Flow Cytometer Study of Anterior-Like Cells in Dictyostelium Discoideum

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 Biochemie, D-8033 Martinsried bet Munchen, Federal Republic of Germany

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 non- prespore 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 2X107 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 mm2 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 anti- mouse 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 ~2xlO6 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 (~6x 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 peroxidase- conjugated 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 3 recognizes only spore surface. MUD1 recognizes a 32Xl0 Mrglycoprotein (formerly 3 reported to be ~30xl0 Mr; Krefft et al. 1983, 1984). MUD3 recognizes a protein 426 L. Voet, M. Krefft, M. Bruderiein and K. L. Williams 3 (~105xl0 Mr), which is present inside prespore cells and on the surface of spores (Figs 1, 2H) . In the flow cytometer 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 dis- tinguishable 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 (~2xlO6); c, spores (-2X106); d, slug cells (-2X106); e, plasma membrane from the 6 3 equivalent of ~2xlO slug cells. The arrow indicates the antigen of ~105xl0 Mr recognized by MUD3. 427

Fluorescence, MUD1 Fluorescence, MUD3

14 h 14h

psp pst

17 h 30min 17 h 30min

-sp

•a 18h 18h

A ^sp

sp • psp psp pst / . pst t sL

24 h 24 h

pst • pst

Fig. 2. For legend see p. 428 428 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 time- course 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

100 11

* • 90 Spore

80 Prespore 70 — | 60 oo c

$ 50

20 Prestalk 10 OL// 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 dual- parameter 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%). Anterior-like cells in D. discoideum 431

Whole slugs

pst

JxX •>;' X' >: >>v V^C X X V

E Hx" pst n , PSP \ . \ ^^-i • \ xi \ r 1 m x m \— X M m V, .X->» X/ ^SHT-SHIK x- m Mr "V y r X\-^ ^^ X V^ K r>rx "x 'X, |! X X / \ xx KX- x x.x. XX . ./'J'>£r %X-x^ X. x^ V ^'^^ X % <* xX x,^ %^ x ^- Xi 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.) Strain NP73 Strain M28 Front 69 ±6-7 69 ±7-6 Middle (front) 11 ±1-7 27 ±3-5 Middle (rear) 7 ±1-3 18 ±1-6 Rear 28 ±6-9 21 ±3-3

B. Volume of slug sections to total slug volume Slug sections (% + S.E.) Strain NP73 Strain M28 Front 14 ±1-2 20 ±0-9 Middle (front) 31 ±1-9 27 ±1-5 Middle (rear) 29 ±1-3 25 ±1-1 Rear 26 ±0-9 28 ±2-1 100 100 C. Prestalk, predisk and anterior-like cells in slug sections to total slug cells Slug sections (% ± S.E.) Strain NP73 Strain M28 Front 9 ±0-7 14 ±1-3 Middle (front) 3 ±0-2 8 ±0-5 Middle (rear) 3 ±0-9 5 ±0-1 Rear 7 ±1-2 6 ±0-7 22 33

D. Prestalk, predisk and anterior-like cells in slug sections to total prestalk slug cells Slug sections (% ± S.E.) Strain NP73 Strain M28 Front 41 ±2-1 43 ±1-5 Middle (front) 15 ±1-5 23 ±1-6 Middle (rear) 12 ±2-2 14 ±0-8 Rear 32 ±1-5 20 ±2-0 100 100

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. Our flowcytomete r studies show that the cells in the tip region remain for several hours in strain NP84, which develops rapidly, before being finally differentiated. 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 cells finally differentiate into basal disk or stalk cells during culmination. We have been unable to distinguish prestalk and anterior- like 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 BONNER, J. T. (1957). A theory of the control of differentiation in the cellular slime molds. Q. Rev. Biol. 32, 232-246. 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 detected between prespore cells of Dictyostelium discoideum and Dictyostelium mucoroides using monoclonal antibodies. Expl Cell Res. 142, 229-233. HAYASHI, M. & TAKEUCHI, I. (1981). Differentiation of various cell types during fruiting body formation of Dictyostelium discoideum. Dev. Growth & Differ. 23, 533-542. KREFFT, M., VOET, L., GREGG, J. H., MAIRHOFER, H. & WILLIAMS, K. L. (1984). Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates. EMBOJ. 3, 201-206. KREFFT, M., VOET, L., MAIRHOFER, H. & WILLIAMS, K. L. (1983). Analysis of proportion regulation in slugs of Dictyostelium discoideum using a monoclonal antibody and a FACS-IV. Expl Cell Res. 147, 235-239. LAEMMLJ, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Land. 227, 680-685. MACWILLIAMS, H. K. (1982). Transplantation experiments and pattern mutants in cellular slime molds. In Developmental Order: Its Origin and Regulation (ed. P. B. Green), pp. 463-483. New York: Alan R. Liss. RAPER, K. B. (1940). Pseudoplasmodium formation and organisation in Dictyostelium discoideum. J. Elisha Mitchell sdent. Soc. 56, 241-282. RAPER, K. B. &FENNELL, D. I. (1952). Stalk formation in Dictyostelium. Bull. Torrey bot. Club 79, 25-51. Anterior-like cells in D. discoideum 435 SMITH, E. & WILLIAMS, K. L. (1981). The age-dependent loss of cells from the rear of a Dictyostelium discoideum slug is not tip controlled. J. Embryol. exp. Morph. 61, 61-67. STENHOUSE, F. O. & WILLIAMS, K. L. (1977). Patterning in Dictyostelium discoideum: The proportions of the three differentiated cell types (spore, stalk and basal disk) in the fruiting body. Devi Biol. 59, 140-152. STENHOUSE, F. O. &WILLIAMS, K. L. (1981). Investigation of cell patterning in the asexual fruiting body oi Dictyostelium discoideum using haploid and isogenic diploid strains. Differentiation 18, 1-9. STERNFELD, J. & DAVID, C. N. (1981). Cell sorting during pattern formation in Dictyostelium. Differentiation 20, 10-21. STERNFELD, J. & DAVID, C. N. (1982). Fate and regulation of anterior-like cells in Dictyostelium slugs. Devi Biol. 93, 111-118. TOWBIN, H., STAEHLIN, T. & GORDON, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natn. Acad. Sd. U.SA. 76, 4350-4354. VOET, L., KREFFT, M., MAIRHOFER, H. & WILLIAMS, K. L. (1984). An assay for pattern formation in Dtctyostelium discoideum using monoclonal antibodies, flow cytometry, and subsequent data analysis. Cytometry 5, 26-33. WILLIAMS, K. L. & WELKER, D. L. (1980). Mutations specific to spore maturation in the asexual fruiting body oi Dictyostelium discoideum. Devi Genet. 1, 355-362.

(Received 10 October 1984 -Accepted 6 December 1984)