Irma Thesleff

Marika Suomalainen

Xiu-Ping Wang

Comptes Rendus Biologies (CRBIOL)

C. R. Biologies 330 (2007) 561–564http://france.elsevier.com/direct/CRASS3/Medical sciences / Sciences médicalesRegulation of epithelial stem cells in tooth regenerationIrma Thesleff a,∗, Xiu-Ping Wang a,b, Marika Suomalainen aa Developmental Biology Program, Institute of Biotechnology, P.O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finlandb Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur,Boston, MA 02115, USAReceived 5 January 2007; accepted after revision 20 March 2007Available online 9 May 2007Presented by Jean-François Bach and Nicole Le DouarinAbstractTeeth form as epithelial appendages and the mechanisms regulating their development share similarities with other organs such ashairs, glands, and gut. However, the regenerative potential of mammalian teeth is generally limited. Stem cells have been identifiedin the epithelium of continuously growing incisors of mice. We have identified a network of signalling molecules that regulatesthe proliferation and differentiation of these stem cells, and that thereby influences the incisors’ growth and enamel formation. Thesignals, including FGFs, BMPs, and Activin, mediate interactions between the mesenchymal and epithelial cells within the stemcell niche and form an integrated network. Follistatin antagonizes the functions of BMPs and Activin, and is a key regulator of theasymmetry of the incisor structure. The evolutionary variation in the growth capacity of teeth and the extent of enamel depositionmay have resulted from fine-tuning of this signal network. In addition, subtle variations in this or in related regulatory networksmay explain the different regenerative capacities of various organs and animal species. To cite this article: I. Thesleff et al., C. R.Biologies 330 (2007).© 2007 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.Keywords: Dental stem cells; Incisors; Enamel; Activin; BMP; FGF; Stem cell nicheEpithelial stem cells are responsible for the continu-ous regeneration of tissues such as hair, skin, and gut.The stem cells reside in specific niches that regulate theself-renewal and differentiation of the adult stem cells[1–3]. The regenerative potential of mammalian teethis generally limited, and in humans as well as in mostother mammals teeth are replaced only once and theirgrowth arrests after the eruption has completed. How-ever, some mammals have continuously growing teeth.Rodents are characterized by sharp incisors that grow* Corresponding author.E-mail address: irma.thesleff@helsinki.fi (I. Thesleff).1631-0691/$ – see front matter © 2007 Académie des sciences. Published bdoi:10.1016/j.crvi.2007.03.005throughout life. A stem cell niche has been identified inmouse incisors at their proximal ends in the so-calledcervical loops [4,5 (Fig. 1)]. The evidence includes datafrom DiI labelling studies, studies on cell cycle kineticsand localization of long-term BrdU label retaining cells[4,6]. This stem cell niche bears anatomical similaritiesto the niches in other epithelial organs, in particular inhairs and intestine.Compared to the niches in other organs, the in-cisor stem cell niche has a powerful property, whichwe have exploited in our studies: the stem cell com-partments on the different sides of the tooth, namelythe labial (anterior) and lingual (tongue-side) cervicalloops vary greatly in size and proliferative capacity.y Elsevier Masson SAS. All rights reserved.562 I. Thesleff et al. / C. R. Biologies 330 (2007) 561–564Fig. 1. Schematic pictures of mouse incisor. (A) The incisor grows continuously from the proximal end towards the apical end, where the tooth isworn down as the asymmetry of enamel coverage maintains the cutting edges of the teeth sharp. (B) Detailed picture from the boxed region in (A).Epithelial stem cells are located in the stellate reticulum (SR) compartment. They proliferate, migrate apically, and differentiate into ameloblaststhat deposit enamel matrix. Dental epithelium (DE), papilla mesenchyme (PM), follicle mesenchyme (FM), inner enamel epithelium (IEE), outerenamel epithelium (OEE), pre-ameloblasts (PA), transient amplifying cells (TA cells).This contributes to the characteristic asymmetric growthand enamel distribution, which together maintain thesharpness of the continuously growing incisor. In thelabial cervical loop, the epithelial stem cells prolifer-ate actively and migrate along the labial surface, differ-entiating into enamel-forming ameloblasts. In contrast,the lingual cervical loop contains fewer proliferatingstem cells, and the lingual surface of the incisor lacksameloblasts and enamel (Fig. 1). Comparisons of thestem cell niches in labial and lingual cervical loops al-lowed us to identify components of the genetic networksregulating the proliferation and differentiation of epithe-lial stem cells.We have shown that epithelial stem cells’ prolifera-tion in the cervical loops is controlled by signals fromthe adjacent mesenchyme. The first mesenchymal sig-nals that were associated with epithelial-cell divisionwere FGF3 and FGF10 [4]. The FGF pathway waslinked with Notch signalling in organ culture experi-ments, in which the dissected cervical loops of mouseembryos were exposed to beads releasing the FGF10 re-combinant protein. The expression of the Notch signalmodulator Lunatic Fringe was markedly upregulated inthe epithelium [4]. Lunatic Fringe is intensely expressedin the basal epithelium of the cervical loop, whereasNotch1 is expressed in the stellate reticulum cell com-partment, in the centre of the cervical loop epithelium.All current evidence supports the idea that the epithe-lial stem cells are located in the Notch1-positive stellatereticulum compartment (Fig. 1).The analysis of the incisor phenotypes in several mu-tant mouse lines has revealed a complex integrated sig-nal network regulating stem cell proliferation (Fig. 3).This network consists of FGFs, BMPs, Activin, andFollistatin, which are all locally expressed within the in-cisor stem cell niche. Fgf3 is asymmetrically expressedand restricted only to the mesenchyme underlying thelabial cervical loop. Activin βA is also asymmetricallyexpressed in the dental mesenchyme with more in-tense signals at the labial aspect than the lingual aspect,whereas Bmp4 and Fgf10 are expressed in the mes-enchyme on both labial and lingual sides with similarintensities. Follistatin, on the other hand, is mainly ex-pressed in the epithelium and restricted to the lingualside [7–9]. The Fgf10 null mutants die at birth, andthe culture of their incisors in organ cultures showedthat the cervical loops were not maintained in the mu-tant teeth [6]. Comparisons of the incisor phenotypes ofFgf3 null mutant mice and the Fgf3;Fgf10 compoundmutants indicated that Fgf3 and Fgf10 interact to main-tain the stem cell pool that provides the continuous sup-ply of ameloblast precursors [7,8].Two mutant mice lines, transgenic mice overexpress-ing Follistatin in the epithelium (under the keratin14promoter), and Follistatin knockout mice have been in-formative in the studies on incisor stem cell prolifera-tion and differentiation [8,9]. Follistatin is a well-knownantagonist of TGFβ signalling, and it inhibits partic-ularly the functions of Activin and BMPs [10]. In theFollistatin overexpressing mice, incisor growth was in-hibited and no enamel formed. The labial cervical loop,was hypoplastic, resembling the lingual cervical loop,and ameloblasts failed to differentiate. In the Follista-tin knockout mice, which die at birth, the analysis ofthe embryonic incisors indicated that the growth of thelingual cervical loop was stimulated and it began to re-semble the labial cervical loop. The stellate reticulumcompartment was large and the entire cervical loop ep-ithelium showed increased BrdU incorporation. In addi-tion, ameloblasts differentiated also on the lingual sideI. Thesleff et al. / C. R. Biologies 330 (2007) 561–564 563Fig. 2. BMP4 induces differentiation of the dental epithelial cells into ameloblasts. Beads releasing the indicated proteins were placed on in-cisor explants and the expression of the enamel protein Ameloblastin was analysed after 24 h. (A) Some endogenous Ameloblastin expression isseen in the incisor explants (BSA (bovine serum albumin) control bead). (B) The BMP4 bead has induced intense expression of Ameloblastin.(C) Ameloblastin expression is downregulated by the BMP inhibitor Noggin.and started to deposit enamel matrix. Fgf3 expressionin the mesenchyme correlated with proliferation of thenearby epithelium. Fgf3 transcripts were absent in theK14-Follistatin incisors, whereas they were ectopicallyexpressed in labial cervical loop mesenchyme in theFollistatin knockout incisors. Hence, the asymmetry ofthe incisor was lost in both Follistatin gain-of-functionand loss-of-function mice, indicating an important rolefor Follistatin in the regulation of incisor asymmetry.Organ culture studies on the effects of the varioussignals in isolated cervical loop tissues further clarifiedthe functions of the different signal pathways and theirintegrations. Beads releasing BMP4 induced the differ-entiation of the epithelial cells into ameloblasts (Fig. 2).These and other observations indicated that Follistatinacts as a BMP antagonist during the differentiation stagein the lingual dental epithelium and prevents enamel for-mation, thereby contributing to the characteristic asym-metry of the incisor [9].Interestingly, at the proliferation stage, Follistatinwas shown to antagonize Activin in the lingual epithe-lium, where it limited the number of lingual stem cellsand contributed to the asymmetric growth of the in-cisor. Activin was shown to be a major stimulator ofepithelial stem cell proliferation and, as it was more in-tensely expressed on the labial than on the lingual sidedental mesenchyme, this conceivably also contributedto the asymmetric growth of the incisor. In vitro ex-periments where BMP4 releasing beads were insertedon cervical loop tissue indicated that BMP4 dramati-cally inhibited the expression of Fgf3, whereas Activin-releasing beads induced ectopic expression of Fgf3 inthe lingual mesenchyme. When Activin-releasing beadstogether with BMP4 beads were inserted on the cervicalloop explants, the repressive effect of BMP4 on Fgf3 ex-pression was inhibited. Finally, when the cervical loopexplants were grown for four days in the presence ofActivin in the culture medium, a dramatic stimulation ofFig. 3. Schematic picture of the signal network regulating epithelialstem cell proliferation and differentiation in the incisor. BMP4 in-duces differentiation of ameloblasts, but inhibits proliferation of thestem cells by negatively regulating the expression of Fgf3. Activincounteracts this effect and stimulates Fgf3 expression and stem cellproliferation. Labial-lingual asymmetry is established in the incisor byhigher Activin expression labially and by lingual expression of Follis-tatin, which acts by inhibiting Activin function in the stem cells andBMP function in the differentiating ameloblasts, respectively.epithelial proliferation was seen, and the loops on bothlabial and lingual sides had developed extra buds. It wasconcluded that Activin counteracts the inhibitory effectof BMP4 on Fgf3 expression and thereby restricts Fgf3expression to labial dental mesenchyme, and that this re-sults in increased epithelial cell proliferation and a largelabial cervical loop with abundant stem cells (Fig. 3).The results of these studies indicated that BMP4 hasopposite roles on the proliferation and differentiation ofthe dental epithelial stem cells: it inhibits the prolifera-tion of stem cells, whereas it stimulates their differenti-ation into ameloblasts (Fig. 3). These conclusions weresupported by the phenotype of the incisors of transgenicmice overexpressing the BMP inhibitor Noggin in theepithelium (K14-Noggin mice). Their incisors are largeand dramatically overgrown, and they lack enamel [11].Several observations indicated that the primary tar-get tissue of mesenchymal Activin and BMP4 is the564 I. Thesleff et al. / C. R. Biologies 330 (2007) 561–564cervical loop epithelium. However, their effects on theproliferation of this epithelium apparently took placesecondarily via the regulation of mesenchymal Fgf3,which stimulates epithelial proliferation. The direct tar-gets of BMP and Activin in the epithelium as well asthe reciprocal epithelial signals regulating Fgf3 in mes-enchyme remain to be identified in future studies.ConclusionsThe work presented here shows how the spatiallyrestricted and balanced effects of specific componentsof a signalling network can regulate stem cell prolif-eration and differentiation. The findings indicate thatthe continuous growth and asymmetry of the incisor, aswell as the formation of enamel, can be significantly af-fected by modulating a complex network of FGF, BMP,and Activin signals that regulates dental epithelial stemcells.Continuously growing teeth are common in many an-imals and there are variations in the extent of enamelcoverage of the teeth. For example, even primatespossess ever-growing incisors, as seen in the Aye-ayelemurs [12], and elephant incisors (tusks) have noenamel. Some rodents such as voles have continuouslygrowing molars in addition to the ever-growing incisors.Work from our laboratory had previously shown thatthe cervical loops of continuously growing molars wereanatomically similar to those of incisors, and that com-ponents of the same molecular signal network were inplace [13]. Hence, it is possible that the evolutionaryvariation in the growth capacity of teeth and the ex-tent of enamel deposition has resulted from fine-tuningof the complex signal network that regulates the main-tenance, proliferation, and differentiation of epithelialstem cells. In addition, subtle variations in this or re-lated regulatory networks may explain the differentregenerative capacities of various organs and animalspecies.References[1] E. Fuchs, T. Tumbar, G. Guasch, Socializing with the neighbors:stem cells and their niche, Cell 116 (2004) 769–778.[2] B. Ohlstein, T. Kai, E. Decotto, A. Spradling, The stem cellniche: theme and variations, Curr. Opin. Cell Biol. 16 (2004)693–699.[3] K.A. Moore, I.R. Lemischka, Stem cells and their niches, Sci-ence 311 (2006) 1880–1885.[4] H. Harada, P. Kettunen, H.S. Jung, T. Mustonen, Y.A. Wang,I. Thesleff, Localization of putative stem cells in dental epithe-lium and their association with Notch and FGF signalling, J. CellBiol. 147 (1999) 105–120.[5] M. Tummers, I. Thesleff, Epithelial stem cell niche in the tooth,in: R.P. Lanza (Ed.), Handbook of Stem Cells, vol. 1, AcademicPress, Boston, MA, USA, 2004, pp. 265–271.[6] W.E. Starkey, The migration and renewal of tritium labelled cellsin the developing enamel organ of rabbits, Br. Dent. J. 115 (1963)143–153.[7] H. Harada, T. Toyono, K. Toyoshima, M. Yamasaki, N. Itoh,S. Kato, K. Sekine, H. Ohuchi, FGF10 maintains stem cellpopulation during mouse incisor development, Connect TissueRes. 43 (2002) 201–204.[8] X.-P. Wang, M. Suomalainen, C.J. Jorgez, M.M. Matzuk,S. Werner, I. Thesleff, Follistatin regulates enamel patterning inmouse incisors by asymmetrically inhibiting BMP signalling andameloblast differentiation, Dev. Cell 7 (2004) 719–730.[9] X.-P. Wang, M. Suomalainen, S. Felszeghy, L.C. Zelarayan, M.T.Alonso, M.V. Plikus, R. Maas, C.-M. Chuong, T. Schimmang,I. Thesleff, An integrated gene regulatory network controls ep-ithelial stem cell proliferation in teeth, PLoS Biol. (2007), inpress.[10] W. Balemans, W. Van Hul, Extracellular regulation of BMP sig-nalling in vertebrates: a cocktail of modulators, Dev. Biol. 250(2002) 231–250.[11] M.V. Plikus, M. Zeichner-David, J.A. Mayer, J. Reyna, P. Brin-gas, J.G. Thewissen, M.L. Snead, Y. Chai, C.M. Chuong, Mor-phoregulation of teeth: modulating the number, size, shape anddifferentiation by tuning Bmp activity, Evol. Dev. 7 (2005) 440–457.[12] R.A. Mittermeier, I. Tattersall, W.R. Konstant, D.M. Meyers,Mast R.B., Lemurs of Madagascar, Conservation International,Washington, DC, USA, 1994.[13] M. Tummers, I. Thesleff, Root or crown: a developmental choiceorchestrated by the differential regulation of the epithelial stemcell niche in the tooth of two rodent species, Development 130(2003) 1049–1057.

Regulation of epithelial stem cells in tooth regeneration

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Regulation of epithelial stem cells in tooth regeneration

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