/. Embryo!, exp. Morph. Vol. 44, pp. 45-51, 1978
Printed in Great Britain © Company of Biologists Limited 1977
45
The stabilization of morphological field size
during slime mold morphogenesis
By MICHAEL PEACOCK 1 AND DAVID R. SOLL1
From the Department of Zoology, University of Iowa
SUMMARY
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 dispersed 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 stabilized 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
1
Authors' address: Department of Zoology, University of Iowa, Iowa City, Iowa 52242
U.S.A.
4
EMB
44
46
M. PEACOCK AND D. R. SOLL
of the axenic medium HL-5 (Cocucci & Sussman, 1970) containing 500 jug 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 temperature amebae multiplied with a generation time of approximately 12 h and
reached a final cell density of approximately 2 x 107 per ml at stationary phase
(Yarger, Stults & Soil, 1974; Soil, Yarger & Mirick, 1976). Cells were diluted
into fresh medium when cell densities reached 5 x 106 per ml. For all experiments
reported in this communication, cells were obtained from mid-log-phase cultures
at densities of 2 x 106 per ml.
Initiating and monitoring morphogenesis
Log-phase cells were washed twice in 10 ml of buffered salts solution
(0-02 M-KCI, 005 M-MgCl2, 0-04 M phosphate buffer, pH 6-5, 35 mM streptomycin sulfate; Sussman, 1966); 5 x 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; Soil & Waddell, 1975) overlaid on two Millipore
prefilters (number AP10037) saturated with buffered salts solution. The developing 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 x 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.
47
Morphological field size in slime mold morphogenesis
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Fig. 1. The distributions of the number of fingers per aggregate at several different
temperatures. Approximately .100 aggregates were scored at each temperature.
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Fig. 2. The relationship between temperature and the average number offingersper
aggregate. The average number offingersat each temperature represents the mean
for five separate experiments.
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
4-2
48
M. PEACOCK AND D. R. SOLL
4 -,
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Temperature after transfer ( C)
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Fig. 3. 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.
1-3 fingers. If amebae are allowed to develop at higher temperatures, the proportion 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 distribution ranging from 1 to 22 fingers per cluster (Fig. 1). The relationship of
fingers per aggregate and developmental temperature is plotted in Fig. 2.
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/mi (± 16S.D., 50 measurements) and at 16 °C
296 jam (±77 s.D., 50 measurements). Since tight aggregates at both temperatures 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 10° /*m3 and
•6-8 x 106 jLtm3 for development at 20 and 16 °C respectively. If one then calculates
Morphological field size in slime mold morphogenesis
49
4 -i
r 100
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- 60
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- 40 a
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Time of transfer (h)
Fig. 4. 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. In 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.
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 x 10G /tm3 and 106 x 106 /tm3 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
50
M. PEACOCK AND D. R. SOLL
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.
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 10-£ and 12yh developmental time, the average number of
fingers per aggregate increased from approximately 1-25 to 30. However, when
shifted at 13 and 13^- 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
13^ 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.
DISCUSSION
Raper (1940) demonstrated years ago that if a migrating slime mold pseudoplasmodium 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 organization 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 morphological field size is regulated by a critical mass value. By depositing cell agglutinates which had formed in suspension on an agar surface, they found that
agglutinates of strain NC4(S2) with diameters less than 370 fim formed one
pseudoplasmodium and agglutinates with diameters greater formed two
Morphological field size in slime mold morphogenesis
51
pseudoplasmodia. Therefore, it is quite likely that decreasing the temperature
of morphogenesis decreases the critical mass value approximately two- to threefold.
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 construction (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.
This investigation was supported by grant PCM77-07193 awarded by the National Science
Foundation toD.R.S.
REFERENCES
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{Received 21 June 1977, revised 11 August 1977)