J. Embryo/, exp. Morph. Vol. 62, pp. 369-318, 1981
Printed in Great Britain © Company of Biologists Limited 1981
369
Dictyostelium amoebae
can differentiate into spores without
cell-to-cell contact
By ROBERT R. KAY1 AND DAVID J. TREVAN
From the Imperial Cancer Research Fund, Mill Hill
Laboratories, London
SUMMARY
Amoebae of sporogenous mutants of Dictyostelium discoideum can differentiate into stalk
cells and spores in the absence of normal morphogenesis when spread on agar containing
cyclic-AMP. The efficiency of differentiation is improved when the amoebae are incubated
as submerged monolayers in plastic petri dishes. Under these conditions spore formation is
density dependent and hence requires some form of cellular interaction. To determine
whether this interaction involves direct cell-cell contact we have made time-lapse films of
cells differentiating at intermediate density. These films show that amoebae can develop into
spores without making contact with any other cells. In addition, although some cells do
divide during incubation, division is not necessary for spore formation. At higher densities
small aggregates form which give rise to mixtures of stalk cells and spores. There is no
detectable patterning of the two cell types within such aggregates.
INTRODUCTION
In the slime mould Dictyostelium discoideum, differentiation of the two major
terminal cell types produced in development (stalk and spore cells) is induced by
a number of different cell interactions. During aggregation the amoebae
generate and experience periodic extracellular pulses of cyclic-AMP in the
nano-molar range (Shaffer, 1975). These are necessary both for directing the
cells towards the cell or group of cells which are the source of the signals and
for stimulating expression of particular aggregative genes (Darmon, Brachet &
Pereira da Silva, 1975; Gerisch, Fromm, Huesgen & Wick, 1975). The effects of
these signals on gene expression can be mimicked by the continuous application
of much higher concentrations of cyclic-AMP (Klein, 1975; Sampson, Town &
Gross, 1978) which also have the effect of blocking normal morphogenesis.
By taking advantage of these effects of high cyclic-AMP concentrations we have
devised in vitro systems in which both stalk and spore differentiation can occur
Bonner, 1970; Town, Gross & Kay, 1976; Kay, Garrod & Tilly, 1978). Stalk
1
Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London, NW7 IAD, U.K.
370
R. R. KAY AND D. J. TREVAN
differentiation requires cyclic-AMP and a low MW diffusible factor (DIF,
Town et al. 1976; Town & Stanford, 1979) whereas spore formation requires
cyclic-AMP (Kay et al. 1978; Kay, 1979) and some other cell interaction. This
interaction appears to be of short range and so might be dependent for transmission on cell-to-cell contact.
The purpose of the present work was to determine whether spore cell differentiation does demand cell contact or not as well as to examine the patterning
of stalk and spore cells differentiating within aggregates in vitro.
MATERIALS AND METHODS
Strain and growth conditions
The sporogenous mutants HM18 (tsg-900, acr-900, cob-900, sci-907) and
HM28 (tsg-901 and/or tsg-903, cyc-900, whi-900, sci-909) derive from V12M2.
They were obtained from their immediate parents by N-methyl-N'-nitro-Nnitrosoguanidine mutagenesis and selection by detergent lysis for the ability
to make spores on cyclic-AMP-containing agar (Town et al. 1976). Since in the
selection conditions the wild type will make prespores but not spores, we
consider that these mutants are affected in spore maturation (Kay et al. 1978).
In normal developmental conditions HM18 usually makes a fruit having a
central stalk with spores at the base and at the top; HM28 makes a fruit
consisting of a mound of stalk and spore cells.
Cells were grown on SM-agar (per 1: Difco Bacto peptone 10 g, Difco yeast
extract 1 g, glucose 10 g, MgSO4.7H2O 1 g, KH 2 PO 4 2-2 g, N a ^ P C ^ 1 g,
agar 15 g) in association with Klebsiella aerogenes. Plates were harvested at the
first sign of clearing and the slime-mould cells freed of bacteria by four centrifugal washes in KK 2 (16-6 mM-KH2PO4, 3-8 mM-K2HPO4, 2mM-MgSO4,
pH ~ 6-1) and a last wash in either 5% Bonners salts (0-5 mM-NaCl, 0-5 mMKC1, 0-lmM-CaCl2) or in NS (20 mM-NaCl, 20 mM-KCl, 1 mM-CaCl2) as
appropriate. The cells were then resuspended in more of the final wash solution
and counted with a Coulter Counter; development was timed from this last
resuspension.
Cell differentiation in vitro.
The standard medium for spore induction consisted of 10 HIM 2-(N-morpholino) ethanesulphonic acid (MES) Na+), 5 mM cyclic-AMP (Na+), NS, 200 /tg/ml
streptomycin SO4, 15//g/ml tetracycline (added from a stock of 7-5 mg/ml in
ethanol) pH 6-2. 4 ml of this medium was normally used per petri dish (Sterilin
5 cm diameter bacteriological plastic, code no. 122). Washed cells at the
densities indicated in the text were pipetted into each petri dish of medium,
where they settled onto the plastic. In some experiments (Table 1) cells were
plated on 2 ml of 1-5 % Oxoid L28 agar made up in the above medium and
Spore differentiation without cell contact
371
again in 5 cm dishes. This agar was pre-washed where indicated in Table 1 with
H 2 0 and ethanol.
The plates were incubated in a moist atmosphere at 22 °C to allow cell
differentiation to occur. Stalk and spore cells were identified by phase-contrast
microscopy at the end of the incubation period or as indicated in the filming
section. Most spores formed at high density were oval shaped as in normal
fruit, but at low density a considerable proportion of round refractile cells
differentiated. These were resistant to detergent lysis and were stained by an
anti-spore antibody so we counted them as spores.
Time-lapse filming
Time-lapse movies were made at 1 frame per 20-30 sec on PanF film (Ilford)
using an inverted phase-contrast microscope with a Bolex HI6 reflex camera
and Bolex-Wild MBF-B and C control units. Low-power films were made at a
cell density of 2-5 x 103 cells cm~2 with a x 10 phase objective and a final
magnification of x 30. High-power films were made using a x 20 phase objective
(final magnification x 100 or x 200) and at a cell density of 2 x 105 cells cm~2.
At this density the cells aggregated to form small balls in which it was impossible
to distinguish many individual cells. It was however possible to follow cells in
small aggregates by holding these flat against the bottom of the dish with
cellophane held in a perspex stretcher; this procedure was still inadequate for
large aggregates. In all the films the image of a clock was projected onto the
film, so as to allow accurate timing of the events filmed. At 20-28 h of low-power
filming the detergent Cemulsol was carefully added to the plate to a final
concentration of 0-3 % and filming contained for another hour or more. In
this time all amoebae were lysed by the detergent but spores were completely
unaffected.
Films were analysed using a Specto Motion Analysis Mk 3 projector. The
optical resolution in the low-power films was such that fine processes projecting
from the cell surface may have been invisible. To allow for this possibility
(and since our interest is in cells that did not make contact) cells whose surfaces
were separated by less than one cell diameter were considered to be in contact.
In fact many of the cells which are listed in Table 2 as having made no cell
contact did not even approach to within two cell diameters of another cell.
RESULTS
Improved conditions for spore cell differentiation
We have shown previously that amoebae of sporogenous strains of Dictyostelium can differentiate into spores, without normal morphogenesis, when they
are plated on agar containing cyclic-AMP (the conditions of Table 1, line 1;
Town et al. 1976; Kay et al. 1978). The efficiency of spore formation can be
improved to that obtained in the earlier work by increasing the concentration
372
R. R. KAY AND D. J. TREVAN
Table 1. Improved conditions for spore differentation in vitro
Conditions of differentiation
Substratum
1.
2.
3.
4.
5.
Agar
Agar
Agar
Plastic
Washed Agar
Submerged
No
No
Yes
Yes
Yes
/o
Salts
5% Bonners
NS
NS
NS
NS
cell type
Stalk
Spore
24-7
17-3
25
28-7
14
6-4
10-7
17-8
45-4
48-1
HM18 cells at a density of 104 cm"2 were plated in the conditions summarised in the table
and below. After 2 days of development the state of differentiation of 100-200 cells per
plate was scored by phase-contrast microscopy. Results are the means from four separate
plates. Where agar was the substratum 2 ml was used per plate and if it was to be submerged,
2 ml of medium was added. The cells on plastic were submerged by 4 ml of medium. Solutions and agar were made up with 10 mM-MES, 5 mM-cyclic-AMP, 200 /tg/ml streptomycin,
15/*g/ml tetracycline, pH 6-2 plus the salts indicated in the Table. 5% Bonners salts is
equivalent to 0-5 mM-NaCl, 0-5 mM-KCl, 01 mM-CaCl2, NS is 20 mM-KCl, 20 mM-NaCl,
1 mM CaCL.
of salts and by submerging the agar (Table 1, lines 2 and 3) A further improvement is obtained when the agar is omitted and the cells plated directly on
plastic with an overlay of buffered salts plus cyclic-AMP (J Gross, personal
communication; Table 1, line 4). It seems that agar contains an inhibitor of
spore differentiation, which can be removed by washing (compare Table 1,
lines 3 and 5).
With these improved conditions, the efficiency of spore and stalk induction
is dependent on the density at which the cells are plated, being very inefficient
at low density for both strain HM18 and strain HM28 (Fig. 1). At high cell
density HM18 produces approximately equal numbers of stalk and spore
cells but HM28 gives mainly spores. Indeed on submerged agar this strain
produces more than 95 % spore cells with most of the remainder being amoebae.
This situation should be convenient for examining spore differentiation virtually
in the absence of stalk cell differentiation.
Neither cell contact nor cell division is essential for spore differentiation
Preliminary experiments showed that amoebae of strain HM28 moved less
actively than those of HM18 in the filming conditions. We therefore used the
former strain for these experiments so as to minimize collisions between cells
and to increase the chances that any particular cell would remain in view throughout the film. Filming started at between 20 and 40min of starvation and
continued for about 24 h; spores normally formed after 10 to 20 h. Cell-to-cell
contacts were rare and of brief duration in the period preceding spore formation;
on average 1-6 contacts per cell in the first 11-12 h of development. Indeed some
Spore differentiation without cell contact
373
Cell density (cells/cm2)
Fig. 1. Density dependence of stalk and spore differentiation by cells of strains
HM18 and HM28. Washed vegetative amoebae of the appropriate strain and
density were plated in bacteriological plastic petri dishes containing 10 ITIM-MES,
NS, 5 mM-cyclic-AMP, 200/*g/ml Streptomycin, 15/tg/ml tetracycline, pH 6-2.
After 2 days incubation at 22 °C about 2 ml of the medium was carefully removed
and cell differentiation scored by phase contrast microscopy. The results are the
means of two experiments, in each of which 100-200 cells weie scored. HM18:
stalk, • ; spore, • . HM28: stalk, O; spore, D-
cells avoided all contact with other cells throughout the period filmed. Some
of these cells became spores as judged by their morphology, lack of movement
and resistance to detergent lysis (Table 2). This proves that in the period filmed
cell contact is not essential for spore differentiation. The films also showed that
cell division during development is not required for spore formation, since
some spores were derived from amoebae that did not divide (Table 2).
Patterning of stalk and spore cells produced by strain HM18 at high cell density
In conditions suitable for normal development of the wild type, cells of strain
HM18 produce aberrant, though clearly patterned, fruit. We wondered
whether this ability to produce a pattern of differentiated cell types might persist
in aggregates formed in vitro. Observation of many such aggregates shows that
large coherent patterns of stalk and spore cells do not form when the cells are
constrained as a monolayer. Given this we sought, without success, for rules
operating on a smaller scale that might relate a cell's fate to its position within
374
R. R. KAY AND D. J. TREVAN
Table 2. Spore differentiation without cell contact or division
Cell type at
end of film
Spore
Amoeba
Spore
Amoeba
Spore
Amoeba
History of cell during filming
No contact, no division
No contact, no division
Contact, no division
Contact, no division
From division
From division
Number of
examples
12
12
20
20
15
6
HM28 cells were at a density of 2-5 x 103 cm~2 plated on bacteriological plastic dishes and
overlayed with 4 ml of the standard medium for spore induction. All of the cells counted in
this table were continuously in view from the start of a film at 20-40 minutes of starvation
until the end. The results from nine films analysed are combined.
an aggregate. For example we noted that clusters of one cell type do sometimes
form but so do mixtures of the two and that stalk and spore cells can be derived
from cells that have held internal or external positions in an aggregate (e.g.
Fig. 2, cells 2,4, 5 and 6). Thus it seems that in the few hours preceding terminal
differentiation the position of a cell bears little relationship to its eventual fate.
DISCUSSION
Developing cells in a solid tissue might interact with each other by two
distinct sorts of mechanism: diffusion-mediated, in which diffusible signal
molecules would be released by the cells and contact-mediated, in which the
signals would remain cell bound. We are analysing the cell interactions required
in vitro (Kay, Town & Gross, 1979) to trigger Dictyostelium amoebae into
spore differentiation. In principle we can disrupt either type of interaction by
sufficient dilution of the cells: in the one case the signal molecules would become
too dilute to be effective and in the other, the cells would not make contact.
By the criterion of dilution, spore formation by amoebae of sporogenous
mutants in the presence of cyclic-AMP is stimulated by some cell interaction.
In this paper we have been able to prove that amoebae can differentiate into
Fig. 2. Frames from a time-lapse film of the differentiation of cells of strain HM18
under cellophane. HM18 cells at a density of 2 x 105 cirr 2 were prepared for differentiation as described in the legend to Fig. 1., except that they were constrained as a
monolayer by a sheet of cellophane held closely against the bottom of the petri dish.
Frames A, B, C and D were taken at 16,18, 20 and 24 h of development respectively.
The arrowed cells are some of those which can be followed for several hours on the
time lapsefilmfrom which these frames are taken. In frame D cells 1 and 3 are stalk
cells (still somewhat immature) and cells 2, 4, 5 and 6 are spores. The bar represents
20 fim.
Spore differentiation without cell contact
\
A
6
\
D
t
y
4
375
376
R. R. KAY AND D. J. TREVAN
spores without cell-to-cell contact (except possibly in the first 20 minutes of
development, which we have been unable to film). Therefore the cell interactions
necessary for spore induction are not contact-mediated and must instead be
diffusion-mediated.
This conclusion prompted a renewed testing of the effects of conditioned
medium, which we can now show will greatly stimulate spore differentiation at
low cell density (Kay, unpublished observations). Tn earlier work (Kay et al.
1978) in which cells were plated at low density on cellophane overlaying 'helper'
cells at high density, we found that spore differentiation in the low density
population correlated strongly with cell-to-cell contact. Although this result
apparently contradicts our present conclusions, in the original work less than
4 % of the cells at low density became spores. Hence the effective concentration
of conditioning factor may have been so low that it only exceeded the threshold
concentration for induction very close to the cells which were its source, giving
an apparent contact dependence of differentiation.
Many workers have shown that physical separation of cells during later development prevents further cell differentiation (Newell, Longlands & Sussman,
1971; Gregg, 1971; Takeuchi & Sakai, 1971). This result points to the existence
of some sort of cell interaction that drives cell differentiation. But in these experiments the cells were both disaggregated and diluted and so it is not possible to
decide whether disruption of a diffusion- or a contact-mediated interaction was
responsible for the inhibition of development. Other workers have reported
that certain post-aggregative gene products are not made in shaken cell suspensions where the cells are largely separate, despite being at a high density
(Rickenberg, Tihon & Giizel, 1977; Okamoto & Takeuchi, 1976; Landfear &
Lodish, 1980). Preliminary experiments suggest that this outcome could be
due to other aspects of the conditions used by these workers, rather than to the
absence of cell contact (M. Peacey and J. Gross, personal communication). On
the other hand we cannot exclude the possibility that our sporogenous mutants
lack a contact-dependent interaction normally present in the wild type. An
analysis of the requirements for prespore cell differentiation in the wild type and
a comparison with other sporogenous isolates (Wilcox & Sussman, 1978;
Ishida, 1980) should help to resolve this matter.
Although slime-mould cells develop in the absence of exogenous nutrients a
certain amount of cell division normally occurs (Bonner & Frascella, 1952) so
that by the completion of morphogenesis there can be approximately a doubling
in cell numbers (Zada-Hames & Ashworth, 1978). These divisions are found
mainly during aggregation and at the slug stage where they are restricted to the
prespore zone (Durston & Vork, 1978). In most other systems development is
accompanied by extensive cell division and it has been suggested that division
may somehow be essential for the expression of new developmental genes
(Holtzer, Weintraub, Mayne & Mochan, 1972). This idea is intrinsically
unlikely in Dictyostelium, given the small amount of cell division that does
Spore differentiation without cell contact
111
occur and the continuous changes in gene expression throughout development.
Sussman & Sussman (1960) have shown that the mutant Fty-1 can fruit
without cell division and Cappuccinelli, Fighetti & Rubino (1979) found that
inhibitors of mitosis delay, but do not prevent development of the wild type.
Likewise our results show that spores can differentiate without cell division.
Thus with three different ways of perturbing development it is possible to
obtain cell differentiation without cell division, making it very unlikely that
normally the former is dependent on the latter.
We are most grateful to Julian Gross, Shuji Ishida and Jenny Brookman for discussions
and improvements in the manuscript.
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