/. Embryo/, exp. Morph. Vol. 47, pp. 195-206, 1978
Printed in Great Britain © Company of Biologists Limited 1978
{95
Colchicine induces
multiple axis formation and stalk cell differentiation
in Dictyostelium discoideum
By DANTON H. O'DAY1 AND ANTONY J. DURSTON 2
From the Hubrecht Laboratory, Utrecht
SUMMARY
Colchicine is shown to have several effects on the development of the pseudoplasmodia of
the cellular slime mould Dictyostelium discoideum At concentrations of 001 M and above
culmination was prevented, while differentiation of cells into stalk cells occurred at the rear
of cell masses. Essentially all cells transformed into stalk cells when slugs were left on colchicine agar for a long time. At concentrations of 001 M normal slug architecture was maintained
while above 0025 M pseudoplasmodia reorganized into multiple mounds. Each of these
mounds developed an apparently normal discrete tip which was devoid of prespore cells as
shown by immunofluorescent staining. The same effects were observed in growing cultures
and in regulating slugs treated with colchicine. The data are consistent with the ideas that
microtubules are involved in the maintenance of slug architecture and in the differentiation
of stalk cells. The modes by which these intracellular structures may operate in these functions
are discussed.
INTRODUCTION
During the development of Dictyostelium discoideum, the aggregation of
individual amoebae leads to construction of a multicellular pseudoplasmodium.
This cell mass ultimately develops into a fruiting body containing a lemonshaped mass of spores, a slender supporting stalk and a basal disc. The pseudoplasmodium contains a polarized axial pattern: future stalk cells occupy its
anterior portion, prespore cells are in the middle and a small population of
pre-basal disc cells completes the rear end. This pattern is at least roughly proportionate (Takeuchi, Hayashi & Tasaka, 1977) and regulative (Sakai, 1973).
At the front of the slug is a nipple-shaped tip, which has been shown to act as
an organizer (Raper, 1940; Durston, 1976). The slug tip is known to contain
a high concentration of cyclic AMP (Rubin & Robertson, 1976) and this cyclic
nucleotide has been shown to induce Dictyostelium discoideum amoebae to
differentiate into stalk cells (Bonner, 1970).
Essentially, there are two schools of thought about the establishment of
1
Author's address: This research was carried out while the author was on research leave
from the Department of Zoology and Erindale College, University of Toronto, 3359
Mississauga Rd., Mississauga, Ontario, Canada.
2
Author's address: Hubrecht Laboratory, Universiteitscentrum 'De Uithof', Uppsalalaan
8, Utrecht, The Netherlands.
196
D. H. O'DAY AND A. J. DURSTON
polarity and pattern in the Dictyostelium discoideum pseudoplasmodium, and
in similar systems. The first supposes that the pseudoplasmodium contains an
axially graded variable, or variables, which provide positional information to
cells and thus define their future differentiated state (see Wolpert, 1969; McMahon, 1973). In most such models the tip would feature as a reference point,
which holds a defined value of the variable. The second supposes the existence
of a non-positional proportioning mechanism, which partitions appropriate
populations of cell types, together with a sorting out process to supply the
spatial pattern (Bonner, 1957; Takeuchi, 1969; Garrod & Forman, 1977;
Durston, York & Weinberger, 1978); the tip may be supposed a focus for the
aggregation of prestalk cells (Durston et ah 1978).
In the course of our investigations into the factors that regulate pattern and
polarity in cellular slime moulds we have begun a series of studies with agents
that are known to affect these developmental aspects in other systems. In this
report, we show that colchicine, which affects polarity and differentiation in
Hydra (Corff & Burnett, 1969), dramatically affects pseudoplasmodial organization and induces differentiation of stalk cells in D. discoideum.
MATERIALS AND METHODS
Culturing
Spores of Dictyostelium discoideum strain NC4 were spread on nutrient agar
with Escherichia coli B/r as a food source (Durston, 1974). After 3 days, at 21 °C
in the dark, the cultures consist of mainly standing and migrating pseudoplasmodia. Pseudoplasmodia were picked from plates using a microscalpel and
were replated on 1 -5 % water agar plates with or without colchicine. Colchicine
from two different sources was employed (lot 53C-1680, Sigma Chemical Co.;
control no. 7474101/1, Boehringer, Mannheim). The agar plates were prepared
by mixing 0-5 ml of warm sterile 3 % water agar with 0-5 ml of a millipore-filtered
(Millex, Millipore Inc.) colchicine solution or sterile distilled water in small
plastic Petri dishes (35 x 10 mm; Corning no. 25000). In one series of experiments D. discoideum spores and bacteria were spread on colchicine-containing
nutrient agar which had been prepared in a similar manner. The plates were
stored in the dark at 21 °C. At selected intervals the gross morphology of the
culture was assessed using a dissecting microscope and cell masses were removed
from each culture, squashed in distilled water, and examined with phase contrast microscopy. All photographs were taken with a Zeiss Photomicroscope
using Panatomic X film.
Neutral red staining
Neutral red stained slugs were prepared by mixing cells of D. discoideum, of
all developmental stages, with bacteria on water agar and adding a drop of
neutral red solution (6 mg/ml) so that a gradient of staining existed through the
cell mixture. The next day darkly stained slugs with clearly defined prestalk
Colchicine and development
in D . discoideum
197
zones were picked up with a microscalpel and plated on control or colchicine
plates. The pattern of neutral red staining in these slugs was examined
with a dissecting microscope using reflected and transmitted light.
Calcofluor staining
Cell masses were placed in a 20 fi\ drop of 0-1 % Calcofluor white ST
(American Cyanamid) on a glass slide and covered with a coverslip. The
fluorescence of stained cellulose was detected usinga Zeiss standard microscope
equipped with a Zeiss IVf 1 H650 epiluminator and appropriate interference
filters (Zeiss combination 4B7703). Photographs were made using Kodak 2475
recording film.
Immunofluorescen ce
Cell masses from control and colchicine plates were placed in a drop of
distilled water on slides that had been previously coated with gelatine (Rogers,
1967). A coverslip was added and the squash preparations were left at room
temperature for about 15-30 min to allow compression and adhesion of the cell
masses. The squash preparations were then placed in a freezer (— 20 °C) until
used. The coverslip was pried from the frozen slide and the squashes were fixed
in cold methanol for 3-5 min. The fixed preparations were then stained with
rabbit antispore serum and goat anti-rabbit FITC-IgG (Nordic Pharmaceuticals). The antispore serum had been prepared against freeze dried spores
of D. mucoroides by Dr S. Brahma. The fluorescence of prespore cells was
examined using the microscopes described above equipped with appropriate
interference filters (Zeiss combination: 487709), and photographed using Kodak
recording film.
RESULTS
Development of pseudoplasmodia on colchicine agar
Within 2 h of plating on 0-05 M colchicine agar each slug reorganized up into
two to five irregular shaped cell masses (Fig. 1B). This subdivision of slugs
continued for at least 24 h (Fig. 1C, D) with the number of mounds produced
generally being related to the original slug length. After 24 h the cell mounds
became very discrete in their morphology, each one possessing a sharply defined
tip. At the base and between each cell mound was a mass of material. The cell
mounds move about continuing to leave this material behind (Fig. 1F). By
7 days very little remained of the cell masses (Fig. 1G). When the material that
lay at the bases and between the cell masses was examined by phase microscopy
it was seen to contain stalk cells and extracellular material. When a group of
cell masses were stained in situ with Calcofluor, the cell mounds exhibited no
fluorescence while the basal and inter-mass material fluoresced brightly
(Fig. 2A). Staining of individual mounds revealed that the fluorescence originated at their bases (Fig. 2B). Stalk cells were present in the fluorescent material,
198
100 ,um
D. H. O'DAY AND A. J. DURSTON
100 HIT!
Fig. 1. The effect of colchicine (005 M) on the morphology of D. discoideum pseudoplasmodia. Pseudoplasmodia from growth plates (A) were placed on water agar
containing colchicine as described in the Methods and Materials. The progressive
gross morphological changes in slug structure were examined by light (transmitted)
microscopy at 2 h (B), 20-24 h (C, D), 4 days (F) and 7 days (G). Phase microscopy
of squashes of the cell masses showed stalk cells in basal material (E) at 20 h and in
all parts of cell mounds (H) at 7 days.
Colchicine and development in D. discoideum
199
Fig. 2. Calcofluor fluorescence of colchicine-treated (005 M) cell masses. After 24 h
on colchicine agar cell masses were stained in situ (A) with calcofluor or single
masses were placed in a drop of stain solution on a slide (B) and examined by fluorescence microscopy (see Methods and Materials for details).
becoming evident after 20 h (Fig. 1 E). By 5-7 days all the cells had transformed
into stalk cells (Fig. 1H) but spores were never observed. Of 276 slugs plated
on 0-05 M colchicine, none deviated from the above description.
Slugs plated on 0-01 M colchicine retained their normal morphology except
that a small posterior protrusion was usually in evidence (Fig. 3 A). The migrating slugs left behind small mounds of stalk cells in their slime trails. The projection from the rear of migrating slugs was also made up of stalk cells (Fig.
3B, C). Calcofluor staining of these slugs revealed the same pattern of
fluorescence as for the cell mounds produced on 0-05 M colchicine (Fig. 2B).
Slugs plated on 001 M colchicine agar rarely fruit, even in the presence of
bright overhead light. Of 187 slugs only 27 culminated, forming fruiting bodies
which lacked a basal disc. Slugs left on the treated agar are mostly transformed
into stalk cells but even by 7 days some undifferentiated cells were still evident.
Slugs plated on 0-025 M colchicine showed one or other of the patterns
described above. Usually both induced phenotypes were expressed on the same
plate by different slugs. Of 187 slugs plated on 0-025 M colchicine only 10
culminated, with each fruit lacking in a basal disc. At 0-005 and 0-0025 M
colchicine did not detectably affect slug morphology but fruiting occurred in
only 41 % (46 of 112) or 70 % (28 of 40) of the cases, respectively. In these cases
fruiting was normal.
Slugs or cell mounds, from all colchicine concentrations, that were replated
onto colchicine-free water agar after 3 days underwent culmination. Fruits
formed from slugs treated with 001 M colchicine or above formed fruiting bodies
that lacked a basal disc and that contained normal stalk cells but small round
spores.
A
.B
Fig. 3. The effects of colchicine (001 M) on the morphology of D. discoideum pseudoplasmodia. After 24 h on 001 M colchicine
agar (see Methods and Materials), slugs revealed a normal morphology except for the presence of a tail of material at their rear ends
(A). Squashes of slugs (B) viewed by phase contrast microscopy revealed that the tail was composed of stalk cells that formed at the
rear of these slugs (C) and appeared to be organized in a manner similar to normal stalk.
O
on
H
c
d
d
O
O
O
Colchicine and development in D. discoideum
201
Morphology and staining pattern
53
24 h
48 h
72 h
001
005
Fig. 4. The effects of colchicine on staining patterns of neutral red-stained slugs.
Stained slugs were plated on water agar or colchicine agar (001 or 005 M) and their
patterns of staining were examined at 24, 48 and 72 h (see Methods and Materials).
The pattern of neutral red staining is indicated by stippling.
Effect of colchicine on neutral red staining patterns
When neutral red-stained slugs are plated on water agar and left in the dark,
their staining patterns become increasingly sharper so that a clearly defined
anterior red region and smaller posterior stained region are evident (Fig. 4).
By 24 h on 0-05 M colchicine, each cell mass had a clearly defined bright red
tip (Fig. 4). No staining was evident in other regions. By 48 h, the tip remained
dark red but red-stained cells also became evident in the main body of the cell
masses. By 72 h the staining was reminiscent of the control staining patterns
except that basal staining was enhanced and red stained stalk cells projected
from the rear of the cell masses. The neutral red staining pattern of slugs plated
on 0-01 M colchicine was markedly different from that of control slugs (Fig. 4).
Within the first 24 h the zone of neutral red staining had begun to extend
two-thirds to three quarters of the way backward and appeared to increase in
intensity. At 48 h most slugs are uniformly stained. By 72 h the staining was
very bright and was limited to the rear end of the slugs. Slugs plated on 0-025 M
showed one or other of the above patterns.
Fig. 5. Immunofluorescence of control and colchicine-treated (001 M) slugs of D. discoideum. Control slugs (A) and treated slugs
(48 h on 001 M colchicine agar) were stained for the presence of prespore cells by a modified immunofluorescence technique of
Takeuchi(1963)as detailed in the Methods and Materials. The immunofluorescence pattern in treated slugs is qualitatively normal,
but the staining of fluorescent cells is weaker.
z
H
O
c
d
o
d
x
d
O
Colchicine and development in D. discoideum
203
Effect of colchicine on immunofluorescence patterns
Squashes of control slugs, of all ages, stained with rabbit antispore serum
and goat anti-rabbit FITC IgG revealed an anterior zone containing cells with
no evident fluorescence and a posterior zone containing cells with bright
particles of fluorescence (Fig. 5 A). In some slugs a small region at the rear end
was also devoid of fluorescence. After treatment of slugs with colchicine, at all
concentrations employed in this study, these regions remained definable
although the fluorescent cells were less bright in their fluorescence. For example,
even after 48 h treatment on 005 M colchicine all mounds revealed a tip region
deficient in fluorescence and a rear region with bright fluorescent cells (Fig. 5B).
Effect of colchicine on growth cultures and regulating slugs
When colchicine (up to 0-05 M) was included in the nutrient agar plates
growth was slowed but was not stopped at any concentration. At 0-025-0-05 M
development proceeded to late aggregation, producing many mounds as in
Fig. 1D. At 0-005-0-01 M development progressed to pseudoplasmodium
formation as described earlier (Fig. 2), but the slugs did not culminate. At
lower drug concentrations development was apparently normal. When slugs
or aggregates from untreated cultures were transferred to and disaggregated
on colchicine agar plates, the developmental results were exactly as described
above.
In a series of experiments, a total of 185 slugs were cut into prestalk and prespore regions and plated on colchicine agar. The results were the same as
described above with both prespore and prestalk pieces developing in the same
manner as whole slugs.
DISCUSSION
Colchicine has been shown to have dramatic effects on the development of
D. discoideum. At 0-01 M (4 mg/ml) colchicine prevents pseudoplasmodia from
culminating. This effect has previously been reported by Cappuccinelli &
Ashworth (1976). In the present study, stalk cell production at the rear of
pseudoplasmodia and cell mounds was also observed at colchicine concentrations of 0-01 M or more. That these were true stalk cells is verified by their
morphology and their fluorescence in the presence of Calcofluor (Bonner, 1970).
During normal development, stalk cell production occurs only at culmination
with the cells for the stalk differentiating at the tip and the basal disc cells at the
base (rear) of the squat culminating cell mass (Raper, 1940; Bonner, 1967). The
induction of stalk cell production at the rear of colchicine-treated slugs suggests
that the future basal disc cells are being prematurely transformed into their
differentiated state. This idea is supported by the evidence that treated slugs
replated on agar lacking colchicine produce a fruiting body lacking a basal disc.
The second major effect on D. discoideum development was the induction of
204
D. H. O'DAY AND A. J. DURSTON
multiple axes. At concentrations above 0-025 M (lOmg/ml) colchicine induces
pseudoplasmodia to divide into many cell mounds, each of which develops a
discrete tip. Immunofluorescent staining of prespore cells revealed the absence
of staining in the mound tips and bright fluorescence in the main body of the
mounds. A distinct prespore-prestalk pattern resembling that seen in normal
pseudoplasmodia (Tacheuchi, 1963) was seen in all treated pseudoplasmodia
and cell mounds, though prespore cells stained less brightly than controls in the
colchicine-treated cultures. Thus colchicine induces the formation of multiple
axes from single pseudoplasmodia.
The third major effect of colchicine was on the neutral red staining patterns
in the slug. Pseudoplasmodia that had been stained with neutral red underwent
unusual changes in stain distribution after plating on 0-01 M colchicine. The
staining of the anterior region of these slugs increased in intensity and progressed backwards so that they exhibited a very bright uniform staining after
48 h. The staining continued backwards until only a bright rear staining was
detectable. In contrast, stained control slugs developed a clearly defined red
anterior region of staining and a smaller but definite posterior region which
continued to sharpen as development progressed. This control pattern has been
observed by other workers (Bonner, 1952; Francis & O'Day, 1971). At 0-05 M,
the staining pattern resembled that of controls, although there was an excessive
accumulation of red stained cells at the slug's rear end. We cannot yet account
for this unusual concentration dependence.
Bonner (1952) first suggested that the neutral red staining pattern in the
Dictyostelium discoideum slug occurs because neutral red is a specific stain for
prestalk cells. Neutral red stains large autophagic vacuoles which are a specific
organelle for the prestalk cell in slugs that have migrated for more than 24 h
(Bluemink, Durston, v. Maurik and Vork, in preparation; Francis & O'Day,
1971). It is, therefore, possible that the neutral red staining change seen in
colchicine-treated slugs reflect an effect of colchicine on the differentiated state
of cells in the slug (so that posterior cells become prestalk-like). This conclusion
is not indicated by our results using immunofluorescent staining since the
immunofluorescent staining pattern is unchanged in colchicine-treated slugs.
The apparent dramatic effects of colchicine on one pattern (neutral red) but
not the other (fluorescence) remains to be resolved.
Corff & Burnett (1969) have previously shown that colchicine at 15-25 mg/ml
has a marked effect on regenerating Hydra, tending to inhibit production of
distal structures (tentacles), while supporting formation of proximal parts
(peduncle). The basis of this effect is unknown. Our findings here indicate that
colchicine can suppress the apical dominance of the Dictyostelium discoideum
slug tip and can also suppress differentiation of one of the two cell types found
in final fruiting body (spores), while causing premature differentiation of the
others (stalk cells). Either or both of these effects may be analogous to the effect
on Hydra, but the analogy remains to be clarified.
Colchicine and development in D. discoideum
205
Cappuccinelli, Hames & Cuccureddu (1977) have found that colchicine binds
specifically to purified D. discoideum tubulin, thus showing that the drug has
its typical effect in cellular slime moulds (Ohmsted & Borisy, 1973). By this
token, the drug can be used as a diagnostic tool for the analysis of microtubular
importance in cellular functions in D. discoideum. From this it can be interpreted that microtubules play a critical role in morphogenesis and cellular
differentiation in this organism. Of the many proposed functions for microtubules (Ohmsted and Borisy, 1973; Snider & Mclntosh, 1976) two may be
relevant: cellular secretion and cell division. The immediate effect of colchicine
(0-25-0-05 M) on slug architecture suggests that the drug is interfering with the
normal signals that maintain slug morphology. Possibly, this effect is due to an
inhibition of the secretion of factors essential for slug structure maintenance. The
immediate effect is unlikely to be due to a prevention of cell division since only
a small number of slug cells undergo division and the cell cycle of D. discoideum
takes over 8 h (Zada-Hames & Ashworth, 1977). The latter effects resulting in
the induction of stalk cells could be due to an effect on cell division. Durston &
Vork (1978) have recently shown a correlation between DNA synthetic patterns
and the prespore-prestalk boundary in D. discoideum. It is also possible that it
is due to an effect on cellular secretion. Rudolph, Greengard & Malawista (1977)
have shown that drug enhancement of cAMP secretion from human leucocytes
is increased in the presence of colchicine. It is conceivable that colchicine
mimicry of cAMP action (i.e. stalk cell induction and multiple tip formation)
may represent an analogous situation.
We would like to thank Peter Poot for his assistance in some of the early work on the
immunofluorescent staining and Carmen Kroon for her photographic and artistic work.
Dr S. Brahma kindly supplied the antispore serum.
REFERENCES
BONNER, J. T. (1952). The pattern of differentiation in amoeboid slime molds. Amer. Nat.
86, 79-89.
BONNER, J. T. (1957). A theory for the control of differentiation in Dictyostelium discoideum.
Quart. Rev. Biol. 32, 232-246.
BONNER, J. T. (1967). The Cellular Slime Molds. Princeton, N. J.: Princeton University Press.
BONNER, J. T. (1970). Induction of stalk cell differentiation by cyclic AMP in the cellular
slime mold Dictyostelium discoideum. Proc. natn. Acad. Sci. U.S.A. 65, 110-113.
CAPPUCCINELLI, P. & ASHWORTH, J. M. (1976). The effect of inhibitors of microtubule and
microfilament function on the cellular slime mould Dictyostelium discoideum. Expl Cell Res.
103, 387-393.
CAPPUCCINELLI, P., HAMES, B. D. & CUCCUREDDU, R. (1977). Tubulin in Dictyostelium
discoideum. In Development and Differentiation in the Cellular Slime Moulds (ed. P.
Cappuccinelli & J. M. Ashworth), pp. 231-241. Elsevier/North Holland.
CORFF, S. C. & BURNETT, A. L. (1969). Morphogenesis in Hydra. I. Penducle and basal disc
formation at the end of regenerating Hydra after exposure to colchicine. /. Embryol. exp.
Morph. 21, 417-443.
DURSTON, A. J. (1974). Pacemaker activity during aggregation in Dictyostelium discoideum.
Devi Biol. 37, 225-235.
206
D. H. O'DAY AND A. J. D U R S T O N
A. J. (1976). Tip formation is regulated by an inhibitory gradient in the Dictyostelium discoideum slug. Nature, Lond. 263, 126-129.
DURSTON, A. J. & VORK, F. C. (1978). The spatial pattern of DNA synthesis in the slug of
Dictyostelium discoideum. Expl Cell Res. (in the Press).
DURSTON, A. J., VORK, FRIDA & WEINBERGER, C. (1978). The control of later morphogenesis
by chemotactic signals in Dictyostelium discoideum. In Biophysical and Biochemical
Information Transfer in Recognition (ed. J. G. Vassileva Popova & E. V. Jensen). New
York: Plenum.
FRANCIS, D. & O'DAY, D. H. (1971). Sorting out in pseudoplasmodium of Dictyostelium
discoideum. J. exp. Zool. 176, 265-272.
GARROD, D. & FORMAN, D. (1977). Pattern formation in the absence of polarity in Dictyostelium discoideum. Nature, Lond. 265, 144-146.
MCMAHON, D. (1973). A cell contact model for cellular position determination in Dictyostelium discoideum. Proc. natn. Acad. Sci. U.S.A. 70. 2396-2400.
OHMSTED, J. B. & BORISY, G. C. (1973). Microtubules. Ann. Rev. Biochem. 42, 507-540.
RAPER, K. B. (1940). Pseudoplasmodial formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Scient. Soc. 56, 241-282.
ROGERS, A. W. (1967). Techniques of Autoradiography. Amsterdam: Elsevier.
RUBIN, J. & ROBERTSON, A. (1976). The tip of Dictyostelium discoideum pseudoplasmodium
as an organizer. / . Embryol. exp. Morph. 33, 227-241.
RUDOLPH, S. A., GREENGARD, P. & MALAWISTA, S. E. (1977). Effects of colchicine on cyclic
AMP levels in human leucocytes. Proc. natn. Acad. Sci. U.S.A. 74, 3404-3408.
SAKAI, Y. (1973). Cell type conversion in isolated prestalk and prespore fragments of the
cellular slime mould Dictyostelium discoideum. Development, Growth and Differertiation
15, 11-19.
SNIDER, J. A. & MCINTOSH, J. R. (1976). Biochemistry and physiology of microtubules.
Ann. Rev. Biochem. 45, 699-720.
TAKEUCHI, I. (1963). Immunochemical and immunohistochemical studies on the development
of the cellular slime mould Dictyostelium discoideum mucoroides. Devi Biol. 8, 1—26.
TAKEUCHI, I. (1969). Establishment of polar organization during slime mould development.
In Nucleic Acid Metabolism, Cell Differentiation and Cancer Growth (ed. E. V. Cowdry &
S. Seno), pp. 297-304. Oxford: Pergamon.
TAKEUCHI, I., HAYASHI, M. & TASAKA, M. (1977). Cell differentiation and pattern formation
in Dictyostelium discoideum. In Development and Differentiation in the Cellular Slime
Moulds (ed. P. Cappuccinelli & J. M. Ashworth), pp. 1-16. Amsterdam: Elsevier/North
Holland.
WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation.
J. theoret. Biol. 25, 1-47.
ZADA-HAMES, I. M. & ASHWORTH, J. M. (1977). The cell cycle during the growth and development of Dictyostelium discoideum. In Development and Differentiation in the Cellular
Slime Moulds (ed. P. Cappuccinelli & J. M. Ashworth), p. 69. Elsevier/North Holland.
DURSTON,
{Received 17 April 1978, revised 2 June 1978)