/. Embryol. exp. Morph. 74, 235-243 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
235
Cell differentiation in a temperature-sensitive
stalkless mutant of Dictyostelium discoideum
By AIKO AMAGAI 1 , SHUJI ISHIDA AND IKUO TAKEUCHI
From the Department of Botany, Faculty of Science, Kyoto University, Japan
SUMMARY
A temperature-sensitive aggregateless and stalkless mutant was isolated from Dictyostelium
discoideum NC-4. The mutant cells cannot aggregate at 27 °C, but aggregate and form normal
fruiting bodies at 21 °C. When the temperature was shifted to 27 °C after aggregation at 21 °C,
almost all of the cells in the aggregate differentiated into spores. Neither stalk cells nor stalk
tubes formed at 27 °C. Inhibition of stalk formation was not lifted by addition of cyclic AMP.
Nevertheless, the proportion of prespore to total cells within the mutant slugs was normal,
at both 21 °C and 27 °C. At 27 °C, a slug was transformed into a spherical cell mass at the end
of migration, within which pre-existing prespore cells differentiated into spores. The remaining prestalk cells were then converted to prespore cells which later became spores. As the celltype conversion continued, formation of a spore mass resulted. The development of the
mutant is thus consistent with the idea that the presumptive cell differentiation is directly
related to the terminal cell differentiation.
During migration at 27 °C, the number of prestalk cells decreased in the anterior part of the
slug but instead increased at the foot or the rear part, whereas the prestalk-prespore pattern
remained normal at 21 °C. The fact that a normal proportion of prespore cells was maintained
in spite of their deranged distribution at 27 °C indicates that the regulation of proportion is
independent of the formation of pattern.
INTRODUCTION
After the vegetative stage, the cells of the cellular slime mould, Dictyostelium
discoideum aggregate and form a cell mass which assumes the shape of a slug.
After a series of morphogenetic movements, each slug eventually forms a fruiting body consisting of stalk cells and spores. During this process, the anterior
part of the slug becomes stalk cells, while the posterior part becomes spores
(Raper, 1940). Prespore cells in the slug are distinguished from prestalk cells by
holding prespore-specific vacuoles (Hohl & Hamamoto, 1969; Maeda &
Takeuchi, 1969), which contain prespore antigen stainable with fluoresceinconjugated antispore serum (Takeuchi, 1963). The prespore-prestalk pattern
observed in the slug is generally regarded as representing an initial step of
differentiation toward the final spore-stalk pattern in the fruiting body (for
review, see Mac Williams & Bonner, 1979). Recently, however, the idea was
1
Author's address: Department of Botany, Faculty of Science, Kyoto University, Kyoto
606, Japan.
236
A. AMAGAI, S. ISHIDA AND I. TAKEUCHI
questioned by Morrissey, Farnsworth & Loomis (1981), on the grounds that
stalky and stalkless mutants which form only stalks and spores respectively in the
fruiting bodies are nevertheless normal in the prespore-prestalk pattern at the
slug stage.
In our attempt to isolate temperature-sensitive aggregateless mutants for the
studies of aggregation, we have found one which cannot aggregate at 27 °C but
aggregate and form fruiting bodies at 21 °C. By shifting the cell masses to 27 °C
after aggregation at 21 °C, differentiation of the mutant could occur but only in
an abnormal fashion, i.e. stalk differentiation did not occur and almost all of the
cells in the aggregate differentiated into a mass of spores. By closely examining
differentiation pattern of prespore cells in this mutant, we found that the spore
mass was derived from prespore cells which not only pre-existed in the slug but
also were converted from the remaining prestalk cells through proportion regulation.
MATERIALS AND METHODS
1. Cultures
Dictyostelium discoideum NC-4 and a mutant KYH-13 isolated from it were
used. Wild-type cells were cultured with Escherichia coli on nutrient agar
(Bonner, 1947), at 21 °C. For the cultures of the mutant, mutant spores and
E. coli were put together in a 300 ml Erlenmyer flask which contained 100 ml of
a lactose-peptone medium (5g lactose, 5g peptone, 1000ml distilled water).
The flask was shaken, at 21 °C, on a rotary shaker (120r.p.m./min). Growthphase cells were collected and washed by centrifugation, twice with Bonner's salt
solution (Bonner, 1947) and once with 20mM-phosphate buffer (pH6-l).
Washed cells were resuspended in the phosphate buffer and spread on nonnutrient or salt (lower pad solution (LPS): Ellingson, Telser & Sussman, 1971)
agar (2 %) at a density of 5 x 105 cells/cm2. After the cells were settled, excess
water was removed. The plates were incubated at 21 °C or 27 °C in the dark.
2. Immunocytochemical and vital staining
Prespore cells were identified by staining with fluorescein-isothiocyanate
(FITC)-conjugated serum globulin produced against spores of Dictyostelium
mucoroides. The preparation of the serum and the staining with it were conducted according to Takeuchi (1963). The proportions of prespore to total cells
within cell masses were determined by the method of Hayashi & Takeuchi
(1976). To know the distribution of prespore cells, slugs were fixed in cold
methanol, embedded in paraffin and sectioned. Sections and cells stained with
the conjugated globulin were observed under a Nikon fluorescence microscope
(OPTIPHOT). Vital staining of cells with neutral red was conducted by the
method of Yamamoto & Takeuchi (1983).
A temperature-sensitive stalkless mutant ofD. discoideum 237
RESULTS
1. Isolation of the mutant
The isolation of mutants was conducted by the method of Fukui & Takeuchi
(1971) using nitrosoguanidine as a mutagen. Temperature-sensitive mutants
which could not aggregate at 27 °C, but aggregate at 21 °C were selected. Among
them, one was found to undergo abnormal differentiation at 27°C, after it
aggregated at 21 °C. This mutant, KYH-13, was used for the work described in
this paper.
2. Development at 21 °C
When plated on non-nutrient agar, KYH-13 aggregated and usually formed
normal fruiting bodies, at 21 °C. Occasionally, during slug migration, some cells
were left in the trail and became stalk cells. The remaining cells differentiated
into a mass of spores.
When the mutant cells were allowed to develop on high salt (LPS) agar, they
always formed normal fruiting bodies without a migration phase.
3. Development at 27 °C
The mutant did not aggregate at 27 °C, but when the temperature was shifted
to 27 °C after aggregation at 21 °C, the cells formed slugs. After some hours of
migration, a slug stopped and formed a spherical cell mass with a tip-like
protrusion on the original anterior side. Most cells within the cell mass differentiated into spores (Fig. 1). The cells which remained undifferentiated at this stage
later became spores as well, thus completing formation of a spore mass (Fig. 2).
In some cases, however, the tip portion remained undifferentiated, or was
separated from the spore mass and undifferentiated. In any case, the mutant
formed neither stalk cells nor stalk tubes at 27 °C, indicating that it is temperature
sensitive for stalk formation as well as aggregation. The mutant took much
longer to form the spore mass than the fruiting body.
During migration, some slugs divided longitudinally or transversely and each
portion produced a spore mass. Dividing also occurred at 21 °C, but more
frequently at 27 °C. Occasionally, during migration, many cells were left behind
inside the slime trail. Although these cells became stalk cells at 21 °C, they
remained undifferentiated at 27 °C.
The effects of the timing of the temperature shift on the development were
examined. After starvation, cells were allowed to develop at 21 °C and shifted to
27°C every 2h. Normally, cells kept at 21 °C became aggregation competent
(judged by their elongate form) after 8 h and began to aggregate after 10 h. Cells
shifted to 27 °C before 8h did not aggregate, while those shifted after 8h
aggregated and produced spore masses. The results suggest that acquisition of
aggregation competence is temperature sensitive in this mutant.
EMB74
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A. AMAGAI, S. ISHIDA AND I. TAKEUCHI
Fig. 1. Culmination of a KYH-13 slug at 27 °C. Cells stained with neutral red were
allowed to aggregate at 21 °C and then shifted to 27 °C. A phase-contrast
photomicrograph of a squashed spherical cell mass formed at the end of migration,
within which spores formed. The tip and the opposite end contained amoeboid cells.
No stalk formation was observed. x590. v
Fig. 2. A phase-contrast photomicrograph of a squashed spore mass formed at 27 °C.
It comprised only spores. Neither stalk cells nor stalk tubes formed. X420.
4. The effects of cyclic AMP
Since cyclic AMP is known to induce stalk-cell differentiation (Bonner, 1970;
Town, Gross & Kay, 1976), the effects of cyclic AMP on the development of the
mutant was examined at both 21 °C and 27 °C. At 21 °C, cyclic AMP had no effect
when cells were incubated after starvation on agar containing 1 mM-cyclic AMP
and LPS. Hence, cells were first incubated, at 21 °C, on a thin layer of nonnutrient agar until cell aggregates produced tips (this was the most effective stage
to cyclic AMP), and then transferred onto cyclic AMP agar and incubated at
either 21 °C or 27 °C.
When incubated at 21 °C, (Fig. 3), a majority of cells in the aggregate became
stalk cells and the rest were undifferentiated. At 27 °C, however, the cells in the
aggregate remained undifferentiated and only a few spore masses formed. Thus,
cyclic AMP induced the mutant to form stalk cells at 21 °C, but not at 27 °C. At
either temperature, wild-type cells formed globular cell masses, within which the
majority of the cells became stalk cells and the rest were either undifferentiated
or became spores.
5. The proportion of prespore cells
We examined the proportion of prespore to total cells within the mutant slugs,
since they eventually produced spore masses. Cells disaggregated from slugs were
stained with fluorescent antispore serum globulin and the number of stained and
A temperature-sensitive stalkless mutant ofD. discoideum 239
Fig. 3. A KYH-13 aggregate on cyclic AMP agar. An aggregate formed at 21 °C was
transferred to cyclic AMP (1 mM) agar and incubated at 21 °C. The majority of cells
in the aggregate were stalk cells. x620.
Table 1. Changes in proportion of prespore {spore) to total cells during the
development of KYH-13
Stages
standing slugs
3 h migrating slugs
6h migrating slugs
spherical cell masses
spherical cell masses after spore
formation
Incubation temperature
27 °C
21 °C
80-0 ±1-9
80-8 ±3-4
78-9 ±2-4
-
79-0 ±1-7
79-6 ±5-1
74-3 ±2-5
92-2 ±1-2*
/prespores 10-8 ± 4-5\
Vspores 81-3 ±4-6/
Cells were allowed to develop at 21 °C until the standing slug stage, when half of the plates
were shifted up to 27°C. At the stages indicated, cells disaggregated from cell masses were
stained with FITC-conjugated antispore serum globulin and stained and unstained cells were
counted. The values indicate the percentages of prespore (stained) cells with standard deviations, except for the asterisked value which shows the sum of prespores and spores. Over 20
cell masses were used for one determination. The values represent the average of six determinations.
unstained cells were scored. Table 1 showed that prespore proportion within the
standing slugs was approximately 80 %, a value equivalent to that of wild-type
slugs (Hayashi & Takeuchi, 1976).
The proportion did not change during migration for at least 6h. Almost the
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A. AMAGAI, S. ISHIDA AND I. TAKEUCHI
same proportion was also observed in the spherical cell masses formed at 27 °C
at the end of migration (Table 1). Proportions of spores and prespore cells were
determined with spherical cell masses after spores formed. In these cell masses,
many additional prespore cells were found besides mature spores (whose ratio
was equivalent to that of prespore cells in the slugs), resulting in a considerable
increase in the ratio of stained cells (spores plus prespores) (Table 1). This
suggests that after spores formed from pre-existing prespore cells, the remaining
prestalk cells redifferentiated into prespore cells.
6. The distribution of prespore cells
Changes in the distribution of prespore cells during the development of the
mutant were examined, both at 21° and 27 °C. A standing slug of the mutant
formed at 21 °C showed the same staining pattern as that of wild type: the
anterior part of the slug was unstained, whereas the posterior part stained except
for the rear-most part. This staining pattern did not change during migration at
21°C(Fig.4).
On the other hand, slugs which had been shifted up to 27 °C at the standing slug
stage showed different staining pattern. During 3 to 6 h of migration, the area of
the anterior prestalk region of these slugs decreased greatly, while prestalk cells
accumulated at the foot (Fig. 5) or the rear (Fig. 6) of the slugs. The mutant slugs
stained with neutral red showed a staining pattern complementary to that of
immunocytochemical staining (Fig. 7).
At the end of migration, a slug was transformed into a spherical cell mass, in
which prespore cells differentiated into spores. The cell mass at this stage contained groups of cells which were unstained by fluorescent antispore serum
globulin but stained by neutral red, i.e., prestalk cells (Bonner, 1952). However,
sections of cell masses at a later stage revealed the appearance of prespore cells
with prespore antigen among the prestalk cells (Fig. 8). During the process such
prestalk cells were never observed dying on and being eliminated from the cell
mass. It was concluded from this and the above sections' results that after the
Fig. 4. A section of a KYH-13 slug migrating at 21 °C. The section was stained with
FITC-conjugated antispore serum globulin. The prestalk (unstained)-prespore
(stained) pattern was the same as in wild type. X270.
Figs 5, 6. Sections of KYH-13 slugs migrating at 27°C, immunocytochemically
stained as in Fig. 4. Prestalk cells (unstained) decreased in the anterior part (toward
the left) of the slug, but accumulated at the foot (Fig. 5) or the rear (Fig. 6). x300
(Fig. 5), X270 (Fig. 6).
Fig. 7. A squashed KYH-13 slug migrating at 27 °C, vitally stained by neutral red.
Prestalk cells in the anterior (toward the left) and rear parts of the slug were stained.
X270.
Fig. 8. A section of a spherical cell mass containing groups of prestalk cells which
remained amoeboid after the majority of cells had become spores. The section was
immunocytochemically stained, as in Fig. 4. Spores and prespore cells were stained.
The latter were observed among unstained prestalk cells. x520.
A temperature-sensitive stalkless mutant ofD. discoideum 241
242
A. AMAGAI, S. ISHIDA AND I. TAKEUCHI
prespore cells pre-existing in the slug had differentiated into spores, the prestalk
cells regulated to become prespore cells. These prespore cells later became
mature spores.
DISCUSSION
A temperature-sensitive aggregateless and stalkless mutant, KYH-13 could
aggregate and form fruiting bodies at 21 °C, but not at 27 °C. When temperature
was shifted to 27 °C after aggregation at 21 °C, the mutant formed masses of
spores. Formation of neither stalk cells nor stalk tubes was observed. In this
respect, the mutant differs from a stalkless mutant, KY19 (Ashworth & Sussman, 1967), which forms small stalk tubes containing unvacuolized amoebae
(Morrissey & Loomis, 1981). The inhibition of stalk cell differentiation in KYH13 at 27 °C was not lifted by cyclic AMP, although it induced stalk cell formation
at 21 °C. Since vital staining of the mutant slugs indicated differentiation of
prestalk cells at 27 °C, the blockage must occur in the maturation of stalk cells.
Although KYH-13 formed only spores at the terminal developmental stage,
the proportion of prespore cells within the slug was the same as in wild-type.
Similar observations have been made not only with KY19 but also with several
stalky mutants which form only stalks (Morrissey, Farnsworth & Loomis, 1981).
From these findings, they questioned the idea that the pattern of differentiation
at the slug stage is related to the pattern of terminal differentiation.
In the case of KYH-13, however, the present work revealed that after the
prespore cells pre-existing in the slug differentiated into spores, the remaining
prestalk cells were converted to prespore cells which then became spores. This
result indicates that even in this mutant, prestalk-prespore differentiation
precedes and corresponds to terminal stalk-spore differentiation and hence is
consistent with the aforementioned idea.
The conversion probably occurred through the mechanism of proportion regulation analogous to that working in a prestalk isolate of a slug. Since the
mutant cells are unable to become stalk cells but only form spores at 27 °C, the
cell-type conversion could continue until almost all the cells differentiate into
spores. This explains the fact that the mutant took much longer to form spore
masses than fruiting bodies. It is possible that a similar type of continual regulation occurs during fruiting body formation of stalky or stalkless mutants
examined by Morrissey et al. (1981) as well, especially since those mutants take
far longer to culminate than the wild type.
The present work showed that during 3 to 6 h of migration at 27 °C, the
distribution pattern of prespore and prestalk cells in the mutant slug was considerably deranged. Because the proportion of prespore cells did not change
during this period, the pattern changes presumably resulted from redistribution
of prespore and prestalk cells. This suggests some alterations at 27 °C of cell
properties involved in cell sorting, such as adhesiveness or motive force of cells.
A temperature-sensitive stalkless mutant o/D. discoideum 243
The fact that the proportion of prespore cells remained normal in spite of their
deranged distribution indicates that the regulation of proportion is independent
of the formation of pattern. This was originally suggested by Forman & Garrod
(1977) and is consistent with the recent finding of Oyama, Okamoto & Takeuchi
(1983) that dissociated aggregative and slug cells shaken in a medium containing
glucose, albumin and cyclic AMP form small cell clumps, in which prespore
proportion can be regulated in the complete absence of the normal prestalkprespore pattern as observed in slugs.
We are grateful to Dr. M. Filosa of University of Toronto for critically reading the
manuscript. This work was supported in part by a grant (57480011) from the Ministry of
Education of Japan.
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MORRISEY,
(Accepted 18 November 1982)