/. Embryol. exp. Morph. Vol. 42, pp. 105-113, 1977
Printed in Great Britain © Company of Biologists Limited 1977
105
Stalk cell differentiation
by cells from migrating slugs of Dictyostelium
discoideum: special properties of tip cells
By C. D. TOWN 1 AND E. STANFORD 1
From the Imperial Cancer Research Fund, Mill Hill Laboratories,
London
SUMMARY
When fragments of migrating slugs of D. discoideum are disaggregated and spread on agar
containing 1 ITIM cyclic AMP, cells from all parts of the slug form stalk cells with high
efficiency. When cyclic AMP is not added to the agar, normal fruiting of dissociated slug
cells can be prevented by overlaying them with cellophane. Under these conditions only cells
from the anterior 10% of the slug (the 'tip') give rise to appreciable numbers of stalk cells,
all other cells remaining amoeboid.
By separating distinct cell populations with cellophane we have shown that tip cells can
induce cells from other parts of the slug to differentiate into stalk cells. The ability of tips to
induce stalk cells is independent of tip age, but the proportion of cells induced depends both
on the age of the slug and the part of the slug from which they are derived. The proportion
induced is greater in older slugs than in newly formed ones, and in the older slugs is greater in
the cells from fronts than from backs.
The active substance released by the tip cells may be cyclic AMP.
INTRODUCTION
Cytological and biochemical differences can be detected between cells comprising approximately the front one third and the rear two thirds of the migrating slug of D. discoideum (Bonner, Chiquoine & Kolderie, 1955; Krivanek,
1956; Newell, Ellingson & Sussman, 1969; Hayashi & Takeuchi, 1976). Under
normal conditions, the cells from the front third of the slug become stalk cells
in the mature fruiting body, while the remainder become spores (Raper, 1940).
Fragments from any part of the slug can regulate to produce normally proportioned fruiting bodies, given sufficient time. However, if fragments are
induced to fruit immediately after cutting, those derived from the front part
produce fruit with disproportionately large stalks, indicating a degree of commitment of front cells to become stalk cells (Raper, 1940; Bonner & Slifkin,
1949; Sampson, 1976).
The apical region or 'tip' corresponding to about the first 10 % of the slug has
1
Authors' address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London, NW7 IAD, U.K.
106
C. D. TOWN AND E. STANFORD
long been recognized as having unique, organizer-like properties (Raper, 1940)
and is often morphologically distinct (Loomis, 1975). There is evidence that it
acts as the receptive and directive centre of migration (Raper, 1940; Poff &
Loomis, 1973) and may be a source of chemotactic signals (Bonner, 1949;
Rubin & Robertson, 1975) which are probably cyclic AMP (Rubin, 1976).
It also specifies anterior-posterior polarity, and is absolutely required for all
development of aggregates (Gerisch, 1960; Farnsworth, 1973). There exists some
gradient down the slug reflected both in the time taken to form a new tip
following removal of the original one (Farnsworth, 1973; Sampson, 1976) and
in the ability of the cut surface at a particular point to inhibit secondary axis
formation (Durston, 1976).
We have recently demonstrated efficient induction of stalk cell differentiation
when vegetative amoebae of strain V12 M2 are plated at high cell density in the
presence of cyclic AMP (1-5 HIM) (Town, Gross & Kay, 1976). Cells spread at
high density in the absence of cyclic AMP can be prevented from normal
development and fruiting by an overlay of cellophane. Under these conditions,
the cells aggregate via streams in the usual way but apparently proceed no
further; no stalk cell differentiation is seen. The evidence for cell commitment
in the slug (Raper, 1940; Bonner & Slifkin, 1949; Sampson, 1976) made it of
interest to use the same technique to examine the behaviour of cells taken from
the fronts and rears of slugs and spread on agar with and without cyclic AMP.
We have found that the ability to form stalk cells in the absence of exogenous
cyclic AMP is largely confined to the tip region and that tip cells are able to
induce cells from other parts of the slug to differentiate into stalk cells.
MATERIALS AND METHODS
Dictyostelium discoideum strain V12 M2 was used. Cells were grown for 24 h
at 22 °C in association with K. aerogenes on SM agar (KH2PO4, 2-25 g;
K2HPO4, 0-67 g; MgSO4.7H2O, 0-5 g; Difco yeast extract, 0-5 g; Difco Bacto
peptone, 5-0 g; glucose 5-0 g; Difco Bacto agar, 15-0 g; per litre water). They
were then freed of bacteria by one wash in KK 2 buffer (KH2PO4, 2-25 g;
K2HPO4, 0-67 g; MgSO4.7H2O, 0-5 g; per litre water, pH 6-1) and three
washes in distilled water. Washed cells were resuspended at 2 x 108 cells/ml in
distilled water and 50 ju\ volumes were dispensed in lines ~ 4 cm long on 9 cm
diameter agar plates (1-5% (w/v) Difco Bacto agar in distilled water). The
plates were incubated in a humid atmosphere at 22 °C under unidirectional
illumination. Slugs formed after ~ 16 h and continued to migrate towards the
light for up to 5 days.
Sections were cut using a microspatula. To examine stalk cell differentiation
at high cell density, three fragments each of one or more type were transferred
to small squares of cellophane (325P; British Cellophane Ltd.) and gently dispersed with a platinum loop. These squares were then transferred to agar with
Stalk cell differentiation in Dictyostelium
107
or without cyclic AMP, so that the cell layer was sandwiched between the agar
and cellophane. For observation at low cell density, 50-100 fragments of each
type were pooled and disaggregated by trituration in 1 % Bonner's salt solution.
Cells were then spread on agar with or without cyclic AMP and examined for
stalk cells after 2-3 days incubation at 22 °C as described previously (Town
et at. 1976).
RESULTS
(a) Stalk cell differentiation in disaggregated fronts and
rears of slugs
The frequencies of differentiation of cells plated at high density (where the
cells are predominantly in contact) in the absence or presence of 1 mM cyclic
AMP are shown in Fig. 1.
In the absence of cyclic AMP, cells from the rears of slugs gave very few
stalk cells, regardless of slug age (Fig. 1 a). In cells from the fronts of slugs,
however, the proportion of stalk cells rose from 20 % for cells from day-0 slugs
to about 50 % for older slugs. The difference between fronts and rears is highly
significant (P < 001) except on day 0 (005 > P > 002).
In the presence of 1 mM cyclic AMP, cells from both fronts and rears of slugs
were induced to form stalk cells (Fig. 1 b). For the pooled data (all time points)
the average frequencies of differentiation for cells from fronts and rears are
74.4 + 5-9 (mean ± standard error of the mean) and 37-1 ±8-3 respectively.
These frequencies are significantly different (0-002 > P > 0001). However, it is
not possible to say from these data whether this average difference reflects a
difference at all slug ages, or is due mainly to a decline in inducibility of cells
from the rears of slugs at later times.
(b) The role of the tip
A second series of experiments was performed some time later using more
refined dissection techniques. The principal object of these was to examine the
possible role of the tip in the differentiation of front quarter cells reported above,
following a suggestion by Dr Marilyn Monk. In these experiments the tip region
was treated as a separate entity and the differentiation of various fragments was
examined either alone or in a number of combinations. The results obtained for
differentiation in the absence of cyclic AMP are shown in Table 1. Columns (a)
and (b) contain the new data for the front and rear fragments, corresponding to
those used in the first series of experiments, and are quantitatively similar to
those in Fig. 1. The effect of the tip can be seen in subsequent columns. Column
(c) shows that when tip cells alone are taken, a rather higher proportion of stalk
cells is formed than when intact front quarters are used. Moreover, front
quarters from which the tip has been removed (fragment 1, column d) yield a
very low frequency of stalk cells, comparable to rear quarters.
Mixtures of tips and fragment-1 or fragment-4 cells gave rise to considerable
108
C. D. TOWN AND E. STANFORD
(a) NocAMP
100 -
80 -
J
60 -
|
40 20 -|
0
100
5-
5-
- tt .__
(b)\ 1 ITIMCAMP
80
8
J£
6 0 -I
1
40
(73
20 -I
Slug age (days)
Fig. 1. Stalk cell differentiation in high density cell populations derived from front
and rear quarters of slugs of various ages, (a) No cyclic AMP, (b) with 1 mM cyclic
AMP. Fronts, —•—; rears,
O
• Data are shown as meanls.E. of the
mean, with three or four independent determinations per point. Points with no
error bars are single determinations. For clarity, some data points have been offset
with respect to the abscissa.
numbers of stalk cells (Table 1, columns (e) and (/)). The fact that the yield was
significantly greater in the former than in the latter mixture (P < 0-01 for pooled
data) suggested that the stalk cells formed in such mixtures might not derive only
from the tip cells. In order to examine this possibility, cells from the tip and
regions 1 or 4 were separately spread on cellophane squares which were then
placed one over the other in pairs, on agar. In this way, the two cell populations
were separated by a layer of cellophane and stalk cell differentiation was scored
in each population separately. The results presented in Table 2 show that: (a)
tip cells are able to induce stalk cell differentiation in cells of fragments 1 or 4
across a cellophane membrane, (b) the inducing 'power' of tip cells is similar
whether they are derived from day-0 or day-2 slugs, (c) the inducibility (or
Stalk cell differentiation in Dictyostelium
109
Table 1. Stalk cell formation by different slug fragments or combinations of
fragments in the absence of exogenous cyclic AMP
Day
Day
Day
Day
0
1
2
3
M e a n of
all <data
(a)
T.I
(b)
4
7-2±l-7
108 ±2-3
23-2 ±4-2
36-4 ± 4 0
18-6±2-8
2-2 ±0-5
2-7 ± 1 0
4-7 ±1-4
5-2±2-6
3-8±O-7
(c)
T
(d)
1
(e)
T+l
15-2 ±2-3 0*
35-7 ±4-4 3-5f
46-6 ±4-4 2 •2±0 •9
35-3 ±7-2 2t
33-8 ±3-1 1•7±0 •6
50±l-7
13-8±3-5
13-3±2-6
110 ±2-4
ll-0±l-5
(h)
CO
(g)
T + 4 T 1 .2.3.4 1.2.3.4
1-8 ±0-5
ll-8±3-2
7-4 ±2-5
5-2 ± 1 0
6-6 ±1-2
4- 7 ±0-7
12-7 ±2-9
8-2 ±1-0
9-2 ±3-1
8-7 ±1-2
0-6 ±0-3
2-7 ±1-5
4-3 ±2-9
0-8 ±0-5
21 ±0-9
The following nomenclature is used to describe the different combinations of cells
examined: T = tip (first 10% of slug length), 1 = front quarter minus tip (i.e. from 10 to
25% of slug length), 4 = rear quarter minus a small 'tail' (i.e. from 75-95% of slug
length), T.I = first quarter including tip (i.e. from 0 to 25% of slug length), T + l = tip
mixed with section 1 from a different slug, T + 4 = tip plus section 4 from another slug,
T.I. 2.3.4 = whole slug, 1.2.3.4 = tipless slug. Data are given ±S.E. of the mean.
* Six determinations.
t Two determinations.
Table 2. Induction of stalk cell differentiation in fragments 1 and 4 by tip cells
For most experiments, slugs were prepared so that slugs of two different ages
(day 0 and day 2) were available on the same day. Fragments T, 1, and 4 were cut as
defined in Table 1 and disaggregated on cellophane squares. These were laid one
over the other in pairwise combinations so that it was possible to examine the
effect of slug age both on the potency of the tip as an inducing source, and on the
sensitivity of the responding population (derived from fragment 1 or 4).
Responders
Day 0
Inducers
1
Day 2
Day 3
1
12
3-3±O-8
2-1 ±0-4 4-5 ±1-4
None
0
Tips, day 0 60 ± 2 0 3-2 ±0-9 19-8±3-8 100±2-5
Tips, day 2 5-5 ±2-3 7-3 ±2-3 20-8 ±3-1 15-4 ±1-4
26
Tips, day 3
15O±l-5
The yields of stalk cells in the layers of tip cells overlaying a test population were comparable to those observed with tip cells alone (column (c), Table 1).
responsiveness) of cells from both fragments 1 and 4 is higher in day-2 slugs
than in day-0 slugs {P < 0-001 in each case), (d) as already suggested by the
results in Table 1, at least with older slugs (day 2 and 3), more cells from
fragment 1 are induced than from fragment 4. This difference is significant at the
1 % level when all the data for 2- and 3-day-old slugs are pooled (21-2 + 2-9
(n = 14) for fragment 1, 14-1 ± 2-0 (n = 13) for fragment 4).
Differentiation of the same fragments and combinations of fragments as in
EMB 42
110
C. D. TOWN AND E. STANFORD
50 -
(a) No cAMP
40 -
§30c/i
7J
£ 20 .2
10 H
0
(b) 1 mM cAMP
60 -
•
•
• •
50 -
•
>
/
'
/
40 -
\
/
30 -
Tl'
20-
.
\
1
\
I
1
10 -
\
\
J^
\
\
b
n
Slug age (days)
Fig. 2. Stalk cell differentiation in low density cell populations. Details as for Fig. 1.
Table 1 was also examined in the presence of 1 mM cyclic AMP. Similar proportions of stalk cells (~ 50 %) were seen in all the cases, irrespective of the age
or origin of the slug fragments. The difference between the response of fronts and
rears was much smaller than in the first series of experiments (Fig. 1) and was
not significant.
(c) Differentiation at low cell density
Stalk cell differentiation was also examined in cells disaggregated from the
fronts and rears of slugs and plated as isolated cells at ~ 5 x 103 cells/cm2, a
cell density much lower than that used above. Vegetative cells plated at this
density give no stalk cells in the absence of cyclic AMP and 15-30 % stalk cells
with 1 mM cyclic AMP (Town et ah 1976). The results for disaggregated slug
cells are shown in Fig. 2.
Stalk cell differentiation in Dictyostelium
111
In the absence of cyclic AMP, cells from the rears of slugs gave rise to only a
very small proportion of stalk cells (Fig. 2 a) and this number remained approximately constant with slug age. In cells from slug fronts, which include tip cells,
the frequency of stalk cell differentiation was higher, and showed a considerable
increase with slug age, rising from ~ 3 % on day 0 to ~ 40 % on day 3. The
maximum frequency observed (40 % at day 3) was comparable to that seen in
the high density preparation from old slug fronts (Fig. 1 a). It therefore appears
that tip cells from old slugs can exert their influence over distances of up to
100 /*m, and that isolated cells of this age can respond as efficiently as cells at
high density.
In the presence of 1 HIM cyclic AMP, cells from fronts and rears of slugs
differentiated into stalk cells with comparable efficiencies (fronts 34-4±60,
rears 36-2 ± 7-4). These values are slightly higher than those observed previously
for vegetative cells at this density (Town et al. 1976).
DISCUSSION
These results demonstrate two new properties of cells from the tips (anterior
10 %) of migrating slugs of Dictyostelium discoideum. Firstly, a substantial
proportion of tip cells spread on agar under cellophane will differentiate into
stalk cells without exogenous cyclic AMP. This proportion increases with slug
age from 15 % for day-0 slugs to 40 % for day-2 to day-3 slugs. We cannot say
whether this increase occurs uniformly throughout a constant 'tip region' or
whether it represents a spreading back of this tip property as the slugs age, in a
manner similar to that described for the pre-stalk enzyme alkaline phosphatase
(Bonner et al. 1955; Krivanek, 1956).
Secondly, tip cells release a dialysable factor which induces stalk cell differentiation in cells from other parts of the slug. The 'strength' of the inducing
factor does not vary with slug age, but the responsiveness of cells to the factor is
greater in older slugs. In addition, cells from the fronts of old slugs are more
responsive to the inducing stimulus than those from the rear. Since 1 mM
exogenous cyclic AMP similarly induces stalk cell differentiation in cells from
any part of the slug, the dialysable inducing factor released by the tip is probably
either cyclic AMP or some other substance capable of elevating the intracellular
level of cyclic AMP. Exogenous cyclic AMP concentrations greater than 10~4 M
are required to cause appreciable stalk cell differentiation, even at low cell
density where there is relatively little hydrolysis (unpublished observations). It
is unlikely that the number of tip cells used in the trans-cellophane induction
experiments (~3 x 104 cells) could produce steady cyclic AMP concentrations
of this magnitude, and even less likely that isolated tip cells could do this at
ranges of up to ~ 100 jum. It is therefore likely that some kind of periodic
signalling and signal amplification is occurring.
It has previously been reported that slug tips are sources of chemotactic
8-2
112
C. D. TOWN AND E. STANFORD
signals (Bonner, 1949; Rubin & Robertson, 1975; Rubin, 1976) and that they
may also contain higher levels of cyclic AMP than the rest of the slug (Pan,
Bonner, Wedner & Parker, 1974; Brenner, 1977). However, even if it should be
true that the substance released by tip cells and inducing stalk cell formation
under our conditions is cyclic AMP, it would be unwise to assume that this
result has a direct bearing on the mechanism of pattern formation. Thus we
know that exposure of a dense preparation of washed cells of various species to
high concentrations of cyclic AMP results in their virtually quantitative conversion to stalk cells, and never to spores (Town et a/. 1976; Hohl, Honegger,
Traub & Markwalder, 1977). However, since no conditions have yet been
found that elicit spore formation in vitro, there can be no certainty that it is the
cyclic AMP that determines the choice of one pathway rather than the other. It
is equally possible that in the intact slug the tip signal is a relayed cyclic AMP
signal (somewhat as it is during aggregation) and that this signalling is required
for gene expression in both pathways of differentiation, the choice of pathway
being determined by other, unknown, factor(s).
We thank Dr Julian Gross for much helpful advice and encouragement.
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(Received 7 October 1976, revised 21 July 1977)