ABSTRACT
The relationship between aggregate size and morphological field size has been investigated in the cellular slime mold Dictyostelium discoideum. Evidence is presented that aggregate size and field size exhibit different temperature sensitivities and that an aggregate can be induced to separate into several morphological fields by a decrease in temperature. In addition, evidence is presented that field size is stabilized at a point in time just prior to tip formation.
INTRODUCTION
When amebae of a log-phase culture of Dictyostelium discoideum are dis-persed as a multicellular carpet on a filter saturated with buffered salts solution, they separate into discrete aggregates. These aggregates then progress through a defined sequence of morphological stages resulting in a fruiting body. Normally, each aggregate forms a single tip at the top which appears to function as an organizer for a single morphological field (Raper, 1940; Farnsworth, 1973; Rubin & Robertson, 1975). Each field then gives rise to a single fruiting body. In this report we have investigated the relationship between the size of the aggregate and the size of the morphological field. Evidence will be presented that aggregate size and field size exhibit different temperature sensitivities and that an aggregate can be induced to separate into several morphological fields by a decrease in temperature. These results indicate that different mechanisms control aggregate size and field size, and that aggregate size does not rigidly determine field size. In addition, it will be demonstrated that field size is stabil-ized at a point in time just prior to tip formation.
MATERIALS AND METHODS
Growth and maintenance of organism
Subclones of the axenic strain of Dictyostelium discoideum, AX-3, clone RC-3, were maintained on lawns of Aerobacter aerogenes on nutrient agar (Sussman, 1966). Axenic cultures were initiated by dispersing spores into 2 ml of the axenic medium HL-5 (Cocucci & Sussman, 1970) containing 500 μg per ml of streptomycin sulfate in sterile test tubes. After several days these initial cultures were in turn inoculated into 1000 ml Erlenmyer flasks containing 130 ml of liquid nutrient medium and rotated at 90 rev./min at 21 °C. At this tempera-ture amebae multiplied with a generation time of approximately 12 h and reached a final cell density of approximately 2 × 107 per ml at stationary phase (Yarger, Stults & Soli, 1974; Soll, Yarger & Mirick, 1976). Cells were diluted into fresh medium when cell densities reached 5 × 106 per ml. For all experiments reported in this communication, cells were obtained from mid-log-phase cultures at densities of 2 × 106 per ml.
Initiating and monitoring morphogenesis
Log-phase cells were washed twice in 10 ml of buffered salts solution (0· 02 M-KCI, 0·05 M-MgCl2, 0·04 M phosphate buffer, pH 6·5, 35 HIM strepto-mycin sulfate; Sussman, 1966); 5 × 107 washed amebae were then resuspended in 0·5–1·0 ml of buffered salts solution and dispersed on a black filter pad (4 cm diameter, Whatman no. 29; Soll & Waddell, 1975) overlaid on two Millipore prefilters (number AP10037) saturated with buffered salts solution. The de-veloping cell culture and pads were centered in a Petri dish which in turn was placed in a humidity chamber at the desired temperature. To change the temperature during development, the black filter supporting the developing culture was transferred to saturated underpads preincubated at the desired temperature. Developmental progress as well as the number of fingers per aggregate were monitored under a Nikon dissection microscope with horizontal lighting.
Measuring aggregate diameters
Aggregate diameters were measured under a Baush and Lomb dissection microscope fitted with a calibrated micrometer in one eye-piece. Measurements were made at 60 × power.
RESULTS
When growing Dictyostelium amebae are washed free of nutrient medium and dispersed on a filter pad saturated with buffered salts solution, they progress through an ordered sequence of stages to the final fruiting body. At 20 °C the amebae begin aggregating after 6 h so that by 9 h they have separated into loose but discrete aggregates. In the next 2 h, each loose aggregate constricts at its perimeter so that by 11 h each aggregate appears as a near-perfect hemisphere referred to as a tight aggregate. After 12·5–13 h, each tight aggregate forms a tip at its top and appears slightly pyramidal, and then elongates rapidly into an inverted cone. By approximately 13·5 h, the height of the cone is twice the diameter at the base. This morphology is referred to as a finger. Each finger then gives rise to a fruiting body after 24 h total developmental time.
The effect of low temperatures upon the number of fingers per aggregate
At 20 °C the majority of aggregates form one finger which in turn develops into one fruiting body. In a standard experiment at 20 °C approximately 75 % of individual aggregates give rise to one finger, 15 % to two fingers, and 10 % to three fingers (Fig. 1); therefore, each aggregate on the average gives rise to 1·3 fingers. If amebae are allowed to develop at higher temperatures, the propor-tion of aggregates giving rise to only one finger is even greater. For instance, at 24 °C, 90 % of the initial aggregates give rise to one finger and only 10 % to two fingers (Fig. 1); therefore, each aggregate on the average gives rise to 1·1 fingers. If amebae are allowed to develop at temperatures below 20 °C, the average number of fingers arising from single aggregates increases dramatically. For instance, at 16 °C each aggregate gives rise on the average to approximately 2·8 fingers, the distribution ranging from one to nine fingers per aggregate (Fig. 1). At 10 °C amebae aggregate into amorphous clusters which then separate into multiple fingers; each cluster gives rise on the average to ten fingers, the dis-tribution ranging from 1 to 22 fingers per cluster (Fig. 1). The relationship of fingers per aggregate and developmental temperature is plotted in Fig. 2.
The distributions of the number of fingers per aggregate at several different temperatures. Approximately .100 aggregates were scored at each temperature.
The relationship between temperature and the average number of fingers per aggregate. The average number of fingers at each temperature represents the mean for five separate experiments.
Although the average number of fingers per aggregate in a culture developing at 16 °C is twice that in a culture developing at 20 °C, the average tight aggregate diameters at the two temperatures are nearly identical. At 20 °C the average tight aggregate diameter is 316 μm (± 16S.D., 50 measurements) and at 16 °C 296 μ m (± 77 S.D., 50 measurements). Since tight aggregates at both tempera-tures appear to be near-perfect hemispheres, one can convert average tight aggregate diameters into average volumes using the formula of a hemisphere. This conversion results in average tight aggregate volumes of 8·3 x 106μm3 and 6·8 x 106μm3 for development at 20 and 16 °C respectively. If one then calculates average field volumes at 20 and 16 °C by dividing the average tight aggregate volumes by the average number of fingers, one obtains average morphological field volumes of approximately 243 × 106μm3 and 106 × 106μm3 respectively. Therefore, the increase in the number of fingers per tight aggregate resulting from a decrease in temperature from 20 to 16 °C cannot be explained by an increase in aggregate volume. Rather, the size of the morphological field is sensitive to temperature and decreases with a decrease in temperature. In addition, it is clear that the size of the morphological field is not strictly dictated by the size of the aggregate.
The effect of a temperature shift at the tight aggregate stage upon the number of morphological fields per aggregate
The sensitivity of field size to temperature can also be demonstrated by decreasing the temperature of a developing culture at the tight aggregate stage. When a culture which has developed to the tight aggregate stage at 20 °C is shifted to 15 °C or less, the average number of fingers per aggregate increases from 1·3 to approximately 3 (Fig. 3). When cultures which have formed more than one finger per aggregate at temperatures below 15 °C are brought back to 20 °C, each finger gives rise to a single fruiting body. Therefore, the size of the morphological field at the tight aggregate stage is not rigidly determined and can still be decreased approximately twofold by a decrease in temperature.
The effect of a decrease in temperature at the tight aggregate stage on the average number of fingers per aggregate. Cultures were developed at 20 °C to the tight aggregate stage and then shifted to the desired temperature and maintained at that temperature until fingers were formed and could be scored.
The effect of a decrease in temperature at the tight aggregate stage on the average number of fingers per aggregate. Cultures were developed at 20 °C to the tight aggregate stage and then shifted to the desired temperature and maintained at that temperature until fingers were formed and could be scored.
Testing when field size is stabilized
To test when field size stabilizes during morphogenesis, developing cultures were shifted at various times after the loose aggregate stage from 20 to 5 °C. In the particular experiment presented in Fig. 4, when the temperature was shifted between and
h developmental time, the average number of fingers per aggregate increased from approximately 1·25 to 3·0. However, when shifted at 13 and
h, at a time when cultures were still in the tight aggregate stage and exhibited virtually no fingers in the population (Fig. 4), the average number of fingers per aggregate remained unchanged at 1·3. When shifted after
h, the average number of fingers per aggregate still remained unchanged at 1·3. Therefore, at a discrete point midway in the interval between completion of the tight aggregate and formation of the finger, an event is completed which stabilizes the size of the morphological field.
The time of morphological field size stabilization. Cultures developing at 20 °C were shifted to 5 °C beginning at the tight aggregate stage. Cultures were then maintained at 5 °C until fingers were formed and could be scored. LA, TA, F, and EC represents the times at which the original population exhibited 50% loose aggregate, tight aggregate, finger, and early culminate morphologies respectively at 20 °C. Tn addition, the percentage fingers in the original population at 20 °C is plotted as a function of time to demonstrate the point that stabilization occurs before completion of the finger morphology.
The time of morphological field size stabilization. Cultures developing at 20 °C were shifted to 5 °C beginning at the tight aggregate stage. Cultures were then maintained at 5 °C until fingers were formed and could be scored. LA, TA, F, and EC represents the times at which the original population exhibited 50% loose aggregate, tight aggregate, finger, and early culminate morphologies respectively at 20 °C. Tn addition, the percentage fingers in the original population at 20 °C is plotted as a function of time to demonstrate the point that stabilization occurs before completion of the finger morphology.
DISCUSSION
Raper (1940) demonstrated years ago that if a migrating slime mold pseudo-plasmodium is bissected into a front and rear portion, each could reorganize and form a well proportioned fruiting body. Therefore, a single slime mold aggregate is not restricted to a single field of organization. In this report, we have demonstrated that decreasing developmental temperature also causes an aggregate, which would normally have formed one fruiting body, to form several fruiting bodies. Therefore, one aggregate possesses the potential for forming several fields of organization and, conversely, several fields of organiza-tion can function in a single aggregate.
We have also presented evidence which indicated that the mechanism which dictates the size of the aggregate may be distinct from the mechanism which dictates the size of the morphological field. Aggregation size appears to be insensitive to a change in temperature from 20 to 16 °C, but field size is sensitive, decreasing by more than twofold. However, it has been argued that both the aggregation process and field organization rely upon the same signaling system, the release of cAMP (Durston, 1974; Rubin & Robertson, 1975; Rubin, 1976). Therefore, it may be the response of cells to the signal rather than the signaling machinery which is different in the establishment of aggregation and field size.
Hohl & Raper (1964) presented evidence that the upper limit of morpho-logical field size is regulated by a critical mass value. By depositing cell aggluti-nates which had formed in suspension on an agar surface, they found that agglutinates of strain NC4(S2) with diameters less than 370 μm formed one pseudoplasmodium and agglutinates with diameters greater formed two pseudoplasmodia. Therefore, it is quite likely that decreasing the temperature of morphogenesis decreases the critical mass value approximately two-to three-fold.
By temperature shift experiments, we have also demonstrated that the size of the morphological field is not rigidly determined in a single aggregate until a point approximately one hour prior to the formation of the finger morphology. Therefore, a relationship may exist between the formation of the tip of the finger and the stabilization of field size. Evidence has accumulated that the tip functions as an organizer of the morphological field in fruiting body construc-tion (Raper, 1940; Farnsworth, 1973; Rubin & Robertson, 1975). Since the stabilization event occurs just prior to tip formation, the event may represent the completion of processes involved in tip formation. In this context, the temperature sensitivity of field size and therefore field number per aggregate indicates that the mechanism dictating size may also dictate the number of tips in a single aggregate. The relationship between the mechanism dictating field size and the formation of the organizing tip is now under investigation.
ACKNOWLEDGEMENTS
This investigation was supported by grant PCM77-07193 awarded by the National Science Foundation to D.R.S.