y. Cell Sci. 75, 423-435 (1985)
493
Printed in Great Britain © The Company of Biologists Limited 1985
FLOW CYTOMETER STUDY OF ANTERIOR-LIKE
CELLS IN DICTYOSTELIUM DISCOIDEUM
LUDWIG VOET, MARIANNE KREFFT*, MARTINA BRUDERLEIN
AND KEITH L. WILLIAMSf
Max-Planck-Institutfur
Germany
Biochemie, D-8033 Martinsried bet Munchen, Federal Republic of
SUMMARY
The Dictyostelium discoideum asexual fruiting body consists of spores, stalk and basal disk cells.
Recently, a fourth cell class has been proposed. It has been suggested that these cells originate from
anterior-like cells that remain undifferentiated. Anterior-like cells are randomly distributed among
prespore cells in the posterior part of the slug. Here monoclonal antibodies that recognize the surface
of prespore cells (MUD1), and spores (MUD3) are used in a quantitative flow cytometer assay to
demonstrate that this fourth cell class does not exist in the mature fruiting body. However, the tip
cells are slow to differentiate, and hence immature fruiting bodies contain a small population of
undifferentiated tip cells. We confirm that anterior-like cells represent a large percentage of the nonprespore cell population in the slug. In this report we were unable to distinguish these anterior-like
cells from prestalk cells on the basis of size or monoclonal antibody staining.
INTRODUCTION
The asexual fruiting body of Dictyostelium discoideum is one of the simplest
multicellular structures formed by a eukaryote organism. The structure arises by
aggregation of starving amoebae, rather than by organized cell division, and
comprises three cell types - spores, stalk and basal disc cells - in roughly similar
proportions in fruiting bodies of various sizes (Raper, 1940; Bonner & Slifkin, 1949;
Stenhouse & Williams, 1977). Many workers ignore the third class (basal disk cells) as
they are somewhat similar to stalk cells and comprise less than 5 % of the total cells
(spores 70-90 % and stalk cells 10-30%; Stenhouse & Williams, 1981). However, as
Raper (1940) showed, the cells forming the basal disk are derived from cells at the rear
of the slug while the stalk cells are derived from cells at the front.
Another cell class, anterior-like cells, has been recognized and characterized in the
slug stage that precedes fruiting body formation (Bonner, 1957; Hayashi&Takeuchi,
1981; Sternfeld & David, 1981, 1982). These cells differ from prestalk cells in their
location at the rear of the slug, which reflects the fact that they are unresponsive to
cAMP (Sternfeld & David, 1981). These authors have shown that anterior-like cells
•Present address for correspondence: Gesamthochschule Wuppertal, Fachbereich 9, 5600
Wuppertal 1, Federal Republic of Germany.
f Present address: School of Biological Sciences, Macquarie University, North Ryde, Sydney,
N.S.W. 2113, Australia.
Key words: Dictyostelium, flow cytometry.
424
L. Voet, M. Krefft, M. Bruderlein and K. L. Williams
make up a considerable proportion of the slug cells and that they remain as a distinct
class of undifferentiated cells in the fruiting body - occupying positions at the base of
the spore mass and the tip of the fruiting body. Hayashi & Takeuchi (1981) have
observed similar undifferentiated cells in mature fruiting bodies, although they said
that these cells arise from prespore cells. Our casual observations of many mutants
suggest that often not all cells mature completely.
However, Sternfeld & David (1982) and Hayashi & Takeuchi (1981) proposed that
these cells may have a role in lifting the spore mass off the substratum. An alternative
explanation for raising the spore mass has been given previously (Raper & Fennell,
1952) and it seems surprising that these early authors should have failed to observe a
class of undifferentiated cells present in great excess of the basal disk cells, which they
documented in some detail. This proposed fourth cell class of cells comprises at least
10 % of the total number of cells in the slug (Sternfeld & David, 1982) and should be
easily observable in a quantitative cytofluorometric assay that we have developed
(Krefft, Voet, Mairhofer & Williams, 1983). In this report we describe experiments
using a combination of prespore-specific, MUD1, and spore-specific, MUD3,
monoclonal antibodies in which we fail to find this fourth cell type in mature fruiting
bodies. The anterior-like cell population is further characterized in the slug stage.
MATERIALS AND METHODS
Growth and development o/D. discoideum
NC4-derived strain M28 (Stenhouse & Williams, 1977), V12-derived strains NP73, NP84
(Gregg, Krefft, Haas-Kraus & Williams, 1982) and strain HU1598, which carries a mutation sprj
359, affecting spore maturation (Williams & Welker, 1980), were developed on SM agar in
association with Websiella aemgenes. For time-course experiments NP84 cells were developed on
black Millipore filters (HABG 04700) as described in detail elsewhere (Krefft et al. 1984).
In order to collect single slugs, amoebae scraped from a growth plate with a toothpick were spread
at one end of a water-agar Petri dish. The plates were incubated at 21 ±1 deg. C in an illuminated
room in black PVC dishes with a 3 mm hole on the side opposite to the cells, as described previously
(Gregg et al. 1982).
Single slugs observed using a binocular microscope were collected by the slime trail with a pin
point. Either four intact slugs were used per sample or, when sections of slugs were examined, nine
slugs were dissected into quarters and the respective quarters combined. The outlines of each slug
and of each fragment were drawn using a camera lucida and analysed with a digitizing tablet
(Summagraphics, Fairfield, CN) connected to a VAX-782 computer (Fisher, Grant, Dohrmann &
Williams, 1983). Slug volumes and their fragments were estimated from their digitized outlines as
the volume of an idealized cylindrical slug with the same projected area and perimeter as the actual
slug (Fisher, unpublished).
Monoclonal antibodies
Spore-specific monoclonal antibody MUD3 was obtained after Balb/c mice (6-18 weeks) were
injected with ~ 1X107 NP73 spores in 0-9% NaCl; twice with 0-5 ml intraperitoneally (i.p.), once
with 0'lml intravenously (i.v.), with intervals of 2 weeks between injections. Three days after the
i. v. boost, spleens of two mice were taken out, pooled, and 1X107 spleen cells were fused with 2X10 7
myeloma cells (cell line Ag8/653, a non-secreting NSl derivative obtained from T. Meo, Institut fur
Immunologie, Universitat Miinchen). Hybridomas were selected in HAT medium and cloned three
times by limiting dilution (de St Groth & Scheidegger, 1980). Tissue culture supernatant or 1:100
diluted ascitic fluid was used for the assays. MUD1, a prespore-specific monoclonal antibody was
Anterior-like cells in D. discoideum
425
obtained as described earlier (Grtgget al. 1982). MUD1 and MUD3 are immunoglobulin G (IgG)type antibodies.
Flow cytometry
For the time-course, samples were prepared at intervals of 30 min over a period from 0 h (initiation
of starvation) to 24 h. A single plate was removed from the incubator at 21 ± 1 deg. C and 3x3 mm 2 of
the black Millipore filter was excised and a representative photograph was taken. Samples were
prepared as described elsewhere (Krefft et al. 1984). Slugs and fragments of slugs were treated as
described previously (Krefft et al. 1983). In both cases, single cell suspensions were obtained by
incubating cells in 0*2 ml of 0' 15 % (w/v) papain and 5 mM-cysteine for 10 min, washed twice, and
incubated with 0-1 ml prespore-specific (MUD1) or spore-specific (MUD3) monoclonal antibody
and 0-1 ml of a 1:40 dilution of goat anti-mouse IgG-F(ab')2-FITC (code 4350 Medac, Hamburg,
FRG). To analyse spore heads four fruiting bodies were collected with tweezers and placed directly
into 0 - l ml of monoclonal antibody in a 0'7 ml Eppendorf tube. Following a short vortex, goat antimouse IgG-F(ab') 2 -FITC was added; neither washing nor centrifugation was necessary for these
samples. After 30 min incubation on ice, samples were analysed directly in the flow cytometer
(model FACS-IV, Becton Dickinson, Sunnyvale, CA) at approximately 1000 cells/second (Voet,
Krefft, Mairhofer & Williams, 1984).
The data analysis techniques used to determine the mean values and percentages of cell
populations have been described elsewhere (Voet et al. 1984).
Immunoblots
Discontinuous sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) was
performed according to Laemmli (1970) using 10% resolving and 4 % stacking gels in
11-5 mmXl3-5 mmX0-1 mm slabs. Using ~ 2 x l O 6 cells per sample, crude plasma membranes from
slug cells (Krefft et al. 1983), whole slug cells and spores (all from strain NP84) were extracted in
2-5% (w/v) SDS and 5 % (w/v) mercaptoethanol at 10b°C for 3 min and applied on the gel.
Samples for stalk cells were prepared from a NP84 growth plate; spores were first removed by
banging them onto the lid. Stalks ( ~ 6 x 107) were collected with a spreader and washed repeatedly
with distilled water on a nylon mesh (180/im) that allowed spores and amoebae to pass through.
When no further spores were observed in the washings, the stalks were then extracted with SDS and
mercaptoethanol as described for the other cell types, and ~2X 106 cells per sample, assuming 50 %
losses of stalks by washing, were applied to each gel track. Gels were run at 10 mA for 1 h followed by
20 mA until the tracking dye was 0'5 cm from the end. Proteins were transferred by blotting onto
nitrocellulose sheet (NC, Schleicher & Schiill, B A83) by the method of Towbin, Staehlin & Gordon
(1979). Blots were first incubated with MUD3 culture supernatant, followed by peroxidaseconjugated staining using 1:1500 dilution of goat anti-mouse IgG peroxidase (code 6450, Medac,
Hamburg, FRG) and visualized by incubation in 1 vol. of 0-3% 4-chloro-l-napthol (Merk, no.
11952) in methanol with 5 vol. Tris-buffered saline (50 mM-Tris, 15 mM-NaCl, pH 7-4) and 001 %
peroxidase. Molecular weight standards (high MW, Biorad) were also transferred to NC sheet and
stained with 1 % Amido Black.
RESULTS
Cell markers
Monoclonal antibodies. In order to distinguish the different cell types by flow
cytometry, suitable cell surface markers are necessary. Two monoclonal antibodies
were used for these studies, MUD1, which recognizes prespore cells, but not
vegetative amoebae, prestalk, anterior-like cells or spore surface, and MUD3, which
recognizes only spore surface. MUD1 recognizes a 32Xl03Mrglycoprotein (formerly
reported to be ~30xl0 3 M r ; Krefft et al. 1983, 1984). MUD3 recognizes a protein
426
L. Voet, M. Krefft, M. Bruderiein and K. L. Williams
(~105xl0 3 M r ), which is present inside prespore cells and on the surface of spores
(Figs 1, 2H) . In theflowcytometer only mature spores, recognized on the basis of their
small size, are labelled (Fig. 2H). The antigen from MUD3 is expressed on the surface
of some mutants, e.g. HU1598, which fail to differentiate spores fully.
Size (forward-angle light scatter). Prestalk and anterior-like cells are not distinguishable from each other on the basis of size. In young slugs, prespore cells are
smaller than the prestalk and anterior-like cells and they can be recognized as a distinct
population in the flow cytometer (Fig. 2A-D; Voet et al. 1984). The light-scatter
pattern of spore cells shows that they are markedly smaller than all amoeboid cells
(Fig. 2E-H).
45-
Fig. 1. Immunoblots from cells of D. discoideum strain NP84 stained with prespore
specific monoclonal antibody MUD3: lanes a, molecular weight standards; b, stalk cells
(~2xlO 6 ); c, spores (-2X10 6 ); d, slug cells (-2X10 6 ); e, plasma membrane from the
equivalent of ~2xlO 6 slug cells. The arrow indicates the antigen of ~105xl0 3 M r
recognized by MUD3.
427
Fluorescence, MUD1
Fluorescence, MUD3
14h
14 h
psp
pst
17 h 30min
17 h 30min
-sp
•a
18h
18h
^sp
A
sp
• psp
psp
. pst
/
pst
t
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24 h
24 h
sp
sp
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Fig. 2. For legend see p. 428
• pst
428
L. Voet, M. Kreffi, M. Bruderlein and K. L. Williams
Fate of differentiated slug cells during culmination
In slugs (Fig. 2A) MUD1 distinguishes two populations of cells (Kreiftet al. 1983):
large unlabelled prestalk, anterior-like and predisk cells, and small labelled prespore
cells. All cells are unlabelled when slug cells are labelled with MUD3 (Fig. 2B).
During culmination the percentage of unlabelled cells decreases as the stalk and basal
disk cells differentiate in a cellulose matrix that cannot be degraded to produce a
single-cell population. Therefore, flow cytometer measurement of these fully differentiated cells is not possible. At the same time prespore cells differentiate to spores
losing MUD1 labelling (Fig. 2C,E) and gaining MUD3 (Fig. 2D,F). By 18 h with
NP84, virtually all prespore cells have lost MUD 1 labelling and been transformed into
spores (Fig. 2E,F; Fig. 3), and the percentage of prestalk and anterior-like cells is
drastically reduced. By 24 h in strain NP84, all prespore cells have differentiated into
spores (Fig. 2G,H; Fig. 3). However, there is a residual population of ~6 % cells with
neither MUD1 nor MUD3 label (Fig. 3). These cells are unlikely to be prespore cells
as they have no MUD 1 labelling and it is known that incompletely differentiated spore
cells still carry MUD3 label (e.g. mutant HU1598). The speed of loss of MUD1 and
gain of MUD3 labelling is extremely rapid as we did not observe an intermediate
population when the bulk of prespore cells were transforming into spores in the time
interval from 17-5 h to 18 h (Fig. 2c,D; Fig. 3), even though we sampled the timecourse at 30-min intervals in three experiments (data not shown). The resulting
changes in percentages of cells in the culminating fruiting body obtained by flow
cytometry are summarized in Fig. 3 (arrows indicate reference to Fig. 2), which shows
that prespore cells disappear and a small residual population of unlabelled cells remain.
We observed that the presence of unlabelled cells in the fruiting body is correlated
with the small nipple, the remaining tip region that is present at the top of a not yet
Fig. 2. Dual-parameter flow cytometer histograms of separated cells from D. discoideum
strain NP84 at different developmental times. The x-axes represent forward-angle light
scatter, which is correlated to the size of single cells. They-axes represent the amount of
fluorescence label on single cells. In A,C,E,G all cells were labelled with monoclonal
antibody MUD1, and in B,D,F,H with monoclonal antibody MUD3. In the histograms,
which are shown as contour plots, single dots indicate events above 1 %, and contour lines
are drawn from S % to 95 % in steps of 10%, with respect to the highest peak in the
histogram, pst, prestalk, predisk and anterior-like cells; psp, prespore cells;sp, spore cells.
At the slug stage (14 h) label with prespore-specific monoclonal antibody MUD 1 (A) shows
two distinct populations on the basis fluorescence and size. Label with MUD3 (B) shows
no fluorescence label but two overlapping populations separated by size. At the early
culmination stage (17 h 30min) label with MUD1 (c) distinguishes a decreasing population of prestalk, anterior-like and predisk cells due to differentiation of stalk and basal
disk cells, and a decreasing population of prespore cells developing into spores that are
apparent as a small unlabelled population. Label with spore-specific monoclonal antibody
MUD3 (D) identifies a clearly separated, small, third population of spores. Only half an
hour later (18 h) the development of prestalk, anterior-like and predisk cells to stalk and
basal disk and prespore cells to spores is nearly complete (E,F). At the fruiting body stage
(24 h) label with MUD 1 (G) shows no fluorescence label but two overlapping populations
of different size. Label with MUD3 (H) identifies a clearly separated spore cell population
containing most of the cells.
Anterior-like cells in D. discoideum
429
fully matured spore head. To determine whether this population of unlabelled cells is
a true fourth cell class in the fruiting body (in addition to stalk, spore and basal disk
cells), further experiments were done on mature fruiting bodies. Fig. 4 shows MUD3
labelling with immature (with nipple) and mature (fully rounded spore head) fruiting
bodies of strain NP84 carefully removed from a growth plate and treated directly
11
100
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Spore
80
Prespore
70
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40
30
20
Prestalk
10
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14
15
16
17
18
19
20
21
22
23
24
Time (h)
Fig. 3. Distribution of different cell types in D. discoideum strain NP84 during
development. The percentages of different cell types are calculated on the basis of dualparameter flow cytometer data (arrows indicate the different developmental stages shown
in Fig. 2). Open symbols indicate percentages of prespore and prestalk (unlabelled) cells
calculated using prespore specific monoclonal antibody MUD1; closed symbols indicate
percentages calculated using the spore-specific monoclonal antibody MUD3. (O—O) and
( • — • ) Prespore population; ( • — D ) and ( • — • ) unlabelled cell population (prestalk,
predisk and anterior-like cells), which are not separable by using these monoclonal
antibodies or on the basis of size; ( A — A ) ( A — • ) spore population. Starting from a
stable prespore/prestalk pattern (—70 % prespore cells and ~30 % prestalk cells) in the
slug stage (14 h) there is a rapid change (between 17 h and 18 h) ending in a stable pattern
of ~94 % spore cells and ~ 6 % prestalk cells in the fruiting body. Note that the percentages
in the slug stage represent all cells of the organism, while in the fruiting body only spore
cells and remaining prestalk cells are included. Stalk cells and basal disk cells are not
separable into single cells and hence could not be examined in the flow cytometer.
430
L. Voet, M. Krefft, M. Bruderlein and K. L. Williams
(without protease treatment) with immunolabelling and flow cytometry. The
protease treatment was omitted so that handling of the cells was minimized to avoid
breakage in case there was a population of fragile cells. The results obtained with the
immature fruiting bodies (Fig. 4A) were identical to those obtained in the time-course
with enzyme treatment (Fig. 2H). There remained ~ 6 % unlabelled cells. In the
mature fruiting bodies this cell population had disappeared, spores being the only
population of cells found in the spore head (Fig. 4B). When such spore heads were
examined microscopically, essentially no undifferentiated cells were observed; in
particular, no significant number of undifferentiated cells were seen adhering to the
stalk.
Mature
Fig. 4. Dual-parameter flow cytometer histograms of immature (A) and mature (B)
fruiting bodies of D. discoideum strain NP84 labelled with monoclonal antibody MUD3.
pst, tip (prestalk) cells; sp, spore cells. Labelling with MUD3 of immature fruiting bodies
(A) identifies a remaining population of ~ 6 % prestalk cells, which have disappeared in
mature fruiting bodies (B).
Fig. 5. Spatial distribution of prestalk, anterior-like and basal disk cells and prespore cells
in the slug of D. discoideum strain NP73 obtained by sectioning and labelling with
monoclonal antibody MUD1. pst, prestalk, predisk, and anterior-like cells; psp, prespore
cells, A. Camera lucida drawings of the slugs used in the experiment shown in this figure;
the arrows indicate the slug tips. B. Dual-parameter flow cytometer histograms from a
parallel measurement of whole slugs (pst, 22%; psp, 78%). C. 1st quarter of the sectioned
slugs (pst, 66%; psp, 34%). D. 2nd quarter (pst, 13%; psp, 87%). E. 3rd quarter (pst,
19%; psp, 81%). F. 4th quarter (pst, 36%; psp, 64%).
431
Anterior-like cells in D. discoideum
Whole slugs
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432
L. Voet, M. Krefft, M. Bruderlein and K. L. Williams
Table 1. Distribution of prestalk, predisk and anterior-like cells in slugs of strain
NP73 (4 measurements) and strain M28 (5 measurements) dissected into four parts
A. Prestalk, predisk and anterior-like cells in slug sections
Slug sections (%± S.E.)
Front
Middle (front)
Middle (rear)
Rear
Strain NP73
69
11
7
28
±6-7
±1-7
±1-3
±6-9
Strain M28
69
27
18
21
±7-6
±3-5
±1-6
±3-3
B. Volume of slug sections to total slug volume
Slug sections (% + S.E.)
Front
Middle (front)
Middle (rear)
Rear
Strain NP73
14
31
29
26
±1-2
±1-9
±1-3
±0-9
Strain M28
20
27
25
28
±0-9
±1-5
±1-1
±2-1
100
100
C. Prestalk, predisk and anterior-like cells in slug sections to total slug cells
Slug sections (% ± S.E.)
Front
Middle (front)
Middle (rear)
Rear
Strain NP73
9
3
3
7
±0-7
±0-2
±0-9
±1-2
22
Strain M28
14
8
5
6
±1-3
±0-5
±0-1
±0-7
33
D. Prestalk, predisk and anterior-like cells in slug sections to total prestalk slug cells
Slug sections (% ± S.E.)
Front
Middle (front)
Middle (rear)
Rear
Strain NP73
41
15
12
32
100
±2-1
±1-5
±2-2
±1-5
Strain M28
43
23
14
20
±1-5
±1-6
±0-8
±2-0
100
Anterior-like cells in slugs
The above results suggest that in V12-derived strain NP84 all cells not labelled with
MUD1 are transformed into stalk cells in the mature fruiting body. Additionally, it
was decided to examine more closely whether or not anterior-like cells exist in slugs of
V12-derived strains. This was achieved by cutting slugs of strain NP73, which are
very large, into four parts of approximately equal length, without regard to the
prestalk/prespore boundary. The volume and percentage of prespore cells (MUD1
labelling) were measured in each segment. Controls were conducted with whole slugs
and results from the combined fractions were compared with these measurements.
A representation of a single experiment is shown in Fig. 5 and a quantitative
summary is given in Table 1. In the front section of strain NP73 most of the cells were
prestalk (Fig. 5c), while in the two middle quarters prespore cells predominated,
Anterior-like cells in D. discoideum
433
although some unlabelled cells were present (Fig. 5D,E). In the rear quarter the
percentage of unlabelled cells increased considerably (Fig. 5F). The sizes of the
unlabelled cells in all sections were similar, hence it is not possible to distinguish
prestalk cells (in the anterior part of the slug) from anterior-like cells (in the posterior
part) on the basis of size (Fig. 5).
When strain M28, which has a high percentage of unlabelled cells in the slug (Krefft
etal. 1983), was tested, more prestalk cells were observed than with NP73. However,
there were still large numbers of anterior-like cells, which were more evenly distributed along the slug than in NP73 (Table 1).
DISCUSSION
Our experiments using a flow cytometer and two monoclonal antibodies: MUD1,
specific to prespore cell surface and MUD3, specific to spore surface, showed that
essentially no undifferentiated amoebae remain in the fully mature fruiting body. We
conclude that anterior-like cells still exist in the D. discoideum slug, but there is no
fourth class of cells in the mature fruiting body. Their role during the slug stage
remains puzzling.
We believe, however, that our results can be reconciled with those of other groups
proposing a fourth cell class (Sternfeld & David, 1982; Hayashi & Takeuchi, 1981).
Figure 4 shown by Sternfeld & David (1982) clearly shows a strongly stained nipple
region on the fruiting body. Our results and studies of time-lapse films indicate that
the nipple (tip region) of the culminating D. discoideum fruiting body is only slowly
resorbed, and it seems probable that Sternfeld & David (1982) terminated their
experiments before final maturation (nipple resorption) occurred. Ourflowcytometer
studies show that the cells in the tip region remain for several hours in strain NP84,
which develops rapidly, before beingfinallydifferentiated. It is of interest to note that
these cells become considerably smaller during the process of culmination — as small as
prestalk cells of slugs that migrate for 3 days (Voet et al. 1984). This is presumably
due to the synthesis of the stalk cylinder by prestalk cells about to enter the stalk
(Bonner, 1982).
The remaining cells observed at the base of the sorus by Sternfeld & David (1982)
were less stained with Neutral Red and may have been immature prespore cells as
shown by Hayashi & Takeuchi (1981). Raper & Fennell (1952) also pointed out that
sometimes all prespore cells do not mature. In our study we used two very robust
strains (NP84 and M28) and prespore cells were essentially all fully differentiated.
Thus we presume that anterior-like cellsfinallydifferentiate into basal disk or stalk
cells during culmination. We have been unable to distinguish prestalk and anteriorlike cells on the basis of size (this study) or with monoclonal antibodies (including
using a prestalk monoclonal antibody: Krefft et al. unpublished). Hence the only
feature of anterior-like cells that distinguishes them from prestalk cells (except their
position at the rear of the slug) is their failure to respond to cAMP and inhibition of
sorting towards the tip (Sternfeld & David, 1981).
434
L. Voet, M. Krefft, M. Bruderlein and K. L. Williams
Why are anterior-like cells found in the slug? Anterior-like cells are capable of
moving from the prespore region to the anterior of the slug during regeneration after
the tip has been removed (MacWilliams, 1982). The apparent increase in the
percentage of anterior-like cells after 3 days (Sternfeld & David, 1982), and the
observation (Smith & Williams, 1981) that slugs begin to drop many cells in their
slime trail after 3 days, is consistent with the idea that anterior-like cells should be
viewed dynamically as cells moving backwards and being left behind. This was an
early hypothesis of Bonner (1957) in which he argues that they are 'worn out' tip cells
no longer able to keep up. The results reported here with NP73 on the concentration
of anterior-like cells in the rear quarter of the slug, together with the observation that
old slugs drop groups of cells (Smith & Williams, 1981), are also consistent with such a
hypothesis. These ideas can be tested when suitable markers to distinguish prestalk
and anterior-like cells are found. Monoclonal antibodies may make this possible and
we are currently seeking an anterior-like-specific or prestalk-specific antibody.
This research was supported by Deutsche Forschungsgemeinschaft, grant Wi 668/1-1. We thank
Helga Mairhofer for technical assistance.
REFERENCES
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BONNER, J. T . (1982). Comparative biology of cellular slime molds. In The Development of
Dictyostelium discoideum (ed. W. F. Loomis), pp. 1-33. New York: Academic Press.
BONNER, J. T . & SLIFKIN, M. K. (1949). A study of the control of differentiation: The proportions
of stalk and spore cells in the slime mold Dictyostelium discoideum. Am. J. Bot. 36, 727-734.
DE ST GROTH, S. F. & SCHEIDEGGER, D. (1980). Production of monoclonal antibodies: Strategy
and tactics. J. Immun. Meth. 35, 1-21.
FISHER, P. R., GRANT, W. N., DOHRMANN, U. & WILLIAMS, K. L. (1983). Spontaneous turning
behaviour by Dictyostelium discoideum slugs. J'. Cell Sci. 62, 161-170.
GREGG, J. H., KREFFT, M., HAAS-KRAUS, A. & WILLIAMS, K. L. (1982). Antigenic differences
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(Received 10 October 1984 -Accepted 6 December 1984)