/ . Embryol. exp. Morph. Vol. 25, 3, pp. 457-465, 1971
457
Printed in Great Britain
Further evidence for the
sorting out of cells in the differentiation of the
cellular slime mold Dictyostelium discoideum
By J. T. BONNER, 1 T. W. SIEJA 1 AND E. M. HALL 1
From the Department of Biology, Princeton University
SUMMARY
The observation of Takeuchi's that the denser cells of Dictyostelium discoideum tend to sort
out towards the anterior end of the migrating slug (and the lighter cells towards the posterior
end) has been confirmed using spore size as a method of identifying cells populations. A
fraction of the anterior and posterior ends of a slug are isolated and allowed to fruit; their
spores are then measured. The same is done for preaggregation cells which have been
separated into heavy and light fractions, using Takeuchi's technique of centrifugation of the
cells in a dextrin solution equal to the mean specific gravity of the cells. Invariably, in three
experiments with different strains of D. discoideum, the spores derived from dense cells
corresponded perfectly with spores derived from the anterior cells of the slug, and a similar
correspondence was found between spores derived from light cells and posterior slug cells.
Contrary to a previous view (Bonner, 1959), cell size did not always correlate with position;
in one strain the anterior cells were larger, in the other two they were smaller.
INTRODUCTION
Some years ago Bonner (1959) put forward the hypothesis that when the
amoebae of the cellular slime mold Dictyostelium discoideum aggregated the cells
were not identical but already possessed tendencies toward the formation of
either stalk cells or spores, and those cells with stalk tendencies sorted out to the
anterior end of the slug, while those with spore tendencies moved to the posterior
end of the slug. The evidence involved the non-random distribution of mutant
marker cells within the slug, and the fact that the anterior cells of the particular
strain of Dictyostelium discoideum investigated were consistently larger than the
posterior cells. Furthermore, it was shown that this size difference was probably
a property the cells possessed before aggregation. This 'sorting out' hypothesis
had the virtue of explaining some curious previous results such as the forward
movement within a slug of grafted anterior, prestalk cells (Bonner, 1952), and
the passing of the cells of a slug of one strain through the cells of a slug of
another in certain grafts between species and strains (Bonner & Adams, 1958).
However, it was not until the work of Takeuchi (1969) that the sorting out
1
Authors'1 address: Department of Biology, Princeton University, Princeton, New Jersey
08540, U.S.A.
458
J. T. BONNER, T. W. SIEJA AND E. M. HALL
hypothesis was given crucial and convincing evidence. First he showed that
when preaggregation amoebae were stained with spore antibodies conjugated
with a fluorescent dye, that some of the cells contained large quantities of spore
proteins, while other cells contained little or none. Furthermore, by the time
cells had formed slugs, all the cells containing the spore proteins were in the
posterior, prespore region.
It is known from the pioneer experiments of Raper (1940) that by cutting a
slug transversely one can induce regulation and cause prestalk cells to become
spore cells and vice versa. Gregg (1965) obtained further insight into these
reversible changes in spore proteins which occurred during such regulation,
also using the fluorescent antibody technique.
More recently Takeuchi (1969 - this is a review of work published previously
in Japanese) has marked the cells using tritiated thymidine and has confirmed
the phenomenon of sorting out in detail. Furthermore, with the same cell
labelling technique he has separated vegetative or preaggregation cells on the
basis of their density, and he has shown that the heavier cells sort out primarily
to the anterior end while the lighter cells move to the posterior end. In other
words prestalk cells are heavier than prespore cells. Finally, Takeuchi (1969) has
dissociated cells from different parts of the slug, and has shown, using the 3H
marker technique, that the cells resume their original position when they reaggregate and form new slugs.
It should be pointed out that Takeuchi (1963) also showed that the sorting out
did not occur during aggregation, as had been previously supposed, but immediately after aggregation. At first the distribution of the two presumptive cell
types is random, but later clearly segregated.
Despite the fact that the evidence for sorting out is becoming increasingly
compelling, there are still a number of workers in the field who cling to the old
view that the position of a cell is determined by the time it enters an aggregate.
The importance of sorting out in animal embryos is now established; there is
little basis, in the light of the accumulated facts, for precluding the sorting out
hypothesis in any consideration of the development of the cellular slime molds.
In the work to be reported here we have additional evidence that the cells in
D. discoideum slugs do sort out and that cell density is a significant correlate
with the ultimate position of the cell within the slug, as Takeuchi (1969) contends.
MATERIALS AND METHODS
Organisms. The following strains of Dictyostelium discoideum Raper were
used in this study: (1) Dd-1, representing the original isolate of this species,
NC-4, was given to one of us (J. T. B.) in 1940 by K. B. Raper. It has been shown
by Bonner & Frascella (1952) to be haploid (7 chromosomes). (2) NC-4(S): this
is a small-spored strain that originated from a single spore isolated in Raper's
laboratory by Dr A. T. Weber. (3) NC-4(L): this represents a large-spored strain
Differentiation of slime mold
459
of NC-4, similarly isolated by Weber. Comparative studies of the two monospore cultures indicate that they are haploid and diploid respectively (Weber,
1967).
Separation of heavy and light amoebae. We have used the technique of Takeuchi
(1969), but since he gives little specific information in his review paper, it
might be helpful if we list some of the details of preparation.
Bacteriological dextrin (Type 1, Sigma Chemical Co.) was mixed with distilled
water and heated to 100 °C to make a concentrated solution. Upon cooling, this
was thinned by adding more water until the specific gravity (measured with a
hydrometer) was 1-070. This solution was centrifuged for 40 min at 16000g
which cleared the solution of particles. It was then autoclaved for 30 min and
stored at 5 °C.
As Takeuchi (1969) points out, different culture conditions give different mean
specific gravities. His value of 1 061 was too high for our amoebae, and by further
dilution with distilled water we found a specific gravity of 1 045 to be satisfactory.
The amoebae were grown at 21 °C on nutrient agar (buffered 1 % peptone and
1 % dextrose - Bonner, 1947) with E. coli B/r (and with some extra water added
to the surface of the Petri dish). Just prior to any signs of aggregation the cells
were harvested in a 1 % salt solution, and centrifuged three times to wash the
amoebae free of the remaining bacteria.
The amoebae were then thoroughly mixed in 12 ml of the dextrin solution in a
15 ml centrifuge tube, and a few drops of 1 % salt solution were gently added to
the upper surface so that the floating cells would not dry up during centrifugation. They were spun for 20 min at 150 g; in some cases this did not seem sufficient and they were spun again at 200 g for 20 min. Both bands were pipetted off
and washed three times in 1 % salt solution, to free them of the dextrin.
Cutting anterior and posterior fractions. In slugs that had migrated less than a
centimeter the anterior and posterior portions (ca. £ of the whole slug) were
isolated with a hair loop and allowed to fruit. Some parallel experiments were
run to see if a smaller segment taken from the middle rather than the distal end
of the prespore region might give different results. It was found that spore sizes
of fruiting bodies derived from the posterior end and the middle of the prespore
region had identical frequency distributions of size.
Statistical significance of results. In a previous study (Bonner, 1959) extensive
significance tests comparing two populations of spores were done. It was shown
that length was as effective a measure as length and width, and in all cases the
means of the spore lengths derived from anterior fractions were significantly
different from those of posterior fragments. To make one key check in this study,
the case in which the means were closest was subjected to a t test. The mean
lengths were 8-2 and 7-7/i (Fig. 2), where t= 3-6, giving a P < 0001. Clearly
the difference between these means is significant, and therefore this must be true
of all those cases where the difference between the means is larger.
460
J. T. BONNER, T. W. SIEJA AND E. M. HALL
RESULTS
The spore length is a particularly useful way to measure cell size; not only is
the length-width ratio constant on the average for spores of different size, but
also one assumes that the degree of hydration of spore protoplasm is constant
and therefore spore length accurately reflects the amount of protoplasm in each
cell (Bonner, 1959). Others have also used spore size as a convenient marker to
50
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Posterior cells
30
8
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»
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8
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Light cells I
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f\
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Spore length (/<)
Fig. 1. D. discoideum-1. A comparison of the spore lengths of .156 spores from
fruiting bodies derived from posterior fractions of slugs and 165 spores derived from
anterior slug fractions. The arrows indicate the mean spore lengths here and in Fig. 2.
Fig. 2. D. discoideum- 1. Preaggregation cells have been separated into light and
heavy fractions by centrifugation in dextrin and the cells allowed to fruit. 155 spores
derived from the light amoebae are compared with 178 spores from the heavy cells.
compare different cell populations (Sussman & Sussman, 1962; Sackin &
Ashworth, 1969). It should be noted that in NC-4(L) the spores had the same
proportions as NC-4(S) and, except in very rare instances (ca. 1 in 50), the same
shape, unlike the diploid strain described by Sussman & Sussman (1962) which
contained many sickle-shaped cells.
The technique employed here was to compare the frequency distribution of
the lengths of spores derived from amputated anterior and posterior fractions of
slugs with those of spores derived from heavy and light preaggregation cells
Differentiation of slime mold
461
which had been separated by dextrin centrifugation following the method of
Takeuchi (1969). This was done for different strains, and combinations of
strains of D. discoideum.
DictyosteUum discoideum Dd-1
This is the same small-spored strain used previously (Bonner, 1959) and the
results of the earlier experiments were confirmed; the spores derived from
anterior cells were significantly larger than those derived from the cells at the
posterior ends of the slugs (Fig. 1).
50
r
40
30
20
10
0
10
11
12
13
14
11
12
13
14
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Light
cells
20
10
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8
9
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Spore length (u)
Fig. 3. D. discoideum - NC-4(S). A comparison of the spore lengths of 282 spores
from fruiting bodies derived from anterior fractions of slugs and 289 spores derived
from posterior slug fractions. Arrows indicate mean spore lengths here and in Fig. 4.
Fig. 4. D. discoideum NC-4(S). Preaggregation cells have been separated into light and
heavy fractions by centrifugation in dextrin and the cells allowed to fruit. 337 spores
derived from the heavy amoebae are compared with 369 spores from light cells.
If these were compared with spores derived from heavy and light preaggregation cells separated by centrifugation with dextrin (Fig. 2), it was clear that the
heavy cells gave a spore length frequency distribution which was almost identical to the anterior cells, and the light cells were similar to the posterior cells.
In other words the anterior cells of a slug of Dd-1 are larger (confirming our
1959 results) and denser (confirming Takeuchi's 1969 results) than the posterior
cells.
462
J. T. BONNER, T. W. SIEJA AND E. M. HALL
Dictyostelium discoideum NC-4(S)
This strain has approximately the same size spores as Dd-1. However, when
we measured the frequency distribution of spore lengths derived from amoebae
in the anterior and posterior ends of the slugs, the results were the opposite from
those of Dd-1. In Dd-1 the anterior cells were larger than the posterior cells;
in NC-4(S) they were smaller (Fig. 3).
50
40
NC-4(S)/
30
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9
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gso
I<-> 40
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30
I12010
c/>
Anterior A
cells
/ \
/
/ \
\
/
Posterior
cells
\
IT
8
9
10
11
12
13
14 15
9
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11
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14
50
40
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0L
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Spore length (/<)
Fig. 5. A comparison of 158 spores from D. discoideum NC-4(S) with 158 of NC4(L). Arrows indicate mean spore lengths.
Fig. 6. Preaggregation cells of NC-4(S) and NC-4(L) were mixed (50/50) and allowed
to form slugs, and anterior posterior fractions were cut from the slugs and allowed
to fruit. 446 spores derived from the anterior fractions of the slugs are compared
with 390 spores from posterior slug fractions.
Fig. 7. Preaggregation cells of NC-4(S) and NC-4(L) were mixed (50/50) and centrifuged in dextrin to separate the light and heavy cells. 270 spores derived from the
heavy fraction are compared with 334 spores from the light cells.
Differentiation of slime mold
463
When the NC-4(S) cells were centrifuged in dextrin, there was again a result
different from that obtained with Dd-1: the heavy cells produced smaller spores
than the light cells (Fig. 4). Therefore in NC-4(S) the anterior cells of a slug are
smaller and denser than the posterior cells. Note that when one compares the
two strains, it is always the heavy cells that lie anteriorly in the slug; in one case
they are the largest cells (Dd-1), while in the other they are the smallest (NC-4(S)).
Mixtures of NC-4(S) and a large spored clone, NC-4(L)
To test these results in another way vegetative cells of NC-4(S) were mixed
approximately 50/50 with another strain derived from NC-4 which contains
spores that are very much larger than NC-4(S). First we compared the two
strains separately (Fig. 5); their mean spore lengths are 7-8 and 11-4/t, which
means that the volume of the larger spores is roughly three times that of the
smaller ones.
If slugs were allowed to form from the 50/50 mixtures of cells of the two
strains, it was possible, as before, to derive spores from anterior and posterior
fractions of the slug. When their frequency distributions were compared, one
can see that cells from the large spore strain predominate in the posterior end of
the slug, and the reverse is true for the anterior end (Fig. 6). By comparing the
two regions where the spore lengths do not overlap for the two strains, one can
compute that 81 % of the spores in the anterior end were of NC-4(S) size, while
in the posterior end 83 % of the spores were of the size of the larger variant
(NC-4(L)).
Again the 50/50 mixture of cells at the end of the vegetative stage was centrifuged in dextrin, and the heavy cells gave rise to fruiting bodies with predominantly small cells, and the light fraction gave the reverse (Fig. 7). In this case
73 % of the NC-4(S) cells were found in the light cell fraction, and 72 % of the
larger NC-4(L) cells in the heavy cell fraction (Fig. 7).
Some preliminary tests for differences in properties between heavy and light cells of
NC-4(S)
We tested the heavy and light fractions to see: (1) if they had different chemotactic properties, using the cellophane square test (Bonner, Kelso & Gillmor,
1966), (2) how they compared with respect to the time of onset of aggregation
after centrifugation, and (3) whether or not they differed in their susceptibility
to cyclic AMP in the induction of stalk cells (Bonner, 1970). No differences
between the two fractions were shown in any of these tests.
DISCUSSION
The experiments reported above offer further evidence that sorting out of
amoebae does take place in the cell mass of D. discoideum. Furthermore, they
give an independent confirmation of Takeuchi's (1969) demonstration that the
heaviest cells sort out to the anterior end.
464
J. T. BONNER, T. W. SIEJA AND E. M. HALL
It is interesting that contrary to expectation from previous results (Bonner,
1959) cell size is not correlated with cell position; in one strain the largest cells
are anterior, while in another they are posterior.
Since cell density correlates consistently with the pattern of sorting out in the
three strains tested, one might ask why this should be so. One answer is obviously
that it might be chance; after all a sample of three is insufficient. Even if it
turns out to be a consistent correlation, it might still be that cell density has no
direct bearing on the mechanism of sorting out, but only an indirect one.
Because no difference can be detected in the rates of movement of anterior
and posterior cells removed from a slug (Samuel, 1961), and because heavy and
light cell fractions give the same values on the cellophane square test, as shown
above, one must tentatively conclude that differences in the rates of individual
cell movement do not account for sorting out in cellular slime mold slugs.
Instead, it might be more profitable to approach the matter in terms of differential cell adhesion, which has been so successful an hypothesis in the analysis of
sorting out in vertebrate embryogenesis (Steinberg, 1970). If this turns out to be
a useful approach for the cellular slime molds as well, then one must ask the
question of whether or not adhesive properties and cell densities could be
related in some way.
Finally, it is clear that the heavy and light cell fractions show no difference,
either in terms of rate of development, or in susceptibility to stalk cell induction
by cyclic AMP. This may simply be a reflexion of the fact that both these tests
take a long time for completion, and by that time regulation may have nullified
any differences between the two populations. This serves further to emphasize
the well-known fact that until the final differentiation into spore or stalk cell
takes place, the differentiation fate of a cell is reversible. It is for this reason that
one must consider the preaggregation cells and the cells in the slug as showing
tendencies towards stalk cell or spore differentiation; the cells have not become
determined in any way and remain labile until they are fully differentiated.
It was not possible to show any functional differences between the light and
heavy cells, stressing the fact that regulation is always possible in either group;
they do not become determined in their differentiation until they form stalk
cells or spores.
The authors wish to acknowledge with gratitude the helpful advice and criticism of
Professor K. B. Raper.
This work was supported in part by funds from research Grant No. GB-3332 of the National
Science Foundation and by funds from the Hoyt Foundation. We also benefited from the
central equipment facilities in the Biology Department, Princeton University, supported by the
Whitehall Foundation and the John A. Hartford Foundation.
Differentiation of slime mold
465
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{Manuscript received 20 October 1970)