J. Cell Sci. 26, 101-117 (1977) IOI

Printed in Great Britain

OUTGROWTH OF HUMAN FIBROBLAST

AGGREGATES ON A SUBSTRATUM TRIGGERS

A WIDE VARIETY OF MORPHOGENETIC

PROPERTIES IN THE CELLS

BERNADETTE VAN DER SCHUEREN, JEAN-JACQUESCASSIMAN AND HERMAN VAN DEN BERGHEDivision of Human Genetics, Department of Human Biology,Minderbroederstraat 12, B-3000, Leuven Belgium

SUMMARYTwenty-four-hour-old aggregates of human diploid skin fibroblasts are allowed to attach to

a glass or plastic substratum. As a result of this attachment the cells in the aggregate demon-strate rapid and generalized changes in cell shape, cell surface and cytoplasm ultrastructureand in their ability to incorporate [3H]thymidine. Within 24 h they grow out on the substratumto attain the regular monolayer configuration. During the process of leaving the aggregate forthe substratum a great number of different morphogenetic properties are displayed by the cells,resembling the properties of embryonic or epithelial cells. The simultaneous occurrence of thisgreat variety of cell shape and cell surface changes, many of them unusual for fibroblasts, aswell as the concurrent formation of organized cytoplasmic structures — microfilaments, micro-tubules - at localized areas of the cells, makes this system a potentially useful tool in the studyof cell behaviour.

INTRODUCTION

Directed migration as seen during morphogenesis and malignant invasion are basedupon such morphogenetic properties as adhesion, deadhesion, and changes in cellshape (Bernfield & Wessells, 1970) and are accompanied by modifications of cellsurface ultrastructure.

Only a limited number of studies have dealt with a description of these complexmorphogenetic events occurring in situ and most of them pertain to embryonalsystems (Trinkaus, 1973; Gustafson & Wolpert, 1961; Bard & Hay, 1975).

The behaviour of fibroblasts growing out of tissue explants has revealed many ofthe fundamental properties of these cells (Abercrombie & Heaysman, 1954;Abercrombie, 1967), although the cellular mechanisms underlying 'monolayering'remain poorly understood and ultrastructural data are absent.

Using single cells or monolayers, specific aspects of cellular behaviour, e.g. celladhesion and spreading (Rajaraman, Rounds, Yen & Rembaum, 1974; Elsdale &Foley, 1969) have been analysed more thoroughly. While the in situ behaviour of thesecells is very complex and makes an interpretation of the events very difficult, theconditions generally used in vitro give only a very simplified and incomplete imageof cellular capabilities.

102 B. Van der Schueren, J.-J. Cassiman and H. Van den Berghe

Aggregates of normal adult skin fibroblasts can be considered to be an alternativeand valid model to study morphogenetic properties and cellular behaviour in vitro.We have previously demonstrated that the fibroblasts in these aggregates expressmorphological features which do not appear under the in vitro culture conditionsgenerally used (Van der Schueren, Cassiman & Van den Berghe, 1976).

The present investigation will demonstrate, using light microscopy correlated withtransmission and scanning electron microscopy, that (i) 24-h-old aggregates of normalhuman skin fibroblasts, which show definite signs of autolysis (Van der Schuerenet al. 1976), can recover and initiate outgrowth on a substratum by simple attachmentto it; (ii) various morphogenetic events, accompanied by changes in intracellularmorphology and in plasma membrane ultrastructure, occur over a fairly short timeperiod; and (iii) under the experimental conditions used, normal human skin fibro-blasts exhibit properties which resemble those of epithelial and embryonic cells,whereas in monolayer culture these properties are rarely observed.

MATERIALS AND METHODS

Normal human diploid fibroblasts were aggregated as previously described by placinga suspension of trypsinized cells in 35-mm bacteriological Petri dishes in Dulbecco's ModifiedEagle's Medium (DME) containing 10 % (v/v) newborn calf serum in a dry incubator at 37 °Con a gyratory shaker at 85 rev/min. Twenty-four-hour-old aggregates, suspended in the samemedium, were placed on a plane surface (glass or plastic) at 37 °C in an incubator with anatmosphere of 95 % air, 5 % CO2 and 100 % humidity. The behaviour and morphology of thecells growing out of the aggregates on to the substratum were examined between o and 48 hafter attachment, using light microscopy, transmission and scanning electron microscopy.

Light microscopy

Aggregates growing out on a glass surface were washed in DME without serum, fixed inCarnoy's solution and coloured with haematin-erythrosin. They were also examined oni-/tm-thick serial sections of Epon-embedded material coloured with 1 % toluidine-bluesolution.

Autoradiographs were performed on outgrowing aggregates incubated for 2-7 h in [3H]-thymidine (S.A. 5 mCi/nuvi) 3 /iCi/ml, which were overlaid with emulsion (Ilford K-2) andkept in the dark at 4 °C for 7 days.

Transmission electron microscopy

Aggregates growing out on a plastic surface (35-mm tissue culture Petri dishes) were fixed in2 % glutaraldehyde in 0-07 M cacodylate buffer (pH 7-4) followed by a postfixation in 1 %osmium tetroxide in o-i M phosphate buffer (pH 7'4). After dehydration by a series of ethanolwashes, the material was embedded in Epon (Epikote 812, Gurr), cut perpendicular to thesurface on a Reichert OM U2 ultramicrotome, contrasted with uranyl acetate and lead citrateand examined with a Hitachi HU-11A electron microscope.

Scanning electron microscopy

For scanning electron microscopy aggregates were grown on glass coverslips, washed inPBS and fixed in glutaraldehyde (2 % in cacodylate buffer, pH 7-4) and osmium tetroxide(1 % in phosphate buffer, pH 7-4). After dehydration with a graded series of ethanols andw-amylacetate solutions they were dried by critical point (E 3000-Polaron) with carbon dioxide,covered with gold in a Polaron sputter coater (E 5000 diode) and examined in a PhilipsPSEM 500.

Outgrowth offibroblast aggregates 103

RESULTS

Human fibroblast aggregates placed on a plane surface (glass or plastic) in completegrowth medium will attach firmly within about 1 h and grow out over the substratumafter about 1—1-5 h- The outgrowth progresses at a high rate, generally faster andsooner with smaller aggregates than with larger ones and will result after 48 h in theformation of a typical monolayer configuration.

General aspects of the outgrowth

Once the aggregates have attached to the substratum, 3 sequential states in theoutgrowth can be observed: (i) the initial outgrowth, restricted to the site of initialattachment or base of the aggregate; (ii) the participation of cells located at theperiphery of the aggregates, and hence not in direct contact with the substratum;(iii) and finally the involvement of cells located all over the aggregate.

As first signs of the outgrowth - after 1-1-5 h - a few cells, at the attachment side,flatten out on the substratum while their trailing end remains enclosed between thecells of the aggregate (Fig. 1 B, top).

Around 3-4 h, the outgrowth no longer remains restricted to the base of theaggregate (Fig. 1 B, middle). The sides of the aggregate gently slope down towards thesubstratum but the aggregate maintains nevertheless a very regular contour. Cellslocated at the free edges of the aggregate have made contact with the substratum,thereby forming several layers of superimposed outgrowing cells. Subsequently (at6 h) (Fig. IB, bottom) the aggregates are surrounded by a very dense corona of cells,lying criss-cross over each other. In vertical sections 3 characteristics are prominent:the irregular outline of the aggregate resulting from the bulging out of cells from theaggregate; the broad and deep intercellular spaces separating the cells; and theelongated cells that are situated, often in tandem, from the top of the aggregatetowards the substratum.

The zone covered by the outgrowing fibroblasts expands gradually and if sufficientfree space is available the piling-up of cells will decrease. After 24 h only a fewremnants of aggregates, recognized by the fact that many cells remain piled-up, arefound. In less than 48 h the cells on the substratum adopt the typical monolayerconfiguration, i.e. they orient themselves parallel to each other. DNA synthesis wasevaluated by [3H]thymidine incorporation; 24-h-old aggregates do not show anythymidine incorporation in suspension. Seven hours after attachment and outgrowthin the presence of [3H]thymidine, label was present as well in cells on the substratumas in cells located at the top of the aggregate (Fig. 1 A).

Changes in cell morphology and in cell surface ultrastructure

The attachment of the aggregate to a substratum and the subsequent outgrowthare accompanied by changes in cell shape and cell surface ultrastructure (Fig. 2).While some cells may form extensive lamellipodia, others bulge out of the aggregateor round up completely; still others acquire an elongated, cylindrical or flattenedshape.

104 -8. Van der Schueren, J.-J. Cassiman and H. Van den Berghe

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Outgrowth of fibroblast aggregates 105

Lamellipodia. Extensive lamellipodia may be formed (Fig. 3 A), initially only bycells located at the adhesion site of the aggregates (1 h), later by cells situated higherup but still in the lower portion of the aggregate (2 h). The lamellipodia may extendin all directions and discrete ruffling activity can be seen at several points of theiredges. When many cells are participating in the outgrowth (4 h) superimposedlamellipodia may be seen (Fig. 3B). At the edges of these lamellipodia new finecytoplasmic extensions make contact with the substratum.

Surface structures. After 2-5 h some cells send out one, sometimes two, long finefilopodia (Fig. 3 c). The cell body from which they originate remains generally com-pletely buried between the other cells of the aggregate. Very often these filopodia havesplit ends and at points of contact with the substratum they form small lamellipodia.From these flat regions, the filopodia can extend further over the substratum, to formhighly ramified structures with multiple small lamellipodia (Fig. 3 D). Similar filopodia- often with bulbous end - lie on top of other spread cells.

The amount of cytoplasm found in the flattened regions on the one hand and in thetrailing part at the aggregate side on the other hand varies. Presumably a flow ofcytoplasm from the cell body into the cell processes towards the substratum occurs.When the cells are completely flattened on the substratum they may remain attachedto the aggregate by very fine fibres (Fig. 3E). Such fibres become very pronounced ata later stage (6 h).

Another type of surface projection, seen concomitantly with filopodia, consists ofthick, fingerlike protrusions which resemble the lobopodia described in embryonictissues (Fig. 3F). The smaller ones suggest that they could originate from blebs. Assoon as the outgrowth starts the aggregates are characterized by the accumulation ofnumerous villi and blebs, especially near the points of outgrowth. They can be seenover the entire outer surface of cells, and they may be very prominent at the cellboundaries.

Typical is the appearance of small regions of the cell surface which are coveredwith villi and blebs mainly near the centre of the cell and sometimes in a moreeccentrical position (Fig. 4A). The edges of these cells may be retracted so thatunderlying cells become apparent.

Rounded cells. A number of different cell types can be found which present somecommon characteristics. Typical for the initial stages (2-3 h) is the presence ofrounded cells (Fig. 4B) which are bulging out from the surface of the aggregate, andwhich are completely covered with villi. These rounded cells are found over the entireaggregate as single cells or in small groups. Some of these cells are attached to theaggregate only at a narrow region by means of so-called retraction fibres; sometimes

Fig. 1. A, DNA synthesis in aggregates. Left, representative autoradiograph of 1 -/tmsection through a 24-h-old suspended aggregate labelled for 24 h with [JH]thymidine;x 675. Right, aggregate grown for 7 h in the presence of [3H]thymidine showingincorporation by cells around and on top of the aggregate; x 250. B, different stages ofaggregate outgrowth. Phase-contrast aspect (left) and i-/tm section (right) of 3 sequen-tial stages in the outgrowth after the aggregate had settled down on a substratum;x 200. Top, after 1-5 h; middle, 3 h; lower, 6 h.

io6 B. Van der Schueren, J.-J. Cassiman and H. Van den Berghe

they are adherent to neighbouring cells by flat and bifurcated extensions (Fig. 4c) orthey form processes which point towards the substratum (Fig. 4D).

Rounded cells are also found on top of cells which have spread on the substratumor in the aggregate. Cone-shaped cells, having the general surface features of the

Fig. 2. General aspect of an outgrowing aggregate. P rominent changes in cell shapeand in cell surface infrastructure, as described in the text, are illustrated. Bar = 1 0 fim.

rounded cells, are also present (Fig. 4E); it is possible that they represent an elongationof the rounded cells through which these gain the substratum. Finally, flattened cellsare seen with localized surface protrusions, oriented towards the substratum. Thesecells often show membrane activity at their periphery (Fig. 4F).

Outgrowth of fibroblast aggregates 107

Elongated cells. As the process goes on (4 h) more and more elongated cells becomeapparent. They are cylindrical or flat. Some do not grow out strictly radially but arearranged criss-cross over each other (Fig. 2). The cells still have part of their cyto-plasm buried between the cells of the aggregate or they are only superficially con-nected with the aggregate by means of numerous short cytoplasmic extensions(Fig. 5 A). Where they meet outgrown cells they may end in a very pronouncedarborization (Fig. 5B) or make intimate contacts by forming very flat irregularcytoplasmic extensions (Fig. 5 c). The aggregate becomes very irregular and evencells in the aggregate clearly participate in the outgrowth. By 8 h the remnant of theaggregate is composed of elongated flattened fibroblasts.

Cells on the substratum. Cells that are breaking out of the aggregate, as well as cellsjust near the boundary of the aggregate, are generally covered with numerous villi.In the spaces between these cells a complex network of filopodia connecting neigh-bouring cells is obvious. In contrast the cells on the substratum become very flat,even if they are accumulating in several layers near the aggregate. They have anelongated or polygonal shape and are devoid of surface structures.

Accumulation of microfilaments and microtubules related to the changes in cell morphologyAs described, the cells at the free aggregate surface start to bulge out of the

aggregate after attachment. A dense accumulation of microfilaments running justbeneath the plasma membrane appears at the free surface of these cells. Sometimesthese microfilaments are accompanied by microtubules (Fig. 6A). A similar localizationof microfilaments is found at places where few blebs are protruded (Fig. 6B).

In a later stage, when the cells are characterized by numerous cell surface structures,the microfilaments form a peripheral band under the blebs (Fig. 6c). The elongationof the fibroblasts towards the substratum is accompanied by an increased accumu-lation of microfilaments, which occupy extensive areas of the cytoplasm underneaththe plasma membrane, and by very long microtubules. In addition, a dense accumu-lation of microfilaments and microtubules may be found at points of contact betweencells in tandem or between the lateral contact sites of cells that have been almostcompletely separated from each other by large intercellular spaces. A similar localizedconcentration of microfilaments can be observed in cells that have completely separatedbut still bear projections pointing towards each other. Extracellular material can benoticed in the intercellular space and on the free surface of the cells. Sometimes thisextracellular material is the only connexion between 2 fully dissociated cells. Thepresence of this material is often accompanied by an intense, localized endo- (or exo-)cytotic activity of the cells.

DISCUSSION

The present investigation has demonstrated that, when 24-h-old fibroblastaggregates are allowed to attach to a substratum, individual fibroblasts leave theaggregate and grow out over the substratum to form a regular monolayer in the next24 h. The initial attachment of the aggregate results in: (1) alterations in cell shape

io8 B. Van der Schueren, J.-J. Cassiman and H. Van den Berghe

Outgrowth of fibroblast aggregates 109

accompanied by the formation of small and large surface projections and cell pro-cesses; (2) the accumulation of microtubules and microfilaments at localized pointsand their subsequent organization over the entire cytoplasm; (3) an increase of theintercellular spaces; and (4) the initiation of [3H]thymidine incorporation, even incells which are not in direct contact with the substratum.

During the outgrowth an increasing number of cells reach the substratum inapparently different modes, while the cells in the aggregates display a variety of shapechanges and cell surface alterations; the cells on the substratum acquire the typicalfibroblast configuration and a smooth surface. Close to the aggregate, multilayeringof the cells is evident but will gradually disappear as the cells move over or undereach other to reach the substratum.

Transmission of information

When the aggregate attaches to the substratum a generalized reaction of the cellstakes place. DNA synthesis, as measured by [3H]thymidine incorporation, is initiatedand the cells loosen their tight contacts with each other while changing shape.Apparently, information is passed along from the cells which are in contact with thesubstratum to the whole aggregate. It is not clear, however, what the nature of thesignal is or how this information is transmitted. Attachment of a few cells mightaffect their contacts with neighbouring cells and a chain reaction might occur withinthe aggregate as soon as the attached cells start forming lamellipodia or change shape.On the other hand we have demonstrated in a previous study (Van der Schuerenet al. 1976) that specialized junctions are present between the fibroblasts in theaggregates. The possibility that information about the availability of a substratum istransmitted through functional junctions must be considered.

Role of the substratum and density-independent behaviour

The requirement for an appropriate substratum for cell spreading, movement,growth and proliferation of fibroblasts has been well documented (Harris, 1973;Dunn & Heath, 1976; Abercrombie & Heaysman, 1954; Abercrombie, 1967; Stoker,O'Neill, Berryman & Waxman, 1968; Martin & Rubin, 1974). In addition the dislikefor multilayering and the density-dependent inhibition of proliferation and movementare standard properties of 'normal' cells (Abercrombie, 1967; Martz & Steinberg,1972; Stoker & Rubin, 1967). The present investigation illustrates that underappropriate circumstances (cell aggregates) fibroblasts may temporarily abandon someof these properties while searching for the appropriate substratum.

Fig. 3. Specific surface projections formed by outgrowing cells. Bars = 10 /im. A, cellsnear the attachment side of the aggregate form extensive lamellipodia. B, superimposedlamellipodia of 2 different cells with cytoplasmic extensions searching for contact withthe substratum, c, a cell, completely buried in the aggregate, sends out a filopodiumtowards the substratum. Near its end a bifurcation can be observed, D, progressionof a filopodium. on the substratum with the formation of numerous flattened regions.E, flattened cell on the substratum still connected with the aggregate by a retractionfibre, F, lobopodia formed by cell(s) at the basal side of the aggregate.

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no B. Van der Schueren, J.-J. Cassiman and H. Van der Berghe

Outgrowth of fibroblast aggregates 111

The ability to induce the observed changes in the aggregates is not limited to aninert substratum. When aggregates are placed on a preformed cell layer they willadhere to it (Cassiman & Bernfield, 1976 a) and similar changes as described on glasswill occur, resulting in the spreading of the cells over the existing cell layer (detailedresults in preparation). Some cells may even bridge the space between 2 adjacentaggregates and adhere to the surface of a neighbouring aggregate.

Although the cells seem to form contacts with the substratum under gravitationalforces, outgrowth can be obtained against gravity on coverslips placed on top of theaggregates. As mentioned, cells may even reach towards a neighbouring aggregate.The only consistent finding therefore is that the cells leave the aggregate for anothersubstratum, cellular or not, wherever they can touch it. This further suggests thatthe cell processes which form the initial contacts are capable of travelling throughspace in all directions until contact is made with the substratum. The question remains,however, why the cells are leaving the aggregate after local attachment.

The behaviour of the aggregated cells resembles somewhat the initial outgrowthfrom a skin biopsy. No growth is observed in suspension, but once the explant isattached to the culture dish, epithelial cells and/or fibroblasts are migrating out overthe substratum. Studies on wound-healing (Elsdale & Foley, 1969) suggest thatfibroblasts tend to cover the available substratum by combined proliferation andmigration, and Abercrombie & Heaysman (1954) have demonstrated the importanceof cell contacts on the radial outgrowth of fibroblasts from explants. None of theseobservations can explain why attachment of epithelial or fibroblastic aggregates or ofan explant can induce outgrowth in the observed fashion.

In a multilayered system, DNA synthesis, loosening of cell contacts and spreadingover other cells, are typical of transformed cells (Stoker et al. 1968). In the presentcase the normally density-inhibited cells which constitute the aggregates also showthese properties. In contrast with aggregates from transformed cells, however(Cassiman & Bernfield, 19766), they do not demonstrate all of these properties insuspension (Van der Schueren et al. 1976) but only when some of the cells are incontact with the substratum.

Whereas normal cells do not spread over other cells (Rajaraman et al. 1974;Di Pasquale & Bell, 1974) many spread cells have been visualized in the presentstudy. A similar multilayering was observed by Cherny, Vasiliev & Gelfand (1975),in dense fibroblast cultures. It seems reasonable to accept that normal fibroblastsunder appropriate circumstances can indeed spread over other fibroblasts withouttouching the substratum.

It is tempting to compare this process of outgrowth to certain processes occurring

Fig. 4. 'Rounded' cells. Bars =10 fim. A, top view of an aggregate characterized bynumerous cells with localized regions of their surface covered with villi. B, cellscovered with villi bulging out of the aggregate, c, round cell adherent to neighbouringcells by flat and bifurcated extensions. D, round cell on the aggregate with cytoplasmicprocess pointing towards the substratum, E, cone-shaped cell, F, flattened cell on theaggregate with localized bulging-out of the surface and membrane activity at itsperiphery.

8-2

U2 B. Van der Schueren, J.-J. Cassiman and H. Van den Berghe

Fig. 5. Elongated cells. Bars = io^m. A, cylindrical cell stretched between aggregateand substratum, B, points of contact between cylindrical cell and a cell growing on thesubstratum. C, flat irregular cytoplasmic extension formed by an elongated cell on topof a cell growing on the substratum.

Outgrowth of fibroblast aggregates 113

during embryonic development (Trinkaus, 1973; Gustafson & Wolpert, 1961) orduring invasion of malignant cells into normal tissues (Abercrombie & Heaysman,1976). Both migration of embryonic and malignant cells occur through poorly knownmechanisms and the event initiating their migration remains completely obscure.Whereas differences in adhesive properties (Steinberg, 1972; Moscona, 1961) or'selective fixation' (Gustafson & Wolpert, 1961) most likely participate in themigration of embryonic, adult and malignant cells, it has not been examined whetherchanges in the adhesive properties of a few cells might not be a trigger for the othercells of the embryonic tissues or of the malignant tumour, as is apparently the case inexplant or aggregate outgrowth.

Cell surface microvilli and blebs

Once the aggregates have attached to the substratum a variety of surface structuresappears. The presence of microvilli and blebs on fibroblasts or epithelial cells hasbeen correlated with different phases of the cell cycle (Rubin & Everhart, 1973;Porter, Prescott & Frye, 1973) or treatment with chemicals such as trypsin, EDTA orcytochalasin (Springer, Hackett & Nelson-Rees, 1976; Dalen & Todd, 1971; Godman,Miranda, Deitch & Tanenbaum, 1975; Witkowski & Brighton, 1971). Their dis-appearance during spreading on the substratum has been documented (Witkowski &Brighton, 1971; Rajaraman et al. 1974; Follet & Goldman, 1970). In certain in vivosituations these surface projections precede and accompany cell locomotion (Trinkaus,1973). The ultrastructure of these surface structures has been analysed in detail(Taylor, 1966; Price, 1967; Cornell, 1969). It seems reasonable to accept that theiroccurrence and function may vary according to the metabolic state of the cell, itslocomotory activity and its change in shape (Erickson & Trinkaus, 1976; Albrecht-Buehler & Goldman, 1976; Knutton, Summer & Pasternak, 1975; Knutton, Jackson,Graham, Micklem & Pasternak, 1976). In the present investigation surface structuresappear concurrently with the change in cell shape, initiation of DNA synthesis andpreparation of locomotor activity as well as the organization of microfilaments andmicrotubules. All of these changes may be responsible or may contribute to theformation of villi and blebs. It should be noted, however, that most of these structuresare absent or reduced in number, on those cells or larger cell processes which havecontacted the substratum. These naked cells or cell processes seem to be particularlytended between the aggregate and the substratum. Since cells on a substratum are alsoactively spread over as great a surface as possible, the absence of villi or blebs might beexplained by the extreme stretching which the plasma membrane undergoes duringthis particular form of outgrowth and during cell spreading.

Lamellipodia, filopodia and lobopodia

The role of large cell processes in locomotion and translocation of cells has been welldocumented (Trinkaus, 1973; Albrecht-Buehler, 1976; Harris, 1973). The significanceof these alternative modes or their relationship remains unknown. In the presentinvestigation lamellipodia were observed only on cells which were in contact with thesubstratum or with cells on the substratum. Filopodia, lobopodia as well as whole

B. Van der Schueren, J.-J. Cassima and H. Van den Berghe

*» . -„

Fig. 6. Intracellular modifications characteristic for the initial outgrowth phenomena.Bars = i /im. A, accumulation of microfilaments (mi) and microtubules (mi) underthe plasma membrane at the free edge of a cell. B, similar localization of microfilaments(mi) as in A accompanied by bleb (b) formation. Ribosomes (r) in spiral associated withendoplasmic reticulum. c, band of microfilaments (mi) in peripheral cytoplasmunderlying numerous blebs (b).

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Outgrowth of fibroblast aggregates 115

cells with small projections at their edges seemed the preferential mode to reach forthe substratum.

It appears therefore that these structures allow the cells to search for the sub-stratum, to scan it and to translocate the cells out of the aggregate on to it. Whilecertain images are suggestive of a cytoplasmic flow into these processes, as describedby Albrecht-Buehler (1976), it cannot be ruled out that certain cells may be pulledabruptly out of the aggregate, or that rounded cells roll off the aggregate on to thesubstratum. To our knowledge, lobopodium formation has never been observed infibroblasts. Their presence, as well as that of all the other surface modifications, furtherstresses the resemblance between the morphogenetic properties of fibroblasts andepithelial cells of adult or embryonic origin (Trinkaus, 1973).

Microfilaments and microtubules

While organized microfilament bundles and microtubules were seldom observedin suspended aggregates (Van der Schueren et al. 1976), they accumulate at specificsites in the early stages of the outgrowth. These cytoplasmic elements are known toaffect the shape of the cells (Spooner, Yamada & Wessells, 1971; Di Pasquale, 1975)and to play a role in cell locomotion (Wessells et al. 1971; Goldman et al. 1973).Their presence at the free edge of the cell as well as at the site of intercellular contacts,before any gross alteration in cell surface has occurred or before the cells have formedlarge processes, suggests a specific role for these elements during outgrowth. Especiallythe position of the microfilaments seems correlated with the direction of movementof search. Whether they anchor the cells tightly to adjoining cells so as to preventtheir premature release from the aggregate or whether these filaments are involvedin breaking down cell contacts, as suggested by Heaysman & Pegrum (1973), cannotbe confirmed or refuted by our observations. Preliminary experiments using cyto-chalasin B and colchicine tend to confirm the primary role of the microfilaments in theobserved processes. During the later stages of the outgrowth bundles of microfilamentsare found underneath plasma membranes covered with blebs. These images resembleremarkably those observed during spontaneous or cytochalasin D-induced zeiosis(Price, 1967; Godman et al. 1975). Blebs are formed at the cell periphery as well asin clusters over the nuclear area. It is not clear, however, whether under the presentcircumstances contraction of these microfilaments or increased cytoplasmic pressureare responsible for the observed blebbing.

In conclusion, the outgrowth of fibroblasts from aggregates attached to a substratumis made possible by a variety of cellular and cell surface properties. This processillustrates the remarkable ability of normal skin fibroblasts to adapt to new in vitrocircumstances.

It further stresses the resemblance between the properties of adult and embryoniccells during directed movements and might be a valuable system to study some ofthose properties in more detail in vitro. This process also calls attention to the role ofthe substratum and of the intercellular contacts in changing the behaviour of the cells,and might give clues about the nature of the initiating signals for directed migrationin embryonic systems and for invasion of malignant cells into normal tissues.

n 6 B. Van der Schueren, J.-J. Cassiman and H. Van den Berghe

The expert technical assistance of Ms Jacobs, Ms De Geest and Mr G. Doucet is gratefullyacknowledged. We thank Dr Trinkaus for helpful suggestions in the preparation of this manu-script. This work was supported by a grant from the Belgian Cancer Fund (ASLK) and bygrant no. 3.0025.75 (FGWO).

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(Received 24 January 1977)