/. Embryol. exp. Morph. Vol. 26, 2, pp. 295-312, 1971
Printed in Great Britain
295
The control of cell adhesion in
a morpho genetic system
By A. S. G. CURTIS 1 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 the
phenomenon of non-coalescence between different strain types {alpha and delta) in the sponge
Ephydatiafluviatilis.The question investigated was whether any process of cell adhesion was
responsible for the phenomenon. Preliminary results suggested that the cells might show
specific adhesion but further results indicated that a more complex system existed. Each
strain produces a soluble factor that increases the adhesiveness of homologous cells but
decreases that of cells of heterologous strains. The adhesion of cells, even in the presence of
these factors, is non-specific but the factors specifically control adhesion and determine its
quantitative value. The adsorption of the factors to the cells was tested for with inconclusive
results. Heterologous factors may irreversibly alter a cell's adhesiveness. It is shown that this
system, particularly by reason of its negative effect on adhesiveness, (a) accounts adequately
for the phenomenon of non-coalescence, (b) provides a model system for many forms of
morphogenesis and (c) allows many apparently contradictory results obtained by other
workers to be reconciled.
INTRODUCTION
The phenomenon of non-coalescence of the tissues of differing species or of
differing 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 two
individuals of the sponge Ephydatia fluviatilis are brought into contact by
experimental 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 strain
type the pinacoderms become confluent, and later the canal systems of the two
sponges join up and the sponges become effectively one organism. If, however,
the two sponges are of different strain type the initial adhesion persists for a
time, after which the two sponge bodies separate from one another leaving a gap
between them. Generally the two sponge bodies move apart. This behaviour has
been termed non-coalescence by van de Vyver (1970). She has suggested that
this behaviour may be due to a specificity of adhesion amongst the cells such
that 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 show
non-coalescence also show specificity of adhesion. This test was carried out
using the collision efficiency method (Curtis, 1970a, b). During this work
results were obtained and independently further information reported by van de
Vyver (1971 b), which now suggest to us an alternative mechanism as the basis
of non-coalescence. Consequently further experiments to test this second
mechanism are described here.
MATERIALS AND METHODS
Sponges of the alpha and delta strains of Ephydatia fluviatilis were grown
from gemmules supplied by Professor Rasmont's laboratory (Universite Libre
de 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 gemmules
and contained about 1 x 105 cells. Cell suspensions were prepared by scraping
the sponge bodies off the culture-dish surface and then transferring the
sponges to the dispersion medium. Each 10~3m3 (approx. 1 litre) of this
medium contained 2-5 xlO" 4 mol. EDTA, 0034 mol. NaCl, 0-00134 mol.
KC1, 000138 mol. glucose, 0-00107 mol. NaHCO 3 , 7xlO- 4 mol. K 2 HPO 4
and was buffered to pH 7-8 with 0006 mol. 2-amino-2-hydroxymethyl, 1-3
propanediol (Tris). After exposing the sponges to this medium at ca. 20 °C
for 7 min they were mechanically dispersed in the same medium by pipetting.
From this point two different methods of cell dispersion were used, namely
the 'washed cell' technique and a second technique in which an appreciable
portion 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 g
for 5 min, the supernatant discarded and the pellet resuspended in Rasmont
medium. In the second technique the cells were dispersed in a very small volume
of EDTA medium and the suspension was then made up with a large volume of
Rasmont medium. Since a small amount of EDTA would be carried over into the
cell suspension by this procedure and because of the possible existence of soluble
factors affecting cell adhesion (see later) the volumes of medium used and
removed at each stage were made standard for all but the preliminary experiments, in which they were roughly standard. The EDTA level in the final cell
suspension was ca. 1 x 10~4mol. (10~3 m 3 )- 1 compared with a Ca + Mg concentration of 1-5 x 10~3 mol. (10~3 m3)"1. The dispersion technique used in each
separate experiment is stated in the appropriate part of the text. The adhesivenesses of the cells (collision efficiencies) were measured with a Couette viscometer, using the method described by Curtis (1969, 1970#, b, c). Measurements
were 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 is
the mean of three sets of measurements each made for five different periods of
aggregation. Counts of cell and aggregate population densities were made using
haemocytometers.
Control of cell adhesion
297
Alpha and delta factors were prepared from alpha and delta sponges
respectively by a modification of the method described by van de Vyver (1971 b).
A precise number of gemmules (usually 30 sponges from 300 gemmules) were
dispersed in the EDTA medium used for cell dispersal after soaking in this
medium 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 suspension of cells and broken cells concentrated and dialysed by vacuum dialysis
against normal Rasmont medium. The dialysis membrane used had an effective
permeation limit for molecules of mass 1000 daltons. The dialysed material
was 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 preparation. A subsidiary experiment showed that this method of preparation of the
factors yielded a product apparently identical with those prepared by the
original 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), of
the two cell types is measured for each type separately and then for mixtures of
various proportions of the two types. If there is no specificity of adhesion the
collision efficiency for any proportion of the two cell types will be the average of
the efficiencies for both types (measured separately) weighted for the proportion
of each cell type in the mixture. If, however, there is complete specificity of
adhesion such that cells derived from different strains will not adhere, then the
measured collision efficiency will be reduced because all collisions between
unlike cells will fail to result in adhesions. The full theory of this test is given
by Curtis (19706). In summary, if there is no specificity of adhesion, the
collision efficiency for a mixture of two types of cells of adhesivenesses Ex and
E2 mixed in proportion rtx and n2 is given by n2E2 + n2E2. If there is complete specificity of adhesion the equivalent relationship is nlE1 + nlE2. If
specificity is incomplete the plots of collision efficiency against proportion of a
cell type in the mixture will lie between those for complete specificity and complete lack of specific adhesion. The difference in collision efficiency for any two
degrees 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 been
published (Curtis, 1970tf, b) and these are used in this work.
PRELIMINARY RESULTS AND DISCUSSION
Tests were made, using the collision efficiency method, to discover whether
specificity of adhesion occurred for cells of the alpha and delta strains. The
results are given in Table 1 and Fig. 1. The results suggest that specific adhesion
is found. The second method of cell dispersion was used.
However, the discovery of factors which promote aggregation, made by
van de Vyver (1971 a), suggests another explanation of these results. An alter-
298
A. S. G. CURTIS AND G. VAN DE VYVER
native hypothesis which would account for the reduced adhesiveness of mixtures
of the cells of two strain types is that these factors promote the adhesiveness of
their own strain but diminish the adhesiveness of other strains. The method of
preparation of these factors is very similar to that used for the preparation
of cell suspensions; indeed van de Vyver used exactly the same method for
factor preparation in her work as used here for cell suspension preparation.
Table 1. Test for specificity of adhesion of alpha and delta
strain cells {see also Fig. 1)
Alpha
cells in
suspension
Collision
efficiency
Standard
deviation
00
101
30-7
49-9
72-6
1000
4-76
3-12
1-90
1-69
204
3-40
0-45
0-29
014
0-24
019
0-65
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. Reduction in sum of squares by curvilinear regression = 21-17. F = 66-15. Highly significant
improvement of fit by use of curvilinear regression, 9 5 % confidence limits at x = 0-500;
0-83-1-32-1-82.
2 -
1 -
50
Alpha strain cells (%)
100
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. The
straight line represents results that will be expected on the hypothesis of non-specific
adhesion. The continuous curve represents the results that will be obtained on the
hypothesis of specific adhesion.
Control of cell adhesion
299
Therefore it is probable that these factors are present in the cell suspensions
unless the cells are very thoroughly washed. If this hypothesis is correct the
especial circumstances of the test for specific adhesion would lead to its
simulation when two equal aliquots of suspensions of cells of unlike type are
mixed. Before mixing, each suspension separately contained a concentration of
the aggregation factor sufficient to maintain a value for adhesiveness E. On
mixing with an equal aliquot of the other cell suspension the concentrations of
both factors would be halved so that the adhesiveness of each cell type would be
reduced (the exact extent of reduction depends upon the nature of the doseresponse curve); moreover each cell type would experience a great increase in
the concentration of the factor derived from its opposite cell type, further
reducing its adhesiveness. In consequence, although the cells do not show
specificity of adhesion the collision efficiency of the mixture is reduced and thus
the existence of specific adhesion might be deduced from the results incorrectly.
The general hypothesis will be termed the factor-specificity theory. It is of
course possible that the above system might exist with specific adhesion occurring
as well. Either in such a case or if the dose-response curves for the factors are
very steep it would be possible to obtain a greater reduction of adhesiveness for
the mixed suspensions than is predicted by the simple theory.
We felt that in order to be sure that the actual mechanism of adhesion is
specific it was necessary to show that the above explanation of specificity in
terms of these factors was incorrect. One simple postulate of the factor specificity
hypothesis is that the delta factor, for example, would increase the adhesiveness
of delta cells and reduce that of alpha cells as the concentration of factor was
increased. An alpha factor would operate in a converse manner. A second postulate is that the dilution of a cell suspension with a bland medium will reduce the
adhesiveness of the cells because the concentration of the aggregation promoting
factor will be reduced. The result of this test may show also that the aggregation
factor is normally present in a cell suspension. These two tests are described in
the next section. However, the results of these tests, even if positive, will only
show that the necessary components of a system of factor-specificity exist, not
that the system operates amongst the cells. A third, more complex, test was
designed to test this latter point, but it is described later because it is best understood 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 of
alpha and delta cells within 1 min of the start of reaggregation. In the absence of
any knowledge of the chemical nature of these factors an arbitrary concentration
scale was used. Concentrations are expressed in terms of the number of sponge
cells disrupted per cc (10~G m3) of medium x 10~5. Thus a 30-unit solution would
300
A. S. G. CURTIS AND G. VAN DE VYVER
contain the yield of 3 x 106 cells/cc. The effect of alpha and delta factors on the
aggregation and adhesiveness of both alpha and delta cells is shown in Table 2
and Fig. 2.
These results confirm the existence of the alpha and delta factors postulated by
van de Vyver and additionally show that the factors diminish the adhesiveness of
cells of the opposite strain type. It should be appreciated that this result with this
test depends upon the accidental or intelligent choice of a suitable level of factors
in terms of the dose-response curve.
The second test examines whether dilution of a cell suspension has any effect
on 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 E
and standard deviations a of alpha and delta strain cells in the presence of homologous and heterologous factors
Concentration
of factor
(units/cc)
Alpha factor
Delta factor
Delta factor
Alpha factor
E
cr
A. Alpha cells
400
12-41
200
8-25
100
606
0-50
3-51
0-40
2-84
0-25
1-96
000
1-43
0125
110
0-67
0-25
0-50
0-43
10
0-21
30
005
3-81
1-75
0-98
110
012
009
0-37
011
0-21
016*
010
010
B. Delta cells
8-30
400
7-56
2-50
1-00
610
0-66
4-59
0-50
2-39
2-34
0-25
1-70
000
0-59
116
0-46
101
019
019
0-29
005
010
0-20
0-25
0-40
0-50
1-46
118
0-50
0-40
0-27
008
0-27
005
0-17
0-20*
014
0-35
Each measurement, with the exception of those marked with an asterisk, is the mean of
four 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 a
few points and the standard deviations have been omitted from the figure.
Control of cell adhesion
301
promoting factor normally present in the cell suspension has been diluted. This
test of course depends upon the accidental or intelligent choice of two suitably
placed points on a dose-response curve. Cell suspensions of both alpha and delta
cells were separately made up at high population densities (l-7xlO 6 cells/
10~6m3) by method 2. An aliquot of each suspension was then placed in the
Couette viscometer for collision efficiency measurement; another aliquot was
diluted with an equal volume of Rasmont medium and then the collision
15r
(a)
Delta factor
(units/cc)
15 r
Alpha factor
(units/cc)
Alpha factor
(units/cc;
(b)
Delta factor
(units/cc)
Fig. 2. Dose-response curves for the adhesiveness of (a) alpha strain and (b) delta
strain cells in the presence of alpha and delta factors. The points are mean values
from experimental observations. See also Table 2.
Table 3. Dilution experiment
Cell strain
type
Experiment
Delta
I
H
III
Alpha
IV
V
Initial
Diluted
Initial
Diluted
Initial
Diluted
Initial
Diluted
Initial
Diluted
Cell population
density ( x 10-6/cc)
Collision
efficiency
2-11
106
1-32
065
1 -81
0-92
1-99
0-99
2-82
0-97
411
2-56
4-97
2-05
3-59
1-46
4-31
2-69
1-34
0-80
0-27
0-26
0-45
008
0-41
015
0-37
0-21
010
004
Each measurement is the mean of five observations.
20
!•: M 1! 2 6
302
A. S. G. CURTIS AND G. VAN DE VYVER
efficiency was measured. The adhesivenesses of concentrated and diluted
suspensions are shown in Table 3. It is clear that dilution of both alpha and
delta cell suspensions diminish their adhesiveness.
These results suggest that it is at least possible that the appearance of specific
adhesion reported in the 'Preliminary Results' is due to the presence of the
alpha and delta factors. In those experiments where a delta strain cell suspension
is mixed with an alpha suspension, the adhesiveness of the delta cells will fall
because of the dilution of delta factor, and because of the presence of the alpha
factor; similarly the adhesiveness of the alpha cells will fall because of the
presence of the delta and dilution of the alpha factors. The main question to be
decided is whether the extent of the reduction of adhesiveness observed in the
preliminary results can be entirely accounted for by the presence of the two
factors together in mixed suspensions. If the reduction in adhesiveness is greater
than can be ascribed to the presence of the factors, it is possible that the hypothesis
of specific cell adhesiveness should be used to explain the results. Examination of
the dose-response curves and the data given in the section on Preliminary Results
suggest that the alpha and delta suspensions in those experiments contained
respectively 0-5 units/cc and 0-8 units/cc of their factors. Mixing equal volumes
of these two suspensions would halve these concentrations in the mixed suspension. Assuming that the collision efficiency of cells in the simultaneous presence
of both factors is the mean of the efficiencies measured in the separate presence
of each factor, the collision efficiency of each strain type in the mixture can be
calculated. In turn the collision efficiency of the mixed cells can be determined
and the question of the specificity of adhesion resolved.
The effect of the simultaneous presence of both factors on the adhesiveness
of each strain type was measured using well-washed cells (method 1), to which
varying concentrations of alpha or delta factors were added. The results are
shown in Table 4. They indicate that the collision efficiency of the cells in the
presence of both factors is slightly lower than a value obtained by taking the
mean of the values found for the separate presence of each factor at the same
concentrations.
We can now calculate the collision efficiencies that would be expected in the
mixed systems described in the Preliminary Results from the operation of the
alpha-delta factor system. The two pure cell strain suspensions contained
respectively 0-5 units/cc and 0-8 units/cc of their respective factors. Talcing into
account the results shown in Table 4 and the dose-response curves, the collision
efficiency of the alpha cells in a 50:50 mixed suspension would be 1 -35 % and that
of the delta cells 1-40%. This would give a mean value of 1-37 % in the mixed
suspension assuming that there is no specificity of adhesion. This is close to the
value determined experimentally of 1-69 % for a mixed suspension. If there was
complete specificity of adhesion in the presence of the alpha-delta factor system
the collision efficiency would be 0-68 % because specificity of adhesion would
further lower the apparent adhesiveness of the cells in the mixture. Since this
r
Control of cell adhesion
303
value lies outside the 99 % confidence limit (lower value 1-13 %; 1-tailed test) of
the measured value there is no reason for concluding that specific adhesion
occurs in this system.
It might be thought at first sight that the alpha-delta factor system is in effect
a mechanism for producing specific adhesion amongst cells which do not possess
it when they are extensively washed. However, it is clear that much of the
effectiveness of the system arises from its ability to diminish the adhesiveness
of 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- and
heterologous factors {means E and standard deviations &)
Concentration of factors
(units/cc)
Alpha strain cells:
Delta strain cells:
Homologous
Heterologous
E
10
0-5
009
0-20
018
018
2-89
1-67
2 04
0-83
0-38
018
1-68
0-28
10
0-5
009
0-20
018
018
3-87
305
2-46
0-83
0-38
112
0-39
0-21
Each measurement is derived from four observations.
adhesion. In addition it is clear that from the experimental data that the kinetics
of aggregation are such that adhesions must form indiscriminately between cells
of different strain type. The alpha-delta factor system is, however, a system in
which there is a strain-specificity in the control of adhesion. In the Discussion
we shall consider other experiments which can be reinterpreted in the knowledge
we now have of the alpha-delta factor system. In some of those experiments
evidence has been put forward for specific mechanisms of cell adhesion involving
the existence of a specific cementing factor. One very simple experiment to test
for the action of a cementing factor is to show that the factor is bound to cells
prior to or during its action in promoting adhesion. Curiously this test does not
appear to have been carried out on any system of cell adhesion by any previous
worker. Since we are interested in the manner in which the alpha-delta factor
system operates and in establishing any similarities with or differences from
systems of specific adhesion, we carried out tests to establish whether these
factors are adsorbed to the cells. It is clear from the dose-response curves that
it is possible to choose concentrations of factor that are below levels that might
saturate any binding sites that may exist on the cells. Cell suspensions prepared
by method 1 (well-washed cells) were treated with non-saturating levels of
304
A. S. G. CURTIS AND G. VAN DE VYVER
heterologous factor. After aggregation the suspensions were centrifuged and the
medium recovered. This medium was used to treat a second batch of cells of the
heterologous strain during their aggregation. (It is undesirable to carry out the
test on cells of a homologous strain type because homologous factor may be
added to the system by the cells.) If the apparent activity of the factor, as shown
by its effect on the adhesiveness of the second batch of cells, is reduced by
exposure to the first batch of cells it would seem probable that the factor is
adsorbed to the cells. The results are given in Table 5. They give no support to
the hypothesis that the factors act by adsorption to the cells, though this cannot
be excluded (see Discussion).
Such a result suggests that the factors are not adsorbed to the cells, and hence
cannot form a part of a cell binding material. The results are consistent with the
factors being enzymic in their action. Related to this point is the question of
whether the effect of each factor is reversed by the opposite factor or otherwise.
Table 5. Factor absorption experiment
Cell strain type
K
i
\
type
on
Assayed
on
Alpha
Delta
Delta
Delta
Alpha
Alpha
Activity (units/cc)
Expected
Found
0-50
0-25
0-25
0-75
0-25
0-60
0-45
0-50
0-25
0-20
0-80
0-20
0-60
0-55
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-washed
cells were treated with factor of the opposite type, and their adhesiveness was
measured over a 30-min period, then the cells were centrifuged out of the medium
and resuspended in a medium containing homologous factor, before measurement
of their adhesiveness. The second method of performing this experiment is to
treat well-washed cells with homologous factor first and then with heterologous
factor, after removal of the homologous factor by centrifugation. This method of
carrying out the experiment is less satisfactory than the first technique because
there is appreciable cell adhesion during the treatment with homologous factor.
In consequence, the measurement of collision efficiencies during the second
treatment is less accurate because the method of measurement presumes that at
the start of aggregation the cell population is monodisperse. However, the second
method of carrying out this experiment was used because there may be differences
Control of cell adhesion
305
in the result depending on whether the cells are exposed first to homologous or
first to heterologous factors. Results are given in Table 6. The values that would
be expected in the absence of a pretreatment step can be read in Table 2. The
results show that the effect of treatment by a homologous factor is reversible by
a factor of the opposite type, but that the reverse is not the case. This suggests
that an active site involved in cell adhesion is irreversibly altered by the heterologous factor so that a subsequent treatment with homologous factor is ineffective.
Table 6. 'Reversed treatments'' -sequential treatment with
homologous and heterologous factors
Second t reatment
First treatment
Cell
strain
type
Factor
concentration
(units/cc)
Collision
efficiency
A. First treatment with heterologous
0-25
Alpha
0-25
0-25
0-25
0-25
0-25
0-25
0-25
0-5
Delta
0-5
0-5
0-5
0-5
0-5
0-5
Factor
concentration
(units/cc)
Collision
efficiency
factor, second with homologous factor
0-21
0-31
1-9
0-31
1-9
009
007
0-42
1-9
0-37
1-9
<005
1-75
<005
0-47
0-42
1-75
<005
1-75
<005
0-49
<0-05
0-47
1-75
0-56
0-89
1-9
1-9
<005
0-29
1-9
<005
0-22
014
010
1-9
1-75
019
015
1-75
013
0-30
0-30
1-75
0-35
B. First treatment with homologous factor, second with heterologous factor
2-64
0-95
0-4
0-5
Alpha
2-49
0-5
0-31
0-4
0-5
0-42
0-4
2-89
0-5
010
0-4
3-36
4-77
<01
0-66
0-5
Delta
5-22
0-5
<01
0-66
4-17
0-66
0-5
<01
3-93
0-5
<01
0-66
Each horizontal line of figures refers to one experiment.
DISCUSSION
The experiments described in this paper start with the apparent demonstration
of the specific adhesion of the cells of the alpha and delta strains of Ephydatia
fluviatilis. The earlier experiments were carried out before the discovery by van
306
A. S. G. CURTIS AND G. VAN DE VYVER
de Vyver (1971 b) of the soluble factors, released during dispersion of the sponge
tissues, which promote the aggregation of cells of the same strain type as the
factor. The existence of these factors suggested that the preliminary experiments
could be reinterpreted as evidence for a system in which strain differences in the
response of the cells to the factors would lead to results simulating specific
adhesion. The experiments reported in the main section of results show that
homologous factors increase and heterologous factors decrease the adhesiveness
of the cells. They also show that the adhesiveness of cells in the presence of both
homo- and heterologous factors is the mean of the values that would be expected
in the separate presence of each factor. Most important of all, the results show
that the extent of adhesion reduction in mixed alpha-delta suspensions is
quantitatively predicted with accuracy from the concentrations of alpha and
delta factors in the mixed suspension, on the assumption that the cells show no
specificity of adhesion. In other words the factor specificity system accounts for
the behaviour, reported in the Preliminary Results, which simulates specific
adhesion.
At this point it is perhaps appropriate to consider the criticism of the test
system for specific adhesion, used in this work, made by Humphreys (1970).
Humphreys suggested that Curtis (1970 c) had not shown that the rate of aggregation has a complete dependence on cell population density and therefore that
the interpretation of the experiments might be incorrect. However, it is implicit
in the Swift & Friedlander (1964) treatment, which is the basis of the method of
measuring cell adhesiveness introduced by Curtis (1969), that the kinetics of
aggregation of any two size classes will follow second order kinetics. (The
aggregation 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 be
found in the measurements of collision efficiency over a time course during
which the total particle concentration falls to about a half of the initial value.
The actual measured standard deviations are small; this suggests that the
aggregation kinetics for one or two size classes are second order. It might at
first sight be felt that the behaviour shown in Table 3, where dilution of a cell
suspension diminishes the collision efficiency, is in fact direct proof that secondorder kinetics apply for any two size classes, but it should be borne in mind that
in calculating the collision efficiency by the Swift and Friedlander treatment a
dimensionless value is obtained, in other words, differences in cell concentration
are removed. Humphreys also suggested that the aggregation rate actually
measured might not be a true measure of adhesiveness, because the rate-limiting
step might be the rate of recovery of the cells from dispersion procedures. If the
cells recover their adhesiveness slowly the rate of aggregation would be determined by this process. Again it is clear that if this occurred, collision efficiencies
would rise during the course of aggregation and in consequence large standard
deviations would be found in the values derived from measurements over the
time course of the experiments. The constancy of collision efficiency values found
Control of cell adhesion
307
for any defined set of conditions in this work argues against the hypothesis that
the measured aggregation rate is limited by a recovery of cellular adhesiveness.
This criticism cannot of course be applied to those experiments where heterologous factors diminish cell adhesiveness.
The next question to be considered is whether the system revealed in this work
can be used to account for the non-coalescence and the sorting out described by
van de Vyver (1971 a, b). It appears from her work that when two sponges of
unlike type make contact the pinacoderms of each sponge form an adhesion.
The cells of each individual do not interpenetrate. Some 15-30 h later (depending on the strain types involved) the adhesion of the sponges breaks down and
they separate. It is possible that contractions in each sponge body help to pull
the sponges apart. When two homologous sponges make contact an adhesion
develops between pinacoderms. Later the pinacoderm cells migrate to the surface
of the fused sponge and the cells from each individual interpenetrate to a
considerable extent. It should be appreciated that if this phenomenon is explained
on the hypothesis that the cells show complete specificity of adhesion (cf.
Humphreys' results on Microciona and Haliclona - Humphreys, 1963) it would
be expected that two sponges of unlike type would not even form a temporary
zone of adhesion.
A more satisfactory explanation of the major features of non-coalescence can
be developed in terms of the idea that the alpha and delta factors control this
phenomenon. The concentration of a sponge's own factor will tend to be maximal
at the centre of the sponge body if it is assumed that all cells of the sponge
produce the substance. The concentration will be minimal at the periphery of the
sponge (under a wide variety of assumptions about the origin, diffusion and
destruction of the factor). Therefore the adhesiveness of the cells of the surface of
the sponge body will be low but not negligible. When two sponges of like type
make contact an adhesion will form. Since the factors are of diffusible nature and
since they may act without being irreversibly bound to the cells, the concentration 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 more
adhesive and a single permanent sponge body will be established.
When two sponges of unlike type make contact an adhesion will form initially
because the concentrations of the two factors will still be low at the original
surface of the sponges along the zone of contact. However, because of the
changed geometry of the sponges the concentrations of the two factors will rise
along the region of contact. Though the measurements of the simultaneous
effect of hetero- and homologous factors on the adhesiveness of cells show a
tendency for the two factors to cancel out (Table 4), it is clear from the sequential
treatment experiments (Table 6) that the heterologous factor will irreversibly
alter the adhesiveness of the cells (unlike the homologous factor) so that eventually the cells exposed to heterologous factor will become non-adhesive. We
can view the process as a competition between homo- and heterologous factors,
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A. S. G. CURTIS AND G. VAN DE VYVER
the heterologous factor being a competitive inhibitor. During a short-term
experiment (30 min) the progressive inactivation of adhesion is not obvious.
The diminution in adhesion will be maximal for the cells lying at the zone of
contact since they are of opposite type on either side. Thus the sponges will cease
to 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 so
non-adhesive that they would separate from the sponge bodies and migrate or
drift to other regions; in this way a gap would as it were be eroded between the
sponges. Though the phenomenon of non-coalescence of unlike strain types can
be adequately explained on the hypothesis advanced in the last paragraph it is
clear from van de Vyver's results (van de Vyver, 19716) that there are more
complex and perhaps secondary features of the phenomenon that are less easy
to explain. When contact is made between homologous sponges the pinacoderm
cells 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 heterologous
contacts the pinacoderms remain intact in the region of contact.
The phenomenon of non-coalescence has certain similarities to that of contact
inhibition of movement of cells (Abercrombie & Heaysman, 1954). In both
phenomena cells make contact and form adhesions with each other and subsequently re-separate. However, contact inhibition of movement is displayed
between homologous and heterologous cells whereas non-coalescence only
occurs between unlike cells. Moreover Abercrombie & Gitlin (1965) have shown
that it is unlikely that any diffusible agent is involved in contact inhibition of
movement. There remains the considerable, if superficial, resemblance between
the two phenomena in that an outgrowth of cells stops movement on contact
with another body of cells. Contact inhibition of movement has been suggested
as being the phenomenon which prevents cells of normal tissues from invading
one another (Abercrombie, 1967). However, it can now be seen that the
strain-specific adhesion factors discovered in this work would, if they acted as
tissue specific adhesion factors, prevent tissues from invading one another.
Moreover such a system would have two further capabilities of great importance
in morphogenesis. First, during morphogenesis, tissue factors of this type could
interact to diminish the adhesiveness of cells in the region of contact of two cell
types. 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 and
gaps between tissues would persist in adult life. It should be remembered that the
vertebrate body develops many cavities within it during development and that
many organs - for example, the liver - lie with the majority of their surface
free and unadherent to the surrounding peritoneum even though there may be
prolonged contact between the two tissues. In a special sense these factors can be
regarded as being the morphogens postulated by Edelstein (1970), their action
being, however, not chemotactic or chemokinetic but as controllers of adhesion.
It is important to emphasise that it is the soluble and therefore presumably
Control of cell adhesion
309
diffusible nature of these factors that makes them of particular utility in explaining morphogenesis.
These factors, if they diffuse from the cells that produce them, could act as a
means of providing positional information and determination of pattern. In
general terms, such a system would be analogous to that proposed by Wolpert
(1969), with the distinction that these factors would control the positioning of
cells rather than the differentiation of cells in fixed positions.
When the results of this work are compared with those obtained on the
reaggregation 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 differing
interpretations. Humphreys (1963) identified a factor which specifically promoted 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 diminished
the adhesiveness of other species types. Such factors were discovered for the
species Haliclona occulata, Microciona prolifera, Halichondria panicea and
C/ione celata. Humphreys suggested that these factors acted as cements which
would attach to binding sites on two apposed cell surfaces, thus binding the
cells together. No evidence was put forward that these factors were adsorbed by
cells. In order to demonstrate the activity of the factors the cells had to be very
thoroughly washed to reduce their adhesiveness for control experiments. On
adding the factors, specific promotion of adhesion could be detected. Curtis
(1970c) repeated part of Humphreys' work using the two species Haliclona
occulata and Halichondria panicea. The cells were very thoroughly washed and
no evidence for specific adhesion could be detected from measurements of the
collision efficiency of mixed suspensions. It can now be seen that Curtis's
experiments can be reinterpreted as showing that the cells, in the absence of any
factor, show non-specific adhesion. Sara (1965) could find no evidence for specific
adhesion in quite a wide range of marine sponges. Unfortunately his papers
give few details of how the cell suspensions were prepared. Curtis (1970a) also
investigated whether a variety of other marine sponge cells showed specific
adhesion. In these experiments the cell suspensions were well washed and no
evidence for specific cell adhesion was found. Curtis's and Humphreys' separate
results can be reinterpreted in the following way as a consequence of the hypothesis advanced above. We shall assume that the factors discovered in E.
fluvial His strains have species-specific counterparts in marine sponges. Curtis's
failure to detect specificity of adhesion or specific control of adhesion would
arise from the fact that his cell suspensions were washed clean of the factors.
Humphreys diminished the adhesiveness of the cells by the method of cell
dispersion and by aggregating the cells at low temperatures. If we assume that
the factors he isolated were identical in general behaviour with those discovered
in E. fluviatilis they would act not as agents of specific adhesion but as substances specifically controlling the appearance or loss of an adhesion whose
310
A. S. G. CURTIS AND G. VAN DE VYVER
direct mechanism would be unspecific. In the experimental design used by
Humphreys such factors of the Ephydatia type would give exactly the same
results as he obtained. In detail the aggregation of cells of a heterologous
type would be prevented and adhesion of cells of a homologous type promoted
in the presence of a single factor. When two species types are aggregated in the
presence of both factors it would be expected that the first adhesions would be
random with respect to the species types joining together. Later as small groups
of cells of one species type began to produce appreciable additional amounts of
their factor they would make their local environment less favourable for interspecific adhesions and more favourable for adhesions with their own species
type; in this way sorting out of the species would occur. This would account for
the fact that it is frequently seen that aggregates involved in sorting-out 'expel'
cells (Curtis, 1967). Thus apparently contradictory results on cell adhesion can
be reconciled by the hypothesis that there are diffusible factors which specifically
control the adhesiveness of cells whose mechanism of adhesion is itself unspecific.
The mode of action of these factors has considerable implications for the
explanation of the mechanism of adhesion of Ephydatia cells. If the factors are
adsorbed by the cells it is, at least in principle, possible that they may act as
binding agents (cements). If they form binding materials they must have at
least two binding sites, one to attach to each cell surface. An alternative explanation of the activity of the factors is that they modify a cell surface component involved in adhesion by enzymic action. The failure of the adsorption test
to detect adsorption of these factors cannot be regarded as anything more than
negative evidence because if the number of binding sites were small and the
binding constant (stability constant) high a very small quantity of factor would
be removed from a given initial concentration of the factor on each successive
exposure to cells. Consequently, since it is improbable that the test would detect
a small reduction in the activity of the factor from one exposure to cells to
another, no clear evidence about adsorption would be obtained. Interestingly
enough other workers (e.g. Humphreys, 1963; Moscona, 1968) who have claimed
to isolate specific cell-binding materials, do not appear to have tested whether
their factors are bound by cell surfaces. The results obtained when cells of either
strain are treated sequentially with both factors are explicable both on the
theory that the factors attack a site involved in adhesion enzymically or that
they adsorb irreversibly to a cell surface binding site. However, if the factors are
adsorbed to the cell surface it is improbable that they act as cements for the
following reason. It is clear that heterologous factors cannot act as binding
agents because they decrease cellular adhesiveness, though they might block
binding sites. The homologous factors increase cell adhesion but do not do so
specifically, yet if they acted as binding agents they would produce specific
adhesion, because they clearly could not bind to heterologous cells. Hence it
seems unlikely that these factors act as specific binding agents, though we
Control of cell adhesion
311
cannot yet conclude whether their action is enzymic or whether they modify
some general method of cell adhesion after surface adsorption.
RESUME
Une nouvelle forme de processus morphogenetique a ete mise en evidence par 1'etude du
phenomene de non-confluence entre deux souches {Alpha et Delta) de l'eponge d'eau douce
E. flu via tilis.
Ce travail a pour but de verifier si le phenomene de non-confluence est en relation avec les
mecanismes de l'adherence cellulaire.
Les resultats preliminaires suggeraient une specificite de l'adherence cellulaire mais des
experiences ulterieures ont montre qu'il existait un systeme plus complexe. Chaque souche
produit un facteur soluble qui augmente l'adherence des cellules homologues et reduit celle
des cellules heterologues. L'adherence des cellules, meme en presence des facteurs n'est pas
specifique, mais les facteurs controlent specifiquement l'adherence et determine sa valeur
quantitative.
L'absorption des facteurs par les cellules a ete testee mais sans resultats. Les facteurs
heterologues 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 de
morphogenese et (c) permet de concilier des resultats apparemment contradictaires obtenus
par differents auteurs.
We should like to express our thanks to Professor R. Rasmont for the supply of sponge
gemmules, 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 one
of us (A. S. G. Curtis), which provided much of the support for this project, and to the
British Council and Le Ministere de 1'Education Nationale et de la Culture de Beige who
provided travel funds. Additional support was provided by the University of Glasgow and
by the Universite Libre de Bruxelles.
REFERENCES
M. (1967). Contact inhibition: the phenomenon and its biological implications.
Natn. Cancer. lust. Monog. 26, 249-277.
ABERCROMBIE, M. & GITLIN, G. (1965). The locomotory behaviour of small groups of fibroblasts. Proc. Roy. Soc. Loud. B 162, 289-302.
ABERCROMBIE, M. & HEAYSMAN, J. E. M. (1954). Observations on the social behaviour of
cells 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.
exp. Morph. 23, 253-272.
CURTIS, A. S. G. (1970c). Re-examination of a supposed case of specific cell adhesion.
Nature, Lond. 226, 260-261.
EDELSTEIN, B. B. (1970). Cell specific diffusion model of morphogensis. /. theor. Biol. 30,
515-532.
HUMPHREYS, T. (1963). Chemical dissolution and in vitro reconstruction of sponge cell
adhesion. I. Isolation and functional demonstration of the components involved. Devi
Biol. 8, 27-47.
HUMPHREYS, T. (1970). Species specific aggregation of dissociated sponge cells. Nature, Lond.
228, 685-686.
ABERCROMBIE,
312
A. S. G. CURTIS AND G. VAN DE VYVER
A. A. (1968). Aggregation of sponge cells: cell-linking macromolecules and their
role in the formation of multicellular systems. From /// vitro, vol. m. Publ. Tissue Culture
Association.
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 fra
Poriferi ed Anemonia sulcata. Boll. Zool. 33, 1067-1077.
SARA, M., LIACI, L. & MELONE, N. (1966). Bispecific cell aggregation in sponges. Nature,
Lond. 210, 1167-1168.
SWIFT, D. L. & FRIEDLANDER, S. K. (1964). The coagulation of hydrosols by Brownian motion
and laminar shear flow. /. Colloid Sci. 19, 621-647.
VAN DE VYVER, G. (1970). La non confluence intraspecifique chez les spongiaires et la notion
d'individu. Ann. Embryol. Morphogen. 3, 251-252.
VAN DE VYVER, G. (1971 a). Analyse de quelques phenomenes d'histoincompatibilite intraspecifique chez l'eponge d'eau douce Ephydatiafluviatilis(Linne). Archs Zool. exp. gen.
(in the Press).
VAN DE VYVER, G. (19716). Mise en evidence d'un facteur d'agregation specifique chez
l'eponge d'eau douce Ephydatiafluviatilis(Linne). Ann. Embryol. Morphogen. (in the Press).
WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation.
/. theor. Biol. 25, 1-47.
MOSCONA,
(Manuscript received 3 March 1971)