/. Embryol. exp. Morph. Vol. 50, pp. 243-25J, 1979
Printed in Great Britain © Company of Biologists Limited 1979
243
Chemotactic cell sorting in Dictyostelium
discoideum
By S. MATSUKUMA 1 AND A. J. DURSTON 1
SUMMARY
We made mixed mounds of vitally stained prestalk cells and unstained prespore cells, from
Dictyostelium discoideum (Dd), and exposed these to external 3',5'-cyclic-AMP (c-AMP)
sources. We show that prestalk cells will sort out by moving directionally toward c-AMP
sources held at < 10~5 M C-AMP. This is evidence for cell sorting via chemotaxis. We present
arguments that the natural cell sorting, observed during Dd development, is also chemotactic
to c-AMP.
INTRODUCTION
Sorting out of different cell types has been observed in organisms ranging
from mammals to sponges, when cells from different embryonic tissues or different species are mixed in vitro (Galtsoff, 1925; Townes & Holtfreter, 1955;
Trinkaus, 1969). Cell sorting may have general importance in vivo, for morphogenesis and pattern formation, but, so far, its importance has been demonstrated
in few cases. There is some evidence that it plays a role in the maintenance of
insect compartments (Morata & Lawrence, 1975). The mechanism of cell
sorting is unknown and the published data do not permit a choice between
fundamentally different hypotheses (Edelstein, 1971; Steinberg, 1975).
This article is concerned with cell sorting in the cellular slime mould Dictyostelium discoideum (Dd). Previous data indicate that sorting occurs when Dd
prestalk and prespore cells are mixed (Takeuchi, 1969) and we have also shown
that these cell types sort out naturally, during formation of the prestalkpresporc pattern (in the late aggregate) and during regulation of it (in pieces
excised from slugs) (Durston, Vork & Weinberger, 1979). Here, we confirm that
sorting occurs when prestalk and prespore cells are mixed and we show that it
then occurs via chemotaxis of prestalk cells to 3',5'-cyclic-AMP (c-AMP).
MATERIALS AND METHODS
Culturing
Dd NC4 cells were grown and harvested as described previously (Durston,
1974).
1
Authors' address; Hubrecht Laboratory, Uppsalalaan 8, 2584 CT Utrecht, the Netherlands.
244
S. MATSUKUMA AND A. J. DURSTON
Neutral red staining
The cells were plated on 1 % distilled water agai (by spreading ~ 5 x 107 cells
in ~ 0-2 ml of 0-1 m potassium phosphate buffer on the surface of each plate),
and incubated in the dark for ^ 2 days at 15 or 17 °C, to induce development
to the slug stage. Cells on one half of the plates were stained with neutral red by
incubating in 0-005% neutral red solution for 10 minutes before spreading.
These plates gave slugs having well stained tips and poorly stained prespore
zones (Bonner, 1952).
Mixed mounds of stained prestalk cells and unstained prespore cells
Front quarters and third quarters (by length) were excised from stained and
unstained slugs respectively and used to make mixed mounds of stained prestalk cells and unstained prespore cells. Our measurements, using immunofluorescent staining show that first and third quarters contain about 10% and
90 % prespore cells respectively. Mixing was usually by stirring two slug pieces
together with a fine tungsten needle, till they formed a rounded mound (0-30-5 mm diameter) of intimately mixed stained and unstained cells. In some
experiments, cells were mixed by repeatedly pipetting a suspension of stained
and unstained pieces until they were dissociated to single cells, dispensing a
droplet of suspension onto an agar surface and letting it dry in. Mixed cell
mounds made in these two ways behaved identically. Experiments using them
were performed on phosphate buffered agar, pH 6-5, at ambient temperature
(~20°C).
Filming
The movements of stained cells were recorded on Kodak 4X reversal 16 mm
film, using a Bolex camera, fitted with a Nikon CFMA autotimer, Zeiss microscope optics, and incident lighting, provided by two Nikon lamps. The films
were projected by an Athena projector, via an angled mirror onto a drawing
pad and cell positions were marked in successive frames, or at desired intervals.
RESULTS
(a) Behaviour of mixed mounds of prespore and prestalk cells on
c-AMP agar
Mixed mounds of stained prestalk cells and unstained prespore cells were
placed on phosphate buffered agar (control) or on phosphate buffered agar
containing c-AMP, at concentrations from 10~3 to 10~8 molar.
In mixed mounds on control (phosphate buffered) agar, neutral red stain
accumulated in the mound's apex, in a distinct nipple-like tip, within 2 h. We
have previously filmed this process and our films show that neutral red accumulation occurs via directional movement of stained cells (Durston et al. 1979).
Chemo tactic cell sorting in D. discoideum
/"'
j* r
I
V
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si*.
B
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FIGURE 1
(A) A mixed cell mound, photographed after 2 h on 10"4 M C - A M P agar. The
mound consists of a peripheral band of stained cells and a central mass of unstained
cells.
(B) Movement of stained cells in a mixed mound on 10~5 M C - A M P agar. Cell
movement was recorded on cine film (p. 244) and the film was analysed to show
trajectories of individual stained cells. The dotted lines show cell trajectories. The
symbols show cell positions after successive intervals of 40 sec (O) or 80 sec (•).
The dashed outline is the periphery of the cell mound. The record covers the period
between 30 and 70 min after the mound was placed on c-AMP agar. The record
shows that cells move directionally to the edge of the mound.
245
246
S. MATUSKUMA AND A. J. DURSTON
Table 1
Glass microelectrodes, filled with c-AMP dissolved in 0-025 M potassium phosphate
buffer (pH 6-5), were inserted into mixed cell mounds and c-AMP was ejected from them by
delivering a 10 sec, 1 /*A, negative pulse every 40 sec. The table shows numbers of experiments,
using various c-AMP concentrations in the electrode, in which stained cells aggregated
within the mixed mound at the electrode tip. In the experiments giving a negative result (i.e.
using 5 x 10~6 M c-AMP or less), the c-AMP electrode had no obvious effect on mound
morphogenesis. Four negative experiments were filmed (using no c-AMP ( x l ) , 5xlO~ 7
c-AMP ( x 2) and 5 x 10"6 c-AMP ( x 1)). Two of these (using 5 x 10"1 and 5 x 10~7 M C - A M P
respectively) showed weak transient attraction of all cells to the microelectrode (i.e. an
appropriate transient deformation of the mixed mound, without any cell sorting).
Number of experiments showing
stained cell
c-AMP
concentration
4
5xlO5 x 10~5
5 x 10~6
5xl0~7
0
Total no. of
aggregation at the
experiments
electrode tip
4
7
6
5
2
4
7
0
0
0
If c-AMP was present in the agar, development of the mounds took a different
course, depending on the c-AMP concentration.
At 10~3 (the highest concentration tried) and 10~4 M, mounds contracted to
a rounded form within 10 min of the start of the experiment and stained
cells began to accumulate at the periphery of the mound within 45 min. Peripheral accumulation of stained cells continued for 2 h, by which time a peripheral band of stained cells and a central, unstained cell mass were distinguishable (Fig. 1A). The central cell mass subsequently remained inert and underwent no further morphogenesis, but the peripheral stained cells became concentrated in a ring of aggregates, each of which made a stalky fruiting body.
At 10~5 c-AMP, peripheral accumulation of stained cells occurred as above,
but contraction of the unstained cell mass was less apparent, and this eventually
flattened. In some cases, the peripheral band of stained cells became separated
from the unstained mass by an indentation or a narrow gap. Large cell mounds
(~ 0-5 mm diameter), as formed by our usual mixing method, typically formed
Fig. 2. (A) A typical positive response, in the experiment described in the Table 1
legend. In this case, the electrode contained 5 x 10~4 M C-AMP, and the photograph
was taken 60 min after the start of the experiment. (B) Cells attracted to microelectrodes make directional movements. The details of thisfigureare as for Fig. 1B.
A microelectrode tip (indicated left) was inserted into the edge of a mixed cell mound
and stained cells moved directionally towards it. The record covers the first 70 min
of the experiment.
Chemotactic cell sorting in D. discoideum
.
.
/
•
.
*
—
-v
\
.
v
*
-
''
100/im
Fig. 2. For legend see opposite page.
247
248
S. MATSUKUMA AND A. J. DURSTON
an outer band of stained cells, an inner band of unstained cells (from which the
outer cells had presumably migrated) and an innermost zone containing both
cell types. At 10~5 M C - A M P , the entire cell mass eventually flattened (within
4 h) and spread outwards, thus dispersing on the agar surface.
At 10"6 and 10~7 M C - A M P , the cell mass flattened and dispersed to a monolayer within 1 h. No sorting of the cell types was observed and further development was prevented.
10~8 M c-AMP and less had no visible effect.
We filmed the movement of stained cells in mixed mounds on c-AMP agar.
The films showed that peripheral accumulation of stain occurs via centrifugal
migration of stained cells along virtually radial trajectories, until they reach
the edges of mixed mounds (Fig. 1 B). These cells then move without obvious
directionality until they enter an aggregate, usually via a spiral trajectory.
Stained cells in regions of mounds showing no sorting (e.g. in the centres of
mounds on 10~8 M agar) moved without obvious directionality.
(b) Behaviour of prestalk and prespore cells, in response to
a localized c-AMP signal
The directional movements of stained cells in mounds on c-AMP agar suggested that the cell sorting observed might be chemotactic to c-AMP. To test
this point directly, we used a c-AMP containing glass microelectrode to deliver
a localized c-AMP signal within cell mounds and recorded the effect on cell
sorting, using time-lapse film. The results of a series of such experiments using
various source amplitudes are summarized in Table 1, and a typical positive
response is illustrated in Fig. 2. In all cases, a superthreshold signal (^ 5 x
10~5 M c-AMP in standard buffer, in the electrode, using a standard electrical
signal) caused aggregation of stained cells at the electrode tip within 60 min
(Fig. 2 A). Lower amplitude signals ( ^ 5 x 10~6 M C - A M P in the electrode) had
no such effect: see Table 1 legend. The stained cells moved directionally
(Fig. 2B). This result was obtained regardless of the position of the electrode tip
in the cell mass (whether in its apex, or base at one edge). The stained aggregate
attracted by the electrode subsequently became the tip of a slug made from the
mound, if the electrode was removed. These results indicate cell sorting via
chemotaxis to c-AMP.
DISCUSSION
We have shown that Dd prestalk and prespore cells can sort out, via directional movements of prestalk cells towards a superthreshold c-AMP source
(pp. 246-7). This is direct evidence that these cells can sort out via a chemotactic
response of prestalk cells to c-AMP. We note that chemotaxis has been dismissed
as a possible factor in vertebrate cell sorting (Steinberg, 1962). In our view, the
evidence against it is not strong (see also Edelstein, 1971). The time may be ripe
for a re-evaluation of its importance. We know of no published data which
Chemotactic cell sorting in D. discoideum
249
show convincingly that a case of sorting out occurs via non-directional movements. We note that, whereas our findings indicate a role for chemotaxis in
a case of cell sorting, they do not show that the Dd cell types sort out because
of a difference in their chemotactic response. These cells sort out by moving
within a tissue and the movement difference leading to sorting could result
from a difference (e.g. in cell adhesiveness), which is not directly connected
with the chemotactic response itself. Our findings give some evidence that this
difference may be dependent on extracellular c-AMP concentratration (see
below).
There is evidence that cell sorting occurs naturally during Dd development.
Takeuchi (1969), Maeda & Maeda (1974) and Forman & Garrod (1977) all
report results suggesting natural cell sorting during slug formation, or during
an equivalent developmental stage seen in liquid culture. We have observed
sorting between neutral red stained and unstained cells at this developmental
stage as well as during regulation in slug pieces (Durston et al. 1979). The
evidence presented here does not show whether this natural Dd sorting is
chemotactic, to c-AMP, but two further findings suggest that it may be.
(1) Films of natural sorting (during slug formation and regulation of slug
pieces) show that this occurs via directional cell movements. It is, therefore,
probably chemotactic (Durston et al. 1979).
(2) The induction of multiple axes in mixed mounds, by c-AMP concentrations ^ 10~4 M resembles a known effect of c-AMP on intact slugs (Nestle &
Sussman, 1972). We observed multiple axis formation in back pieces excised
from neutral red stained slugs, and placed on 10~4 M C - A M P agar. These pieces
contain scattered, neutral red stained, prestalk-like cells, and these show similar
behaviour as stained cells in mixed mounds (i.e. they accumulate at the periphery
of the piece and then aggregate to make new axes). This observation suggests
that neutral led stained cells in intact Dd tissues can sort out in response to
c-AMP.
The possibility that natural Dd sorting occurs via chemotaxis to c-AMP
raises the question whether c-AMP concentrations in natural tissues are in the
right range to induce it. Unfortunately, the appropriate data (the extracellular
free c-AMP concentrations during late aggregation and regulation) are not
available. Also, we do not know the extracellular c-AMP concentration required
to induce cell sorting artificially - only that this is less than, or equal to, 10~5 M
(the minimum concentration required in agar under a mixed mound). Brenner
(1977) has measured the mean c-AMP level in Dd slugs. This is about 1040 p mole c-AMP/mg protein. Based on a cell volume estimate of 250 /tm3
(Bonner & Frascella, 1953) and a cell protein estimate of 4 mg/108 cells (White &
Sussman, 1961), this implies a mean c-AMP concentration of 1-6-6-4 x 10~6 M
in a slug composed of packed cells. Slugs contain very little extracellular space
(Maeda & Takeuchi, 1969). The c-AMP concentration range calculated for
intracellular c-AMP in aggregating cells is comparable (~ 1-20/on; Gerisch
250
S. MATSUKUMA AND A. J. DURSTON
et al. 1977). Extracellular c-AMP concentrations in the range 10~5-10~6 M do
not seem implausible.
The hypothesis that Dd cell sorting is chemotactic provides a possible
explanation of why the prestalk zone forms in the apex of a mixed mound and
in the apices of natural Dd aggregates and regulating slug pieces. c-AMP
secretion by the mound and c-AMP loss to the agar substrate should set up an
apico-basal gradient of extracellular c-AMP, and this could direct prestalk cells
towards its apex. We note that, if a c-AMP electrode is used to set up a c-AMP
gradient within a mixed mound, artificially, this can lead to formation of a prestalk zone at any chosen point (p. 5). We also note that non-induced sorting, in
mixed mounds, takes 2 h, whereas induced sorting takes ^ 1 h. We suggest
that the time course of non-induced sorting is determined by the time course of
c-AMP secretion.
Our staining procedure enabled direct observation of the movement of prestalk cells, but not of prespore cells (which do not stain well with neutral red).
However, the behaviour of mixed mounds gave indirect information about
prespore cell behaviour. The fact that, on 10~3 and 10~4 M C - A M P agar, unstained cell mounds remained motionless while stained cells dispersed suggests
that prespore cells within tissues are not chemotactically responsive or not
motile at high c-AMP concentrations. The fact that, on 10~5-10~7 M C - A M P
agar, all cells eventually dispersed suggests that prespore cells are chemotactically
responsive and motile within a lower c-AMP concentration range. These
findings may have relevance for understanding later Dd morphogenesis. They
are being pursued.
We thank Pieter Nieuwkoop and Job Faber for their comments, Dorothy Parsons for
typing the manuscript and Carmen Kroon and Leen Boom for the figures.
REFERENCES
J. T. (1952). The pattern of differentiation in ameoboid slime moulds. Am. J.
Botany 31, 175-182.
BONNER, J. T. & FRASCELLA, E. B. (1953). Variations in cell size during the development of
the slime mould Dictyostelium discoideum. Biol. Bull. 104, 3, 297-300.
BRENNER, M. (1977). Cyclic AMP gradient in migrating pseudoplasmoida of the cellular slime
mould Dictyostelium discoideum. J. biol. Chem. 252, 4073-4077.
DURSTON, A. J. (1974). Pacemaker activity during aggregation in Dictyostelium discoideum.
Devi Biol. 37, 225-235.
DURSTON, A. J., VORK, F. C. & WEINBERGER, C. (1979). The control of later morphogenesis
by chemotactic signals in Dictyostelium discoideum. In Biophysical and Biochemical Information Transfer in Reproduction and Ageing (ed. J. G. Vassileva-Popova & E. V. Jensen).
(In the Press.)
EDELSTEIN, B. (1971). A cell-specific diffusion model of morphogenesis. /. theor. biol. 30,
515-532.
FORMAN, D. & GARROD, D. R. (1977). Pattern Formation in Dictyostelium discoideum. II.
Differentiation and pattern formation in non-polar aggregates. /. Embryol. exp. Morph.
40, 229-243.
BONNER,
Chemotactic cell sorting in D. discoideum
251
P. S. (1925). Regeneration after dissociation (an experimental study on sponges).
Behaviour of dissociated cells of Microciona prolifera under normal and altered conditions.
J. exp. Zool. 42, 183-221.
GERISCH, G., MAEDA, Y., MALCHOW, D., ROOS, W., WICK, U. & WURSTER, B. (1977).
Cyclic AMP signals and the control of cell aggregation in Dictyostelium discoideum. In
Development and Differentiation in the Cellular Slime Moulds (ed. Cappucinelli & Ashworth), pp. 105-124. Elsevier/North Holland Biomedical Press.
MAEDA, Y. & TAKEUCHI, I. (1969). Cell differentiation and fine structures in the development
of the cellular slime moulds. Development, Growth and Differentiation 11, 232-245.
MAEDA, Y. & MAEDA, M. (1974). Heterogeneity of the cell population of the cellular slime
mould Dictyostelium discoideum before aggregation and its relation to the subsequent
locations of the cells. Expl Cell Res. 84, 88-94.
MORATA, G. & LAWRENCE, P. (1975). Control of compartment development by the engrailed
gene in Drosophila. Nature, Lond. 255, 614-617.
NESTLE, M. & SUSSMAN, M. (1972). The effect of cyclic AMP on morphogenesis and enzyme
accumulation in Dictyostelium discoideum. Devi Biol. 28, 545-553.
STEINBERG, M. S. (1962). On the mechanism of tissue reconstruction by dissociated cells. I.
Population kinetics, differential adhesiveness and the absence of directed migration. Proc.
natn. Acad. Sci. U.S.A. 48, 1577-1582.
STEINBERG, M. S. (1975). Adhesion-guided multicellular assembly: a commentary on the
postulates, real and imagined, of the differential adhesion hypothesis, with special attention
to computer simulations of cell sorting. /. theor. Biol. 5, 431-443.
TAKEUCHI, I. (1969). Establishment of polar organisation during slime mould development.
In Nuclei Acid Metabolism Cell Differentiation and Cancer Growth (ed. X. Cowdry &
Y. Seno). Oxford: Pergamon Press.
TOWNES, P. & HOLTFRETER, J. (1955). Directed movements and selective adhesion of embryonic
amphibian cells. J. exp. Zool. 128, 53-120.
TRINKAUS, J. P. (1969). Cells into Organs; The Forces that Shape the Embryo. New Jersey:
Prentice-Hall.
WHITE, G. J. & SUSSMAN, M. (1961). Metabolism of major cell components during slime
mould morphogenesis. Biochimica et Biophysica Acta 53, 285-293.
GALTSOFF,
{Received 3 October 1978, 29 November 1978)