tissue interaction isn embryonic mouse tooth germs · these conclusions indicate that tooth...

/ . Embryol exp. Morph. Vol. 24, 1, pp. 173-186, 1970 173

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Tissue interactions in embryonic mousetooth germs

II. The inductive role of the dental papilla

By EDWARD J. KOLLAR1 AND GRACE R. BAIRD2

From the Department of Anatomy and Zoller Dental Clinic,The University of Chicago

SUMMARYThe response of embryonic mouse dental epithelium and mesoderm to tissues of ectopic

origin was examined. Isolated molar or incisor mesoderm was confronted with epitheliumisolated from the plantar surface of the embryonic mouse foot plate or from the snout.

Harmoniously structured teeth were formed from the foot epithelium and incisor or molarmesoderm. These data are interpreted as an unequivocal demonstration of the inductive roleof the dental mesenchyme.

Teeth were absent in confrontations of dental mesenchyme and snout epithelium. Thepresence of hair follicles in these explants is described and discussed with reference to otherintegumental epithelio-mesenchymal interactions.

Dental epithelium forms keratinizing surface-like epithelium and invading bands ofepithelium in association with foot mesoderm; definitive structures are not formed.

On the other hand, when incisor or molar epithelium is associated with snout mesoderm,hair follicles are seen in addition to keratinizing surface-like epithelial configurations.

The roles of the epithelial and mesenchymal tissues and the nature of epithelio-mesen-chymal interactions in the developing mouse integument are discussed.

INTRODUCTION

Previous studies of tooth development have provided a number of importantconclusions. (1) Both epithelium and mesenchyme must be present if toothdevelopment is to proceed (Huggins, McCarroll &Dahlberg, 1934; Koch, 1967;Kollar & Baird, 1969). (2) The epithelium and mesoderm will develop typicalmatrices if physically separated by Millipore filters (Koch, 1967). (3) The struc-tural specificity for the shape of the tooth germ resides in the mesoderm; typicalincisiform and molariform patterns of cytodifferentiation are directed by themesoderm (Kollar & Baird, 1969, 1970a, b). (4) The enamel organs from em-bryonic mice (Kollar & Baird, 1970#) and rabbits (Glasstone, 1952; Slavkin &

1 Author's address: Department of Anatomy, The University of Chicago, Chicago,Illinois 60637, U.S.A.

2 Author's address: Zoller Dental Clinic, The University of Chicago, Chicago, Illinois60637, U.S.A.

174 E. J. KOLLAR AND G. R. BAIRD

Bavetta, 1968) remain plastic and will regulate far into the developmentalperiod. (5) The lip-furrow epithelium, an epithelium temporally and spatiallyrelated to the incisor enamel organ, is able to regulate into harmonious toothgerms in the presence of incisor or molar mesoderm. Thus, the dental papillacan elicit new developmental expressions from the lip-furrow epithelium. Thesedata imply an inductive interaction between the epithelium and the dentalpapilla (Kollar & Baird, 1970a).

These conclusions indicate that tooth development is the product of anepithelio-mesenchymal tissue interaction similar to interactions described formany other structures. However, an unequivocal demonstration that dentalpapillae induce tooth germs has been lacking.

This investigation describes experiments in which dental papillae fromembryonic mice are associated with epithelium from embryonic snout and footplate (plantar surface). The dental papillae induce recognizable tooth structuresin the plantar surface epithelium from the foot plates of embryonic mice;matrix synthesis is induced in this surface epithelium. In addition, the previouslydescribed versatile developmental performance of the enamel organ (Kollar &Baird, 1970«) is confirmed and is extended to include the ability of the enamelepithelium to reorganize into0 surface epithelium and hair follicles when snoutmesoderm is present.

MATERIALS AND METHODS

Tissue sources

Molar and incisor tooth germs, snout skin (upper lip) containing the mys-tacial vibrissae primordia, and hairless plantar surface of posterior foot plateswere dissected from embryonic C57/10 mouse embryos. Embryonic stages,spanning 12-16 days of gestation, were determined by the appearance of avaginal plug and confirmed by the staging criteria of Griineberg (1943).

Molar and incisor tooth germs from the mandibles of 14- and 16-day-oldembryonic mice were used in this investigation. The in vivo and in vitro develop-ment of these tooth germs have been repeatedly described (Cohn, 1957; Hay,1961; Glasstone, 1967; Kollar & Baird, 1968, 1969, 1970a). The reader shouldconsult these papers.

The structure and development of mystacial vibrissae follicles (Davidson &Hardy, 1952; Hardy, 1949, 1951, 1968; Kollar, 1966) as well as the response ofthese follicles in tissue culture (Hardy, 1968; Kollar, 1966) have been describedin detail. At 12 and 13 days of embryonic development, the upper lip containsa number of mystacial follicle primordia. The epithelium thickens to formplacodes in association with mesodermal condensations. The epithelium con-tinues to invade the mesoderm during subsequent days of development andeventually gives rise to hair follicles with keratinizing hair shafts. The meso-dermal condensations are incorporated into the epithelial follicles as dermalpapillae.

Mouse tooth germs. II 175The plantar surfaces of posterior foot plates of 14- and 15-day-old embryos

were chosen because this epithelium is hairless, and, at this developmentalperiod, the mesodermal condensations associated with foot pads are beginningto develop. The dissection of this integumental site was performed so that onlycentral portions of the plantar surfaces were excised. The tissue was trimmedso that contaminating skin containing hair follicle primordia was excluded.

Tissue separation and recombination

Trypsinization was accomplished by incubating excised tissue fragmentswith a 1 % solution of trypsin (Bacto-Difco, 1:250) in Tyrode's solution at4 °C for 1-2 h. The 12-day snout skin required 1 h of incubation; 16-dayincisor germs required a minimum of 2 h incubation. This procedure is describedin detail elsewhere (Kollar & Baird, 1969). The epithelial and mesodermal com-ponents separate at the basement membrane and can be handled as uncontami-nated sheets of epithelium and mesoderm (Kollar, 1966; Kollar, 1970; Kollar& Baird, 1969, 1970a).

The isolated components were recombined as control and experimentalcombinations. The ability of the isolated tooth components to develop inculture and to produce type specific structures has already been described(Kollar & Baird, 1968, 1969). Control recombinants of snout epithelium(SE12_13) and snout mesoderm (SM12_13) as well as the control combinations offoot epithelium (FE14_15) associated with its homologous foot mesoderm(FM14_15) were reconstructed. These data are described elsewhere (Kollar, 1970).

Culture methods

The isolated tissue fragments were stored in a solution of foetal bovineserum and Tyrode (1:1, v/v) and were transferred to Falcon organ culturedishes containing 1 ml of Eagle's basal medium containing 10 % foetal bovineserum, 1 % glutamine, 0-4 % agar and 50 units/ml each of penicillin andstreptomycin. The explants were incubated at 37 °C in a humidified 5 % CO2-95 % air-gas mixture and were allowed to cohere for 1 or 2 days on this medium.

When the explants had re-established a stable junction between the epitheliumand mesoderm and could be transferred safely, they were grafted into theanterior chambers of homologous mice eyes. The grafted explants were allowedto grow for 1-2 weeks before harvesting.

Histology

The explants were fixed in Zenker's acetic acid and decalcified with Versene(Schmidt, 1956). The tissue was paraffin-embedded, serially sectioned at 7 /i,and stained with hematoxylin and eosin (H and E) or Masson's trichrome(MT).


RESULTS

Recombinations of dental mesoderm and foreign epithelium

(1) Dental mesenchyme combined with foot epithelium

The plantar surface of the posterior foot plate does not produce structurallycomplex epithelial adnexa. Thus, this set of experiments provided a crucial testof the inductive capabilities of the dental mesenchyme. The foot epithelium con-sistently produced surface epithelium characterized by extensive surface kera-tinization. The presence of tooth structures in these explants provided anunequivocal and dramatic demonstration of the inductive properties of themesenchyme and the developmental plasticity of the epithelium.

When 15-day-old molar mesoderm was confronted with foot epitheliumfrom 15-day-old embryos (MM15FE15) and allowed to grow in the eye for 2weeks unmistakable teeth were produced (Fig. 1A-C). Similarly, when youngertissue components were combined (IM14FE14) tooth structures were recoveredafter 2 weeks of growth (Fig. ID).

The tooth structures could be serially traced to the heavily keratinizingsurface-like epithelial cysts and did not occur in isolation from the grafted epi-thelium. The deposition of matrix and the formation of dentin and enamel wereharmoniously structured.

(2) Dental mesenchyme combined with snout epithelium

The development of snout epithelium confronted by dental mesenchyme wasrestricted to a more characteristic surface-like epithelial pattern. Stratified

Scale line represents 50/* throughout except Fig. 1E.

FIGURE 1

(A) An harmonious tooth developing after 2 weeks in the anterior chamber froman explant of molar mesoderm and plantar surface foot epithelium (MM15FE15)from a 15-day-old embryo. Note the keratinizing epithelium (k) in close associationwith the tooth germ. MT. x 300.

(B) A tooth-germ construction formed after 2 weeks in the anterior chamber. Thisgraft was composed of molar mesenchyme and foot epithelium (MM15FE15) froma 15-day-old embryo. H and E. x 600.(C) An harmonious tooth germ contiguous with a heavily keratinizing epithelium(k). This explant (MM15FE15) was grown in the anterior chamber for two weeks.MT. x300.(D) A tooth primordium developing in an explant (IM14FE14) after 2 weeks inthe anterior chamber. Note the keratinizing epithelium (k) and an additional areasuggestive of a tissue interaction (/). H and E. x 300.(E) Integumental adnexa developing in a graft of dental mesenchyme and snoutepithelium (IM14SE12) 2 weeks after explantation. Note the surface-like epithelium(se), the hair follicle with papilla (p) and keratinizing hair shaft (ks), and thesebaceous gland (sg). H and E. x 1000. Scale line = 15//.(F) Hair follicles developing in an explant of molar mesenchyme and snout epi-thelium (MM14SE12) after 1 week in culture. MT. x 750.

Mouse tooth germs. II 111

EM B 24


keratinizing epithelium and hair follicles were present in these grafts (Fig. 1E,F). Sebaceous glands were associated with the hair follicles. Because the presenceof hair follicles in grafts composed of snout epithelium and dental mesenchymewere unexpected and of unusual interest, these experiments were repeated. Thedental mesenchyme was severely trimmed in order to ensure that no othermandibular mesenchyme was present except the papilla and a narrow strip ofsurrounding mesenchyme. These experiments confirmed our earlier finding;hair follicles were present in these explants as well.

Recombinations of dental epithelium and foreign mesenchyme

(1) Dental epithelium combined with foot mesenchyme

The dental epithelium developed surface-like epithelial sheets; stratificationand heavy keratinization were present. In addition, the basal layer of theseepithelial sheets underwent extensive downgrowths into the foreign foot meso-derm (Fig. 2 A, B). The pattern of collagen deposition was of interest in thesegrafts. The invading epithelium appeared to be walled off by heavy collagendeposition around the invading epithelium (Fig. 2C, D).

Several grafts provided insights into the source of the epithelial proliferation(Fig. 2C, E). When the plane of section was favorable, it was possible to tracethe epithelial downgrowths serially to a section that appeared to be the originalgrafted enamel organ. For example, extensive epithelial proliferation appearedto originate from a tissue configuration similar to the enamel organ (Fig. 2E).Note that in the enamel organ-like configuration the cellular pattern includesa stellate reticulum. Similar observations were made when the incisor enamelorgan (IE15FM15) was the source of the epithelium confronted by ectopic footmesoderm (Fig. 2C).

Of unusual interest in these observations was the source of the proliferationfrom these enamel organ-like tissue patterns. The proliferation was mostextensive from the outer enamel epithelium. Similar proliferations were seenalong the inner enamel epithelium, but they were not as extensive as thoseapparently derived from the outer enamel epithelium.

(2) Dental epithelium combined with snout skin mesenchyme

The dental epithelium confronted with snout mesenchyme behaved in afashion similar to its response to foot mesenchyme. Incisor or molar enamelorgan epithelium from 14- and 15-day embryonic tooth germs displayed re-markable proliferative and invasive properties when associated with mesen-chyme from 12- or 13-day-old embryonic snout skin. The dental epitheliumproduced keratinizing surface-like epithelium and deeply invaginating tonguesof epithelium. The pattern of epithelial invasion into this ectopic mesenchymeresembled abortive enamel organ formations (Fig. 3A-C). At no time, how-ever, did the dental epithelium or the snout mesenchyme suggest cellular


FIGURE 2

(A) This explant consists of molar epithelium and foot mesoderm and displays theextensive and random invasion of the mesoderm by dental epithelium. MT. x 750.

(B) Incisor enamel organ epithelium invades the foot mesoderm incorporatingsmall clusters of mesodermal cells. MT. x 750.

(C) Heavy deposition of collagen (c) at the interface between incisor epitheliumand foot mesoderm. MT. x 750.

(D) Collagen deposition (c) at the interface between molar epithelium and footmesoderm. MT. x 500.

(E) An enamel organ-like epithelial configuration. Note the surface epithelium(se), the stellate reticulum-like (sr) epithelium and the overall shape of the epithelium.Epithelial proliferation can be seen budding from the enamel organ-like structure.This explant (ME15FM15) was grown for 2 weeks. MT. x 750.


patterns typical of the inner enamel organ or an odontoblast layer of the mesen-chyme; tooth structures were never seen in these explants. However, hairfollicles were observed (Fig. 3D-F). The follicles were both pelage- and vibrissal-like. Occasionally, the follicle structure appeared aberrant, but, even in thesecases, keratinizing hair shafts were present (Fig. 3E, F). In addition to thehair follicles, occasional small clusters of sebaceous glands were observed.

DISCUSSION

Induction by the dental papilla

The murine dental papilla elicits new developmental expressions fromectopic embryonic mouse epithelium. The deposition of dentin and enamelmatrices in histotypic patterns clearly recognizable as tooth constructions indi-cates that the dental papillae act inductively during the ontogeny of teeth. Thus,these data provide a stringent test in that an unrelated non-dental epithelium inthe presence of mesodermal tissue produces structural equivalents of the epi-thelium homologous to the inducing mesoderm. These data extend the obser-vations of Lillie & Wang (1941, 1944), Wang (1943), Cairns & Saunders (1954),Gomot (1958), Rawles (1963), Sengel (1964), and others who have demonstratedthe inductive nature of the local mesodermal components in the integument,[ndeed, studies of epithelio-mesenchymal interactions in general confirm thenotion that the mesodermal component is necessary for the induction andmaintenance of ectodermal or endodermal structures during the early stages ofdevelopment (see Wessells (1967), Grobstein (1967) and McLoughlin (1968)for reviews of the properties of epithelio-mesenchymal interactions in em-bryonic systems).

These data confirm our earlier data (Kollar & Baird, 1970 a) which showedthat a more closely related epithelium, lip-furrow epithelium, can participatewith the dental mesoderm to produce perfectly harmonious teeth. It is of interest

FIGURE 3

(A) This explant (ME15SM12) demonstrates the invasion of the molar epitheliuminto snout mesoderm. MT. x 625.

(B) Incisor epithelium incorporating snout mesoderm in an explant (IE14SM12)grown for 2 weeks. H and E. x 625.

(C) Incisor epithelium invading snout mesoderm (IE14SM12) in a fashion reminis-cent of an enamel organ. H and E. x 750.

(D) Hair follicles and surface-like keratinizing epithelium derived from an ex-plant of incisor epithelium and snout mesoderm (IE14SM12). H and E. x 300.

(E) An aberrant hair follicle developing in an explant (ME15SM12) composed ofmolar epithelium and snout mesoderm. Note the papilla (p) and keratinizing hairshaft (ks). H and E. x 750.

(F) A detail from Fig. E demonstrating the keratinized hair shaft. H and E. x 2800.

182 E. J. KOLLAR AND G. R. BA1RD

that the structural harmony of the tooth germs induced in the lip-furrow epi-thelium is often more obvious than that produced in the foot epithelium. Thereis no doubt, however, that tooth structures are produced from the ectopic sur-face epithelium of the foot plate, although dental constructions easily scoredas incisiform or molariform are less obvious in these latter experimental teeth.None the less, the production of dentin and enamel matrices in normal con-figurations, cytodifferentiation of the epithelium into a basal layer of cells withtall columnar cells that undergo a reversal in nuclear polarity, and specializedsecretory activity are compelling demonstrations of mesodermal induction ininteracting embryonic systems.

In contrast, the absence of tooth structures in experimental confrontationsconsisting of dental mesoderm and snout epithelium requires cautious interpre-tation. Despite the optimal culture conditions afforded by the intraocular site,the snout epithelium remained refractory to the inductive influence of the dentalmesoderm. Certainly, the data of Rawles (1963) that clearly establish the im-portance of tissue age and the establishment of developmental stability in theepithelium must be considered here. It may be inferred from the present datathat snout epithelium at 12 and 13 days of gestation has already stabilized andcannot respond to the inductive activity of dental mesoderm. Clearly, thesenegative data do not diminish the impact of the demonstration of the inductiverole of dental mesoderm; rather, this series of experiments suggests that snout-skin epithelium must be examined at earlier ages.

In addition, this series of experiments can be viewed as an additional controlfor other combinations of dental mesoderm and ectopic epithelium. Since thedental mesoderm from a single litter was combined with snout and foot epi-thelium, the ectopic teeth formed by the foot epithelium cannot be the result ofrandom contamination of the dental mesoderm with dental epithelial frag-ments. The complete absence of tooth structures or developing fragments ofcontaminating dental epithelia in the snout epithelium-dental mesoderm con-frontations provides additional confirmation of the effectiveness and reliabilityof tissue separations after tryptic digestion.

The development of hair follicles in those explants composed of snout epi-thelium and dental mesenchyme provides further insights into the develop-mental performance of these two tissues. The persistence of hair follicles incarefully controlled experiments that excluded mandibular mesenchyme withpotential hair-follicle-inducing qualities and the absence of hair follicles inother combinations of dental mesenchyme with homologous or heterologousepithelia confirm the view that snout epithelium and dental mesenchyme canparticipate in hair follicle development. At first view these data appear contra-dictory; however, these data are supported by similar observations in developingchick skin.

Rawles (1963) demonstrated that in the closely related avian feather-bearingdorsal skin and scaled metatarsal skin, developmentally advanced and stabilized

Mouse tooth germs. II 183back skin produces typical feathers when confronted with mesoderm from thescale-bearing mesoderm from the metatarsal region. In contrast, metatarsalmesoderm induces scales in younger feather-producing back skin. These datalead to the conclusion that a stabilized epithelium can produce type-specificstructures when associated with an ectopic but related mesoderm.

The similarities between the snout hair follicle and dental structures must beconsidered in this context. The developmental origins of the snout and man-dibular mesenchyme from the cranial neural crest, the similar trigeminal inner-vation to vibrissae follicles and teeth, and the anatomically similar develop-mental sequence in the initial development of the primordia of these dissimilarstructures suggest that the phenomenon reported here may be similar to thatdescribed in avian skin. Thus, if 12- and 13-day-old embryonic snout epitheliumhas indeed stabilized and lost its regulative capabilities, the response of thisepithelium to dental mesenchyme would be to produce type specific structures:hair follicles. This view must await confirmation from the response of 11-daysnout epithelium confronted by dental mesenchyme. Younger, and thereforemore plastic, epithelium should respond in predictable fashion; in the presence ofdental mesenchyme, the more labile epithelium should produce dental structures.

These data demonstrate the inductive and supportive qualities of the mesen-chyme in developing integumental systems and, once again, emphasize theimportance of examining the temporal and spatial parameters that influenceepithelio-mesenchymal interactions.

The response of the dental epithelium to foreign mesoderm

The dental epithelium does not produce recognizable structures when asso-ciated with foot mesoderm. Instead, the epithelium invades the mesenchymeand proliferates as crenulated epithelial bands. The observation that the epi-thelial proliferation originates, in part, from the outer epithelium of the enamelorgan supports the view that this epithelium retains the properties of the stratumgerminativum, and adds further support to the notion that during recon-struction of harmonious teeth from fragments of the enamel organ (Kollar &Baird, 1970tf, b) or in heterologous combinations of dental mesenchyme andenamel organs (Kollar & Baird, 1969) the outer enamel epithelium may con-tribute to the reorganization of the explanted dental epithelium.

The dental epithelium can participate in hair-shaft production when con-fronted by snout mesoderm containing the mystacial vibrissae dermal condensa-tions. This ability of the dental epithelium to organize into new histotypicpatterns, to suppress enamel matrix synthesis, and, instead, to produceorganized keratinizing hair shafts and sebaceous glands, corroborates theinductive role of the vibrissae dermal condensations (Kollar, 1966, 1970) andthe plasticity of the enamel organ epithelium. Our previous findings (Kollar& Baird, 1970 a) that the enamel organs of incisor germs from 16-day-oldembryos can reconstruct complete tooth germs and keratinizing surface epithelia


are supported by these new observations of regulative ability in the dentalepithelium.

The pattern of epithelial invasion into an ectopic mesoderm also providessome insight into the nature of the interactions between the epithelium and themesoderm. In those cases in which the dental epithelium is confronted withsnout mesoderm the topography of the epithelium resembles an enamel organrather than the basal layer of surface epithelium with follicle adnexa. The epi-thelium invades the mesoderm as crenulated tongues of epithelium incorporat-ing mesodermal cells into the invaginations of the epithelium. This pattern ofepithelial invasion is suggestive of the invasion pattern of the enamel organ.Often these structures were induced to form keratinizing hair shafts andsebaceous glands. Although some of these tissue configurations are not normalhair follicles, many other follicle structures are harmonious in all respects.Similarly, the dental epithelium invades the mesoderm of the foot plate in afashion reminiscent of the enamel organ.

These data suggest that the invasiveness of the epithelium in relation to themesoderm may be determined, in part, by inherent properties of the epithelium.On the other hand, the random nature of the epithelial invasion in these hetero-logous combinations suggests that the usual incisiform, molariform and folliclepatterns are stabilized by some properties of homologous or closely relatedmesoderm. Thus, appropriate structural relationships are established by aninterplay between the invasive properties of the epithelium and the modelingproperties of the local inductive mesoderm. The depth of epithelial invasion, thedefinitive shape of the invading epithelium, and the spatial relationship of theepithelium to the inductive papilla appear to be determined by the mesoderm.

The data discussed here suggest that, as suspected, the development of teethis the result of an epithelio-mesenchymal interaction not unlike many otherdeveloping systems in the avian and mammalian embryo. Once again, the lackof information concerning the processes involved in the inductive event is themost intriguing aspect of this problem.

RESUME

Interactions tissulaires des germes dent air es de Souris.If. Le role indueteur de la pap i lie dent a ire

La reponse de l'epithelium dentaire de l'embryon de Souris ainsi que du mesoderme al'egard de tissus d'origine ectopique a ete examinee. Du mesoderme molaire ou incisif isolea ete combine a de l'epithelium isole de la surface de la plaque plantaire d'embryon de sourisou a partir du museau.

Des dents harmonieusement constitutes ont ete formees a partir de l'epithelium de la pattecombine a du mesoderme incisif ou molaire. Ces resultats sont interpretes comme une demon-stration non equivoque du role inducteur du mesenchyme dentaire.

Des dents ne se sont pas formees a la suite de la combinaison de mesoderme dentaire et del'epithelium du museau. La presence de follicule pileux dans ces explants est decrite et dis-cutee en relation avec les autres interactions epithelium-mesenchyme.

Mouse tooth germs. II 185L'epithelium dentaire forme un epithelium a keratinisation de surface et des travees

epitheliales profondes en reponse a la combinaison avec le mesoderme du pied; des structuresdeflnies ne sont pas formees.

Par ailleurs, lorsque l'epithelium incisif ou molaire est associe a du mesoderme du museau,on voit des follicules pileux s'ajouter aux formations epitheliales keratinisees en surface.

Les roles des tissus epitheliaux et mesenchymateux et la nature des interactions epithelium-mesenchyme dans le developpement du tegument de la souris sont discutes.

The authors wish to thank Dr Benson E. Ginsburg who generously made available hisanimal colony and animal care facilities. This research was supported by a grant from theAmerican Cancer Society (ACS-IN-41-H). Finally, we wish to thank the Inland Steel-Ryerson Foundation for a Faculty Fellowship to E.J.K.

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(Manuscript received 11 August 1969)

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