/. Emoryol. exp. Morph. Vol. 61, pp. 61-67, 1981
Printed in Great Britain © Company of Biologists Limited 1981
The age-dependent loss of
cells from the rear of a Dictyostelium
discoideum slug is not tip controlled
By ELIZABETH SMITH 1 AND KEITH L.WILLIAMS 2
From the Genetics Department, Research School of Biological Sciences,
Australian National University, Canberra City
SUMMARY
Young slugs of the cellular slime mould Dictyostelium discoideum drop small numbers of
individual amoebae (~ 10/mm) in the slime trail. With increased time of migration, slugs
develop trailing tails and leave clumps of cells in their slime trails. Using reciprocal transplants between tips of young and old slugs and between a wild-type strain and an ' aged''
mutant it was shown that this age-dependent cell loss is due to changes in the bulk of cells
comprising the slug, rather than to changes in the effectiveness of the tip (organizer region).
Another property of the slug, the decision to continue migrating or form a fruiting body
which is controlled by the tip, was less affected by age. This raises the possibility that cell
autonomous properties of the slug are more subject to ageing than is the tip.
INTRODUCTION
The cellular slime mould, Dictyostelium discoideum, is a simple eukaryote
whose asexual life cycle can be divided into two separate phases, i.e. a growth
phase and a differentiation phase. The organism is a soil amoebae which,
during the vegetative phase, feeds on bacteria. When the food supply is depleted
the amoebae aggregate to form a multicellular organism which, after a nonobligatory period of migration (slug stage), differentiates into three cell types,
viz. spores, stalk cells and basal disc cells. During the migratory stage of the
life cycle the slug moves towards light or heat. It is an elongated mass of cells
enclosed by a layer of slime, the slime sheath, through which the mass moves
leaving behind the collapsed sheath, or slime trail (Raper, 1940). Under
conditions of high humidity and low ionic strength, the migratory stage lasts
for several days in wild-type strains of D. discoideum. Both biochemical studies
on developmental^ regulated enzymes (Newell & Sussman, 1970) and studies
of cell patterning of slugs (Takeuchi, Hayashi & Tasaka, 1977; Sampson, 1976)
1
Present address: Department of Biochemistry, Faculty of Science, P.O. Box 4, Canberra
City, A.C.T., Australia.
2
Present address: Max-Planck-Institut fur Biochemie, D-8033 Martinsried bei Munchen,
Federal Republic of Germany.
3
EMB 6l
62
E. SMITH AND K. L. WILLIAMS
Fig. 1. Camera-lucida drawings of slugs at various times after commencing
migration. (A) NP84, 5 h; compact rear, few cells dropped in trail. (B) NP84, 76 h.
(C) AX3 < 5 h and (D) AX3, 76 h; trailing rears, large numbers of cells dropped in
trails. Slugs are approximately 1 mm long.
suggest that, although there are some age-related changes, development reaches
a steady state in the slug and there are no major differences between young and
aged slugs. By contrast, Bonner (1957) reported that the number of cells left
in the slime trail increases with time of migration, suggesting that there is an
age-related component to the integrity of the slug. In this report we investigate
the loss of cells from the rear of the slugs of D. discoideum using young and
aged slugs and a mutant strain, and we show that while the loss of small
numbers of single cells occurs independently of age, the loss of clumps of
cells is age-dependent. By the use of transplant experiments we show that the
age-dependent characteristic is a property of the bulk cells in the slug rather
than the organiser region in the tip. We also demonstrate that a slug property
controlled by the tip, the 'slug/fruit' switch (Smith & Williams, 1980) is not
very different in young and aged slugs.
MATERIALS AND METHODS
Preparation of young and aged slugs ofD. discoideum
Slugs of D. discoideum of strains NP84 (North & Williams, 1978) and AX3
(Loomis, 1971) were prepared as described previously and migrated towards
light on non-nutrient agar plates (Smith & Williams, 1979). Aged slugs were
prepared by allowing slugs to migrate for 5 days after the vegetative amoebae
were put on water agar. During this time the light source was moved twice
so that after 5 days the slugs were moving across a fresh area of agar. Because
a slug which lies across the slime trail of another slug cannot be lifted without
breaking at the point of contact with that slime trail, most of the aged slugs
used were from the front of the field.
Cell autonomous property o / D . discoideum slugs
63
Transplant experiments
Transplant experiments were performed on slugs approximately 24 h after
washed vegetative amoebae had been put on to non-nutrient agar, except
when aged (5-day-old) slugs were used. The experiments involved the transfer
of matched pairs of slugs to fresh non-nutrient agar plates, followed by
reciprocal transplants of the intact rear portions (70-80 %) between two slugs,
as previously described (Smith & Williams, 1980). The chimeras were replaced
in standard slugging conditions (Smith & Williams, 1980).
Observation of slugs
The degree of contamination of slime trails with cells and the morphologies
of the rears of slugs were noted, and in some cases slugs were drawn using
a camera-lucida attachment on a Leitz dissecting microscope so that their
morphologies could be easily compared. Numbers of cells left in slime trails
were determined by marking the position of slugs at intervals and counting
the number of cells in the slime trails between these points. This was possible
because we have developed a technique (to be described elsewhere, Fisher &
Williams [in preparation]) for attaching slime trails to transparent PVC discs
and staining them with Coomassie Brilliant Blue (Sigma Chemical Co.), which
stains both the slime trails and individual cells.
RESULTS AND DISCUSSION
Morphology of slugs and contamination of trails
We have previously reported that slugs of strain NP84 leave only a few single
cells in their slime trails over the first 4 to 5 days after vegetative amoebae are
put under conditions which induce slug formation (Smith & Williams, 1979).
Over this early period, the rear of the slug is well-defined and there are few
cells in the slime trail (Fig. 1 A), but as the time of migration increases the rear
becomes more trailing and clumps of cells, as well as slightly increased numbers
of single cells, are left in the slime trail. The numbers of cells left during different
periods of migration are given in Table 1 for five individual trails, and these
results are consistent with the less detailed observations we have made on
a large number of slime trails of strain NP84. It is clear that the numbers are
fairly constant (~ 10 cells/mm) over the first 70-80 mm (52 h) of migration
and increase slightly over the next 24 h (~ 15 cells/mm). These results are
consistent with those of Bonner (1957), who observed 10 cells/mm dropped by
young slugs. After 76 h this level of cell loss is markedly increased, with large
numbers of cells (> 100/mm) being left in clumps in the slime trail and the
rear of the slug trailing (Fig. 1B), suggesting that the integrity of the NP84 slug
is breaking down on prolonged migration.
Young slugs of strains AX3 have a phenotype which is similar to that of
3-2
64
E. SMITH AND K. L. WILLIAMS
Table 1. Numbers of single cells and clumps of cells left in slime trails of migrating
slugs of strain NP84
Total cells left
Slug
*Time interval
(h)
Distance
moved
(mm)
Single
cells
t Clumps of cells
f
Small
Medium
Large
Cells/mm
1
0-28
28-52
52-76
176-130 +
50-3
33-1
37-1
48-8
703
425
686
577
0
0
0
100
0
0
0
38
0
0
0
11
14
13
18
100 +
2
0-28
28-52
52-76
76-120
47-1
330
37-4
26-8
601
300
561
562
0
0
0
40
0
0
0
10
0
0
0
2
13
9
15
100 +
3
0-28
28-52
52-76
76-140
46-5
34-5
36-9
46-6
469
0
0
0
322
573
997
4
0-28
28-52
52-76
$76-130 +
401
31-3
34-6
390
5
0-28
28-52
52-76
76-156
43-6
34-9
32-5
52-9
0
0
10
9
0
88
0
0
33
0
15
15
100 +
295
270
492
501
0
0
0
87
0
0
0
22
0
0
0
0
7
9
14
100 +
310
260
402
630
0
0
0
52
0
0
0
6
0
0
0
0
7
8
12
50 +
* NP84 slugs were transferred to fresh water agar 22 h after putting vegetative amoebae
onto water agar, and this is taken as zero time.
t Small 10-50 cells, medium 50-100 cells, large > 100 cells.
% These times are not exact, since a fruiting body formed at some unknown time.
aged NP84 slugs, but much exaggerated. The rears of AX3 slugs have a trailing
appearance (Fig. 1C) and there is an increasingly high level of cell loss into
the slime trail throughout the period of slug migration (Fig. 1D), which lasts
only for up to 2, or occasionally 3, days before fruiting bodies are formed. As
well as many single cells there are compact clumps of cells, which sometimes
form fruiting bodies, in the trails of AX3 slugs. Based on these observations,
we regard AX3 as showing premature and exaggerated ageing. The increased
trailing with age could result from changes in cell speed (lnouye & Takeuchi,
1979) such that slower cells drop back despite end-to-end adhesion, resulting
in a trail of cells behind the slug.
Cell autonomous property ofT>. discoideum slugs
65
' Slug /fruit' developmental switch
Slugs of strain NP84 have a high propensity to continue migrating under
conditions of high humidity, low ionic strength and unilateral light. We have
observed slugs which have migrated for up to 11 days, and at this time are
leaving a continuous stream of cells in the slime trail, so that eventually no
fruiting body is formed. This suggests that the switch from continued migration
to fruiting body construction is not age-related. However, there is an age-related
component since we have noted that disturbance of NP84 slugs by subjecting
them to overhead light and dry air for a few minutes is more likely to cause
fruiting in aged slugs than in young slugs.
Slugs of strain AX3 do not need to be disturbed but have a high incidence
of spontaneous fruiting body formation over the first two days of migration.
Thus the 'slug/fruit' switch may be age-related, but this is convincingly
demonstrated only in strain AX3 which we regard as an ' aged' mutant.
Use of transplant experiments to determine the location within the slug of the
age-dependent characteristics
Heterotypic transplant experiments were carried out to determine whether
the loss of cells in the slime trail and trailing rear described here are determined
by the tip or by the cells in the bulk of the slug.
Morphology of slugs and contamination of trails
Since 1-day-old AX3 slugs have similar but exaggerated slugging characteristics to those of aged (5-day-old) NP84 slugs, we carried out tip transplants
between young slugs of these two strains to determine whether the increased
dropping of cells in the slime trail is a tip-controlled or a cell autonomous
function. Control transplants between slugs of the same strain resulted in slugs
with the characteristics of the original slugs. Reciprocal transplants between
young NP84 and AX3 slugs, however, yielded slugs in which the shape of the
rear and the degree of contamination of the trail were characteristic of the
slug cells comprising the back portion of the chimeric slug (Fig. 2 A and 2B).
Slugs composed of NP84 front/AX3 rear have trailing rears and many cells
left in the slime trails (Fig. 2B). Slugs were rarely formed from transplants
involving AX3 tips and NP84 rears, since the chimera almost always fruited
immediately (Smith & Williams, 1980). However, in seven exceptional chimeras
which migrated (Smith & Williams, 1980), the rear was characteristic of strain
NP84, i.e. compact with little cell loss (Fig. 2 A). Thus the cells in the tip do not
control the trailing of cells at the rear. Similar, but less extreme, results were
obtained from transplants between young and aged NP84 slugs, in which the
shape of the rear of the chimeric slug and the level of cell loss in the slime trail
were characteristic of the slug cells comprising the back portion; i.e. young
rear, few cells in trail; old rear, larger number of cells in trail.
66
E. SMITH AND K. L. WILLIAMS
B
Fig. 2. Chimeric slugs 24 h after transplanting rear portions. (A) AX3 front/NP84
rear, showing compact rear and few cells dropped. (B) NP84 front/AX3 rear,
showing a continuous stream of cells dropped in the trail. The tip of this slug is
raised off the agar surface. Slugs are approximately 1 mm long.
The tip of the D. discoideum slug secretes cAMP (Rubin, 1976) and this may
be involved in maintaining polarity and cohesion in the slug (Durston & Vork,
1979). While a tip is necessary for the cell mass to remain organised (Raper,
1940), our experiments show that it does not entirely control the integrity of
the slug; an NP84 tip will not maintain the integrity of an NP84 front/AX3
rear chimera, nor will a young NP84 tip maintain the integrity of an aged
NP84 rear. Also an aged NP84 tip which comes from a slug that has a trailing
rear does not prevent the young NP84 rear portion of a chimeric slug from
being well defined. This is consistent with the hypothesis that the integrity
of the slug is maintained, at least in part, by cell autonomous factors such as
cell surface components.
Understanding of cell loss from the rear of the D. discoideum aggregate may
provide insight into the mechanism of branching in the more complex fruiting
bodies of some species of cellular slime moulds. If D. discoideum strain AX3
formed stalk continuously during migration, as do most species of cellular
slime moulds (e.g. D. mucoroides), the fruiting bodies formed from clumps of
cells left in the slime trail would be branches along the stalk. There are now
Cell autonomous property o/D. discoideum slugs
67
known to be species of cellular slime moulds which have irregularly spaced
fruiting bodies along the stalk caused by leaving behind clumps of cells from
the rear of the aggregate (e.g. D. aureostipes; Cavender, Raper & Norberg,
1979), as well as those in which loss of cells is more regular and evenly
proportioned branches are formed (e.g. P. violaceum, P.pallidum; Harper, 1932;
Bonner, 1967).
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{Received 4 August 1980, revised 26 August 1980)