/ . Embryol. exp. Morph. Vol. 29, 3, pp. 647-661, 1973
Printed in Great Britain
647
Cell sorting out during the
differentiation of mixtures of metabolically
distinct populations of Dictyostelium discoideum
By C. K. LEACH, 1 J. M. ASHWORTH 2 AND
D. R. GARROD 3
From the Biochemistry Department, Leicester University
SUMMARY
The behaviour, during the multicellular phase of the life-cycle, of amoebae of Dictyostelium
discoideum grown indifferent media is described. Amoebal populations were marked by growthtemperature-sensitive genetic lesions which do not interfere with developmental phenomena.
The fate of cell populations was determined by measuring the relative number of mutant and
wild-type cells at various stages in the life-cycle. Cells sort out during development in such a
way that they may be ordered in a sequence in which those given early in the following list
preferentially appear in the spore population when mixed with those given later in the list:
cells grown in axenic medium + 86 ITIM glucose and harvested when in the exponential phase of
growth; cells grown in axenic medium and harvested when in the exponential phase of growth;
cells grown on bacteria and harvested when in the exponential phase of growth; cells grown
in axenic medium + 86 niM glucose and harvested when in the stationary phase of growth.
Chemotactic aggregation and grex migration are not essential for sorting-out to occur but, in
the normal life-cycle, the cells of a grex formed from amoebae grown in different media have
sorted out anteroposteriorly. The relationship between this sorting out behaviour and the
mechanism of pattern formation in fruiting-body morphogenesis is discussed. Differences in
density of the amoebae cannot account for the sorting out predispositions we observe.
INTRODUCTION
The development of Dictyostelium discoideum is a seemingly simple example
of pattern formation. Vegetative amoebae from an apparently homogeneous
population aggregate and eventually form an elongate, migrating grex in which
pre-spore cells are positioned posteriorly and pre-stalk cells anteriorly (see
Bonner, 1967). These two cell types may be distinguished on the basis of cytological, density and enzyme differences (see Garrod & Ashworth, 1973). However, since cut grexes regulate, cells in the grex are not yet determined in the
embryological sense. It seems clear that determination only occurs when the
1
Author's address: Corporate Laboratories, P.O. Box 11, The Heath, Runcorn, Cheshire
WA7 4QE, U.K.
2
Author's address: Biochemistry Department, School of Biological Sciences, Leicester
University, Leicester LEI 7RH, U.K.
3
Author's address: Department of Zoology, The University, Southampton, SO9 5NH, U.K.
42
12MB 29
648
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
stalk cells and the spores are finally formed in the fruiting body and that there
is therefore no clear temporal distinction in this system between cell determination and cell differentiation.
Bonner (1959) suggested that pre-spore and pre-stalk tendencies existed
amongst the amoebae and that the amoebae sorted out during aggregation
according to these tendencies. This suggestion was based on studies of the
behaviour of cells marked with vital dyes, such as Nile blue and cresyl violet
(Bonner, 1957), and on the behaviour of mixtures of cells carrying morphologically distinguishable genetic lesions. Farnsworth & Wolpert (1971) have questioned the reliability, as cell marking agents, of the dyes used by Bonner (1957)
and they have reported that there is no mass relative movement of cells in the
migrating grex. It is also possible to argue that the morphological markers used
by Bonner (1959) affected the sorting out behaviour of the cells carrying them
since this behaviour is postulated as forming an integral part of the morphogenesis. More recently Takeuchi (1969) has shown that vegetative amoebae
differ in density and that the denser cells of a population sort out to the
anterior position in the grex. Bonner, Sieja & Hall (1971) have confirmed this
observation using cells derived from strains containing genetic markers (such as
spore size) which are unlikely to affect the behaviour of the cells in the grex.
It thus seems established that amongst populations of amoebae there exist
variations in a cell property (which may be related to density) as a consequence
of which certain cells sort out to the anterior and others to the posterior end of
the grex. Thus certain cells are more likely to become spores and others stalk
cells. However, since the developmental fate of every cell in the grex is reversible
until fruiting body formation commences (the culmination stage) it is clear that
we must be dealing with two pattern-forming processes. The first of these is
associated with the sorting-out behaviour of the cells and the second is imposed
on the cells of the grex during culmination and results in the correct morphogenetic relationship between the stalk and the spore mass. These two patternforming processes could, but need not necessarily, be related.
In this paper we confirm that sorting out of cells can occur and that the developmental fate of an amoeba can thus be predicted. We also show that the density
per se of a cell is unlikely to be the determining parameter and that sorting out
can occur in the absence of chemotactic aggregation and grex migration.
MATERIALS AND METHODS
Organisms and growth conditions
Dictyostelium discoideum strain Ax-2 was isolated, and has been described, by
Watts & Ashworth (1970). Two mutants of this strain, designated G-2 and G-8,
have been isolated by our colleague, Dr E. Gingold, after treatment of strain
Ax-2 with the mutagen Af-methyl-iV-nitroso-N'-nitroguanidine and as a result of
selecting for organisms unable to grow at 27 °C. Dr Gingold has designated the
Sorting out in D. discoideum
649
presumed single mutations carried by these strains as gts-2 and gts-8 respectively.
The amoebae of both mutant strains will grow at 22 °C (the optimal temperature
for growth of D. discoideum) but G-2 has a longer generation time than either
the wild type or G-8. Both G-2 and G-8 strains differentiate in the same time
and their differentiation follows the same morphological sequence as does the
wild type at 22 or 27 °C. Loomis (1969) has described similar mutants in which
growth but not development is temperature-sensitive.
D. discoideum amoebae were grown on bacterial lawns as described by Sussman (1966). Plaques formed from Ax-2 and G-8 amoebae were scored after
4 days incubation at 22 °C and from G-2 amoebae after 5 days incubation at
22 °C. Only strain Ax-2 amoebae grow at 27 °C and plaques formed by these
amoebae were scored after 7 days incubation at this temperature. The reversion
rates of the gts markers to the gts+ condition are less than 1 in 105.
Two different kinds of axenic medium have been used in this study. One
(NS medium) contains yeast extract, peptone and inorganic salts and the other
(glucose medium) contains, in addition, 86 mM glucose. Cells grown in these
media (which have been described in detail with the exact growth conditions by
Garrod & Ashworth, 1972) are described as NS cells and glucose cells respectively. Exponential phase amoebae were harvested when at a cell density of less
than 6 x 106 amoebae/ml and stationary phase cells grown in glucose medium
were harvested when the cell density had risen by less than 10% over a 24 h
period (this corresponds to a cell density of 1-2 x 107 amoebae/ml).
Differentiation
Amoebae were harvested and washed in cold distilled water and resuspended
in cold distilled water at a final cell density of 108 amoebae/ml. Portions (0-5 ml)
of this suspension were placed on Millipore filters resting on filter paper pads as
described in detail by Garrod & Ashworth (1972). Fruiting bodies are formed
after 25 h incubation in a moist environment at 22 °C but the filters were usually
left for 36 h before the spores were harvested and plated out clonally.
When it was necessary to perform manipulations on intermediate stages of the
life-cycle, amoebae were spread over the surface of a 2 % (w/v) solution of
ionagar in distilled water (non-nutrient agar) at a cell density of approximately
7 x 104 amoebae/cm2 and incubated in a moist atmosphere at 22 °C. Examination of the plates after overnight incubation enabled the appropriate stages to be
identified and picked for experimental manipulation.
Aggregation of amoebae in suspensions of 00167 M phosphate buffer were
carried out as described by Garrod (1972).
Determination of the density of amoebae
Density gradients were made using the commercially available colloidal silica
preparation Ludox (available as a 40% suspension from Du Pont Chemical
Co.) essentially as recommended by Pertoft (1966). A 10 % aqueous suspension
42-2
650
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
Table 1. Viability of Dictyostelium discoideum strain Ax-2 and its growthtemperature-sensitive mutant strains G-2 and G-8 when grown in bacterial lawns at
different temperatures
% Viability at
Strain
Ax-2
G-2
G-8
Stage
Exponentially growing amoebae
16 h*
Spores
Exponentially growing amoebae
16 h*
Spores
Exponentially growing amoebae
16 h*
Spores
22 °C
27 °C
98-100
95-100
78-85
78-98
81-92
10-18
89-98
88-92
75-88
90-98
88-96
55-65
0
0
0
0
0
0
* Late grex stage. The time refers to time after initiation of development on Millipore
filters (Sussman, 1966).
of Ludox was adjusted to 1 mM final concentration in EDTA and pH 6-5. The
resulting solution was centrifuged at 20000 rev/min in the 3 x 25 ml swing-out
rotor of an MSE 50 centrifuge for 90 min to create a very stable density
gradient. Portions (1-0 ml) of cell suspension were then layered over the gradient
and the tubes centrifuged for a further 10 min at 2500 rev/min. After such centrifugation the cells are found in a very narrow band and are 100% viable.
RESULTS
Characterization of mutant strains. The viability of the wild-type strain Ax-2
and the two growth-temperature-sensitive mutants G-2 and G-8 derived from it
varied slightly from experiment to experiment but these viabilities were always
consistent within any one experiment. In Table 1 the highest and the lowest
viabilities that we have obtained are recorded for the permissive (22 °C) and the
discriminating (27 °C) temperature. The viabilities obtained were independent
of the medium used to grow the amoebae.
Nature of the spores formed by mixtures of cells grown in various media.
Exponentially growing amoebae were grown in axenic media in the presence
(glucose medium) or absence (NS medium) of glucose. The amoebae were harvested, washed in cold distilled water, mixed in various proportions and allowed
to form fruiting bodies on Millipore filters. The spores from such fruiting bodies
were collected and plated clonally and in duplicate with A. aerogenes and the
number of clones which grew at 22 °C was compared to the number which
grew at 27 °C. After correction for the variable viabilities at these two temperatures (Table 1) the results were expressed as the percentage of mutant spores
Sorting out in D. discoideum
651
100 i -
20
40
60
80
",, mutant cells initially
100
Fig. 1. Spores formed by mixtures of amoebae grown in different media. O, G-2
glucose grown + Ax-2 NS grown; • , G-2 MS grown + Ax-2 glucose grown; A,
G-2 and Ax-2 glucose grown; A, G-2 and Ax-2 NS grown. All amoebae were
harvested when in the exponential phase of growth.
20
40
60
80
"„ m u t a n t cells initially
20
40
60
80
",, mutant cells initially
100
Fig. 2. Spores formed by mixtures of amoebae grown in different media, (a) O,
G-8 glucose grown + Ax-2 bacterial grown; • , G-8 bacterial grown + Ax-2
glucose grown; D, G-8 and Ax-2 bacterial grown, (b) • , G-8 NS grown + Ax-2
bacterial grown; • , G-8 bacterial grown + Ax-2 NS grown. All amoebae were harvested when in the exponential phase of growth.
formed as a function of the percentage of mutant amoebae present in the initial
mixture. In Fig. 1 it can be seen that growth of cells in glucose containing medium
leads to the preferential appearance of those cells in the spore population. Similar
results were obtained when G-8 amoebae were used in mixtures with Ax-2
instead of G-2 amoebae.
Similar experiments were done with mixtures of cells grown on bacteria on
652
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
3 60 -
E 4 0 -
20
40
60
80
"„ mutant cells initially
100
40
60
-SO
mutant cells initiallv
100
Fig. 3
Fig. 4
Fig. 3. Spores formed by mixtures of amoebae grown in different media. • , G-8
exponential amoebae grown in NS medium + Ax-2 stationary phase amoebae grown
in glucose medium; +, Ax-2 exponential amoebae grown in NS medium-+ G-8
stationary amoebae grown in glucose medium.
Fig. 4. Spores formed by mixtures of amoebae grown in different media. • , G-8
exponential amoebae grown on bacteria + Ax-2 stationary phase amoebae grown
in glucose medium; +,G-8 stationary phase amoebae grown on bacteria + Ax-2
exponential amoebae grown in glucose medium.
solid media and harvested before complete lysis of the bacterial lawn had
occurred and cells grown in axenic medium. Fig. 2 gives the results of such experiments using G-8 amoebae. Here it can be seen that cells grown in axenic media
preferentially appear in the spore population when mixed with cells grown on
bacteria whether the axenic medium contained glucose or not.
The experiments described in Figs. 1 and 2 were done with amoebae harvested
when in the exponential phase of their growth cycle. When amoebae grown in
glucose medium were allowed to go into the stationary phase of the growth
cycle then the results shown in Figs. 3 and 4 were obtained. Similar results were
obtained when G-2 amoebae were used instead of G-8 amoebae. Thus cells
harvested in exponential growth preferentially appear in the spore population
whether they were grown on bacteria or in axenic medium when mixed with
stationary phase cells. Experiments similar to those of Figs. 3 and 4 are difficult to
carry out with cells harvested in the stationary phase of growth in NS medium
since in these circumstances the amoebae have little glycogen and cell lysis occurs
quite rapidly (Weeks & Ashworth, 1972). Similarly it is difficult to obtain bacterial grown cells in the stationary phase of growth using solid media since such
cells aggregate and begin differentiation.
The results shown in Figs. 1-4 demonstrate that cells may be arranged in a
sequence so that those given early in the following list preferentially appear in the
Sorting out in D. discoideum
653
Table 2. Analysis of the percentage of mutant spores found in individual fruiting
bodies formed from various mixtures of Ax-2 and G-8 amoebae (these analyses
were carried out on fruiting bodies on Millipores also used to obtain the data of
Figs. 2 and 3)
Strain and
growth conditions*
Ax-2 NS
G-8 glucose
Ax-2 glucose
G-8NS
Ax-2 NS
G-8NS
Ax-2 glucose
G-8 glucose
Ax-2 bacteria
G-8 glucose
Ax-2 glucose
G-8 bacteria
Ax-2 bacteria
G-8NS
Ax-2 NS
G-8 bacteria
Ax-2 bacteria
G-8 bacteria
% of mutant
cells initially
in mixture
% of mutant spores
in fruiting body
30
60±10-7(6)
42
5-2 ± 4-3 (6)
42
44 ±9-4 (6)
48
49-5 + 6-6 (6)
51
85-2± 11-4 (6)
44
3 ± 2-8 (6)
33
63-7 ±5-9 (6)
61
18-3 ±7-7 (6)
68
61 ±12-5 (6)
Results are expressed as mean + S.D. with the number of individual fruiting bodies analysed
in parentheses.
* All amoebae were harvested when in the exponential phase of growth.
spore population when mixed with those given later in the list: exponential
phase cells grown in glucose medium; exponential phase cells grown in NS
medium; exponential phase cells grown on bacteria; stationary phase cells grown
in glucose medium.
The results shown in Figs. 1-4 were obtained from an analysis of pooled spores
from many fruiting bodies and are thus open to two alternative interpretations;
either cells grown in different media form separate fruiting bodies and the spore
analyses merely reflect the proportions of different fruiting body types or, cells
grown in different media form composite fruiting bodies and the spore analyses
therefore reflect the behaviour of cells within individual grexes. Analysis of individual fruiting bodies (Table 2) shows thatjhe latter alternative is correct and
that each fruiting body is composed of both types of cell.
Analysis of migrating grexes. This demonstration of sorting out of the two cell
populations in the fruiting body poses the question of whether this sorting out is
654
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
* * &
Ax -2
\
Mix
Rear
14 d\%
f • •
G-2 or G-8
Amoebae
Front
Grcx
Fig. 5. Analysis of front and rear portions of migrating grexes formed from cells
grown in different media.
Table 3. Sorting out of the component cells of the migrating grex
Strain and
growth
conditions*
Ax-2 glucose
G-8NS
Ax-2NS
G-8 glucose
Ax-2NS
G-8NS
Ax-2 glucose
G-8 glucose
Ax-2 glucose
G-2NS
% of mutant cells present
Initially
54
Rear of grex
9-5
Front of grex
82-5
52
75
22
49
52
57
39
49
43
59
17
93
* All amoebae were harvested when in the exponential phase of growth.
manifest in the migrating grex, as implied by the studies of Takeuchi (1969) and
Bonner et al. (1971).
Amoebae were grown in different media and allowed to aggregate together on
non-nutrient agar. After 16 h one large migrating grex from each mixture was
cut at right angles to its longitudinal axis into three sections. The front and back
sections were dissociated by trituration in cold distilled water and the cells
plated clonally with A. aerogenes to determine the percentage of mutant cells in
each section (Fig. 5). From the results of such experiments (Table 3) it is clear
that the sorting out of the amoebae had occurred at or before the migrating grex
stage. Hence the question arises of which stage in the life-cycle is essential for
sorting out.
Analysis of aggregates made in suspension. In order to see if the chemotactic
aggregation stage was essential for sorting out Ax-2 and G-2 or G-8, amoebae
Sorting out in D. discoideum
655
V
w
S
Ax-2
Mix in
roller
culture
•
Aggregate
Fruiting
bodies
Ci-2 or G-8
Amoebae
Fig. 6. Analysis of sorting out of cells in aggregates made in roller cultures.
Table 4. Sorting out of cells in aggregates made in suspension
without any chemotactic aggregation stage
Strain and
growth conditions*
Ax-2 NS
G-8 glucose
Ax-2 glucose
G-8NS
Ax-2 NS
G-2 glucose
Ax-2 glucose
G-2NS
% of mutant
cells initially
in mixture
53
% of mutant
spores in
fruiting body
82-8 ± 8 (5)
44
13 ±8-4 (4)
44
80+13(5)
40
15 ±11 (3)
Results are expressed as mean ± S.D. with the number of aggregates examined in parentheses.
* All amoebae were harvested when in the exponential phase of growth.
were grown in various media and then mixed in 0-0167 M phosphate buffer, pH
6-0, in stoppered tubes. These tubes were then rotated about their long axis at 18
rev/min at 22 °C overnight (Gerisch, 1968; Garrod, 1972) so that spherical balls of
cells formed. When these were placed on a solid surface they gave rise to fruiting
bodies whose spores were analysed for mutant and wild-type cells (Fig. 6). As
can be seen in Table 4 the cells in such aggregates sorted out during fruiting body
construction.
Mixing of cells at the migrating grex stage. It is known that when cut grexes
regulate the regulation is optimal if the cut tip is allowed to migrate before it
constructs a fruiting body (Raper, 1940). This suggests that the migration of the
grex might be in some way necessary for sorting out. In order to test this hypothesis amoebae grown in various conditions were allowed to form migrating
grexes on non-nutrient agar. Grexes of different provenances were then placed
side by side on the agar and stirred with a fine needle so that the component cells
656
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
* <2>
Mix with
needle
/
-p/
—'
r - ~>
r- <J
G-2 or G-8
Fruiting
bodies
-f
Grex
Amoebae
Fig. 7. Analysis of sorting out of grex cells grown in different media.
Table 5. Sorting out of grex cells in the absence of any
migration of the grex
Strain and growth conditions*
of grexes whose cells were mixed
% of mutant spores in the
resulting fruiting bodies
Ax-2 NS + G-8 glucose
Ax-2 glucose + G-8 NS
Ax-2 NS + G-2 glucose
Ax-2 glucose + G-2 NS
Ax-2 NS + G-2 glucose
88 ± 2 (4)
17 ± 20 (4)
76-8 ± 8-4 (4)
18-5±12(4)
88-5 ± 9 (4)
Results are presented for five separate experiments as mean ± S.D. with the number of pairs
of grexes mixed in each experiment in parentheses.
* All amoebae were harvested when in the exponential growth phase.
of the grexes were mixed thoroughly. It is not possible to determine accurately the
number of cells of each type mixed in this experiment. However, care was taken to
select grexes of similar size and shape so that there were approximately equal
numbers of glucose and NS cells present after mixing of the two grexes. From the
resulting mass of cells a number (usually 3-6) of small fruiting bodies are formed
directly without any further migration (Fig. 7). The spores of these fruiting
bodies were collected and analysed for mutant and wild-type spores. The results
described in Table 5 show that considerable sorting out occurs and hence migration of the grex is not essential for sorting out to occur.
Grafting experiments. Raper (1940) showed that when the tip of one grex was
placed on the back of another whose tip had been removed the two pieces of grex
fused to form a composite grex in which the grafted cells largely retained their
relative positions. We have shown that it is possible to predispose cells to sort
out to the back of a grex (Table 3) and so it is of interest to place the tip of a
grex composed of cells which are known to sort out posteriorly on to a grex
(whose original tip has been removed) composed of cells known to sort out
Sorting out in D. discoideum
657
Fruiting
body
NS-grown
Grex
Fig. 8. Grafting experiment.
Table 6. Sorting out of cells in grafted grexes
Amoebae grown in glucose medium were allowed to form grexes and the front
portion (•£-•»•) was cut off and grafted on to the back portion of a grex formed from
amoebae grown in NS medium, thus forming a composite grex of approximately the
same size as the two donor grexes. After 1-3 h at 22 °C the composite grexes stopped
migrating, formed fruiting bodies, and the spores of these were analysed for mutant
and wild-type cells (Fig. 8).
Strain and growth conditions* of
A
r
N
Recipient (Back) grex. Donor (Front) grex.
Ax-2 NS
G-2NS
G-2 glucose
Ax-2 glucose
% of mutant spores
in the resulting
fruiting body
69-4116-6 (7)
301 ±17-3 (8)
Results are presented as mean ± S.D. with the number of composite grexes analysed in
parentheses.
* All amoebae were harvested when in the exponential phase of growth.
anteriorly (Fig. 8). In this way it is possible to determine if cells 'remember'
their position or their sorting out predisposition when these two are in conflict.
In all cases examined (Table 6) the sorting out predisposition is dominant and
grafted tip cells can be found preferentially amongst the spore population.
Determination of the density of cells. Density gradients made from Ludox are
much easier to make, less toxic, more reproducible and more discriminating than
those we have used previously (Miller, Quance & Ashworth, 1969). Amoebae
grown in various media were analysed and found to have densities in the order:
stationary-phase cells grown in glucose medium > exponential-phase cells
grown in glucose medium > exponential-phase cells grown on bacteria >
exponential-phase cells grown in NS medium. Dissociated grex cells formed two
distinct bands of different density as found before (Miller et al. 1969) and both
these bands were at higher densities than that characteristic of the stationary
phase cells grown in glucose medium.
658
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
DISCUSSION
In this paper we have studied the behaviour, during the multicellular phase of
the life-cycle, of mixed cell populations the component cells of which have been
grown in different media. In each mixture one of the cell populations has been
genetically marked such that its cells were incapable of growth at 27 °C. It is
important to establish that the genetic markers we have used have no effect on
the observed sorting out behaviour. We believe that there is no such effect because
(a) Ax-2, G-2 and G-8 cells do not sort out from one another when they are
grown in the same media (Figs. 1, 2), (b) G-2 and G-8 were isolated as a consequence of independent mutagenic events, are phenotypically distinguishable,
yet have identical sorting out behaviour, (c) development of strains Ax-2, G-2 and
G-8 is in all respects identical at 22 and 27 °C, (d) genetic lesions which specifically affect growth processes have been isolated before (Loomis, 1969) and in this
organism growth and development are mutually exclusive phenomena, and (e)
our results are consistent with previous work using other methods of marking
cell populations (Takeuchi, 1969; Bonner et al. 1971).
It is not easy to estimate the degree of precision shown by the sorting out process which we report here. The sharpness of the points of inflexion in Figs. 1-4
(particularly in Fig. 1 and Fig. 4, crosses) suggests that there is a very high
degree of precision. However, this is difficult to express quantitatively since it is
known that whereas the spore: stalk cell ratio is 3-95:1 for cells grown in glucose
media it is only 2-7:1 for cells grown in NS medium (Garrod & Ashworth, 1972).
We do not know what the spore: stalk cell ratio is for mixtures of cells of the type
we have been studying here but it is unlikely to be linearly related to the cell
composition of the mixture. Further, it is technically difficult to measure accurately small numbers of one cell type in the presence of a great excess of the other.
However, we feel that it is possible to postulate that all, to a first approximation,
of the' vacancies' for spore cells are filled by those amoebae with the appropriate
sorting out predisposition, and only then when all these vacancies have been
filled, are amoebae predisposed by their sorting out behaviour to become spore
cells forced, by the necessities of the morphogenetic pattern, to become stalk
cells.
We must therefore now examine the question of whether there is any mechanistic connexion between sorting out and pattern formation. There are essentially two possibilities: (1) either glucose cells have a greater predisposition to
form spores than NS cells, even in the vegetative stage, and once incorporated
into a composite grex such cells sort out according to their predisposition, thus
giving rise to the spore:stalk pattern, or (2) vegetative amoebae are merely predisposed by their growth conditions to sort out from each other in such a way
that glucose cells preferentially adopt the posterior position in the grex and NS
cells the anterior. In the latter case the cellular spore:stalk pattern would be
imposed on the grex cells by some pattern-specifying mechanism within the grex
Sorting out in D. discoideum
659
which is distinct from the sorting out predispositions of its component cells. In
the first case there would be a causal connexion between the pattern and the
sorting out whereas in the latter case the connexion would be merely fortuitous.
We have presented arguments against believing that there is any causal connexion between sorting out and pattern formation previously (Garrod & Ashworth,
1973) and the work we report here provides no reason for changing this view.
Whilst we cannot discount entirely a causal connexion between sorting out and
pattern formation, at present there seems no evidence which would lead us to
conclude that the connexion is other than fortuitous.
Our results show that during the normal life-cycle sorting out of cell populations has taken place by the migrating grex stage (Table 3). This does not
enable us to say at which stage sorting out normally occurs. However, we can
say with some confidence that certain stages in the life-cycle are not essential for
sorting out to be manifest. Two experiments, making mixed aggregates in suspension and grex grafting (Tables 4, 6), allow the development of heterogeneous
cell populations without a chemotactic aggregation stage. In both cases sorting
out of the two cell populations took place, demonstrating that the aggregation
stage is not essential for sorting out. When two homogeneous grexes each composed of a different cell type were stirred together fruiting bodies were formed in
which sorting out had occurred without the intervention of a migrating grex
stage, showing that a migration stage itself is also not essential for sorting out.
Thus, provided only that a culmination stage intervenes between mixing of the
cell populations and fruiting body formation, sorting out can occur.
Our grex grafting and mixing experiments (Figs. 7, 8) require special discussion in relation to a conflict which has arisen between the results of various
workers. On the one hand Bonner (1952) and Takeuchi (1969) suggest that cells
of the migrating grex 'remember' their positions, and if displaced therefrom will
return, whilst on the other hand Farnsworth & Wolpert (1971) have demonstrated
that cells displaced within a grex (e.g. tip cells placed at the back of a grex)
remain where they are put and they conclude 'that there is no cell sorting out in
the migrating grex'. Our experiments show that grex cells sort out according to a
hierarchy determined by the medium used for their growth rather than according
to the position in the grex from which they were taken. We cannot, though, say
whether this sorting out takes place during the migration period following the
graft or during the subsequent culmination stage. It appears that the medium in
which a cell is grown is more important than the position from which it was
taken when these two influences are in opposition in grex grafts.
The fact that tip cells move to the back of the grex (Table 6) in our grafts
makes it most unlikely that the tip of the grex can usefully be said to act in a
manner analogous to the autonomous 'organizer' region postulated to regulate
pattern formation during early vertebrate embryogenesis(Spemann, 1938). Rather
we would suggest that the importance of the tip is a morphogenetic one in that
it leads and directs the movement of the cell mass (Raper, 1940; Garrod, 1969).
660
C. K. LEACH, J. M. ASHWORTH AND D. R. GARROD
The physiological basis for the sorting out process remains obscure. Takeuchi
(1969) suggested that the cells sort out on the basis of their density such that the
lightest cells tend to become spores, but we find that the amoebae of strain Ax-2
have densities in the following order: stationary-phase cells grown in glucose
medium > exponential-phase cells grown in glucose medium > exponentialphase cells grown on bacteria > exponential-phase cells grown in NS medium.
This order is the same as that of the glycogen content of the amoebae (Weeks &
Ashworth, 1972; Ashworth & Watts, 1970) but is quite different from the sorting
out order of preferential appearance in the spore population of cells: exponential-phase cells grown in glucose medium; exponential-phase cells grown in NS
medium; exponential-phase cells grown on bacteria; stationary-phase cells
grown in glucose medium.
From a physiological point of view the most revealing experiment is that
shown in Fig. 4, where it is clear that the predisposition of cells grown in
glucose medium alters as the time at which they were harvested alters. It is
known that as such cells leave the exponential phase of the growth curve and
enter the stationary phase they synthesize and excrete into the medium relatively large amounts of adenosine 3'55'-cyclic monophosphate (cAMP) (Malkinson & Ashworth, 1972). Preliminary experiments have shown that the rate of
change in the predisposition of the glucose-grown cells can be markedly accelerated by incubating exponential-phase cells with cAMP and it is known that high
concentrations of cAMP can cause isolated amoebae to become similar in
appearance to stalk cells (Bonner, 1970). If these observations can be confirmed
then the most likely explanation for the sorting out phenomena that we have
reported here is that it is due to changes in the cell membrane catalysed by
enzymes released from amoebae as a consequence of an increase in the extracellular concentration of cAMP (Ashworth, 1971).
Finally, the emergence of an order of preferential appearance of physiologically distinct populations of cells in the spore population is reminiscent of
the finding of an order for preference for the internal position in the sorting out
of embryonic chick cells (Steinberg, 1970). We have no indication, of course,
of whether this apparent similarity is trivial or significant.
We thank Mrs Janet Kwasniak and Miss Julia Johnson for technical, and the Science
Research Council for financial, assistance. One of us (C. K.L.) thanks the Medical Research
Council for the award of a fellowship.
REFERENCES
J. M. (1971). Cell development in the cellular slime mould Dictyostelium discoideum. In Control Mechanisms of Growth and Differentiation (ed. D. D. Davies & M.
Balls). Symp. Soc. exp. Biol. 25, 27-49.
ASHWORTH, J. M. & WATTS, D. J. (1970). Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture. Biochem. J. 119, 175-182.
ASHWORTH,
Sorting out in D. discoideum
661
BONNER, J. T. (1952). The pattern of differentiation in amoeboid slime moulds. Am. Nat. 86,
79-89.
BONNER, J. T. (1957). A theory of the control of differentiation in the cellular slime moulds.
Q. Rev. Biol. 32, 232-246.
BONNER, J. T. (1959). Evidence for the sorting out of cells in the development of the cellular
slime moulds. Proc. natn. Acad. Sci. U.S.A. 45, 379-384.
BONNER, J. T. (1967). The Cellular Slime Moulds. Princeton: Princeton University Press.
BONNER, J. T. (1970). Induction of stalk cell differentiation by cyclic AMP in the cellular
slime mould Dictyostelium discoideum. Proc. natn. Acad. Sci. U.S.A. 65, 110-113.
BONNER, J. T., SIEJA, T. W. & HALL, E. M. (1971). Further evidence for the sorting out of
cells in the differentiation of the cellular slime mould Dictyostelium discoideum. J. Embryol.
exp. Morph. 25, 457-465.
FARNSWORTH, P. A. & WOLPERT, L. (1971). Absence of cell sorting out in the grex of the slime
mould Dictyostelium discoideum. Nature, Lond. 231, 329-330.
GARROD, D. R. (1969). The cellular basis of movement of the migrating grex of the slime
mould Dictyostelium discoideum. J. Cell Sci. 4, 681-698.
GARROD, D. R. (1972). Acquisition of cohesiveness by slime mould cells prior to morphogenesis. Expl Cell Res. 72, 588-591.
GARROD, D. R. & ASHWORTH, J. M. (1972). Effect of growth conditions on development of
the cellular slime mould Dictyostelium discoideum. J. Embryol. exp. Morph. 28, 463-479.
GARROD, D. R. & ASHWORTH, J. M. (1973). Development of the cellular slime mould
Dictyostelium discoideum. In Microbial Differentiation (ed. J. M. Ashworth and J. E.
Smith). Symp. Soc. gen. Microbiol. 23, 407-435.
GERISCH, G. (1968). Cell aggregation and differentiation in Dictyostelium. In Current Topics
in Developmental Biology (ed. A. A. Moscona & A. Monroy), pp. 157-196. New York:
Academic Press.
LOOMIS, W. F. JR. (1969). Temperature-sensitive mutants of Dictyostelium discoideum. J. Bact.
99, 65-69.
MALKINSON, A. & ASHWORTH, J. M. (1972). Extracellular concentrations of adenosine
3': 5'-cyclic monophosphate during the axenic growth of myxamoebae of the cellular slime
mould Dictyostelium discoideum. Biochem. J. 127, 711-612.
MILLER, Z. I., QUANCE, J. & ASHWORTH, J. M. (1969). Biochemical and cytological hetero-
geneity of the differentiating cells of the cellular slime mould Dictyostelium discoideum.
Biochem. J. 114, 815-818.
PERTOFT, H. (1966). Gradient centrifugation in colloidal silica and polysaccharide media.
Biochem. biophys. Acta 126, 594-596.
RAPER, K. B. (1940). Pseudoplasmodium formation and organisation in Dictyostelium discoideum. J. Elisha Mitchell scient. Soc. 56, 241-282.
SPEMANN, H. (1938). Embryological Development and Induction. New Haven: Yale University
Press.
STEINBERG, M. S. (1970). Does differential adhesion govern self-assembly in histogenesis?
Equilibrium configurations and the emergence of a hierarchy among populations of embryonic cells. J. exp. Zool. 173, 395-433.
SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mould
development. Meth. Cell Physio!. 2, 397-410.
TAKEUCHT, I. (1969). Establishment of polar organisation during slime mould development. In
Nucleic Acid Metabolism, Cell Differentiation and Cancer Growth (ed. E. V. Cowdry & S.
Seno), pp. 298-304. Oxford: Pergamon Press.
WATTS, D. J. & ASHWORTH, J. M. (1970). Growth of myxamoebae of the cellular slime mould
Dictyostelium discoideum in axenic culture. Biochem. J. 118, 505-512.
WEEKS, G. & ASHWORTH, J. M. (1972). Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during growth. Biochem. J.
126, 617-626.
(Received 27 September 1972, revised 2 November 1972)