DEVEJLOPMENTAL
BIOLOGY
149,235-237
(1992)
BRIEF NOTE Cell-Type-Specific Responsiveness to CAMP in Cell Differentiation of Dictyos telium discoideum YOHKO~AMADAAND Department
KOJIOKAMOTO
of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan Accepted October 2, 1991
In Dictyostelium diswideum, both prespore and prestalk differentiation require extracellular CAMP. We investigated the difference in inducibility of the two cell types by CAMP. Previous studies indicate that CAMP added in the early stage of development inhibits prespore differentiation, and this was confirmed using three species of prespore specific mRNAs. By contrast, early treatment with CAMP did not inhibit, but induced the expression of prestalk-specific mRNA. These results indicate that differentiation pathways of the two cell types have different processes in the early stage of development. o 1992 Academic PWS, h. INTRODUCTION
In the cellular slime mould, Dictyostelium discoideum, cells differentiate into two cell types, prespore and prestalk in multicellular agglomerates. Extracellular CAMP is known to be required for differentiation of both cell types (Gerisch, 1987; Firtel et ak, 1989). Another secreted factor, DIF-1, acts in the stage later than CAMP (Sobolewski et ab, 1983) and specifically induces prestalk differentiation and inhibits prespore differentiation (Jermyn et aL, 1987; Early and Williams, 1988; Berks and Kay, 1990). These two factors are considered to act on developing cells and play a role in diversification of cell types. However, it is not clear whether earlier events in development are shared by both prespore and prestalk differentiation pathways. One of the ways to clarify this is to investigate if there is any difference between the two cell types in responsiveness to CAMP in the early stages of development. As for prespore differentiation cells become inducible by CAMP only after passing through several steps of development. The addition of CAMP in the early stages rather inhibits or delays the differentiation (Oyama and Blumberg, 1986; Schaap et aZ., 1986; Mehdy and Firtel, 1985; Yamada and Okamoto, 1990). Prestalk differentiation is also induced by CAMP, but it is not known whether its early addition causes an inhibition. Mehdy and Firtel (1985) and Oyama and Blumberg (1986) examined the inducibility of prestalk mRNAs with CAMP at various stages of development. However mRNA markers used in these studies have now been argued against their cell-type specificity; they have only a few times higher expression in prestalks 235
than in prespores (Jermyn et aL, 1987). Thus it is necessary to reexamine the inducibility using the markers with higher prestalk specificity, such as ecmA or ecmB, whose expression is DIF-1 dependent (Jermyn et aL, 1987). In this report we examined responsiveness of cells to CAMP in expressing prespore and prestalk mRNAs at different developmental stages. Cells of early stages show completely different inducibility of mRNAs of the two cell types, suggesting that prespore and prestalk pathways have different processes in the early stage of development. MATERIALS
AND METHODS
Growth and develqment. D. discoideum NC4 cells were grown in association with washed Eschem’chia coli B/r in 20 mMNa/K, phosphate buffer, pH6.4, in a shaking culture. Cells in an exponential growth phase (2-5 x lo6 cells/ml) were washed free from bacteria, resuspended in 20 mM Mes-KOH, pH6.6, at a density of 1 x 10’ cells/ml, and developed by shaking at 175 rpm. At indicated times cells were collected by centrifugation; resuspended in the original volume of 20 mMMes-KOH, pH6.6, containing 1 mMMgSO,, 1 mMEDTA, and 1 mM CAMP; and shaken. After 4 hr 1 mMcAMP was further added. Where indicated 100 nM DIF-1 (Molecular Probes) was added. The time after starvation (i hr) was indicated as ti. RNA analysis. Cells were lysed by 1% SDS, and RNA was extracted by phenol:chloroform, size fractionated on a 1.2% formaldehyde denaturing gel, transferred to a nylon filter (Gene Screen, New England Nuclear), and 0012-1606/92$3.00 Copyright All rights
0 1992 by Academic Press, Inc. of reprodudion in any form reserved.
236
DEVELOPMENTAL
t9
BIOLOGY
(time al
starved for various periods were supplied with CAMP
andafter 6 hr supplementedwith DIF-1 (seebelow)and for the expression
of DIF-1
dependent
prestalk mRNAs,ecmA,andecmB(Fig. 1B).In contrast
SP96
ti
B
to i-5
t3 m--m
15
t7
8101281012810128
ts
Mm. a)
1012
(tima b)
FIG. 1. Developmental changes in inducibility of prespore-specific (A) and prestalk-specific (B) mRNAs. In (A), cells starved until indicated times (time a) were transferred to medium containing CAMP. After incubation for different periods with CAMP (time b, indicated as the time after CAMP supply), RNA was extracted and analyzed as described under Materials and Methods. (2H3 encodes the SP70 protein.) In (B), cells were treated as in (A) except that 100 nMDIF-1 was added 6 hr after the transfer.
hybridized with cDNA inserts labeled by a randomprimed method (Sambrook et al., 1989). Probes were kindly given by the following persons: 2H3 (SP70), Dr. R. A. Firtel; SP96, Dr. W. F. Loomis; D19 (prespore antigen), Dr. M. Oyama; pDd63 (ecmA) and pDd56 (ecmB), Dr. J. G. Williams. RESULTS of induction
AND DISCUSSION
of prespore mRNAs by CAMP
in early stages of development. Prespore differentiation requires a high concentration of CAMP, but it is known that, in the case of the strain NC4, CAMP inhibits or delays the differentiation if added at early stages of development. We confirmed this by examining the induction of several prespore-specific mRNAs by CAMP with cells at different developmental stages. Cells were starved by shaking for different periods and then transferred to medium containing CAMP. After further incubation for various periods RNA was prepared and analyzed for the expression of prespore-specific mRNAs such as D19, SP70, and SP96 (Fig. 1A). When CAMP was added to ts cells, all species of prespore mRNAs were detected after 8 hr. However if t, or t, cells were treated with CAMP, only a very slight expression was seen up until 16 hr. Therefore CAMP added to cells at early stages inhibited the induction of all species of prespore mRNAs examined. Inducibility
l&,1992
then analyzed
2H3
Inhibition
VOLUME
of prestalk mRNAs with CAMP and DIF-
1. We next asked whether the inhibition tion of CAMP also occurs for prestalk
by early addimRNAs. Cells
to prespore mRNAs, strong induction of both mRNAs was observed in response to DIF-1 when CAMP was added to ts or t, cells. With both cells the expression was detected 8-10 hr after the addition of CAMP. Somewhat lower expression was seen with ts and & cells than with t5 or t, cells in Fig. 1, but in other repeated experiments, the same level of expression occurred with all of these cells. These results indicate that CAMP did not inhibit, but induced, the expression of the prestalk mRNAs even if supplied to cells at early developmental stages. However if CAMP was added to to cells, only very little expression was seen at 12 hr. The above results indicate that even in the early stages of development, differentiation pathways of the two cell types have different processes (Fig. 2); there exists a specific stage in prespore differentiation which is inhibited by CAMP before cells acquire the competence for CAMP dependent induction, but this stage is not involved in the induction of prestalk mRNAs. Early in the development many mRNAs and proteins are regulated by pulsatile signals of CAMP, and the regulation is halted when a high concentration of CAMP is provided (Gerisch, 1987; Firtel et al, 1989). However, prestalk differentiation can proceed even if CAMP was added in early stages. Time course of induction of prestalk mRNAs. We further examined kinetics of the appearance of ecmA and ecmB in response to DIF-1 at different developmental stages. & cells were supplied with CAMP, and after 2,4, 6, or 8 hr DIF-1 was added. Regardless of the time of DIF-1 addition, the expression of ecmA and ecmB was always detected 8-10 hr after CAMP addition (Fig. 3A). Without CAMP, no expression occurred (Fig. 3B). These results indicate that incubation with CAMP for 6-8 hr is required for cells to acquire responsiveness to DIF-1 and that incubation with DIF-1 for 2 hr is sufficient for DIF-1 responsive cells to express these mRNAs. We performed the same kind of experiments using t, cells and obtained essentially the same results (data not shown), suggesting that there was no significant difference be-
CAMP Jfy
PRESPORE
VEGETATIVE
-c
CAMP,
DIF
PRESTALK
FIG. 2. Scheme for differentiation pathway of two cell types. Open arrow: induction by CAMP or by CAMP and DIF-1.
BRIEF
A 2 6slb
4
6
6sO
670
8 6810
(DIF-1 addition (sampling)
1
ecmAecmB-
237
NOTE
The results presented here show that the acquisition of the competence for CAMP dependent induction greatly differs in the two cell types. We are now attempting to investigate the mechanisms involved in these processes. REFERENCES
+ 83
8%
(CAMP) Lsampling)
FIG. 3. Time course of appearance of ecmA and ecmB in response to DIF-1. (A) Cells starved till 4 were transferred to medium containing CAMP. At indicated times after the transfer 100 nMDIF-1 was added. (B) Cells starved till 4 were transferred to medium with (+) or without (-) CAMP. After 6 hr 100 n&f DIF-1 was added. Both in (A) and (B), RNA was prepared at each time point (indicated as the time after the transfer) and analyzed as described under Materials and Methods.
tween ts and & cells in the times needed for acquisition of DIF-1 responsiveness and for mRNA induction. Berks and Kay (1990) reported that cells acquired the competence to express ecmA and ecmB in response to CAMP and DIF-1 at 6-8 hr of development on filters. This is much later than the time when cells become competent in our study (t&. This discrepancy might have arisen from the difference in sampling modes. In our time-course experiments t3 cells acquire DIF-1 responsiveness after 6-8 hr incubation with CAMP, and after 2 more hr the expression was induced in response to DIF1. By contrast, Berks and Kay (1990) examined the expression only l-2 hr after the addition of DIF-1. Therefore, it seems likely that in their study the cells developed for 6-8 hr have already acquired responsiveness to DIF-1 by the action of endogenous CAMP. Wang et al. (1990) reported that treating cells with CAMP in a pulsatile manner was more effective than treating with high concentrations of CAMP to induce responsiveness to DIF-1; hence, it is probable that CAMP pulses emitted during the aggregation phase induce the responsiveness to DIF-1 in normal development.
Berks, M., and Kay, R. R. (1990). Combinatorial control of cell differentiation by CAMP and DIF-1 during development of Dictyostelium discoideum. Development 110,977-984. Early, A. E., and Williams, J. G. (1988). A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro. Develop mf?nt 103,519-524. Firtel, R. A., Van Haastert, P. J. M., Kimmel, A. R., and Devreotes, P. N. (1989). G protein linked signal transduction pathways in development: Dictyostelium as an experimental system. Cell 58,235-239. Gerisch, G. (1987). Cyclic AMP and other signals controlling cell develAnnu. Rev. B&hem. opment and differentiation in Dictyostelium. 56,853-879. Jermyn, K. A., Berks, M., Kay, R. R., and Williams, J. G. (1987). Two distinct classes of prestalk-enriched mRNA sequences in Dictye stelium discoideum. Development 100,745-755. Mehdy, M. C., and Firtel, R. A. (1985). A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictycstelium discoideum Mol. CelL BioL 5, 705-713. Oyama, M., and Blumberg, D. D. (1986). Changes during differentiation in requirements for CAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum. Dev. BioL 117,550-556. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). In “Molecular Cloning” (C. Nolan, Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schaap, P., Van Lookeren Campagne, M. M., Van Driel, R., Spek, W., Van Haastert, P. J. M., and Pinas, J. (1986). Postaggregative differentiation induction by cyclic AMP in Dictyostelium: Intracellular transduction pathway and requirement for additional stimuli. De-u. BioL 118,52-63. Sobolewski, A., Neave, N., and Weeks, G. (1983). The induction of stalk cell differentiation in submerged monolayers of Dktyostelium discoideum Characterization of the temporal sequence for the molecular requirements. D&?rentiation 25,93-100. Wang, M., Roelfsema, J. H., Williams, J. G., and Schaap, P. (1990). Cytoplasmic acidification facilitates but does not mediate DIF-induced prestalk gene expression in Dictyostelium discoideum. Dev. BioL 140,182-188. Yamada, Y., and Okamoto, K. (1990). Three steps in prespore differentiation in Dictyostelium disctideum with different requirements of cellular interaction. Dijbrentiation 44,81-87.
BIOLOGY
149,235-237
(1992)
BRIEF NOTE Cell-Type-Specific Responsiveness to CAMP in Cell Differentiation of Dictyos telium discoideum YOHKO~AMADAAND Department
KOJIOKAMOTO
of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan Accepted October 2, 1991
In Dictyostelium diswideum, both prespore and prestalk differentiation require extracellular CAMP. We investigated the difference in inducibility of the two cell types by CAMP. Previous studies indicate that CAMP added in the early stage of development inhibits prespore differentiation, and this was confirmed using three species of prespore specific mRNAs. By contrast, early treatment with CAMP did not inhibit, but induced the expression of prestalk-specific mRNA. These results indicate that differentiation pathways of the two cell types have different processes in the early stage of development. o 1992 Academic PWS, h. INTRODUCTION
In the cellular slime mould, Dictyostelium discoideum, cells differentiate into two cell types, prespore and prestalk in multicellular agglomerates. Extracellular CAMP is known to be required for differentiation of both cell types (Gerisch, 1987; Firtel et ak, 1989). Another secreted factor, DIF-1, acts in the stage later than CAMP (Sobolewski et ab, 1983) and specifically induces prestalk differentiation and inhibits prespore differentiation (Jermyn et aL, 1987; Early and Williams, 1988; Berks and Kay, 1990). These two factors are considered to act on developing cells and play a role in diversification of cell types. However, it is not clear whether earlier events in development are shared by both prespore and prestalk differentiation pathways. One of the ways to clarify this is to investigate if there is any difference between the two cell types in responsiveness to CAMP in the early stages of development. As for prespore differentiation cells become inducible by CAMP only after passing through several steps of development. The addition of CAMP in the early stages rather inhibits or delays the differentiation (Oyama and Blumberg, 1986; Schaap et aZ., 1986; Mehdy and Firtel, 1985; Yamada and Okamoto, 1990). Prestalk differentiation is also induced by CAMP, but it is not known whether its early addition causes an inhibition. Mehdy and Firtel (1985) and Oyama and Blumberg (1986) examined the inducibility of prestalk mRNAs with CAMP at various stages of development. However mRNA markers used in these studies have now been argued against their cell-type specificity; they have only a few times higher expression in prestalks 235
than in prespores (Jermyn et aL, 1987). Thus it is necessary to reexamine the inducibility using the markers with higher prestalk specificity, such as ecmA or ecmB, whose expression is DIF-1 dependent (Jermyn et aL, 1987). In this report we examined responsiveness of cells to CAMP in expressing prespore and prestalk mRNAs at different developmental stages. Cells of early stages show completely different inducibility of mRNAs of the two cell types, suggesting that prespore and prestalk pathways have different processes in the early stage of development. MATERIALS
AND METHODS
Growth and develqment. D. discoideum NC4 cells were grown in association with washed Eschem’chia coli B/r in 20 mMNa/K, phosphate buffer, pH6.4, in a shaking culture. Cells in an exponential growth phase (2-5 x lo6 cells/ml) were washed free from bacteria, resuspended in 20 mM Mes-KOH, pH6.6, at a density of 1 x 10’ cells/ml, and developed by shaking at 175 rpm. At indicated times cells were collected by centrifugation; resuspended in the original volume of 20 mMMes-KOH, pH6.6, containing 1 mMMgSO,, 1 mMEDTA, and 1 mM CAMP; and shaken. After 4 hr 1 mMcAMP was further added. Where indicated 100 nM DIF-1 (Molecular Probes) was added. The time after starvation (i hr) was indicated as ti. RNA analysis. Cells were lysed by 1% SDS, and RNA was extracted by phenol:chloroform, size fractionated on a 1.2% formaldehyde denaturing gel, transferred to a nylon filter (Gene Screen, New England Nuclear), and 0012-1606/92$3.00 Copyright All rights
0 1992 by Academic Press, Inc. of reprodudion in any form reserved.
236
DEVELOPMENTAL
t9
BIOLOGY
(time al
starved for various periods were supplied with CAMP
andafter 6 hr supplementedwith DIF-1 (seebelow)and for the expression
of DIF-1
dependent
prestalk mRNAs,ecmA,andecmB(Fig. 1B).In contrast
SP96
ti
B
to i-5
t3 m--m
15
t7
8101281012810128
ts
Mm. a)
1012
(tima b)
FIG. 1. Developmental changes in inducibility of prespore-specific (A) and prestalk-specific (B) mRNAs. In (A), cells starved until indicated times (time a) were transferred to medium containing CAMP. After incubation for different periods with CAMP (time b, indicated as the time after CAMP supply), RNA was extracted and analyzed as described under Materials and Methods. (2H3 encodes the SP70 protein.) In (B), cells were treated as in (A) except that 100 nMDIF-1 was added 6 hr after the transfer.
hybridized with cDNA inserts labeled by a randomprimed method (Sambrook et al., 1989). Probes were kindly given by the following persons: 2H3 (SP70), Dr. R. A. Firtel; SP96, Dr. W. F. Loomis; D19 (prespore antigen), Dr. M. Oyama; pDd63 (ecmA) and pDd56 (ecmB), Dr. J. G. Williams. RESULTS of induction
AND DISCUSSION
of prespore mRNAs by CAMP
in early stages of development. Prespore differentiation requires a high concentration of CAMP, but it is known that, in the case of the strain NC4, CAMP inhibits or delays the differentiation if added at early stages of development. We confirmed this by examining the induction of several prespore-specific mRNAs by CAMP with cells at different developmental stages. Cells were starved by shaking for different periods and then transferred to medium containing CAMP. After further incubation for various periods RNA was prepared and analyzed for the expression of prespore-specific mRNAs such as D19, SP70, and SP96 (Fig. 1A). When CAMP was added to ts cells, all species of prespore mRNAs were detected after 8 hr. However if t, or t, cells were treated with CAMP, only a very slight expression was seen up until 16 hr. Therefore CAMP added to cells at early stages inhibited the induction of all species of prespore mRNAs examined. Inducibility
l&,1992
then analyzed
2H3
Inhibition
VOLUME
of prestalk mRNAs with CAMP and DIF-
1. We next asked whether the inhibition tion of CAMP also occurs for prestalk
by early addimRNAs. Cells
to prespore mRNAs, strong induction of both mRNAs was observed in response to DIF-1 when CAMP was added to ts or t, cells. With both cells the expression was detected 8-10 hr after the addition of CAMP. Somewhat lower expression was seen with ts and & cells than with t5 or t, cells in Fig. 1, but in other repeated experiments, the same level of expression occurred with all of these cells. These results indicate that CAMP did not inhibit, but induced, the expression of the prestalk mRNAs even if supplied to cells at early developmental stages. However if CAMP was added to to cells, only very little expression was seen at 12 hr. The above results indicate that even in the early stages of development, differentiation pathways of the two cell types have different processes (Fig. 2); there exists a specific stage in prespore differentiation which is inhibited by CAMP before cells acquire the competence for CAMP dependent induction, but this stage is not involved in the induction of prestalk mRNAs. Early in the development many mRNAs and proteins are regulated by pulsatile signals of CAMP, and the regulation is halted when a high concentration of CAMP is provided (Gerisch, 1987; Firtel et al, 1989). However, prestalk differentiation can proceed even if CAMP was added in early stages. Time course of induction of prestalk mRNAs. We further examined kinetics of the appearance of ecmA and ecmB in response to DIF-1 at different developmental stages. & cells were supplied with CAMP, and after 2,4, 6, or 8 hr DIF-1 was added. Regardless of the time of DIF-1 addition, the expression of ecmA and ecmB was always detected 8-10 hr after CAMP addition (Fig. 3A). Without CAMP, no expression occurred (Fig. 3B). These results indicate that incubation with CAMP for 6-8 hr is required for cells to acquire responsiveness to DIF-1 and that incubation with DIF-1 for 2 hr is sufficient for DIF-1 responsive cells to express these mRNAs. We performed the same kind of experiments using t, cells and obtained essentially the same results (data not shown), suggesting that there was no significant difference be-
CAMP Jfy
PRESPORE
VEGETATIVE
-c
CAMP,
DIF
PRESTALK
FIG. 2. Scheme for differentiation pathway of two cell types. Open arrow: induction by CAMP or by CAMP and DIF-1.
BRIEF
A 2 6slb
4
6
6sO
670
8 6810
(DIF-1 addition (sampling)
1
ecmAecmB-
237
NOTE
The results presented here show that the acquisition of the competence for CAMP dependent induction greatly differs in the two cell types. We are now attempting to investigate the mechanisms involved in these processes. REFERENCES
+ 83
8%
(CAMP) Lsampling)
FIG. 3. Time course of appearance of ecmA and ecmB in response to DIF-1. (A) Cells starved till 4 were transferred to medium containing CAMP. At indicated times after the transfer 100 nMDIF-1 was added. (B) Cells starved till 4 were transferred to medium with (+) or without (-) CAMP. After 6 hr 100 n&f DIF-1 was added. Both in (A) and (B), RNA was prepared at each time point (indicated as the time after the transfer) and analyzed as described under Materials and Methods.
tween ts and & cells in the times needed for acquisition of DIF-1 responsiveness and for mRNA induction. Berks and Kay (1990) reported that cells acquired the competence to express ecmA and ecmB in response to CAMP and DIF-1 at 6-8 hr of development on filters. This is much later than the time when cells become competent in our study (t&. This discrepancy might have arisen from the difference in sampling modes. In our time-course experiments t3 cells acquire DIF-1 responsiveness after 6-8 hr incubation with CAMP, and after 2 more hr the expression was induced in response to DIF1. By contrast, Berks and Kay (1990) examined the expression only l-2 hr after the addition of DIF-1. Therefore, it seems likely that in their study the cells developed for 6-8 hr have already acquired responsiveness to DIF-1 by the action of endogenous CAMP. Wang et al. (1990) reported that treating cells with CAMP in a pulsatile manner was more effective than treating with high concentrations of CAMP to induce responsiveness to DIF-1; hence, it is probable that CAMP pulses emitted during the aggregation phase induce the responsiveness to DIF-1 in normal development.
Berks, M., and Kay, R. R. (1990). Combinatorial control of cell differentiation by CAMP and DIF-1 during development of Dictyostelium discoideum. Development 110,977-984. Early, A. E., and Williams, J. G. (1988). A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro. Develop mf?nt 103,519-524. Firtel, R. A., Van Haastert, P. J. M., Kimmel, A. R., and Devreotes, P. N. (1989). G protein linked signal transduction pathways in development: Dictyostelium as an experimental system. Cell 58,235-239. Gerisch, G. (1987). Cyclic AMP and other signals controlling cell develAnnu. Rev. B&hem. opment and differentiation in Dictyostelium. 56,853-879. Jermyn, K. A., Berks, M., Kay, R. R., and Williams, J. G. (1987). Two distinct classes of prestalk-enriched mRNA sequences in Dictye stelium discoideum. Development 100,745-755. Mehdy, M. C., and Firtel, R. A. (1985). A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictycstelium discoideum Mol. CelL BioL 5, 705-713. Oyama, M., and Blumberg, D. D. (1986). Changes during differentiation in requirements for CAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum. Dev. BioL 117,550-556. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). In “Molecular Cloning” (C. Nolan, Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schaap, P., Van Lookeren Campagne, M. M., Van Driel, R., Spek, W., Van Haastert, P. J. M., and Pinas, J. (1986). Postaggregative differentiation induction by cyclic AMP in Dictyostelium: Intracellular transduction pathway and requirement for additional stimuli. De-u. BioL 118,52-63. Sobolewski, A., Neave, N., and Weeks, G. (1983). The induction of stalk cell differentiation in submerged monolayers of Dktyostelium discoideum Characterization of the temporal sequence for the molecular requirements. D&?rentiation 25,93-100. Wang, M., Roelfsema, J. H., Williams, J. G., and Schaap, P. (1990). Cytoplasmic acidification facilitates but does not mediate DIF-induced prestalk gene expression in Dictyostelium discoideum. Dev. BioL 140,182-188. Yamada, Y., and Okamoto, K. (1990). Three steps in prespore differentiation in Dictyostelium disctideum with different requirements of cellular interaction. Dijbrentiation 44,81-87.
