/. Embryol. exp. Morph. Vol. 28, 2, pp. 463-479, 1972
463
Printed in Great Britain
Effect of growth conditions on development of the
cellular slime mould, Dictyostelium discoideum
By D. R. GARROD 1 AND J. M. ASHWORTH 2
From the Biochemistry Department, Leicester University
SUMMARY
The effect of growth in the presence and absence of 86 m\i glucose on fruiting-body number
and size, fruiting-body proportions and morphogenesis in the slime mould Dictyostelium
discoideum, strain Ax-2, has been investigated. Cells grown in the absence of glucose (N.S. cells)
formed 2-1 limes more fruiting bodies than glucose-grown cells allowed to differentiate under
the same conditions and at the same cell density. Glucose fruiting bodies were 2-6 times
larger than N.S. fruiting bodies.
During aggregation, N.S. aggregation streams generally broke up into numerous secondary
aggregation centres. Glucose streams generally did not break up but moved into the initial
aggregation centre. Each secondary centre formed one small grex, whereas initial centres
fragmented into several grexes which were larger than those formed from secondary centres.
The preponderance of secondary centres in N.S. aggregation and of initial centres in glucose
aggregation accounts for the difference in size and number of fruiting bodies. We speculate
about the mechanism giving rise to the morphogenetic difference.
The spore: stalk ratio was 3-95:1 in glucose fruiting bodies and 2-70:1 in N.S. fruiting bodies.
This difference is not related to the difference in fruiting-body size because proportions are
size invariant for both fruiting-body types. Some difference in the physiological mechanism
which determines proportions is suspected.
INTRODUCTION
The life-cycle of Dictyostelium discoideum consists of two distinct phases
which are mutually exclusive in the sense that cell growth and almost all cell
multiplication are confined to the solitary feeding phase, whereas morphogenesis
and differentiation occur in the absence of growth during the subsequent multicellular phase. (For a detailed description of the life-cycle see Bonner, 1967.)
However, conditions pertaining during the growth phase can influence differentiation. Variations in the composition of the growth medium of the axenic
strain, D. discoideum Ax-2, cause alterations in the levels of key metabolites and
enzymes in the growing cells (Weeks & Ashworth, 1972; Ashworth & Quance,
1972). These differences in metabolite and enzyme levels are frequently preserved
during differentiation (Hames, Weeks & Ashworth, 1972). Further, variation in
1
Author's address: Department of Zoology, The University, Southampton SO9 5NH, U.K.
Author's address: Biochemistry Department, School of Biological Sciences, Leicester
University, Leicester LEI 7RH, U.K.
2
30-2
464
D. R. GARROD AND J. M. ASHWORTH
the glucose content of the axenic growth medium affects the size and number of
fruiting bodies formed. At the same cell density, cells grown in the presence of
86 mM glucose form fewer, larger fruiting bodies than cells grown in the absence
of added carbohydrate (Quance & Ashworth, 1972). Thus it is possible to study
the morphogenetic and biochemical behaviour of two physiologically distinct but
genetically identical cell populations.
In this paper we describe the difference in size and number of fruiting bodies
formed by cells grown in the presence and absence of glucose, the proportions
(i.e. the spore: stalk ratios) of the two types of fruiting body, and the morphogenetic event which gives rise to the difference in fruiting-body size and number.
MATERIALS AND METHODS
Growth of cells
D. discoideum Ax-2 cells were grown at 22 °C in suspension in 2 1 flasks containing approximately 700 ml of medium. The flasks were shaken on a rotary
shaker at 140 rev/min with a radius of rotation of 2-75 cm. The media contained
peptone, 15-2 g/1.; yeast extract, 7-6 g/1.; Na 2 HPO 4 , 5 mg/1.; KH2PO4, 5mg/l.;
pH 6-7. Cells grown in this medium, which contains no added carbohydrate,
will be referred to as N.S. cells. Glucose cells were grown in the same medium
with the addition of glucose to a final concentration of 86 mM.
Flasks were inoculated from stock cultures of cells (not spores), the inoculation
densities being 7x 103/ml for N.S. and 1-4 x 104/ml for glucose. Growth was
continued for approximately 65 h, after which time cells of each type reached
a concentration of about 2 x 106/ml.
Differentiation
Cells were harvested from growth flasks and put on to the surfaces of black
Millipore filters (catalogue no. AABP 04700) to allow them to differentiate.
These filters contain a filtration aid (probably a detergent) which may affect
morphogenesis and which must be removed before use by extensive washing in
tap and distilled water.
Harvesting was done by spinning the cell suspension in culture medium at
350 g for 15 min at 4 °C. The cells were then washed once with cold distilled
water and resuspended in cold distilled water at 5x 107/ml; 0-5 ml of this suspension was spread over the entire surface of each 47 mm Millipore filter, giving
an average cell density of 1-76 x 106/cm2. The Millipore filters were placed in
60 mm plastic Petri dishes on support pads saturated with 1-6 ml of pad diluting
fluid (PDF). The PDF contained in 11 of 50 mM-Na/K phosphate buffer at
pH 6-5; KC1, 1-5 g; MgCl 2 .6H 2 O, 0-5 g; streptomycin sulphate 0-5 g. Dishes
were incubated at 21 °C in a dark, humid container. Under these conditions
aggregation began 9 h after the beginning of incubation and fruiting-body construction was complete after 24 h (Sussman, 1966).
Development o/D. discoideum
465
Assessment of fruit ing-body population density
This was done with a binocular microscope by counting the number of
fruiting bodies in four fields, chosen at random from each Millipore filter. The
area of each field was 15-9 mm2.
Preparation offruiting bodies for measurement
Fruiting bodies were fixed by exposure to the vapour of 40 % formalin solution
for 1 h and then to the vapour of 2 % OsO4 for a further hour. The Millipore with
attached, fixed fruiting bodies was then immersed in 50 % ethanol for 10 min,
then in distilled water. The spore heads of many fruiting bodies were damaged
during immersion in ethanol, but it was easy to select only undamaged fruiting
bodies at the next stage of preparation.
Next, a few drops of 1 % agar in distilled water were put on to a warm microscope slide and the agar kept liquid by placing the slide in the beam of an infrared lamp supported about 2 ft (0-6 m) above the bench surface. Using a pair of
needle forceps, undamaged fruiting bodies were dislodged from the Millipore
surface and placed in the agar on the slide. When about 20 fruiting bodies had
been transferred to the slide it was removed from the infra-red beam to allow the
agar to solidify. A few more drops of liquid agar were then added and a coverslip
placed over the preparation. When the second lot of agar had solidified, the
preparation was sealed with vacuum grease. A glucose fruiting body prepared
by this method is shown in Fig. 1.
Measurement offruiting bodies and volume calculation
A Projectina microscope was used to measure the fruiting bodies. At x 60
magnification the fruiting bodies were projected on to the instrument's screen,
which has a graduated linear scale. Four measurements were taken on each
fruiting body (Fig. 2): (a) the major axis of the spore head; (b) the minor axis of
the spore head; (c) the diameter of the base of the stalk; (d) the length of the
stalk between points A and B in Fig. 2. As stalks were invariably curved they
were divided by eye into a number of straight sections the lengths of which were
added to give the total stalk length. The basal disc of the stalk was not measured.
The volumes of the spore heads were calculated by treating them as prolate
spheroids (Bonner & Slifkin, 1949). The volume of a prolate spheroid is given
by the formula
%-nab\
where a is the major semi-axis and b the minor semi-axis (Turrell, 1946). The
volumes of the stalks were calculated by treating them as cones, using the
formula
30-3
466
D. R. GARROD AND J. M. ASHWORTH
f
Fig. 1. A glucose fruiting body prepared for measurement as described in Materials
and Methods. The arrowed region is where the stalk was slightly damaged during
removal from the Millipore filter.
Fig. 2. Diagram showing the measurements taken from each fruiting body Stalk
length was measured between points A and B as described in the text.
Development o/D. discoideum
467
where r is the radius of the base and h the height. Some comments on the use of
these methods are given in the Discussion.
Photography of developing cultures
Photographs were taken at 15 min intervals from the beginning of aggregation
to the early standing grex stage, using a 24 mm Repro Sum mar lens attached to
a 35 mm camera body. Illumination was provided by an electronic flash lamp
(intensity 400 J) supported at an angle 30° to the horizontal.
During an experiment the culture was kept at 21 °C in a dark, humid container which was opened for the minimum possible time for each exposure. Each
culture was allowed to complete development after photography to ensure that
its differentiation was normal.
RESULTS
Fruiting-body density
The average fruiting-body density obtained with N.S. cells was 247-6/cm2.
(S.E. ±13-8, range 170-6-407-7), whereas for glucose cells it was 118-5/cm2
(S.E. ± 7-9; range 51-3-181-2). While the difference between N.S. and glucose was
statistically significant (t = 8-12 for 41 degrees of freedom; P < 0-001), the
extreme variability of fruiting-body density within each group was noteworthy.
The average ratio of number of N.s. to glucose fruiting bodies was 2-1:1. Cultures
with N.S. and glucose fruiting bodies are shown in Fig. 17. Assuming that
aggregation was 100 % efficient, these results would mean that these were on
average approximately 7 x 103 cells/N.s. fruiting body and 1-5 x 104 cells/glucose
fruiting body.
Fruiting-body size and proportions
One hundred and seventeen fruiting bodies of each type were measured. These
were taken from two cultures of each type. The average volume of N.s. fruiting
bodies was 1-3 x 106 /6m3 (S.E. ± 1-2 x 105; range 5-3 x 10* to 8-1 x 106), whereas
for glucose fruiting bodies it was 3-2 x 106 /tm3 (S.E. ± 2-0 x 105; range 3-4 x 105
to 1-5 x 107). Here too the variability was noteworthy. The ratio of average
volumes was 2-6:1 (glucose:N.S.). This is slightly different from the ratio of
fruiting-body numbers, but because of the range in fruiting-body numbers
between cultures of cells of the same type represents reasonably good agreement.
Histograms showing the distribution of fruiting-body volumes for N.S. and
glucose cells are given in Fig. 3. It will be noted that the ranges of fruiting-body
volumes were almost identical.
The ratios of spore volume to stalk volume for N.S. and glucose fruiting bodies
were obtained from the slopes of the respective regression lines (Figs. 4, 5).
A marked difference in the proportions of the fruiting bodies was found. For
N.S. fruiting bodies the spore: stalk ratio was 2-70:1, whereas for glucose fruiting
468
D. R. GARROD AND J. M. ASHWORTH
40-i
((/) M.S.
f
30-
20-
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i
i
i
i
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i
\n
i
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i
i
i
i
10 15 20 25 30 35 40 45 50 70 75 80 85
Fruiting body total volume (^m3 x 10s)
20 -,
(/>) Glucose
10-
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015
i
10
i
i
i
i
i
i
i
i
i
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i
i
15 20 25 30 35 40 45 50 55 60 65
i
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70 75 80 145 150
Fruiting body total volume (//m 3 x |()5)
Fig. 3. Distribution of fruiting body total volumes (a) for N.S. and (b) for glucose.
Arrows indicate average values.
bodies it was 3-95:1. Two things stand out from these figures. First, the scatter
of points for glucose fruiting bodies was greater than that for N.S. fruiting bodies,
as is indicated by the respective correlation coefficients, r = 0-75 for glucose and
r = 0-89 for N.S. Both N.S. and glucose fruiting bodies had greater spore: stalk
ratios than the fruiting bodies measured by a similar technique by Bonner &
Slifkin (1949), who obtained a spore:stalk ratio (taken from the slope of their
line) of about 1-75:1.
469
Development ofD. discoideum
40 r
4
5
6
7
8
9
Stalk volume (//m 3 x 105)
10
I
12
13
Fig. 4. Graph of spore head volume against stalk volume for N.S. fruiting bodies.
The solid line was obtained by regression analysis of the data. Its slope is 2-70. The
correlation coefficient (r) was 089 for 115 degrees of freedom (P < 0001). For comparison, the lines for glucose fruiting bodies (C) and for wild-type fruiting bodies
(Bonner & Slifkin, 1949) (BS) are shown.
There seemed to be two possible reasons why the proportions of glucose and
N.s. fruiting bodies were different. First, there might be some physiological
difference between the two cell populations which affects the mechanism responsible for determining proportions. Secondly, proportions might in some way be
determined by the absolute size of fruiting bodies so that glucose fruiting bodies
may have a greater spore:stalk ratio than N.S. fruiting bodies because they are
larger, and vice versa. In Fig. 6, spore:stalk ratio (calculated directly by dividing
spore volume by stalk volume) has been plotted against fruiting-body volume
for both glucose and N.S. cells. Regression analysis of these data gave, in both
cases, lines which were almost parallel to the volume axis: the slope for N.S. was
- 001 and the slope for glucose was + 003. Thus it appeared that, in both cases,
spore: stalk ratio was constant over the entire range of volumes. Further, the
correlation coefficients were small (r = -0-08 for N.S., r = 0-22 for glucose),
indicating no dependence of proportions on total volume.
470
D. R. GARROD AND J. M. ASHWORTH
80 r-
70
^ 60
o
X
I 50
o
"o 40
30
20
10
I
5
I
l
l
l
l
l
I
I
I
l
10
15
Stalk volume (//m 3 x 105)
I
18
Fig. 5. Graph of spore head volume against stalk volume for glucose fruiting bodies.
The solid line was obtained by regression analysis of the data. Its slope is 3-95. The
correlation coefficient (r) was 0-75 for 115 degrees of freedom (P < 0001). For comparison, the lines for N.S. fruiting bodies (MS) and wild-type fruiting bodies (Bonner
& Slifkin, 1949) (BS) are shown.
Morphogenetic origin of size difference
It seemed likely that the morphogenetic event giving rise to the difference in
fruiting-body size and population density between N.S. and glucose might occur
at one of two stages in the life cycle, either (a) at the aggregation stage or (b) at
the stage the aggregate breaks up into individual grexes. Accordingly, we investigated these stages by photographing developing cultures at 15 min intervals,
starting at the beginning of aggregation and continuing until the standing grex
stage.
A distinct difference between the patterns of aggregation of N.S. and glucose
cells was discovered. Early aggregation of N.s. cells was characterized by highly
branched streams around an aggregation centre (Fig. 7). The streams then
developed nodular swellings (Fig. 8). Stream breakage then took place at the
sides of swellings proximal to the original aggregation centre. Each swelling then
acted as a secondary aggregation centre and absorbed distal streams (Fig. 9).
Secondary aggregation centres were usually smaller than the initial centre.
Early glucose aggregation streams were less branched than those of N.S. cells
(Fig. 11). Moreover, what branches there were coalesced to form broad streams
Development o/D. discoideum
14
13
12
11
.2 10
2 9
2 8
471
N.S.
I 7
gj 6
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a 5
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3
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10
15
20
25
30
35
40
Fruiting body volume (//nv1 x 105)
1
18
17
16
15
14
13
.2 12 -
1
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1
1 1
(b) Glucose
I 11
i± 10
B 9
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1
1
1
1
1
20
30
40
50
60
Fruiting body volume (/mi3 x 105)
70
80
1
1
Fig. 6. Graphs of spore:stalk ratio against fruiting body total volume. The lines
were obtained by regression analysis, (a) N.S. fruiting bodies: slope, —0-01; correlation coefficient, - 008; P > 0-1. (b) Glucose fruiting bodies: slope, 003; correlation
coefficient, 0-22; P^ 002.
which tapered evenly towards the aggregation centre (Fig. 12). Stream swelling
was rare with glucose cells. The streams were usually absorbed by the initial
aggregation centre, which either remained as a single aggregate until all the
streams had been absorbed or itself fragmented into a number of smaller
aggregates of roughly equal size (Figs. 13, 14).
Standing grex formation from aggregation centres of N.S. and glucose cultures
are shown in Figs. 15 and 16. These photographs demonstrate the difference
between the two very clearly. In both cases there were large centres which broke
up into several standing grexes. Standing grexes from large centres appeared
472
D. R. GARROD AND J. M. A S H W O R T H
Figs. 7-10. Aggregation of N.S. cells.
Fig. 7. Early aggregation showing highly branched streams.
Fig. 8. One hour later, showing nodular stream swellings.
Fig. 9. Seventy-five minutes after Fig. 8, showing break-up of streams and formation
of secondary centres.
Fig. 10. Two hours after Fig. 9, showing early standing grexes.
Development ofD. discoideum
473
Figs. 11-14. Aggregation of glucose cells.
Fig. 11. Early aggregation.
Fig. 12. Seventy-five minutes later, showing broad streams. Note that the two initial
centres roughly in the middle of the picture have become much larger.
Fig. 13. Two hours after Fig. 12. The streams have almost been absorbed by the
centre which is beginning to break up.
Fig. 14. One hour after Fig. 13, showing aggregates just before standing grex
formation.
474
D. R. GARROD AND J. M. ASHWORTH
Fig. 15. N.S. standing grexes. There are groups of large grexes formed from initial
centres (arrows /, for example), and numerous small grexes formed from secondary
centres (arrows S).
Fig. 16. Glucose standing grexes. Almost all are large and grouped together. Figs. 15
and 16 are different preparations from those shown in Figs. 7-14.
Fig. 17. Millipore filters showing NS and glucose (G) fruiting bodies. The black
filters are 47 mm in diameter.
Development o/D. discoideum
475
roughly the same size in both cases. The difference between the two was in the
number of small centres between the large ones, each of which gave rise to only
one grex. Such centres were numerous in N.S. cultures and almost non-existent
in glucose cultures. The grexes formed singly from small centres were, almost
without exception, smaller than those formed by the break-up of large centres.
The small centres originated from swellings on aggregation streams.
DISCUSSION
Proportions offruiting bodies
It is important to know whether addition of glucose to the growth medium of
the cells affects the physiological mechanism for determining proportions at the
differentiation stage. Compared with the method of Bonner & Slifkin (1949),
our method of proportion measurement was slightly less laborious - enabling
more fruiting bodies to be examined - but also less accurate. The inaccuracy
lay in our treating stalks as cones, whereas Bonner and Slifkin treated them as
tapered cylinders. In general, stalks seemed to taper slightly less rapidly than
would cones of similar height and basal radius, so that our measurement gave
an underestimate of stalk volume. However, since there was no obvious difference
in the shapes of glucose and N.s. stalks, the method seemed reasonable as a means
of comparing fruiting bodies of the two types. We conclude that there was
a distinct difference in the spore: stalk proportions of glucose and N.s. fruiting
bodies, the ratios being 3-95:1 for glucose and 2-7:1 for N.S.
Because glucose fruiting bodies were on average larger than N.s. fruiting
bodies, it was possible that the proportions of fruiting bodies were in some way
dependent on the size of the grexes from which they were formed. This would be
contrary to the view (Bonner & Slifkin, 1949; Bonner, 1957) that proportions
are constant over a wide range of fruiting-body sizes. Our analysis, by plotting
spore:stalk ratio against fruiting-body volume, strongly supports the views of
previous workers. The slopes of our lines from these plots are very nearly
parallel to the volume axis, indicating that proportions remained the same
irrespective of volume, and the regression coefficients are small, indicating no
dependence of proportions on fruiting-body volume. In view of this, it seems
that the different proportions of glucose and N.s. fruiting bodies could not be
related to the difference in absolute volume. Hence, we suggest that some physiological difference between the two affects the mechanism responsible for determining proportions, but we have no indication from biochemical studies of what
this difference might be.
The fact that both N.S. and glucose fruiting bodies appeared to have greater
spore: stalk ratios than the fruiting bodies measured by Bonner & Slifkin (1949)
deserves specific comment. Part of the reason for this was undoubtedly that we
underestimated stalk volume, but in order to bring our glucose fruiting bodies
into line with Bonner & Slifkin's, their stalks would have to be roughly 167 %
476
D. R. GARROD AND J. M. ASHWORTH
larger than our measurements suggested. So large an error could only have
arisen if the stalks were approximately cylindrical (a cylinder is 200 % greater in
volume than a cone of equal height and basal radius), whereas in fact all stalks
tapered considerably (Fig. 1). We conclude that our fruiting bodies did, indeed,
have greater spore:stalk ratios than those of Bonner & Slifkin. This may have
been for several reasons: (1) We used a different strain of D. discoideum. (2) Our
strain was grown axenically in suspension and not on an agar surface with
a bacterial food supply. (3) Differentiation took place on the surface of a Millipore filter, not on agar. (4) Differentiation took place at 22 °C, compared with
17 °C in the study by Bonner and Slifkin, who produced evidence that temperature affects proportions. All of these factors may affect proportions to some
degree. Indeed, our results appear to demonstrate that conditions pertaining
during the growth phase of the life-cycle affect the proportions of differentiated
fruiting bodies.
Difference in size of glucose and N.s. fruiting bodies
During aggregation the initial streams which formed around an aggregation
centre were either absorbed into the intial centre or broke up, forming numerous
secondary centres. This breaking up of the stream was extensive during aggregation of N.S. cells and uncommon during aggregation of glucose cells. Secondary
centres were generally smaller than initial centres and gave rise to only one grex.
Initial centres broke up into several grexes which were larger than these formed
from secondary centres. We found no evidence for break-up of grexes at later
stages of development. The smaller average size of N.S. fruiting bodies was due
to their formation predominantly from small grexes from secondary centres,
whereas the larger glucose fruiting bodies were formed predominantly from
larger grexes from initial centres. The greater number of N.S. than glucose
fruiting bodies arose because the difference in aggregation pattern resulted in the
formation of more grexes per unit area, i.e. per unit number of cells.
Although the above argument seems plausible, it was not immediately obvious
that aggregation was the only developmental stage at which the size of grexes
and hence of fruiting bodies was determined. In particular, was it true that
initial centres of glucose and N.S. broke up to form grexes of roughly equal size?
Hohl & Raper (1964) showed that there was an upper limit, the 'critical mass',
to the size of grexes formed from aggregates. If an aggregate was smaller than
the critical mass, one grex formed from it, but if it exceeded the critical mass,
two grexes formed. Was the critical mass for glucose and N.S. the same ? Because
the ranges of fruiting-body size for glucose and N.S. were almost identical, we
feel that the critical masses were probably the same. If the critical mass for
glucose had been greater than that for N.S., we would have expected the upper
size limit of glucose fruiting bodies to be greater than that for N.S. In fact only
one glucose fruiting body was found which was larger than the upper N.S. size
limit. We conclude that initial centres of both glucose and N.S. broke up to give
Development o/D. discoideum
477
grexes of about the same size. Secondary centres were smaller than the critical
mass and thus gave only one grex. The smaller average size of N.s. fruiting bodies
may be accounted for by the preponderance of secondary centres in N.S. cultures,
and vice versa for glucose.
Hohl & Raper reported that the critical mass for 'wild type' fruiting bodies
was 15 x 106/tm3. Taking the volume of a wild-type cell as about 7 x 102/im3
(Garrod & Born, 1971), this critical mass would represent about 2x 104 cells.
(This is probably an over-estimate because the figure of 15 x 106 jam3 for critical
mass must include some extracellular space.) We have estimated that the average
size of glucose fruiting bodies was about 1-5 x 104 cells. Since this average must
be somewhat below the critical mass, we conclude that the critical mass for
strain Ax-2 was of roughly the same order as that of the wild type in terms of
numbers of cells involved. A calculation based on our volume measurements
reinforced this conclusion, suggesting that the critical mass did not exceed
4x 104 cells, except in one case which may have been aberrant. Because our
experiments were not designed to test them specifically, all conclusions relating
to critical mass must be regarded as provisional.
Shaffer (1957) described in detail the formation and behaviour of swellings on
aggregation streams of D. discoideum. This behaviour was extremely variable,
but in itself constituted only one aspect of the even greater variability of stream
development. It seems that the different behaviour of both N.S. and glucose
aggregation streams may each represent a slight reduction in overall variability,
and therefore a simplification, in that each shows a preponderance of one type
of stream behaviour. However, the wide ranges of fruiting-body population
density for both glucose and N.S. indicates considerable variability in the degree
to which their aggregation streams exhibit the different types of behaviour.
Shaffer suggested that variability is occasioned by the simplicity of the mechanisms controlling development (Shaffer, 1957, p. 405). This may well be correct,
but it also seems possible that developmental controlling mechanisms may be
complex in that there may be many controlling factors, variation in any one of
which may alter the behaviour of aggregation streams in one or a number of
different ways. It may be more accurate to say that controlling mechanisms may
be imprecise or imprecisely co-ordinated, rather than simple.
Clearly, speculation as to the physiological difference which is responsible for
the difference in behaviour of N.S. and glucose aggregation streams must be
cautious, N.S. streams develop swellings which become secondary aggregation
centres much more commonly than glucose streams. This could be because N.S.
centres are less efficient inhibitors of centre formation than glucose centres.
Inhibition of centre formation by existing centres has been demonstrated in
Polysphondylium viofoceum (Shaffer, 1963) and in D. discoideum (Bonner &
Hoffman, 1963). Theoretically, inhibition of centre formation within the aggregation territory of an existing centre may occur if the centre efficiently entrains the
surrounding cells, i.e. if it maintains the highest frequency of emission of acrasin
478
D. R. GARROD AND J. M. ASHWORTH
pulses (Cohen & Robertson, 1971). So it is possible that glucose centres entrain
the surrounding cells more efficiently than N.S. centres. It is also possible to put
forward an explanation of the different behaviour in terms of cellular cohesiveness (cell-cell adhesion) or cellular adhesiveness (cell-substatum adhesion), or
both. Briefly, either a decrease in cohesiveness or an increase in adhesiveness
might in principle, account for the reduced tendency of glucose streams to form
swellings. Both effects would tend to make the cells less likely to round up into
aggregates. On the other hand, a decrease in cohesiveness might cause glucose
streams to fragment more easily, which they clearly do not do. We hope that
further experimentation will allow us to assess the relative importance of the
factors mentioned in the control of aggregative behaviour.
CONCLUSION
It is clear that conditions pertaining during the growth phase of the slime
mould life-cycle can influence both morphogenesis and pattern formation
(spore:stalk ratio) during the differentiation phase. Because the slime mould,
grown axenically, is amendable to biochemical analysis, it provides a system in
which morphogenesis and pattern formation may be correlated with biochemical
variables.
We thank Mr G. Asquith for advice on photography and for preparation of the figures.
The work was supported by the Science Research Council.
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601-608.
BONNER, J. T. (1957). A theory of the control of differentiation in the cellular slime moulds.
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BONNER, J. T. (1967). The Cellular Slime Moulds, 2nd ed. New Jersey: Princeton University
Press.
BONNER, J. T. & HOFFMAN, M. E. (1963). Evidence for a substance responsible for the spacing
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{Manuscript received 23 March 1972)