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Development of Teeth in Chick Embryos after Mouse Neural Crest Transplantations Author(s): Thimios A.

Mitsiadis, Yvonnick Chéraud, Paul Sharpe and Josiane Fontaine-Pérus Reviewed work(s): Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, No. 11 (May 27, 2003), pp. 6541-6545 Published by: National Academy of Sciences Stable URL: . Accessed: 25/09/2012 11:13
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PNAS I May 27. 11 | 6541-6545 . but a number of genes that initiate odontogenesis continue to be expressed in their jaws (10. Floor 28 Guy's Tower. Classical tissue recombination experiments and more recent molecular analysis have identified the oral epithelium as providing the instructive information for the initiation of mouse tooth development. Pitx2 is a homeobox gene that is Thispaper was submitteddirectly(Track to the PNAS office. and which remain silent in modern birds. France Edited by N. involves reciprocal series of epithelial-mesenchymal interactions (1). To ascertain the presence of donor tissue in hosts. Isolated chick epithelium cultured in combination with mouse tooth mesenchyme produced dental structures. nestin) were used. and Josiane Fontaine-Perus? after mouse *Department of Craniofacial Development. Heads of older chimeras were prepared for histological examination and in situ hybridization on sections. Currentthinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity. the initiation period of tooth development extends from embryonic day (E) 8. MK. To investigate the odontogenic capacity of ectomesenchyme. cFGF8. the odontogenic potential shifts to the mesenchyme. can be reactivated upon appropriate signaling (8. Institut d'Embryologie Cellulaire et Moleculaire du Centre Nationale de la Recherche Scientifique. we grafted mouse cranial neural crest in place of chick. we have used neuraltube transplantations from mice to chickembryos to replace the chickneural crest cell populations with mouse neural crest cells. digoxigenin-labeled mouse and chick specific riboprobes (mMK. To determine whether teeth can form in the developing jaws of avian embryos in ovo. Vector Laboratories) staining was performed as previously described (18. Chimera embryos were incubated in ovo for different periods (1-18 days). King's College London. which may be due to the origin of the neural crest cells and/or regional differences in the oral epithelium. then removed from the host and replaced by the donor graft (Fig. In the mouse embryo. Unite Mixte de Recherche 5665. The epithelium then invaginates into the underlying mesenchyme to form the tooth bud (E12. Ecole Normale Superieure de Lyon. BMP. The mouse/chick chimeras obtained show evidence of tooth formation showing that avian oral epithelium is able to induce a nonavian developmental program in mouse neural crestderived mesenchymal cells. which can instruct any kind of epithelium to form tooth-specific structures (7-9).) In Situ Hybridization Immunohistochemistry Tissue Sections. Although these epithelial ingrowths share similarities in organization and morphology with mouse tooth fibroblast growth chick Developmentof teeth embryos crest neural transplantations Thimios A. 13). Paul Sharpe**. whereas the oral epithelium retains the signaling properties requiredto induce odontogenesis. Immunoperoxidase (ABC Kit. Birds lost their dentition almost 70-80 million years ago. The E9-11 presumptive dental epithelium can elicit tooth formation even in neural crest-derived mesenchyme that does not normally form teeth. sonic hedgehog. mBarxl. but is not able to induce tooth formation in a nonneural crest-derived mesenchyme. M. United Kingdom. whereas the oral epithelium www. it is difficult to eliminate completely contamination of mouse mesenchyme with residual epithelium. Positive peroxidase staining produces red color on light microscopy. Nogent-sur-Marne Cedex. France. During avian embryonic development. ?Centre National de la Recherche Scientifique Unite Mixte de Recherche 6018. Materials and Methods Preparationof Mouse/ChickChimeras. Guy's Hospital. 3).Chimeras killed 1-2 days after surgery were prepared for whole-mount in situ hybridization with mouse probes to check mouse neural crest cell migration into the facial territories of the chick host embryos. Reciprocal exchanges of precisely defined regions of the neural tube were performed between chick and mouse embryos as previously described (16).bone morphogenetic protein. cShh) were *To whom correspondence should be addressed. Unfortunately. affinity-purified antibodies against several proteins (dentin sialoprotein. 11).1073/pnas.mitsiadis@ens-lyon. For immunohistochemistry.5-13). FGF.JA657 chick embryos at 1 day of incubation (seven somites) and Swiss mice embryos at E8 (four to six somites) were used for the microsurgery. 1 Upper).sharpe@kcl. cBMP4. as described (16). such recombination approaches suffer from being performed in vitro. and on For in situ hybridization.2003 | vol. France. Mitsiadis*t.Shh. 2002) Teeth were lost in birds 70-80 million years ago. 44322 Nantes Cedex 3. Faculte des Sciences et des Techniques. 100 I no. such as the limb mesenchyme (4. mPax9. 19). cPitx2. mMsxl. Whole-mount in situ hybridization and in situ hybridization on cryosections were performed according to Wilkinson (17). and approved April 1. London SE1 9RT. to Ell. The distribution of mouse neural crest cells in the head of chimeras was visualized by a DNA repartition that differs in the mouse and chick nuclei. 14. and tLaboratoire de Biologie Moleculaire et Cellulaire. and moreover.1 137104100 retains the signaling properties to induce odontogenesis in competent mesenchyme (8. the molecular mechanisms regulating their outgrowth appear to be distinct because their development is arrested at this stage. Several in vitro studies have suggested that a number of genes involved in tooth formation. (Mouse cells had a brighter appearance after Hoechst staining. 7). 2003 (received for review November 21. En. and St. Guy' These experiments point to the importance of epithelial signals in the initiation of mouse tooth formation. when local thickenings of the oral epithelium are formed (4-6). 46 Alle d'ltalie. several sections of each chimera were stained with bis-benzimide (Hoechst staining). E-mail: paul. Thomas' Dental Institute. Universite de Nantes. These inductive interactions are mediated by diffusible factors between oral epithelium and neural crest-derived mesenchyme (2. 69364 Lyon Cedex 07. Results and Discussion Several molecules are well known to be involved in the initiation of murine tooth formation. 2 Rue de la Houssiniere. Visualizationof Neural Crest Cell Migration. King's. 15). II) Abbreviations: embryonicday n. Centre National de la Recherche Scientifique. rudimentary structures consisting of local epithelial thickenings are formed transiently in the mandibular arch (12. BP 92208. Le Douarin. in common with the formation of many other vertebrate organs. Tooth development. The cephalic region to be grafted was first delimited in the mouse donor and the chick host. when crest cells first emerge from the cranial neural folds.pnas. By E12. and these thickenings closely resemble the murine dental thickenings. Yvonnick Cheraud?.midkine. suggesting that it is the cranial neural crest-derived mesenchyme of modern Aves that has lost odontogenic or efthimios. 14).

oral epithelium.Rhomb .the chickprosencephalon-mesencephalon-rhombencephalon (ProsWe undertooka molecularanalysisto examineexpressionof Mes-Rhomb. Inseries (series B) and A. Expression initiallyexpressedthroughoutthe mouse oral epitheliumand transcriptionfactors becomes restrictedto the dental epithelium(20). Depending on the series (A-C).whereas no tooth structureswere obimplanted into a chick host embryo (homotopic and homochronicgraft).md. in transversesections of the head region of mouse/ chickchimeras.Mouse Embryo i Chick Embryo Pros. was transplanted into an avian host survived Chimeras 10 38 26 74 (41%) from which the equivalenttissues had been surgically removed 14 8 22 (30%) Casesof toothstructures 0 Mouse neuralcrestcells had alreadyinvadedthe (Fig.and D). Mouse/Chick Chimeras whole dorsoventralaspect of the rostral murine neural tube. Subsequently. in series B. These cells contribute to the Experimental procedureof mouse neuraltube transplantationinto chickhost formationof tooth-likegerm structures differenttime points at embryos. cells expressingPax9 and Msxl with MK possess odontogenic determination ectomesenchyme Expressionof BMP4 and FGF8 has been documentedin chick potential. MKwasfoundto be expressedin a largepopulationof embryos. 3 A and C). 74 in series C. To investigatethe odontogeniccapacityof murineectomes. 26). Although Shh is expressedin areas of facial genesis (21. fibroblastgrowthfacor tor-8 (FGF8). 3 D-F). 25). aboralepithelium. The restrictedexpressionof mouse and localized regions of the chick oral epitheliumare these molecules to dental placodes in oral epitheliumduring responsiblefor the subsequentelaborationof the specificform mouse developmentis thus an indicationof the inductionof and structureof the teeth describedabove.1073/pnas. Note the invaginations of the chick oral epithelium (arrowsin B.the grafts involved15 chimerasin series stagesof mouseembryonic development (18). The implantationlevel in sixsomite-stage chickhost embryosisshown Transplantsof prosencephalon-mesencephalon in red. One hundredseventy-eight chickembryos tor that is expressedin all neural crest cells during the early were grafted at the cephalic level and 74 (41%)survivedbetween 1 and 18 MK daysafter surgery. with 10 survivals. maxillaryand mandibular processesof the chick host by 1 to 2 series C. is not expressedin early interactionsbetween neural crest-derivedmesenchyme from (st21) dental epithelium(11.(Upper) days after surgery (Fig. 2 B-D and Fig. In tained from transplants prosencephalon of (series A).The chimeraswere removed at various developmental stages (betissues and organs.with 26 expression becomes down-regulatedin the vast majority of survivals.pnas.different zones were grafted. Mitsiadis al. the prosencephalon(Pros)of the chickembryowas replaced by the equivprosencephalon-mesenchephalon-rhombencephalon (series C) alent encephalic region of the mouse embryo (homotopic and homochronic both contributedto the formation of tooth structuresin the was graft). 89 and A. mandibularprocess. in blue.four to six somites) shown after graftingin the chickhosts (Fig.(A-D)Migrations and localization of mouse neural crest cells (brightercells) in maxillaryand mandibularprocesses. of the mouse homeodomain-containing progressively in Msxl and Pax9was far more restricted.Pros C Mes . A B C Total 1137104100 6542 | www. enchymalcells and also to examinewhetheravian oral epithe.Mes ikC T. 1 Upper). MPros IJMes Rhomb UPros TnMes W . ae.rRhomb Fig. mouse encephalon disposed one behind the other (homocoronic graft). These findingssuggestthat neuralcrest of into dental papilla (21-25). performeda series The tooth rudimentsis responsiblefor their failureto progressinto of interspecifichomotopic neural-tubetransplantations.MPros B .3 (B). and sonic hedgehog (Shh) are involved in the those cell populationssurrounding contactingthe chickoral determinationof tooth-formingsites in mice and the stepwise epithelium( mesenchymefrom the earlieststages of murineodontovelopment (10).1.the mouse prosencephalon-mesencephalon(Pros-Mes) mouse/chick chimeras. maxillary process. and then prepared for different murine teeth until E18 (late bell stage) (19).Mouse cells detected after Hoechst staining.crest-derived signaling molecules involved in epithelialinteractions.but its expressionpersistsin the developing tween 1 and 18 days of incubation). InseriesB.oe. et .mesenchymal It had been suggestedthat loss of BMP4 in chick "vestigial" we liumcan undergofull tooth development. 2.5 (C). 1 Lower).fixed.becausethe expressionof Pax9 andMsxl is limitedto oral epitheliumin broad domainsduringearly embryonicde. 22. 2A). C.2 (A).and 9 (D)daysafter grafting.The loss of teeth in Aves is thus probablydue to the lack of appropriateneural Rhomb.Rhomb Pros Rhomb f A. before its closure (when it still containsall of the premigratory Chimeras realized 74 178 15 89 cranialneural crest cells). In chimeric analyses. Midkine(MK) is a heparin-binding facgrowth/differentiation (Lower)Numberof cases studied. We conclude that the reciprocalsignaling it epitheliumduringchickdevelopment. Mouse neuraltube transplantationinto chickhost embryos. and the acquisitionof a tooth-bud configuration (D).the whole red region) was replaced by two half parts of the both early and late markersof tooth developmentin chimeras. A . mouse neuralcrest cells migratinginto the maxillaryand mandibularprocessesof the chickhost (Fig.with 38 survivals. hMes. Visualization of mouse ectomesenchymal cells surrounding the ingrowths of the oral chick epithelium in chimerasand formation of a mineralized dental-likestructurein a mouse/chick chimera.Graftlevel in mouse donor embryos(E8.Forthethree series.occurring specifically Bone morphogenetic protein-4(BMP4).

aboral epithelium.D). and BMP4(cBMP4. Fig. aboralepithelium. in vivo. MK expression. became restricted around the invaginated chimera epithelium (Fig. and M)markers. we performed in situ hybridization in chimeras. oe cFmE8 . (B) Detection of mouse Msxl (mMsxl) transcripts in the grafted area (arrowhead) and the facial process (arrow) in a chimera 1 day after grafting.d oe oe . 3. ae. Whole-mount digoxigenin in situ hybridization(A and B)and in situ hybridizationon sections (C-F). These localized regions of epithelial expression correspond to the sites overlying mouse neural crest cells (Fig. in restrictedareas of the oral epithelium. Seven to nine days after grafting. 5 A and B). mandibularprocess. . D. cShh. 100 I no. and BMP4 expression was restricted to those areas that form the epithelial placodes described above (Fig.1 to 5 daysafter grafting. eye. 5 C. (M) Mouse Msxl (mMsxl) expression in a population of mesenchymal cells in contact with the oral epitheliumof the maxilla. and facial region (arrow) of a mouse/chick chimera 2 days after grafting.mMK r ae ? oe cFGF8 cShh mx mx ae '" md oe cBMP4 . the chick oral epithelium had invaginated into the underlying ectomesenchyme and acquired a bud configuration (Fig. (E and F) Mouse Msxl expression limited to mesenchymal cell populations in contact with the oral epithelium in two different chimeras(3 and 5 days after grafting).oe. Visualizationof mouse neural crest cell populations in the forming and maxillary mandibularprocessesof mouse/chickchimeras. mandibularprocess. Barxl. md I mx cPiA2 J md mMsxl mMsxl x oeV ae md 5 L ae md M . (G) RestrictedcShh expression in oral epithelium. . Shh( The differential growth and subsequent folding of the dental epithelium is thought to be directed by a transient signaling center known as the enamel knot (2. and K)and digoxigenin in situ hybridization on sections of 2 (A-F)to 4 (G-M)days after grafting chimeras. and G). ~ oe m oe-j md ae m G md H ae . (F)Expression E) of chickPitx2(cPitx2)in all cells of the oral epithelium. showing the arrivalof mouse neural crest cells at the sites of cFGF8. 4 F and L).oe. Formationof dental placodes in the oral epithelium of mouse/chick chimeras. and MK genes (Fig. Similarly. 11 | 6543 . Although each of these genes is expressed in cells other than dental mesenchyme.(A) Expressionof mouse MK(mMK)in the grafted area (arrowhead). 26). and Barxl (Fig. 4 H. . (cFGF8. and F with A and B. and K) that express the mouse Msxl. neural crest cells may play a role in the activation of BMP4 and Shh expression in tooth-forming sites of the murine oral epithelium. . eye. G.e. oe D e mx md mMsxl oe . ectopic chick Shh expression was seen in a restricted population PNAS I May27. The asterisk shows the mesenchymalarea in which the mouse of Barxlgene (mBarxl)was detected in an adjacentsection. and I). As during the bud stage of Mitsiadis et al.J.1 *. 3. and M). B. Shh expression has been reported in the enamel knot of developing mouse teeth (27). mouse ectomesenchymal cells surrounding the chick epithelial invaginations expressed the toothspecific genes Msxl. maxillaryprocess. oral and cBMP4expression.D.(C-E)Expression chickFGF8 C). mouse tooth development. Pax9.and L)and mouse mesenchymal(H. asterisk in /). teeth (13). dental mesenchyme cells are the only cells in the embryo that express all three genes.I. oe md C ae mPax9 mx I D ae md md E cShh e* F mx mBarxl .-1 .Hoechststaining (A. which was initially widespread in the migrating neural crest cells. 5E).2003 | vol.. Note the absence of the hybridization signal in the aboral epithelium. These results indicate that. (C)Widespread mMKexpression in migrating cells of a chimera 3 days after grafting. (L) Expressionof cPitx2 to the oral epithelium of maxillaryand mandibularprocesses. ectopic chick using chick (A epithelial (C-G.. 2 to 4 days after surgery. 4 C-E. E. chick epithelium expressing cFGF8 asteriskin J) is in contact with the mesenchyme in which the mMK (I.(I and J) Similarly. gene was expressed (J. Shh. Although Pitx2 was expressed throughout the oral epithelium ( To test if mouse cranial neural crest contains specific signals that can ectopically induce localized BMP4 and Shh expression in the chick oral epithelium. The oral origin of the epithelium was evident from the detection of the chick Pitx2 gene (Fig. CompareC.4. md. 4A. J. mMK "~~~~~~~~~~~~~ :L. 5F). IAA"f . maxillaryprocess. (H)Expression the mBarxl gene in mesenchymalcells that are in contact with cShh-expressing the epithelial cells (asterisk). and B) Mouse cells appear brighter after Hoechst staining in the maxillaryand of mandibularprocesses. oral epithelium. md E F Fig. (D) Restrictedmouse Pax9 (mPax9) expression in mesenchymalcells surroundingthe chick oral epithelium of 3 days after grafting chimera.

. oe. 5.pnas. cPitx2 . m. in mesenchymal cells surroundingthe epithelial bud structures. Formationof dental-like epithelial bud structuresin mouse/chick chimeras. J of epithelial cells in chimera tooth germs (Fig.formationof a single cusp (conical or incisalshape).'-' oe ' t . nestin .1073/pnas. brown color. mandibularprocess. showing mineralmatrixdeposition (arrow). i y . Chick Pitx2 was expressed in the epithelium of the 6544 | www. .D).-::.G)expressionin the epithelium. maxillaryprocess. md.A. and G) Detection of mouse Msxl (mMsxl. epithelium.and Arrowsindicate (H-J)by usingtooth-specific markers. 6 C and E) and in several unusual structures formed at the anterior part of the oral epithelium (Fig.: cPitx2 m cPitx2 oye. and mouse Msxl (mMsx. immunohistochemistry the sites of mineralmatrixdeposition.(A and B)Visualizationof mouse neural crest cells surroundingchickepithelial bud/cap structures(arrows) in the maxillaryprocesses. mx.7-9 days after grafting. which starts at the tip of the cusps. nestin .1.G)transcripts F). oe. dental papilla. arrow). p ".6.digoxigenin in situ hybridization (C-J). . . The lack of enamel formation may be due to the early termination of the interaction between the epithelium and mesenchyme. m. dental epithelium. This possibility can be supported by the fact that the dentin matrix is not as mature as in teeth when enamel deposition is occurring in a deeper layer of dentin. '" '. de.. } . b. (J)Highermagnificationof /.Aj.!. arrow) that represents the equivalent population of cells of forming the "enamel knot" in developing mouse teeth.f*~t ' ** :* ^ ''' '~. I . (E-G) Higher magnification of the tooth germ. oral epithelium.: .The chick oral epithelium invaginates into the underlying mouse neural crest-derivedmesenchymeand acquiresbud configurations. MK(mMK. jaw mesenchyme.. Significantly. Hoechststaining (Aand B)and in situ hybridizationon sections by using tooth-specific molecular markers(C-G). Fourteen days after grafting. Fig.I oe oe G mBarxl cSlhh tc . (E)Expression the chickPitx2gene (cPitx2)in the bud epithelium.C).** F ~~~1 ~ mMK t C *. (H) Dentin sialoprotein (DSP)distribution(red color) in cells of the dental mesenchyme and in mineralized matrix of the chimera tooth. and the absence of enamel matrix and polarized chickPitx2(cPitx2) epithelial cells. bone. brown color) or digoxigenin-labeled (C-G.t. 6D). tooth-like germ. tooth bud. which is reminiscent of Pitx2 Mitsiadis et al. (A) Histologicalsection of the mineralized structure resembling a tooth germ in a mouse/chick chimera. These tooth-like structures were clearly distinguishable from the surrounding mineralized bone. oe oe . No dentin tubules were evident because these teeth represent an early stage of mineral deposition.' L-t. DSP . t . ."' . The deposition of the mineral matrix in the tooth-like structures was typical of the dentin matrix deposition observed in developing teeth of murine embryos. the epithelial-derived enamel matrix was absent from these structures. enamel knot. D.A C mMsxl D Atb oe mPax9 t E oe- cPitx2 mftb ?' cPitx2 -' *' oetb '. mesenchyme.. Examinationof the chick originand dental potential of the epithelial ingrowthsby in situ hybridization by using either fluorescein-labeled (G. proceeds apically and follows odontoblast differentiation.tb. chimera tooth germs (Fig.(B)Highermagnificationof Fig. p. tooth cap-like invagination. (/) Nestin expression (red color) in odontoblast-like cells responsible for mineral matrix production in the chimera teeth. Chick Pitx2 was not expressed in the epithelial cells contacting the mineralized matrix. o.2A. 6 A and B).. D mIMsxl cShh .violet and blue colors)species-specificprobes. 'm I. 5G). Histology(Aand B).V .. (C)Exclusive expressionin the epitheliumof the tooth germ. . The mineralizedmatrixhas a blue color after Masson'strichromestaining. and mineralized structures resembling tooth germs were observed beneath the oral epithelium (Fig.Pax9(mPax9. Restricted expressionof the chickShh gene (cShh)to cells of the epithelial bud structure (G. . . 1137104100 Fig... . indicating the presence of a tooth-shape regulator in these structures. Formationof tooth-like structuresin mouse/chick chimeras. showing cPitx2 (E) and chick Shh (cShh. H I.(D)An unusual structureexpressingcPitx2in the anterior part of the oral and Barxl (mBarxl.. expression F) in the o *'.. Spongy bone hasformed aroundthe dental-likestructure. cPitx2 ? .. odontoblast-like cells. F.14 days after grafting. .Note that the cPitx2gene is down-regulated in epithelial cells contacting the mineralized matrix (E. . Note that the gene isabsent in oral epithelium."' 'e '-!" oe .. (C. multiple ingrowths of the oral epithelium were evident. ".

Kollar. Ch6raud. 4. J. T. U. 6.. (1980) Science207.H. (1996)Mech. Zimmerman. (1999) Trends Genet. Dev. Schneider.. 533-539. & Mina. Dyn. The mouse Msxl (Fig. & Butler.. J. R.Upholt. A. (1983)in Epithelial-Mesenchymal in Interactions Development. (1998) Development 3-28.. 11-24.K) 126. Barlow. 30. to their skeletal fates. C.C. & Rieger. J. M. E. Halgand. C. Hu. Vainio. T.J. the laboratory C.. 287-295. Nichols.. 11. (1998)Mech. T. 993-995.. C. Chen. A.. A.2003 I vol. About. D. 123-127.I. 28. (2000) Proc. G.A.Proust. M. Tucker. E. 9. The research supported grantsfromthe Association was by pour la Recherche le Cancer. A. (Cambridge. R. 111-123. Muramatsu. Peters. Houston) for the dentin U.. a marker of neurons.M. 14. 24..296. 12. Biol.New York). Heaney..V. (2002)J.N.T. 386-397. 7.. Biol. 6G) were expressed. Cadena. A.. D. 19. U. (OxfordUniv. sur Association contreles Myopathies. A. Laurent-Maquin. was also detected in cells producing dentin matrix in the chimera tooth germs (Fig.-M. Nagai. 11. Embryol. G.K) 124. (1991) J.& Goridis M. (1993) Cell 75. & Sharpe.. (1999) A. 2803-2811.. Vaahtokari. 45-58. 6F) and the chick Shh genes (Fig. Biol. Thesleff. T. H. & McMahon.. D.. 17.. the Wellcome Trust. 31. A. (Praeger. Somerman.D. P. H... Muramatsu. Velasco. USA 97.. ed. 8. 11 I 6545 . Barlow. A.K) 125. Mann. Lemaire. Schrohenloher. Mo. & Maas.U. F. 105-120. Mitsiadis et al. Farach-Carson..M. C. 15. 25.. 22.100 I no. & Martin.-P... Kollar.Y.B.J. (1997) Development (Cambridge. R. Brunn. Rouaud. D'Souza.pp.. & Graveson. M. St. (Cambridge. they do contain the information to interpret generic epithelial signals and to behave in a speciesspecific way. We thank W. S.. E. Fontaine-Perus. Pourqui6for valuable comments. 26. E.. & Sharpe. P. 64. H. 10..J. 155-169. Aberg. R.R.. (1997)Cell90. 3. L.R. Jernvall... and the University of Nantes...P T. (1997)Mech.. P. Karavanova. Medical Research Council.New York). PNAS I May27. 2.A. 16.Stockholm) sialoprotein antibody. H. (1998)Dev. & Sharpe.15. H. et al. Chuong. Vassileva. 81. A.-P.OralBiol. (1986)Arch.I. 5. & Balling. Press.T. & Helms. & Sharpe.P. G. 21. 103-109. Raffo.K) 103.J. R. Wang.M. Jalkanen. Hui. A..Oxford). S. (1988) Development (Cambridge. pp.W. muscles (30) and differentiating and mature odontoblasts (31). J. (1960) The Avian Embryo(Macmillan. & Fallon. Dev. Lumsden.75. S.. Seyer. Sharpe. T. 459-460. Jiang. Cell T. M. A. Francis-West. 213. C. 129.. & Thesleff. N. A. 13. and 0. 10044-10049. Muramatsu. Pachnis. Dentin sialoprotein.. A. Bhown. J. R. S. Zhang. T. S. 20. C. D. A... 37-51.E. U. Ridall.. 32. Neubiiser. (1992)Matrix 343-351.-L. These results show that. Happonen. respectively. M. Wilkinson. 59-65. Salmivirta. 3025-3036.B.. 189.Y.. Acad. J. Francis-West. Khamis.Natl.. J..K) 127. although within a species cranial neural crest cells do not appear to be prepatterned with respect 1..I.E. & Thesleff. & Sharpe. T. (Cambridge. Maroto.. H. J. Sawyer. 267-281.157. Ferguson. Brunet. (1995)J. Rauvala. 67.. Qin.Mitsiadis.P. C.Y. Genet. P. A. Gavin. Wilkinson. for Institute.. was detected in ectomesenchymal cells and the mineralized matrix (Fig. 54. 392-394.. Francaise Centre National de la Recherche Scientifique. Dent.J. (1999)Development U. Development U. A. Parr.Z. B.pp. Cunningham.. Le Douarin. 223-228.T. A.I. P. T. N. Ladher. Grigoriou. 12. G.. Exp. Amand. The intermediate filament protein nestin. J. G. I.R. Artavanis-Tsakonas.Dev. (Cambridge. Keranen.OralBiol. & Mina. & Fisher. Muramatsu.P. U. Hu. T...J.S. (1987)Arch. (1995) Development (Cambridge.. & Thesleff.R..W. M. Suppl. & Helms. (1992) in In Situ Hybridization:A Practical Approach. 27. H. Y. (2000)Am.Thisworkwaspartlyrealizedat the Institut de la Biologie du D6veloppement Marseille.C. R.-F. G. Mucchielli. Dale for commentsandimproving manuscript. M. a noncollagenous extracellular matrix protein produced by odontoblasts (28) and osteoblasts (29). J..Dyn..-X. 247-255.A. 23. Butler (Universityof Texas. 212. & Thesleff. & Kollar. F. McKay. D.G. Peters. 275-284. Lumsden.Res.. Nagatsuka.M. C. Kollar. T. A. J. Hardcastle. 39-43.R.Morphol.S.H..T. 6H). M. Tsujigiwa.J. A. & Mitsiadis.. 27-74.. Jowett. Mitsiadis. Sci.K.K) 126. Yamada. I. Lendahl. S. Kollar. G. 51-61. Butler... Lendahl(Karolinska the nestinantibody.down-regulation in ameloblasts observed in mouse teeth.U. P. eds. C.Y. 403-412. Lendahl.Dev.. 29. Craniofac. Hu.W. 18. U.I. Pathol. Romanoff. Mitsiadis. in dental mesenchyme and epithelium of the chimera teeth. Brunn. Balling.. Foster. (1981)J. S.E.K) 121. M.R.-H. 301-311. M. Tucker. (1999)Cell Tissue R. L.E. 6 1 and J). Zampieri helpwith the and for Masson's trichrome staining.E. S. Res. 75-83.A. J. Cifuentes Diaz.... (1997)Dev. Lehtonen. The presence of both these markers together in the same cell layer provides a strong indication that these cells are odontoblasts. 31. Mina. Tucker.4873-4884. (1994)Neuron12.P.J. (1998)Dev. de of Goridis. (2000)Development A. & Buchanan.