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/ . Embryol. exp. Morph. Vol. 26, 2, pp. 295-312, 1971 2 9 5

Printed in Great Britain

The control of cell adhesion ina morpho genetic system

By A. S. G. CURTIS1 AND GYSELE VAN DE VYVER2

From the Department of Cell Biology, University of Glasgow

SUMMARY

A new general type of morphogenetic process has been revealed by experiments on thephenomenon of non-coalescence between different strain types {alpha and delta) in the spongeEphydatia fluviatilis. The question investigated was whether any process of cell adhesion wasresponsible for the phenomenon. Preliminary results suggested that the cells might showspecific adhesion but further results indicated that a more complex system existed. Eachstrain produces a soluble factor that increases the adhesiveness of homologous cells butdecreases that of cells of heterologous strains. The adhesion of cells, even in the presence ofthese factors, is non-specific but the factors specifically control adhesion and determine itsquantitative value. The adsorption of the factors to the cells was tested for with inconclusiveresults. Heterologous factors may irreversibly alter a cell's adhesiveness. It is shown that thissystem, particularly by reason of its negative effect on adhesiveness, (a) accounts adequatelyfor the phenomenon of non-coalescence, (b) provides a model system for many forms ofmorphogenesis and (c) allows many apparently contradictory results obtained by otherworkers to be reconciled.

INTRODUCTION

The phenomenon of non-coalescence of the tissues of differing species or ofdiffering strains of the same species of sponge discovered by van de Vyver (1970)appears to form a remarkable system of morphogenesis. When the tissues of twoindividuals of the sponge Ephydatia fluviatilis are brought into contact byexperimental means or when they come into contact through natural processes,they form an adhesion in the zone of contact. If the tissues are of the same straintype the pinacoderms become confluent, and later the canal systems of the twosponges join up and the sponges become effectively one organism. If, however,the two sponges are of different strain type the initial adhesion persists for atime, after which the two sponge bodies separate from one another leaving a gapbetween them. Generally the two sponge bodies move apart. This behaviour hasbeen termed non-coalescence by van de Vyver (1970). She has suggested thatthis behaviour may be due to a specificity of adhesion amongst the cells suchthat cells from different strains cannot stick to one another. The first aim of the

1 Author's address: Department of Cell Biology, University of Glasgow, Glasgow W.2,U.K.

2 Author's address: Zoologie Generate, Faculte des Sciences, Universite Libre de Bruxelles,Avenue F.-D. Roosevelt, 50, Bruxelles 5, Belgium.

296 A. S. G. CURTIS AND G. VAN DE VYVER

work described here was to test whether or not those strains which shownon-coalescence also show specificity of adhesion. This test was carried outusing the collision efficiency method (Curtis, 1970a, b). During this workresults were obtained and independently further information reported by van deVyver (1971 b), which now suggest to us an alternative mechanism as the basisof non-coalescence. Consequently further experiments to test this secondmechanism are described here.

MATERIALS AND METHODS

Sponges of the alpha and delta strains of Ephydatia fluviatilis were grownfrom gemmules supplied by Professor Rasmont's laboratory (Universite Librede Bruxelles). The sponges were cultivated in Rasmont medium (Rasmont, 1961)for 1-2 weeks before use, at ca. 20 °C. Each sponge was grown from ten gemmulesand contained about 1 x 105 cells. Cell suspensions were prepared by scrapingthe sponge bodies off the culture-dish surface and then transferring thesponges to the dispersion medium. Each 10~3m3 (approx. 1 litre) of thismedium contained 2-5 xlO"4 mol. EDTA, 0034 mol. NaCl, 0-00134 mol.KC1, 000138 mol. glucose, 0-00107 mol. NaHCO3, 7xlO-4mol. K2HPO4

and was buffered to pH 7-8 with 0006 mol. 2-amino-2-hydroxymethyl, 1-3propanediol (Tris). After exposing the sponges to this medium at ca. 20 °Cfor 7 min they were mechanically dispersed in the same medium by pipetting.From this point two different methods of cell dispersion were used, namelythe 'washed cell' technique and a second technique in which an appreciableportion of the disaggregation medium is included in the final cell suspension.In the washed cell method the cell suspensions were then centrifuged at ca. 200 gfor 5 min, the supernatant discarded and the pellet resuspended in Rasmontmedium. In the second technique the cells were dispersed in a very small volumeof EDTA medium and the suspension was then made up with a large volume ofRasmont medium. Since a small amount of EDTA would be carried over into thecell suspension by this procedure and because of the possible existence of solublefactors affecting cell adhesion (see later) the volumes of medium used andremoved at each stage were made standard for all but the preliminary experi-ments, in which they were roughly standard. The EDTA level in the final cellsuspension was ca. 1 x 10~4mol. (10~3 m3)-1 compared with a Ca + Mg con-centration of 1-5 x 10~3 mol. (10~3 m3)"1. The dispersion technique used in eachseparate experiment is stated in the appropriate part of the text. The adhesive-nesses of the cells (collision efficiencies) were measured with a Couette visco-meter, using the method described by Curtis (1969, 1970#, b, c). Measurementswere made at shear rates between 8 and 9 sec"1 and at room temperature(20-25 °C). Each value for a collision efficiency quoted in the Results section isthe mean of three sets of measurements each made for five different periods ofaggregation. Counts of cell and aggregate population densities were made usinghaemocytometers.

Control of cell adhesion 297

Alpha and delta factors were prepared from alpha and delta spongesrespectively by a modification of the method described by van de Vyver (1971 b).A precise number of gemmules (usually 30 sponges from 300 gemmules) weredispersed in the EDTA medium used for cell dispersal after soaking in thismedium for 7 min. The cell suspensions were then exposed to ultrasound(100 W at 20 kHz for 2 min). The gemmule cases were removed, and the suspen-sion of cells and broken cells concentrated and dialysed by vacuum dialysisagainst normal Rasmont medium. The dialysis membrane used had an effectivepermeation limit for molecules of mass 1000 daltons. The dialysed materialwas filtered through a Millipore HA filter and made up in a volume of 3 ml.The factors were stored under refrigeration and used within 2 days of prepara-tion. A subsidiary experiment showed that this method of preparation of thefactors yielded a product apparently identical with those prepared by theoriginal method (van de Vyver, 1971 a).

Specificity of adhesion was tested by the method described by Curtis(1970(7, b). In this test the collision efficiency, is (written as a in Curtis, 1969), ofthe two cell types is measured for each type separately and then for mixtures ofvarious proportions of the two types. If there is no specificity of adhesion thecollision efficiency for any proportion of the two cell types will be the average ofthe efficiencies for both types (measured separately) weighted for the proportionof each cell type in the mixture. If, however, there is complete specificity ofadhesion such that cells derived from different strains will not adhere, then themeasured collision efficiency will be reduced because all collisions betweenunlike cells will fail to result in adhesions. The full theory of this test is givenby Curtis (19706). In summary, if there is no specificity of adhesion, thecollision efficiency for a mixture of two types of cells of adhesivenesses Ex andE2 mixed in proportion rtx and n2 is given by n2E2 + n2E2. If there is com-plete specificity of adhesion the equivalent relationship is nlE1 + nlE2. Ifspecificity is incomplete the plots of collision efficiency against proportion of acell type in the mixture will lie between those for complete specificity and com-plete lack of specific adhesion. The difference in collision efficiency for any twodegrees of specificity is at a maximum for a 50:50 mixture of the two cell types.Appropriate statistical tests for the treatment of experimental data have beenpublished (Curtis, 1970tf, b) and these are used in this work.

PRELIMINARY RESULTS AND DISCUSSION

Tests were made, using the collision efficiency method, to discover whetherspecificity of adhesion occurred for cells of the alpha and delta strains. Theresults are given in Table 1 and Fig. 1. The results suggest that specific adhesionis found. The second method of cell dispersion was used.

However, the discovery of factors which promote aggregation, made byvan de Vyver (1971 a), suggests another explanation of these results. An alter-


native hypothesis which would account for the reduced adhesiveness of mixturesof the cells of two strain types is that these factors promote the adhesiveness oftheir own strain but diminish the adhesiveness of other strains. The method ofpreparation of these factors is very similar to that used for the preparationof cell suspensions; indeed van de Vyver used exactly the same method forfactor preparation in her work as used here for cell suspension preparation.

Table 1. Test for specificity of adhesion of alpha and deltastrain cells {see also Fig. 1)

Alphacells in

suspension

0 010130-749-972-6

1000

Collisionefficiency

4-763-121-901-692043-40

Standarddeviation

0-450-290140-240190-65

Each measurement is the mean of four observations.Linear regression y = l-08x+ 3-34. Sum of squares of deviations from regression = 28-23,

where x is percentage expressed as a proportion.Curvilinear regression y = 3-612x2 + 0-424. Sum of squares of deviations from regression

= 7054, where x is the proportion of the major component in the mixed suspension. Re-duction in sum of squares by curvilinear regression = 21-17. F = 66-15. Highly significantimprovement of fit by use of curvilinear regression, 9 5 % confidence limits at x = 0-500;0-83-1-32-1-82.

2 -

1 -

50 100Alpha strain cells (%)

Fig. 1. Test of specificity of adhesion for mixtures of alpha and delta strain cells.Experimental results are shown as means (o) with standard deviations. Thestraight line represents results that will be expected on the hypothesis of non-specificadhesion. The continuous curve represents the results that will be obtained on thehypothesis of specific adhesion.


Therefore it is probable that these factors are present in the cell suspensionsunless the cells are very thoroughly washed. If this hypothesis is correct theespecial circumstances of the test for specific adhesion would lead to itssimulation when two equal aliquots of suspensions of cells of unlike type aremixed. Before mixing, each suspension separately contained a concentration ofthe aggregation factor sufficient to maintain a value for adhesiveness E. Onmixing with an equal aliquot of the other cell suspension the concentrations ofboth factors would be halved so that the adhesiveness of each cell type would bereduced (the exact extent of reduction depends upon the nature of the dose-response curve); moreover each cell type would experience a great increase inthe concentration of the factor derived from its opposite cell type, furtherreducing its adhesiveness. In consequence, although the cells do not showspecificity of adhesion the collision efficiency of the mixture is reduced and thusthe existence of specific adhesion might be deduced from the results incorrectly.The general hypothesis will be termed the factor-specificity theory. It is ofcourse possible that the above system might exist with specific adhesion occurringas well. Either in such a case or if the dose-response curves for the factors arevery steep it would be possible to obtain a greater reduction of adhesiveness forthe mixed suspensions than is predicted by the simple theory.

We felt that in order to be sure that the actual mechanism of adhesion isspecific it was necessary to show that the above explanation of specificity interms of these factors was incorrect. One simple postulate of the factor specificityhypothesis is that the delta factor, for example, would increase the adhesivenessof delta cells and reduce that of alpha cells as the concentration of factor wasincreased. An alpha factor would operate in a converse manner. A second postu-late is that the dilution of a cell suspension with a bland medium will reduce theadhesiveness of the cells because the concentration of the aggregation promotingfactor will be reduced. The result of this test may show also that the aggregationfactor is normally present in a cell suspension. These two tests are described inthe next section. However, the results of these tests, even if positive, will onlyshow that the necessary components of a system of factor-specificity exist, notthat the system operates amongst the cells. A third, more complex, test wasdesigned to test this latter point, but it is described later because it is best under-stood in the light of the results of the first two tests.

RESULTS

Effect of aggregation factors on cell adhesiveness

In the first test alpha and delta factors were added to 'washed' suspensions ofalpha and delta cells within 1 min of the start of reaggregation. In the absence ofany knowledge of the chemical nature of these factors an arbitrary concentrationscale was used. Concentrations are expressed in terms of the number of spongecells disrupted per cc (10~G m3) of medium x 10~5. Thus a 30-unit solution would


contain the yield of 3 x 106 cells/cc. The effect of alpha and delta factors on theaggregation and adhesiveness of both alpha and delta cells is shown in Table 2and Fig. 2.

These results confirm the existence of the alpha and delta factors postulated byvan de Vyver and additionally show that the factors diminish the adhesiveness ofcells of the opposite strain type. It should be appreciated that this result with thistest depends upon the accidental or intelligent choice of a suitable level of factorsin terms of the dose-response curve.

The second test examines whether dilution of a cell suspension has any effecton its adhesiveness. If an effect is found it can be concluded that an aggregation

Table 2. Dose-response curve for the adhesiveness (collision efficiency) means Eand standard deviations a of alpha and delta strain cells in the presence of homo-logous and heterologous factors

Concentrat ionof factor

(units/cc) E cr

Alpha factor

Delta factor

Delta factor

Alpha factor

A. Alpha4002001000-500-400-25000

01250-250-501030

cells12-418-256063-512-841-961-431100-670-430-21005

B. Delta cells

4002-501-000-660-500-25000

0050100-200-250-400-50

8-307-566104-592-392-341-70

1-461180-500-400-27008

3-811-750-981100120090-37

0110-21016*010010

0-591160-461010190190-29

0-270050-170-20*0140-35

Each measurement, with the exception of those marked with an asterisk, is the mean offour measurements. The asterisked measurements are the mean of 11 observations.

The majority of mean collision efficiencies are shown in Fig. 2 but for reasons of clarity afew points and the standard deviations have been omitted from the figure.

Control of cell adhesion 301promoting factor normally present in the cell suspension has been diluted. Thistest of course depends upon the accidental or intelligent choice of two suitablyplaced points on a dose-response curve. Cell suspensions of both alpha and deltacells were separately made up at high population densities (l-7xlO6 cells/10~6m3) by method 2. An aliquot of each suspension was then placed in theCouette viscometer for collision efficiency measurement; another aliquot wasdiluted with an equal volume of Rasmont medium and then the collision

(a) 15 r (b)1 5 r

Delta factor(units/cc)

Alpha factor(units/cc)

Alpha factor(units/cc;

Delta factor(units/cc)

Fig. 2. Dose-response curves for the adhesiveness of (a) alpha strain and (b) deltastrain cells in the presence of alpha and delta factors. The points are mean valuesfrom experimental observations. See also Table 2.

Table 3. Dilution experiment

Cell straintype

Delta

Alpha

2 0

Experiment

I

H

III

IV

V

InitialDiluted

InitialDiluted

InitialDiluted

InitialDiluted

InitialDiluted

Each measurement is

Cell populationdensity (x 10-6/cc)

2-11106

1-32065

1 -810-92

1-990-99

2-820-97

Collisionefficiency

4112-56

4-972-05

3-591-46

4-312-69

1-340-80

the mean of five observations.

0-270-26

0-45008

0-41015

0-370-21

010004

!•: M 1! 2 6


efficiency was measured. The adhesivenesses of concentrated and dilutedsuspensions are shown in Table 3. It is clear that dilution of both alpha anddelta cell suspensions diminish their adhesiveness.

These results suggest that it is at least possible that the appearance of specificadhesion reported in the 'Preliminary Results' is due to the presence of thealpha and delta factors. In those experiments where a delta strain cell suspensionis mixed with an alpha suspension, the adhesiveness of the delta cells will fallbecause of the dilution of delta factor, and because of the presence of the alphafactor; similarly the adhesiveness of the alpha cells will fall because of thepresence of the delta and dilution of the alpha factors. The main question to bedecided is whether the extent of the reduction of adhesiveness observed in thepreliminary results can be entirely accounted for by the presence of the twofactors together in mixed suspensions. If the reduction in adhesiveness is greaterthan can be ascribed to the presence of the factors, it is possible that the hypothesisof specific cell adhesiveness should be used to explain the results. Examination ofthe dose-response curves and the data given in the section on Preliminary Resultssuggest that the alpha and delta suspensions in those experiments containedrespectively 0-5 units/cc and 0-8 units/cc of their factors. Mixing equal volumesof these two suspensions would halve these concentrations in the mixed suspen-sion. Assuming that the collision efficiency of cells in the simultaneous presenceof both factors is the mean of the efficiencies measured in the separate presenceof each factor, the collision efficiency of each strain type in the mixture can becalculated. In turn the collision efficiency of the mixed cells can be determinedand the question of the specificity of adhesion resolved.

The effect of the simultaneous presence of both factors on the adhesivenessof each strain type was measured using well-washed cells (method 1), to whichvarying concentrations of alpha or delta factors were added. The results areshown in Table 4. They indicate that the collision efficiency of the cells in thepresence of both factors is slightly lower than a value obtained by taking themean of the values found for the separate presence of each factor at the sameconcentrations.

We can now calculate the collision efficiencies that would be expected in themixed systems described in the Preliminary Results from the operation of thealpha-delta factor system. The two pure cell strain suspensions containedrespectively 0-5 units/cc and 0-8 units/cc of their respective factors. Talcing intoaccount the results shown in Table 4 and the dose-response curves, the collisionefficiency of the alpha cells in a 50:50 mixed suspension would be 1 -35 % and thatof the delta cells 1-40%. This would give a mean value of 1-37 % in the mixedsuspension assuming that there is no specificity of adhesion. This is close to thevalue determined experimentally of 1-69 % for a mixed suspension. If there wascomplete specificity of adhesion in the presence of the alpha-delta factor systemthe collision efficiency would be 0-68 % because specificity of adhesion wouldfurther lower the apparent adhesiveness of the cells in the mixture. Since this

rControl of cell adhesion 303

value lies outside the 99 % confidence limit (lower value 1-13 %; 1-tailed test) ofthe measured value there is no reason for concluding that specific adhesionoccurs in this system.

It might be thought at first sight that the alpha-delta factor system is in effecta mechanism for producing specific adhesion amongst cells which do not possessit when they are extensively washed. However, it is clear that much of theeffectiveness of the system arises from its ability to diminish the adhesivenessof cells of unlike strain types. This is not a characteristic of any system of specific

Table 4. Collision efficiency in the simultaneous presence of homo- andheterologous factors {means E and standard deviations &)

Concentration of factors(units/cc)

Alpha strain cells:

Delta strain cells:

Each

Homologous Heterologous

100-5

018

100-5

018

measurement is derived

0090-20

018

0090-20

018

E

2-891-672 040-83

3-873052-460-83

from four observations.

0-380181-680-28

0-381120-390-21

adhesion. In addition it is clear that from the experimental data that the kineticsof aggregation are such that adhesions must form indiscriminately between cellsof different strain type. The alpha-delta factor system is, however, a system inwhich there is a strain-specificity in the control of adhesion. In the Discussionwe shall consider other experiments which can be reinterpreted in the knowledgewe now have of the alpha-delta factor system. In some of those experimentsevidence has been put forward for specific mechanisms of cell adhesion involvingthe existence of a specific cementing factor. One very simple experiment to testfor the action of a cementing factor is to show that the factor is bound to cellsprior to or during its action in promoting adhesion. Curiously this test does notappear to have been carried out on any system of cell adhesion by any previousworker. Since we are interested in the manner in which the alpha-delta factorsystem operates and in establishing any similarities with or differences fromsystems of specific adhesion, we carried out tests to establish whether thesefactors are adsorbed to the cells. It is clear from the dose-response curves thatit is possible to choose concentrations of factor that are below levels that mightsaturate any binding sites that may exist on the cells. Cell suspensions preparedby method 1 (well-washed cells) were treated with non-saturating levels of


heterologous factor. After aggregation the suspensions were centrifuged and themedium recovered. This medium was used to treat a second batch of cells of theheterologous strain during their aggregation. (It is undesirable to carry out thetest on cells of a homologous strain type because homologous factor may beadded to the system by the cells.) If the apparent activity of the factor, as shownby its effect on the adhesiveness of the second batch of cells, is reduced byexposure to the first batch of cells it would seem probable that the factor isadsorbed to the cells. The results are given in Table 5. They give no support tothe hypothesis that the factors act by adsorption to the cells, though this cannotbe excluded (see Discussion).

Such a result suggests that the factors are not adsorbed to the cells, and hencecannot form a part of a cell binding material. The results are consistent with thefactors being enzymic in their action. Related to this point is the question ofwhether the effect of each factor is reversed by the opposite factor or otherwise.

Table 5. Factor absorption experiment

Cell strain typei K \ Activity (units/cc)

Assayedtype on on Expected

0-500-250-250-750-250-600-45

Found

0-500-250-200-800-200-600-55

Alpha Delta Delta

Delta Alpha Alpha

The regression of measured activity on expected activity is given by y = 1-159.x:-0-062,sb = 0091. Testing the hypothesis that factor adsorption has diminished or raised its activity,t = 1 -75, D.F. = 5. The hypothesis is rejected.

The reversal test was carried out by two methods: in the first, well-washedcells were treated with factor of the opposite type, and their adhesiveness wasmeasured over a 30-min period, then the cells were centrifuged out of the mediumand resuspended in a medium containing homologous factor, before measurementof their adhesiveness. The second method of performing this experiment is totreat well-washed cells with homologous factor first and then with heterologousfactor, after removal of the homologous factor by centrifugation. This method ofcarrying out the experiment is less satisfactory than the first technique becausethere is appreciable cell adhesion during the treatment with homologous factor.In consequence, the measurement of collision efficiencies during the secondtreatment is less accurate because the method of measurement presumes that atthe start of aggregation the cell population is monodisperse. However, the secondmethod of carrying out this experiment was used because there may be differences


in the result depending on whether the cells are exposed first to homologous orfirst to heterologous factors. Results are given in Table 6. The values that wouldbe expected in the absence of a pretreatment step can be read in Table 2. Theresults show that the effect of treatment by a homologous factor is reversible bya factor of the opposite type, but that the reverse is not the case. This suggeststhat an active site involved in cell adhesion is irreversibly altered by the hetero-logous factor so that a subsequent treatment with homologous factor is in-effective.

Table 6. 'Reversed treatments'' -sequential treatment withhomologous and heterologous factors

Cellstraintype

A. First treatment

Alpha

Delta

First treatment

Factorconcentration

(units/cc)Collisionefficiency

Second t

Factorconcentration

(units/cc)

reatment

Collisionefficiency

with heterologous factor, second with homologous factor

0-250-250-250-250-250-250-250-25

0-50-50-50-50-50-50-5

0-310-310-420-370-470-420-490-47

0-890-290-220100150-300-35

B. First treatment with homologous factor,

Alpha

Delta

Each

0-40-40-40-4

0-660-660-660-66

horizontal line

2-642-492-893-36

4-775-224-173-93

1-91-91-91-91-751-751-751-75

1-91-91-91-91-751-751-75

0-21009007

<005<005<005<005<0-05

0-56<005<005

0140190130-30

second with heterologous factor

0-50-50-50-5

0-50-50-50-5

0-950-310-42010

< 0 1< 0 1< 0 1< 0 1

of figures refers to one experiment.

DISCUSSION

The experiments described in this paper start with the apparent demonstrationof the specific adhesion of the cells of the alpha and delta strains of Ephydatiafluviatilis. The earlier experiments were carried out before the discovery by van


de Vyver (1971 b) of the soluble factors, released during dispersion of the spongetissues, which promote the aggregation of cells of the same strain type as thefactor. The existence of these factors suggested that the preliminary experimentscould be reinterpreted as evidence for a system in which strain differences in theresponse of the cells to the factors would lead to results simulating specificadhesion. The experiments reported in the main section of results show thathomologous factors increase and heterologous factors decrease the adhesivenessof the cells. They also show that the adhesiveness of cells in the presence of bothhomo- and heterologous factors is the mean of the values that would be expectedin the separate presence of each factor. Most important of all, the results showthat the extent of adhesion reduction in mixed alpha-delta suspensions isquantitatively predicted with accuracy from the concentrations of alpha anddelta factors in the mixed suspension, on the assumption that the cells show nospecificity of adhesion. In other words the factor specificity system accounts forthe behaviour, reported in the Preliminary Results, which simulates specificadhesion.

At this point it is perhaps appropriate to consider the criticism of the testsystem for specific adhesion, used in this work, made by Humphreys (1970).Humphreys suggested that Curtis (1970 c) had not shown that the rate of aggre-gation has a complete dependence on cell population density and therefore thatthe interpretation of the experiments might be incorrect. However, it is implicitin the Swift & Friedlander (1964) treatment, which is the basis of the method ofmeasuring cell adhesiveness introduced by Curtis (1969), that the kinetics ofaggregation of any two size classes will follow second order kinetics. (Theaggregation rate integrated over all size classes will show first order kinetics.)If the cells did not aggregate in this way very large standard deviations would befound in the measurements of collision efficiency over a time course duringwhich the total particle concentration falls to about a half of the initial value.The actual measured standard deviations are small; this suggests that theaggregation kinetics for one or two size classes are second order. It might atfirst sight be felt that the behaviour shown in Table 3, where dilution of a cellsuspension diminishes the collision efficiency, is in fact direct proof that second-order kinetics apply for any two size classes, but it should be borne in mind thatin calculating the collision efficiency by the Swift and Friedlander treatment adimensionless value is obtained, in other words, differences in cell concentrationare removed. Humphreys also suggested that the aggregation rate actuallymeasured might not be a true measure of adhesiveness, because the rate-limitingstep might be the rate of recovery of the cells from dispersion procedures. If thecells recover their adhesiveness slowly the rate of aggregation would be deter-mined by this process. Again it is clear that if this occurred, collision efficiencieswould rise during the course of aggregation and in consequence large standarddeviations would be found in the values derived from measurements over thetime course of the experiments. The constancy of collision efficiency values found


for any defined set of conditions in this work argues against the hypothesis thatthe measured aggregation rate is limited by a recovery of cellular adhesiveness.This criticism cannot of course be applied to those experiments where hetero-logous factors diminish cell adhesiveness.

The next question to be considered is whether the system revealed in this workcan be used to account for the non-coalescence and the sorting out described byvan de Vyver (1971 a, b). It appears from her work that when two sponges ofunlike type make contact the pinacoderms of each sponge form an adhesion.The cells of each individual do not interpenetrate. Some 15-30 h later (depend-ing on the strain types involved) the adhesion of the sponges breaks down andthey separate. It is possible that contractions in each sponge body help to pullthe sponges apart. When two homologous sponges make contact an adhesiondevelops between pinacoderms. Later the pinacoderm cells migrate to the surfaceof the fused sponge and the cells from each individual interpenetrate to aconsiderable extent. It should be appreciated that if this phenomenon is explainedon the hypothesis that the cells show complete specificity of adhesion (cf.Humphreys' results on Microciona and Haliclona - Humphreys, 1963) it wouldbe expected that two sponges of unlike type would not even form a temporaryzone of adhesion.

A more satisfactory explanation of the major features of non-coalescence canbe developed in terms of the idea that the alpha and delta factors control thisphenomenon. The concentration of a sponge's own factor will tend to be maximalat the centre of the sponge body if it is assumed that all cells of the spongeproduce the substance. The concentration will be minimal at the periphery of thesponge (under a wide variety of assumptions about the origin, diffusion anddestruction of the factor). Therefore the adhesiveness of the cells of the surface ofthe sponge body will be low but not negligible. When two sponges of like typemake contact an adhesion will form. Since the factors are of diffusible nature andsince they may act without being irreversibly bound to the cells, the concentra-tion of the factors will rise in the region of contact between the sponge bodies.As the homologous factors increase in concentration the cells will become moreadhesive and a single permanent sponge body will be established.

When two sponges of unlike type make contact an adhesion will form initiallybecause the concentrations of the two factors will still be low at the originalsurface of the sponges along the zone of contact. However, because of thechanged geometry of the sponges the concentrations of the two factors will risealong the region of contact. Though the measurements of the simultaneouseffect of hetero- and homologous factors on the adhesiveness of cells show atendency for the two factors to cancel out (Table 4), it is clear from the sequentialtreatment experiments (Table 6) that the heterologous factor will irreversiblyalter the adhesiveness of the cells (unlike the homologous factor) so that even-tually the cells exposed to heterologous factor will become non-adhesive. Wecan view the process as a competition between homo- and heterologous factors,


the heterologous factor being a competitive inhibitor. During a short-termexperiment (30 min) the progressive inactivation of adhesion is not obvious.The diminution in adhesion will be maximal for the cells lying at the zone ofcontact since they are of opposite type on either side. Thus the sponges will ceaseto adhere to each other some time after the initial contact is made.

It is possible that the cells in the original zone of contact might become sonon-adhesive that they would separate from the sponge bodies and migrate ordrift to other regions; in this way a gap would as it were be eroded between thesponges. Though the phenomenon of non-coalescence of unlike strain types canbe adequately explained on the hypothesis advanced in the last paragraph it isclear from van de Vyver's results (van de Vyver, 19716) that there are morecomplex and perhaps secondary features of the phenomenon that are less easyto explain. When contact is made between homologous sponges the pinacodermcells in the region of contact eventually migrate to the free surface of the sponge,thus allowing the inner regions of the two sponges to unite. In heterologouscontacts the pinacoderms remain intact in the region of contact.

The phenomenon of non-coalescence has certain similarities to that of contactinhibition of movement of cells (Abercrombie & Heaysman, 1954). In bothphenomena cells make contact and form adhesions with each other and sub-sequently re-separate. However, contact inhibition of movement is displayedbetween homologous and heterologous cells whereas non-coalescence onlyoccurs between unlike cells. Moreover Abercrombie & Gitlin (1965) have shownthat it is unlikely that any diffusible agent is involved in contact inhibition ofmovement. There remains the considerable, if superficial, resemblance betweenthe two phenomena in that an outgrowth of cells stops movement on contactwith another body of cells. Contact inhibition of movement has been suggestedas being the phenomenon which prevents cells of normal tissues from invadingone another (Abercrombie, 1967). However, it can now be seen that thestrain-specific adhesion factors discovered in this work would, if they acted astissue specific adhesion factors, prevent tissues from invading one another.Moreover such a system would have two further capabilities of great importancein morphogenesis. First, during morphogenesis, tissue factors of this type couldinteract to diminish the adhesiveness of cells in the region of contact of two celltypes. As a result, gaps and cavities between tissues could be formed. Second,such systems for the control of adhesion would act to ensure that cavities andgaps between tissues would persist in adult life. It should be remembered that thevertebrate body develops many cavities within it during development and thatmany organs - for example, the liver - lie with the majority of their surfacefree and unadherent to the surrounding peritoneum even though there may beprolonged contact between the two tissues. In a special sense these factors can beregarded as being the morphogens postulated by Edelstein (1970), their actionbeing, however, not chemotactic or chemokinetic but as controllers of adhesion.It is important to emphasise that it is the soluble and therefore presumably


diffusible nature of these factors that makes them of particular utility in explain-ing morphogenesis.

These factors, if they diffuse from the cells that produce them, could act as ameans of providing positional information and determination of pattern. Ingeneral terms, such a system would be analogous to that proposed by Wolpert(1969), with the distinction that these factors would control the positioning ofcells rather than the differentiation of cells in fixed positions.

When the results of this work are compared with those obtained on thereaggregation of other species of sponge, in particular by Humphreys (1963),Sara (1965), Sara, Liaci & Melone (1966), Humphreys (1970) and Curtis(1970<7, c), it is clear that it is now possible to reconcile a number of differinginterpretations. Humphreys (1963) identified a factor which specifically pro-moted the adhesion of cells of the species type from which the factor was derived.He appears to have carried out no test to discover whether the factor diminishedthe adhesiveness of other species types. Such factors were discovered for thespecies Haliclona occulata, Microciona prolifera, Halichondria panicea andC/ione celata. Humphreys suggested that these factors acted as cements whichwould attach to binding sites on two apposed cell surfaces, thus binding thecells together. No evidence was put forward that these factors were adsorbed bycells. In order to demonstrate the activity of the factors the cells had to be verythoroughly washed to reduce their adhesiveness for control experiments. Onadding the factors, specific promotion of adhesion could be detected. Curtis(1970c) repeated part of Humphreys' work using the two species Haliclonaocculata and Halichondria panicea. The cells were very thoroughly washed andno evidence for specific adhesion could be detected from measurements of thecollision efficiency of mixed suspensions. It can now be seen that Curtis'sexperiments can be reinterpreted as showing that the cells, in the absence of anyfactor, show non-specific adhesion. Sara (1965) could find no evidence for specificadhesion in quite a wide range of marine sponges. Unfortunately his papersgive few details of how the cell suspensions were prepared. Curtis (1970a) alsoinvestigated whether a variety of other marine sponge cells showed specificadhesion. In these experiments the cell suspensions were well washed and noevidence for specific cell adhesion was found. Curtis's and Humphreys' separateresults can be reinterpreted in the following way as a consequence of the hypo-thesis advanced above. We shall assume that the factors discovered in E.fluvial His strains have species-specific counterparts in marine sponges. Curtis'sfailure to detect specificity of adhesion or specific control of adhesion wouldarise from the fact that his cell suspensions were washed clean of the factors.Humphreys diminished the adhesiveness of the cells by the method of celldispersion and by aggregating the cells at low temperatures. If we assume thatthe factors he isolated were identical in general behaviour with those discoveredin E. fluviatilis they would act not as agents of specific adhesion but as sub-stances specifically controlling the appearance or loss of an adhesion whose


direct mechanism would be unspecific. In the experimental design used byHumphreys such factors of the Ephydatia type would give exactly the sameresults as he obtained. In detail the aggregation of cells of a heterologoustype would be prevented and adhesion of cells of a homologous type promotedin the presence of a single factor. When two species types are aggregated in thepresence of both factors it would be expected that the first adhesions would berandom with respect to the species types joining together. Later as small groupsof cells of one species type began to produce appreciable additional amounts oftheir factor they would make their local environment less favourable for inter-specific adhesions and more favourable for adhesions with their own speciestype; in this way sorting out of the species would occur. This would account forthe fact that it is frequently seen that aggregates involved in sorting-out 'expel'cells (Curtis, 1967). Thus apparently contradictory results on cell adhesion canbe reconciled by the hypothesis that there are diffusible factors which specificallycontrol the adhesiveness of cells whose mechanism of adhesion is itself un-specific.

The mode of action of these factors has considerable implications for theexplanation of the mechanism of adhesion of Ephydatia cells. If the factors areadsorbed by the cells it is, at least in principle, possible that they may act asbinding agents (cements). If they form binding materials they must have atleast two binding sites, one to attach to each cell surface. An alternative ex-planation of the activity of the factors is that they modify a cell surface compo-nent involved in adhesion by enzymic action. The failure of the adsorption testto detect adsorption of these factors cannot be regarded as anything more thannegative evidence because if the number of binding sites were small and thebinding constant (stability constant) high a very small quantity of factor wouldbe removed from a given initial concentration of the factor on each successiveexposure to cells. Consequently, since it is improbable that the test would detecta small reduction in the activity of the factor from one exposure to cells toanother, no clear evidence about adsorption would be obtained. Interestinglyenough other workers (e.g. Humphreys, 1963; Moscona, 1968) who have claimedto isolate specific cell-binding materials, do not appear to have tested whethertheir factors are bound by cell surfaces. The results obtained when cells of eitherstrain are treated sequentially with both factors are explicable both on thetheory that the factors attack a site involved in adhesion enzymically or thatthey adsorb irreversibly to a cell surface binding site. However, if the factors areadsorbed to the cell surface it is improbable that they act as cements for thefollowing reason. It is clear that heterologous factors cannot act as bindingagents because they decrease cellular adhesiveness, though they might blockbinding sites. The homologous factors increase cell adhesion but do not do sospecifically, yet if they acted as binding agents they would produce specificadhesion, because they clearly could not bind to heterologous cells. Hence itseems unlikely that these factors act as specific binding agents, though we

Control of cell adhesion 311cannot yet conclude whether their action is enzymic or whether they modifysome general method of cell adhesion after surface adsorption.

RESUME

Une nouvelle forme de processus morphogenetique a ete mise en evidence par 1'etude duphenomene de non-confluence entre deux souches {Alpha et Delta) de l'eponge d'eau douceE. flu via til is.

Ce travail a pour but de verifier si le phenomene de non-confluence est en relation avec lesmecanismes de l'adherence cellulaire.

Les resultats preliminaires suggeraient une specificite de l'adherence cellulaire mais desexperiences ulterieures ont montre qu'il existait un systeme plus complexe. Chaque soucheproduit un facteur soluble qui augmente l'adherence des cellules homologues et reduit celledes cellules heterologues. L'adherence des cellules, meme en presence des facteurs n'est passpecifique, mais les facteurs controlent specifiquement l'adherence et determine sa valeurquantitative.

L'absorption des facteurs par les cellules a ete testee mais sans resultats. Les facteursheterologues peuvent alterer l'adherence cellularie de maniere irreversible.

Le systeme mis en evidence, notamment par son action negative sur l'adherence cellulaire(a) explique le phenomene de non-confluence, (b) fournit un modele pour diverses formes demorphogenese et (c) permet de concilier des resultats apparemment contradictaires obtenuspar differents auteurs.

We should like to express our thanks to Professor R. Rasmont for the supply of spongegemmules, to Miss Rose McKinney for her expert assistance and to Mr Graeme Ferguson for'planting' gemmules. We are most grateful to the Science Research Council for a grant to oneof us (A. S. G. Curtis), which provided much of the support for this project, and to theBritish Council and Le Ministere de 1'Education Nationale et de la Culture de Beige whoprovided travel funds. Additional support was provided by the University of Glasgow andby the Universite Libre de Bruxelles.

REFERENCES

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ABERCROMBIE, M. & HEAYSMAN, J. E. M. (1954). Observations on the social behaviour ofcells in tissue culture. II. 'Monolayering' of fibroblasts. Expl Cell Res. 6, 293-306.

CURTIS, A. S. G. (1967). The Cell Surface. London: Logos Press, Academic Press.CURTIS, A. S. G. (J969). The measurement of cell adhesiveness by an absolute method.

/. Embryo/, exp. Morph. 22, 305-325.CURTIS, A. S. G. (1970a). Problems and some solutions in the study of cellular aggregation.

Symp. Zool. Soc. Lond. 25, 335-352.CURTIS, A. S. G. (19706). On the occurrence of specific adhesion between cells. /. Embryol.

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MOSCONA, A. A. (1968). Aggregation of sponge cells: cell-linking macromolecules and theirrole in the formation of multicellular systems. From /// vitro, vol. m. Publ. Tissue CultureAssociation.

RASMONT, R. (1961). Une technique de culture des eponges d'eau douce en milieu controle.Annls Soc. r. zool. Belg. 91, 147-156.

SARA, M. (1965). Aggregazione cellulare interspecifica fra specie diverse di Poriferi e fraPoriferi ed Anemonia sulcata. Boll. Zool. 33, 1067-1077.

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VAN DE VYVER, G. (1970). La non confluence intraspecifique chez les spongiaires et la notiond'individu. Ann. Embryol. Morphogen. 3, 251-252.

VAN DE VYVER, G. (1971 a). Analyse de quelques phenomenes d'histoincompatibilite intra-specifique chez l'eponge d'eau douce Ephydatia fluviatilis (Linne). Archs Zool. exp. gen.(in the Press).

VAN DE VYVER, G. (19716). Mise en evidence d'un facteur d'agregation specifique chezl'eponge d'eau douce Ephydatia fluviatilis (Linne). Ann. Embryol. Morphogen. (in the Press).

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(Manuscript received 3 March 1971)

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