DEVELOPMENTALBIOLOGY 140,182-188(1990)
Cytoplasmic Acidification Facilitates but Does Not Mediate DIF-Induced Prestalk Gene Expression in Dictyostelium discoideum MEIWANG,*JEROENH.ROELFSEMA,*JEFFREYG.WILLIAMS,SANDPAULINESCHAAP* *Cell Biology and Genetics Unit, Zoological Laboratory, L&den University, Kaiserstraat 63,2511 GP Leiden, The Netherlands; and slmperial Cancer Research Fund, Glare Hall kborataries, South Mimms, Herts. EN6 SL, England Accepted March 5,1990 Stalk cell differentiation in Dictyostelium can be induced by the differentiation-inducing factor, DIF, or by conditions that decrease intracellular pH (pHi). We have investigated whether cytoplasmic acidification acts directly to induce expression of pDd56 and pDd63, two DIF-regulated genes, specifically expressed in prestalk cells. The weak base methylamine, which increases pHi, inhibits DIF-induced transcription. The weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi, stimulates DIF-induction of the two prestalk genes. After relatively long incubation periods, DMO also induces a low level of prestalk gene expression in the absence of added DIF. However, unlike DIF-mediated induction, the apparent DMO-mediated induction decreases to undetectable levels when the cell density is reduced from 10’ to 106cells/ml. This indicates that DMO does not itself induce gene expression, but acts to enhance the effects of an autonomously secreted stalk-inducing factor, presumably DIF. These results suggest that the effects of DIF on gene expression are regulated by intracellular PH. _ but do not support a role for protons as direct intermediates in the DIF signal transduction pathway.- 0 1990 Academic Press, Inc. INTRODUCTION
posed pH, changes directly affect the earliest events in cellular differentiation, the expression of genes specifiDuring Lktyostelium discoideum development, unically expressed in prestalk or prespore cells. cellular amoebae aggregate to form multicellular strucWe recently found that CAMP induction of prespore tures, which enter into a program of stage and cell typegene expression was accompanied by a sustained inspecific gene expression, controlled by intercellular sigcrease in pH,. However, artificial elevation of pH, could naling. This program results in the differentiation of a not induce prespore gene expression, and inhibition of mass of spores supported by a column of stalk cells. Two the CAMP-induced pH, increase did not inhibit prespore morphogenetic signaling molecules, responsible for the gene expression (Van Lookeren Campagne et aL, 1989). induction of differentiation, have been identified. Cyclic These results show that CAMP-induced cytoplasmic alAMP, acts as a chemoattractant during aggregation, kalinization does not mediate the effect of CAMP on preand also induces spore cell differentiation (Kay, 1982; spore gene expression. Mehdy et aa, 1983; Wang et al, 1988), while DIF, a chloriIn this study, we have investigated the effect of pH, nated alkyl phenone (Morris et ah, 1987), induces the modulation on the expression of the prestalk genes, differentiation of stalk cells (Town et ah, 1976; Kay and pDd56 and pdD63 (Jermyn et ah, 1987). These genes are Jermyn, 1983; Williams, et aZ.,1987). It is not yet known first expressed in the tight aggregate stage of develophow these two molecules act to control cell type-specific ment (Williams et aZ., 1989) and their transcripts are gene expression. However, several studies have sugspecifically localized to prestalk and stalk cells (Jermyn gested that morphogen-induced intracellular pH (pHi) et al, 1987). In vitro, expression of these genes requires changes may determine the choice between differentiaexogenous DIF (Jermyn et al., 1987), and also depends on tion pathways: a period of prior exposure to millimolar CAMP, to induce Agents which supposedly increase pHi, such as high responsiveness to DIF. Thereafter expression of the extracellular pH, and treatment with weak bases such pDd56 gene is repressed by CAMP, while that of the as ammonia, promote spore and inhibit stalk cell differpDd63 gene is relatively unaffected (Berks and Kay, entiation, while agents which decrease pHi, such as low 1988). extracellular pH or addition of weak acids, favor stalk We show that DIF induction of pDd56 and pDd63 gene and inhibit spore differentiation (Gross et al., 1983; expression responds to changes in pHi, but our data do Town, 1984; Dominov and Town, 1986; Bradbury and Gross, 1989). All these experiments utilized prolonged not support a direct role for protons as intracellular secincubation of cells with the various agents (12 hr up to ond messengers in the process. We furthermore show several days) and hence do not show whether the pro- that responsiveness to DIF is more efficiently induced 0012-1606/90 $3.00 Copyright All rights
Q 1990 hy Academic Press, Inc. of reproduction in any form reserved.
182
WANGETAL.
by nanomolar CAMP pulses than by millimolar concentrations. MATERIALS
AND
183
pHi Regulation of Prestalk Gene Expression
CAMP
METHODS
Materials [14C]Chloramphenicol, [‘4C]acetyl-coenzyme A (CoA) and [a-32P]dATP were from Amersham (UK). 5,5dimethyl-2,4-oxazolidinedione (DMO), methylamine, CAMP, and G418 were from Sigma (St. Louis, MO). Acetylcoenzyme A was from Pharmacia (USA). Thin-layer chromatography plates were from Kodak (USA). Gene Screen was from New England Nuclear (UK). Chemically synthesized DIF-1 was a kind gift of Dr. R. Kay (MRC, Cambridge, UK). Cell Lines and Incubation Conditions D. discoideum AX-2 cells were transformed with the integrating vector PBlOTPl, containing a fusion of either the pDd63 or the pDd56 promotor and the bacterial chloramphenicol transferase (CAT) gene. These two prestalk-specific promotors direct correct temporal and cell type-specific CAT expression. (Ceccarelli et al, 1987; McRobbie et ab, 1988; Jermyn et aZ.,1989). The transformants were grown in axenic medium containing G418 at 10 pg/ml to a cell density of 2 X lo6 cells/ml. Cells were harvested and washed three times with 10 mM phosphate buffer, pH 6.5 (PB), resuspended to 5 x lo6 cells/ml, and shaken at 120 rpm and 21°C in PB. After 1 hr of incubation, the cells were shaken for a further 4 hr with 1 mM CAMP. Alternatively, the cells were treated during 4 hr with 20 nM CAMP pulses added at 6-min intervals. Following preincubation with CAMP, the cells were washed, resuspended in fresh PB to 3 x lo6 cells/ml, and incubated for variable time periods with DIF, DMO, or methylamine as indicated in the figure legends. The cells were collected for the assay of total cytosolic CAT activity or for the analysis of stalk-specific mRNAs.
toradiography of the TLC plates, the labeled spots were scratched off and measured by scintillation counting (Gorman et al., 1982). Method 2: The extract from 3 X lo6 cells was incubated for 2 hr at 37°C in a total volume of 40 ~10.25 MTris, pH 7.8, containing 1 mM chloramphenicol and 0.1 &i (12 nmole) [14C]acetyl-CoA. The reaction was stopped by adding 500 ~1 ice-cold ethylacetate. The layers were mixed by vigorous vortexing and separated by centrifugation for 3 min at 10,OOOg. The radioactivity of 300 ~1of the organic phase was measured by scintillation counting (Sleigh, 1986). RNA Isolation and Analysis Total cellular RNA from 3 X 10’ lysed cells was isolated and purified according to Mann and Firtel (1987). RNA (10 pg) was size fractionated on formaldehyde gels, transferred to Gene Screen membranes, and hybridized to 32P-labeled cDNA inserts as described by Peters et al. (1989). After hybridization and washing, the membranes were exposed to X-ray films. RESULTS
Induction of Competencefor Prestalk Gene Expression
In order to facilitate measurement of their expression, the promoters and 5’ proximal regions of the pDd56 and pDd63 genes were fused to the bacterial CAT gene. During normal development, the expression of pDd63 and pDd56 is first evident at the late aggregate stage (Williams et aZ., 1989). In vitro, the expression of both genes can be induced by DIF (Williams et al, 1987), provided that the cells have been made competent by pretreatment with millimolar CAMP for several hours (Berks and Kay, 1988). Figure 1 shows that DIF rapidly induces the expression of the pDd63-CAT and pDd56-CAT in competent cells, to reach a maximum after about 2 hr of stimulation. In this experiment, the induction of competence was achieved by treating preaggregative cells for 4 hr CAT Assay with 1 mM CAMP. This is somewhat unphysiological, lo7 cells were pelleted and resuspended in 100 ~1 of since during early aggregation, cells are not exposed to 0.25 M Tris, pH 7.8. Cells were lysed by three cycles of high constant CAMP stimuli. We measured whether freezing-thawing and centrifuged for 15 min at 10,OOOg. competence for DIF induction can also be achieved by The supernatant was heated for 15 min at 65°C to inac- the physiological stimulus of this developmental stage, tivate endogenous CAT inhibitors. CAT activity was i.e., nanomolar CAMP pulses. measured by two different methods: Cells were first starved for 1 hr in PB and were subseMethod 1: The extract from 5 X lo6 cells was incubated quently incubated in 1 mM CAMP or stimulated every 6 for 2 hr at 37°C in a total volume of 100 ~10.25 M Tris, min with a 20 nM CAMP pulse for 1,2,3, or 4 hr. SubsepH 7.8, containing 1 &i (0.02 pmole) [14C]chlorampheni- quently cells were incubated for a fixed period (2.5 hr) co1 and 0.5 mM acetyl coenzyme A. Acetylated with 50 nM DIF-1 (Fig. 2). It appeared that nanomolar [14C]chloramphenicol was separated from the nonacety- CAMP pulses induce subsequent responsiveness to DIF lated substrate by thin-layer chromatography. After au- faster and to higher levels than millimolar CAMP con-
184
DEVELOPMENTALBIOLOGY
vOLUME140,1090 B
POd56
PDd63
m3
0
30
60
90
120
160
30
60
Control
90 100 nM
120
160
Time (mid
/-• 1'
OF
0
/~/,-o~I 1
DIF-1
2
/
-
3 TIMEEhl
,*A
0
1
2 &,h,
FIG. 1. Time course of pDd63 and pDd56 expression in response to DIF. Vegetative pDd63 and pDd56 CAT transformants were starved for 1 hr
in PB and subsequently incubated for 5 hr with 1 mMcAMP to induce DIF competence. Cells were resuspended at 3 X 10scells/ml in fresh PB and incubated at 150 rpm and 20°C in the presence (0) or absence (0) of 100 nM DIF-1. At the indicated time periods, cytosolic CAT activity was assayed, using [“CJchloramphenicol as substrate (method 1). (A) pDd63-CAT expression, autoradiograph of the different acetylated forms of [“C]chloramphenicol separated by TLC. (B) Quantitated data expressed as percentage of the highest level of CAT induction during incubation.
centrations. In all subsequent experiments, we therefore used a 4-hr pretreatment with 20 nM CAMP pulses to induce competence. Eflects of Extracellular
pH on Prestalk Gene Expression
Incubation of cell monolayers at relatively low pH has been reported to induce the differentiation of stalk cells (Gross et al., 1983; Town, 1984; Town and Dominov, 1986). We investigated the effect of extracellular pH (pH,) on the expression of the pDd63 and pDd56 genes in the presence and absence of DIF (Fig. 3). Expression of the pDd56 and pDd63 genes requires DIF and cannot be induced by reducing the extracellular pH to 5.0. (Below this pH, cell viability is seriously affected.) The induction of pDd56 and pDd63 expression by DIF occurs equally efficient at pH 5,6, and 7, but is markedly inhibited at pH 8.
ml
W56
POd63
Eflects of Intracellular
pH on Prestalk Gene Expression
pHi can be manipulated by treating cells with weak acids or weak bases (Aerts et al, 1985; Inouye, 1988a). The weak acid DMO causes a 0.2-unit decrease in pH, at a concentration of 5 mM, while the weak base methylamine at 2.5 mM causes a 0.3-unit increase (Van Lookeren Campagne et u& 1989; Van Duijn, unpublished data). We measured the effects of DMO and methylamine on the expression of pDd56 and pDd63 in the presence and absence of DIF. Figure 4 shows that the DIFinduced expression of both genes is inhibited by methylamine. pDd56 expression seems to be somewhat more sensitive to this inhibitory effect than pDd63 expression. In contrast, the weak acid DMO stimulates the DIF-induced expression of pDd63 and pDd56; the effect saturates at about 2 mM DMO (Fig. 5). We confirmed that the CAT constructs mirror the behavior of the endogenous genes by analyzing mRNA accumulation in the presence of methylamine and DMO (Fig. 6).
1 POd63
f 0
1
0
2 Odion
of4pre+rea+men+
12
3
4
(h)
FIG. 2. Acquisition of competence for DIF-induced prestalk gene expression. Vegetative pDd63 or pDd56 transformants were starved for 1 hr in PB and subsequently incubated with either 1 m&f CAMP added as a single dose (open symbols), or with 20 nMcAMP added as pulses with 6-min intervals (closed symbols). At the indicated time periods, cells were collected and resuspended in fresh PB. Cells were subsequently incubated for 2.5 hr with 50 nM DIF and assayed for cytosolic CAT activity according to method 2.
O$ e
FIG. 3. Effect of extracellular pH on prestalk gene expression. Competent pDd56 or pDd63 cells were incubated in PB of the indicated pH values in the presence or absence of 50 n&f DIF-1. After 2.5 hr cytosolic CAT activity was assayed. Means and SE of two experiments performed in triplicate are presented.
WANG
PDd56 I
PDd63
185
pHi Regulation of Prestulk Gem Expressim
ET AI..
PB DMO PH6.6 DIF DMO+DlF
I
PB CHSNHZ pH7.4 0
2
4
6
E mM CH$H2
0
10
-6
2
6
4
8
I3 10
mM CH+H2
4. Effect of methylamine on prestalk gene expression. Competent pDd63 and pDd56 cells were incubated in PB of pH 7.4 in the presence (closed symbols) or absence (open symbols) of 50 nM DIF-1 with the indicated methylamine concentrations. After 2.5 hr cytosolic CAT activity was assayed. Means and SE of three experiments performed in triplicate are presented. FIG.
In the absence of added DIF, DMO induces prestalk gene expression (Figs. 5, ‘7,8), but much more slowly and to a lower level than DIF. The most obvious alternative interpretations of this result are: (i) DMO induces prestalk gene expression, but at a much lower efficiency than DIF. (ii) DMO acts to enhance the effect of endogenously produced DIF. (iii) DMO induces DIF synthesis, which subsequently induces prestalk gene expression. To discriminate between these possibilities, we measured the effects of DMO and DIF at different cell densities. If the first supposition were true, then a reduction of cell density should not diminish the effects of DMO, but if either of the two last suppositions are true, the effect of DMO should be much lower at low cell densities, where less DIF is produced. When the cell density is reduced from 10' to lo5 cells/ml, DMO cannot induce pDd56 or pDd63 expression anymore (Fig. 8). The effect
PH7.4 CH~NHZ+DIF
DIF
-
FIG. 6. Effects of DMO and methylamine on prestalk mRNA levels. Competent cells were incubated in PB of the indicated pH to which 2.5 mM DMO, 5 mM methylamine, 50 nM DIF, or combinations of DIF with either weak acid or base had been added. After 2.5 hr of incubation, total RNA was isolated. Northern transfers were probed with q-labeled pDd63 or pDd56 cDNA inserts, respectively.
of DIF is unaffected by reducing the cell density. Although reduction of cell density might change some variable, which is necessary for induction by DMO, this variable is evidently not important for induction by DIF. It therefore seems more likely that DMO cannot itself induce prestalk gene expression, but acts to either induce DIF synthesis or to stimulate the effect of endogenously produced DIF. We next measured the effects of a fixed concentration of DMO at different DIF concentrations. In the absence of DIF, the effect of DMO is smaller than when DIF is present (Fig. 9). At saturating DIF concentrations, DMO induces a twofold increase of pDd63 expression. This suggests that DMO does not act by inducing the production of DIF, because if this were the case the effect of DMO would be highest at the lowest DIF concentration and should become negligible at saturating DIF concentrations. We therefore conclude that DMO acts as a DIF synergist, but not as a DIF agonist.
PDd56
POd63 I -.----I
TX-
-
1
I-----
z5 I/ i= / %wA t
p
0
12
1/
3 mtl OMO
4
5
0
12
3
4
5
mMMO
5. Effects of DMO on prestalk gene expression. Competent pDd63 or pDd56 cells were incubated in PB of pH 6.6 in the presence (0) or absence (0) of 50 n2MDIF-1 with the indicated DMO concentrations. After 2.5 hr cytosolic CAT activity was assayed. Means and SE of three experiments performed in triplicate are presented. FIG.
TIME(h) FIG. 7. Time dependency of DMO and DIF effects on pDd63 expression. Competent pDd63 cells were incubated in PB pH 6.6, without further additions (O), with 5 mMDM0 (O), with 50 nMDIF-1 (a), or with 5 mil4 DMO plus 50 nM DIF-1 (w). At the indicated time periods, cytosolic CAT activity was assayed. Means of two experiments are presented.
DEVELOPMENTALBIOLOGY
VOLUME 140.1990
lar Ca’+ efflux; although it could be that the pHi drop produced by DMO is insufficient to affect the Ca2+chann’ nels. A much simpler possibility is that the binding of DIF to its presumptive intracellular receptor is pH sensitive. Earlier data showed that weak acids and bases, respectively, promote the differentiation of stalk and spore cells, as measured by the ratio of mature cell types, or the accumulation of spore and stalk-specific 0 5 6 7 0 5 6 1 gene products. (Gross et al., 1983; Town, 1984; Dominov logkells/mli logkellr/mll and Town, 1986; Bradbury and Gross, 1989). Weak acids FIG. 8. Effect of cell density on DMO and DIF induced gene expresand bases neither inhibit nor promote the transcription sion. Competent pDd56 and pDd63 cells were incubated at the indiof prespore genes and the synthesis of spore antigens cated cell density in PB pH 6.6 (0), with 5 mMDMO (0) or with 50 r&f DIF-1 (m). After 3.5 hr of incubation cytosolic CAT activity was mea- (Van Lookeren Campagne et al, 1989). The present data sured. Means of three experiments are presented. show that a pHi decrease promotes DIF-induced prestalk gene expression, but cannot induce expression in the absence of DIF. DISCUSSION The main difference between the previous experiments and our studies is the time-scale over which the We investigated, the effects of pHi modulation by weak acids and bases on prestalk gene expression and effects of PHi modulation were measured. In our experifound that the weak base methylamine inhibits DIF- ments prestalk gene expression was measured 2-3 hr induced expression of the prestalk genes pDd63 and after exposure to weak acids or bases and prespore gene pDd56. Inhibition of pDd56 was half-maximal at 5 mM expression after 4-6 hr. All previous experiments inmethylamine. At this concentration, methylamine does volved exposure from 12 hr up to 4 days (Gross et al, not affect CAMP-induced expression of the prespore 1983; Dominov and Town, 1986; Bradbury and Gross, gene D19, or of the cysteine proteinase-2 gene (Van Loo- 1989). The fact that weak acids increase DIF responsivekeren Campagne et ab, 1989 and unpublished data), sug- ness may explain their long-term stimulatory effects on gesting that the inhibitory effect of methylamine on stalk cell differentiation. The observation that weak acids also stimulate DIF accumulation (Kwong and stalk gene expression is not due to a general inhibition of transcription. Weeks, 1989) may be an additional factor affecting cell The weak acid DMO stimulates the DIF-induced ex- fate over the long term. pression of pDd56 and pDd63 and induces a low level of In addition to inducing prestalk gene expression, DIF expression of both genes in the absence of added DIF. In also inhibits CAMP-induced prespore gene expression at contrast to the effect of DIF, the inductive effect of the transcription level (Wang et ah, 1986; Early and WilDMO is strongly cell density dependent. Stimulatory ef- liams, 1988). This means that DIF synergists, as weak fects of weak acids on DIF production have been reported (Kwong and Weeks, 1989). However, the stimulatory effects of DMO are much stronger in the presence than in the absence of added DIF and remain high at saturating DIF concentrations. This suggests that under our conditions DMO does not induce gene expression by stimulating DIF secretion. We conclude that DMO acts to enhance the effect of DIF, but cannot, itself, induce transcription of prestalk genes. These data suggest that the induction of prestalk gene expression is not mediated by protons. This conclusion is supported by the observation that DIF does not induce a sustained decrease in pHi (Kay et al., 1986; Inouye, 1988a). It appears rather, that the pHi deternM DIF mines the responsiveness of cells to DIF. The observa9. Effects of DMO at increasing DIF concentrations. Competion that cytoplasmic acidification is ineffective in the tentFIG.pDd63 cells were incubated during 2.5 hr in the presence (0) or absence of DIF does not support’s recent model (Gross absence (0) of 5 mMDM0 with 20,35,50,70 &of DIF-1. After 2.5 hr, et al., 1988), wherein DIF and protons both act to open a cytosolic CAT activity was assayed. Means and SE of two experiments vesicular chloride channel resulting in reduced vesicu- are presented. ‘Dd63
‘Dd56
m------m
m-------m
0%---r-x
WANG ET AL.
pHi Regulation
acids, and DIF antagonists, as weak bases, will respectively inhibit or promote prespore gene expression under conditions where DIF is added, or allowed to accumulate. The conclusion that pHi modification alone does not induce the expression of cell type-specific genes does not mean that pH, is not an important factor. Developing cells secrete fairly large amounts of the weak base ammonia (Schindler and Susman, 1977), which inhibit stalk cell differentiation in vitro (Gross et ak, 1983) and furthermore inhibit the transition from migration to culmination (Schindler and Sussman, 1977). Local ammonia depletion may occur during normal development at the apex and base of the culminating structure during early fruiting body formation (Sussman and Schindler, 1978). We recently showed that DIF can only induce stalk cell differentiation in viva when endogenously produced ammonia is removed (Wang and Schaap, 1989). Stimulatory effects of ammonia depletion or CO,induced acid load on stalk cell differentiation in migrating slugs were also shown by Inouye (1988b), but in these experiments it is not clear whether the effects result directly from inducing stalk cell differentiation or indirectly from inducing the slug/fruit switch. The effects of NH, may not be confined to culmination, but may also be involved in cell type-regulation in the slug. A class of mutants, which are hypersensitive to the inhibitory effects of NH, upon culmination (Newell and Ross, 1982), display a reduced ratio of prestalk to prespore cells in the migratory slug (MacWilliams and David, 1984). Cyclic AMP induces a sustained increase in pH, (Van Lookeren Campagne et al, 1989) and prespore cells have a higher intracellular pH and higher resistance to acid load than prestalk cells (Inouye, 1985, 1988a). It is therefore possible, that prestalk cell differentiation is dictated in part by factors, such as ammonia and CAMP, which control responsiveness to DIF, rather than to the specific spatial distribution of DIF itself. Such a model would help to explain the apparent paradox, that the concentration of DIF in the back of the slug is higher than that in the front. (Brookman et ah, 1987). REFERENCES AERTS,R. J., DURSTON,A. J., and MOOLENAAR,W. H. (1985). Cytoplasmic pH and the regulation of the Dictyostelium cell cycle. Cell 43, 653-657. BERKS,M., and KAY, R. R. (1988). Cyclic AMP is an inhibitor of stalk cell differentiation in Dietyostelium discoideum. Dev. Biol. 126, 108-114. BRADBURY,J. M., and GROSS,J. D. (1989). The effect of ammonia on cell-type-specific enzyme accumulation in Dictyostelium discoideum. Cell LX&r. Dev. 27,121-128. BROOKMAN,J. J., JERMYN, K. A., and KAY, R. R. (1987). Nature and distribution of the morphogen DIF in the Dietyostelium slug. De?,&opment 100,119-124.
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BROOKMAN,J. J., Tows, C. D., JERMYN,K. A., and KAY, R. R. (1982). Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum Dev. Biol. 91,191-196. CECCARELU,A., MCROBBIE,S. J., JERMYN,K. A.,DUFFY,K., EARLY, A., and WILLIAMS, J. G. (1987). Structural and functional characterization of a Dictyostelium gene encoding a DIF inducible, prestalkenriched mRNA sequence. Nucleic Acids Res. 15,7463-‘7476. DOMINOV, J. A., and TOWN,C. D. (1986). Regulation of stalk and spore antigen expression in monolayer cultures of Dtctyostelium discaideum by pH. J. Embryol. Exp. Merphol. 96,131-150. EARLY, A. E., and WILLIAMS, J. G. (1988). A Dictyostelium presporespecific gene is transcriptionally repressed by DIF in vitro. Development, 103,519-524. GORMAN,C. M., MOFFAT,L. F., and HOWARD,B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol 2,1044-1051. GROSS,J. D., BRADBURY,J., KAY, R. R., and PEACEY, M. J. (1983). Intracellular pH and the control of cell differentiation in DictyosteRum disccrideum Nature
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GROSS,J. D., PEACEY,M. J., and POGGEVON STRANDMANN,R. (1988). Plasma membrane proton pump inhibition and stalk cell differentiation in Dictyostelium diswideum. Dz&rentiatiun 38,91-98. INOUYE,K. (1985). Measurements of intracellular pH and its relevance to cell differentiation in Dictyostelium discoideum. J. Cell Sci. 76, 235-245. INOUYE,K. (1988a). Differences in cytoplasmic pH and the sensitivity to acid load between prespore cells and prestalk cells of Dictyostelium. J. Cell Sci. 91,109-115. INOUYE,K. (198813).Induction by acid load of the maturation of prestalk cells in Dictyostelium discoideum. Development 104,669-681. JERMYN,K. A., BERKS,M., KAY, R. R., and WILLIAMS,J. G. (1987). Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium disco&urn.
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KAY, R. R., and JERMYN,K. A. (1983). A possible morphogen controlling differentiation in Dictyostelium. Nature (London) 303,242-244. KAY, R. R., GADIAN, G. D., and WILLIAMS, S. R. (1986). Intracellular pH in Dictyostelium: A alP nuclear magnetic resonance study of its regulation and possible role in controlling cell differentiation. J. Cell Sci. 83,165-179.
KWONG,L., and WEEKS, G. (1989). Studies on the accumulation of the differentiation-inducing factor (DIF) in high-cell-density monolayers of Dictyostelium diswideum De-v. Biol. 132,554-558. MACWILLIAMS, H. K., and DAVID, C. N. (1984). Pattern formation in Dictyostelium In “Microbial Development” (R. Losick and L. Shapiro, Eds.), Vol. 255, pp 255-274. Cold Spring Harbor Press, Cold Spring Harbor, NY. MANN, S. K. O., and FIRTEL, R. A. (1987). Cyclic AMP regulation of early gene expression in Dictyostelium discaideum: Mediation via the cell surface cyclic AMP receptor. Mol. Cell Biol. 7,458-469. MCROBBIE,S. J., TILLY, R., BLIGHT, K., CECCAREUI, A., and WILLIAMS, J. G. (1988). Identification and localization of proteins encoded by two DIF inducible genes of Didyostelium. Dev. Biol. 125,59-63. MEHDY, M. C., RATNER, D., and FIRTEL, R. A. (1983). Induction and modulation of cell-type-specific gene expression in Dictyostelium disurideum.
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NEWELL, P., and ROSS,F. (1982). Genetic-analysis of the slug stage of LXctyostelium discoideum, J. Gen Microbial 128,1639-X52. PETERS,D. J. M., VAN LOOI(EREN-CAYPAGNE,M. M., VAN HAASTERT, P. J. M., SPEK, W., and SCHAAP,P. (1989). Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Didyostelium discoideuwz. J. Cell Sci 93,205210. SCHINDLER,J., and SUSSMAN,M. (1977). Ammonia determines the choice of morphogenetic pathways in Dictyostelium diswideum J. Mol. Biol 116,161-169. SLEIGH, M. J. (1986). A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eukaryotic cells. Anal. Biochem 156,251~256. SUSSMAN,M., and SCHINDLER,J. (1978). A possible mechanism of morphogenetic regulation in Dictyostelium disco&urn B&mntiation 10, l-5. discoideum in TOWN, C. D. (1984). Differentiation of Dictyostelium monolayer cultures and its modification by ionic conditions. werentiation 27,29-35. TOWN,C. D., GROSS,J. D., and KAY, R. R. (1976). Cell differentiation without morphogenesis in Dictyostelium diskdeum. Nature @ondon) 262,717-719. VAN LOOKERENCAMPAGNE,M. M., AERTS, R. J., SPEK, W., FIRTEL, R. A. F., and SCHAAP, P. (1989). Cyclic AMP induced elevation of
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intracellular pH precedes, but does not mediate, the induction of prespore differentiation in Dictyostelium discoideum Devekpment 105,401-406. WANG, M., and SCHAAP,P. (1989). Ammonia depletion and DIF trigger stalk cell differentiation in intact Dictyostelium discoideum slugs. Development 105,569-574. WANG, M., VAN DRIEL, R., and SCHAAP, P. (1988). Cyclic AMPphosphodiesterase induces dedifferentiation of prespore cells in Dictyostelium discaideum slugs: Evidence that cyclic AMP is the morphogenetic signal for prespore differentiation. Development 103,611-618. WANG, M., VAN HAASTERT, P. J. M., and SCHAAP,P. (1986). Multiple effects of differentiation-inducing factor on prespore differentiation and cyclic-AMP signal transduction in Dictyostelium. D$wentiation
33,24-28.
WILLIAMS, J. G., CECCARELLI,A., MCROBBIE,S., MAHBIJBANI,H., KAY, R. R., EARLY, A., BERKS,M., and JERMYN,K. A. (1987). Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen. Cell 49,185-192. WILLIAMS, J. G., DLJFFY,K. T., LANE, D. P., MCROBBIE,S. J., HARWOOD, A. J., TRAYNOR,D. T., and JERMYN, K. A. (1989). Origins of the development. Cell 59, prestalk-prespore pattern in Dictyostelium 1157-1163.
Cytoplasmic Acidification Facilitates but Does Not Mediate DIF-Induced Prestalk Gene Expression in Dictyostelium discoideum MEIWANG,*JEROENH.ROELFSEMA,*JEFFREYG.WILLIAMS,SANDPAULINESCHAAP* *Cell Biology and Genetics Unit, Zoological Laboratory, L&den University, Kaiserstraat 63,2511 GP Leiden, The Netherlands; and slmperial Cancer Research Fund, Glare Hall kborataries, South Mimms, Herts. EN6 SL, England Accepted March 5,1990 Stalk cell differentiation in Dictyostelium can be induced by the differentiation-inducing factor, DIF, or by conditions that decrease intracellular pH (pHi). We have investigated whether cytoplasmic acidification acts directly to induce expression of pDd56 and pDd63, two DIF-regulated genes, specifically expressed in prestalk cells. The weak base methylamine, which increases pHi, inhibits DIF-induced transcription. The weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi, stimulates DIF-induction of the two prestalk genes. After relatively long incubation periods, DMO also induces a low level of prestalk gene expression in the absence of added DIF. However, unlike DIF-mediated induction, the apparent DMO-mediated induction decreases to undetectable levels when the cell density is reduced from 10’ to 106cells/ml. This indicates that DMO does not itself induce gene expression, but acts to enhance the effects of an autonomously secreted stalk-inducing factor, presumably DIF. These results suggest that the effects of DIF on gene expression are regulated by intracellular PH. _ but do not support a role for protons as direct intermediates in the DIF signal transduction pathway.- 0 1990 Academic Press, Inc. INTRODUCTION
posed pH, changes directly affect the earliest events in cellular differentiation, the expression of genes specifiDuring Lktyostelium discoideum development, unically expressed in prestalk or prespore cells. cellular amoebae aggregate to form multicellular strucWe recently found that CAMP induction of prespore tures, which enter into a program of stage and cell typegene expression was accompanied by a sustained inspecific gene expression, controlled by intercellular sigcrease in pH,. However, artificial elevation of pH, could naling. This program results in the differentiation of a not induce prespore gene expression, and inhibition of mass of spores supported by a column of stalk cells. Two the CAMP-induced pH, increase did not inhibit prespore morphogenetic signaling molecules, responsible for the gene expression (Van Lookeren Campagne et aL, 1989). induction of differentiation, have been identified. Cyclic These results show that CAMP-induced cytoplasmic alAMP, acts as a chemoattractant during aggregation, kalinization does not mediate the effect of CAMP on preand also induces spore cell differentiation (Kay, 1982; spore gene expression. Mehdy et aa, 1983; Wang et al, 1988), while DIF, a chloriIn this study, we have investigated the effect of pH, nated alkyl phenone (Morris et ah, 1987), induces the modulation on the expression of the prestalk genes, differentiation of stalk cells (Town et ah, 1976; Kay and pDd56 and pdD63 (Jermyn et ah, 1987). These genes are Jermyn, 1983; Williams, et aZ.,1987). It is not yet known first expressed in the tight aggregate stage of develophow these two molecules act to control cell type-specific ment (Williams et aZ., 1989) and their transcripts are gene expression. However, several studies have sugspecifically localized to prestalk and stalk cells (Jermyn gested that morphogen-induced intracellular pH (pHi) et al, 1987). In vitro, expression of these genes requires changes may determine the choice between differentiaexogenous DIF (Jermyn et al., 1987), and also depends on tion pathways: a period of prior exposure to millimolar CAMP, to induce Agents which supposedly increase pHi, such as high responsiveness to DIF. Thereafter expression of the extracellular pH, and treatment with weak bases such pDd56 gene is repressed by CAMP, while that of the as ammonia, promote spore and inhibit stalk cell differpDd63 gene is relatively unaffected (Berks and Kay, entiation, while agents which decrease pHi, such as low 1988). extracellular pH or addition of weak acids, favor stalk We show that DIF induction of pDd56 and pDd63 gene and inhibit spore differentiation (Gross et al., 1983; expression responds to changes in pHi, but our data do Town, 1984; Dominov and Town, 1986; Bradbury and Gross, 1989). All these experiments utilized prolonged not support a direct role for protons as intracellular secincubation of cells with the various agents (12 hr up to ond messengers in the process. We furthermore show several days) and hence do not show whether the pro- that responsiveness to DIF is more efficiently induced 0012-1606/90 $3.00 Copyright All rights
Q 1990 hy Academic Press, Inc. of reproduction in any form reserved.
182
WANGETAL.
by nanomolar CAMP pulses than by millimolar concentrations. MATERIALS
AND
183
pHi Regulation of Prestalk Gene Expression
CAMP
METHODS
Materials [14C]Chloramphenicol, [‘4C]acetyl-coenzyme A (CoA) and [a-32P]dATP were from Amersham (UK). 5,5dimethyl-2,4-oxazolidinedione (DMO), methylamine, CAMP, and G418 were from Sigma (St. Louis, MO). Acetylcoenzyme A was from Pharmacia (USA). Thin-layer chromatography plates were from Kodak (USA). Gene Screen was from New England Nuclear (UK). Chemically synthesized DIF-1 was a kind gift of Dr. R. Kay (MRC, Cambridge, UK). Cell Lines and Incubation Conditions D. discoideum AX-2 cells were transformed with the integrating vector PBlOTPl, containing a fusion of either the pDd63 or the pDd56 promotor and the bacterial chloramphenicol transferase (CAT) gene. These two prestalk-specific promotors direct correct temporal and cell type-specific CAT expression. (Ceccarelli et al, 1987; McRobbie et ab, 1988; Jermyn et aZ.,1989). The transformants were grown in axenic medium containing G418 at 10 pg/ml to a cell density of 2 X lo6 cells/ml. Cells were harvested and washed three times with 10 mM phosphate buffer, pH 6.5 (PB), resuspended to 5 x lo6 cells/ml, and shaken at 120 rpm and 21°C in PB. After 1 hr of incubation, the cells were shaken for a further 4 hr with 1 mM CAMP. Alternatively, the cells were treated during 4 hr with 20 nM CAMP pulses added at 6-min intervals. Following preincubation with CAMP, the cells were washed, resuspended in fresh PB to 3 x lo6 cells/ml, and incubated for variable time periods with DIF, DMO, or methylamine as indicated in the figure legends. The cells were collected for the assay of total cytosolic CAT activity or for the analysis of stalk-specific mRNAs.
toradiography of the TLC plates, the labeled spots were scratched off and measured by scintillation counting (Gorman et al., 1982). Method 2: The extract from 3 X lo6 cells was incubated for 2 hr at 37°C in a total volume of 40 ~10.25 MTris, pH 7.8, containing 1 mM chloramphenicol and 0.1 &i (12 nmole) [14C]acetyl-CoA. The reaction was stopped by adding 500 ~1 ice-cold ethylacetate. The layers were mixed by vigorous vortexing and separated by centrifugation for 3 min at 10,OOOg. The radioactivity of 300 ~1of the organic phase was measured by scintillation counting (Sleigh, 1986). RNA Isolation and Analysis Total cellular RNA from 3 X 10’ lysed cells was isolated and purified according to Mann and Firtel (1987). RNA (10 pg) was size fractionated on formaldehyde gels, transferred to Gene Screen membranes, and hybridized to 32P-labeled cDNA inserts as described by Peters et al. (1989). After hybridization and washing, the membranes were exposed to X-ray films. RESULTS
Induction of Competencefor Prestalk Gene Expression
In order to facilitate measurement of their expression, the promoters and 5’ proximal regions of the pDd56 and pDd63 genes were fused to the bacterial CAT gene. During normal development, the expression of pDd63 and pDd56 is first evident at the late aggregate stage (Williams et aZ., 1989). In vitro, the expression of both genes can be induced by DIF (Williams et al, 1987), provided that the cells have been made competent by pretreatment with millimolar CAMP for several hours (Berks and Kay, 1988). Figure 1 shows that DIF rapidly induces the expression of the pDd63-CAT and pDd56-CAT in competent cells, to reach a maximum after about 2 hr of stimulation. In this experiment, the induction of competence was achieved by treating preaggregative cells for 4 hr CAT Assay with 1 mM CAMP. This is somewhat unphysiological, lo7 cells were pelleted and resuspended in 100 ~1 of since during early aggregation, cells are not exposed to 0.25 M Tris, pH 7.8. Cells were lysed by three cycles of high constant CAMP stimuli. We measured whether freezing-thawing and centrifuged for 15 min at 10,OOOg. competence for DIF induction can also be achieved by The supernatant was heated for 15 min at 65°C to inac- the physiological stimulus of this developmental stage, tivate endogenous CAT inhibitors. CAT activity was i.e., nanomolar CAMP pulses. measured by two different methods: Cells were first starved for 1 hr in PB and were subseMethod 1: The extract from 5 X lo6 cells was incubated quently incubated in 1 mM CAMP or stimulated every 6 for 2 hr at 37°C in a total volume of 100 ~10.25 M Tris, min with a 20 nM CAMP pulse for 1,2,3, or 4 hr. SubsepH 7.8, containing 1 &i (0.02 pmole) [14C]chlorampheni- quently cells were incubated for a fixed period (2.5 hr) co1 and 0.5 mM acetyl coenzyme A. Acetylated with 50 nM DIF-1 (Fig. 2). It appeared that nanomolar [14C]chloramphenicol was separated from the nonacety- CAMP pulses induce subsequent responsiveness to DIF lated substrate by thin-layer chromatography. After au- faster and to higher levels than millimolar CAMP con-
184
DEVELOPMENTALBIOLOGY
vOLUME140,1090 B
POd56
PDd63
m3
0
30
60
90
120
160
30
60
Control
90 100 nM
120
160
Time (mid
/-• 1'
OF
0
/~/,-o~I 1
DIF-1
2
/
-
3 TIMEEhl
,*A
0
1
2 &,h,
FIG. 1. Time course of pDd63 and pDd56 expression in response to DIF. Vegetative pDd63 and pDd56 CAT transformants were starved for 1 hr
in PB and subsequently incubated for 5 hr with 1 mMcAMP to induce DIF competence. Cells were resuspended at 3 X 10scells/ml in fresh PB and incubated at 150 rpm and 20°C in the presence (0) or absence (0) of 100 nM DIF-1. At the indicated time periods, cytosolic CAT activity was assayed, using [“CJchloramphenicol as substrate (method 1). (A) pDd63-CAT expression, autoradiograph of the different acetylated forms of [“C]chloramphenicol separated by TLC. (B) Quantitated data expressed as percentage of the highest level of CAT induction during incubation.
centrations. In all subsequent experiments, we therefore used a 4-hr pretreatment with 20 nM CAMP pulses to induce competence. Eflects of Extracellular
pH on Prestalk Gene Expression
Incubation of cell monolayers at relatively low pH has been reported to induce the differentiation of stalk cells (Gross et al., 1983; Town, 1984; Town and Dominov, 1986). We investigated the effect of extracellular pH (pH,) on the expression of the pDd63 and pDd56 genes in the presence and absence of DIF (Fig. 3). Expression of the pDd56 and pDd63 genes requires DIF and cannot be induced by reducing the extracellular pH to 5.0. (Below this pH, cell viability is seriously affected.) The induction of pDd56 and pDd63 expression by DIF occurs equally efficient at pH 5,6, and 7, but is markedly inhibited at pH 8.
ml
W56
POd63
Eflects of Intracellular
pH on Prestalk Gene Expression
pHi can be manipulated by treating cells with weak acids or weak bases (Aerts et al, 1985; Inouye, 1988a). The weak acid DMO causes a 0.2-unit decrease in pH, at a concentration of 5 mM, while the weak base methylamine at 2.5 mM causes a 0.3-unit increase (Van Lookeren Campagne et u& 1989; Van Duijn, unpublished data). We measured the effects of DMO and methylamine on the expression of pDd56 and pDd63 in the presence and absence of DIF. Figure 4 shows that the DIFinduced expression of both genes is inhibited by methylamine. pDd56 expression seems to be somewhat more sensitive to this inhibitory effect than pDd63 expression. In contrast, the weak acid DMO stimulates the DIF-induced expression of pDd63 and pDd56; the effect saturates at about 2 mM DMO (Fig. 5). We confirmed that the CAT constructs mirror the behavior of the endogenous genes by analyzing mRNA accumulation in the presence of methylamine and DMO (Fig. 6).
1 POd63
f 0
1
0
2 Odion
of4pre+rea+men+
12
3
4
(h)
FIG. 2. Acquisition of competence for DIF-induced prestalk gene expression. Vegetative pDd63 or pDd56 transformants were starved for 1 hr in PB and subsequently incubated with either 1 m&f CAMP added as a single dose (open symbols), or with 20 nMcAMP added as pulses with 6-min intervals (closed symbols). At the indicated time periods, cells were collected and resuspended in fresh PB. Cells were subsequently incubated for 2.5 hr with 50 nM DIF and assayed for cytosolic CAT activity according to method 2.
O$ e
FIG. 3. Effect of extracellular pH on prestalk gene expression. Competent pDd56 or pDd63 cells were incubated in PB of the indicated pH values in the presence or absence of 50 n&f DIF-1. After 2.5 hr cytosolic CAT activity was assayed. Means and SE of two experiments performed in triplicate are presented.
WANG
PDd56 I
PDd63
185
pHi Regulation of Prestulk Gem Expressim
ET AI..
PB DMO PH6.6 DIF DMO+DlF
I
PB CHSNHZ pH7.4 0
2
4
6
E mM CH$H2
0
10
-6
2
6
4
8
I3 10
mM CH+H2
4. Effect of methylamine on prestalk gene expression. Competent pDd63 and pDd56 cells were incubated in PB of pH 7.4 in the presence (closed symbols) or absence (open symbols) of 50 nM DIF-1 with the indicated methylamine concentrations. After 2.5 hr cytosolic CAT activity was assayed. Means and SE of three experiments performed in triplicate are presented. FIG.
In the absence of added DIF, DMO induces prestalk gene expression (Figs. 5, ‘7,8), but much more slowly and to a lower level than DIF. The most obvious alternative interpretations of this result are: (i) DMO induces prestalk gene expression, but at a much lower efficiency than DIF. (ii) DMO acts to enhance the effect of endogenously produced DIF. (iii) DMO induces DIF synthesis, which subsequently induces prestalk gene expression. To discriminate between these possibilities, we measured the effects of DMO and DIF at different cell densities. If the first supposition were true, then a reduction of cell density should not diminish the effects of DMO, but if either of the two last suppositions are true, the effect of DMO should be much lower at low cell densities, where less DIF is produced. When the cell density is reduced from 10' to lo5 cells/ml, DMO cannot induce pDd56 or pDd63 expression anymore (Fig. 8). The effect
PH7.4 CH~NHZ+DIF
DIF
-
FIG. 6. Effects of DMO and methylamine on prestalk mRNA levels. Competent cells were incubated in PB of the indicated pH to which 2.5 mM DMO, 5 mM methylamine, 50 nM DIF, or combinations of DIF with either weak acid or base had been added. After 2.5 hr of incubation, total RNA was isolated. Northern transfers were probed with q-labeled pDd63 or pDd56 cDNA inserts, respectively.
of DIF is unaffected by reducing the cell density. Although reduction of cell density might change some variable, which is necessary for induction by DMO, this variable is evidently not important for induction by DIF. It therefore seems more likely that DMO cannot itself induce prestalk gene expression, but acts to either induce DIF synthesis or to stimulate the effect of endogenously produced DIF. We next measured the effects of a fixed concentration of DMO at different DIF concentrations. In the absence of DIF, the effect of DMO is smaller than when DIF is present (Fig. 9). At saturating DIF concentrations, DMO induces a twofold increase of pDd63 expression. This suggests that DMO does not act by inducing the production of DIF, because if this were the case the effect of DMO would be highest at the lowest DIF concentration and should become negligible at saturating DIF concentrations. We therefore conclude that DMO acts as a DIF synergist, but not as a DIF agonist.
PDd56
POd63 I -.----I
TX-
-
1
I-----
z5 I/ i= / %wA t
p
0
12
1/
3 mtl OMO
4
5
0
12
3
4
5
mMMO
5. Effects of DMO on prestalk gene expression. Competent pDd63 or pDd56 cells were incubated in PB of pH 6.6 in the presence (0) or absence (0) of 50 n2MDIF-1 with the indicated DMO concentrations. After 2.5 hr cytosolic CAT activity was assayed. Means and SE of three experiments performed in triplicate are presented. FIG.
TIME(h) FIG. 7. Time dependency of DMO and DIF effects on pDd63 expression. Competent pDd63 cells were incubated in PB pH 6.6, without further additions (O), with 5 mMDM0 (O), with 50 nMDIF-1 (a), or with 5 mil4 DMO plus 50 nM DIF-1 (w). At the indicated time periods, cytosolic CAT activity was assayed. Means of two experiments are presented.
DEVELOPMENTALBIOLOGY
VOLUME 140.1990
lar Ca’+ efflux; although it could be that the pHi drop produced by DMO is insufficient to affect the Ca2+chann’ nels. A much simpler possibility is that the binding of DIF to its presumptive intracellular receptor is pH sensitive. Earlier data showed that weak acids and bases, respectively, promote the differentiation of stalk and spore cells, as measured by the ratio of mature cell types, or the accumulation of spore and stalk-specific 0 5 6 7 0 5 6 1 gene products. (Gross et al., 1983; Town, 1984; Dominov logkells/mli logkellr/mll and Town, 1986; Bradbury and Gross, 1989). Weak acids FIG. 8. Effect of cell density on DMO and DIF induced gene expresand bases neither inhibit nor promote the transcription sion. Competent pDd56 and pDd63 cells were incubated at the indiof prespore genes and the synthesis of spore antigens cated cell density in PB pH 6.6 (0), with 5 mMDMO (0) or with 50 r&f DIF-1 (m). After 3.5 hr of incubation cytosolic CAT activity was mea- (Van Lookeren Campagne et al, 1989). The present data sured. Means of three experiments are presented. show that a pHi decrease promotes DIF-induced prestalk gene expression, but cannot induce expression in the absence of DIF. DISCUSSION The main difference between the previous experiments and our studies is the time-scale over which the We investigated, the effects of pHi modulation by weak acids and bases on prestalk gene expression and effects of PHi modulation were measured. In our experifound that the weak base methylamine inhibits DIF- ments prestalk gene expression was measured 2-3 hr induced expression of the prestalk genes pDd63 and after exposure to weak acids or bases and prespore gene pDd56. Inhibition of pDd56 was half-maximal at 5 mM expression after 4-6 hr. All previous experiments inmethylamine. At this concentration, methylamine does volved exposure from 12 hr up to 4 days (Gross et al, not affect CAMP-induced expression of the prespore 1983; Dominov and Town, 1986; Bradbury and Gross, gene D19, or of the cysteine proteinase-2 gene (Van Loo- 1989). The fact that weak acids increase DIF responsivekeren Campagne et ab, 1989 and unpublished data), sug- ness may explain their long-term stimulatory effects on gesting that the inhibitory effect of methylamine on stalk cell differentiation. The observation that weak acids also stimulate DIF accumulation (Kwong and stalk gene expression is not due to a general inhibition of transcription. Weeks, 1989) may be an additional factor affecting cell The weak acid DMO stimulates the DIF-induced ex- fate over the long term. pression of pDd56 and pDd63 and induces a low level of In addition to inducing prestalk gene expression, DIF expression of both genes in the absence of added DIF. In also inhibits CAMP-induced prespore gene expression at contrast to the effect of DIF, the inductive effect of the transcription level (Wang et ah, 1986; Early and WilDMO is strongly cell density dependent. Stimulatory ef- liams, 1988). This means that DIF synergists, as weak fects of weak acids on DIF production have been reported (Kwong and Weeks, 1989). However, the stimulatory effects of DMO are much stronger in the presence than in the absence of added DIF and remain high at saturating DIF concentrations. This suggests that under our conditions DMO does not induce gene expression by stimulating DIF secretion. We conclude that DMO acts to enhance the effect of DIF, but cannot, itself, induce transcription of prestalk genes. These data suggest that the induction of prestalk gene expression is not mediated by protons. This conclusion is supported by the observation that DIF does not induce a sustained decrease in pHi (Kay et al., 1986; Inouye, 1988a). It appears rather, that the pHi deternM DIF mines the responsiveness of cells to DIF. The observa9. Effects of DMO at increasing DIF concentrations. Competion that cytoplasmic acidification is ineffective in the tentFIG.pDd63 cells were incubated during 2.5 hr in the presence (0) or absence of DIF does not support’s recent model (Gross absence (0) of 5 mMDM0 with 20,35,50,70 &of DIF-1. After 2.5 hr, et al., 1988), wherein DIF and protons both act to open a cytosolic CAT activity was assayed. Means and SE of two experiments vesicular chloride channel resulting in reduced vesicu- are presented. ‘Dd63
‘Dd56
m------m
m-------m
0%---r-x
WANG ET AL.
pHi Regulation
acids, and DIF antagonists, as weak bases, will respectively inhibit or promote prespore gene expression under conditions where DIF is added, or allowed to accumulate. The conclusion that pHi modification alone does not induce the expression of cell type-specific genes does not mean that pH, is not an important factor. Developing cells secrete fairly large amounts of the weak base ammonia (Schindler and Susman, 1977), which inhibit stalk cell differentiation in vitro (Gross et ak, 1983) and furthermore inhibit the transition from migration to culmination (Schindler and Sussman, 1977). Local ammonia depletion may occur during normal development at the apex and base of the culminating structure during early fruiting body formation (Sussman and Schindler, 1978). We recently showed that DIF can only induce stalk cell differentiation in viva when endogenously produced ammonia is removed (Wang and Schaap, 1989). Stimulatory effects of ammonia depletion or CO,induced acid load on stalk cell differentiation in migrating slugs were also shown by Inouye (1988b), but in these experiments it is not clear whether the effects result directly from inducing stalk cell differentiation or indirectly from inducing the slug/fruit switch. The effects of NH, may not be confined to culmination, but may also be involved in cell type-regulation in the slug. A class of mutants, which are hypersensitive to the inhibitory effects of NH, upon culmination (Newell and Ross, 1982), display a reduced ratio of prestalk to prespore cells in the migratory slug (MacWilliams and David, 1984). Cyclic AMP induces a sustained increase in pH, (Van Lookeren Campagne et al, 1989) and prespore cells have a higher intracellular pH and higher resistance to acid load than prestalk cells (Inouye, 1985, 1988a). It is therefore possible, that prestalk cell differentiation is dictated in part by factors, such as ammonia and CAMP, which control responsiveness to DIF, rather than to the specific spatial distribution of DIF itself. Such a model would help to explain the apparent paradox, that the concentration of DIF in the back of the slug is higher than that in the front. (Brookman et ah, 1987). REFERENCES AERTS,R. J., DURSTON,A. J., and MOOLENAAR,W. H. (1985). Cytoplasmic pH and the regulation of the Dictyostelium cell cycle. Cell 43, 653-657. BERKS,M., and KAY, R. R. (1988). Cyclic AMP is an inhibitor of stalk cell differentiation in Dietyostelium discoideum. Dev. Biol. 126, 108-114. BRADBURY,J. M., and GROSS,J. D. (1989). The effect of ammonia on cell-type-specific enzyme accumulation in Dictyostelium discoideum. Cell LX&r. Dev. 27,121-128. BROOKMAN,J. J., JERMYN, K. A., and KAY, R. R. (1987). Nature and distribution of the morphogen DIF in the Dietyostelium slug. De?,&opment 100,119-124.
of Prestalk
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Gene Expression
BROOKMAN,J. J., Tows, C. D., JERMYN,K. A., and KAY, R. R. (1982). Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum Dev. Biol. 91,191-196. CECCARELU,A., MCROBBIE,S. J., JERMYN,K. A.,DUFFY,K., EARLY, A., and WILLIAMS, J. G. (1987). Structural and functional characterization of a Dictyostelium gene encoding a DIF inducible, prestalkenriched mRNA sequence. Nucleic Acids Res. 15,7463-‘7476. DOMINOV, J. A., and TOWN,C. D. (1986). Regulation of stalk and spore antigen expression in monolayer cultures of Dtctyostelium discaideum by pH. J. Embryol. Exp. Merphol. 96,131-150. EARLY, A. E., and WILLIAMS, J. G. (1988). A Dictyostelium presporespecific gene is transcriptionally repressed by DIF in vitro. Development, 103,519-524. GORMAN,C. M., MOFFAT,L. F., and HOWARD,B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol 2,1044-1051. GROSS,J. D., BRADBURY,J., KAY, R. R., and PEACEY, M. J. (1983). Intracellular pH and the control of cell differentiation in DictyosteRum disccrideum Nature
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GROSS,J. D., PEACEY,M. J., and POGGEVON STRANDMANN,R. (1988). Plasma membrane proton pump inhibition and stalk cell differentiation in Dictyostelium diswideum. Dz&rentiatiun 38,91-98. INOUYE,K. (1985). Measurements of intracellular pH and its relevance to cell differentiation in Dictyostelium discoideum. J. Cell Sci. 76, 235-245. INOUYE,K. (1988a). Differences in cytoplasmic pH and the sensitivity to acid load between prespore cells and prestalk cells of Dictyostelium. J. Cell Sci. 91,109-115. INOUYE,K. (198813).Induction by acid load of the maturation of prestalk cells in Dictyostelium discoideum. Development 104,669-681. JERMYN,K. A., BERKS,M., KAY, R. R., and WILLIAMS,J. G. (1987). Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium disco&urn.
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