Alternative Developmental Pathways Determined by Environmental Conditions in the Cellular Slime Mold Dictyostelium discoideum

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1 JOURNAL OF BACTERIOLOGY, Nov. 1969, p Copyright a 1969 American Society for Microbiology Vol. 100, No. 2 Printed in U.S.A. Alternative Developmental Pathways Determined by Environmental Conditions in the Cellular Slime Mold Dictyostelium discoideum PETER C. NEWELL, ALVIN TELSER, AND MAURICE SUSSMAN Departmenit of Biology, Brandeis University, Waltham, Massachusetts Received for publication 31 July 1969 The cellular slime mold Dictyostelium discoideum grows in the soil as a population of independent, uninucleate amoebae. Upon entrance to the stationary phase, the amoebae collect in multicellular aggregates to form organized fruiting bodies composed of spores and stalk cells. Depending upon environmental conditions, the developing aggregate either constructs the fruiting body at the site of aggregation or transforms into a structure that can migrate to a more favorable location. Environmental conditions that favor migration are (i) the accumulation of metabolite(s) produced by the aggregate and (ii) a low ionic strength in the substratum. Conditions that prevent migration or that stop a migrating slug are (i) the presence of buffer and (ii) illumination by overhead light. Cellular slime molds exist in the soil as solitary, uninucleate amoebae, ingesting the bacteria surrounding them and multiplying until the food supply is exhausted. The amoebae then collect into multicellular aggregates composed of as many as 105 cells, and each aggregate subsequently undergoes a complex and precisely regulated development leading to the construction of a fruiting body. This is composed of two types of specialized cells: spores and stalk cells, the latter serving to lift the former off the substratum (9). The morphogenetic sequence is accompanied by an equally precise progression of biochemical events, which includes drastic changes in ribonucleic acid (RNA) and protein composition (12, 14). This report correlates the morphogenetic changes in the developing cell aggregate with changes in the immediate environment. It appears that at least part of the complexity involved in fruiting body construction arises as a result of responses of the aggregate to environmental alterations. Depending upon these conditions, the aggregate can either remain stationary and construct a fruiting body in situ or transform into a migrating slug and move to a more suitable environment before making the fruiting body. The environmental conditions affecting the selection of the developmental pathways are described in this paper. The responses of the aggregate to the environment appear to have an ecologically relevant function, namely, to insure 763 the most advantageous conditions for spore formation and dispersal. MATERIALS AND METHODS Organism and culture conditions. Dictyostelium discoideum strain NC-4 (haploid) was grown with Aerobacter aerogenes, a prototrophic strain (no. 1033) generously provided by B. Magasanik of the Massachusetts Institute of Technology. The solid growth medium contained, per liter: NZCase peptone (Sheffield Chemical, National Dairy Products Corp., Norwich, N.Y.), 10 g; glucose, 10 g; K2HPO4, 2.2 g; KH2PO4, 1.0 g; MgSO4-7H20, 1.0 g; and Difco agar, 20 g; the ph was 6.4. This medium replaced the previously used SM medium containing Difco peptone and Difco yeast extract (5, 11), because cells grown on current batches of the Difco products have been found to develop aberrantly and asynchronously when dispensed on filter papers as described below. The absence of yeast extract in this medium necessitated the replacement of the previously employed auxotrophic A. aerogenes with the prototroph. Cells were grown to the beginning of the stationary phase and harvested. Development on Whatman filters. Samples (0.5 ml) of a suspension of washed amoebae (2 X 108 cells/ml) were pipetted uniformly onto a circle of Whatman no. 50 filter paper (42.5 mm in diameter) resting on an absorbent pad (Millipore Corp., Bedford, Mass.; no. AP ) saturated with a solution containing streptomycin (0.5 mg/ml) and various concentrations of phosphate buffer, ph 6.5 (Na2HPO4/KH2PO4), all contained in a 60-mm plastic petri dish. For some experiments, further buffering capacity was provided by employing two absorbent pads saturated with 1.0 M Na2HPO4/KH2PO4 (ph 6.0), held a few milli-

2 764 NEWELL, TELSER, AND SUSSMAN J. BACTERIOL. meters above the developing aggregates by being attached to the underside of the petri dish cover. RESULTS Effect of the buffer capacity of the substratum on the decision to migrate. Cells were grown, harvested, and dispensed on Whatman filters for development as described in Materials and Methods. Under these conditions, 108 cells can construct about 103 fruiting bodies synchronously over a 24-hr period. Figure 1 summarizes the developmental programs followed when the substratum was unbuffered, lightly buffered (0.01 M phosphate, ph 6.5), or well buffered (0.04 M phosphate, ph 6.5; in addition, two absorbent pads saturated with 1.0 M Na2HP04/KH2PO4, ph 6.0, were held a few millimeters above the developing aggregates by being cemented to the petri dish cover). If well buffered, an aggregate remained stationary and constructed a fruiting body in situ without ever assuming the shape of a migrating slug. If lightly buffered, an aggregate began the characteristic transformation into a migrating slug, but did not complete it and instead reverted to the program followed by wellbuffered cells, constructing a fruiting body in the same time period as the latter. If unbuffered, the aggregate completed the transformation into a migrating slug and remained so for several days. Slifkin and Bonner (10) reported slug migrations for periods up to 10 days. It should be noted that the conventional morphogenetic sequences previously described in the literature have tended to be intermediate between the unbuffered and lightly buffered systems (8, 12, 15). Nature of the migratory stimulus. Several salient features of the migratory stimulus can be deduced from the experiments summarized below. c~ A A series of experiments showed that the stimulus is not due to the ph value itself. When cells developed in a well-buffered system for 18 hr, the ph of the underlying pad solution remained at 6.5. In contrast, if they developed over the same period in a lightly buffered system, the ph of the pad solution rose to 6.9, and in the unbuffered system to 7.2. It might therefore be supposed that the migrating stimulus is simply an elevated ph value. This is not the case. When cells were allowed to develop for 15 hr in the well-buffered system and the filter papers bearing them were then switched to fresh support pads, saturated with 0.04 M phosphate at ph 6.5, 7.0, 7.2, or 7.4, all continued to construct fruiting bodies as if they had not been switched at all. If, however, the aggregates were switched to unbuffered pads on which cells had previously been allowed to develop, they uniformly stopped fruit construction and transformed into slugs. The stimulus was shown to be appreciably volatile when cells were allowed to develop in the unbuffered system but with an absorbent pad saturated with 1 M phosphate buffer, ph 6, cemented to the petri dish cover. Unlike the controls with no overlying pad, these aggregates began to transform into migrating slugs but then quickly reverted and constructed fruiting bodies in situ. When the overlying pad was saturated with water, it was much less effective (though a few migrating slugs did revert and construct fruits). When saturated with dilute NaOH solution, it was completely ineffective in preventing slug formation. Finally, it was shown that the stimulus is produced by the cell aggregates and migrating slugs. The previous experiments were all performed with populations of 108 cells per filter. Under all F-Iro-- 12 NOT BUFFERED HOURS LIGHTLY BUFFERED WELL BUFFERE 20 FIG. 1. Schematic representation of the developmental programs observed without buffer, with a lightly buffered substratum (0.01 M phosphate, ph 6.5), and with a well-buffered substratum [0.04 M phosphate, ph 6.5 with, in addition, two absorbent pads saturated with 1.0 M phosphate buffer (ph 6.0) held a few millimeters above the developing aggregates]. HOURS

3 VOL. 100, 1969 DEVELOPMENTAL PATHWAYS IN D. DISCOIDEUM 765 buffering conditions, the lower the cell density (5 X 106 to 5 X 107 cells/filter), the less readily inducible was the transformation into migrating slugs. Cells at low population density produced fewer and smaller aggregates (13). At densities just above the threshold that permits any aggregates to form, all construct fruits; none form migrating slugs even if unbuffered. The transformation into slugs is also influenced by the porosity of the substratum. In increasing order of effectiveness in this respect are the following: Whatman no. 50 filter disc over a water-saturated pad; Millipore filter over a water-saturated pad; 2% agar. Since this is also the decreasing order of porosity, it further supports the notion that the migratory stimulus is a diffusible substance produced by the developing aggregate. The previous results suggest that the metabolite is also basic and somewhat volatile. One possible candidate is ammonia, produced during morphogenesis as a product of amino acid oxidation (3, 16). However, addition of ammonium hydroxide to the substratum or to a pad cemented to the petri dish cover over a wide concentration range (10-1 to 5 X 10-4 M) did not stimulate slug formation. Effect of salt concentration. Slifkin and Bonner (10) showed that inorganic salts decreased the time over which slugs migrated on agar prior to constructing fruiting bodies. In the unbuffered system used here, KCl and NH4Cl were equally effective in inducing a significant proportion of migrating slugs to stop and construct fruits. Addition of either salt to the weakly buffered system significantly decreased the tendency to form migrating slugs. However, even at a concentration of 0.06 M, this ability was not nearly so great as that of 0.01 M (ph 6.5) phosphate buffer in influencing the decision to migrate. Effect of light. Bonner et al. (1) reported that unidirectional light parallel to the substratum caused migrating slugs to orient and move toward the light source. This was confirmed by Francis (4), who obtained an action spectrum showing peaks at 425 and 550 nm. It should be noted, however, that the light gradient does not influence the decision to begin or to continue migrating, but merely the direction of movement. In contrast, we have found that illumination from overhead does affect this decision profoundly. A line of washed amoebae (4 X 107 cells in 0.1 ml) was laid down with a pipette on an unbuffered 2% agar substratum (containing no salt) set in a covered rectangular plastic box wrapped in aluminum foil. In some cases, a small hole was introduced in the foil so that it was at one end of the box and the line of cells was at the other. The remaining boxes were left dark. In confirmation of Bonner et al. (1), the slugs moved on a straight course toward the light in the first set of boxes and randomly in the second. However, if at any time the aluminum foil was removed, the slugs immediately stopped migrating, reared back in a characteristic pose (see Fig. 2), and constructed fruiting bodies within a few hours. If the overhead light was present prior to slug formation, it prevented the transformation, even though the system was unbuffered. Thus, the presence of overhead light can override the migratory stimulus. Relatively low light intensities (ordinary interior illumination or dull daylight) are sufficient to create the full effect. The light need be imposed for only 15 to 30 min because if the slugs are then returned to darkness the majority stop and construct fruits. Thus, it must act as a trigger. Reversibility of the decision to migrate. A migrating slug can stop migrating and construct a fruit at any time. Whether this occurs depends upon ionic strength and buffering capacity of the environment and the presence of overhead light, either individually or in combination. Individually, the three conditions increasingly influence fruit formation in the order named. The combination of buffer and light has been found to be particularly useful for biochemical studies because the reversion of the slugs is rapid and highly synchronous. Even if the external parameters are not changed, the migratory period is eventually terminated by fruiting. Thus, cells on 2% agar in the dark or oriented in a light gradient will migrate for days and most will cover 10 to 20 cm. During this time, an occasional slug will stop migrating and fruit, until eventually all have done so. Their size during the migration diminishes until they can be a mere 10 to 20% of their original size, and the fruiting bodies they form are proportionately small. Reversibility of the decision to construct a fruit. Up to a critical morphogenetic stage, a developing aggregate can be induced to stop, revert, and transform into a migrating slug by shifting the filter disc from a buffered absorbent pad to an unbuffered pad on which other cells had previously been developing. In the experiment summarized in Fig. 3, two sets of replicate cell samples were allowed to develop, one set on pads saturated with 0.04 M phosphate (ph 6.5) and the second on pads saturated with water. At hourly intervals from 13 to 20 hr, a filter disc from an unbuffered pad was interchanged with one from a buffered pad and incubated further. All were examined at 24 hr. The migrating slugs that were switched from unbuffered to buffered pads had constructed fruits by 24 hr regardless of the time of the switch. However, the develop-

4 766 NEWELL, TELSER, AND SUSSMAN J. BACTERIOL. FIG. 2. Morphology of slugs after exposure to overhead light. Photographs were take,i at: (1) zero time; (2) I hr; (3) 1.5 hr; (4) 2.5 hr; (5) 3.5 hr; (6) 4.5 hr; (7) 5.5 hr; (8) 6.5 hr.

5 VOL. 100, 1969 DEVELOPMENTAL PATHWAYS IN D. DISCOIDEUM 767 HOURS OF DEVELOPMENT MORPHOLOGY BEFORE SWITCH FROM BUFFERED TO UNBUFFERED PADS MORPHOLOGY AFTER SWITCH (24 HOURS) MORPHOLOGY BEFORE SWITCH FROM UNBUFFERED TO BUFFERED PADS ~~ Q ==> <==4o~Z:Z FIG. 3. Effect of changing buffer conditions at various times of development. Two sets of replicate cell samples were allowed to develop, one set on pads saturated with 0.04 M phosphate (ph 6.5) and the second on pads saturated with water. At hourly intervals from 13 to 20 hr, a filter from an unbuffered pad was interchanged with one from a buffered pad, and morphology was recorded at 24 hr. ing aggregates switched from buffered to unbuffered pads reverted and transformed to migrating slugs only if the switch occurred prior to 17 hr. Once the developing aggregate is in the process of transforming into the characteristic "Mexican hat" (17 to 18 hr), the decision to fruit is irrevocable under these conditions. DISCUSSION Previous descriptions of the developmental cycle of D. discoideum have tended to include the migrating slug stage as an inevitable interlude between cell aggregation and fruit construction (6, 15). The results described here demonstrate that the transformation of a cell aggregate into a migrating slug is not in fact a necessary preamble to fruit construction. The migratory stimulus appears to be a somewhat volatile, diffusible metabolite produced by the cell aggregate itself. Whether an aggregate became a migrating slug or a fruit was determined by the buffering capacity of the immediate vicinity, the ionic strength of the substratum, and the presence or absence of overhead light (Fig. 4). The natural habitat of D. discoideum is in decaying forest leaves or in very shallow positions in loose soil (6). The growth stage can only occur where bacteria are found that the organism can feed on, and once the food supply is exhausted a new supply must be located. The migratory stage is clearly of importance in moving to new areas. Of all the factors affecting the migratory period, it is the accumulation of an excess of some metabolite that seems to be dominant in initiating the migration. In the absence of this product, or under laboratory conditions where it is efficiently absorbed by buffer, the aggregate does not go through a migration stage. Excess excretory metabolites, however, could indicate to the organism that the habitat was overcrowded and migration to fresh areas was desirable. To aid them in finding the best location for spore dispersal, the slugs are phototactic and can move toward a unidirectional light source of extremely feeble intensity (1, 4). Moreover, they can also move toward a source of heat, even when the heat gradient is as little as 0.05 C/cm (1, 4). Both of these responses would lead the slug from its location in soil or under leaves into the open sunlight. Once there, the slug would respond to the overhead light and stop migration and start forming a fruiting body. The organism, in the open sunlight, can form spores and raise them off the substratum by its stalk, in a position with a far better chance for dispersal than in their original underground position. The effect of a drop in humidity also stimulates this response (2). The responses of the developing slime mold to a number of environmental factors therefore appear not to be arbitrary, but can be seen as those most suited for survival and propagation under the circumstances prevailing.

6 768 NEWELL, TELSER, AND SUSSMAN J. BACTERIOL. Downloaded from FIG. 4. Schematic representation of the alternative pathways of development open to the developing aggregate. The aggregate may form a migrating slug (A) or start to fruit without migration (B). At any time up to 16 hr that the developing fruit receives a migration stimulus, it can suspenid the fruiting process and revert to a slug but after reaching the 17-hr stage (E) it is committed to completing the fruit (F). If a migrating slug is formed, it migrates toward the light source in a light gradient parallel with the substratum, or in darkness it migrates randomly. If it is illuminated by overhead light, it stops migrating and starts fruit formationi (C, D). ACKNOWLEDGMENTS This work was supported by grant GB5976X from the National Science Foundation. M. S. is a recipient of a Career Development Award from the National Institutes of Health. P. C. N. gratefully acknowledges receipt of a Wellcome Trust Research Travel Grant. A. T. is a fellow of the Helen Hay Whitney Foundation. LITERATURE CITED 1. Bonner, J. T., W. W. Clarke, Jr., C. L. Neely, Jr., and M. K- Slifkin The orientation to light and the extremely sensitive orientation to temperature gradients in the slime mold Dictyostelium discoideum. J. Cell. Comp. Physiol. 36: Bonner, J. T., and M. J. Shaw The role of humidity in the differentiation of the cellular slime molds. J. Cell. Comp. Physiol. 50: Cohen, A. L The effect of ammonia on morphogenesis in the Acrasieae. Proc. Nat. Acad. Sci. U.S.A. 39: Francis, D. W Some studies on phototaxis of Dictyostelium. J. Cell. Comp. Physiol. 64: Newell, P. C., and M. Sussman UDP-glucose pyrophosphorylase in Dictyostelium discoideum: stability and developmental fate. J. Biol. Chem. 244: Raper, K. B Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agr. Res. 50: Raper, K. B Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Sci. Soc. 56: Raper, K. B Developmental patterns in simple slime molds. Growth. Third Growth Symp. 5: Raper, K. B., and D. I. Fennell Stalk formation in Dictyostelium. Bull. Torrey Bot. Club 79: Slifkin, M. K., and J. T. Bonner The effect of salts and organic solutes on the migration time of the slime mold Dictyostelium discoideum. Biol. Bull. (Woods Hole) 102: Sussman, M Biochemical and genetic methods in the study of cellular slime mold development, p : In D. M. Prescott (ed.), Methods in cell physiology. Academic Press Inc., New York. 12. Sussman, M Evidence for temporal and quantitative control of genetic transcription and translation during slime mold development. Fed. Proc. 26: Sussman, M., and R. R. Sussman Aggregative performance. Exp. Cell Res. Suppl. 8: Sussman, R. R RNA metabolism during cytodifferentiation in the cellular slime mold Polysphondelium pallidum. Biochim. Biophys. Acta 149: Wright, B. E Multiple causes and controls in differentiation. Science 153: Wright, B. E., and M. L. Anderson Protein and amino acid turnover during differentiation in the slime mold: utilization of endogenous amino acids and proteins. Biochim. Biophys. Acta 43: on October 14, 2018 by guest