Neonatal seminal vesicle mesenchyme induces a new morphological and
functional phenotype in the epithelia of adult ureter and ductus deferens
GERALD R. CUNHA1*, PETER YOUNG1, STEPHEN J. HIGGINS2 and PAUL S. COOKE3
1 Department of Anatomy and Center for Reproductive Sciences, University of California, San Francisco, CA 94143, USA2Department of Biochemistry, University of Leeds, Leeds LS2 9JT, UK3Department of Veterinary Biosciences, University of Illinois, Urbana, IL 61801, USA
* To whom correspondence and reprint requests should be addressed
Summary
Mesenchyme from neonatal mouse and rat seminalvesicles (SVM) was grown in association with postnatal(adult) epithelial cells from the ureter (URE) and ductusdeferens (DDE) in chimeric tissue recombinants com-posed of mouse mesenchyme and rat epithelium or viceversa. Functional cytodifferentiation was examined inthese SVM+URE and SVM+DDE tissue recombinantswith antibodies against major androgen-dependentseminal-vesicle-specific secretory proteins. Adult DDEand URE were induced to express seminal cytodifferen-tiation and produced the complete spectrum of majorseminal vesicle secretory (SVS) proteins. The SVSproteins produced were specific for the species thatprovided the epithelium. In the case of SVM+URErecombinants, the URE, which normally lacks androgenreceptors (AR), expressed AR. These results demon-
strate that adult epithelial cells retain a developmentalplasticity equivalent to their undifferentiated fetalcounterparts and are capable of being reprogrammed toexpress a completely new morphological, biochemicaland functional phenotype.
Mesenchyme, defined as loose embryonic connectivetissue, is critically involved in a myriad of secondaryinductions involved in the development of the integu-mental (Kratochwil, 1987; Sawyer, 1983), urinary(Ekblom, 1984; Saxe"n, 1970), gastrointestinal (Haffenetal. 1987; Kedinger et al. 1986), skeletal (Hall, 1987;Wolpert, 1981) and urogenital systems (Cunha, 1976a;Cunha etal. 1980a, 1983a, 1987). Mesenchyme inducesspecific patterns of epithelial morphogenesis resultingin the formation of a broad spectrum of epithelial formssuch as branched ductal networks, planar epithelialsurfaces, simple epithelial ducts and tubules and manyhighly unique epithelial patterns found in teeth,feathers and sense organs, to name but a few (Bernfieldet al. 1973, 1984; Cunha, 19766; Cunha et al. 19806,19836; Higginser al. 1989a,6; Rawles, 1963). Extensiveevidence suggests that during these morphogeneticprocesses epithelial proliferation is regulated by themesenchyme, perhaps via paracrine factors (Alescioand Piperno, 1967; Bigsby and Cunha, 1986; Chung and
Cunha, 1983; Cooke et al. 1986; Goldin and Wessells,1979; Norman et al. 1986; Rutter et al. 1978).Mesenchyme-induced epithelial development culmi-nates in the emergence of specific types of epithelialcytodifferentiation and the expression of tissue-specificmacromolecules (Cunha et al. 19806, 19836; Haffen etal. 1982; Higgins et al. 1989a,6; Kedinger et al. 1986;Kollar and Baird, 1970).
The adult counterpart of embryonic mesenchyme isstroma, which constitutes the non-epithelial componentof an organ. The predominant cells of stroma arefibroblasts and smooth muscle. The role of epithelial—stromal interactions in adulthood has received consider-ably less attention than embryonic mesenchymal-epithelial interactions. In part this is because adultepithelial cells are thought to be irreversibly deter-mined and terminally differentiated (Slack, 1985).Nonetheless, various lines of evidence support the ideathat adult epithelial cells remain responsive to theinductive influences of stromal cells.
Many examples of developmental plasticity in adult-hood concern the induction of regional variation in
146 G. R. Cunha and others
adult epidermal differentiation by heterotypic dermis(Bernimoulin and Schroeder, 1980; Billingham andSilvers, 1968; Karring et al. 1975; Mackenzie and Hill,1984; Spearman, 1974). However, these findings arebest characterized as connective tissue-induced modu-lations in epidermal differentiation since such minorchanges in epidermal thickness and patterns of keratin-ization are encompassed within the basic stratifiedsquamous epidermal phenotype. In other experimentalmodels, mesenchymal or stromal cells have also elicitedchanges in morphology, differentiation or growth ofadult epithelial cells (Dudek and Lawrence, 1988;Haslam, 1986; McGrath, 1983; Norman et al. 1986;Sakagami et al. 1984; Sakakuraef al. 1979a,6; Sugimuraet al. 1986). On the other hand, the profound changeselicited by urogenital sinus mesenchyme in epitheliumof the adult urinary bladder provides one of the moststriking examples of a mesenchyme-induced alterationin adult epithelial differentiation. In this model,embryonic urogenital sinus mesenchyme elicited pros-tatic differentiation in epithelial cells of the adulturinary bladder, which entailed the morphogenesis of abranched ductal network, a marked stimulation inepithelial proliferation, the differentiation of a simplecolumnar secretory epithelium and the expression ofseveral prostate-specific markers (Cunha et al. 1983i>;Neubauer et al. 1983).
Is such a profound developmental reprogramming asrepresented by this last example more widely applicableto other adult epithelia? Recently, a closely relatedmesenchyme derived from the seminal vesicle (SVM)has been reported to be able to induce seminal vesicle(SV) differentiation from embryonic epithelium fromthe middle and upper Wolffian duct (prospective ductusdeferens and epididymis, respectively). Significantly,the induced epithelial cells expressed the full comp-lement of seminal vesicle secretory (SVS) proteins(Higgins et al. 1989a,6). Since the embryonic Wolffianducts normally give rise to the epithelia of theepididymis, SV, ureter (UR) and ductus deferens(DD), this system provides a suitable model in whichmorphological and functional aspects of the reprogram-ming of postnatal epithelia can be studied.
Materials and methods
AnimalsBalb/c mice and Sprague-Dawley rats were obtained from theCancer Research Laboratory (University of California,Berkeley) and local vendors (Simonsen, Gilroy, CA andBantin-Kingman, Fremont, CA). Adult athymic mice wereobtained from Harlan, Inc. (Indianapolis, IN). Neonatal malerats and mice were used within 24 h of birth (day 0). Allanimals received water and laboratory chow ad libitum andwere housed under standard laboratory conditions.
MaterialsThe preparation and characterization of polyclonal rabbitantibodies (IgG fraction) monospecific for the androgen-dependent proteins (proteins I-V) of rat SV have beendescribed before (Fawell and Higgins, 1986; Fawell etal. 1986,
1987). The antibody to mouse SVS (anti-mouse SVS) hasbeen described by Higgins et al. (1989a). Sources anddescriptions of all reagents used will be found in Higgins et al.(1989a,b)
Preparation of tissue recombinantsSV were excised from 0-day-old neonatal rats and mice. URand DD were excised from 0- to 60-day-old rats and mice. Theepithelium and mesenchyme or stroma of these organs wereseparated following tryptic digestion and recombined asdescribed earlier (Cunha, 1976c; Cunha et al. 1983ft;Sugimura et al. 1986). In homospecific recombinants, themesenchyme and epithelium were from the same species,either mouse or rat. In heterospecific recombinants, tissuesfrom both rat and mouse were combined, eg., mouseSVM+rat DDE or rat SVM+mouse DDE. After overnightculture, the tissue recombinants were grafted under the renalcapsule of adult male hosts anesthetized with Avertin (tertiaryamyl alcohol plus tribromoethanol). For homospecific tissuerecombinants, syngeneic male hosts were used. For hetero-specific recombinants, athymic nude mice were used. Up tosix grafts were placed on each kidney. This study is based onthe analysis of 279 tissue recombinations.
Recovery and processing of tissue recombinantsHosts were killed 4 weeks after grafting and the tissuerecombinants were dissected from the renal capsule. Se-cretion was recovered from within the cystic recombinantssolubilized in SDS and stored as described (Higgins et al.1989a). The remaining tissue was then fixed by immersionovernight in 4% paraformaldehyde, embedded in paraffin,sectioned at 6/mi and air-dried onto poly-L-lysine-coatedslides (Higgins et al. 1989a). For histologic analysis sectionswere stained with hematoxylin and eosin. Nuclear stainingwith Hoechst dye 33258 was as described (Cunha andVanderslice, 1984).
ImmunocytochemistryAnti-mouse SVS and the antibodies against rat SVS proteinsIV and V were used in immunocytochemistry. As describedearlier (Higgins et al. 1989a) the anti-mouse SVS reacts withmouse but not rat SV, while anti-rat SVS IV or V reacts withrat but not mouse SV. The methodology has been described indetail earlier (Higgins et al. 1989a). Briefly, deparaffinizedtissue sections were reacted successively with the primaryantisera, biotinylated donkey anti-rabbit IgG, PBS-Tweenand the Vectastain peroxidase ABC reagent. The coloredreaction product was developed using diaminobenzidine andH2O2 as described (Cunha et al. 1989).
Polyacrylamide gel electrophoresisProtein samples prepared in PBS-SDS were analyzed byelectrophoresis in polyacrylamide (10-20% linear gradient)slab gels using a discontinuous buffer system (Laemrnli, 1970)with 0.1% SDS throughout as described by Brooks andHiggins (1980). Proteins (20-50//g per lane) resolved by thismethod (SDS-PAGE) were visualized by staining for 1 h in0.1 % Coomassie blue in acetic acid:methanol:H2O (1:3:6 byvol) followed by prolonged destaining in the same solutionwithout the dye.
Immunoblotting of SV proteinsMouse SVS proteins were identified immunologically usingWestern blotting (Burnette, 1981) with anti-mouse SVS.However, for the rat SVS proteins the large number ofsamples was more conveniently screened with each of the fiverat SVS antibodies by immunodot blot procedures rather than
Seminal vesicle induction of adult epithelia 147
five separate Western blots on each occasion (Higgins et al.1989a).
Western blotsImmediately after SDS-PAGE, resolved proteins weretransferred electrophoretically from the unstained gels tonitrocellulose sheets using a BioRad transblot apparatuscontaining 0.15 M glycine, 20mM Tris (Fawell et al. 1986).Electrophoretic transfer of proteins to the nitrocellulosesheets was then checked by staining the nitrocellulose paperwith India ink (Hancock and Tsang, 1983).
lmmunodot blotsProtein samples in PBS-SDS were spotted (2/il aliquotscontaining 2//g protein unless otherwise indicated) ontonitrocellulose sheets or strips and air-dried (Higgins et al.1989a).
Processing of blotsWestern and immunodot blots were incubated successively atroom temperature in PBS containing 5 % BSA, PBS, and 5 %BSA-PBS-Tween containing the primary antibody in heat-sealed plastic bags as described by Higgins et al. (1989a). Theblots were then incubated in PBS-Tween, 5% BSA-PBS-Tween, 5% BSA-Tween containing [125I]iodo-Protein A(lxlO6disintsmin~1mr1), PBS-Tween. Finally, they wereair-dried, mounted on cardboard and autoradiographed at-70°C with Kodak X-Omat film (Eastman-Kodak, Roches-ter, NY) and Cronex Lightning Plus intensifying screens(Dupont, Wilmington, DE).
Steroid autoradiographyFor steroid autoradiography, specimens were incubated forlh in 10 nM of [3H]dihydrotestosterone (3H-DHT, Amer-sham, specific activity 100-150 Cimmol"1) with or without a300-fold excess of radioinert DHT (Steraloids, Wilton, NH) at37 °C. After labelling they were placed into nylon mesh bags(Tetko, Elmsford, NY) and washed for 3 to 4h with constantstirring in a 1 liter flask of PBS which was changed at 30minintervals. After washing, the tissues were then embedded inOCT medium (Miles, Elkhart, IN) and frozen in liquidpropane as described by Shannon et al. (1982). Frozensections (4 mm) were cut, thaw-mounted onto emulsion-coated slides (NTB-II emulsion, Eastman Kodak) andexposed autoradiographically. The autoradiograms wereprocessed photographically and stained with hemotoxylin andeosin.
Results
ControlsIn all cases (19/19), SVM grafted beneath the renalcapsule by itself formed masses of fibroblastic cells(Fig. 1) devoid of SVE morphology. SDS-PAGE,immunocytochemistry, Western blot and protein dotblot analysis demonstrated the complete absence ofSVS proteins in these SVM grafts (Figs 5 and 7).Deletion of the primary antisera (not illustrated)completely eliminated immunocytochemical staining asdescribed earlier (Fawell and Higgins, 1986; Higgins etal. 1989a).
Homotypic tissue recombinantsHomotypic recombinations of UR, DD and SV
Fig. 1. Graft of 0-day rat SVM grown for 1 month in amale athymic mouse (80x).Fig. 2. Adult mouse URM+URE recombinant grown for 1month in a male host (320x).Fig. 3. Adult mouse DDM+DDE recombinant grown for1 month in a male host. Normal epithelialhistodifferentiation is maintained. Note stereocilia indicatedby arrow (200x).
148 G. R. Cunha and others
Fig. 4. Homotypic tissue recombinant of neonatalSVM+SVE grown for 1 month (320x).
developed as expected. Namely, transitional uro-thelium differentiated in URM+URE recombinants(Fig. 2). DDM+DDE tissue recombinants formedsimple ductal structures having a star-shaped luminalcontour lined by tall columnar epithelial cells withstereocilia (Fig. 3). SVM+SVE recombinants devel-oped the complex morphology characteristic of SV(Fig. 4). Neither URM+URE nor DDM+DDErecombinants expressed SVS proteins as judged byimmunocytochemistry (not illustrated), SDS-PAGE orimmunoblotting (Figs 5 and 7). As expectedSVM+SVE recombinants formed SV tissue whichproduced the full complement of SVS proteins(Figs 5-7). When tissue recombinants were constructedwith rat SVM+mouse SVE, the 6 major SVS proteinscharacteristic of the mouse SV were observed(Figs 5-6), whereas tissue recombinants constructedwith mouse SVM+rat SVE expressed the 5 majorsecretory proteins of the rat SV (Figs 5,7).
SVM+ URE tissue recombinantsAlmost all of the SVM+URE tissue recombinants inthis study were heterospecific (rat mesenchyme+mouseepithelium or vice versa). In this way, staining withHoechst dye 33258 (Cunha and Vanderslice, 1984)
68-
43-
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8 10 12
Fig. 5. Analysis of secretoryproteins from grafts ofhomotypic tissue recombinants.Grafts were prepared withneonatal tissues and grown for1 month in male hosts.Secreted proteins wereanalyzed by SDS-PAGE. lanel=molecular weight standards;lane 2=mouse SVS proteins;lane 3=rat SVM+mouse SVE;lane 4=rat SVS proteins; Lane5=mouse SVM+rat SVE; lane6=rat SVM; lane 7=mouseSVM; lane 8=mouse serum;lanes 9 and 10=mouseUrM+UrE; lanes 11 and12=mouse DDM+DDE. Noteprotein bands 1-6,characteristic of mouse SVSproteins, and bands I-V,characteristic of rat SVSproteins. Homotypic SVrecombinants (ratSVM+mouse SVE and mouseSVM+rat SVE) express theSVS proteins characteristic ofthe donor epithelium.Abbreviations as per text: r,rat; m, mouse.
Seminal vesicle induction of adult epithelia 149
Table 1. Developmental response of tissuerecombinations prepared with SVM and epithelium
•Number of tissue recombinants analyzed,t As assessed by immunocytochemistry with anti-mouse SVS and
anti-rat SVS-IV and SVS-V.Abbreviations as per text, r=rat; m=mouse.
could be used to verify that any morphological orfunctional changes that occurred in the epithelium ofthe tissue recombinants was due to inductive actions ofthe mesenchyme on the heterospecific epithelium andnot due to contaminating homospecific epithelial cells.Tissue recombinants composed of rat SVM+0- to 60-day-old mouse URE (Table 1) formed histologicallyrecognizable SV tissue (Fig. 8A). Such recombinantsexhibited intense staining of the apical portion of theepithelial cells with anti-mouse SVS but not withantibodies to rat SVS (Fig. 8B-C). Staining with theHoechst dye verified that the epithelium was mouse inorigin (not illustrated). Reciprocal tissue recombinantscomposed of mouse SVM+rat (0 to 60 day) URE(Table 1) also differentiated into SV tissue (Fig. 8D). Inthis case, the epithelium was rat in origin based uponHoechst dye staining (not illustrated), a findingcorroborated by the fact that the induced SVE cellsstained immunocytochemically with antibodies to rat,but not mouse, SVS proteins (Figs 8E,F). Epithelialage did not influence the outcome of the experiments.
Functional cytodifferentiation of the induced UREcells was also analyzed by SDS-PAGE, Western blotand protein dot blot using antibodies specific to rat ormouse SVS proteins (Figs 7, 9-10). Based upon theanalysis of secretory proteins extracted from 43individual tissue recombinants the SVS proteinsdetected were found to correspond to the species of theepithelium utilized. Thus, in rat SVM+mouse URErecombinants, the proteins characteristic of mouse SVSwere detected (Figs 9,10). Likewise, in mouseSVM+rat URE recombinants, the five major rat SVSproteins were expressed. For all SVM+URE tissuerecombinants analyzed by SDS-PAGE (n=ll) inwhich neonatal epithelium was used, the entire spec-trum of SVS proteins was detected by SDS-PAGE,Western blot and dot blot methods. However, when
f?
1 "
2 -3 "
Fig. 6. Western blot analysis of homotypic SV tissuerecombinants. Mouse SVS proteins (1-6) separated bySDS-PAGE were probed with the anti-mouse SVS andvisualized with 125I-protein A (labelled according toMarkwell [1982]). Note that the anti-mouse SVS reactsintensely with mouse SVS proteins 3-6 (lane 1) andminimally with proteins 1 and 2. Reactivity was observedin rat SVM+mouse SVE recombinants (lane 2) for mouseSVS proteins 2-6, but not for recombinants composed ofmouse SVM+rat SVE (lane 3).
adult rat URE was utilized, rat SVS protein I was oftenpresent in exceedingly low levels as judged bySDS-PAGE (Fig. 9), even though its presence could beverified by more sensitive dot blot methods (Fig. 7).
One feature unique to SVE (not shared with URE) isthe expression of androgen receptors (AR). Neitherepithelial nor stromal cells of the UR exhibit nuclearconcentration of 3H-DHT (Fig. 11A). By contrast,nuclear androgen binding (indicative of AR) is aprominent feature in both the epithelium and stroma ofthe SV (Fig. 11B). When URE was induced by SVM todifferentiate into SV tissue, the induced epitheliumexpressed nuclear 3H-DHT binding sites (Fig. 11C).This nuclear binding was abolished by coincubationwith a 300-fold excess of radioinert DHT (Fig. 11D).
SVM+DDE tissue recombinantsDDE from 0- to 60-day old rats or mice (Table 1) was
150 G. R. Cunha and others
• •B
rSVS •
5ug 2ug 0.8ug
o-SVM by itself-
o o0.32ug
o
mSVS
o orSVS mSVS
• omSVM + rUrE(Od) mSVM + rUrE(Ad) mSVS
o omSVM +DDBpd) mSVM + rDDBAd) mSVS
mSerum rSerum UrM + UrE
•rSVM + rSVE-
DDM + DDE
o o o o o omSVM + mSVE
• • • • o o* Secretory proteins - 2jig in 2ml except whereindicated
Fig. 7. Immunodot blotting of rat SVSproteins collected from authentic ratSVS and tissue recombinants. Secretoryprotein samples (2 A*g/2 il except whereindicated) were applied to nitrocellulosepaper and probed with anti-rat SVSproteins I-V (A to E, respectively)followed by [ I]iodo-Protein A andautoradiography. The layout of thesamples is given in the template (solidand open circles indicate positive andnegative immunologic reactions,respectively). Note the similarity in thepatterns of reactivity with all five of theantibodies.
also induced by SVM to differentiate into SV tissue inboth rat SVM+mouse DDE and mouse SVM+ratDDE recombinants (Fig. 12A,D). The species origin ofthe induced SVE was verified as above with Hoechstdye staining and was appropriate for the types of tissuerecombinants constructed (not illustrated). The induc-tive effect of SVM on DDE was further corroborated bySDS-PAGE, Western blotting and through use ofspecies-specific antibodies on tissue sections, whichshowed that the appropriate SVS proteins wereexpressed in these tissue recombinants (Figs 9,10,12B,-C,E,F). Thus, rat SVM+mouse DDE recombinants
were stained with antibodies to mouse (but not rat) SVS(Fig. 12B.C), whereas mouse SVM+rat DDE recombi-nants were stained with antibodies to rat (but notmouse) SVS (Fig. 12E,F). Once again these immuno-cytochemical findings were verified by SDS-PAGE,Western blot or protein dot blot analysis of the secretedproteins (Figs 7, 9, 10). Tissue recombinants con-structed with rat SVM+mouse DDE expressed the 6major mouse SVS proteins, while the reciprocal tissuerecombinants (mouse SVM+rat DDE) expressed the 5major rat SVS proteins (Figs 7, 9, 10). The fullspectrum of SVS proteins was observed in all tissue
Seminal vesicle induction of adult epithelia 151
Fig. 8. SVM induces SV development in adult URE. Rat SVM+mouse URE and mouse SVM+rat URE tissuerecombinants were grown in male athymic hosts for 1 month. The characteristic complex morphology of seminal vesicle canbe recognized in both types of tissue recombinants (A, D). Immunocytochemical staining with anti-mouse SVS wasobserved in rat SVM+mouse URE (B) but not mouse SVM+rat URE recombinants (F). Immunocytochemical stainingwith anti-rat SVS-V was observed in mouse SVM+rat URE (E) but not rat SVM+mouse URE recombinants (C)(A,B,D,E=250x; C and F=320x).
recombinants (n=23) analyzed irrespective of the initialage of the epithelium.
Discussion
Mesenchyme-induced changes in epithelial differen-
tiation described herein were examined from severalstandpoints: gross morphological organization, epi-thelial cytodifferentiation, and the expression of ARand tissue-specific secretory proteins. In SVM+URErecombinants, epithelial cytodifferentiation was radi-cally changed from the transitional phenotype charac-teristic of a urothelium, which lacked AR (Cunha et al.
152 G. R. Cunha and others
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- 4 3
- 2 5
Fig. 9. Analysis of secretoryproteins of SVM+DDE andSVM+URE recombinantsusing 1-day (Id) or adult (Ad)epithelium. Tissuerecombinants prepared withmouse epithelium (lanes 1 and2) express mouse SVS proteins(1-6) while tissuerecombinants prepared with ratepithelium (lanes 3 and 4)express rat SVS proteins(I-V). Note albumin (arrow),an unavoidable contaminant inthe tissue recombinants. Lane3 (m SVM+r URE [AD]) issomewhat overloaded to showfaintly rat SV protein I. Notethat when neonatal rat URE isused (lane 4) that all 5 rat SVSproteins are present in roughlyequivalent amounts.
1980c), to a simple columnar secretory epithelium(Brandes, 1974; Price and Williams-Ashman, 1961),which expressed both AR and SVS proteins. It is likelythat the expression of AR preceded, and is aprerequisite for, the production of SVS proteins. In thenormal course of SV development, epithelial ARappear on 2 to 3 days postpartum (Cooke, 1988; Shimaet al. 1990), whereas SVS protein synthesis begins afterday 10 (Fawell and Higgins, 1986). This striking changein epithelial cytodifferentiation in SVM+URE recom-binants is directly comparable to the induction ofprostatic differentiation in epithelium of the urinarybladder (Cunha et al. 19806, 19836; Neubauer et al.1983). Here, too, a stratified AR~ urothelium differen-tiated into an AR+ simple columnar secretory epi-thelium. Such profound changes in cytodifferentiation
Fig. 10. Western blot analysis of heterotypic tissuerecombinants. Secretory proteins separated by SDS-PAGEwere probed with anti-mouse SVS and visualized with[l25I]iodo-Protein A and autoradiography. Note expressionof mouse SVS proteins in individual rat SVM+mouse URErecombinants (lanes 1-2) and rat SVM+mouse DDErecombinants (lanes 3-4) but not in homotypic mouseDDE+mouse DDE or mouse URE+mouse URE tissuerecombinants (lanes 7-8). Abbreviations as per text andfigures 5-6.
s? s? f?
8
154 G. R. Cunha and others
M2A
Fig. 12. SVM induces SV development in adult DDE. Rat SVM+mouse DDE and mouse SVM+rat DDE tissuerecombinants were grown in male athymic hosts for 1 month. Note the induction of the characteristic complex morphologyof seminal vesicle (A, D). Immunocytochemical staining with anti-mouse SVS was observed in rat SVM+mouse DDE (B)but not mouse SVM+rat DDE (F) recombinants. Anti-rat SVS-IV stained the epithelium of mouse SVM+rat DDE (E)but not rat SVM+mouse DDE (C) recombinants (A,D,E: 250x; B,C,F: 400x).
are accompanied by major reprogramming of epithelialbiochemistry and functional differentiation. The induc-tion of SV differentiation from adult DDE involves theconversion of a pseudostratified epithelium, which hasprominent stereocilia, to a simple columnar epitheliumlacking stereocilia. Again these changes in epithelialcytodifferentiation resulted in a complete change infunctional differentiation of the epithelial cells, and theexpression of SVS proteins. However, in this case theDDE already possessed AR prior to the construction ofthe tissue recombinants (Cooke, 1988) and presumablycontinued to express AR during the induction anddifferentiation of SV tissue.
The inductions described in this paper represent thefirst examples where complete functional reprogram-
ming has occurred in adult epithelial cells. In otherexamples of tissue interactions in which epithelialcytodifferentiation is minimally altered, changes infunctional cytodifferentiation did not take place, e.g.tissue recombinants composed of salivary glandmesenchyme+embryonic mammary epithelium (Sak-akura et al. 1976). In this case, the embryonic mammaryepithelium formed branched ductal networks resem-bling salivary gland, but continued to produce a-lactalbumin, a major constituent of milk. In other cases,a mixed response has been obtained, e.g. in interactionsbetween salivary gland mesenchyme and embryonicpituitary epithelium where the outcome (production ofACTH or ir-amylase) is usually mixed and dependentupon the age of the epithelium (Kusakabe et al. 1985).
Seminal vesicle induction of adult epithelia 155
The experimental data in this paper show that UREand DDE from 1- to 60-day old rats and mice can beinduced by neonatal SVM to undergo SV differen-tiation and to express the full complement of thesecretory proteins characteristic of the SV. There aretwo possible interpretations of these findings whichcannot be distinguished at present. The induction offully functional SVE from adult URE and DDE mayindicate that determined fully differentiated adultepithelial cells first dedifferentiated and then werereprogrammed to express the SV phenotype. Alterna-tively, these adult epithelia may contain undeterminedand undifferentiated stem cells that were the source ofthe induced SV tissue. Based upon histological,ultrastructural and imrnunocytochemical observationssuch uncommitted embryonic-like cells would bepresent in adult epithelia in exceedingly small numbersso that SV differentiation would likely to be inducedfocally by SVM in scattered sites throughout the adultepithelia. This is not supported by preliminary timecourse studies with SVM+URE recombinants grownfor 6, 9 and 12 days in culture which have shown that theconversion of URE (originally organized as a simpletubular structure) into the complex folded andbranched SV mucosa occurred globally throughout thetissue recombinant (Cunha and Young, unpublished).Thus, the alternate mechanism, dedifferentiation andsubsequent reprogramming, is favored. Since adultepithelial cells are clearly capable of expressingalternative phenotypes when associated with inductivemesenchymes, this suggests that the stability of adultepithelial differentiation in intact glandular organs mustbe due to ongoing influences of the adult stroma.
Differentiated adult epithelial cells in situ expressqualitatively distinct histotypes and faithfully maintaintheir characteristic histodifferentiation even in rapidlyrenewing tissues. These features describe the stabilityof the differentiated state (Ursprung, 1968). How is thisstability of the differentiated state maintained in adultepithelial cells? For some epithelia, e.g. from mammarygland and liver, functional differentiation can bemaintained by various extracellular materials withoutthe need for living stromal cells (Blum etal. 1987; Bisseland Barcellos-Hoff, 1987; Reid and Jefferson, 1984).For other epithelia, adult stromal cells may be involvedin maintaining adult epithelial differentiation as exper-imental recombination of adult epithelium with hetero-typic stromas can lead to profound changes in adultepithelial cytodifferentiation and function (Cunha et al.1985). The studies reported herein and many others(Bernimoulin and Schroeder, 1980; Billingham andSilvers, 1966, 1968; Briggaman, 1982; Karring et al.1975; Mackenzie and Hill, 1984; Spearman, 1974;Cunha, 1975, 19766; Cooke et al. 1987; Cunha, 19766;Cunha et al. 19806, 19836; Neubauer et al. 1983; Danieland DeOme, 1965; Daniel et al. 1965; Hoshino, 1967;Hoshino, 1978; Dudek and Lawrence, 1988; Norman etal. 1986; Sakagami et al. 1984) demonstrate that adult(or at least postnatal) epithelia can be induced toundergo changes in cytodifferentiation and in somecases biochemical function. While all of these studies
emphasize the responsiveness of postnatal epithelialcells to inductive mesenchyme, they do not prove thatadult stromal cells themselves have inductive proper-ties. However, adult mammary stroma (the fat pad) hasbeen shown to induce mammary development in bothadult and fetal mammary epithelial cells (Daniel andDeOme, 1965; Daniel et al. 1965; Sakakura et al.19796), and adult vaginal stroma can induce neonataluterine epithelium to express vaginal differentiation(Cunha, 19766). Adult dermal cells possess region-specific inductive activities (Billingham and Silvers,1966, 1968; Briggaman, 1982; Bernimoulin andSchroeder, 1980; Karring et al. 1975; Mackenzie andHill, 1984; Spearman, 1974). Moreover, epidermalappendages such as feather and hair are induced toform and grow by cells of the adult dermal papillae(Ibrahim and Wright, 1977; Jahoda et al. 1984; Lillieand Wang, 1943; Oliver, 1968; Wang, 1943). Morerecently it has been shown that neonatal uterine andvaginal mesenchymes, following culture for 1 to 2months, retain the ability to instructively and permissi-vely induce responsive epithelia (Cooke et al. 1987).Thus, adult stromal cells do seem capable of function-ing either as permissive or instructive inductors.
The chemical mediators of stromal effects upon adultepithelial growth and differentiation have not yet beendefined even though growth-promoting activities havebeen described in conditioned medium from mammaryfibroblasts (Enami et al. 1983; Howard et al. WI6;Kawamura et al. 1986) and adult corneal fibroblasts(Chan and Haschke, 1983). Based upon data reportedherein and by others, mesenchymal effects on epithelialcells are not species specific (Cunha et al. 1983c;Fukamachi et al. 1986; Haffen et al. 1983; Kedinger etal. 1981; Kollar and Fisher, 1980; Lacroix et al. 1984),which suggests that the mediators of these cell-cellinteractions are highly conserved in higher vertebrates.Previously characterized growth factors such as EGF,TGFar, TGF-/?, FGF and KGF are likely candidates.Other evidence suggests that extracellular matrix iscertainly involved in maintenance of epithelial differen-tiation (Bissell and Barcellos-Hoff, 1987; Blum et al.1987; Reid and Jefferson, 1984; Michalopoulos andPitot, 1975; Chen and Bissell, 1989).
These striking mesenchyme-induced changes in adultepithelial differentiation and function have importantimplications for understanding the etiology of abnormalcellular proliferation including carcinogenesis. In hu-mans, benign prostatic hyperplasia and prostatic adeno-carcinoma are common proliferative lesions of thegenital tract (Coffey etal. 1987; McNeal, 1983). Benignprostatic hyperplasia is thought to be due to a re-awakening of inductive activity of prostatic stroma cells(McNeal, 1978), an idea that has received a certaindegree of experimental support (Cunha et al. 1987).Likewise, mesenchymal-epithelial interactions havebeen implicated in the genesis and modulation ofcarcinomas (Cooper and Pinkus, 1977; DeCosse et al.1973, 1975; Fujii et al. 1982; Hodges et al. 1977;Mackenzie et al. 1979). Of particular interest is therecent report that SVM can modify ductal morphogen-
156 G. R. Cunha and others
esis and elicit secretory cytodifferentiation in carcinomacells of the Dunning prostatic adenocarcinoma(Hayashi et al. 1990). Similarly, digestive tract mesen-chymes from fetal rats can elicit glandular morphogen-esis and differentiation in human colon carcinoma cells(Fukamachi et al. 1986, 1987). Since most carcinomasarise postnatally, ongoing epithelial-stromal interac-tions in adulthood may play an important role in thedifferentiation, proliferation and malignant propertiesof emerging carcinomas.
This work was supported by NIH grants: HD 21919, DK32157 and HD 17491.
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