/ . Embryol. exp. Morph. Vol. 40, pp. 229-243, 1977
Printed in Great Britain © Company of Biologists Limited 1977
229
Pattern formation in Dictyostelium discoideum
II. Differentiation and pattern formation in non-polar aggregates
By D. FORMAN 1 AND D. R. GARROD 2
From the Department of Biology, University of Southampton
SUMMARY
Cells of the cellular slime mould D. discoideum were allowed to form into spherical aggregates, by shaking vegetative cells as a suspension in phosphate buffer. In such conditions,
grex polarity is never established and surface sheath is not formed (Loomis, 1975 a). Despite
the absence of such characteristics of normal development, differentiation of prespore cells,
as tested for by immunofluorescent staining, and the organization of such cells into a patterned structure still occurred within the aggregates.
Differentiation of prespore cells was found to occur within the cultures at times equivalent
to those in the normal life cycle; such differentiation could be advanced by pulsation of the
cultures with cyclic-AMP. When cell contact and aggregate formation was prevented,
differentiation never occurred within the single cells. Our results suggest that the prespore
cells develop randomly within the aggregate and that a pattern is subsequently formed as a
result of sorting out of cell types within the cell mass. Aggregates shaken for extended periods
of time showed development into cyst-like structures.
The process of pattern formation that occurred within these aggregates which possess
neither polarity nor a grex tip, would be unlikely to involve any mechanism of positional
information signalling. The relevance of polar organization in the generation of pattern in
the normal life cycle may therefore be questionable. We present a model of pattern formation
in the slime mould in which sorting out of predetermined cell types is viewed as the major
mechanism in bringing about patterned organization of the grex precursor cells.
INTRODUCTION
In the preceding paper (Forman & Garrod, 1977) we reported evidence that
there is a direct correspondence between the prestalk: prespore cell proportions
in the slime mould grex and the stalk:spore ratio of the fruiting body. Thus
pattern formation in the slime mould is largely a problem of the emergence of
the anterior-posterior prestalk-prespore pattern amongst the grex cells.
One possible explanation as to how this pattern arises is that there is a
pattern specifying mechanism within the grex. Such a mechanism would
involve the anterior-posterior polarity of the grex being responsible for
the establishment of some type of morphogenetic gradient within the grex.
1
Author's address: Department of Bacteriology and Immunology, University of Glasgow,
Western Infirmary, Glasgow, Gil 6NT, U.K.
2
Author's address: (for reprints) Department of Biology, University of Southampton,
Medical and Biological Sciences Building, Southampton SO9 3TU, U.K.
230
D. FORMAN AND D. R. GARROD
According to their position within the grex and their relative place in the
gradient, cells would be directed along different pathways or differentiation
towards either prespore or prestalk cells. Several suggestions have been advanced
along these lines (Bonner, 1957; Ashworth, 1971; Loomis, 1972, 19756;
McMahon, 1973; Pan, Bonner, Wedner & Parker, 1974; Farnsworth & Locmis,
1974, 1975; Robertson & Cohen, 1972; Rubin & Robertson, 1975; Durston,
1976).The idea of positional information as a mechanism for pattern formation
has been outlined for other organisms by Wolpert (1969, 1971). In principle
it could provide an explanation for the size invariance and regulatory properties
of the pattern in the slime mould.
Morphological polarity is an essential requirement for a model based on
positional information, so that reference points for the gradient can be defined.
In the multicellular phase of the life cycle of D. discoideum the first evidence of
any polarity is at about 3-4 h after aggregation when the cell mass forms a tip.
This tip will eventually become the anterior end of the grex. Recent work has
suggested that differentiation of prespore cells is already in progress by the
time that this tip becomes established (Hayashi & Takeuchi, 1976; Forman &
Garrod, 1977). If this is the case then differentiation cannot be a process which
sequentially follows tip formation and the establishment of polarity.
It is therefore important to investigate the exact nature of the interrelationships between anterior-posterior polarity, differentiation and pattern formation.
The extent to which polar organization is an essential feature of patterning
processes is critical to the analysis of models concerning slime mould development. We have investigated this problem, using a system which allows the
formation of non-polar multicellular aggregates of D. discoideum cells. A brief
report of this work has been published previously (Garrod & Forman, 1977).
MATERIALS AND METHODS
Growth of cells
In all experiments D. discoideum strain Ax-2 cells were used, grown in axenic
conditions (Watts & Ashworth, 1970) with 86 HIM glucose. Cells were grown
on a rotary shaker (140 rev./min; radius of rotation 2-75 cm) at 22 °C and
were harvested while in the exponential growth phase at a density of 2-4 x 106
cells per ml.
Formation of aggregates in suspension
Cells were harvested from growth medium by centrifugation at 350 g for
5 min at 4 °C. They were then washed twice in cold distilled water and resuspended in sterile 0-0167 M phosphate buffer (pH 6-0) at a density of 106 cells
per ml. (In some experiments the density was increased to 107 cells per ml
with no noticeable effect.) Four or 40 ml samples were then shaken in 25 or
250 ml respectively Erlenmeyer flasks on a rotary shaker (140 rev./min; radius
Pattern formation in D. discoideum
231
of rotation J in.) at 22 °C. The flasks were siliconized prior to use and sealed
while shaking. In such conditions visible aggregates formed within an hour.
Maintenance of single cell suspension
When cells were shaken in the above conditions, but at the increased speed
of 260 rev./min, they usually did not form aggregates but remained almost
entirely single. Occasionally, this technique failed to work and small aggregates
were formed, in which case the cell suspension was discarded.
Dissociation of aggregates into single cell preparations
Aggregates were spun out of phosphate buffer at 700 g for 5 min and resuspended in 1 ml of cold distilled water. They were then triturated with a
drawn out Pasteur pipette until a single cell suspension was produced. Such
trituration worked reliably up until 24 h of shaking, after which it became difficult to dissociate the aggregates even with chemical methods used on migrating
grexes (Takeuchi & Yabuno, 1970). This is probably due to the formation of a
non-cellular coat around the aggregates.
Cells were allowed to settle on coverslips and fixed for immunofluorescent
staining as in the previous paper (Forman & Garrod, 1977). Single cell suspensions and cells dissociated from aggregates were assayed for chemotactic
aggregation competence by allowing them to settle onto coverslips for 30 min
from a suspension at 5 x 105/ml. The cells on the coverslips were then examined
for the characteristic chaining patterns formed by end-to-end cell contacts.
Cyclic-AMP pulsation of aggregates in suspension
This was carried out using the technique previously reported (Darmon,
Brachet & Perera da Silva, 1975). After cells, at a density of 107 per ml, had
been shaking for 2 h, they were given a pulse of cyclic-AMP every 5 min. The
cyclic-AMP was delivered in 20 /i\ drops using a peristaltic pump. The cyclicAMP concentration in the flask after the first pulse was 10~7 M.
Testing viability of aggregates
The developmental capacity of aggregates, after shaking for varying lengths
of time, was tested for by allowing them to settle out from the buffer and
plating out either on non-nutrient agar (2 % w/v in distilled water) or on standard agar medium (Sussman, 1966) with E. coli B/r. The plates were incubated
at 22 °C and observed daily to examine the developmental progress of the
aggregates.
Histological preparations
Aggregates were allowed to settle out of phosphate buffer and then fixed in
95 % ethanol at 4 °C. They were then processed for the cold wax embedding
method of Sainte-Marie (1962). The aggregates could be easily transferred by
232
D. FORMAN AND D. R. G A R R O D
Fig. 1. Multicellular aggregates, formed by shaking vegetative cells in phosphate
buffer for 24 hours (x 300).
allowing them to settle out, or lightly centrifuging, decanting off one solution
and adding the next. The aggregates were embedded in plastic e.m. embedding
capsules, the wax being kept molten for 5-10 min in order to allow the aggregates to sink to the tip. The wax was then solidified at room temperature and
the blocks stored at 4 °C. Prior to sectioning, the plastic capsule was stripped
off from the wax block. Sections were cut at 5 /«n.
Staining with fluorescent antispore serum
Single cell and sections were stained and microscopically examined as previously described (Forman & Garrod, 1977). For staining sections it was found
preferable to use the following timings:
Reaction with D. mucoroides antispore serum absorbed with D. discoideum
vegetative cells 30 min; P.B.S. wash 30 min; reaction with fluoresceinconjugated anti-rabbit 15 min; immunoglobulin P.B.S. wash 60 min.
Control staining was carried out as previously described and found to be
negative.
RESULTS
Differentiation in suspension aggregates
When D. discoideum (strain Ax-2) cells were shaken in phosphate buffer at
140 rev./min, they rapidly formed multicellular aggregates of variable size,
20-200 jLim in diameter. These spherical aggregates (Fig. 1) showed no overt.axis
of polarity nor any sign of tip development. Differentiation of prespore cells
in these aggregates was tested for by staining single cell preparations made
Pattern formation in D. discoideum
233
'**'"'
. • • !
(a)
Fig. 2. Cells dissociated from aggregates, formed after shaking vegetative cells in
phosphate buffer for 18 h, stained with fluorescent labelled antispore serum. Note
that only a proportion of the cells shown in (a) possess the specific prespore vesicles
seen in (b). x 760.
after dissociating the aggregates at various time intervals, with fluorescent
antispore serum. There was no sign of prespore cells until 12-13 h of shaking,
after which stained vesicles became apparent in the cytoplasm of a proportion
of the aggregate cells. Initially the vesicles were stained very weakly but by
17-18 h of shaking, the staining was quite intense (Fig. 2), with vesicles found
234
D. FORMAN AND D. R. GARROD
Fig. 3. Single cells, shaken at 260 r.p.m., for 18 h (a), exhibiting no specific staining
with antispore serum (b). x 760.
in approximately 60 % of the cells. The vesicles appeared identical to those
found in prespore cells in the normal life cycle.
Such cells also passed through a phase of aggregation competence after 8 h
of shaking. On glass coverslips the cells exhibited characteristic end-to-end
cell contacts and the formation of 'chains' of cells (Gerish, 1968). (Such competence is a chemotactic parameter and should not be confused with the fact
Pattern formation in D. discoideum
235
Fig. 4. Cells, dissociated from aggregates, formed after shaking vegetative cells in
phosphate buffer for 13 h with (a) and without (b) cyclic-AMP pulsation. Note the
clearer definition and brighter intensity of prespore vesicles in cells from pulsed
aggregates on staining with antispore serum, x 480.
that in shaking suspension cells form aggregates within an hour by mutual
adhesion.)
When cells were shaken in suspension at 260 rev./min, aggregates did not
form; the cells remained single with the occasional formation of doubles
or triplets (Fig. 3). Such cells never showed any sign of specific staining
with anti-spore serum even when shaken for up to 36 h. These cells did,
236
D. FORMAN AND D. R. GARROD
Fig. 5. Section through aggregates, shaken in buffer for 18 h, and stained with
antispore serum. Note apparently random distribution of the PSV containing cells.
x300.
however exhibit aggregation competence at 8 h of shaking and also developed into normal fruiting bodies when transferred from suspension to an agar
surface.
Differentiation in suspension aggregates with c' AMP pulse
Cells of D. discoideum shaken in suspension and pulsed at 5 min intervals
with 10~7 M c' AMP showed a 3 h advancement in aggregation competence
(Darmon et al. 1975). The development of prespore cells was advanced by
some 2 h compared with unpulsed controls. By 13 h of shaking the intensity
of PSV staining in the pulsed cells was much greater than in the controls (Fig. 4)
and about twice the number of cells possessed vesicles as compared with the
controls. By 18 h of shaking both pulsed and unpulsed cells were similar in
both intensity of vesicle staining and proportions of prespore cells.
Pattern formation in suspension aggregates
Sections through 18 h aggregates, on staining with antispore serum, did not
show spatial localization of the differentiated prespore cells (Fig. 5). On the
contrary, the fluorescently stained cells appeared to be scattered randomly
throughout the cell mass. The analysis of many such sections revealed no areas
exhibiting concentrations of either the stained or the unstained cell types. By
24 h of shaking there were several changes within the aggregates. There were
more prespore cells present (72% as compared to 60% at 18 h) and the intensity of fluorescence was considerably brighter. The major change, however,
Pattern formation in D. discoideum
237
Fig. 6. Section through aggregate, shaken in buffer for 24 h, and stained with antispore serum. Note separation within the aggregate of the PSV containing cells
from the others, x 160.
was that at 24 h there was a patterned spatial arrangement of the two cell
types (Fig. 6). The unstained cells were invariably confined to a small region at
the periphery of the aggregate. All the aggregate sections examined were
consistent with a structure of a mass of stained prespore cells partially surrounded by a 'cap' of unstained cells.
Both 18 and 24 h aggregates developed into normal fruiting bodies within
6-8 h of placing on a non-nutrient agar surface. When aggregates were maintained in suspension for 4 days each gave rise to an unusual cyst-like structure
(Garrod & Forman, 1977). An account of the structure and development of
these cysts will be presented in a future paper.
DISCUSSION
By studying the differentiation of prespore cells in spherical aggregates of
D. discoideum cells, we have shown the following:
(1) The appearance of PSVs in prespore cells follows at a set time (4-5 h)
after the chemotactic aggregation phase provided that cells are in contact.
(2) At the time when PSVs first appear there is no obvious patterned arrangement of prespore cells within aggregates. Instead their distribution appears to
be spatially random.
(3) Some time after the initial appearance of PSVs, a patterned spatial
distribution of prespore cells and unstained (prestalk) cells arises within aggregates. This pattern is similar to the prespore-prestalk pattern found in the
normal migrating grex.
l6
EMB 40
238
D. FORMAN AND D. R. GARROD
Being spherical these aggregates exhibit none of the external morphological
features usually taken to indicate the polarity of the grex: they possess neither
an axis of elongation, nor a distinct posterior end nor an anterior tip. Thus
there is an absence of the major features which many previous suggestions
have implicated in formation of the spore-stalk pattern. It seems unlikely
therefore that the tip and morphological polarity of the normal grex are
essentially requirements for pattern formation. We cannot exclude the possibility that cells in spherical aggregates are in some way polarized prior to cell
differentiation and pattern formation, but at present we see no reason to
suppose that they are. Of course once pattern formation has taken place, the
aggregates possess overall polarity because the pattern itself is polarized. We
suggest that, prior to pattern formation, neither the individual cells nor the
aggregate as a whole are polarized, and that polarity is not instrumental in
forming the pattern.
An essential prerequisite for the onset of differentiation in these aggregates
is that the cells should be in mutual contact for some time following depletion
of the food supply. Cyclic-AMP pulsation of the suspension cultures advances
both the onset of chemotactic aggregation competence and prespore differentiation. This indicates that it is probably a combination of contact and changes
in cell surface properties accompanying or following aggregation competence
that is responsible for initiating the formation of PSV in prespore cells.
Our results are consistent with the view that the transition from the early
apparently random arrangement of prespore cells to the spatially organized
pattern seen in aggregates of 24 h occurs by sorting out of the two cell types
such that homogeneous groupings of like cells arise through cell translocation.
Although not providing direct proof of sorting out, the results seem inconsistent with the alternative possibility that differentiation of the cell types occurs
in position. This is because we have never observed in 18 h aggregates a region
completely devoid of prespore cells comparable in size to the non-staining
region of 24 h aggregates.
Two crucial questions need to be answered in order to confirm the role of
sorting out in this developmental system. Firstly, do the prespore cells really
differentiate at random within the cell mass, and secondly, is the transition to
an organized pattern due to cell translocation within the aggregate? A more
detailed study of the events taking place between 18 and 24 h of shaking is
being undertaken in order to answer these problems, for although all our evidence suggests an initial randomness followed by sorting, the use of fluorescent
antispore serum to stain prespore cells has limitations in the early stages of
differentiation when staining is weak and diffuse.
A model for pattern formation in D. discoideum
Our confirmation that PSVs are a reliable marker of prespore cells (Forman
& Garrod, 1977) and our finding that pattern formation can occur in the
Pattern formation in D. discoideum
239
absence of both anterior-posterior polarity and the grex tip have led us to
formulate a model to explain pattern formation during normal development of D. discoideum. The model incorporates some features which have
been suggested previously and, while being speculative in many ways, takes
account of the known evidence. We present the model as a basis for further
experimentation, as a crystallization of views and as a stimulus to discussion.
The model is as follows:
(1) Differentiation of prespore and prestalk cells is initiated as a result of
the establishment of cell to cell contacts during chemotactic aggregation.
(2) The two cell types differentiate at random within the early grex and then
sort out to give the prespore-prestalk pattern.
(3) Differentiation, sorting out and pattern formation begin before the
formation of a grex tip. Thus the grex tip does not play an organizing role in
any of these processes.
(4) The early prestalk region itself gives rise to the grex tip. It is the formation
of the pattern which gives rise to polarity of the grex rather than polarity which
gives rise to the pattern.
(5) Control of the ratio of prestalk to prespore cells and thus the size invariance of the fruiting body pattern is independent of the mechanism which
determines the spatial arrangement of cell types.
We now briefly discuss the main points of this model. The suggestion that
cell contact is an essential 'trigger' for differentiation is not new. Cell contact
has been shown to mediate the synthesis of several enzymes in D. discoideum
(Newell, Longlands & Sussman, 1971; Newell, Franke & Sussman, 1972), and
results in membrane changes (Aldrich & Gregg, 1973; Yu & Gregg, 1975).
PSV formation is also dependent on cell association (Gregg, 1971; Gregg &
Badman, 1970; Sakai & Takeuchi, 1971) and a scheme of the possible mechanisms involved has been presented by Brackenbury & Sussman (1975). It
should be stressed that our model uses PSV formation as the criterion for
prespore differentiation. This does not preclude the idea that prespore or
prestalk tendencies may be present in the cell population before aggregation
as is suggested by the experiments of Takeuchi (1963, 1969), Bonner, Seija &
Hall (1971), Leach, Ashworth & Garrod (1973) and Maeda & Maeda (1974).
However, any such differentiation prior to aggregation cannot progress without
the additional stimulus provided by cell contact.
The notion of random differentiation followed by a sorting out process is
implicit in the work cited above on pre-aggregation differentiation and it is also
consistent with the results of Bonner (1957, 1959), Francis & O'Day (1971),
and Miiller & Hohl (1973). The mechanism for such a process is still very much
an open question. However, differential speed of movement (Bonner, 1959;
Garrod, 1974) would seem an unlikely candidate because the pattern can be
formed in spherical aggregates in the absence of polarized movement of the
cell mass. Indeed Leach et al. (1973) have shown that sorting out can occur in
16-2
240
D. FORMAN AND D. R. GARROD
the absence of the grex migration phase. It seems more likely that differential
adhesiveness of the two cell types could be responsible for sorting out, as has
been suggested for vertebrate embryonic cells by Steinberg (1964). The arrangement of the two cell types within the suspension aggregates prespore cells being
partially surrounded by unstained prestalk cells, is consistent with such a
mechanism and would imply that the prestalk cells are less cohesive than the
prespore cells.
In addition to our present results, there is now abundant evidence that the
grex tip and the overall morphological polarity of the grex are not required for
the differentiation of spore and stalk cells. For example, differentiation in the
absence of normal morphogenesis can be induced by plating vegetative cells
on agar in the presence of cyclic-AMP (Bonner, 1970; Town, Gross & Kay,
1976). Differentiation within tipless cell masses occurs in the mutants FR-17
(Sonneborn, White & Sussman, 1963) and P4 (Chia, 1975) and in the wild type
if cell masses are treated with EDTA (Gerisch, 1968) or if an impermeable
barrier is inserted to the correct depth into the early culminating grex (Farnsworth, 1974).
During normal development, it seems that differentiation of prestalk and
prespore cells begins within the early aggregate before a tip develops. We
suggest therefore that the tip plays no role in guiding or 'organizing' the course
of differentiation. We further suggest that sorting out begins at the time of
differentiation, generating the prestalk-prespore pattern. Thus it seems possible
that a prestalk region may be generated within the aggregate either just before
or simultaneously with the formation of the grex tip. We suggest that this early
prestalk region becomes the tip of the grex. This is contrary to the view that
the tip forms first and then the prestalk region appears at the tip.
An important question is that if the tip has no role in differentiation and
pattern formation, then what is its function ? We contend that the role of the
tip is solely to organize morphogenetic movement of the cell mass. This is
suggested by many experiments both early and recent (Raper, 1940; Rubin &
Robertson, 1975; Durston, 1976) and a mechanism by which it does so has
been suggested (Garrod, 1969) and supported by some evidence (Loomis, 1972;
Farnsworth & Loomis, 1975). The grex tip therefore undoubtedly plays an
important role in the construction of the normal fruiting body, but there is
no evidence whatsoever that it functions in relation to determination of differentiation, proportionality or formation of the prestalk-prespore pattern.
So far we have said nothing about the mechanism which controls the spore:
stalk ratio. Indeed, very little is known about this mechanism, except that both
the proportions of fruiting bodies and the proportions of prespore and prestalk
cells during the grex stage can be altered by temperature, growth conditions
and certain mutations (see Forman & Garrod, 1977). It is not sufficient to
propose that proportions are determined at the preaggregation stage and that
the correct numbers of each type of cell come together at the aggregation stage
Pattern formation in D. discoideum
241
(Garrod, 1974), because regulation of proportions can take place if the grex is
cut (Raper, 1940; Sampson, 1977), and because proportions can be altered if
the temperature is altered during the early culmination stage (Farnsworth,
1975). We feel that the most likely situation is the third possibility suggested
by Garrod (1974), namely that the proportions and arrangement of different
cell types are roughly laid down initially by sorting out of cells with different
predispositions. Some internal mechanisms then adjust the proportions of the
two cell types and are also involved in regulation if the grex is cut. We would
add, however, that it is possible that regulation of proportions begins when the
cells first differentiate and when they appear to be randomly arranged. This
would mean that the mechanism of regulation of proportions is not critically
dependent on a patterned and polarized arrangement of the two cell types.
We thank Lynette Banham, Alastair Nicol and Sherilee Taylor for technical assistance.
This work was supported by the Science Research Council. D. Forman was in receipt of a
S.R.C. Studentship.
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