Journal of Dental Research - CiteSeerX

http://jdr.sagepub.com/Journal of Dental Research

http://jdr.sagepub.com/content/77/8/1589The online version of this article can be found at:

 DOI: 10.1177/00220345980770080601

1998 77: 1589J DENT RESC. Sahlberg, M. Hormia, T. Airenne and I. Thesleffl

Intense in AmeloblastsLaminin y2 Expression is Developmentally Regulated during Murine Tooth Morphogenesis and is

Published by:

http://www.sagepublications.com

On behalf of: 

International and American Associations for Dental Research

can be found at:Journal of Dental ResearchAdditional services and information for    

  http://jdr.sagepub.com/cgi/alertsEmail Alerts:

http://jdr.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

http://jdr.sagepub.com/content/77/8/1589.refs.htmlCitations:  

What is This? 

- Aug 1, 1998Version of Record >>

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

J Dent Res 77(8): 1589-1596, August, 1998

Laminin y2 Expression is DevelopmentallyRegulated during Murine ToothMorphogenesis and is Intense in AmeloblastsC. Sahlbergl*, M. Hormia2, T. Airenne3, and I. Thesleffl

lInstitute of Biotechnology, P.O. Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland; 2Institute of Dentistry, University ofTurku, Lemminkaisenkatu 2, FIN-20520 TURKU, Finland; and 3Biocenter Oulu and Department of Biochemistry, University of Oulu,FIN-90570 OULU, Finland; *corresponding author

Abstract. Mutations in the laminin y2 gene cause junctionalepidermolysis bullosa, and enamel hypoplasias arefrequently seen in these patients. Laminin y2 is one of thethree polypeptide chains forming the basement membraneglycoprotein laminin-5. We have localized the expression ofthe laminin y2 gene by in situ hybridization during mousetooth development from early morphogenesis to completionof crown development. The expression was restricted toepithelial cells. During the early morphogenesis of the toothgerm, laminin y2 was expressed by the outer dentalepithelium and by the stellate reticulum cells. No expressionwas detected in the cells of the inner dental epitheliumgiving rise to ameloblasts. The pre-ameloblasts remainednegative during the early bell stage, but, interestingly,expression was very prominently upregulated as the cellsdifferentiated into ameloblasts. This upregulation appearedto coincide with the start of enamel matrix secretion. Theameloblasts expressed laminin y2 intensely throughout theperiod of active enamel deposition. The expressioncontinued at a lower level in the maturation-stage amelo-blasts covering the enamel surface. Immunolocalization oflaminin-5 with polyclonal antibodies indicated that theprotein formed a continuous lining at the basal surfaces ofthe cells expressing the laminin y2 transcripts. We suggestthat the role of laminin-5 during enamel formation may beto strengthen the anchorage of the ameloblasts to theenamel matrix, and that the pathogenesis of enamelhypoplasias in cases of laminin-5 mutations could beassociated with detachment of the ameloblast cell layer fromthe enamel surface.

Key words: tooth development, basement membrane,laminin-5, enamel hypoplasia, ameloblasts.

Received June 6, 1997; Last Revision October 16, 1997;Accepted October 28, 1997

Introduction

The basement membranes are special forms of extracellularmatrix connecting epithelial and endothelial cell layers tothe underlying mesenchymal stroma. The major compo-nents of the basement membrane are laminins, type IVcollagens, proteoglycans, nidogen, perlecan, and SPARC(Secreted Protein Acid and Rich in Cystein) (osteonectin)(for reviews, see, e.g., Kleinman et al., 1993; Tryggvason,1993; Burgeson et al., 1994; Timpl and Brown, 1994).

Laminin is already expressed in the vertebrate embryo atthe two-cell stage, and it has important functions in celldifferentiation, adhesion, migration, and cell proliferation(Kleinman et al., 1993). Laminin is a large heterotrimericglycoprotein consisting of an a-, a 3, and a y chain coiledtogether, forming a cross-like molecule. So far, 11 lamininsformed by different trimeric combinations of five a-, three f,and two y chains have been described. Laminin-5 (kalinin,nicein, epiligrin) was first described in 1991 (Rousselle et al.,1991; Carter et al., 1991), and it is composed of an cx3, a 33,and a y2 chain. Laminin-5 is unique in being the only onehaving a y2 chain (Engvall and Wewer, 1996).

Tooth development starts as a thickening of the oralepithelium, which subsequently forms a bud around whichmesenchymal cells condense. During morphogenesis, itdevelops into a cap-like structure enclosing the mesenchymaldental papilla cells which later give rise to odontoblasts anddental pulp. The morphology of the tooth crown isestablished during the bell stage, when the cusps of thetooth develop as a result of folding of the epithelial-mesenchymal interface. Ameloblasts differentiate from theinner dental epithelium and start secretion of the enamelmatrix after a layer of predentin has been deposited by theunderlying odontoblasts. After having secreted the fullthickness of enamel, the ameloblasts change in morphologyand function as so-called maturation-stage ameloblastsregulating the mineral composition of the enamel matrix.

The morphogenesis and cell differentiation during tooth

1589 at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1590 Sahlberg et al.

development are regulated by epithelial-mesenchymalinteractions. Several soluble signaling molecules-includinggrowth factors in the TGF3, BMP, FGF, and EGF families-have been implicated as mediators of these interactions(Thesleff and Sahlberg, 1996). In addition, many extra-cellular matrix components like fibronectin, and differentcollagens and laminins as well as integrin receptors havebeen implicated in morphogenetic regulation (Ruch, 1987;Ruch et al., 1995; Thesleff et al., 1995; Salmivirta et al., 1996).

Laminin has been localized in the developing tooth bythe use of polyclonal antibodies against EHS-laminin. Theseantibodies are now known to recognize the chains ocl, P1,and -yl and thus several laminin isoforms but not laminin-5(Langhofer et al., 1993). With these antibodies, lamininstaining was uniform in all dental basement membranes,from cap to late bell stage. However, in postnatal teeth,laminin disappeared from the basement membrane betweenthe newly deposited predentin and the differentiatedameloblasts, and only weak staining could be seen inameloblasts (Thesleff et al., 1981). Aberdam et al. (1994a)reported expression of laminin-5 cL3, P3, and y2 chains inteeth of newborn mice. The expression of other lamininsduring tooth development has so far not been reported.

Mutations in the laminin y2 gene have recently beenlinked to Herlitz' junctional epidermolysis bullosa (JEB), oneof the more severe forms of blistering skin disorders(Aberdam et al., 1994b; Pulkkinen et al., 1994). Defectivestructure of dental enamel but normal dentin developmenthas been reported in JEB patients (Wright et al., 1993). Thissuggests that laminin-5 might be of importance for enamelformation.

In this study, we have analyzed the expression of lamininy2 by in situ hybridization and immunohistochemistrythroughout tooth morphogenesis, starting from embryonicday 13 to postnatal day 12.

Materials and methods

Preparation of tissue sectionsThe embryonic age of the hybrid mice (CBA x NMRI) was setaccording to the day of the vaginal plug, which was designatedday 0. The mice were anesthetized with CO2 and killed bycervical dislocation. The use of animals was approved by theInstitutional Animal Care and Use Committee (IACUC) of theFaculty of Science of the University of Helsinki.

For in situ hybridization assays, whole heads, lower jaws, orlower first molar tooth germs from 13-day-old embryos to 12-day-old postnatal mouse pups were dissected under a stereomicroscope in PBS, fixed in 4% paraformaldehyde (PFA) in PBS(pH 7.2) at 4°C overnight, dehydrated, and embedded inparaffin wax. In addition, postnatal tissues were demineralizedin 2.5% PFA + 12.5% EDTA in PBS for up to 2 wks before beingdehydrated and embedded in paraffin wax. Sections 7 pm thickwere placed on silanized glass slides, dried overnight at 37°C,and stored in tight boxes at 4°C until used.

For immunostainings, whole heads or jaws of 13- to 17-day-old embryos were dissected and transferred to 20% sucrose forone day, frozen in TissueTec O.C.T. compound (MilesLaboratories, Elkhart, IN, USA), sectioned at 7 pm, and fixed in

ice-cold methanol. Molars from seven-day-old postnatal micewere demineralized as for in situ hybridization.

ProbesThe probe for the laminin y2 chain has been describedelsewhere (Hormia et al., 1998). It is a 352-bp fragmentcorresponding to bases 3530 to 3882 in mouse cDNA (Sugiyamaet al., 1995) and cloned into a pGEM 3z vector.

The plasmids were linearized with Hind III for the senseprobe and with Eco RI for the antisense probe. [35S]-uridineS'-triphosphate (= 1000 Ci/nmol; Amersham)-labeled RNA-probes were transcribed by means of a transcription kit fromPromega. The labeled probes were precipitated with ethanol,dissolved in hybridization buffer, and used at 50,000 to 60,000cpm/pL.

In situ hybridizationThe in situ hybridization of sections was according to Wilkinsonand Green (1990). In short, the deparaffinized sections were pre-treated with proteinase K (Sigma), hybridized with the labeledprobes in a humid chamber overnight at 50°C, and washedunder high-stringency conditions. The dried slides were dippedin autoradiographic emulsion (Kodak NTB2) and exposed for 10days at 4°C. After developing the film, we stained the sectionsin hematoxylin, mounted them, and examined them in anOlympus AX70 Provis microscope.

AntibodiesThe rabbit polyclonal antibody J18 was raised againstsolubilized extracellular matrix of 804G cells and has previouslybeen shown to recognize laminin-5 in both human and rodenttissues (Langhofer et al., 1993). The rabbit polyclonal antibody torat laminin-1 was purchased from Telios Pharmaceuticals(Gibco, Grand Island, NY). This antibody has a reactivitysimilar to that of anti-EHS-laminin antibodies, thus reactingwith laminin-1 chains otl, 31, and y1.

Immunofluorescence stainingFrozen sections were first incubated in 0.2% BSA in PBS for 10min and then incubated at 37°C with the polyclonal antibody(diluted 1:100 for EHS-laminin and 1:50 for laminin-5 in 0.2%BSA in PBS) for 40 min, followed by three five-minute washesin PBS. After this, the sections were incubated for 40 min at37°C with the secondary antibody, diluted 1:200 in 0.2% BSA inPBS (FITC-conjugated donkey anti-rabbit IgG; JacksonImmunoResearch Laboratories, Inc., West Grove, PA, USA).Controls were carried out by replacement of the primaryantibody with unspecific rabbit IgG (Technical Grade, Sigma, St.Louis, MO, USA). After washes in PBS, the specimens wereembedded in Immu-Mountg (Shandon, Pittsburgh, PA, USA)mounting medium and examined in an Olympus AX70 Provismicroscope equipped with a UV-light source and a filtermodule for FITC-fluorescence.

Image processingPhotography was done with a CCD digital camera and Image-

J Dent Res 77(8) 1998

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

Laminin-5 Expression in Anieloblasts

C.

Nk N

K"I

Figure 1. In situ hybridization analysis of lamiiinin y2 expression during murine tooth development. Bright- and dark-field illuminations ofsagittal (A-H) and frontal (I-L) sections through mandibular molar tooth germs. (A,B) Bud-stage tooth germ (E13). Laniimnn y2 expression isseen in the dental and oral epithelium, but is absent from some cells of the epithelial bud (arrow). (C,D) E15 cap-stage tooth and (E,F) E19first (right) and second molars. Expression is continuing in the oral and dental epithelia, except in the inner dental epitheliumn (arrows). (G,H)One-day postnatal first (right) and second molars. Lanininn y2 has been intensely upregulated in the newly differentiated ameloblasts. In therest of the inner dental epithelium, no lamninin py2 expression is detected. In the oral epithelium, the laniiinii y2 expression is restricted to thebasal cells (arrows). (I,J) Three-day postnatal first molar. Laminin y2 expression is very intense in secretory ameloblasts. (K,L) Twelve-daypostnatal first molar. Expression is reduced in maturation-stage ameloblasts, but remains high in cervical secretory ameloblasts. Bars: A-D =

200 pm; E-L 501) pm.

Pro Plus 1,3 software. Figure plates were made with AdobePhotoshop 4.0 and Micrografx Designer.

ResultsLaminin y2 mRNA expression was analyzed by in situhybridization in first molar and incisor tooth germs fromembryonic day 13 (E13) to 12 days after birth.

At E13, laniinin y2 expression was seen in all cells of theoral epithelium as well as in most epithelial cells of theinvaginating tooth bud. The future inner dental epitheliumat the base of the bud, however, had lost the expression oflaminin y2 (arrow) (Figs. 1A, 1B). Mesenchymal tissues werenegative at this stage as well as at all subsequentdevelopmental stages.

During the cap stage (E14 and E15), the expression in

1591j Dent Rcs 77(8) 1998

'K41!W.

.-.' r, -

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1592 Sahlberg et al.

V

am>

" i

aI0

C

'. -.

r

E

,r.;,, . .:Cz a-E ,, s ?f tP ,, q

* 92t TiC; y;

- ., \ . .s a ,= M.

b. -, ,. ,. :1

0 '' <, t: :,:

. j; -b Q

it -

e

Figure 2. In situi hybridization analysis of laniinin y2 expression in ameloblasts. (A,B) E19. Nolaminini y2 is detected in differentiated but not yet secreting ameloblasts. (C,D) In one-day postnatalameloblasts which are secreting, enamel matrix proteins also intensely express latilinill y2(arrowhead). (E,F) Three-day postnatal ameloblasts. Expression of latninin y2 is seen in allameloblasts except in nonsecreting cells in the cervical loop (arrow). (G,H) In 12-day postnatalmolars, the ameloblasts are in the maturation stage and show less intense expression of laninin y2.a = ameloblasts; e = enamel; o = odontoblasts. Bar: 100 pm.

both molar and incisor tooth germs was concentrated at theouter dental epithelium, and it was particularly intense inthe cervical loops. The inner dental epithelium did notexpress lamninin y2 (Figs. IC, ID; Figs. 3A, 3B).

During the bell stage (E17-19) of first molar tooth germs,expression of laminitn y2 continued in the outer dentalepithelium, the stellate reticulum, and in the dental lamina.The inner dental epithelium and the stratum intermediumwere negative (Figs. 1E, IF, arrows).

11" 4-;. t 1,

1,I A 11

.11. 4.*

pattern was evident. During early morphogenesis, expres-sion was seen mainly in the outer dental epithelium and inthe cervical loops. The inner dental epithelium (arrow) didnot show laminin y2 expression (Figs. 3A, 3B). Oral epithe-lium was positive for laminin y2 expression. A very intenseexpression was already seen in newly differentiated amelo-blasts in the labial aspect of the incisor at E17 (Figs. 3C, 3D).

The distribution of laminin-5 protein was analyzed byimmunofluorescence staining and compared with that of

j Dent Res 77(8) 1998

At E19, odontoblasts are dif-ferentiating, but pre-ameloblastshave not yet polarized. The ex-pression in the stellate reticulumwas decreased in comparisonwith earlier stages. This is seen inFigs. IE and IF, showing both thefirst and second molars. Nolaminin y2 expression was seen inthe pre-ameloblasts (Figs. 1E, lF,arrows; Figs. 2A, 2B) or in thestratum intermedium.

Dramatic upregulation oflaminin y2 expression in theapparently differentiated amelo-blasts was seen at the mesial cuspslopes in the first molars one daypostnatally (Figs. 2C, 2D, arrow-head). Weak expression was stilldetected in the stellate reticulumand the outer dental epithelium.In the oral epithelium, laEininii y2expression was restricted to thebasal cells adjacent to the base-ment membrane (Figs. 1G, 1H,arrow). The expression patternswere identical in the molars inlower and upper jaws.

Intense expression of lamni7iny2 continued in the secretoryameloblasts of the first molar atpostnatal day 3 (Figs. 11, 1J). Faintexpression was seen in the rest ofthe dental epithelium and in oralepithelium. In the cervical loopepithelium, where ameloblastswere not differentiated, nolaminin y2 transcripts could bedetected (Figs. 2E, 2F, arrow).

After the full thickness of ena-mel has been secreted, the amelo-blasts enter the maturation stage.The cells were smaller, and lanti-nin y2 expression was markedlyless intense than at earlier stages(Figs. 1K, 1L, 2G, 2H). In thecervical part of the crown, whereenamel secretion continues, thecells expressed laniinitn y2.

During the development ofthe incisor, a similar expression

v -

.' lk_'"k-'v .'-

I.,

I'.

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

Laminin-5 Expression in Ameloblasts

EHS-laminin. Both laminins were localized to the epithelialbasement membranes, but only EHS-laminin protein wasfound in the basement membranes of the endothelial wallsof blood vessels (Figs. 4B, 4D, 4F). Laminin-5 protein wasseen in the basement membranes of the oral epithelium at allstages of tooth development. At E13, it began to disappearfrom the basement membranes of the dental epithelium (Fig.4A), and at E17, laminin-5 was no longer detected in theinner dental epithelium (Fig. 4C). EHS-laminin protein wasseen in both inner and outer dental epithelia during allembryonic stages. It disappeared from the inner dentalepithelium only after enamel secretion had started (Figs. 4B,4D, 4F). In newborn mouse teeth, no laminin-5 was detectedat either inner or outer dental epithelium (Fig. 4E). Later, atpostnatal day 7, laminin-5 was detected between thesecretory ameloblasts and the enamel matrix (Fig. 4G,arrow). At this stage, no EHS-laminin was seen in eitherbasal or apical parts of the ameloblasts (Fig. 4H, arrow).Hence, the distribution of laminin-5 protein correlated withthe expression of the laminin y2 transcripts.

DiscussionThe expression of the laminin y2 gene was shown to beconfined to epithelial cells in the developing jaws. The basalcells of oral epithelium expressed laminin y2 at allembryonic and postnatal stages studied. Of the dentalepithelial cells, the outer dental epithelium and the stellatereticulum cells expressed the laminin y2 gene throughouttooth development. Remarkable changes in expression wereseen in the ameloblast cell lineage. During the morpho-genesis of the tooth germ from the bud to the bell stage, noexpression of laminin y2 was detected in the cells of theinner dental epithelium, which terminally differentiates intoameloblasts. Interestingly, the expression of laminin y2 wasvery intensely upregulated as the cells differentiated intoameloblasts. This upregulation appeared to coincide withthe start of enamel matrix secretion.

Immunohistological analysis showed that the laminin-5protein is localized as a continuous line along the basalaspect of the oral epithelium, and from there it extendsalong the dental lamina to the outer dental epitheliumbetween the epithelium and the dental follicle cells. Thespecific role of laminin-5 in this location during early toothdevelopment is not known, but it can be speculated that itregulates dental epithelial morphogenesis by affecting theanchorage of the outer dental epithelium to the mesen-chymal cells of the dental follicle. Laminin-5 protein wasdetected neither in blood vessels nor in the basementmembrane of the inner dental epithelium. Hence, thedistribution of the protein correlates well with theexpression of the laminin y2 transcripts. As has also beenreported earlier, antibodies against the EHS-laminin stainedall basement membranes in the developing teeth, includingthose of blood vessels (Thesleff et al., 1981). The expressionof laminin-5 was, hence, more restricted.

The expression of laminin y2 in ameloblasts started at astage when the basement membrane between the pre-ameloblasts and odontoblasts had just been degraded. Thisdegradation has been evidenced by ultrastructural exami-

A

.Isp

C

Figure 3. In sitiu hybridization analysis of lanminin y2 expression insagittal sections of developing murine incisors. (A,B) E14 upperincisor. (C,D) Sagittal section of an E17 incisor. Lanimnin, y2 isintensely upregulated in the secretory ameloblasts of the incisor. a= ameloblasts. Bars: A,B = 200 pm; C,D = 500 pm.

nation, as well as in immunohistochemical studies withantibodies against basement membrane molecules,including type IV collagen, heparan sulfate proteoglycan(perlecan), fibronectin, and EHS-laminin (Thesleff et al.,1981). Hence, the laminin-5 expressed by the secretoryameloblasts appears not to be part of a basement-membrane-like structure. This is in line with ultrastructuralstudies in which no basement membrane has been observedin this region. Our immunohistological analysis using bothantibodies against EHS-laminin and laminin-5 showed thatonly laminin-5 was present during secretory amelogenesisat the surface of the enamel matrix.

Some insights into the regulation and possible functionsof laminin-5 during tooth development may come fromcomparison with the expression patterns of other genes("Gene expression in tooth", a www database, 1996).Although the pattern of the lanminin y2 gene expression isunique, there is apparent co-expression with some genes atcertain stages of development. Like laInminin y2, the Notcli 1, -2, and -3 genes are expressed in the outer dental epitheliumand stratum intermedium during tooth development. TheNotch genes are already downregulated in the future innerdental epithelial cells prior to the bud stage, which is earlier

j Dent Res 77(8) 1998 1593

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1594 Sahllberg et al.

IFigure 4. Immunohistochemical analysis of laminin-5 (A,C,E,G) and EHS-laminin (B,D,F,H) expression in developing murine molars. (A,B)Frontal sections of an E13 mouse embryo head showing both mandibular and maxillary tooth buds. Laminini-5 is disappearing from thebasement membranes of the dental epithelium (arrow). EHS-laminin is seen in all the basement membranes, including blood vessels. (C,D)Sagittal sections of E17 mandibles. EHS-laminin expression is seen in the basement membranes of both inner (arrows) and outer (arrowheads)dental epithelium, whereas laminin-5 is seen only in the outer dental epithelium. (E,F) Frontal section of one-day postnatal molar. Laminin-5protein is detected only in the basement membranes of the dental lamina and the oral epithelium. EHS-laminiin is seen in all basementmembranes, including blood vessels. (G,H) Higher magnification of ameloblasts at postnatal day 7. Laminin-5 but not EHS-lamirnin is locatedon the basal surfaces of ameloblasts (arrows). EHS-laminin is intensely expressed in the stellate reticulum. Enamel matrix shows nonspecificstaining (arrowhead). a = ameloblasts; e = enamel.

than laminin y2 (Mitsiadis et al., 1995). In many embryonictissues, the downregulation of Notch genes is associatedwith the acquisition of new cell fates (Artavanis-Tsakonas etal., 1995). The Notch genes are not expressed in theameloblasts at any stage.

The expression of laminin y2 in the ameloblasts correlatesobviously with the expression of the structural componentsof the enamel matrix. These genes include amelogenin,tuftelin, and ameloblastin (Fong et al., 1996; Inage et al., 1996;Krebsbach et al., 1996; Zeichner-David M. et al., 1997). Ofthese three genes, amelogenin shows the closest similarity tolaminin y2 expression in mature secretory ameloblasts. Inaddition, the expression of the Bmp-5 gene in theameloblasts appears to correlate closely with that of laminin-5 (Aberg et al., 1997). Bmp-5 may be involved in autocrineregulation of gene expression in ameloblasts, perhaps alsoregulating lamiinin y2 in the ameloblasts. Several growthfactors in the TgfI superfamily are expressed in theodontoblasts prior to the terminal differentiation ofamelob]asts. These include Tgfp-l, -2, and -3 as well asBmps-2, -4, and -7 (Pelton et al., 1991; Vaahtokari et al., 1991;

Aberg et al., 1997). These signal molecules could act asparacrine signals secreted by the odontoblasts, regulatingameloblast differentiation and perhaps also controllinglaminin y2 gene expression.

In developing as well as in adult tissues, laminin-5 ispredominantly associated with hemidesmosomes joining thebasal surfaces of epithelial cells to the underlying basallamina, and it is expressed widely in basal cells of the skin(Masunaga et al., 1996). Interestingly, the lainminii yy2 gene isspecifically expressed by the junctional epithelium joiningthe tooth crown to the gingiva (Hormia et al., 1998). At thislocation, the epithelium has a unique function as it connectsgingival tissue to the inorganic enamel.

Mutations in the genes encoding the different subunits oflaminin-5 have been identified as causes in the humandisease epidermolysis bullosa, specifically its junctional type(Aberdam et al., 1994b; Pulkkinen et al., 1994). The symp-toms in the skin involve severe blistering, which resultsfrom detachment of the epidermis from the underlyingdermis. Hence, laminin-5 is necessary for the stability of theepidermal-dermal junction.

j Deiit Res 77(8) 1998

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

Laminin-5 Expression in Ameloblasts 1595

We speculate that laminin-5 expressed by secretoryameloblasts and deposited to their secretory ends may haverelated unique functions during enamel formation andprovide special attachment of the seceretory cells to theenamel matrix. This attachment apparently differs from thatof the basal surfaces of other epithelial cells, becausehemidesmosomes are not seen in ameloblasts before thematuration stage (Ten Cate, 1994). However, lung epitheliaalso lack hemidesmosomes, although they contain laminin-5(Aberdam et al., 1994a).

Aberdam et al. (1994a) have previously localizedtranscripts of the cx3, 33, and y2 chains of laminin-5 inameloblasts. They also analyzed the protein expression of allthree subunits of laminin-5, and localized them both at thebasal and apical surfaces of ameloblasts. There is adiscrepancy with our study, since we saw only the proteinat the enamel surface corresponding to the basal ends of theameloblasts.

Interestingly, patients with the junctional type of epider-molysis bullosa have hypoplastic defects in their enamel(Wright et al., 1993). Our observations on the expression oflaminin-5 at the RNA and protein levels strongly suggestthat, as at the epidermal-dermal junction, laminin-5 also hasa necessary function at the ameloblast-enamel junction.Interestingly, when enamel hypoplasia is caused byfluorosis in experimental animals, it is associated withdetachment of the ameloblast layer from the enamel surface(Ekstrand et al., 1981). This could be the pathogeneticmechanism in the enamel hypoplasia caused by mutationsin laminin-5 genes. In conclusion, we suggest that thefunction of laminin-5 may be to strengthen the anchoring ofthe secretory and/or maturation ameloblasts to the enamelmatrix.

AcknowledgmentsThe skillful technical assistance of Ms. Maire Holopainen,Ms. Merja Makinen, and Ms. Riikka Santalahti is gratefullyacknowledged. The work was supported by grants from theFinnish Academy and the Human Frontier Science ProgramNo. RG 558/95.

ReferencesAberdam D, Aguzzi A, Baudoin C, Galliano MF, Ortonne JP,

Meneguzzi G (1994a). Developmental expression of niceinadhesion protein (laminin-5) subunits suggests multiplemorphogenic roles. Cell Adhes Commun 2:115-129.

Aberdam D, Galliano MF, Vailly J, Pulkkinen L, Bonifas J,Christiano AM, et al. (1994b). Herlitz's junctionalepidermolysis bullosa is linked to mutations in the gene(lam (2)) for y2 subunit of nicein/kalinin (LAMININ-5).Nature Genet 6:299-304.

Aberg T, Wozney J, Thesleff I (1997). Expression patterns ofBMPs in the developing mouse tooth suggest roles inmorphogenesis and cell differentiation. Dev Dynam210:383-396.

Artavanis-Tsakonas S, Matsuno K, Fortini ME (1995). Notchsignaling. Science 268:225-232.

Burgeson RE, Chiquet M, Deutzmann R, Ekblom P, Engel J,Kleinman H, et al. (1994). A new nomenclature for thelaminins. Matrix Biol 14:209-211.

Carter WG, Ryan MC, Gahr PJ (1991). Epiligrin, a new celladhesion ligand for integrin 03031 in epithelial basementmembranes. Cell 65:599-610.

Ekstrand J, Lange A, Ekberg 0, Hammarstrom L (1981).Relationship between plasma, dentin and bone fluorideconcentrations in rats following long-term fluorideadministration. Acta Pharmacol Toxicol 48:433-437.

Engvall E, Wewer UM (1996). Domains of laminin. J CellBiochem 61:493-501.

Fong CD, Hammarstrom L, Lundmark C, Wurtz T, Slaby I(1996). Expression patterns of RNAs for amelin andamelogenin in developing rat molars and incisors. AdvDent Res 10:195-200.

Gene expression in tooth (www-database), http://honeybee.helsinki.fi/toothexp. Developmental BiologyProgramme of the University of Helsinki, 1996.

Hormia M, Sahlberg C, Thesleff I, Airenne T (1998). Theepithelium-tooth interface-a basal lamina rich in laminin-5 and lacking other known laminin isoforms. J Dent Res77:1481-1487.

Inage T, Shimokawa H, Wakao K, Sasaki S. (1996). Geneexpression and localization of amelogenin in the rat incisor.Adv Dent Res 10:201-207.

Kleinman HK, Weeks BS, Schnaper HW, Kibbey MC,Yamamura K, Grant DS (1993). The laminins: a family ofbasement membrane glycoproteins important in celldifferentiation and tumor metastases. Vitamins andHormones 47:161-186.

Krebsbach PH, Lee SK, Matsuki Y, Kozak CA, Yamada KM,Yamada Y (1996). Full-length sequence, localization, andchromosomal mapping of ameloblastin. A novel tooth-specific gene. J Biol Chem 27:4431-4435.

Langhofer M, Hopkinson SB, Jones JC (1993). The matrixsecreted by 804G cells contains laminin related componentsthat participate in hemidesmosome assembly in vitro. J CellSci 105:753-764.

Masunaga T, Shimizu H, Ishiko A, Tomita Y, Aberdam D,Ortonne JP, et al. (1996). Localization of laminin-5 in theepidermal basement membrane. J Histochem Cytochem44:1223-1230.

Mitsiadis TA, Lardelli M, Lendahl U, Thesleff I (1995). Titleexpression of notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in thedeveloping mouse tooth and associated with determinationof ameloblast cell fate. J Cell Biol 130:407-418.

Pelton RW, Saxena B, Jones M, Moses HL, Gold LI (1991).Immunohistochemical localization of TGF,B-1, TGF,B-2 andTGF3-3 in the mouse embryo: expression patterns suggestmultiple roles during embryonic development. J Cell Biol115:1091-1105.

Pulkkinen L, Christiano AM, Airenne T, Haakana H,Tryggvason K, Uitto J (1994). Mutations in the gamma 2chain gene (LAMC2) of kalinin/laminin 5 in the junctionalforms of epidermolysis bullosa. Nature Genet 6:293-297.

Rousselle P, Lunstrum GP, Keene DR, Burgeson RE (1991).Kalinin: an epithelium-specific basement membraneadhesion molecule that is a component of anchoring

j Dent Res 77(8) 1998

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from

1596 Sahlberg et al.

filaments. J Cell Biol 114:567-576.Ruch JV (1987). Determinisms of odontogenesis. Revisiones Sobre

Biologia Celular 14:1-112.Ruch JV, Lesot H, Begue-Kirn C (1995). Odontoblast

differentiation. Int J Dev Biol 39:51-68.Salmivirta K, Gullberg D, Hirsch E, Altruda F, Ekblom P (1996).

Integrin subunit expression associated with epithelial-mesenchymal interactions during murine tooth develop-ment. Dev Dynam 205:104-113.

Sugiyama S, Utani A, Yamada S, Kozak CA, Yamada Y (1995).Cloning and expression of the mouse laminin gamma 2(B2t) chain, a subunit of epithelial cell laminin. Eur IBiochem 228:120-128.

Ten Cate AR (1994). Oral histology. Development, structure,and function. 4th ed. St. Louis: Mosby Year Book Inc.

Thesleff I, Sahlberg C (1996). Growth factors as inductivesignals regulating tooth morphogenesis. Semin Cell Dev Biol7:185-193.

Thesleff I, Barach HJ, Foidart JM, Vaheri A, Pratt RM, MartinGR (1981). Changes in the distribution of type IV collagen,laminin, proteoglycan, and fibronectin during mouse toothdevelopment. Dev Biol 81:182-192.

Thesleff I, Vaahtokari A, Partanen AM (1995). Regulation oforganogenesis. Common molecular mechanisms regulatingthe development of teeth and other organs. Int J Dev Biol39:35-50.

Timpl R, Brown JC (1994). The laminins. Matrix Biol 14:275-281.Tryggvason K (1993). The laminin family. Curr Opin Cell Biol

5:877-882.Vaahtokari A, Vainio S, Thesleff I (1991). Associations between

transforming growth factor beta 1 RNA expression andepithelial-mesenchymal interactions during toothmorphogenesis. Development 113:985-994.

Wilkinson DG, Green J (1990). In situ hybridization and thethree-dimensional reconstruction of serial sections. In:Postimplantation mammalian embryos. Copp AJ, CockroftDL, editors. Oxford: Oxford University Press, pp. 155-171.

Wright JT, Johnson LB, Fine JD (1993). Developmental defects ofenamel in humans with hereditary epidermolysis bullosa.Arch Oral Biol 38:945-955.

Zeichner-David M, Vo H, Tan H, Diekwisch T, Berman B,Thiemann F, et al. (1997). Timing of the expression ofenamel gene products during mouse tooth development.Int J Dev Biol 41:27-38.

j Dent Res 77(8) 1998

at PENNSYLVANIA STATE UNIV on March 5, 2014 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from