J. Embryo!, exp. Morph. Vol. 67,pp. 181-193, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Studies of early stages of differentiation of the
cellular slime mould Dictyostelium discoideum
By P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS1
From the Biochemistry Department, University of Sheffield
SUMMARY
Countercurrent distribution in a polymer, two-phase system has been used to study changes
in the cell surface properties of amoebae of Dictyostelium discoideum. Amoebae harvested
during exponential growth in axenic culture and during the subsequent first six hours of
development on Millipore filters were distributed as a single peak. However, the position of
the peak changed during the period of early development which showed that changes in cell
surface properties were occurring. At aggregation (8 h), the peak markedly broadened, indicating considerable increase in cell surface heterogeneity amongst the amoebae, and heterogeneity was so great by 9-10 h that the amoebae distributed as two peaks. Amoebae from
one peak were shown to be precursors of spores while amoebae from the other peak appeared
to be precursors of stalk cells. Similarly, amoebae from the trailing and leading edges of the
broad peak, formed from amoebae beginning to aggregate (8 h), were found to have different
fates. Thus cell differentiation had been found at times of development prior to formation of
aggregates having apical tips or anterior-posterior polarity and neither of these features of
aggregates can be essential for initiation of cell differentiation. It is therefore concluded that
differentiation is not initiated in D. discoideum in response to 'positional information'.
INTRODUCTION
The life-cycle of Dictyostelium discoideum is much studied as a 'model
system' of development and differentiation (Loomis, 1975). Development is
initiated by starvation when the Dictyostelium amoebae, which have remained
solitary during growth, aggregate to form multicellular masses that eventually
differentiate into fruiting bodies containing essentially only two cell types, i.e.
stalk cells and spores.
In standard laboratory conditions (Sussman & Lovgren, 1965), aggregates
begin to form after 8 h development and then pass synchronously, and at
known times, through successive morphogenetic stages. The pseudoplasmodium
stage is attained first as erect 'finger-like' structures at 12 h and then continues
as slug-like masses when the 'fingers' bend over to lie down horizontally on the
substratum after 14-15 h development (Treffry & Watts, 1976). The 'slugs'
migrate towards light and higher temperature and so have an easily recognized anterior-posterior polarity. Migration soon ceases and fruiting bodies,
1
Author's address: Biochemistry Department, University of Sheffield, Sheffield S10 2TN,
U.K.
182
P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS
comprising a spore mass surmounting a thin, cellular stalk, are formed at 24 h.
The stalk cells are derived from the anterior, and the spores from the posterior,
of the slugs (Raper, 1940; Bonner, 1944). Amoebae at the anterior of the slug
differ from amoebae at the posterior in ultrastructure (Miiller & Hohl, 1973;
Gregg & Badman, 1970), enzyme composition (Newell, Ellingson & Sussman,
1969) and in their pattern of protein synthesis (Alton & Brenner, 1979). Hence
differentiation has begun by the slug stage of development to give pre-spore
and pre-stalk cells. Any attempt to explain how development and differentiation
take place in D. discoideum must therefore account for both the formation of
pre-spore and pre-stalk cells within 14-15 h development and for the pattern
the two cell types assume in the migrating slug.
Several theories (reviewed by MacWilliams & Bonner, 1979) have been
proposed to explain how the population of apparently unspecialized amoebae
at the beginning of development can eventually give rise to two populations of
specialized cells, but all these theories seem to depend on adoption of one or
other of two assumptions. Most commonly, it is assumed that differentiation
does not begin until aggregates having anterior-posterior polarity have formed
subsequent to aggregation. It is then proposed that, because of this polarity,
amoebae within aggregates can respond to some form of 'positional information' (Wolpert, 1969) which ensures that cells at the anterior become pre-stalk
cells whilst cells at the posterior become pre-spore cells. The alternative
assumption suggests that differentiation has to begin before aggregate polarity
is established if unspecialized amoebae are to have time to differentiate into
pre-spore and pre-stalk cells by the slug stage of development. Hence it is
proposed that the two cell types begin to form early in development and that,
after aggregation, sorting-out of the two cell types leads to establishment of
polarity in slugs as pre-stalk cells collect at the anterior and pre-spore cells at
the posterior.
The two accounts of slime mould development differ markedly concerning
the time at which they predict that cell differentiation begins, and it would be
possible to decide which is the more accurate if it were known whether cell
differentiation precedes, or follows, establishment of polarity in aggregates. A
sequence of events consistent with the proposal that differentiation precedes
establishment of polarity has been observed during D. discoideum development
(Forman & Garrod, 1977; Tasaka & Takeuchi, 1981) but only in abnormal
conditions, where aggregates were allowed to form from amoebae suspended in
phosphate buffer and where aggiegates would not eventually develop into
fruiting bodies. It has not been possible to determine whether differentiation
precedes formation of polar aggregates in the normal conditions of development on a solid substratum because of a lack of criteria for recognizing cell
differentiation if it occurs at such early stages of development. However, it
seemed that, if two cell types are formed early in development, they must have
different surface properties in order to be able to sort out subsequently to give
Early differentiation o/D. discoicleum
183
the pre-spore-pre-stalk pattern found in slugs. We have therefore made use of a
technique (countercurrent distribution in a polymer, two-phase system), that
separates cells having different surface properties (see Walter, 1977; Fisher,
1981), to determine whether two cell types are present early in development
of D. discoideum and prior to formation of polar aggregates.
The technique depends on the ability of mixtures of aqueous solutions of
dextran and poly (ethylene glycol) to separate, on standing, into two phases,
the upper phase being poly (ethylene glycol)-rich and the lower phase dextranrich (Albertsson, 1971). When mixed with such a phase system, cells with
different surface properties have different affinities for the two phases and
therefore separate. However, it is usually necessary to repeat such cell partitioning many times in a countercurrent fashion with fresh phases to obtain
good separation.
MATERIALS AND METHODS
Chemicals
Dextran T 500 (batch 4094) was obtained from Pharmacia Fine Chemicals
and poly (ethylene glycol) 4000 (batch 6444220) from BDH Chemicals Limited.
Empigen BB was a gift from Albright & Wilson Limited, Marchon Division,
Whitehaven, England.
Dictyostelium discoideum
Amoebae of D. discoideum strain Ax-2 were grown at 22 °C in HL 5 medium
containing 86 mM glucose (Watts & Ashworth, 1970) and were harvested
either during exponential growth at approximately 106 amoebae ml" 1 or during
stationary phase of growth at densities greater than 107 amoebae ml"1. Amoebae
were washed once with distilled water at 5°.
Development was at 22° on Millipore filters (Sussman, 1966). At various
times of development, amoebae were washed off the filters with distilled water
at 5°. Single-cell suspensions were obtained from aggregates by vigorous
mixing on a Vortex mixer.
A mutant resistant to acriflavin was isolated by spreading amoebae of
strain Ax-2 and Aerobacter aerogenes NCTC 418 on nutrient agar plates
(Sussman, 1966) containing 100/tg ml" 1 acriflavin. A colony that grew rapidly
was recloned before being maintained in HL 5 glucose medium. Frequent
checks confirmed that the mutant did not revert to being acriflavin-sensitive.
The growth rate in axenic culture and the time-course of development were the
same as for the parental Ax-2 wild-type strain.
Partitioning
The phase system comprised 5-5% (w/w) dextran and 5-5% (w/w) poly
(ethylene glycol) to which had been added, per 200 g final mixture, 10 ml
1-0 M-NaCl, 10 ml 0-2 M-Na2SO4 and 1 ml 0-2 M phosphate (KH 2 PO 4 /K 2 HPO 4 )
buffer pH 7-8. This gave a 'zero-potential' phase system, i.e. one in which
184
P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS
there was essentially no potential difference between the two phases (Johansson,
1974). The phases were kept at 4° and were at pH 6-8.
Partitioning was at 4° in a thin-layer, countercurrent distribution apparatus
similar to that described by Albertsson (1965), and cells were partitioned 59
times between the two phases. Each time, cells were shaken with the phases for
30 sec and the phases were then left for 10 min to separate. After completion
of the countercurrent distribution either 1 % (v/v) Empigen or 50 mM phosphate
(NaH2PO4/K2HPO4) buffer pH 6-5 was added to convert each fraction into
a single phase. Cell density was determined in fractions by counting cells in a
haemocytometer or, for samples that had bsen lysed in Empigen, by measurement of absorbance at 280 nm. It had previously been found that absorbance
at 280 nm was linearly related to cell density.
Determination of spore character
In some experiments, amoebae that were acriflavin-resistant were mixed
with wild-type amoebae (see text for further details) and allowed to form
fruiting bodies on Millipore filters. Spores were collected and resuspended in
water. Samples (0-1 ml) of the spore suspension containing about 1000 spores
ml" 1 were spread with A. aerogenes on ten nutrient agar plates, and on ten
other nutrient agar plates containing 100 /ig ml" 1 acriflavin. The plates were
left four days at 22°. The number of spores of the acriflavin-resistant strain was
determined by counting colonies growing on the plates containing acriflavin
whilst the total number of spores of wild-type and acriflavin-resistant character
was given by counting colonies on the plates lacking acriflavin. The viability
on nutrient agar plates of spores from the acriflavin-resistant strain was always
found to be the same as the viability of spores of the wild-type strain. Spores
of the acriflavin-resistant strain had the same viability on nutrient agar plates
in the presence or absence of acriflavin.
RESULTS
Selection of a polymer, two-phase system
It has been usual to make use of phase systems containing a high (e.g. 0-1 M)
phosphate concentration to effect cell separation (Walter, 1977). In such
phase systems there is an electrostatic potential difference between the two
phases, with the upper phase positive with respect to the lower phase. It is
therefore believed that cell separation achieved with these phase systems largely
reflects differences in surface charge between the separated cells. Attempts to
use charged phase systems to study Dictyostelium discoideum amoebae were
unsuccessful, and studies were continued with a 'zero-potential' system where
there was virtually no potential difference bstween the two phases. Thus
D. discoideum cells were separated on the basis of differences in surface
properties that were not dependent on cell surface charge.
Amoebae were found to bs between 85 and 95 % viable after partitioning.
185
Early differentiation ofD. discoideum
A (Oh)
0-5
B(2h)
0-5
C(4h)
0-5
D(6h)
0-5
10
20
30
40
Fraction number
50
60
Fig. 1. Partitioning of amoebae harvested during exponential, axenic growth and
allowed to develop on Millipore filters. (A) Amoebae at 0 h development.
(B) Amoebae at 2 h development. (C) Amoebae at 4 h development. (D) Amoebae
at 6h development. After partitioning, the amoebae were lysed and the absorbance
at 280 nm of each fraction was measured. This was proportional to cell density.
In the absence of any cell lysate, fractions gave an absorbance at 280 nm of approximately 0 1 .
Development changes in partitioning of amoebae harvested during
exponential growth
Amoebae grown in axenic culture were harvested during exponential growth
and allowed to develop on Millipore filters for up to 11 h. Partitioning distributed amoebae harvested at any time during the first six hours of development
as a single peak (Fig. 1 A, B, C, D). However, the position of the peak changed
with time of development as cell affinity for the upper phase of the two-phase
system gradually increased (0-4 h) and then decreased at 6 h. This would
indicate that changes were occurring during development in the surface properties of the amoebae. No change in cell partitioning was detected between 6 h
186
P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS
A(8h)
0-5
B (9h)
0-5
C(10h)
0-5
D (11 h)
0-5
10
20
30
40
50
60
Fraction number
Fig. 2. Partitioning of amoebae harvested during exponential axenic growth and
allowed to develop on Millipore filters. (A) Amoebae at 8 h development.
(B) Amoebae at 9 h development. (C) Amoebae at 10 h development. (D) Amoebae
at 11 h development.
and 7 h development, but marked change was apparent at 8 h (Fig. 2 A) when
the amoebae had begun to aggregate. Then the distribution became extremely
broad and indicated that the population of amoebae was extremely heterogeneous in cell surface properties. By 9 h development, heterogeneity was so
great that the amoebae were divided by partitioning into two populations
(Fig. 2 B). A similar distribution was found at 10 h, and the two populations
have bsen designated as peak I and peak II in Fig. 2 C. Two populations were
still apparent at 11 h development (Fig. 2 D) but amoebae were distributed
between them in the approximate ratio 1:1 (amoebae in peak I: amoebae in
peak II), whereas at 9 h the ratio was 1:3 and at 10 h 1:1-5. Thus from 8 h
development onwards there was a gradual tendency for amoebae to regain
affinity for the lower phase of the two-phase system, and thus to distribute in
Early differentiation o/D. discoideum
187
Table 1. Developmental fate of amoebae isolated by partitioning
at 10 h development
Percentage of spores
derived from
Nature of amoebae from
A
Experiment
peak I
1
Wild-type
Mutant
Wild-type
Mutant
Wild-type
Mutant
2
3
Means
peak II
Mutant
Wild-type
Mutant
Wild-type
Mutant
Wild-type
t
peak I
peak II
67
60
61
88
60
80
69
33
40
39
12
40
20
31
Wild-type and mutant (acriflavin-resistant) amoebae were harvested during exponential
growth and allowed to develop on Millipore filters for 10 h. Amoebae were then separated
into peaks I and II by partitioning (see Fig. 2C).
lower-numbered fractions of the countercurrent distribution (i.e. to the left
in Fig. 2).
After 11 h development amoebae were so cohesive that they tended to aggregate during partitioning unless at very low density. Partitioning of amoebae at
later stages of development than 11 h has not therefore been studied.
Fate of the two populations present at JO h development
During differentiation D. discoideum amoebae differentiate to give two
populations of specialized cells, i.e. the stalk cells and spores. Since partitioning
studies indicated that amoebae at 10 h development were also divided into two
populations, it seemed possible that one of these populations might eventually
differentiate into spores and the other into stalk cells. In order to investigate
this possibility an experiment was designed which depended on use of both
wild-type amoebae and amoebae of a mutant strain, derived from the wild type
and resistant to acriflavin. The time course of development was the same for
both strains.
Wild-type amoebae at 10 h development were separated by partitioning into
the two populations (peak I and peak II in Fig. 2C). Similarly, the two populations were isolated after amoebae of the mutant strain had been subjected to
partitioning after 10 h development. Wild-type amoebae from peak I were
then mixed with an equal number (approximately 107) of mutant amoebae
from peak II and the mixture was allowed to form fruiting bodies on Millipore
filters. It was then possible to identify the origin of the spores of the fruiting
bodies by determining whether the spores were of wild-type or mutant character.
188
P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS
Table 2. Developmental fate of amoebae isolated by partitioning
at 8 h development
Percentage of spores
derived from
t
Experiment
trailing edge
leading edge
1
Wild-type
Mutant
Wild-type
Mutant
Wild-type
Mutant
Wild-type
Mutant
Mutant
Wild-type
Mutant
Wild-type
Mutant
Wild-type
Mutant
Wild-type
2
3
4
Mean
trailing
edge
leading
edge
59
83
59
84
62
60
60
76
68
41
17
41
16
38
40
40
24
32
Wild-type and mutant (acriflavin-resistant) amoebae were harvested during exponential
growth and allowed to develop on Millipore filters for 8 h. Amoebae from the trailing
edge (fractions 10-20) and from the leading edge (fractions 31-41) of the peaks produced
by partitioning (see Fig. 2 A) were isolated.
The converse experiment was also carried out, in which mutant amoebae from
peak I were mixed with wild-type amoebae from peak II.
It was found (Table 1) that the majority of spores were of wild-type character
when wild-type amoebae from peak I were mixed with mutant amoebae from
peak II. In the converse experiment, where mutant amoebae from peak I were
mixed with wild-type amoebae from peak II, it was also found that the majority
of spores were formed from amoebae in peak I. Thus it appeared that amoebae
from peak I were the precursors of the spores of the fruiting bodies. Similar
results (not shown) were obtained in a few experiments where amoebae of
mutant strain G8 which is temperature-sensitive for growth (Gingold &
Ash worth, 1974) were used in place of the mutant amoebae that were
acriflavin-resistant.
Wild-type amoebae from peak I were also allowed to develop on Millipore
filters without being mixed with amoebae from peak II. Fruiting bodies were
formed in the same time as, and having a similar appearance to, fruiting bodies
formed from the mixtures of amoebae from peaks I and II. However, when
amoebae from peak II were allowed to develop alone, fruiting body formation
was delayed by 24 h and the fruiting bodies had much longer stalks than
fruiting bodies formed from peak I amoebae alone or from mixtures of peak I
and II amoebae.
Amoebae from peak I also appeared to differ from amoebae from peak II
in cohesiveness. Thus pellets of amoebae from peak II were easily resuspended
189
Early differentiation ofD. discoideum
A (Oh)
B(2h)
0-5
C(4h)
0-5
D(6h)
0-5
10
20
30
40
Fraction number
50
60
Fig. 3. Partitioning of amoebae harvested during stationary phase of axenic growth
and allowed to develop on Millipore filters. (A) Amoebae at Oh development.
(B) Amoebae at 2 h development. (C) Amoebae at 4 h development. (D) Amoebae
at 6 h development.
in water to give single-cell suspensions, but amoebae from peak I could be
similarly resuspended only after vigorous mixing for several minutes on a Vortex
mixer.
Studies of heterogeneity at 8 h development
Amoebae were isolated from the leading and trailing edges of the broad
peak (Fig. 2 A) obtained by partitioning amoebae at 8 h development. It was
then possible, in experiments similar to those described in the previous section,
to determine the fate of these amoebae when mixed together and allowed to
form fruiting bodies. It was found that spores tended to be formed from
amoebae from the trailing edge of the peak and not from amoebae from the
leading edge (Table 2).
7
EMB 67
190
P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS
20
30
40
50
60
Fraction number
Fig. 4. Partitioning of amoebae harvested during stationary phase of axenic growth
and allowed to develop on Millipore filters. (A) Amoebae at 8 h development.
(B) Amoebae at 10 h development.
Developmental changes in partitioning of amoebae harvested
during the stationary phase of growth
The distribution of amoebae harvested during the stationary phase of growth
and allowed to develop for various times is shown in Figs. 3 and 4. Changes in
distribution, when related to time of development, were qualitively similar to
those previously described for amoebae harvested during exponential growth.
Thus a marked broadening in cell distribution by partitioning was first observed
at 8 h development for amoebae harvested in both growth phases (compare
Fig. 4 A and Fig. 2 A). However, whilst this change was associated with formation of aggregates by amoebae harvested during exponential growth, it was not
associated with the beginning of aggregation of amoebae harvested during the
stationary phase of growth, since the latter amoebae formed aggregates after only
6 h of development.
DISCUSSION
Amoebae harvested during exponential growth were distributed by partitioning as a fairly sharp peak during early development. However, the position of
the peak changed with time of development so that changes in the cell surface
properties of the amoebae were occurring. The nature of these changes is not
known, but the changes may be related to the extensive changes in glycoprotein
composition of the cell plasma membranes that occur during early development
(Toda, Ono & Ochiai, 1980).
After 8 h development the distribution of amoebae by partitioning became
extremely broad, and it would appear that the amoebae were then extremely
heterogeneous in cell surface properties. Nevertheless, it was possible to discern
Early differentiation ofD. discoideum
191
that amoebae at 9-11 h development were divided into two, albeit heterogeneous,
populations and it was found that during subsequent development amoebae
from the two populations had different fates.
When amoebae from the two populations at 10 h (designated as peak I and
peak II in Fig. 2]C) were mixed in equal numbers and allowed to form fruiting
bodies, about 69% of the spores formed were derived from amoebae from
peak I. This would have been possible for fruiting bodies comprising about
75% spores and 25% stalk cells only if the amoebae from peak I had all
differentiated into spores. Thus it is concluded that the population designated
as peak I contained amoebae that were all the precursors of spores.
The majority of amoebae from peak II did not form spores. Since stalk cells
are the only other major cell type formed during differentiation of D. discoideum,
it would seem reasonable to conclude that the preferred fate of amoebae from
peak II was stalk formation. Consistent with this conclusion was the observation
that amoebae from peak II that were allowed to develop in the absence of
amoebae from peak I formed fruiting bodies having much longer stalks than
the fruiting bodies formed from mixtures of amoebae from peaks I and II.
At 10 h development the two separable populations of amoebae are in
aggregates that appear merely to be rounded masses of cells. Differentiation
can therefore occur to give the two cell populations with ultimately different
developmental fates before aggregates gain apical tips or anterior-posterior
polarity. Thus anterior-posterior polarity is not required to initiate cell differentiation during development of D. discoideum and hence it would appear that
neither can 'positional information' be required. It would now seem more
probable that polarity is a consequence of differentiation and, as proposed by
Forman & Garrod (1977), is established as a result of sorting-out of the two
different cell populations. It was also found that precursors of spore cells are
more cohesive than precursors of stalk cells, and Steinberg (1964) has discussed,
with reference to vertebrate cells, how differences in cohesiveness may permit
cell sorting-out.
Although amoebae that had been harvested during exponential growth were
not clearly separable into two populations on the basis of cell surface properties
until 9-10 h development, it was apparent that differential change in amoebal
cell surface properties had begun at 8 h development when amoebae began to
aggregate. Furthermore, amoebae from the trailing edge of the distribution
produced by partitioning amoebae at 8 h development differentiated into spores
whilst amoebae from the leading edge tended not to differentiate into spores
and, instead, presumably differentiated into stalk cells. Clearly differentiation
in D. discoideum begins prior to formation of aggregates, and this proves
unequivocally that neither aggregate polarity nor 'positional information' is
essential to initiate differentiation of amoebae in standard, laboratory conditions
of development that allow formation of fruiting bodies. However, it is probable
that the correlation between initiation of differential change in cell surface
7-2
192
P. T. SHARPE, T. E. TREFFRY AND D. J. WATTS
properties and initiation of aggregation was fortuitous. Certainly aggregation
is not in itself the stimulus initiating this differentiation, since amoebae harvested during the stationary phase of growth formed aggregates at 6 h development, but differential change in cell surface properties was not detected until
two hours later.
We thank Irene Donnelly and Katrina Longmore for technical assistance. We are grateful
to the Royal Society (T.E.T.) for a grant to build the counter-current distribution apparatus,
to the Wellcome Trust (D. J. W.) for a grant to purchase dextran and to the SRC for a research
studentship (P.T.S.).
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(Received 7 August 1981, revised 7 October 1981)