/ . Embryol. exp. Morph. Vol. 36, 3, pp. 663-668, 1976
Printed in Great Britain
663
Cell patterning in migrating slugs of
Dictyostelium discoideum
By J. SAMPSON 1
From the Imperial Cancer Research Fund, Mill Hill Laboratories, London
SUMMARY
When front quarters of migrating slugs of Dictyostelium discoideum are isolated by
surgery and induced to fruit immediately they produce fruiting bodies with disproportionately
large stalks (Raper, 1940). The data in this communication show that the 'stalky' character
of fruits derived from front quarters persists even if the cells of the front quarters are disaggregated and hence have to reaggregate before fruiting. The data also demonstrate that
fruits derived from rear quarters of slugs have disproportionately large spore heads, and that
this effect becomes more pronounced with increasing age of the slugs. These observations
support the view that the cells of the front and rear of migrating slugs are to some extent
committed to different fates.
INTRODUCTION
Under conditions of high humidity and low ionic strength aggregates of
Dictyostelium discoideum form slugs which can migrate for days before transforming into mature fruiting bodies (Newell, Telser & Sussman, 1969). Both
slug migration and the events associated with fruiting body formation
(culmination) appear to be under the control of the apical 'tip' (Raper, 1940;
Farnsworth, 1973). The cells in the front third of the slug give rise to the stalk
of the fruiting body while the remainder become spores (Raper, 1940). Striking
differences have been observed in the distribution of certain enzymes and cell
organelles between the front and back region of slugs (for a review see Loomis,
1975). In addition Raper (1940) has shown that if a slug is cut transversely the
front quarter produces a fruiting body with a disproportionately large stalk
when it is induced to fruit immediately; whereas if front quarters are permitted
to migrate for various periods of time before fruiting the proportions of the
resulting fruiting bodies become progressively more normal. These findings have
led most authors to the view that the cells of the front and back of the slug are,
to some extent, committed to different fates. However, this view has been
challenged recently by Farnsworth who has proposed instead that the fate of
cells is determined uniquely at the time of culmination when the slug becomes
transformed into a fruiting body, under the control of the tip (Farnsworth,
1
Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London NW7 IAD, U.K.
664
J. SAMPSON
1973, 1974 and 1975). On this theory the 'stalky' character of fruits derived
from front quarters of slugs would presumably result from perturbations in the
ultimate transformation process due to the fact that front regions have a tip
which is too large for the truncated body. In order to distinguish between
these two views I have performed experiments similar to those of Raper under
conditions where the cells of the front quarter of the slug must reform a new tip
before culmination.
MATERIALS AND METHODS
Dictyostelium discoideum strain V12/M2 (kindly supplied by Dr G. Gerisch)
was grown at 22 °C with Aerobacter aerogenes on a medium containing peptone,
yeast extract, magnesium sulphate, glucose, potassium phosphate (pH 6-1) and
agar. Vegetative cells were harvested during exponential growth and freed of
bacteria by two washes in KK 2 buffer (16mM-KH2PO4 4 mM-K2HPO4,
2 mM-MgSO4, pH 6-1) and two washes in distilled water. Washed cells were
resuspended at a concentration of 2 x 108 cells/ml in distilled water and 50 /A
aliquots were applied along a 4 cm line on an agar surface (9 cm Petri dishes
containing 25 ml of 2 % agar in water were routinely used). The Petri dishes
were then placed in a humid atmosphere in unidirectional light so that the slugs
would migrate along the agar surface toward the light source, a 60 W bulb
placed 3 ft away from the Petri dish (Raper 1940; Bonner, Clarke, Neely &
Slifkin, 1950). Under these conditions over 95 % of aggregates form slugs which
migrate for up to 4 days.
Pieces of slugs were cut using a microspatula. In experiments which involved
the removal of the tip from front quarters, the front quarter was first cut and
approximately 10 % of this quarter was removed from the anterior end. After
the sections had been cut the surrounding agar surface was cleared of unwanted
cells and cell debris, and a block of agar containing the sections was removed to
a new Petri dish. If cut pieces were to be allowed to migrate prior to fruiting the
Petri dish containing the agar blocks was placed in a humid atmosphere in
unidirectional light, as described above. Induction of fruiting was achieved by
removing the Petri dish lid and placing the sections under an overhead light for
\ h. The lid was then replaced and the sections were allowed to culminate at
22 °C. The resulting fruiting bodies were fixed in formaldehyde vapour for 4-6 h
after which time they were transferred onto a microscope slide and either
photographed or traced with a camera lucida. Volumes were calculated
assuming that the stalk is a cone (volume: \Trr2h) and the spore head a prolate
sphere (volume: \-nab2, where a is the major semi-axis and b the minor semiaxis) (Garrod & Ashworth, 1972). The results are expressed as the ratio of spore
head volume to stalk volume which has been abbreviated in the text to ' spore
to stalk ratio' for brevity.
Cell patterning in Dictyostelium
665
Table 1. Spore: stalk ratios offruits from whole slugs and slug pieces
Pieces were cut from slugs which had been migrating for 35 h and were induced to
fruit immediately. The ratios are expressed ± standard error. The P values shown in
parentheses are the results of a statistical analysis, by the Student's t test, of the
differences between fruits derived from whole slugs and the fruits derived from each
of the pieces. Analysis of the difference between front quarters and front quarters
minus a tip gave a value off = 0 1 . Other experimental details were as described in
the Materials and Methods section.
Piece of slug
No. of
pieces used
Spore:stalk ratio
Whole slug
Front quarter
Front quarter minus tip
Back quarter
9
6
7
11
3 ±0-27
0-26 + 0-03 (P < 0001)
0-33±003 (P < 0001)
8-3±0-8 (P < 0001)
RESULTS
The results of experiments in which cut pieces of slugs were induced to fruit
immediately after operation are shown in Table 1. The data confirm Raper's
observations that front quarters produce very 'stalky' fruits (Raper 1940).
Removal of the tip region of the front quarters so that new tips had to form
before culmination (see Materials and Methods) had little effect on the outcome;
the fruits were still very 'stalky' though perhaps slightly less so than those from
intact front quarters. Fruiting bodies derived from back quarters of slugs had
a spore:stalk ratio higher than control fruits (P = < 0-001) (see below). When
intact front quarters or those with tips removed were permitted to migrate for
a time they produced progressively more normal fruits (Table 2), as reported
by Raper (1940).
There was little difference in the sequence of morphological events observed
when front quarters or those with their tips removed were induced to fruit
immediately after operation. In both types of fragments the cut surface at the
rear quickly became covered in slime, and within about 30 min a new tip
appeared at the anterior end of those whose tip had been removed. Thereafter
all the transected pieces rounded up and proceeded through the normal events
of culmination. Sections from the rear end of slugs behaved differently. The cut
surface rapidly became covered with slime and the mass of cells rounded up to
form a small hemisphere. There was then a delay of 2-4 h before a tip became
visible, and when it appeared it was at the top of each hemisphere. This then
rose upwards from the agar surface and culminated in the normal fashion.
It could be argued that the new tip formed when the anterior region of a front
quarter is removed is not necessarily appropriate to the reduced size of the cell
mass but instead depends, say, on the cross-sectional area of the exposed
anterior surface. In that case the 'stalky' character of the resulting fruits need
not be inconsistent with the model of Farnsworth (1975). In order to eliminate
666
J. SAMPSON
Table 2. Effect of post-surgical migration on the fruits derived from front
quarters
Pieces were cut from slugs which had been migrating for 35-40 h. Other details are
described in the legend to Table 1.
Piece of slug
Whole slug
Front quarter
Front quarter minus tip
Front quarter
Front quarter minus tip
Front quarter
Front quarter minus tip
Duration
of
migration
after
surgery
00
No. of
pieces
used
Spore: stalk ratio
0
0
0
6
6
12
12
10
10
8
7
6
6
6
2-22 ±0-17
01 ±002 (P < 0001)
015±002 (P < 0001)
0-75±0-17 (P < 0001)
1-24±014 (P < 0001)
1-9 ±0-28 (P> 0-1)
2-5 ±0-28 (P> 01)
Table 3. Effect of disaggregation on spore:stalk ratios
Pieces were cut from slugs which had been migrating for 40 h and were induced to
fruit immediately after operation. Other details are described in the legend to
Table 1.
Piece of slug
Treatment
No. of
pieces
Spore:stalk ratio
Whole slug
Whole slug
Front quarter
Front quarter
Front quarter minus tip
Front quarter minus tip
None
Disaggregated
None
Disaggregated
None
Disaggregated
10
9
6
8
6
7
2-22 ±014
2-28 ±0-16
0-46±008 (P < 0001)
0-37±007 (P < 0001)
0-51 ± 0 1 (P < 0001)
0-48 ± 006 (P < 0001)
this objection I examined the effect of disaggregating the cut sections so that the
cells had to reaggregate before fruiting (Table 3). Cell masses were gently
disaggregated on the agar surface, taking care to ensure that the area covered
by the disaggregated cells was kept to a minimum. Disaggregated cell masses
reaggregated and formed new tips within 2 h of disaggregation. Whole,
disaggregated slugs reaggregated to form two or three new fruits; front quarters
and front quarters without tips in general reaggregated to form a single fruit.
Disaggregation had no effect on the spore:stalk ratios; fruits from disaggregated slugs had normal ratios whereas fruits from disaggregated front
quarters still had their' stalky' character whether the tip region had been removed
prior to disaggregation or not (Table 3).
Finally the effect of slug age on the behaviour of pieces of slugs was examined
(Fig. 1). The youngest slugs operated on were obtained 20 h after plating
washed amoebae and had been migrating for no more than 5-6 h. Front
Cell patterning in Dictyostelium
667
12-
10-
6-
4-
I
20
-AA--—6
40
I
60
1
100
Time after starvation (h)
Fig. 1. Pieces were cut from migrating slugs at the times indicated and were induced
to fruit immediately after operation. O—O, Back quarters; • — # , whole slugs;
A
A, front quarters minus a tip; •
• , front quarters.
quarters of these slugs, whether intact or with tips removed, gave results
indistinguishable from older slugs (Fig. 1). On the other hand the fate of rear
quarters depended markedly on the age of the slugs. The spore: stalk ratio was
approximately normal at 20 h and increased gradually to three times normal in
3-day-old slugs. The regression lines through the data for intact slugs and for
back quarters (Fig. 1) intersect at 10 h after starvation.
DISCUSSION
In this communication the effect of various treatments on the proportion of
spore and stalk cells has been inferred from the ratio of the volume of the stalk
to that of the spore head. This same method has been employed frequently by
other investigators (Raper, 1940; Bonner & Slifkin, 1949; Garrod & Ashworth,
1972) and the ratios observed for control fruiting bodies in the present work are
similar to those reported previously. This method will of course be misleading
if the surgical manipulations produce changes in the volume of spore head or
stalk that are not simply due to changes in the number of cells in the two stuctures.
However, changes in relative cell numbers appears the simplest explanation for
the differences in relative volumes observed after surgery, particularly in view
668
J. SAMPSON
of the fact that the change in volume ratios in fruits derived from anterior slug
segments is the converse of that observed in fruits from the posterior segments.
With this methodological proviso in mind, the data argue strongly against the
view that the fate of cells in the slug is determined uniquely at the time of
culmination under the influence of the tip and favour the view that the cells of
the front and back of a migrating slug are to some extent at least committed to
different fates.
Interestingly, the present results point for the first time to a commitment of
cells in the rear of the slug to form spore cells. The data in Fig. 1 show that
whereas the 'stalkiness' of fruits obtained from front quarters is independent of
slug age, the 'sporiness' of rear quarters increases gradually over a period of
3 days. This latter behaviour could mean that, with time, an increasing proportion of the cells in the rear of the slug begin to express the genetic programme
corresponding to prespore cells. Alternatively, all cells may express this programme from an early time but their ability to regulate and switch to the stalk
programme may decline progressively. The fact that the majority of the cells in
the rear of slugs contain prespore vacuoles early after slug formation (Muller
& Hohl, 1973) favours the latter explanation.
I should like to thank Dr Julian Gross for his encouragement and helpful advice.
REFERENCES
J. T., CLARKE, W. W., Jr., NEELY, C. L., Jr. & SLIFKIN, M. K. (1950). The
orientation to light and the extremely sensitive orientation to temperature gradients in the
slime mould Dictyostelium discoideum. J. cell comp. Physiol. 36, 149-158.
BONNER, J. T. & SLIFKIN, M. K. (1949). A study of the control of differentiation: the
proportions of stalk and spore cells in the slime mould Dictyostelium discoideum. Am. J.
Bot. 36, 727-734.
FARNSWORTH, P. A. (1973). Morphogenesis in the cellular slime mould Dictyostelium discoideum: the formation and regulation of aggregate tips and the specification of developmental axes. /. Embryol. exp. Morph. 29, 253-266.
FARNSWORTH, P. A. (1974). Experimentally induced aberrations in the pattern of differentiation in the cellular slime mould Dictyostelium discoideum. J. Embryol. exp. Morph.
31, 435-451.
FARNSWORTH, P. A. (1975). Proportionality in the pattern of differentiation of the cellular
slime mould Dictyostelium discoideum and the time of its determination. J. Embryol. exp.
Morph. 33, 869-877.
GARROD, D. R. & ASHWORTH, J. M. (1972). Effect of growth conditions on development of
the cellular slime mould, Dictyostelium discoideum. J. Embryol. exp. Morph. 28, 463-479.
LOOMIS, W. F. (1975). Dictyostelium discoideum: A Developmental System. New York and
London: Academic Press.
MULLER, U. & HOHL, H. R. (1973). Pattern formation in Dictyostelium discoideum:
temporal and spacial distribution of prespore vacuoles. Differentiation 1, 267-276.
NEWELL, P. C , TELSER, A. & SUSSMAN, M. (1969). Alternative developmental pathways
determined by environmental conditions in the cellular slime mould Dictyostelium discoideum. J. Bad. 100, 763-768.
RAPER, K. B. (1940). Pseudoplasmodium formation and organisation in Dictyostelium
discoideum. J. Elisha Mitchell Scient. Soc. 56, 241-282.
BONNER,
{Received 21 May 1976, revised 6 August 1976)