/. Embryol. exp. Morph. 75, 293-301 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
293
Proportion regulation without pattern formation in
Dictyostelium discoideum
By MASAKAZU OYAMA 1 , KOJI OKAMOTO AND
IKUO TAKEUCHI
From the Department of Botany, Kyoto University
SUMMARY
When shaken in a glucose-albumin-cAMP medium, dissociated aggregative cells formed
small clumps, in which both prespore and prestalk cells differentiated in essentially the same
proportions as in a slug. Immunocytochemical staining of sections of such clumps revealed that
the two types of cells showed no particular pattern of distribution, unlike the two-zoned
prestalk-prespore pattern as observed in the slug.
Cells dissociated at stages later than the onset of aggregation always produced a constant
proportion of prespore cells, irrespective of the initial proportion when transferred to the
culture. Furthermore, prestalk cells fractionated from slugs and transferred to the culture
restored almost the normal prespore proportion through conversion of the cell types, whereas
proportion of unfractionated slug cells remained unchanged. We conclude from these findings
that the normal prestalk-prespore pattern is not required for proportion to be regulated.
INTRODUCTION
After depletion of food supply, cellular slime mould Dictyostelium discoideum
amoebae enter a developmental stage in which they aggregate with chemotaxis
and form cell masses. The cell mass transforms itself into a pseudoplasmodium
of a slug shape and finally forms a fruiting body which consists of two cell types,
spores and stalk cells.. During the slug formation, two types of presumptive cells
appear and are arranged along the slug axis. Later, anterior prestalk cells in the
slug differentiate into stalk cells and posterior prespore cells into spores (Raper,
1940).
It is well known that the proportion between the two presumptive cells is
roughly constant irrespective of the slug size (Bonner, 1957; Williams, Fisher,
MacWilliams & Bonner, 1981) and that any prestalk or prespore fragment
isolated from a slug restores normal proportion through conversion of the cell
type (Bonner, Chiquoine & Kolderie, 1955; Sakai, 1973). The regulation of
proportion among different cell types in a tissue is a general and important aspect
of development. Different models were proposed for pattern formation and
proportion regulation in the cellular slime moulds (for review, see MacWilliams
1
Author's address: Department of Botany, Faculty of Science, Kyoto University, Kyoto
606, Japan.
294
M. OYAMA, K. OKAMOTO AND I. TAKEUCHI
& Bonner, 1979). Some models require the existence of the two-zoned
prestalk-prespore pattern for the proportion to be regulated. It is, however,
difficult to analyse the relationship between pattern formation and proportion
regulation, since both appear to proceed concurrently during the normal
development.
In this paper, we investigated proportion regulation in a liquid shaking culture
in which prespore cells differentiate (Okamoto, 1981), to know whether or not
pattern formation is prerequisite to proportion regulation. We found that no
particular prestalk-prespore pattern formed in small cell clumps obtained in this
culture, but that prespore proportion was nevertheless regulated as in a slug.
These results indicate that proportion regulation is independent of pattern
formation.
MATERIALS AND METHODS
Culture
D. discoideum NC4 cells were grown in a nutrient medium (1/75 M-phosphate
buffer, pH6-2, containing 1 % glucose and 1 % Bactopeptone) with Escherichia
coli B/r, at 21 °C. Early stationary cells were collected and washed with a
solution of 35mM-NaCl and 35mM-KCl. Washed cells were deposited on a
Millipore filter (AABG04700) (5-8 x 104 cells/mm2) and incubated, at 21 °C, in
a moist chamber (Oyama, Okamoto & Takeuchi, 1982). Under the culture
conditions employed, the development after aggregation was somewhat
delayed, but entirely normal. Aggregating cells (1 h after the onset of aggregation) were dissociated with a 0-9% NaCl solution containing 2mM-EDTA
(Takeuchi & Yabuno, 1970). Dissociated cells were washed, filtered through
nylon mesh (32/im openings) and resuspended at 107 cells/ml in 20mM-KK2phosphate buffer, pH6-0, containing 5 % glucose, 2% albumin, lmM-cAMP
and 2 mM-EDTA (G AC medium). A 20 ml vial containing 1 ml of the cell suspension was rotary shaken at 120r.p.m., at 21 °C.
Fractionation of presumptive cells
Early stationary phase cells which had been grown with washed E. coli in
20 mM-KK2-phosphate buffer pH6-0 were washed, deposited on a Millipore
filter placed on pads soaked with 40 mM-phosphate buffer, pH6-2, containing
2-5 mM-MgCb and 1% glucose and incubated at 21 °C, in a moist chamber.
Migrating slugs were dissociated and dissociated cells were fractionated in a
Percoll (Pharmacia Fine Chemicals) density gradient, essentially according to
Tsang & Bradbury (1981). The cells were suspended in a Percoll solution with
a density of 1-09g/ml, and centrifuged at 15000r.p.m., for 30min, at 4°C, in a
Hitachi RPR18B rotor. After centrifugation, light and heavy cell fractions were
collected with pipettes and washed with 20 mM-KK2-phosphate buffer, pH6-0,
Proportion regulation in Dictyostelium
295
containing 2mM-EDTA and 5 % glucose. Washed cells were suspended in a
GAC medium.
Determination of prespore proportion
Cell clumps formed in the culture were dissociated and fixed with cold absolute
methanol (Oyama et al. 1982). Fixed cells were air dried on a cover glass and
stained with FITC-conjugated anti-D. mucoroides spore serum globulin
(Hayashi & Takeuchi, 1976). Stained and non-stained cells in fields were counted under a Nikon OPTIPHOT fluorescence microscope.
Histological preparations
Cell clumps formed in the culture were collected and fixed with cold absolute
methanol. Fixed clumps were washed and mixed with melted 2 % agar. Clumps
embedded in agar blocks were dehydrated with ethanol-benzene series and
embedded in paraffin. Serial sections were stained with the FITC-conjugated
antispore serum globulin and observed.
Vital staining
One drop of the culture medium was diluted into 50 mM-phosphate buffer,
pH7-0, containing 0-001% neutral red and dissociated with pipetting. After
being placed on a slide glass, dissociated cells were observed under a microscope.
RESULTS
Differentiation of presumptive cells
Shaken slowly in a glucose-albumin-cAMP (GAC) medium as described in the
preceding section, dissociated aggregative cells formed small clumps, in which
cells containing prespore specific antigen differentiated, confirming the previous
studies (Okamoto, 1981; Oyama et al 1982). The presence of cAMP (10" 3 10~4 M) and albumin (higher than 1 %) was essential for the prespore differentiation as detected by the presence of prespore antigen (data not shown). When
cultured with no or low concentrations of added glucose, cells tended to form
clumps partially enclosed by slime sheath and gave a lower yield of prespore
cells. Changes in cell density within the range of 5 x 105-2 x 107 cells/ml did not
affect the ratio of prespore to total cells (data not shown). The conditions employed in the present work (5 % glucose, 2 % albumin and 1 mM-cAMP) gave rise
to the most effective prespore differentiation, where cells with and without the
antigen were clearly distinguishable.
Under the above conditions, prespore cells rapidly increased in number after
3 h of culture and reached the maximum at 6h, where about 80 % of cells contained the prespore antigen (Fig. 1), the value equivalent to that found in an
ordinary slug. The prespore proportion thereafter remained constant throughout
296
M. OYAMA, K. OKAMOTO AND I. TAKEUCHI
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Fig. 1. Time course of differentiation of prespore cells in the liquid shake culture.
D. discoideum NC4 cells dissociated at the early aggregation stage were shaken
slowly (120r.p.m.), at 21 °C, in 20ITIM KK2-phosphate buffer (pH6-0) containing
5 % glucose, 2 % albumin, 1 ITIMCAMP and 2mMEDTA. Prespore cells were identified as those containing prespore antigen detected by FITC-conjugated anti-spore
globulin, as described in Materials and Methods. Bars indicate standard deviation.
the culture, although the amount of the antigen as indicated by the intensity of
fluorescence after immunocytochemical staining continued to increase up until
ca. 20 h.
It is known that when vegatative cells stained with neutral red are allowed to
form slugs, only anterior prestalk cells become strongly stained (Bonner, 1952).
When cells harvested after 11-17 h of the present culture were stained with
neutral red, as described in the Materials and Methods section, ca. 20 % of cells
contained strongly stained granules. This suggests that not only prespore cells
but prestalk cells may differentiate in the culture. This was confirmed by the fact
that the prestalk specific isozyme of acid phosphatase (Oohata, 1982) became
detectable during the culture (data not shown). It is noteworthy that the proportion of stained cells in the culture was equal to that of prestalk cells in an ordinary
slug.
Distribution of prespore cells
Since normal proportion of prestalk and prespore cells was attained in the
culture, we investigated the distribution pattern of these cells within cell clumps
formed in the culture. After dissociated aggregative cells were cultured for 18 h,
Proportion regulation in Dictyostelium
297
<•*
Fig. 2. A section of a cell clump stained with FITC-conjugated anti-spore globulin.
Dissociated aggregative cells were rotary shaken slowly, at 21 °C, in a glucosealbumin-cAMP-EDTA medium for 18 h. Cell clumps formed in the culture were
embedded in paraffin and serial sections were stained with FITC-conjugated antispore globulin. Prespore cells were strongly stained with their granules. The clump
is 50 jum long.
cell clumps which had formed were embedded in paraffin. At this stage, all the
prespore cells which had differentiated accumulated enough prespore antigen.
Serial sections were stained with FITC-conjugated antispore globulin and observed. Unlike the two-zoned prestalk-prespore pattern in an ordinary slug, prespore cells in the cell clumps showed no particular pattern, but were distributed
almost randomly (Fig. 2).
Effect of dissociation stage
During normal development, the proportion of prespore cells increases from
the late-aggregation to the standing-slug stage (Hayashi & Takeuchi, 1976; see
Fig. 3). We examined whether or not cells dissociated and transferred to the
liquid culture at different developmental stages give rise to the same proportion
of prespore cells. Cells dissociated before and at the onset of aggregation
(rippling stage) gave no and small yield of prespore cells respectively (Fig. 3A,
B), confirming the previous study by Okamoto (1981). However, cells harvested
at stages later than this always gave rise to normal proportion of prespore cells,
irrespective of the proportion when transferred to the liquid culture (Fig. 3C, D,
E, F). This suggests that the proportion of prespore cells may be regulated in this
culture.
298
M. OYAMA, K. OKAMOTO AND I.
TAKEUCHI
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299
Proportion regulation in Dictyostelium
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Fig. 4. Cell type conversion in the liquid culture of fractionated slug cells. Prestalk
(•) and prespore (A) cells were fractionated from dissociated slug cells in a Percoll
density gradient. Fractionated and unfractionated (O) cells were transferred to the
liquid shake culture and prespore proportions were determined at times. Open and
closed marks show independent experiments.
Regulation of prestalk cell fraction
To examine whether or not the prespore proportion is regulated in this culture
as in normal slugs, prestalk and prespore cells were fractionated from dissociated
Fig. 3. Effect of dissociation stages on proportion of prespore cells. Cell aggregates
developing on Millipore filters were dissociated at (A) 1 h before aggregation, (B)
beginning of aggregation, (C) l h after aggregation, (D) tight aggregate stage, (E)
tip stage and (F) slug stage. Dissociated cells were transferred to the liquid shake
culture and proportions of prespore to total cells were determined after various times
of incubation. Each point represents the average and the standard deviation of three
independent experiments. The abscissa indicates times after the onset of aggregation
(arrows). The dotted lines indicate the changes in prespore proportion when the cells
were allowed to develop on Millipore filters.
300
M. OYAMA, K. OKAMOTO AND I. TAKEUCHI
slug cells and transferred to the culture. While the prespore fraction obtained
was 100 % pure, the prestalk fraction was contaminated by ca. 10 % prespore
cells, as pointed out by Tsang & Bradbury (1981). When cultured, the prestalk
fraction gradually increased in the percentage of prespore cells to almost the
normal level, while unfractionated slug cells showed constant proportion
throughout the culture (Fig. 4). On the other hand, the prespore fraction gave
a slight but significant decrease during the culture.
DISCUSSION
The present work showed that both prestalk and prespore cells differentiated
within cell clumps formed in a liquid shake culture of dissociated aggregative
cells, in essentially the same proportions as in the slug. Nevertheless, no particular pattern of distribution of the two cell types was observed in the clumps,
unlike the two-zoned pattern of the slug. These indicate that although cell differentiation normally occurred, sorting out of differentiated cells was somehow
blocked in this culture. Matsukuma & Durston (1979) demonstrated that prestalk cells (but not prespore cells) within a cell mass showed chemotactic movement toward cAMP and argued that this may bring about sorting out of the two
cell types. Furthermore, Sternfeld & David (1981) reported that, when prestalk
and prespore cells are mixed in a submerged aggregate, prestalk cells become
enclosed by prespore cells (confirming Tasaka & Takeuchi (1981)), but that
when the aggregate is immersed in 10~7-5 x 10~6 M-CAMP the distribution pattern is reversed. The random distribution of prespore cells (instead of the
inside-outside pattern) which formed in our culture is probably due to the fact
that a cAMP gradient fails to be established within cell clumps, since (1) the
culture medium contains a high level of cAMP, (2) cells have a low level of
cellular and extracellular cAMP-phosphodiesterase activity (Okamoto,
Takemoto, Kato & Takeuchi, 1982; Okamoto, unpublished data) and (3) cAMP
is most likely to be freely diffusible into cell clumps which are small in size and
not surrounded by slime sheath (Okamoto, 1981).
An important aspect of the present finding is that although no particular
pattern of prestalk and prespore cells formed within cell clumps, the proportions
of both types of cells were regulated. A constant proportion of prespore cells was
always obtained, irrespective of the initial prespore proportion of cells
dissociated at various developmental stages (Fig. 3). Furthermore, prestalk cells
fractionated from slugs restored almost normal proportion through conversion
of the cell type from prestalk to prespore cells, whereas proportion of unfractionated slug cells remained unchanged (Fig. 4). These indicate that the twozoned prestalk-prespore pattern as observed in the slug is not required for
proportion to be regulated.
In conclusion, the present findings are consistent with the view that the differentiation pattern of prestalk and prespore cells was constructed through sorting
Proportion regulation in Dictyostelium
301
out of the cells which have beforehand differentiated randomly within a cell
aggregate (Forman & Garrod, 1977; Tasaka & Takeuchi, 1981; Takeuchi et al.
1982).
This work was supported in part by Grants-in-Aid (nos. 444003, 56108008) from the
Ministry of Education of Japan.
BONNER,
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(Accepted 7 October 1982)