The differentiation-inducing factor, DIF, was previously shown to induce stalk cell differentiation in Dictyostelium discoideum cells incubated as submerged monolayers. We investigated the mechanism that regulates the differentiation of stalk cells in the intact organism. It was found that in migrating or submerged slugs DIF cannot induce stalk cell differentiation, which is most likely due to the presence of a DIF antagonist. Cyclic AMP and ammonia were earlier reported to act as DIF antagonists in vitro. We show here that ammonia, but not cAMP, acts as an antagonist for DIF-induced stalk cell differentiation in vivo. DIF can induce stalk cell differentiation when ammonia levels in the slug are enzymically depleted. However, depletion of cAMP levels does not increase the efficacy of DIF. We propose that the induction of stalk cell differentiation during early culmination may be triggered by a drop in ammonia levels inside the organism.

The life cycle of the cellular slime mould Dictyostelium discoideum represents a comparatively simple model system to study the regulation of cell differentiation and pattern formation. The system becomes increasingly attractive, because several compounds that can induce differentiation in vitro have been identified (see Williams, 1988).

D. discoideum development can be subdivided into several major events of stage- or cell-type-specific gene expression. Starvation initiates the synthesis of a large number of gene products involved in the aggregation process (see Kessin, 1988). This type of gene expression is accelerated by the chemotactic signal itself, i.e. nanomolar cAMP pulses (Darmon et al. 1975; Gerisch et al. 1975).

Spore-specific gene expression is first evident after aggregation and remains restricted to the basal part of the aggregate and later to the posterior region of the slug (Hayashi & Takeuchi, 1976; Morrissey et al. 1984; Krefft et al. 1984). Spore-specific gene expression can be induced in vitro by cAMP concentrations in the micromolar range (Kay, 1982; Schaap & Van Driel, 1985; Qyama & Blumberg, 1986) and elevated extra cellular cAMP levels are essential for prespore differentiation in vivo (Wang et al. 1988). Adenosine, a cAMP degradation product, inhibits prespore differentiation (Weijer & Durston, 1985; Schaap & Wang, 1986; Spek et al. 1988) and is considered to be essential for the establishment of the anterior prespore-free region in slugs (Schaap & Wang, 1986; Wang et al. 1988).

Stalk cell differentiation is specifically initiated during early fruiting body formation at the apex of the culminating structure. The process involves the synthesis of at least 10 new proteins, (Kopachik et al. 1985; Morrissey et al. 1984), extreme vacuolization of the cells and the synthesis of a fibrous cellulose cell wall (Raper & Fennell, 1952). Two stalk-specific genes are expressed several hours earlier and are specifically present at the anterior region of migrating slugs (Williams et al. 1987; Jermyn et al. 1987).

The synthesis of stalk-specific proteins as well as the expression of the two early stalk genes can be induced in vitro by the stalk-inducing factor DIF (Kay & Jermyn, 1983; Kopachik et al. 1985; Williams et al. 1987; Sobolewski & Weeks, 1988). However, the regulation of stalk cell differentiation in vivo is not immediately evident from the spatiotemporal distribution of this factor. DIF levels increase after aggregation and are maximal at the slug stage of development (Brookman et al. 1982), which is several hours before culmination takes place. Furthermore, no evidence for increased DIF levels at the apex of slugs and fruiting bodies has yet been found; it rather appears that DIF concentrations are somewhat higher at the posterior than at the anterior region of the slug (Brookman et al. 1987).

The induction of stalk cell differentiation by DIF is inhibited in vitro by cAMP (Berks & Kay, 1988) and by ammonia, which was proposed to act as a natural DIF antagonist (Gross et al. 1983). It was shown much earlier by Schindler & Sussman (1977) that a drop in ammonia levels triggers the culmination process. These investigators proposed that stalk cell differentiation was induced by a combination of high cAMP and low ammonia levels (Sussman & Schindler, 1978). The role of cAMP in this model is contradicted by the observed inhibitory effects of cAMP on stalk cell differentiation (Berks & Kay, 1988), but if cAMP is replaced by DIF, the proposed role of ammonia may still be valid.

In order to gain insight in control mechanisms operative in the intact organism, we investigated whether DIF can induce stalk cell differentiation in migrating and submerged slugs and whether either cAMP or ammonia levels in the slug antagonize the effects of DIF. Our results show that ammonia, but not cAMP, acts as an natural DIF antagonist for stalk cell differentiation.

Materials

DIF-1 was obtained as a kind gift from Dr Robert R. Kay, or isolated and HPLC purified as described by Kay et al. (1983). One unit of DIF is defined as the amount of DIF that induces one percent of D. discoideum V12M2 cells to differentiate as stalk cells in the DIF bioassay (Brookman et al. 1982). Calcofluor, 4’,6’-diamidino 2-phenyl-indole (DAPI), L-gluta-mate dehydrogenase and α-ketoglutarate were from Sigma (St Louis, USA). Beef heart cAMP-phosphodiesterase and NADH were from Boehringer (FRG), cellulase Onozuka was from Serva (FRG) and FITC-conjugated swine anti-rabbit IgG was from Dakopatts (Denmark).

Culture and incubation conditions

D. discoideum NC4 cells were grown in association with Escherichia coli on glucose-peptone agar (Schaap & Spek, 1984). Vegetative cells were freed from bacteria by repeated washing with 10mm-Na/K phosphate pH 6-5 (PB) and plated on non-nutrient agar (1-5% agar in PB) or on dialysis membrane supported by PB agar. The cells were incubated at 22°C until migrating slugs had formed. Membranes carrying slugs were then transferred to agar containing various DIF concentrations and allowed to migrate further for an additional 6h period. Alternatively about 10 migrating slugs were carefully transferred from agar to 1 ml screw cap septum vials containing 400 µl PB pH 7-0 or 400µ1 10 mm-imidazole buffer pH7-5 with various additives. The tubes were flushed for 30s with O2, closed and rotated at 10 rev min−1 at 21 °C during 6 to 12 h. Every hour the tubes were replenished with fresh O2.

Histological procedures

In order to study effects of the different treatments on pattern formation, intact slugs were fixed in ice-cold methanol, embedded in paraplast and cut into serial sections of about 5 qm thickness. In the case of slugs migrating on dialysis membranes, slugs plus membranes were embedded to preserve the original orientation of the slugs. Slug sections were either stained with prespore-specific rabbit IgG (PSRI) and FITC-conjugated swine anti-rabbit IgG (SARFITC) or with 0·02% Calcofluor in phosphate-buffered saline (freshly diluted before use from 1% Calcofluor in ethanol).

To study effects on cell-type proportions, slugs were dissociated into single cells and small cell clumps by repeated aspiration through a 25-gauge needle. The cells were then allowed to adhere to glass slides, fixed in methanol and respectively stained with PSRI and SARF1TC or with 0-02% Calcofluor. Cells stained with prespore antiserum were counterstained with 0·2 µg ml−1 DAPI. Using a Leitz fluorescence microscope equipped with appropriate filters, the ratio of prespore cells (cell containing at least three FITC-stained vacuoles) versus total cells (cells stained with DAPI) was determined by counting. Calcofluor and DAPI fluorescence cannot be separated by filters; to determine the proportion of stalk cells, stalk cells were identified by Calcofluor fluorescence, while the total number of cells was measured by using the phase-contrast facility on the same microscope.

Transmission electron microscopy

Slugs were fixed in a mixture of 83 mm-glutaraldehyde, 26 mm-OsO4 and 60mm-sucrose in 100 HIM sodium-cacodylate buffer pH 7-4. After postfixation in 1% OsO4, the slugs were dehydrated, embedded in Agar 100 epoxy resin, sectioned and observed with a Jeol CX100 transmission electron microscope as described before (Schaap et al. 1982).

Effects of DIF on cell differentiation in migrating and submerged slugs

We first studied the effects of DIF on migrating D. discoideum NC4 slugs. Early migrating slugs were transferred to agar which contained 3000 units ml−1 of DIF. After 6h of migration, the slugs were fixed in methanol, embedded in paraffin and sectioned. Fig. 1A show a section of a DIF-treated slug, which was stained with prespore-specific antiserum. DIF treatment has induced the disappearance of prespore antigen in the region proximal to the DIF agar, but no formation of mature stalk cells were observed. Similar effects of DIF have also been found by Kay and co-workers (personal communication).

Fig. 1

Effects of DIF on slug pattern. (A) D. discoideum NC4 slugs, developed on dialysis membrane, were transferred with the membrane to agar that contained 3000units ml−1 of DIF. After 6h of migration, the slugs were sectioned and stained with prespore antiserum. DIF induced a loss of prespore antigen at the side of the slug, proximal to the DIF agar. Alternatively, slugs were submerged in phosphate buffer (B) or buffer containing 5000 units ml−1 DIF (C) and incubated in roller tubes for 6h at 10 rev min−1 in an oxygen atmosphere. In control slugs, the prespore-staining pattern remained preserved under these conditions (B). In slugs submerged in DIF, a general decrease in prespore staining occurred. However, a complete loss of prespore antigen was never observed and neither could any differentiation of vacuolated stalk cells be detected (A: ×190; B: ×160; C: ×180).

Fig. 1

Effects of DIF on slug pattern. (A) D. discoideum NC4 slugs, developed on dialysis membrane, were transferred with the membrane to agar that contained 3000units ml−1 of DIF. After 6h of migration, the slugs were sectioned and stained with prespore antiserum. DIF induced a loss of prespore antigen at the side of the slug, proximal to the DIF agar. Alternatively, slugs were submerged in phosphate buffer (B) or buffer containing 5000 units ml−1 DIF (C) and incubated in roller tubes for 6h at 10 rev min−1 in an oxygen atmosphere. In control slugs, the prespore-staining pattern remained preserved under these conditions (B). In slugs submerged in DIF, a general decrease in prespore staining occurred. However, a complete loss of prespore antigen was never observed and neither could any differentiation of vacuolated stalk cells be detected (A: ×190; B: ×160; C: ×180).

To obtain a more efficient penetration of DIF, intact slugs were submerged in oxygenated buffer (Sternfeld & Bonner, 1977) containing DIF (Fig. 1C). Submersion in buffer did not affect the anteroposterior pattern significantly in the absence of DIF (Fig. 1B). After 6h of incubation with DIF, a general reduction of prespore antigen was observed (Fig. 1C). It must, however, be noted that the effects of DIF on prespore pattern as represented in Fig. 1A and C represent the more extreme examples of loss of prespore antigen.

To quantify the effects of DIF on prespore proportions, slugs were dissociated into single cells after DIF treatment. After 6h of submersion in 10 000 units ml−1 DIF the proportion of prespore cells was reduced from 66 to 56%, while 30000 units ml−1 DIF caused a reduction to 38%. At higher DIF concentrations, cell viability was affected. Prolonged incubation with DIF induced a somewhat further decrease in the proportion of prespore cells, but even after 10h of DIF treatment, virtually no mature stalk cells could be detected (Table 1).

Table 1

Cell-type proportions in submerged slugs after various treatments

Cell-type proportions in submerged slugs after various treatments
Cell-type proportions in submerged slugs after various treatments

Effect of elimination of possible DIF antagonists on pattern formation in slugs

It was previously reported that both cAMP and ammonia antagonize DIF-induced stalk cell differentiation in vitro (Gross et al. 1983; Berks & Kay, 1988). To investigate whether secretion of cAMP or ammonia by slug cells inhibits the induction of stalk cell differentiation by DIF in vivo, we depleted extracellular cAMP levels by treatment with cAMP-phosphodiesterase, while ammonia levels were depleted by means of a glutamate dehydrogenase mixture which use ammonia as a substrate (Schindler & Sussman, 1977). It was necessary to perform these experiments on slugs submerged in buffer, since application of the glutamate dehydrogenase mixture to migrating slugs was reported to rapidly induce the transition of migrating slugs into early fruiting structures (Schindler & Sussman, 1977).

Treatment of slugs with increasing amounts of cAMP-phosphodiesterase induced a progressive reduction of the proportion of prespore cells (Table 1) as was reported before (Wang et al. 1988), but no significant differentiation of stalk cells could be observed. The small amount of stalk cells, which appears after 10 h treatment with 15 000 units ml−1 DIF is not further increased by addition of cAMP-phosphodiesterase.

More striking results were obtained when slugs were incubated with DIF in combination with the glutamate dehydrogenase mix. Irregularly shaped clumps of stalk cells appeared within 6h of incubation. After 10h of incubation, DIF treatment (in the presence or absence or glutamate dehydrogenase mix) generally caused a severe disruption of slug morphology. In the presence of glutamate dehydrogenase large amounts of fully differentiated stalk cells had been formed (Fig. 2A-D), as was evident by Calcofluor staining (Harrington & Raper, 1968) and by ultrastructural characteristics as large vacuoles and a fibrous cell wall (Fig. 2E,F). As far as could be judged from the disrupted cell masses, the stalk cells tended to be formed preferentially at the periphery of the cell mass (Fig. 2A,B). Isolated small groups of mature stalk cells were also often observed (Fig. 2B,C).

Fig. 2

Effects of DIF and ammonia depletion on slug pattern. Migrating slugs were submerged during 10 h in 400µl of 70mm-α-ketoglutarate, 01 mm-NADH, 0 03 units ml−1 L-glutamate dehydrogenase in lOmm-imidazole buffer pH 7-5 to which 6000units DIF were added. (A-D) Sections of methanol-fixed slugs stained with 0-02% calcofluor (A,B: ×50; C: ×240; D: ×280). (E,F) TEM preparations of slugs fixed in glutaraldehyde and OsO4, showing the extreme vacuolization and fibrous cell wall that is characteristic for mature stalk cells (E: ×13000; F: ×52 000).

Fig. 2

Effects of DIF and ammonia depletion on slug pattern. Migrating slugs were submerged during 10 h in 400µl of 70mm-α-ketoglutarate, 01 mm-NADH, 0 03 units ml−1 L-glutamate dehydrogenase in lOmm-imidazole buffer pH 7-5 to which 6000units DIF were added. (A-D) Sections of methanol-fixed slugs stained with 0-02% calcofluor (A,B: ×50; C: ×240; D: ×280). (E,F) TEM preparations of slugs fixed in glutaraldehyde and OsO4, showing the extreme vacuolization and fibrous cell wall that is characteristic for mature stalk cells (E: ×13000; F: ×52 000).

Control experiments, in which cells were incubated with DIF in combination with boiled glutamate dehydrogenase, or in which the substrate α-ketoglutarate was replaced by the product L-glutamate did not result in any significant differentiation of stalk cells.

Quantification of the effect of the different treatments on the proportion of stalk cells and prespore cells showed that ammonia depletion combined with DIF addition causes 22% of the cells to differentiate into stalk cells (Table 1). Both treatments alone yielded about 3% stalk cells. Ammonia depletion does not affect the proportion of prespore cells, while 15 000 units ml−1 DIF reduces the proportion of prespore cells from 62 to 40% after 10 h of incubation.

We investigated whether DIF can induce stalk cell differentiation in vivo and whether cAMP and ammonia function as DIF antagonists. It was found that DIF causes a moderate reduction in the proportion of prespore cells in migrating and submerged slugs, but causes only 2% of the cells to differentiate into stalk cells. In combination with enzymic depletion of ammonia, DIF induces stalk cell differentiation in 22% of the cells within 10 h of incubation. Enzymic depletion of ammonia in the absence of added DIF leads to stalk cell differentiation in about 3% of the cells, without affecting the proportion of prespore cells. Depletion of cAMP, another putative DIF antagonist, does not promote stalk cell differentiation, but results in the almost complete disappearance of prespore cells as was previously reported (Wang et al. 1988). Apparently, despite its antagonistic effects on stalk cell differentiation in vitro (Berks & Kay, 1988), extracellular cAMP is not critically involved in the regulation of this type of differentiation in vivo. Possibly, the cAMP levels in the prestalk region of the slug are not sufficiently high to inhibit stalk cell differentiation.

During normal development, DIF levels are maximal at the early slug stage (Brookman et al. 1982), but the synthesis of the majority of stalk-specific proteins does not start earlier than the culmination stage (Kopachik et al. 1985). Our data suggest that ammonia depletion during early culmination may trigger the differentiation of stalk cells during normal development.

A sudden drop in ammonia levels could be a simple consequence of the culmination process and the position of the tip region during culmination, as was earlier suggested by Sussman & Schindler (1978). As soon as the slug stops migrating and erects itself, the exchange of volatile compounds between the cell mass and the atmosphere becomes more efficient. The tip region, which is obviously at the best position to exchange volatile compounds is furthermore relatively narrow compared to the rest of the cell mass, which results in a relatively large surface-to-volume ratio. Loss of ammonia due to evaporation may therefore occur most efficiently at this region. The expression of two stalkspecific genes, pdD63 and pDd56, coincides with the increase in DIF levels during normal development (Williams, 1988). It is possible that the expression of these genes is less sensitive to inhibition by ammonia than the expression of the majority of stalk genes.

The process of stalk formation does not only involve the differentiation of stalk cells, but also the construction of a cellulose stalk tube at the centre of the prestalk region. (Raper & Fennell, 1952). It is as yet difficult to imagine that such a topologically complicated process could simply result from local ammonia depletion. It is likely that a drop in ammonia levels permits the transition of prestalk into stalk cells but that the actual formation of the stalk, is controlled by a more intricate regulatory mechanism.

We are grateful to Gerda Lamers for skilful transmission electron microscopy.

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