/. Embryol. exp. Morph. 86, 19-37 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
Influence of cyclic AMP and hydrolysis products on
cell type regulation in Dictyostelium discoideum
CORNELIS J. WEIJER* AND ANTONY J. DURSTON
Hubrecht Laboratorium, Uppsalalaan 8, 3584CT Utrecht, The Netherlands
SUMMARY
We describe the effect of cyclic AMP on regulation of the proportion of prespore and prestalk
cells in Dictyostelium discoideum. Prespore and prestalk cells from slugs were enriched on Percoll
density gradients and allowed to regulate in suspension culture under 100 % oxygen. The transition of prespore to prestalk cells is blocked by cAMP, while cAMP phosphodiesterase and
caffeine cause a decrease in the number of prespore cells. This suggests that extracellular cAMP
plays a role in cell type proportioning by inhibiting the conversion of prespore to prestalk cells.
Low concentrations of cAMP prevent the conversion of prestalk to prespore cells; the same
effect is seen with hydrolysis products of cAMP, 5 AMP, adenosine and also adenine. We suggest
that, when low concentrations of cAMP are added to regulating cells, the cAMP itself is quickly
broken down and the breakdown products thereafter inhibit the prestalk-to-prespore conversion.
The relevance of these findings is discussed in the context of an non-positional double-negative
feedback model for cell type homeostasis.
INTRODUCTION
The life cycle of the cellular slime mould Dictyostelium discoideum (Dd) involves
a simple differentiation process in which cells of one type, vegetative amoebae,
differentiate into at least two cell types, stalk cells and spores. The ratio of the two
cell types is almost constant over a large size range, under specified environmental
conditions (Bonner & Slifkin, 1949; Stenhouse & Williams, 1977).
Prestalk and prespore cells are first recognizable at the late aggregate stage and
are organized in a one-dimensional pattern in the slug stage. At this stage the
prestalk cells are localized in the front quarter of the slug and the prespore cells in
the back three quarters. Under normal circumstances prestalk and prespore cells
convert quantitatively into stalk and spores respectively (Tsang & Bradbury, 1981).
The prespore-prestalk pattern at the slug stage is still regulative, i.e., when a slug
is cut into two pieces, separating the prestalk and prespore zones, both pieces, have
the ability to regulate and reform the missing parts and finally give rise to normally
proportioned fruiting bodies (Raper, 1940; Sakai 1973). This indicates the existence of a well-developed cell type homeostatic mechanism. There is good evidence
* Present address Zoologisches Institut, Universitat Miinchen, Luisenstrasse 14, 8000 Miinchen
2, West Germany
Key words: Dictyostelium discoideum, cyclic AMP, regulation, cell type regulation, hydrolysis
products, feedback model.
20
C. J. WEIJER AND A. J. DURSTON
that the pattern-forming mechanism in Dd consists of a largely positionindependent cell type homeostasis mechanism and another mechanism, chemotactic cell sorting, that puts the cells into the right place (Takeuchi, 1969; Forman &
Garrod, 1977; Durston & Vork, 1979; Tasaka & Takeuchi, 1981; Sternfeld &
David, 1981; Weijer, McDonald & Durston, 1984a; Weijer, Duschl & David,
19846).
One possible way to investigate the cell type homeostasis mechanism is to study
the cell type transitions under conditions of regulation, a method first used by Sakai
to quantitatively study the regulation of prespore and prestalk pieces of slugs
(Sakai, 1973). The normal proportion of prespore and prestalk cells is perturbed
and the kinetics with which both cell types return to their equilibrium proportion
is followed.
We have modified this procedure by taking gradient-purified cell types (Weijer
et al. 1984fl) instead of prestalk and prespore pieces cut out of slugs. We let the cell
types regulate in oxygenated suspensions (Sternfeld & Bonner, 1977), instead of on
water agar, which allows better control over the extracellular environment. Under
these conditions both the purified prestalk and prespore cells rapidly reaggregate,
i.e. they form submerged aggregates, and regulate their proportions. At various
times aliquots of the aggregates were collected, dissociated and the percentage of
prespore cells and prestalk cells determined.
This procedure allowed us to follow the regulation of the prestalk and prespore
cells with and without the addition of putative morphogens. In this study we have
concentrated on the role of cyclic AMP and metabolites in cell type regulation.
MATERIALS AND METHODS
Strains and developmental conditions
All experiments were performed with Dictyostelium discoideum strain Ax2, which was grown
in axenic medium as described earlier (Watts & Ash worth, 1970). Cells were harvested when they
had reached a density between 2-7 x 106 cells. ml"1 and collected by centrifugation (2 min 400g).
Cells were washed three times in 20mM-potassium phosphate buffer pH6-8 (KK2) and resuspended in the same buffer to a density of 2 x 107 cells. ml"1. Plates of synchronous slugs were
prepared by pouring 5 ml of the cell suspension onto levelled agar plates (1 % Difco bacto agar
in KK2) 9 cm in diameter. The cells were allowed to settle for 15 min after which the excess liquid
was carefully decanted and the plates air dried for 10 min. This resulted in an evenly distributed
multilayer of cells which gaveriseto synchronous standing slugs after 16 h of development at 22 °C
in the dark.
Gradient separation of prespore and prestalk cells
The gradient-separation procedure used is described in detail elsewhere (Weijer et al. 1984a).
The relevant points from this procedure are described below. After 16-18 h slugs were scraped
off the plates with an microscope slide and resuspended in KK2 buffer. The slugs were separated
from the unaggregated cells by filtration through afinenylon sieve (mesh size 40 ym). The slugs
were dissociated, either in 0-6% NaCl containing 2mM-EDTA or in a pronase/BAL mixture
according to published procedures (Takeuchi & Yabuno, 1970). After this the cells were washed
twice in KK2 buffer and once in a salt solution containing 0-6 % NaCl, 2mM-EDTA and 20 mMKK2. The density of the cells was adjusted to 5 x 107 ml and 2 ml were layered on top of a 10 ml
Cyclic AMP and cell type regulation in Dictyostelium
21
Percoll gradient (linear 30-90 %, preformed) containing the same salt mixture as the solution
used for the last cell wash. The gradients were centrifuged for lOmin at 2000g in a Hettich K2S
table-top centrifuge at 10 °C. The gradients were fractionated and the fractions containing the
majority of the prespore (bottom 1 ml) and the prestalk cells (2nd to the 6th ml from the top) were
collected by hand with a Pasteur pipette. Fractions from three such gradients were pooled,
washed three times in KK2 buffer and resuspended to a density of 5 x 106 cells ml"1.
The purity of the fractions was determined by staining cells with prespore antiserum. The
prestalk (lightest) fraction normally contained 10 % of total cells put on the gradient and consisted of 10-25 % prespore cells and 75-85 % neutral-red-staining cells. This fraction also showed
the prestalk-specific acid phosphatase isoenzyme. Based on these criteria this fraction was enriched about fourfold for prestalk cells. The dense fraction normally contained about 50 % of
total cells and consisted of 90-95 % prespore cells, 2-7 % neutral-red-staining cells and was
negative for the prestalk-specific acid phosphatase. This fraction therefore consisted almost
entirely of prespore cells.
Roller tube cultures
3 ml of cell suspension were added to a 15 ml screwcap test tube with or without added test
substances and the tubes were flushed out with ten tube volumes of pure oxygen and sealed. The
test tubes were put on a test tube rotator where the tubes were rotated around their own long axis
at20r.p.m. at22°C.
Aggregate dissociation and prespore cell staining
The cells in the roller tubes aggregated rapidly and the aggregates developed to tight aggregates
within 4-6 h. At this stage they were very difficult to dissociate with protease/BAL, therefore we
tried a number of different enzymes and found Cellulase Onozuka R10 (10 mg. ml"1) in KK2
pH 6-0 containing 2 mM-EDTA and 0-6 % NaCl to be very effective. Incubation of the aggregates
for 10 min at room temperature and then pipetting the suspension two to three times through an
Eppendorf pipette resulted essentially in a single-cell suspension. Cells were pelleted by
centrifugation for 8 sec in an Eppendorf microcentrifuge and the pellet was resuspended in
50 jid KK2 and fixed in 60 % methanol. 5-10 jwl of the fixed cell suspension was put on a microscope
slide and air dried. The cells were postfixed with 25 fA 100 % methanol and air dried again. Then
the cells were stained for 30 min with lO/il of a rabbit anti-spore antiserum, which had been
adsorbed with aggregation competent cells (Takeuchi, 1963). The final dilution of the antiserum
was 500-fold in KK2 buffer pH6-8 and the staining was done in a moist chamber. After this the
slides were rinsed by immersing in KK2 buffer and then stained for another 30 min with a 100-fold
dilution of a FITC-labelled goat anti-rabbit IgG. (Nordic Immunological Laboratories, Tilburg,
the Netherlands). After this the cells were rinsed again in KK2 and mounted in a 50 % dilution
of glycerin in 100 mM-Tris/HCl pH 8-0. Prespore cells are defined as those cells that show staining
of more than two prespore vacuoles per cell. There did not seem to be an appreciable loss of cells
during the staining procedure. At least 400 cells/time point were counted, resulting in counting
errors less than 4 %.
Neutral red staining
Dissociated cells were stained for 5 min in 0-02 % neutral red in KK2. The fraction of neutralred-stained cells was determined by counting strongly stained cells with large vacuoles under
Nomarski optics. In all cases at least 400 cells/sample were counted.
Acid phosphatase gel electrophoresis
Acid phosphatase isoenzymes were separated on non-denaturing polyacrylamide gradient gels
modified according to existing procedures (Oohata, 1983 and Loomis & Kuspa, 1984). Linear
gradient gels were made with a gradient mixer from 3 % acrylamide (3 % acrylamide, 0-08 %
bis acrylamide, 0-06% ammonium persulphate and 0-03% N,N,N,N:-tetramethyl
ethylenediamine) solution in 100 mM-imidizole/HCl pH6-8 and 6% acryl/bis acrylamide in
100mM-imidizole/HCl pH7-8. The running buffer consisted of 4g.l" 1 imidizole and 4g.l" 1
22
C. J. WEIJER AND A. J. DURSTON
sodium acetate pH7-0. The samples were lysed in 0-2 % Triton X100 in imidizole/HCl pH6-8
for 30 min at 4 °C. The samples were made up to 15 % sucrose and 20 jtd were layered onto the
gels. The gels were run for about 4 h at 4°C at a constant voltage of 100 V after which time the
tracking dye bromphenolblue had reached the bottom. To visualize the isoenzymes the gels were
incubated with 0-5 mg. ml"1 a-naphtyl acid phosphate sodium salt and 0-5 mg. ml"1 Fast Garnet
GBC in lOOmM-citrate buffer pH4-8. The gels were stained for 60min at 25 °C.
Protein determination
Protein was determined using the Coomassie Blue method according to Bradford (1976), with
bovine serum albumin as a standard.
RESULTS
The regulation ofprespore andprestalk cells in roller tubes
Under our culture conditions cell type regulation occurred from prestalk to
prespore cells and from prespore to prestalk cells. At equilibrium about 50 % of the
cells were prespore cells, in contrast to about 80 % prespore cells in slugs. This ratio
shift is a property of our in vitro culture system, and is in agreement with the
observation of an enlarged neutral-red-stained area in submerged aggregates
(Sternfeld& David, 1981).
The regulation of prespore cells isolated on density gradients gives the following
picture. Over the first 2-6 h a significant proportion of the prespore cells lose their
prespore vacuoles and a new equilibrium concentration of about 50 % prespore
cells is established. This condition is then stable (Fig. 1A). In order to investigate
Time (h)
Fig. 1. Regulation of prespore and prestalk cell populations in suspension culture under
100 % oxygen.
Prespore and prestalk cells were purified on Percoll density gradients, resuspended
in Bonner salts at a density of 2-5 x K^ml" 1 and allowed to regulate under 100%
oxygen. At successive time points the percentage of prespore and neutral-red-stained
cells was determined. Closed symbols, % ofprespore cells; open symbols, % of neutralred-stained cells. (A) Regulation of prespore population; (B) regulation of prestalk
population.
Cyclic AMP and cell type regulation in Dictyostelium
23
the differentiation state of the newly formed non-prespore cells we determined the
fraction of cells containing large neutral-red-staining vacuoles, a marker for prestalk and anterior-like cells at the slug stage (Bonner, 1959; Durston & Vork, 1979;
Sternfeld & David, 1981; Tasaka & Takeuchi, 1981). Figure 1A indicates that
during the regulation of prespore cells neutral-red-stained cells are formed at a rate
comparable with the disappearance of prespore cells.
During regulation of prestalk cells isolated from density gradients prespore cells
are newly formed. At the same time the fraction of neutral-red-staining cells
diminishes (Fig. IB).
In both regulation experiments (Fig. 1A,B) the change in neutral-red-stained
cells roughly equals the change in prespore cells since the sum of the percentages
of prespore cells and neutral-red-stained cells is approximately 100 % during regulation. Thus the changes in cell type proportions must be due to conversion of one
cell type into the other. As is the case for slug cells, the neutral-red and presporestaining properties of cells appear to be mutually exclusive states (Yamamoto &
Takeuchi, 1983).
In order to further characterize the neutral-red-stained cells we investigated
another prestalk-specific marker, the prestalk-specific acid phosphatase isoenzyme
(Oohata, 1983; Loomis & Kuspa, 1984). In contrast to previous observations we
found that under our conditions there are at least three distinct acid phosphatase
1
2
Fig. 2. Acid phosphatase isoenzymes of prestalk and prespore cells.
Prespore and prestalk cells were isolated on density gradients. Lane 1, prestalk cell
fraction (containing 10 % prespore cells), 60 fig protein per lane; Lane 2, prespore cell
fraction (95 % prespore cells).
24
C. J. WEIJER AND A. J. DURSTON
0
8
20
0
8
20
Fig. 3. Acid phosphatase isoenzymes in regulating populations of prespore and prestalk
cells.
Prespore and prestalk cells were allowed to regulate in suspension culture under
standard conditions. Samples were taken at 0, 8 and 20h (40/xg protein per lane). (A)
Regulating prestalk cells; (B) regulating prespore cells.
isoenzymes (Fig. 2). The band with the highest mobility is present in all cells from
the vegetative stage onwards. Slug-stage cells normally contain three bands of
which the one with the lowest mobility is most strongly represented in old and
culminating slugs. Prestalk cells contain all three bands, while prespore cells contain only the two fastest bands.
Figure 3 shows the changes in acid phosphatase isoenzymes during prestalk
and prespore regulation experiments. During regulation of prestalk cells all three
bands remain present at about the same intensity from the beginning of the
experiment onwards (Fig. 3A), despite the decrease in the number of neutralred-stained cells (Fig. IB), thus the isoenzymes are relatively stable under our
conditions. During regulation of prespore cells the slowly migrating prestalkspecific band is only formed after 20 h of regulation (Fig. 3B), while neutral-redpositive cells are formed during the first 4-6 h in the regulation process (Fig. 1).
Thus, under these conditions, the differentiation of prespore cells and neutralred-staining cells appears to be uncoupled from the expression of the prestalkspecific acid phosphatase isoenzyme. This uncoupling is also found during normal
development of vegetative cells in our in vitro system. Although 50 % of the cells
differentiate to prespore cells after 20 h of development, the prestalk-specific acid
phosphatase does not appear until 24-28 h of development (Fig. 4). Although
the expression of the acid phosphatase during regulation experiments confirms
the identification of neutral-red-stained cells as prestalk cells, it is an inconvenient marker due to the delay of its expression (in prespore-to-prestalk
Cyclic AMP and cell type regulation in Dictyostelium
25
regulation) and its long-term stability (in prestalk-to-prespore regulation). Hence
we have used neutral-red-staining and prespore staining to identify prestalk and
prespore cells in most experiments.
32
12
16
20
Time (h)
24
28
Fig. 4. Time course of the appearance of prespore cells and acid phosphatase
isoenzymes during development in suspension culture.
Cells were developed at 2-5 x 106 cells/ml in 20 mM potassium phosphate buffer (pH
6-8). (A) The expression of the acid phosphatase isoenzyme (60/xg protein/lane); (B)
appearance of prespore cells.
26
C. J. WEIJER AND A. J. DURSTON
The effect of cAMP on prespore regulation
We first investigated the influence of cyclic AMP (cAMP) on prespore regulation. Several cell differentiation systems have been described in which cAMP is
needed to obtain differentiation of prespore cells (Kay, Garrod & Tilly, 1978;
Okamoto, 1981; Abe, Saga, Okada & Yanagisawa, 1981; Chung etal. 1981; Kay,
1982; Mehdy, Ratner & Firtel, 1983). From earlier experiments it is known that
when slug cells are shaken in suspension under conditions where they are not able
to make cell contact, they dedifferentiate, but that dedifferentiation can be inhibited by the inclusion of cAMP in the medium (Takeuchi & Sakai, 1971;
Okamoto & Takeuchi, 1976; Tasaka et al. 1983). We found that in our system
cAMP blocks the regulation of prespore cells, irrespective of the initial prespore
cell 'concentration': 95 % prespore cells remain 95 % prespore cells under the
influence of cAMP (Fig. 5), while untreated prespore cells regulate to around 50 %
prespore cells (Figs 1,5). The effect of cAMP is concentration dependent, higher
concentrations have a stronger effect (Fig. 5). When cAMP is added at millimolar
concentrations it blocks the regulation of prespore cells almost completely for at
least 6-8 h and during this time there are no appreciable numbers of neutral-redstaining cells formed.
The effect of cAMP on the regulation of prestalk to prespore cells is more
complicated. At high concentrations of cAMP there is little effect on the conversion
Fig. 5. Regulation of prespore and prestalk cell populations in the presence of various
cAMP concentrations.
Conditions of the experiment are the same as described in Fig. 1. Closed symbols,
prespore cell population; open symbols, prestalk cell population. Control (diamonds),
10~ 3 M-CAMP (triangles), 10" 5 M-CAMP (circles).
Cyclic AMP and cell type regulation in Dictyostelium
27
of prestalk to prespore cells (Fig. 5), but at lower concentrations there is a
pronounced inhibition of prespore cell formation and a stabilization of neutral-redstaining cells. This rather surprising result is further characterized below.
The effect of cAMP hydrolysis products on cell type regulation
Slug-stage cells contain appreciable extracellular activities of phosphodiesterase
and 5'nucleotidase (Brown & Rutherford, 1980; Armant & Rutherford, 1980;
Tsang & Bradbury, 1981; Weijer et al 1984a). Therefore, when cAMP is added to
a slug cell suspension, it will be degraded at least partially to 5'AMP and adenosine
and possibly to adenine. Therefore we tested the effect of cAMP breakdown
products on the cell type transitions.
The effect of two possible breakdown products of cAMP, adenosine and
adenine, on cell type regulation is shown in Fig. 6. At concentrations up to
10~ 5 M there is no effect upon the conversion of prespore to prestalk cells. High
concentrations of adenosine however, increase the rate of disappearance of
prespore cells and lead to a shift in the equilibrium proportions, an effect also
seen with caffeine (see below). The conversion of prestalk to prespore cells is
inhibited by 10~5 M-adenosine and adenine (Fig. 6). The results in Fig. 7 confirm
the stabilization of prestalk cells by 10~5 M-adenosine, scored as neutral-redstained cells. The effect of 5'AMP is similar to that of adenosine (data not
shown).
2
4
6
Time (h)
8
Fig. 6. Regulation of prespore and prestalk cell populations in the presence of cAMP
and breakdown products.
Conditions of the experiment are the same as in Fig. 1. Closed symbols, prespore cell
population; open symbols, prestalk cell population. Control (diamonds), 10" 4 M-CAMP
(circles), 10~5M-adenosine (triangles), 10~5 M-adenine (squares).
28
C. J. WEIJER AND A. J . DURSTON
Fig. 7. The influence of 1CT5 M-adenosine on the regulation of prespore and neutralred-stained cells in suspension culture. Conditions as in Fig. 1. Closed symbols, % of
prespore cells; open symbols, % of neutral-red-stained cells. (A) Regulation of the
prespore cell population; (B) regulation of the prestalk cell population.
Protease
Cellulase
Salt/EDTA
90;
Fig. 8. Effect of various dissociation procedures on the time course of cell type regulation.
Closed symbols, prespore cell population; open symbols, prestalk cell population.
(A) Slugs were dissociated with pronase/BAl. (B) Slugs were dissociated with cellulase
(1 mg.ml"1) in KK2. (C) Slugs were dissociated by tituration in an EDTA/salt solution
as described in Methods. Control, diamonds; 10~3 M-CAMP, squares; 10~ 5 M-CAMP,
triangles. Conditions of regulation are the same as in Fig. 1.
Cyclic AMP and cell type regulation in Dictyostelium
29
The effect of the dissociation procedure on cell type regulation and its inhibition by
cAMP
We used pronase dissociation of slug tissue in order to achieve good cell type
separation on Percoll gradients. It has however, been shown that pronase treatment
of slug-stage cells of certain mutants directs those cells into the stalk pathway
(Peacy & Gross, 1981). Therefore we compared the effect of three different
dissociation procedures on cell type regulation in our culture system. Slugs were
dissociated either with pronase or with cellulase or dissociated mechanically by
tituration in a salt/EDTA solution. The results in Fig. 8 show that the dissociation
method does not greatly influence the results, although pronase dissociation may
slightly increase the sensitivity of prespore cells to cAMP.
Evidence for a prespore-stabilizing action of the cAMP produced endogenously by
the regulating cells
The results above indicate that cAMP blocks the prespore-to-prestalk conversion
in our culture system. To test the possibility that cAMP produced endogenously by
regulating cells stabilizes prespore cells under our culture conditions we added
cAMP phosphodiesterase in order to lower the concentration of extracellular
cAMP. Under these conditions the prespore cells disappeared faster than normal
and the equilibrium of the cell types shifted to fewer prespore cells (Fig. 9). This
result is consistent with the idea that, under conditions of regulation, some cells
1001
Time (h)
Fig. 9. Effect of exogenous beefheart cAMP phosphodiesterase on the regulation of
prespore and prestalk cell populations.
cAMP beefheart phosphodiesterase (Sigma P 0134,0-2 units.mg"1) was added to the
regulating cell populations at a concentration ofO-2mg.ini"1. Closed symbols, prespore
cell population; open symbols, prestalk cell population. Control, diamonds;
phosphodiesterase, triangles.
30
C. J. WEIJER AND A. J. DURSTON
A
100
lOOi
80-
60
60
=g 40
40-
U
20
0
4
8
0
4
8
Time (h)
Fig. 10. Effect of caffeine on the regulation of prespore and prestalk cell populations.
Caffeine was added at a concentration of 5 mM, ether conditions as in Fig. 1. Closed
symbols, % prespore cells; open symbols, % neutral-red-stained cells. (A) prespore cell
population. (B) prestalk cell population.
secrete cAMP into the extracellular space, where it stabilizes prespore cells. The
added phosphodiesterase hydrolyses this external cAMP and therefore destabilizes
prespore cells resulting in a more rapid decrease in the proportion of prespore cells.
An alternative method for reducing endogenously produced cAMP is to treat
cells with caffeine. It has been shown that caffeine blocks the relaying response of
aggregating cells by inhibiting the activation of adenylate cyclase and thus the
production of cAMP (Theibert & Devreotes, 1983; Brenner & Thorns, 1984). We
therefore examined the effect of caffeine on cell type regulation. Caffeine blocks
the conversion of prestalk to prespore cells (Fig. 10A) and accelerates the conversion of prespore to neutral-red-stained cells (Fig. 10B). Thus caffeine also leads to
a shift in the ratio of the cell types in the direction of prestalk-like cells. Millimolar
concentrations of adenosine and adenine have the same destabilizing effect on
prespore cells as caffeine. The effect of caffeine can only be partially antagonized
by the addition of millimolar concentrations of cAMP. Thus the activation of the
adenyl cyclase and therefore elevated levels of cAMP appear to be involved in the
stabilization of prespore cells.
DISCUSSION
Cell type regulation
We have shown that, in suspension culture, regulation from prestalk to prespore
and from prespore to prestalk occurs on the time scale of a few hours to an
equilibrium situation of 50 % prespore cells and 50 % prestalk cells (Fig. 1). Since
essentially all cells can be accounted for as prespore (antigen-positive) or prestalk
(neutral-red-stained) cells, these results indicate that cell type conversion is occurring under regulation conditions.
Cyclic AMP and cell type regulation in Dictyostelium
31
Expression of the prestalk-specific acid phosphatase isoenzyme however is
delayed until 20 h during regulation (Fig. 3). A delay in timing of differentiation in
suspension culture can also be seen at the level of prestalk/prespore sorting. Although prespore cells form at 16 h of development in suspension culture, sorting
only occurs at 24 h (Forman & Garrod, 1978; Tasaka & Takeuchi, 1981). Together
these results suggest that the neutral-red-stained cells initially formed in regulation
experiments are anterior-like cells, which do not sort (Sternfeld & David, 1981).
Thus the delay in suspension cell culture seems to be at the level of the conversion
of anterior-like (early prestalk) cells to prestalk cells. This suggests that the acid
phosphatase isoenzyme may be a marker which distinguishes prestalk cells from
anterior-like cells.
The reason for this difference in the timing of differentiation in suspension culture
and on agar or filters remains to be elucidated. One possibility is that the timing of
prestalk differentiation is markedly different due to the inability of the cells to condition the medium with factors required for later stages of differentiation (Town,
Gross & Kay, 1976; Kopachick et al. 1983). Preliminary experiments using conditioned media of various types, however, have not shown any substantial effect on
the rate of appearance of the prestalk specific isoenzyme during in vitro development.
Effect of cAMP and hydrolysis products on cell type regulation
We have found that cAMP added to roller tube cultures of prespore cells blocks
the regulatory process in which about half of these cells convert to neutral-redstaining cells. The effect is concentration dependent; higher concentrations have a
stronger effect. Treatments with caffeine or cAMP phosphodiesterase, which lower
endogenous cAMP, destabilize prespore cells. It is difficult to determine the effective extracellular cAMP concentrations since there is no simple way to estimate the
extracellular cAMP concentration in the aggregates. However micromolar concentrations of cAMP show a short-term stabilization of prespore cells and it is
therefore likely that the in vivo concentrations lie well below this value. It thus
appears that extracellular cAMP stabilizes prespore cells.
Millimolar concentrations of cAMP do not affect the conversion of prestalk to
prespore cells, although lower concentrations (10~ 5 -10~ 7 M) do inhibit this process.
Furthermore low concentrations of cAMP hydrolysis products block conversion of
prestalk to prespore cells. These seemingly self-contradictory findings can be explained by the following assumptions:
1. Added cAMP is rapidly broken down and the hydrolysis products inhibit the
prestalk-to-prespore conversion.
2. cAMP itself has no effect on prestalk-to-prespore conversion.
3. High concentrations of cAMP antagonize the inhibitory action of cAMP
hydrolyses products, for instance by competition for binding sites and therefore
relieve the inhibition of the prestalk-to-prespore conversion.
It is at present not known whether cAMP hydrolysis products normally play
a role in proportion regulation in slugs. However, it has recently been shown that
32
C. J. WEIJER AND A. J. DURSTON
aggregation stage cells, at least, have both low- and high-affinity adenosine receptors which are distinct from their receptors for cAMP (Newell, 1982; Newell &
Ross, 1982; van Haastert, 1983) and that cAMP when added in a 100-fold excess
suppresses adenosine binding effectively (Newell & Ross, 1982). In our in vitro
regulation system adenine was also found to inhibit the prestalk-to-prespore conversion as effectively as adenosine and 5'AMP. The possibility that the adenosine
receptors also bind adenine or that there are special adenine receptors has to our
knowledge, not yet been investigated.
The mechanism by which added cAMP stabilizes prespore cells remains to be
elucidated. The finding however that external cAMP is only partially effective in
antagonizing the caffeine effect on prespore cell differentiation would indicate that
the activation of adenylate cyclase is important for the stabilization of prespore
cells.
Effect of high and low cAMP concentrations on cell type differentiation in a mutant
Ishida (1980) has described a mutant in which differentiation of single cells
depends on the concentration of added cAMP; at low cAMP levels the mutant
forms stalk cells, while at higher levels spores are formed. This effect can be
explained under the assumption that, in the presence of cAMP, any prespore cells
formed are stable, but that cells, in the absence of added cAMP, cannot form
sufficient cAMP to stabilize prespore cells but can produce sufficient cAMP or
hydrolysis products to form stalk cells. This result indicates a clear concentrationdependent cell-type-specific function for cAMP in cell differentiation and
qualitatively agrees with our findings.
Timing differences in prestalk-to-prespore regulation in vitro and in vivo
Sakai (1973) found that there is a timing difference in the regulation of prespore
and prestalk pieces of slugs: in prestalk-to-prespore regulation there is lag of at least
3 h before any cell type conversion occurs, while in the opposite direction cell type
conversion begins immediately. We observe no such difference under our regulation conditions. This suggests that the cell type switch from prestalk to prespore is
not the limiting step in prestalk-to-prespore regulation in slugs. Possibly the delay
seen in slugs is a feature of the dynamics of the cell-type-regulating signal, which
could be quantitatively different in vivo and in vitro.
A model for cell type regulation
To place our findings in a conceptual framework we note that it is possible to have
a pattern-forming mechanism that consists of two processes, a cell type homeostasis
mechanism and a sorting process that puts cells in the right place. The easiest way
to imagine a cell type homeostasis process that regulates the proportions of two cell
types is to assume that each cell has at any given time a certain probability to convert
into the other cell type, i.e. cycles through two differentiation states (Durston &
Weijer, 1980). Analogous to cells traversing different cell cycle phases, where the
Cyclic AMP and cell type regulation in Dictyostelium
33
entry in the DNA synthesis phase can be described as regulated by a random
transition probability (Smith & Martin, 1973).
If both cell types can switch, this will lead to an equilibrium situation where the
ratio of the cell types is given by the ratio of the transition probabilities. Disturbance of this equilibrium will lead to an exponential return to this equilibrium state.
When one applies this idea to the regulation of the prespore and prestalk cell types
one is confronted with the fact that prestalk and prespore cells are spatially
separated. To maintain this separation in the presence of continuous cell type
switching there would have to be continuous sorting out of the cell types. This does
not seem to be in agreement with the observation that during slug migration there
is not much cell sorting of neutral-red-stained cells going on; sorting mainly takes
place during slug formation and regulation of slug pieces (Durston & Vork, 1979).
It also does not agree with the finding that it is possible to label subpopulations of
cells at the vegetative stage and find the labelled cells back with a non-random
distribution at the slug stage (Takeuchi, 1969; Weijer etal. 1984).
To solve this problem we propose that each cell type produces an inhibitor that
inhibits its own formation from cells of the other type (Fig. 11). This is equivalent
to saying that the transition probability is a function of the inhibitor (cell type)
concentration, i.e. at higher inhibitor concentrations the transition probability gets
smaller leading to a drastic reduction of the cell type transitions as soon as the
inhibitor concentration increases. This results in a situation where the cells are
stable once in equilibrium and therefore no sorting is required to maintain the
pattern in the slug once it is formed. The possibility that cell type conversion
continues at a low rate (Durston & Vork, 1979) is not excluded. Cells will start to
convert to the other cell type only when the equilibrium is disturbed (i.e. the
inhibitor level falls below effective inhibition) and the population will regulate until
equilibrium is reached again.
Prestalk
^"^-
«~^
0 \
stalk
cAMP
hydrolysis
products
Prespore ' ^ ^ ^ - spore
Fig. 11. Schematic representation of the double negative feedback model for cell type
proportioning.
Both cell types are assumed to have a certain probability of converting to the other
cell type. The transition probabilities are regulated by inhibitors produced by the two
cell types. Prespore cells produce an inhibitor of the prestalk-to-prespore transition and
prestalk cells produce an inhibitor of the prespore-to-prestalk transition. We propose
that cAMP is a prespore-stabilizing agent, i.e. an inhibitor of the prespore-to-prestalk
transition, whose synthesis is a measure of the prestalk population. cAMP hydrolysis
products are candidates for the stabilization of prestalk cells.
34
C. J. WEIJER AND A. J. DURSTON
An essential feature of this model is that the production of the substance that
stabilizes the prespore cells is dependent on prestalk cells. The concentration of the
inhibitor is expected to be highest close to the main prestalk mass and to fall off
towards the end of the slug. We predict therefore that prespore cells are likely to
be most stable at the boundary between the prespore and prestalk zone and most
unstable at the posterior end of the slug, where the inhibitor concentration is
lowest. This prediction is contrary to the one made by positional information
models which would say that cells are most unstable at the border between two cell
type domains (MacWilliams & Bonner, 1979). Our proposal that prespore cells are
most unstable in the rear of the prespore zone appears to be supported by the
observation that the anterior-like cells, which are scattered in the prespore zone and
most likely are a differentiation state between prespore and prestalk cells, are more
frequent in the posterior part of the prespore zone than in the anterior part (Durston & Vork, 1976; Voet & Williams, personal communication). The anterior-like
and rearguard cells might form preferentially at the distal end of the slug due to a
lower extracellular concentration of cAMP, whose synthesis is dependent on prestalk cells and is therefore expected to be the highest near the prestalk zone and
lowest in the posterior prespore zone.
Possible molecular realizations of the model
We have shown that cAMP inhibits the conversion of prespore to prestalk cells
and we thus suggest that cAMP is the prespore stabilizing factor (whose synthesis
is dependent on prestalk cells). It can now easily be seen why it was difficult to
obtain evidence for a function for cAMP in cell type regulation. When cAMP is
added from the beginning of development onwards it does not result in a noticeable
shift in the prestalk-to-prespore ratio as might be expected for an activator (Kay etal.
1978; Abe et al. 1981, own unpublished observations). Any prespore cells formed
are stabilized by the added cAMP, but as soon as enough prespore cells are formed,
the conversion of prestalk to prespore cells will be blocked by the presporecontrolled inhibitor of the prestalk-to-prespore-cell conversion (Fig. 11). The effect
can only be seen under conditions where due to low cell density no cell interactions
can occur (Ishida, 1980; Kay, 1982), or where prespore cells are studied under
regulation conditions (see above).
It is clear that the transition from prestalk to prespore cells has to be regulated
as well. In our model it is suggested that this transition is also regulated by an
inhibitor. Our experiments indicate that cAMP hydrolysis products at low concentrations inhibit the transition of prestalk to prespore cells, while they do not
appreciably affect the prespore-to-prestalk conversion. Hence these hydrolysis
products are potential candidates for the inhibitors of the prestalk-to-prespore
conversion.
Another factor that has been shown to influence cell type regulation is DIF
(Town, Gross & Kay, 1976; Kay et al. 1978). It has recently been shown that, in the
presence of cAMP, DIF can convert isolated prespore cells to stalk cells (Kay &
Cyclic AMP and cell type regulation in Dictyostelium
35
Jermyn, 1983). One possible way to explain the action of DIF under those conditions is to suppose that it desensitizes prespore cells to cAMP for instance by
inhibition of the activation of adenylate cyclase (like caffeine) and therefore results
in the loss of prespore cells in the presence of cAMP. At the moment we are
investigating the effect of DIF on prespore and prestalk regulation in our in vitro
system.
The finding that under certain conditions cells can form stalk cells in the presence
of cAMP (Bonner, 1970; Town & Stanford, 1978; Kay & Jermyn, 1984) seems at
first sight to contradict our findings that cAMP stabilizes prespore cells. We however think it is possible that there is more than one signal involved in prespore and
spore differentiation. Cells are only sensitive to cAMP for a limited period of time
and that during this limited period of time further signals are required (Sternfeld
& David, 1979; Wilkinson, Wilson & Hames, 1984) for spore maturation. Such a
sequence of stimuli has recently been shown to be necessary for the differentiation
of stalk cells (Sobolewski, Neave & Weeks, 1983).
It has recently been shown that by treatment of isolated cells of sporogenous
mutants with weak acids or bases cell differentiation can be shifted into the stalk
or spore pathway (Gross, Bradbury, Kay & Peacy, 1983). It therefore would be
interesting to investigate whether these treatments affect the internal cAMP
concentration by altering the internal pH or altering the associated membrane
potential.
We thank Dr S. K. Brahma for the preparation of the prespore-specific antiserum and Charles
N. David and Harry K. MacWilliams for helpful suggestions in the preparation of the manuscript.
Rob Bleumink for expert technical assistance in the initial experiments and Gerdi Duschl for
making the phosphatase gels work. We also wish to thank the referees for constructive criticism.
This work was supported by the Foundation for Fundamental Biological Research (BION),
which is subsidized by the Netherlands Organization for the Advancement of Pure Research
(ZWO).
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(Accepted 16 November, 1984)