm, scale bars (I, L, and O)=80 μm, scale bars (J, K, M, N, P, and Q)=

described in murine cementoblasts using in situ hybridization(MacNeil et al., 1996; Sommer et al., 1996; D'Errico et al., 1997). Weexamined the expression of type I collagen and Bsp in K14-Cre;R26Rmice and compared them with the expression of K14 and β-gal. Wedetected expression of type I collagen in odontoblasts, PDL, andcementoblasts (Figs. 5L–M). Bsp was expressed strongly in cemento-blasts, pre-odontoblasts, and osteoblasts (Figs. 5O–Q and Figs. 6I–L).Comparing the percentage of β-gal-positive cells and Bsp positive cellsin different areas of the surface of the root at PN 21.5, we found thatmore than 37.62% of the cells on the furcationwere β-gal-positive andmore than 87.67% of the cells expressed Bsp. This data suggests that at

g for K14 (A–D) and X-gal staining (E–H) of tooth root sections from different stages of(A, E). After PN 7.5, the number of LacZ positive cells is greater than that of K14 positivein situ hybridization of Bsp of PN13.5 K14-Cre;R26R mice. β-gal, Bsp and type I collagenmentum, CC: cellular cementum, D: dentin, DP: dental pulp, PDL: periodontal ligament.20 μm.

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 ofthe 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, scalebars (B, F, J)=80 μm, scale bars (C, D, G, H, K, and L)=40 μm.

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least 25% of the β-gal-positive cells expressed Bsp, which indicatesthat some of the cells derived from dental epithelium express Bsp, themarker for mesenchymal cells and cementoblasts. Thus, these resultslend support to the conclusion that HERS cells may participate incementogenesis.

ALPase expression in HERS

Traditional methods such as ALPase detection for calcium depositshave been used to identify cells with osteogenic potential. Previousstudies have reported high ALPase activity in alveolar bone andcementum (Groeneveld et al., 1995). The function of ALP is related tomineralization. In order to confirm that HERS cells can differentiateinto cementoblasts and participate in mineralization, we performeddouble staining of ALPase and LacZ. ALPase could be detected inameloblasts but not in HERS at PN 3.5, and 13.5 (Figs. 7A, B, E, F).ALPase and LacZ could both be detected in the cells on the lateralsurface, furcation and apical area of the root at the different stages,including PN 13.5, and 21.5, (Figs. 7C, D, G–J, L). For the double stainingprocess, we first documented the X-gal staining (Fig. 7K), thenperformed the ALPase assay (Fig. 7L). β-gal-positive cells clearly alsoexpress ALPase. These results further support the conclusion thatHERS cells may participate in mineralization and cementogenesis.

Discussion

Using the two-component Cre/loxp strategy (Chai et al., 2000), wehave generated K14-Cre;R26R mice to follow HERS cells during toothroot development. The progeny of dental epithelial cells can betracked using LacZ staining in these mice. Although Keratin has beenused as amarker for dental epithelium, we have found thatmost of thecells adjacent to the root did not express K14 during root developmentat PN 21.5. To date, the patterning and function of HERS has remainedunknown (Diekwisch, 2001; Luan et al., 2006), although HERS isclearly important during root formation. The canonical theory of rootdevelopment suggests that mesenchymal cells of the dental folliclebecome cementoblasts and secrete cementum after they transitthrough the barrier of the HERS (Paynter and Pudy, 1958; Lester,1969; Chai et al., 2000; Diekwisch, 2001). Previously, the widelyaccepted theory of root development and cementogenesis suggeststhat cementum is a dental follicle-derived connective tissue that isformed subsequent to HERS disintegration (Diekwisch, 2001). Thistheory was developed from morphological observations thatmesenchymal cells from the dental follicle penetrate the HERS bi-layer and deposit an initial cementummatrix, although HERS cells areseparated 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

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). ALPaseand 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 byarrows). 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 (asindicated 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.

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from the beginning of root formation to PN 60.5. Using TEM, some pre-cementum or cementummatrix is present between the epithelial cellsand the outer surface of dentin at PN 13.5 when acellular cementum isformed. Moreover, we determined that HERS cells might havedifferentiated into cementocytes in cellular cementum based on ourfinding that some of the cementocytes are β-gal-positive and areembedded in the cellular cementum. In addition, HERS cells expresstype I collagen, Bsp, and ALPase, consistent with our hypothesis thatdental epithelial cells may differentiate into cementocytes. CNC-derived-mesenchymal cells from the dental follicle are critical for rootformation. They penetrate into the HERS, attach to the surface of thedentin, and form the extracellular matrix. Interestingly, HERS ispartially dissociated, but still maintains a network of connectionsduring root development. The HERS epithelial network provides spacefor penetration by the dental follicle cells, which are then able tocontact the surface of the root. Some of the cells of the dental follicledifferentiate into slender fibroblasts, which secrete collagen and otherproteins to form fibers. Our data suggests that both HERS cells anddental follicle cells form cementum-like tissue. The fibroblasts derivedfrom dental follicle cells form the fibers (also known as Sharpey'sfibers) that are embedded in the developing cementum. In cellularcementum, which can be found in the apical and inter-radicularregions of the tooth, the majority of the cementocytes in the apicalarea of the root is derived from the dental follicle. We detected noHERS cells on the surface of the apical cellular cementum.

Our results suggest that the two regions of cellular cementum (theapical area of the root and the furcation) are different. The HERS in theinter-radicular region of the first molar is disassociated after PN 10.5.However, unlike the HERS cells on the surface of the acellularcementum, the epithelial cells in the inter-radicular region are stillactive until PN 60.5. Some of the HERS cells in the inter-radicularregion are embedded in the cellular cementum and may becomecementoblasts, but others remained on the root surface. The layer of

the cellular cementum in the molar furcation is not as thick as in theapical region. Taken together, these observations indicate that themechanism of cellular cementogenesis is likely different in the apicalroot region versus the furcation. The mechanism of cementogenesis inthe furcation is similar to acellular cementogenesis. Most HERS cellsare active in the furcation and some of them are embedded incementum to become cementocytes. However, cellular cementogen-esis in the apical region of the root is similar to osteogenesis. Mostcementocytes and almost all of the cementoblasts on the surface ofthe cellular cementum are β-gal-negative cells. This indicates thatthese cementocytes and cementoblasts in the apical root are notprimarily derived from HERS, but from dental follicle cells.

HERS provides a structural boundary between two mesenchymalstructures, the dental follicle and the dental papilla, suggesting that itmay play a critical function in tissue–tissue interactions. Previousstudies suggested that HERS cells expressed not only epithelialmolecules such as cytokeratin, E-cadherin, ameloblastin, but alsomesenchymal molecules such as Bsp, vimentin, and N-cadherin (Fonget al., 1996; Fong and Hammarström, 2000; Zeichner-David et al.,2003; Yamamoto et al., 2004; Sonoyama et al., 2007). Previous geneexpression studies during mouse molar root development havesuggested that some growth factors, including bone morphogeneticproteins (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 ofodontoblasts and/or cementoblasts and in the mineralization ofdentin and/or cementum (Nakatomi et al., 2006, Yamashiro et al.,2002, Yamashiro et al., 2003). The transcription factor, Nfic, is essentialfor root development, because the root fails to form in Nfic−/− mice(Steele-Perkins et al., 2003). Nevertheless, the mechanism of interac-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 andSharpe, 1997; Miletich and Sharpe, 2003). There are many intriguingquestions that remain to be answered: (1) How is the HERS formed?(2) How are odontoblasts induced to differentiate during root dentinformation? Is HERS the inducer? (3) Do infiltrating dental follicle cellsreceive reciprocal signal from the dentin or the surrounding HERScells? (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 thedifference between acellular and cellular cementum? (8) Whatdetermines the number of roots formed?

In this study, we have found that HERS cells remain on the surfaceof the root throughout root formation and can differentiate intocementoblasts and cementocytes to participate in cementogenesis.We also detected a network structure of HERS that suggests the HERScells may be a signal center of interaction between the dentalepithelium and mesenchyme during root elongation, odontogenesis,and cementogenesis. Future studies will help to elucidate the processof root development, the interaction between epithelial andmesench-ymal cells, and the signaling pathway in this process. This knowledgemay eventually lead to our ability to promote tooth or rootregeneration with obvious clinical applications.

Acknowledgments

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

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