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.

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 & 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.

Culture

D. discoideum NC4 cells were grown in a nutrient medium (1/75 M-phosphate buffer, pH 6·2, 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 × 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 (32μm openings) and resuspended at 107 cells/ml in 20mM-KK2-phosphate buffer, pH6·0, containing 5% glucose, 2% albumin, ImM-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 pH 6·0 were washed, deposited on a Millipore filter placed on pads soaked with 40 mM-phosphate buffer, pH 6·2, containing 2·5mM-MgC12 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, pH 6·0, 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, pH 7·0, containing 0·001% neutral red and dissociated with pipetting. After being placed on a slide glass, dissociated cells were observed under a microscope.

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 (10−310−4 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 × 105–2 × 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 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.

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 20HIM KK2-phosphate buffer (pH6·0) containing 5 % glucose, 2 % albumin, 1 mMcAMP 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.

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 20HIM KK2-phosphate buffer (pH6·0) containing 5 % glucose, 2 % albumin, 1 mMcAMP 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.

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, 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).

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 glucose-albumin-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 μm long.

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 glucose-albumin-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 μm long.

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.

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) 1 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.

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) 1 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.

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 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.

Fig. 4.

Cell type conversion in the liquid culture of fractionated slug cells. Prestalk (▫) and prespore (▵) cells were fractionated from dissociated slug cells in a Percoll density gradient. Fractionated and unfractionated (○) cells were transferred to the liquid shake culture and prespore proportions were determined at times. Open and closed marks show independent experiments.

Fig. 4.

Cell type conversion in the liquid culture of fractionated slug cells. Prestalk (▫) and prespore (▵) cells were fractionated from dissociated slug cells in a Percoll density gradient. Fractionated and unfractionated (○) cells were transferred to the liquid shake culture and prespore proportions were determined at times. Open and closed marks show independent experiments.

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 × 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 two-zoned 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 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.

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