Spore maturation occurs during normal development in Dictyostelium when environmental influences induce a migrating slug to transform into a fruiting body. As the amoeboid prespore cells turn into refractile spores there is a burst of enzyme accumulation, including UDP-galactose epimerase, and at a later stage the exocytosis of preformed components of the spore coat. Evidence is presented here that this process is triggered by an elevated intracellular cAMP concentration.

First, a number of rapidly developing (rde) mutants, whose cAMP metabolism had been investigated previously, are shown to be able to form spores in submerged monolayers, whereas wild-type strains are not. The phenotypes of these mutants are best explained by a derepression of the signal transduction pathway utilizing intracellular cAMP.

Second and more direct, it is shown that the permeant cAMP analogues 8-Br-cAMP and 8-chlorophenylthio-cAMP, but not cAMP itself, can rapidly induce spore differentiation in wild-type amoebae incubated in submerged monolayers. These analogues also stimulate accumulation of UDP-galactose epimerase in slug cells transferred to shaken suspension.

The ability to induce spore differentiation with Br-cAMP in wild-type strains provides a new technique that can be exploited in various ways. For instance, spore differentiation in strain V12M2 is induced by 8-Br-cAMP at very low cell densities, suggesting that neither cell contact nor additional soluble inducers are necessary in these conditions. In contrast NC4 cells may require an additional inducer. Spore differentiation is inhibited by the stalk-specific inducer DIF-1 suggesting that DIF-1 inhibits a target downstream of intracellular cAMP in the signal transduction pathway inducing spore differentiation.

The migrating slug of Dictyostelium discoideum is an arrested stage of development which can persist for days before it is triggered by suitable environmental conditions to transform into a mature fruiting body. As this transformation proceeds, there is a radical reorganization of the slug, accompanied by the overt differentiation of stalk and spore cells from their amoeboid precursors. The prestalk cells sequentially vacuolate and lay down cellulose as they move into the growing tip of the stalk. The prespore cells are carried aloft by the stalk and transform synchronously into refractile-walled spores in a process that involves a burst of accumulation of enzymes such as UDP-galactose epimerase (Newell & Sussman, 1970) and, at a later stage, the exocytosis of preformed components of the spore coat (Hohl & Hamamoto, 1969; Maeda & Takeuchi, 1969).

The transformation of a slug into a fruiting body (culmination) can be triggered by overhead light, a drop in humidity or the loss of ammonia from the aggregate (Newell et al. 1969; Schindler & Sussman, 1977). In addition the slugs appear to accumulate a low Mr metabolite, possibly a weak acid, that is necessary for culmination (Sussman et al. 1978). Presumably these influences must be transduced into the individual cells within the slug and integrated to trigger changes in both gene expression and cellular morphogenesis.

The initial differentiation of prespore cells appears to be induced by extracellular cAMP (Kay et al. 1978; Kay, 1979) and, since there is evidence that cAMP signalling persists to later stages of development (Bonner, 1949; Nestle & Sussman, 1972; Schaap & Wang, 1984), some modulation of cAMP signalling may be involved in triggering spore maturation. cAMP signals are transduced into at least three intracellular second messengers: cAMP itself, cGMP and inositol phosphates (Gerisch, 1987; Europe-Finner & Newell, 1987). However, the individual roles of the second messengers in producing changes in gene expression, cell movement and morphogenesis have not yet been clearly distinguished.

The hypothesis will be advanced here that spore maturation is triggered by elevated levels of intracellular cAMP. This idea was first suggested by the phenotypes of certain mutants facilitated in spore maturation and then powerfully supported by the use of permeant cAMP analogues to induce spore formation in wild-type strains. The techniques for spore induction then allowed further questions to be asked about the possible role of signals, such as cell-cell contact, in wild-type spore differentiation.

8-(4-chlorophenylthio)-cyclic AMP was from Boehringer, cAMP (cAMP), 2′deoxy-cyclic-AMP and 8-bromo-cAMP (Br-cAMP) were from Sigma. Br-cAMP was also synthesized by bromination of cAMP with bromine water (Muneyama et al. 1971). The precipitate was purified by first dissolving in water by bringing to pH∼8 with KOH and then reprecipitating with HC1. The product remained pale brown after 3 cycles of purification but did not contain detectable impurities on TLC (Muneyama et al. 1971, system B) and behaved identically with the commercial material in the experiments to be described. DIF-1 was synthesized as described (Masento et al. 1988), and synthetic discadenine was a kind gift from Dr Y. Tanaka (Abe et al. 1976).

The sporogenous mutant HM15 derives ultimately from strain V12M2 and was selected from its immediate parent, HM2, by virtue of its ability to form detergent-resistant spores when incubated in a monolayer with cAMP (Town et al. 1976; Kay et al. 1978; Kay, 1987). The rapidly developing mutants HTY 217, HTY 506, HTY 507 and HTY 509 were a kind gift from Drs K. Abe and K. Yanagisawa (Abe & Yanagisawa, 1983) and Frl7 (Sonneborn et al. 1963) from Dr C. D. Town. Cells were grown on Klebsiella aerogenes and prepared for development as previously described (Kay, 1987). Slugs were obtained by allowing cells to develop on 1·8% L28 agar (Oxoid) containing 20 mm-KCl, 20mm-NaCl, 1 mm-CaCl2 and after 18 h harvested, partially disaggregated by syringing through a 19-gauge needle and resuspended at a nominal cell density of 2×107cellsml−1. Suspensions were shaken in conical flasks at 180 revs min−1. The medium for development, in suspension or in monolayers in Sterilin tissue culture dishes, was 10mm-2-(N-morpholino)-ethanesulphonic acid, 20 mm-KCl, 20mm-NaCl, Imm-MgCb, Imm-CaCl2 pH 6·2 containing 15 μg ml−1 tetracycline, 200 μg ml−1 streptomycin sulphate and cyclic nucleotides as indicated (‘spore medium’, Kay, 1982, 1987). Cell differentiation was monitored by phase-contrast microscopy.

UDP-galactose-4-epimerase (EC 5.1.3.2) was assayed by a coupled spectrophotometric assay (Telser & Sussman, 1971) at 35°C and protein by a dye-binding assay (Bradford, 1976).

Mutants in spore maturation

The initial clue linking intracellular cAMP to spore maturation came from the phenotypes of two sets of independently isolated mutants in which spore maturation is facilitated or ‘derepressed’. The sporogenous mutants were isolated because they are able to form spores in submerged monolayers with cAMP, whereas their parents arrest as amoeboid prespore cells and never (<1 in 105) form spores in the same conditions

(Town et al. 1976; Kay et al. 1978). These mutants are therefore considered to be facilitated in the maturation of prespore cells into spores. The rapidly developing (rde) mutants were isolated because they form spores prematurely in normal development (Sonneborn et al. 1963; Kessin, 1977; Abe & Yanagisawa, 1983) and were of interest because their lesion had been linked to an altered intracellular cAMP metabolism (see below) and because of their phenotypic resemblance to some of the sporogenous mutants. For instance, in conditions suitable for normal development, both the sporogenous mutant HM15 and the rdeC mutants arrest as mounds and produce spores several hours earlier than their respective parents. Similarly HM18 and the rdeA mutants arrest as early culminates and spore differentiation is again premature. These similarities suggested that some of the rde and sporogenous mutants might be allelic. Unfortunately a direct genetic test of this idea is difficult, because the two groups of mutants were isolated in the V12 and NC4 backgrounds, which are incompatible in parasexual crosses (Robson & Williams, 1979). However, it has already been shown that the rdeA mutant Fr17 is sporogenous (Town et al. 1976) and Table 1 shows that all rde mutants of both available complementation groups (rdeB is lost) are sporogenous, that is they make spores in monolayers when incubated with cAMP. Kessin (1977) suggested that the rde phenotype might be due to an overproduction of intracellular cAMP, which in turn acted as an inducer of developmental gene expression. Altered cAMP metabolism in the rde mutants was subsequently confirmed by direct measurement (Coukell & Chan, 1980; Abe & Yanagisawa, 1983). Rde A mutants have elevated intracellular cAMP levels as expected, but surprisingly rdeC mutants have very low levels. However, this paradoxical property of rdeC mutants can be explained within the original hypothesis, since in both yeast and mammalian cells cAMP levels are controlled by negative feedback acting through the cAMP-dependent protein kinase (Nikawa et al. 1987; Gettys et al. 1987). Thus a constitutively active protein kinase would feed back to inhibit adenyl cyclase and produce low cAMP levels, as seen in the rdeC mutants, while producing the downstream effects of elevated cAMP levels.

Table 1
graphic
graphic

The results with the spore maturation mutants suggest that elevated intracellular levels of cAMP induce spore maturation, but a more direct test of this idea was required.

Spore induction in wild-type strains by permeant cAMP analogues

In mammalian cells, many of the effects of hormones that use intracellular cAMP as a second messenger can be mimicked using high extracellular concentrations of certain cAMP analogues. These analogues can bypass the relevant surface receptor by penetrating the plasma membrane and activating cAMP-dependent protein kinase directly. The most potent analogues, such as those with an 8-substitution of the adenine ring, are effective because they are both more resistant to hydrolysis by cAMP-phosphodiesterase and better able to activate the cAMP-dependent protein kinase than cAMP itself (Simon et al. 1973; Miller et al. 1975). The most promising analogue for Dictyostelium cells seemed to be 8-bromo-cAMP (Br-cAMP) which has about a 7-fold increased Km for the phosphodiesterase and a 3-fold decreased KA for the protein kinase compared to cAMP (Van Haastert et al. 1983; de Wit et al. 1982). Concentrations of 0·1–1 mm-Br-cAMP are usually necessary with mammalian cells but higher concentrations were also explored with Dictyostelium cells, because of their relative impermeability.

It is apparent from Fig. 1 that high concentrations of Br-cAMP can induce greater than 70% spore formation amongst amoebae of strain V12M2 incubated from the start of development with the inducer in submerged monolayers. In these experiments, spore formation started after about 16 h. Spores could also be induced in strain NC4 though less efficiently (see later). The induced spores stain with a spore-specific antibody (Takeuchi, 1963) and retain full viability after detergent treatment, which kills all amoebae (0·3% cemulsol for 2h; not shown). Spore formation can be detected at 5 mm-Br-cAMP and is half-maximal at 11mm-Br-cAMP. Of a number of other analogues tested over a range of concentrations only 8-chlorophenylthio-cAMP was active. It was roughly as potent as Br-cAMP but unfortunately it could not be used above 8 mm due to precipitation in the incubation medium. The following were inactive at up to 40mm: cAMP, dibutyryl-cAMP, 8-bromo-cGMP, dibutyryl-cGMP, 2-deoxy cAMP.

Fig. 1

Induction of spore differentiation by Br-cAMP in submerged monolayers of cells of strain V12M2. Left, dose-response curve with cells at a density of 5 × 103cm−2. Right, phase-contrast micrographs of cells at 104cm−2 incubated without Br-cAMP (top) or with 20 mm-Br-cAMP (bottom). Amoebae were incubated for 48 h in tissue culture plates containing spore medium plus 100 μ g ml−1 BSA and the appropriate concentrations of Br-cAMP. Results from 2 dose-response experiments are pooled.

Fig. 1

Induction of spore differentiation by Br-cAMP in submerged monolayers of cells of strain V12M2. Left, dose-response curve with cells at a density of 5 × 103cm−2. Right, phase-contrast micrographs of cells at 104cm−2 incubated without Br-cAMP (top) or with 20 mm-Br-cAMP (bottom). Amoebae were incubated for 48 h in tissue culture plates containing spore medium plus 100 μ g ml−1 BSA and the appropriate concentrations of Br-cAMP. Results from 2 dose-response experiments are pooled.

The experiments described so far show clearly that Br-cAMP can induce starving cells to differentiate into spores, but do not indicate when Br-cAMP (rather than cAMP) acts to do this. Three observations suggest that it is the maturation of prespore cells into spores that can be specifically promoted by Br-cAMP but not by cAMP. First, cAMP is able to induce starving cells to differentiate as far as prespores but not spores in similar monolayer incubation conditions (Kay et al. 1978; Kay, 1982). Second, Br-cAMP induces prespore cells, taken from migrating slugs, to differentiate into spores with a delay of only 3–4 h compared to the 16 h delay with vegetative cells. Again, spores do not form with cAMP (not shown). Finally a biochemical marker for spore maturation, UDP-galactose epimerase (Newell & Sussman, 1970), is rapidly induced when Br-cAMP is added to slug cells in shaken suspension (Fig. 2). In these conditions cAMP does not induce the enzyme, though it does stabilize existing levels.

Fig. 2

Induction of UDP-galactose epimerase, a marker for culmination, by Br-cAMP. Migrating slugs at t18 were partially disaggregated in spore medium and the suspension shaken at 180 rev min−1 with the additions indicated (cyclic nucleotides were 15 mm). Duplicate 1·5 ml portions were assayed for enzyme activity and proteins as described in Materials and methods. The experiment is representative of 4.

Fig. 2

Induction of UDP-galactose epimerase, a marker for culmination, by Br-cAMP. Migrating slugs at t18 were partially disaggregated in spore medium and the suspension shaken at 180 rev min−1 with the additions indicated (cyclic nucleotides were 15 mm). Duplicate 1·5 ml portions were assayed for enzyme activity and proteins as described in Materials and methods. The experiment is representative of 4.

Mode of action of Br-cAMP

Several arguments indicate that Br-cAMP cannot be inducing spore maturation solely by occupation of the known surface cAMP receptor: (1) receptor saturating concentrations of Br-cAMP (2 mm, about 20 times the receptor KD for Br-cAMP; Van Haastert & Kein, 1983) do not induce spore maturation (Fig. 1); (2) high concentrations of agonists (cAMP, 2′-deoxy-cAMP) with a much greater affinity for the surface receptor than Br-cAMP are without effect; (3) spore induction by Br-cAMP is not inhibited by equimolar cAMP, though this should displace nearly all the Br-cAMP from the surface receptor (the KD for cAMP is about 450-fold lower than that for Br-cAMP; van Haastert & Kein, 1983; result not shown). It therefore seems most likely that Br-cAMP works by penetration of the cell membrane and activation of the intracellular response machinery in Dictyostelium, as in mammalian cells.

Involvement of other signals

The technique just described for inducing wild-type cells to differentiate into spores allows a number of further questions to be asked about the factors controlling spore differentiation. For instance, spore differentiation might require, in addition to Br-cAMP, some form of interaction between the cells in the monolayer. The interaction might require either cell-cell contact or the accumulation of a diffusible inducer but in either case it would be attenuated at low compared to high cell density. Fig. 3 shows that spore differentiation in strain V12M2 is in fact very efficient at low densities, where the cells are all single. This result is similar to that obtained previously with various sporogenous mutants (Kay, 1982) and seems to preclude any essential role in spore induction for cell-cell contact or diffusible inducers in these conditions. The reduced efficiency of spore formation at high cell density is probably due to accumulated DIF diverting the amoebae to stalk formation (see Fig. 4). Spore formation by cells of strain NC4 is always less efficient than with V12M2 cells, being rarely greater than 30% at high cell density and falling to zero at 103 cells cm−2 (not shown). One contributing factor is that the NC4 spores tend to hatch out to give amoebae soon after they form. Hatching can be reduced by including 10 μM-discadenine (a spore germination inhibitor, Abe et al. 1976) in the medium, but even in this case NC4 cells do not form spores at low density, suggesting that an additional factor is necessary (see Grabel & Loomis, 1978; Mehdy & Firtel, 1985; Berks & Kay, 1988). The putative factor has not been characterized but preliminary experiments indicate that it is not methionine or ammonia, which do not improve the efficiency of spore formation by low-density NC4 cells at 5mm and 20 mm, respectively (not shown; Gibson & Hames, 1988; Gross et al. 1983).

Fig. 3

Cell-density dependence of spore differentiation in strain V12M2. Cells were plated at the stated densities in tissue culture dishes containing spore medium plus 15 mm-Br-cAMP, 100 μg ml−1 BSA and 10 μg ml−1 of the spore germination inhibitor discadenine. Cell differentiation was scored microscopically at t48. At low cell densities most cells become spores, whereas at high density DIF accumulates and stalk cells differentiate in consequence. Spores: • — •; stalk cells: ▴ — ▴.

Fig. 3

Cell-density dependence of spore differentiation in strain V12M2. Cells were plated at the stated densities in tissue culture dishes containing spore medium plus 15 mm-Br-cAMP, 100 μg ml−1 BSA and 10 μg ml−1 of the spore germination inhibitor discadenine. Cell differentiation was scored microscopically at t48. At low cell densities most cells become spores, whereas at high density DIF accumulates and stalk cells differentiate in consequence. Spores: • — •; stalk cells: ▴ — ▴.

Fig. 4

DIF-1 diverts cells of strain V12M2 from spore to stalk cell differentiation. Vegetative cells were incubated in tissue culture dishes at a density of 5 × 103 cm−2 in spore medium supplemented with 100 μg ml−1 BSA, 20mm-Br-cAMP and DIF-1 as indicated and spores scored microscopically after 40 h. The results of 2 experiments, each done with duplicate plates, are combined. • — • spore cells; ▴ — ▴ stalk cells. DIF-1 also suppressed spore formation when slug-stage cells were incubated under the same induction conditions (not shown).

Fig. 4

DIF-1 diverts cells of strain V12M2 from spore to stalk cell differentiation. Vegetative cells were incubated in tissue culture dishes at a density of 5 × 103 cm−2 in spore medium supplemented with 100 μg ml−1 BSA, 20mm-Br-cAMP and DIF-1 as indicated and spores scored microscopically after 40 h. The results of 2 experiments, each done with duplicate plates, are combined. • — • spore cells; ▴ — ▴ stalk cells. DIF-1 also suppressed spore formation when slug-stage cells were incubated under the same induction conditions (not shown).

DIF-1 (l-[3,5-dichloro-2,6-dihydroxy-4-methoxy-pheny1]hexan-1-one; Morris et al. 1987) is an endogenous stalk-specific inducer which has been shown to inhibit prespore and spore differentiation, diverting the cells to differentiate instead into stalk cells (Kay & Jermyn, 1983). It has been suggested that the inhibition of spore differentiation by DIF-1 is a consequence of an inhibition of cAMP binding to its surface receptor (Wang et al. 1986). Such an inhibition would be bypassed by Br-cAMP if it acts intracellularly. However, since spore formation induced by Br-cAMP is still sensitive to inhibition by DIF-1 (Fig. 4), it appears that DIF-1 must also have a target further down the signal transduction pathway than intracellular cAMP, at least at the time of spore maturation.

The hypothesis underlying the experiments described here is that spore maturation can be triggered by an especial elevation in intracellular cAMP levels. This hypothesis was first suggested by the altered cAMP metabolism in mutants where spore maturation occurs more readily than in the wild type and is strongly supported by the induction of spore maturation in wild-type strains by permeant cAMP analogues. There are several further consequences of this hypothesis.

First, elevated intracellular cAMP levels may trigger spore maturation during normal development as well as during monolayer incubation. In support of this, several studies, including one where the aggregates were individually staged, show that cAMP levels increase 2- to 3-fold as spores mature during culmination (Brenner, 1978; Abe & Yanagisawa, 1983; Merkle et al. 1984). It is possible that in the single exception, where only a small rise in cAMP levels was detected, the strain A3 used did not develop with sufficient synchrony to produce a strong increase in cAMP levels (Brenner, 1978). The rise in cAMP levels during culmination could be brought about by a modulation of the basic cAMP signalling system by some other signal. For instance, a drop in ammonia levels can trigger culmination (Schindler & Sussman, 1977) and would be expected to produce an elevation in intracellular cAMP levels (Williams et al. 1984).

Second, the hypothesis suggests a number of lesions that might account for the sporogenous and rde phenotypes. Since all the mutants tested are genetically recessive, they could represent the knock-out of different inhibitory elements in the cAMP signal transduction pathway. Targets might include a Gi protein affecting adenyl cyclase, the regulatory subunit of cAMP-dependent protein kinase and intracellular cAMP phosphodiesterase.

Finally, intracellular cAMP may stimulate differentiation at other stages of development apart from during culmination (Sampson et al. 1978; Kessin, 1977). This idea is attractive even though it has been shown that the expression of certain aggregative and postaggregative genes can be induced without the normal oscillatory increases in intracellular cAMP (Wurster & Bumann, 1981; Oyama & Blumerg, 1986). Even in these cases, adenyl cyclase is sufficiently active to produce intracellular concentrations of cAMP in the pM range, which should be adequate to stimulate fully the cAMP-dependent protein kinase or other cAMP-binding proteins (Sampson, 1977; de Gunzberg & Veron, 1982; Tsang & Tasaka, 1986). A role for intracellular cAMP at earlier stages of development is further suggested by the acceleration of early gene expression in the rde mutants (Sonneborn et al. 1963; Kessin, 1977; Abe & Yanagisawa, 1983).

Spore differentiation by monolayers of wild-type cells has not been described before (a preliminary report appeared in Kay et al. 1988) and this technical advance allows a number of further questions to be asked about the control of spore differentiation. One question is whether cell-cell contact is necessary for cell differentiation, as has been suggested by indirect experiments (Mehdy et al. 1983; Chisholm et al. 1984). Cell contact is clearly not necessary in strain V12M2 since isolated cells at great dilution form spores efficiently in the Br-cAMP medium (see also Kay & Trevan, 1981; Kay, 1982, for similar results with sporogenous mutants). Strain NC4 differs from V12M2 in that both spore and stalk cell differentiation (Berks & Kay, 1988) are very inefficient at low cell density. The reason for this is not known but it could be due to a stringent requirement for a soluble factor early in development whereas in V12M2 cells this requirement is more relaxed (Grabel & Loomis, 1978; Mehdy & Firtel, 1985). A second question is where is the target for the inhibition of spore cell differentiation by DIF-1 (Kay & Jermyn, 1983). In principle, this target could be at any point in the signal transduction pathway leading from extracellular cAMP to overt spore differentiation and the cAMP receptor has been suggested as a potential target (Wang et al. 1986). However, the present results suggest an additional target below intracellular cAMP in the pathway. Finally it has been suggested that cell-cycle phase at the time of starvation may determine whether an individual cell differentiates toward a stalk or a spore cell (Gomer & Firtel, 1987). In the experiments described here amoebae can be switched from about 90% spore to 90% stalk cell differentiation merely by adding DIF-1 to the starvation medium. This, and many other results showing cell-type regulation during development (e.g. Raper, 1940), indicate that cell-type differentiation is regulated by interactions between the cells and is not predetermined by intrinsic differences between them.

I should like to thank Jeff Williams, Mary Berks, David Traynor, Ines Carrin, Robert Insall, Jenny Brookman and Mark Bretscher for comments on the manuscript.

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