Development 101. 491-500 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

491

Mechanical aspects of the mesenchymal influence on epithelial

branching morphogenesis of mouse salivary gland

HIROYUKI NOGAWA1 and YASUO NAKANISHI2

lBiological Laboratory, College of Arts and Sciences, Chiba University, Yayoi-cho, Chiba 260, Japan2Department of Chemistry, Faculty of Science, Nagoya University, Chikusa, Nagoya 464, Japan

Summary

Three activities of mesenchymes from mouse embry-onic submandibular gland, lung, stomach, mandibleand skin were comparatively studied. The first abilitywas the induction of branching of submandibularepithelial lobes. Epithelial lobes branched well inrecombination with submandibular or lung mesen-chyme, less well with stomach mesenchyme, but neverwith mandibular or dermal mesenchyme. The secondbehavioural aspect studied was the contraction ofcollagen gels. When respective mesenchymal cellswere dispersed at 2-0xlO5 cells ml"1 in collagen gels(l-5mgml~') and incubated, dermal mesenchymalcells had the highest gel-contracting activity. The gel-contracting activity of submandibular or lung mes-enchymal cells was two thirds as high as that of dermalcells and that of stomach or mandibular mesenchymalcells was much lower. The last activity was to separatethree plastic beads that were recombined with mesen-chymes in place of epithelial lobes. Salivary or lung

mesenchyme effected a large separation of the beads,whereas dermal mesenchyme left beads contacting oneanother. There was a positive correlation between thebranch-inducing activity and the beads-separatingactivity within the five kinds of mesenchymes. In time-lapse cinematography of recombinates, cells of sub-mandibular and lung mesenchyme were observedmoving (or flowing) around, and their property wasdifferent from that of dermal mesenchyme. In thepresence of cytochalasins, both the contraction ofcollagen gels and separation of plastic beads bysubmandibular mesenchymal cells were completelyinhibited. These results suggest the importance ofmechanical influences of the mesenchyme in salivarybranching morphogenesis.

Key words: mouse submandibular gland, branchingmorphogenesis, tissue interaction, collagen-gelcontraction, flowing movement, cytochalasins.

Introduction

Branching morphogenesis of mouse salivary epi-thelium does not proceed without mesenchyme ofsalivary glands, accessory sexual glands or lungs(Grobstein, 1953; Cunha, 1972; Lawson, 1974). Themouse salivary mesenchyme is able to supportbranching morphogenesis of lung epithelium (Law-son, 1983); to induce mammary epithelium to branchin salivary-like fashion (Kratochwil, 1969) and furtherto induce nonbranching epithelium of quail anteriorsubmaxillary glands to branch (Nogawa & Mizuno,1981). These results suggest that mechanisms ofbranching morphogenesis of salivary glands are never

understood without making the nature of instructiveinfluences of the mesenchyme clear.

There are some different explanations for theprocesses of branching morphogenesis of salivaryglands. The first model by Spooner & Wessells (1972)and Spooner (1973) showed that the contraction ofepithelial microfilaments, which causes changes incell shapes of epithelia, takes place in a specific areaand forms a cleft. According to this model, themesenchymal cells that stimulate epithelial micro-filaments to contract should be prelocalized in thespecific area, but no evidence has been given to showthe prelocalization of the specific mesenchymal cells.Second, Bernfield (1981) and Bernfield & Banerjee(1982) reported that the epithelium whose basal

492 H. Nogawa and Y. Nakanishi

lamina was degraded by the mesenchyme had ahigher cell proliferation rate than the other epi-thelium whose basal lamina was stabilized by col-lagen, and they discussed a possibility that thedifferential cell proliferation rates cause cleft forma-tion. However, recently, it was proved by Nakanishi,Morita & Nogawa (1987) that clefts were initiated anddeepened in vitro when cell proliferation was in-hibited with X-ray irradiation and aphidicolin treat-ment of salivary rudiments. The third model byNogawa (1983) showed that the mesenchyme has anability to determine the curvature of epithelial sur-faces, and clefts are formed on the epithelial surfacewhen the curvature increases. In the last model byNakanishi, Sugiura, Kishi & Hayakawa (1986c), mes-enchymal cells exert traction forces on collagenbundles at the epitheliomesenchymal interface andclefts are formed on the consequently deformedepithelial surface. In the first two models, it isepithelial cells that generate shape-changing forcesand mesenchymal cells only modify the way theepithelial cells work. In the latter two models, incontrast, mesenchymal cells generate shape-changingforces and exert them on the epithelial surface. Todetermine which mechanism mainly functions in thebranching morphogenesis of salivary glands, epi-thelial or mesenchymal forces, it is necessary tounderstand the physiological characteristics of thesalivary mesenchyme.

In the present study, using mesenchymes of sali-vary gland, lung, stomach, mandible and skin, wecomparatively studied relations between the branch-inducing activity and two mechanical activities ofthese mesenchymes, and tried to elucidate mechan-ical aspects of the mesenchymal influence on salivarybranching morphogenesis. One is the ability to con-tract collagen gels and the other is the movement ofplastic beads that are placed in mesenchymal massesinstead of epithelial lobes. Collagen-gel contraction israther commonly possessed by fibroblastic cells: hu-man skin fibroblasts (Bell, Ivarsson & Merrill, 1979;Grinnell & Lamke, 1984), rat skin fibroblasts (Buttle& Ehrlich, 1983), chick embryonic ventricle, skeletalmuscle and dermis (Stopak & Harris, 1982) andbovine vascular smooth muscle cells (Ehrlich, Gris-wold & Rajaratnam, 1986). However, it is importantto study this activity of the salivary mesenchyme,because collagens have a crucial role in salivarybranching morphogenesis (Nakanishi, Sugiura, Kishi& Hayakawa, 1986a,b,c).

Materials and methods

Organ rudimentsICR mice were mated during the night and the day of thediscovery of vaginal plug was counted as day 0. Rudiments

of lung and stomach were isolated from 11-day fetuses inHanks' balanced salt solution (HBSS). Rudiments of sub-mandibular gland, sheets of lateral body skin and mesen-chymes of mandible were isolated from 13-day fetuses inHBSS.

Separation of epithelia and mesenchymesFour kinds of rudiments excluding mandibular mesen-chyme were treated with dispase (1000 protease units ml"1

in HBSS; Godo Shusei Co., Tokyo, Japan) at 37-5°C for30 min and epithelia and mesenchymes were separated withfine forceps. After the separated tissue fragments werewashed with HBSS, one part of the mesenchymes wassubmitted to collagen-gel-contraction experiments, and theother part of the mesenchymes and lobes of submandibularepithelium were stored in HBSS with 20% horse serum(Gibco Lab.) at room temperature for recombinationexperiments.

Recombination experimentsTo equalize the volume within five kinds of mesenchymes,we cut mesenchymal sheets of skin and stomach down tothe same size as an isolated submandibular mesenchyme,mesenchymal pieces of lung and mandible being as large asa submandibular mesenchyme when isolated. Three homo-typic pieces of each mesenchyme were assembled onsemisolid medium composed of medium 199 Earle's BSS(Gibco Lab.) with 20% horse serum, 0-5% agar (DifcoLab.) and penicillin G potassium (100 units ml"1), andthese mesenchymal assemblies were preincubated at 37-5°Cin 5 % CO2 for 4 h t o allow compaction to occur. Threesubmandibular epithelial lobes with diameters from 140 to160f/m, keeping mutual contact, were placed on the mes-enchymal mass and cultivated.

In other recombination experiments, plastic beads wereused in place of epithelial lobes. Cytospheres (Lux Scien-tific Corp.) with specific gravity 1-04 were available. Beadswith a diameter of 150/im were picked out and stored inHBSS with 20 % horse serum. Three beads were placed ona mesenchymal mass in the same manner as with epitheliallobes and incubated.

Time-lapse cinematographyMesenchymes were recombined with epithelial lobes orplastic beads on the above-described agar media, whichwere prepared with HBSS instead of Earle's BSS in thehollow (diameter 31mm and depth 4 mm) of a thick glassslide. The hollow was covered with a cover-glass whoseinner surface was coated with horse serum to avoid collect-ing moisture and sealed up with paraffin. The glass slide wasset on the stage of an Olympus BHS microscope in a warmbox adjusted to 37-5°C and recombinates were photo-graphed with Kodak Plus-X 16 mm film at intervals of 2 minusing an Olympus PM-16mm time-lapse cinematographicapparatus.

Collagen gel contractionMesenchymal pieces were dispersed in trypsin solution(0-25% in Ca2+-, Mg2+-free HBSS; Difco Lab., 1:250),and magnetically stirred for 30 min at 20°C. Suspensions ofsingle mesenchymal cells were obtained through a nylon

mesh with 20/im square pores, and horse serum was addedto it in order to stop the remaining activity of trypsin.Larger mesenchymal fragments that were caught in nylonmesh were re-treated in the same manner and the obtainedcell suspension was added to the former. After the cellnumber was counted with a haemocytometer, mesenchymalcells were washed twice with a basal medium (medium 199Earle's BSS with 20% horse serum and 100 units mP1

penicillin G potassium) and finally suspended at l-0x106 cells ml"' in the basal medium. Five kinds of mesenchy-mal cells prepared by this method consisted of more than95 % single cells and seemed intact since the aggregatedmass of the submandibular mesenchymal cells was able tosupport branching morphogenesis of a submandibular epi-thelial lobe (Nogawa & Nakanishi, 1986).

A collagen solution was purchased from Nitta GelatineCo. (Osaka, Japan; Cellmatrix type I-A: acid-solublefraction of type I collagen from bovine tendon). Thecollagen solution, lOx medium 199 Earle's BSS, 200 mM-Hepes buffer solution and horse serum were mixed in aratio of 16:2:2:5, and kept on ice to prevent immediategelation. 4 vol. of the collagen mixture were added to 1 vol.of the cell suspension. The final concentrations of collagenand cells were l-5mgml~' and 2-0xlO5cellsmr1, respect-ively. 0-5 ml of the final mixture was allowed to gel in eachwell of Falcon 24-well tissue-culture plate for 1 h of incu-bation at 37-5°C in 5% CO2. After 2 ml of the basalmedium was added to each well, the gels were let float byinserting a needle around the corner of the well andincubated. Changes of the diameters of the gels weremeasured with the eye-piece micrometer of a dissectionmicroscope.

Biochemical and histological techniques for collagengelsOne gel or 0-5 ml of the medium was placed in a screw-capped tube, hydrolysed with 2-5 ml of 6 M-HC1 at 110°C for24 h and neutralized with 10M-KOH. Hydroxyproline con-tents of a gel or the medium were assayed by the method ofKivirikko, Laitinen & Prockop (1967).

Gels were fixed in a 10% neutral formalin solution,embedded in paraffin and sectioned at 5^m thickness.Sections were stained with Fast green FCF for cells andSirius red F3BA (Schmidt GmbH) for collagen fibrilsaccording to the procedure of L6pez-De Le6n & Rojkind(1985).

CytochalasinsCytochalasin B (Aldrich Chemical Co.) was dissolvedat lOmgmP1 in dimethylsulfoxide (DMSO) and stored.When used, cytochalasin B was added to the basal mediumor the agar medium at a concentration of lO^gml"1 withDMSO at 0-1%. In the control experiments, a mediumcontaining only DMSO at 0 1 % was used. The concen-tration of cytochalasin B in the present experiments wassimilar to that in experiments by Spooner & Wessells (1970,1972). Cytochalasin D (Aldrich Chemical Co.), which wasknown not to inhibit hexose transport of cells, in contrast tocytochalasin B (Jung & Rampal, 1977), was also used at aconcentration of 1/igml"1.

Mechanical influence of salivary mesenchyme 493

Results

Branching morphogenesis of epithelial lobesrecombined with mesenchymes

A set of three lobes of submandibular epithelium wascultivated in recombination with five kinds of mesen-chymes (Figs 1-7; Table 1). When three epitheliallobes were cultivated without any mesenchymes, theyfused to form one spherical lobe within 8 h of culti-vation (Fig. 2). In all the recombinates with subman-dibular mesenchymes, the three lobes fused mutuallyand further branched typically 1 day after cultivation(Fig. 3). Lung mesenchyme had the ability to inducesubmandibular epithelial lobes to branch, though alittle inferior to submandibular mesenchyme (Fig. 4).Half of the recombinates with stomach mesenchymesshowed signs of branching morphogenesis 1 day aftercultivation when four or five clefts were present(Fig. 5A) and all the recombinates conspicuouslybranched on the 2nd day (Fig. 5B). The submandibu-lar epithelium never branched in recombinates withmandibular or dermal mesenchyme for 2 days ofcultivation, and three clefts that had been formed bythe fusion of three lobes (Fig. 2A) disappeared inrecombinates with mandibular mesenchyme (Fig. 6),while they were partly present in recombinates withdermal mesenchyme (Fig. 7). The area of epitheliumin recombinates with mandibular or dermal mesen-chyme 1 day after cultivation did not expand largerthan those at the beginning of cultivation (compareFig. 6 or 7 with Fig. 1).

Contraction of collagen gels by mesenchymesThe diameters of collagen gels (l-5mgml~') contain-ing submandibular mesenchymal cells (2-OxlO5cellsml"') became 88 ± 3 % of the starting diameter 1 dayafter incubation, 69 ± 7 % after 2 days and 59 ± 6 %after 3 days. Two possibilities were considered as thecause of the decrease in size of the collagen gels. Onewas the decomposition of collagen molecules by the

Table 1. Branching morphogenesis of submandibularepithelial lobes recombined with mesenchymes 1 day

after cultivation

Mesenchymes

13-day submandibular gland11-day lung11-day stomach13-day mandible13-day skin

Branching

+

2012800

(no.

±

03619

of recombinates)*

-

004

134

(total)

(20)(15)(18)(14)(13)

* Recombinates were classified according to the extent ofbranching morphogenesis. +, having more than three clefts;± , having one to three clefts which seemed to have been formedby the fusion of three lobes; - , having no clefts.

494 H. Nogawa and Y. Nakanishi

5A .\

5BI

Fig. 1. Three submandibular epithelial lobes before recombination.Fig. 2. An epithelial lobe formed by the fusion of three epithelial lobes without any mesenchymes (A) 4h and (B) 8hafter cultivation.Fig. 3. A recombinate of three epithelial lobes with submandibular mesenchyme 1 day after cultivation. Epithelial lobescontact one another in the centre of the recombinate.Fig. 4. A recombinate of three epithelial lobes with lung mesenchyme 1 day after cultivation.Fig. 5. An isolated epithelium from a recombinate of three epithelial lobes with stomach mesenchyme cultivated for(A) 1 day and (B) 2 days. Since the contrast between epithelium and mesenchyme was low in recombinates withstomach mesenchyme, epithelial parts were isolated by treatment with dispase.Fig. 6. A recombinate of three epithelial lobes with mandibular mesenchyme 1 day after cultivation. Three lobes fusedtogether to form a spherical lobe (arrow).Fig. 7. A recombinate of three epithelial lobes with dermal mesenchyme 1 day after cultivation. Three clefts (arrows),which seemed to have been formed by the fusion of three lobes, were left. Bar, 200^m.

collagenase activity of mesenchymal cells and theother was changes in the meshes of the collagenlattice. The hydroxyproline contents were assayed innoncontracting gels without cells and contractinggels with 2X105 cells ml"1 3 days after incubation(Table 2). Although the contracting gels were threefifths as large as the noncontracting gels in diameter,they had almost the same contents of hydroxyproline.The slightly lower value of the contracting gels thanthe noncontracting gels seemed to be due mainly tothe fact that the contracting gels had less intragelspace which retained the hydroxyproline-containingmedium. Next, paraffin sections of the gels werestained with Fast green FCF and Sirius red F3BA to

Table 2. Hydroxyproline contents of gel and medium3 days after incubation

Gel(Diameter of gel. %)*

MediumGig/ml)'

noncontractingcontractingfresh medium!

(100 ±0)(59 ± 4)

80±375 ±4

25±426 ±313 ±3

* Each datum was obtained from six samples of three timeexperiments. Mean±s.D.

t Contents of hydroxyproline which was originally present inthe basal medium were assayed.

Mechanical influence of salivary mesenchyme 495

to-} • • * • ' - * • '

\ \ * * '

r-T ^^ m: ~ r

y -if

,. V B

Fig. 8. Sections of gels 3 days after incubation stained with Fast green FCF and Sirius red F3BA: (A) containing nocells and (B) containing 2xl05cellsmrl. Collagen fibres gathered around cells (arrows). Bar, 50j<m.

show the orientation of collagen fibres (Fig. 8). Anoncontracting gel without mesenchymal cells wasconstituted with a loose meshwork of thin collagenfibrils. In a contracting gel, thick collagen fibres wereobserved tightly gathering around mesenchymal cells.These results indicate that the decrease in size ofcollagen gels was caused not by the degradation ofcollagen matrix but by the traction of collagen fibresby mesenchymal cells.

Collagen gels (l-5mgml~') containing respectivemesenchymal cells at l-OxlC^ml"1 were incubatedand activities of gel contraction were expressed as thepercentage of the reduced length to the startingdiameter (Table 3). Dermal mesenchymal cells,

which failed to induce submandibular epithelium tobranch in the state of a cell mass, had the strongestgel-contracting activity. The gel-contracting activityof submandibular or lung mesenchymal cells was twothirds as strong as that of the dermal mesenchymalcells and that of stomach or mandibular mesen-chymal cells was much weaker. Five kinds of dis-persed mesenchymal cells contracted collagen gelsin different degrees irrespective of the degreesof their branching-morphogenesis-inducing activities(Table 1). These results suggest that the collagen-gel-contracting activity of cells is a necessary butinsufficient condition for the branch-inducing mesen-chyme.

Table 3. Contraction of collagen gels and separation of plastic beads by mesenchymes 1 day after cultivation

Mesenchymes

13-day submandibular gland11-day lung11-day stomach13-day mandible13-day skin

Activities ofcontracting gels

(%)•

12 ±3 (12)11 ±2 (14)4±1 (12)3±1 (12)

18 ±1 (14)

3

63100

Activities of separatinj

Separated points!(no. of recombinates)

2

94710

1

29

11100

0

3348

18

(total)

(20)(19)(23)(19)(18)

5 beads

— Separated distanceOwn)*

200 ±190170 ±22040±6020 ±200 ± 0

'These values expressed percentage of the reduced length to the starting diameter of gels. Mean ± S.D. (no.).t Recombinates were classified according to the number of the separated points among three contact points of beads. The £2-test

showed that there was significant difference in 'separated points' according to the kinds of mesenchymes (P<001).X These values express the sum of three distances away from each bead per explant. Mean ± s D. of the same recombinates as in

'separated points'.

496 H. Nogawa and Y. Nakanishi

9 10 11

• ."v

• > •

12 13 14

Fig. 9. Three plastic beads with diameter 150/im before recombination.Figs 10-14. A set of three plastic beads recombined with mesenchymes and cultivated for 1 day: (Fig. 10)submandibular mesenchyme, (Fig. 11) lung mesenchyme, (Fig. 12) stomach mesenchyme, (Fig. 13) mandibularmesenchyme and (Fig. 14) dermal mesenchyme. Bar, 200/<m.

Separation of plastic beads by mesenchymesWe also tried to study specificity of the branch-inducing mesenchymes when in a mesenchymal mass.A set of three plastic beads as large as the epitheliallobes was recombined with the mesenchymes and themovement of the beads in the mesenchymal mass wasexamined (Figs 9-14). The three beads were separ-ated well in submandibular or lung mesenchyme (Figs10, 11), but never separated in dermal mesenchyme(Fig. 14). Beads-separating activities of the mesen-chymes were quantified both by counting the numberof the separated points among three contact pointsand by measuring the sum of three distances awayfrom each bead in individual explants (Table 3). Thebeads-separating activity proved to correlate posi-tively with the branching-morphogenesis-inducing ac-tivity. The values of 'separated distance' had largerdeviations, but we observed in some recombinatesthat the beads moved closer to each other after onceparting, which would explain the larger deviations.

The behaviour of mesenchymes in living recombi-nates was checked with time-lapse cinematography.Submandibular and lung mesenchymal cells wererecognized moving (or flowing) around in groups not

only near the surface of the beads but also far awayfrom the beads. Since this type of cell movement wasobserved in the mesenchymes recombined with epi-thelial lobes, it was not abnormal cell movementcaused by the plastic beads as foreign matter. Incontrast, most dermal mesenchymal cells movedlittle, but some cells moved at random in short steps.

Effects of cytochalasins on submandibularmesenchymeIn the presence of cytochalasin B (CB, 10/igmP1),neither contraction of collagen gels (Fig. 15) norseparation of plastic beads by submandibular mesen-chyme were observed. Since the same results wereobtained when cytochalasin D (1/zgml"1) was used,inhibitory effects of CB on the submandibular mes-enchymal behaviour seemed to be caused not byinhibition of hexose transport but by disruption ofmesenchymal microfilaments. We then examined theeffect of CB on the gels that had contracted to 84 % ofthe starting diameter (Fig. 15). When the mediumwas replaced with the CB medium, the gels wereobserved increasing in size as early as 1 h aftertreatment and they relaxed to 86% of the starting

Mechanical influence of salivary mesenchyme 497

0 l 2Incubation days

Fig. 15. Effects of cytochalasin B (CB) on contraction ofgels with 2xl05cellsmr1. Five groups of samples:• , control —> control; O, controls CB for 6 h—> control;• , control ->CB for 24 h -»control; D, control ->CB,and • , CB —• CB were expressed ( , in the controlmedium and , in the CB medium). Each pointrepresents mean ± S.D. of six samples.

diameter 6h after treatment, and continuing to relaxgradually thereafter. The gels, however, resumedcontracting when CB was washed out with the controlmedium 6h or 1 day after treatment.

Discussion

Earlier studies of mesenchymal influences on epi-thelial morphogenesis have mainly taken account ofthe biochemical aspect of the mesenchymal influ-ences, for instance, the growth factor that stimulatesproliferation of epithelial cells (Ronzio & Rutter,1973; Goldin & Wessells, 1979; Goldin & Opperman,1980) and the activity of degrading basal lamina(Bernfield & Banerjee, 1982; Smith & Bernfield,1982), and have not considered the mechanical as-pect. As for mechanical influences, Oster, Murray &Harris (1983) and Harris, Stopak & Warner (1984)pointed out the possibility that traction of collagenfibrils by dermal cells may cause the clumping ofdermal fibroblasts and the interconnecting polygonalnetwork of collagen bundles in the pattern formationof avian feather papillae. Nogawa (1983) and Naka-nishi et al. (I986a,b,c, 1987) have suggested thepossibility that the mesenchyme may exert shape-changing forces on the epithelial surface and cleftsmay be initiated on the deformed surface in thebranching morphogenesis of mouse salivary gland.The present study submits more reliable evidence

that shows the importance of the mechanical influ-ences of the mesenchyme on branching morphogen-esis of salivary epithelium.

The present study showed that the mesenchymesthat were able to induce the salivary epithelium tobranch had a higher beads-separating activity andthat the mesenchymal cells moved (or flowed) aroundin groups. The separation of beads by the mesen-chymes seems to come from the fact that mesenchy-mal cell current pushes or pulls the beads and fromthe invasion of mesenchymal cells into gaps betweenthe beads. Since the flowing movement of mesenchy-mal cells was also observed in the mesenchymesrecombined with epithelial lobes, these forces arethought to work in the process of cleft widening inwhich each lobe is separated while keeping mutualconnections.

Lung mesenchyme induced the submandibular epi-thelium to branch, which is consistent with Lawson's(1974) results. Stomach mesenchyme proved to beable to induce the submandibular epithelium tobranch, though inferior to lung mesenchyme, and toseparate plastic beads away to a degree correspond-ing to its branch-inducing activity. Often clefts wereobserved in recombinates of epithelial lobes withdermal mesenchyme, which never separated plasticbeads. We, however, confirmed with time-lapse cine-matography that the clefts were not newly formed butremained because the lobes had not fused, suggestingthat immotile, stiff dermal mesenchymal cells physi-cally stopped the lobes fusing into one spherical lobe.This may be supported by the fact that most of thelobes recombined with mandibular mesenchyme,which was slightly motile, fused to give one sphericallobe. Furthermore, when the epithelium was tightlypacked with stiff mesenchyme, the epithelium with-out any space to grow might cease cell proliferation,probably due to 'postconfluence inhibition of celldivision' (Martz & Steinberg, 1972). We observedthat the area of submandibular epithelium did notexpand in recombination with dermal and mandibu-lar mesenchyme which were both stiff. The presentstudy, however, gives no evidence of whether theepithelial expansion was inhibited mechanically bythe stiff mesenchymes or by lack of growth factorsproduced by these mesenchymes. Lawson (1984)reported that the lung epithelium that failed tobranch in recombination with submandibular mesen-chyme began to proliferate actively and to branchwhen the mesenchyme was removed from it and thenrecombined with it. This recovery can be explainedby the change of the surrounding mesenchyme from astiff to a flowing mass, which was discussed as thethird among four possibilities by Lawson (1984): shereferred to 'packed' and 'diffuse' mesenchymes.

498 H. Nogawa and Y. Nakanishi

All the five kinds of mesenchymal cells examinedhad the activity of contracting collagen gels, and thedermal mesenchyme, which failed to induce theepithelium to branch, had the highest gel-contractingactivity. The lack of correlation between branch-inducing and gel-contracting activities appears tocontradict our suggestion for the mechanisms ofbranching morphogenesis (Nakanishi et al. 1986c),but it is highly probable that the values of gel-contracting activities may not always reflect thestrength of traction forces of mesenchymes in vivobecause the density of mesenchymal cells and thecontent of extracellular matrix components contain-ing collagen vary with each organ in vivo. Bernfield &Wessells (1970) and Nakanishi et al. (1986c) reportedthat collagen bundles are aligned at the epithelio-mesenchymal interface along shallow and deep clefts.Since aligned collagen fibres guide moving cells andconversely moving cells align collagen fibres (Oster etal. 1983; Harris et al. 1984), traction forces generatedby the mesenchyme can be amplified to shape-changing forces where collagen fibres happen to bealigned and bundled by moving mesenchymal cells.Initial shallow clefts may be deepened with both thetraction force and flowing movement of the mesen-chyme. However, there is another possibility that, atthe basal lamina, collagen may cross-link proteo-glycans which associate with the actin cytoskeleton inthe epithelium, and modify the morphology of theepithelium through this cross-linking (Rapraeger,Jalkanen & Bernfield, 1986).

In the present study, we examined the interactionof mesenchymal cells with gels of type I collagen.Recently, Kratochwil et al. (1986), using mouse em-bryos deficient in type I collagen, reported thatsalivary rudiments undertook branching morphogen-esis normally in vitro without detectable synthesis oftype I collagen. Since collagens have a crucial role insalivary branching morphogenesis (Nakanishi et al.19S6a,b,c), type III or V collagen, which is fibrillar(Mayne, 1984), may substitute for type I collagen innormal branching morphogenesis. In addition to acollagen-mediating mechanism, there can be anothermechanism in which mesenchymal cells exert shape-changing forces on the epithelial surface by interact-ing with some insoluble materials to which they areattached, as shown by Harris, Wild & Stopak (1980)that chick heart fibroblasts were able to exert tractionforces on the silicon rubber and make it wrinkle.

Spooner & Wessells (1970, 1972) and Spooner(1973) reported that shallow clefts disappeared anddeep clefts, where collagen fibres were abundantlypresent, remained when submandibular rudimentswere treated with cytochalasin B (CB). From the

results, they presented the model of branching mor-phogenesis in which epithelial microfilaments con-tribute to the formation of clefts and accumulatingcollagen fibres stabilize the clefts. However, thepresent study, demonstrating inhibitory effects of CBon both the gel contraction and beads separation bysubmandibular mesenchyme, necessitates taking intoaccount the effects of CB on mesenchymal as well asepithelial microfilaments. If the stability of shallowclefts is balanced with the traction forces of themesenchyme, shallow clefts will disappear with CB inthe same way as the contracting gels relax with CB,and deep clefts may remain due to the presence ofrearranged collagen fibres and invading mesenchymalcells which become immotile with CB.

Epitheliomesenchymal specificity in salivarybranching morphogenesis (Grobstein, 1953) mayoriginate from the mesenchymal nature of motiletissue, but the question how the flowing movementof cells occurs in the specific mesenchymes is un-answered. Comparative studies of submandibularmesenchyme with dermal mesenchyme will give cluesfor approaching the problem. Pieces of dermal mes-enchymes, which were incubated for 4h before re-combination in the present experiments, seemed tocontact more tightly with one another than piecesof submandibular or lung mesenchyme (data notshown), and the area of dermal mesenchyme becamesmallest during 1 day of incubation (Figs 7, 13). Thecell-to-cell adhesion may have an important role inthe flowing movement of the cell mass, and somay the adhesion of cell to extracellular matrices ofthe mesenchymal tissue (Funderburg & Markwald,1986). It remains to be determined what cell adhesionmolecules and extracellular matrix materials are es-sential to the characteristic movement of the salivarymesenchyme.

The authors wish to express their gratitude to Prof. T.Mizuno of the University of Tokyo for his continuousencouragement during the course of this work, to Dr S.Takeuchi (the University of Tokyo) for his helpful ad-vice on time-lapse cinematography, and to Dr J. Enami(Dokkyo University School of Medicine) and Dr N. Shiojiri(Shizuoka University) for their technical advice. This workis supported in part by grants to Y. Nakanishi from theMinistry of Education, Science and Culture of Japan andfrom the Mitsubishi Foundation.

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Mechanical influence of salivary mesenchyme 499

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(Accepted 13 July 1987)