Archives of Oral Biology (2005) 50, 131—136

www.intl.elsevierhealth.com/journals/arob

Dental epithelial histo-morphogenesis in themouse: positional information versus cell history

Bing Hua,b, Amal Nadiria, Sabine Bopp-Kuchlera,Fabienne Perrin-Schmittc, Songlin Wangb, Herve Lesota,*

aINSERM U595, Faculty of Medicine, 11, rue Humann, 67085 Strasbourg Cedex, FrancebFaculty of Stomatology, Capital University of Medical Sciences, Beijing, PR ChinacLGME, CNRS-ULP UMR 7104, INSERM U596, Faculty of Medicine, Strasbourg, France

Accepted 17 September 2004

KEYWORDSEnamel organ;Epithelial histogenesis;Positional information;Enamel knot;Odontogenesis

Abbreviations: BrdU, 5-bromo-2-deoxyuridine; IDE, innerdental epithelium; ODE, outer dental epithelium; PEK, primaryenamel knot; SR, stellate reticulum

* Corresponding author. Tel.: +33 3 90 24 33 78/31 11;fax: +33 3 90 24 35 64.

E-mail address: Herve.Lesot@odonto-ulp.u-strasbg.fr(H. Lesot).

0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights resedoi:10.1016/j.archoralbio.2004.09.007

Introduction

Reciprocal epithelial—mesenchymal interactionscontrol all steps of odontogenesis. Crown morpho-

Summary Reciprocal epithelial—mesenchymal interactions control odontogenesisand the cap stage tooth germmesenchyme specifies crownmorphogenesis. The aim ofthis work was to determine whether this mesenchyme could also control epithelialhistogenesis. Dental mesenchyme and enamel organ were dissociated from mousefirst lower molars at E14. At this early cap stage, the enamel organ consists of four celltypes forming the inner dental epithelium (IDE), primary enamel knot (PEK), outerdental epithelium (ODE) and the stellate reticulum (SR). Pelleted trypsin-dissociatedsingle dental epithelial cells, which had lost all positional information, were reasso-ciated to either dental mesenchyme or dissociated mesenchymal cells and cultured invitro. Although with different timings, teeth developed in both types of experimentsshowing a characteristic dental epithelial histogenesis, cusp formation, and thedifferentiation of functional odontoblasts and ameloblasts. The rapid progressionof the initial steps of histogenesis suggested that the cell history was not memorized.The dental mesenchyme, as well as dissociated mesenchymal cells, induced theformation of a PEK indicating that no specific organisation in the mesenchyme isrequired for this step. However, the proportion of well-formed multicusped teeth wasmuch higher when intact mesenchyme was used instead of dissociated mesenchymalcells. The mesenchymal cell dissociation had consequences for the functionality ofthe newly-formed PEK.# 2004 Elsevier Ltd. All rights reserved.

rved.

132 B. Hu et al.

genesis (i.e. cusp number, size and position) isspecific for each molar in the upper and lower jaws.Tissue reassociation experiments have shown thatcrown morphogenesis is controlled by the dentalmesenchyme.1,2

Epithelial histogenesis is already initiated atthe bud stage since two cell types can be distin-guished morphologically. Cells in contact withthe basement membrane are elongated, whileinternal epithelial cells are small and round.Positional information might thus be involved inspecifying the cell shape and cell fate as illustratedfor other models.3—6 At the early cap stage, epithe-lial histogenesis becomes much more complexwhen the inner dental epithelium (IDE) and outerdental epithelium (ODE), the stellate reticulum(SR) and the primary enamel knot (PEK) becomedistinct. Themolecular mechanisms, which controldental epithelial histogenesis, involve cell—celland cell—matrix interactions as well as diffusiblesignaling molecules.7—15 The aim of the presentwork was to determine whether the mesenchymecould control epithelial histogenesis. For this pur-pose, the dental mesenchyme from the cap stage(E14) was reassociated with pelleted single dentalepithelial cells, which had lost all positional infor-mation, and cultured in vitro. In complementaryexperiments, dissociated single dental mesenchy-mal cells were pelleted and reassociated withepithelial cells to test whether the potentialitiesof the mesenchyme required any specific tissueorganisation.

Figure 1 Schematic representation

Materials and methods

ICR mice were mated overnight and the detection ofthe vaginal plug was determined as Embryonic day(E)0. The first lower molars (n = 2420) were dis-sected from embryos at E14 under a stereomicro-scope (Leica MZ9).

Tissue dissociation

The dental epithelium and mesenchyme weredissociated by using 1% trypsin in Hanks’ at 4 8C.2

After separation of the two tissues, the dentalepithelium, and sometimes mesenchyme, weredissociated into cells by sequentially using fineneedle and filtered through 70 mm nylon filters.Epithelial cells were then pelleted by centrifugationat 9000 � g, the pellets were cut into fragments,reassociated with either intact mesenchyme orpelleted mesenchymal cells and cultured (Fig. 1).

Cultures

The reassociations were cultured for up to 12 dayson a semi-solid medium, which consisted of DMEM/F-12 (Gibco) containing 20% fetal bovine serum(CAMBREX), and supplemented with ascorbic acid(0.18 mg/ml, Merck), L-glutamine (2 mM, Gibco),penicilline/streptomycine (50 units/ml, Gibco)and agar (0.36%, Sigma). Cultures were performedat 37 8C in a humidified atmosphere of 5% CO2. Themedium was changed every 2 days. In this work, 234

of the experimental procedures.

Epithelial cell positional information 133

Figure 2 In vitro development of reassociations from first lower molars at ED14: (A—R) reassociations between dentalmesenchyme and dissociated epithelial cells; (S—AJ) reassociations between dissociated dental mesenchymal cells andepithelial cells. The reassociations were cultured for 12 h (A, J, S, AB); 24 h (B, K, T, AC); 2 days (C, L, U, AD); 3 days (D, M, V,AE); 4 days (E, N, W, AF); 6 days (F, O, X, AG); 8 days (G, P, Y, AH); 10 days (H, Q, Z, AI) and 12 days (I, R, AA, AJ). AM:ameloblast; BM: basement membrane; D: Dentin; DE: dental epithelium; DM: dental mesenchyme; DP: dental papilla; IDE:inner dental epithelium; OD: odontoblast. ODE: outer dental epithelium; PEK: primary enamel knot; SEK: secondary enamelknot; SI: stratum intermedium; SR: stellate reticulum. Bar = 40 mm.

134 B. Hu et al.

reassociations were performed between mesench-yme and dissociated epithelial cells and 56 betweenepithelial and mesenchymal cells.

Histology

All samples were fixed in Bouin—Hollande,embedded in paraffin, and serial sections (5 mm)were stained with Mallory’s stain.

Results

Mouse first lower molars were trypsin-dissociated inorder to separate the mesenchyme from the epithe-lium at E14, when the PEK was formed andexpressed transcripts for signaling molecules.16

Reassociations between the dental mesenchymeand dissociated single epithelial cells were culturedin vitro (Fig. 1). Complementary experiments werealso performed by reassociating pelleted disso-ciated mesenchymal cells and pelleted dissociatedepithelial cells (Fig. 1). In both experimentalgroups, the epithelial histo-morphogenesis was suc-cessfully achieved. The epithelial—mesenchymaljunction was restored after 12 h in tissue—cell reas-sociations despite an initial high frequency of celldeath, especially in the internal epithelial cells (Fig.2A and J). During the first 12 h, the deposition of anew basement membrane progressed more slowly incell—cell reassociations (compare Fig. 2S and ABwith Fig. 2A and J). Tissue—cell reassociationreached the bud stage after 24 h (Fig. 2B and K),instead of 48 h for cell—cell reassociations (Fig. 2Uand AD). At this stage, the epithelial cells in contactwith the basement membrane started to elongate. A24 h delay was observed for cell—cell reassociationsto reach the early cap stage (Fig. 2V and AE) whencompared to tissue—cell reassociations (Fig. 2C andL). A clear epithelial histogenesis, with a stellatereticulum intercalated between the inner and outerdental epithelium was achieved after 3 days fortissue—cell reassociations (Fig. 2D and M) insteadof 4 days for cell—cell reassociations (Fig. 2W andAF). After 4 days, the PEK were still visible in cell—cell reassociations (Fig. 2AF) while secondaryenamel knots (SEKs) were formed in tissue—cellreassociations (Fig. 2E and N). Cusp formationoccurred in both types of reassociations, but againwith a delay in cell—cell reassociations (compareFig. 2F and O with Fig. 2Y and AH). Odontoblastdifferentiation was initiated at the tip of the cuspsand progressed, forming gradients in both types ofcultures (Fig. 2G, P, Z and AI) and then ameloblastdifferentiation was initiated with the same pattern(Fig. 2H, Q, I, R, AA and AJ). In terms of cell

differentiation, cell—cell reassociations were alsoretarded compared to tissue—cell reassociations.

Discussion

When epithelial cells were dissociated, the enamelorgan already comprised four distinct cell popula-tions (IDE, PEK, ODE and SR). Their specific fate maybe determined by positional information,3—5 whichcan be specified by differential cell—cell interac-tions,8,9,15,17 cell—matrix interactions7,10—12,14,18

and signaling molecules.13,19,20 After trypsin treat-ment, the breakdown of the basement membraneand cell-surface molecules resulted in the loss ofpositional information. For each experiment, tissuesfrom about 60 teeth were used. The dissociatedepithelial cells were mixed together and pelletedby centrifugation. In a first set of experiments,fragments of the pellet were reassociated to dentalmesenchyme and cultured. During the first 12 h inculture, an important cell death, probably a con-sequence of trypsin treatment, took place whilesurviving cells deposited a new basementmembraneat the epithelial—mesenchymal junction. After24 h, most of the dead cells had disappeared andthe epithelial cells in contact with the newly depos-ited basement membrane started to elongate, a firstsign of histogenesis. This was similar to thatobserved at the bud stage in vivo.8 Cell elongationwas found to be slightly faster when using tissuesfrom E13 instead of E14 (data not shown). Althoughwith different timings, the same was observed whenepithelial cells were cultured in contact with eithera dental mesenchyme or dental mesenchymal cells.The cap stage including the formation of a PEK wasachieved after 2 days when epithelial cells werereassociated to a dental mesenchyme, instead of 3days when they were reassociated with mesenchy-mal cells. The PEK was characterized by (1) itsspecific cell arrangement, (2) the absence of BrdUincorporation, (3) the expression of Shh as observedby in situ hybridisation, (4) a positive staining forWNT5a and Frizzled receptor and the absence ofWNT10b, as observed in cap stagemolars.20 This PEKwas functional since cusps formed in both types ofexperiments. The two types of reassociations led toteeth with functional odontoblasts and polarizedameloblasts.

Epithelial cells showed a remarkable plasticity.They rapidly adjusted to their new environment andrestored the histological characteristics of IDE, ODE,PEK and SR cells. During the first 12 h, differentprocesses might take place: epithelial cell selectionsince cell death was important, segregation21 pos-sibly mediated by cell migration as reported in the

Epithelial cell positional information 135

dental papilla during reparative processes22 orcell reprogramming. A detailed study of the re-expression of surface molecules will have to beperformed during the initial stages of the cultureto follow cadherins,8,15,23 integrins,7,14 andchanges in the basement membrane compositionduring the bud to cap transition, as investigatedduring tooth development in vivo. The fast pro-gression in the initial steps of epithelial histogen-esis in the reassociations suggested that the cellhistory4 was not memorized. New positional infor-mationwas acquired at the bud stage, since epithe-lial cells adapted their morphology to being eitherin contact with a basement membrane or not, andthen changing again at the cap stage when beinglocalized in the region of either the IDE or ODE.This latter change is controlled by themesenchymeas shown by heterotopic tissue reassociationexperiments,24 suggesting that regional specifici-ties might exist in the mesenchyme (dental versusperidental). It was shown that the changes inthe basement membrane composition during thebud to cap transition, depending on whether it isreassociated to the ODE or IDE, might be a conse-quence ofMMPand TIMPexpression in themesench-yme.11 The dental mesenchyme, as well asmesenchymal cells from E14, induced the forma-tion of a PEK indicating that no specific organisationin the mesenchyme is required for this step.Furthermore, mesenchyme from E13 had thesame potentialities to support tooth development(not shown) showing that a specification by signal-ing molecules from the PEK is not required. How-ever, the proportion of multicusped teeth wasmuch higher when epithelial cells were reasso-ciated with intact mesenchymes instead ofmesenchymal cells, suggesting that the dissocia-tion of the mesenchyme had consequences on thefunctionality of the PEK. Further investigation atthe molecular level will have to be performed tocompare the PEK, which forms in the two types ofreassociations.

Acknowledgement

The authors wish to thank Mr. A. Ackermann forhistology.

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