An analysis of culmination in Dictyostelium using prestalk and stalkspecific cell autonomous markers

Transcription

1 Development 111, (1991) Printed in Great Britain The Company of Biologists Limited An analysis of culmination in Dictyostelium using prestalk and stalkspecific cell autonomous markers K. A. JERMYN and J. G. WILLIAMS The Imperial Cancer Research Fund, Clare Hall Laboratory, South Mimms, Herts, EN6 3LD Summary The ecma (pdd63) and ecmb (pdd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstb cells, which express the ecmb gene, surrounded by psta cells, which express the ecma gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, psta cells transform into pstb cells by activating the ecmb gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecma and ecmb genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devi Biol. 93, ) and we find just such a distribution for pstb cells. We believe that the ecmb protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, psto cells, which lie behind psta cells in the slug anterior and which appeared to express neither the ecma nor the ecmb gene. Using fi-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecma gene but at a much lower level than psta cells. We also show that expression of the ecma gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecma gene in the pst 'O' region is a preparatory step in the process. Key words: Dictyostelium, /?-gal fusion genes, multiple prestalk cell types, anterior-like cells, overhead light. Introduction Morphogenesis is typically the result of a combination of cellular differentiation and directed cell movement and the latter process is not well understood in any developmental system. In Dictyostelium, cellular motility and chemotaxis have been the subject of intensive study but, thus far, most effort has concentrated on the behaviour of separate cells during aggregation. Cell movement during terminal morphogenesis has been analyzed largely at a descriptive level. This is partly due to the difficulties of working with a three-dimensional structure and partly because of the relative complexity of the cell movements that occur during culmination. The spores of the mature fruiting body are held aloft by a tapering stalk, which is expanded at its base to form a supporting disc. The stalk is composed of dead, highly vacuolated cells which are surrounded by a protein and cellulose-containing extracellular matrix. Stalk cells are further encased in an outer layer of an apparently similar matrix termed the stalk tube. The migratory slug is sensitive to both light and temperature gradients and these signals direct it to the surface for culmination. Overhead light induces culmination, probably indirectly by attracting the tip of the slug to the air-water interface (Bonner et al. 1982). When culmination is triggered, prestalk cells, located very near the slug tip, synthesize stalk tube material and migtate into it (Raper and Fennell, 1952; Bonner et al. 1955). The stalk tube is pushed downwards through the aggregate by accretion of prestalk cells at its apex. When it meets the base it fuses with a separate population of prestalk cells, derived from the rear of the slug, to form the basal disc (Bonner, 1957; Hayashi and Takeuchi, 1981; Raper, 1940). Continued entry of prestalk cells into the entrance of the tube extends the stalk and the spore mass is lifted off the substratum. This process continues until all the prestalk cells, which initially constitute the

2 780 K. A. Jermyn and J. G. Williams anterior one-fifth of the slug, are consumed into the stalk. Vital dyes, such as neutral red, selectively stain prestalk cells in the anterior region. They also stain scattered amoebae in the rear of the slug, termed anterior-like cells (ALC; Bonner, 1957; Sternfeld and David, 1981), which have most of the biochemical characteristics of prestalk cells (Devine and Loomis, 1985). By grafting the front of a Nile blue-labelled slug on to the rear of a neutral red-labelled slug and inducing culmination, Sternfeld and David (1982) showed that some ALC migrate upwards to form a cup over the spore head and some downwards to form a cup under the spore head. These cells appeared to remain as undifferentiated amoebae but this conclusion was subsequently questioned by Voet et al. (1985), who concluded that ALC must eventually differentiate into stalk cells because they could not detect, by flow cytometry, a population of undifferentiated amoebae in fully mature culminants. This confused situation illustrates the other great difficulty in studying culmination, the lack of definitive stalk cell markers. Most gene products specifically expressed in prestalk cells are not retained in stalk cells (Borth and Ratner, 1983; Morrissey et al. 1984). Two exceptions to this rule are the products of the pdd56 and pdd63 genes. They are inducible by the stalk cell inducer, DIF, and their transcripts are highly enriched in both prestalk and stalk cells (Jermyn et al. 1987; Williams et al. 1987). They encode closely related proteins, primarily composed of multiple copies of a highly conserved, cysteine-rich, 24 amino acid repeat (Ceccarelli et al. 1987; Williams et al. 1987). Immunoelectron microscopy locates the proteins to the slime sheath of the migratory slug and the stalk tube, and stalk cell walls, of the mature culminant (McRobbie et al. 1988a,b). Thus their structure and location suggests that they are extracellular matrix proteins, with a structural role in the slug and the mature culminant. By fusing the promoters and 5' non-coding regions of each gene to an immunologically detectable reporter gene, and isolating stable Dictyostelium transformants containing one or other of the two constructs, we generated cell autonomous markers and showed that the two genes display different patterns of expression (Jermyn et al. 1989). The pdd63 fusion gene is expressed at a high level in most or all cells in the front half of the prestalk zone, while the pdd56 gene is expressed only in a central, funnel-shaped region near the slug tip. We termed cells expressing the pdd63 gene psta cells, those expressing the pdd56 gene pstb cells, and cells in the rear half of the prestalk zone, which appeared to express neither marker, psto cells (Jermyn et al. 1989). The genes can now be renamed in a more meaningful way. Since the pdd63 gene encodes an extracellular matrix protein expressed in psta cells it will be termed the ecma gene. Similarly, the pdd56 gene will be termed the ecmb gene. By fusing the promoters of the ecma and ecmb genes to /3-galactosidase (/J-gal), we have created more convenient, and much more sensitive, reporter constructs and used them to re-analyze the structure of the slug. Our previous study was performed using slugs migrating towards a low-level, uni-directional light source. In the present study we also used slugs migrated in the light and find that they show a different structure from dark-migrated slugs. Because the two fusion genes encode proteins that are retained within stalk cells, we have also been able to determine their pattern of expression during culmination. This has yielded new insights into the architecture of the fruit. Materials and methods Fusion gene constructs The ecma(63) and ecmb(56) /3-gal constructs were made by insertion of BgHl fragments containing their promoters into the BgUI site of pddlac-1 (Dingermann et al. 1989). The Bglll fragments derive from analogous constructs, in which the promoters were fused to a reporter containing a region from within the SV40 T-Antigen gene inserted into the bacterial CAT gene (Jermyn et al. 1989). Double transformants were constructed by co-transformation of the ecma /Sgal and ecmb T-Ag CAT constructs into Ax2 cells. Growth and development of transformants Cells were grown in axenic medium containing G418 at 20^gmr'. They were washed in KK2 (16.5 DIM KH 2 PO 4, 3.8mM K 2 HPO 4, ph6.2) and developed on 2% Bacto Agar (Difco) plates. These were incubated at 22 C either in clear humid boxes or in dark, humid chambers with a single slit to allow light entry. Migrating slugs were induced to culminate by temporary removal of the plate lid and exposure to overhead light. Neutral red staining was performed as described by Sternfeld and David (1982). Histology Where staining for /J-gal was to be performed, whole mounts were fixed in 1 % glutaraldehyde in Z buffer (Dingermann et al. 1989) for 10 min, washed twice with Z buffer and incubated in staining buffer (Z buffer containing 5mM K 3 [Fe(CN) 6 ], 5mM K 4 [(CN) 6 ], lmm X-gal) for various times before mounting in Gelvatol. Sections were cut from stained whole mounts embedded in OCT compound (Tissue Tek) as described previously (Williams et al. 1989). Where double staining for prespore and prestalk gene expression was to be performed, sections, prestained as above to detect /3-gal, were first permeabilized in methanol for 10 min and then incubated for lh with Mudl, a monoclonal antibody directed against the PsA protein of prespore cells (Krefft et al. 1984) It was detected by incubation for 1 h with goat anti-mouse immunoglobulin (Biorad, diluted 1:500) conjugated to horseradish peroxidase (Williams et al. 1989). Where double staining for ecma and ecmb gene expression was to be performed, whole mounts were fixed in acetone for 2 min. They were incubated overnight at 4 C with a polyclonal antiserum to /3-galactosidase and a panel of monoclonal antibodies directed against the T-antigen of SV40 (Williams et al. 1989). The secondary antibodies were FITC-conjugated anti-mouse (Tago diluted 1:500) and rhodamine-conjugated anti-rabbit (Tago diluted 1:250) and incubations were in PBS for 3-5 h at room temperature. The slides were mounted in a 2.5% solution of 'DABCO' (Janssen) in glycerol. The primary and secondary polyclonal antibodies were preabsorbed with acetone-fixed cells for 4 h at room temperature before dilution.

3 An analysis of Dictyostelium culmination 781 Results New reporter constructs for ecma and ecmb gene expression Using an immunologically detectable reporter construct, we have shown that the upstream regions of the ecma and ecmb genes direct correct temporal and celltype-specific expression (Jermyn etal. 1989). Sensitivity is, however, limiting; an overnight incubation with antibody being required for efficient prestalk cell detection. Also, we find very poor staining within cells in the lower half of the stalk of ecma and ecmb transformants (data not shown). Since we know that the ecmb gene is maximally expressed during culmination (Jermyn et al. 1987) we infer that antibodies penetrate the more basal region of the stalk tube poorly. Fusion genes, in which the promoters drive expression of the E. coli ^3-galactosidase gene, were therefore constructed, and these allow detection of ecma and ecmb expression in the slug with only very short periods of incubation in X-gal. There is also strong staining down the entire length of the stalk, indicating that the stalk tube must be readily permeable to X-gal. We have used these transformants to compare slugs migrating under differing conditions of illumination and to analyze culmination. Expression of the ecma and ecmb genes in migrating slugs Using the /3-gal fusion constructs, we have re-analyzed the structure of the slug. The /8-gal assay is very convenient in that it is easy to vary the time of incubation with X-gal to discriminate different levels of expression. In the experiment shown in Fig. 1, ecma transformant slugs, migrating towards a dim unidirectional light source ('dark'-migrated slugs), were fixed, stained for /5-gal activity and photographed after increasing incubation times. Initially staining is confined to the extreme tip (Fig. 1A), at intermediate times there is staining in the approximate front 10% (Fig. 1B,C) but eventually the entire prestalk region (the front 20 % of the slug) becomes detectably stained (Fig. ID). Previously, we observed staining only in the front 10% of the length of the slug, even after prolonged incubation with the primary antibody. We assume that this difference reflects a lower sensitivity of the immuno-histochemical detection procedure. Thus pst 'O' cells express the ecma gene, but at a much lower level than psta cells. (N.B. This anterioposterior gradient is not due to a higher permeability of the sheath at the tip, a similar gradient of j3-gal staining is seen in sectioned material (data not shown). If slugs are allowed to migrate in the presence of overhead light, then the pattern of expression of the ecma gene is altered. Individual ecma transformant slugs, which were clearly still migrating, were fixed and stained with X-gal for increasing periods of time. This experiment was performed in parallel with the above analysis of dark-migrated slugs, so that the relative levels of staining can be directly compared. At each time point, the apparent level of expression is higher than at the corresponding time of staining in the darkmigrated slugs (Fig. 1E-H). This overall quantitative difference is the consequence of a much shallower anterioposterior gradient of gene expression in the anterior zone of light-migrated slugs, with no distinct 'pst O' region visible even at very short times of incubation. When transformant slugs expressing the ecmb gene are stained, using short periods of exposure to X-gal, the pattern of staining is identical to that observed previously (Jermyn et al. 1989): a central funnel of cells in the anterior part of the prestalk zone (Fig. 2). In slugs migrated in the presence of overhead light, the intensity is generally stronger than in dark-migrated slugs but the level is variable and it is possible to find the occasional dark-migrated slug showing a higher level of ecmb staining than the average light-migrated slug (data not shown). When ecma or ecmb transformant slugs are incubated with X-gal for longer periods of time additional staining cells are detected in the prespore zone (Fig. 3A-C). The distribution of pstb cells outside the central cone is variable. In some slugs (Fig. 3A), they are scattered throughout the entire prestalk and prespore regions while in others (Fig. 3D-F) some of the pstb cells are concentrated at the prestalk-prespore boundary. This clustering of pstb cells is observed in both dark- and light-migrated slugs and we have not been able to correlate it with any other variable. However, this pattern presages the re-distribution that we observe for these cells at culmination and may indicate that, despite the fact that they were still migrating, these slugs were preparing to culminate. In some, but not all, slugs we detect a 'rearguard' zone by staining with neutral red and the ecma and ecmb genes are expressed at a low level in cells in such regions (data not shown). These may be cells that are about to be shed into the slime trail. After overnight incubation with X-gal, at least 50 % of cells within the trail stain for the expression of one or other fusion gene (Fig. 3B,3C). Prestalk cells constitute only % of cells in the slug, hence they are considerably overrepresented in the cells deposited in the trail. The ecma and ecmb cells within the prespore region could be ALC or they could be a subtraction of the prespore cells that express, or at some time have expressed, the ecma or ecmb genes. In order to distinguish these possibilities, cells were disaggregated from the rear of neutral-red-stained slugs, allowed to attach to a surface and photographed for neutral red before staining with X-gal (Fig. 4). About two thirds of cells expressing the ecma gene are ALC, i.e they stain with neutral red, and an approximately similar fraction of ecm B-expressing cells are ALC (Table 1). We cannot estimate the extent to which there is overlap (i.e. the fraction of cells expressing both genes), hence we do not know what fraction of the ALC population the pst A and pstb cells together constitute. We have shown that there is some co-expression, by immunohistochemical double staining of co-transformants expressing the ecma gal and ecmb T-Ag CAT fusion genes

4 782 K. A. Jermyn and J. G. Williams (Jermyn and Williams, unpublished results), but the relative insensitivity of this technique prevents accurate quantitative analysis at this early stage. Further evidence that the ecmb cells are ALC is their predominant localization on the ventral surface of the slug (Fig. 3E,F), where ALC are known to be enriched (Sternfeld and David, 1982). Expression of the ecma gene during culmination Culminants show expression of the ecma gene throughout the apical prestalk region, down the entire length of the stalk tube and in the base (Figs 5A-C, 6). This is as expected, because fruiting is induced by overhead light and, in light-migrated slugs, there is an essentially invariant level of expression of the ecma gene throughout the prestalk zone (Fig. 1E-F). We have confirmed that the psta and prespore regions are juxtaposed, by double staining mexican hat sections for /S-galactosidase and for the PsA protein, a surface marker of prespore cells (Fig. 5B). Fate of the central core of ecmb cells in the tip of the migrating slug at culmination The central core of ecmb cells in the tips of transformant slugs accumulate /3-galactosidase during slug formation. As we will show, there are new sites of accumulation during culmination but, by manipulating the level of staining, we can select against the visualization of these and determine the fate of the 'original' ecmb cells. They lie in the position where formation of the stalk tube is initiated, they are the first cells to enter the stalk tube (Fig. 5D) and they maintain their 'pathfinder' postion during the Mexican hat stage (Fig. 5E, F, 6A). They embed themselves into the rearguard cells eventually to form part of the basal disc (Fig. 6B). We have repeated the grafting experiments performed by Raper (1940), fusing the anterior region of an ecmb slug with a non-transformant slug, and culminants derived from such slugs show strong staining confined to the central region of the basal disc (Fig. 5G). Fig. 1. High level expression of the ecma genes in slugs migrating towards a uni-directional light source or under overhead light. (A-D) Transformant slugs containing the ecma /3-galactosidase fusion gene were allowed to migrate towards a uni-directional light source for one day, fixed and then incubated with X-gal for increasing lengths of time before photography. This example is typical of the different slugs examined and shows the staining pattern when incubated for the indicated times in X-gal. (E-H) Transformant slugs containing the ecma /3- galactosidase fusion gene were allowed to migrate for one day, under over-head light. In these conditions, many slugs enter culmination. Slugs that were clearly still migrating were individually fixed and then incubated with X-gal for increasing lengths of time before photography. This is a typical example of the results obtained, again by the analysis of different slugs and shows the staining pattern of two slugs incubated for the indicated times in X-gal. The staining and photography were performed in parallel with the experiment presented in A-D, hence the relative levels of staining can be directly compared. Times of incubation were: A,E 20min; B,F 40min; C,G 60min; and D, H 80min. Fig. 4. Correlation of psta and B cells in the rear of the slug with ALC. The rear halves of 2 day old neutral-redstained migrating slugs were dissociated by trituration in cold '20/20' buffer (Devine and Loomis, 1985) and resuspended in KK2 on a poly-l-lysine coated haemacytometer grid or, in some cases, on a scored tissue culture dish. Selected fields were photographed (A), the cells were fixed and incubated overnight with X-gal and the fields rephotographed (B). Individual cells arrowed are: (a) positive for neutral red and /3-gal, (b) positive for neutral red only, (c) positive for /3-gal only. visible at the boundary between the prestalk and prespore region, in the base of the aggregate and randomly scattered throughout the prespore region. (Fig. 5H-K). These cells derive, in part at least, from ALC. There are two pieces of evidence to support this conclusion. Induction of the ecmb gene in anterior psta cells and in ALC at culmination and morphogenetic movements of the resultant pstb cells Culmination proceeds by the influx of psta cells into the stalk tube. They do not express the ecmb gene until they are just within the entrance, where they become heavily stained (Figs 5H, 6A). The simplest interpretation is that there is an inductive signal located at the mouth of the tube, which activates the ecmb gene. During culmination strongly staining pstb cells are also Table 1. Correlation of ALC with ecma- and ecmbexpressing cells Source of cells ecma gal transformant ecmb gal transformant Percentage of cells in each class Neutral red only /S-gal only Neutral red +/3-gal Fig. 2. High level expression of the ecmb gene in migrating slugs. Transformant slugs, containing the ecmb /3-galactosidase fusion gene, were allowed to migrate towards a uni-directional light source, fixed and then incubated with X-gal for 5h.

5 H

6 LU

7 Fig. 5. The differentiation and morphogenetic movements of psta and pstb cells during culmination. (A-C) PstA cells. (A) A longitudinal section of a mexican hat derived from an ecma reporter gene transformant. (B) Longitudinal section of a mexican hat stage preculminant derived from an ecma reporter gene transformant and double stained for /3-gal and for a prespore specific marker. This is a central, longitudinal section derived from an aggregate which was stained as a whole mount with X-gal. After sectioning it was stained using the Mudl antibody, that detects the PsA cell surface protein of prespore cells. (C) A whole mount of a late stage ecma culminant. (D-K) PstB cells. (D) A whole mount of a very early mexican hat stage ecmb transformant. (E) Whole mounts of mexican hat stage preculminants derived from an ecmb reporter gene transformant. (F) Longitudinal section of a mexican hat stage pre-culminant derived from an ecmb reporter gene transformant and double stained for /?-gal and for the prespore-specific marker as described in B. (G) Whole mounts of the base of two mature cuhninants derived from grafting the anterior regions of ecmb transformant slugs onto the prespore regions of non-transformed slugs. The approximate front 10 % of ecmb transformant slugs was grafted onto the approximate posterior 80 % of nontransformant slugs and the slugs were induced to culminate. They were then fixed and stained for approximately 16 h with X-gal. (H) The anterior region of a whole mount of a mid preculminant derived from an ecmb reporter gene transformant. (I) Low power view of a whole mount of an ecmb preculminant at a stage equivalent to that shown in H. (J) Longitudinal section through an ecmb aggregate at an approximately equivalent stage to that shown in H and I. (K) Whole mount of a very late stage culminant of an ecmb transformant.

8 Fig. 9. Simultaneous detection of the ecma and ecmb fusion gerie products in culminants. This is a whole mount of a late culminant derived from a double transformant expressing the ecma gal and ecmb T-Ag CAT fusion genes. Only the upper portion of the fruit, proximal to the spore head, is shown with the papilla at the top. The two reporter gene proteins were detected as described in the methods section. A shows the location of the ecma gal product and B and C the location of the ecmb T-Ag CAT product. 0-galactosidase accumulates in the cytoplasm and T-Ag CAT accumulates in the nucleus.

9 An analysis of Dictyostelium culmination 783 3A B Fig. 3. Low level expression of the ecma and ecmb genes in slugs detected using prolonged periods of incubation with X-gal. (A) Transformant slugs containing the ecmb ^-galactosidase fusion gene were allowed to migrate towards a uni-directional light source, fixed and floated onto chrome-alum-coated slides. They were then incubated with X-gal for 10 h. (B) A high power view of the back of an ecmb slug with its adherent slime trail treated exactly as described in A. (C) The back of an ecma transformant slug treated exactly as described in A. (D-F) Individual ecmb transformant slugs, migrating in the presence of overhead light, were picked up, deposited in fixative and stained with X-gal for 18 h. The examples shown in E and F were the same two slugs, photographed initially with their ventral surface upwards (E) and then with their dorsal surface upwards (F). After fixation with glutaraldehyde, the cells become relatively opaque and it is therefore possible to discriminate on which surface cells are located. It is clear that most of the scattered cells are localized on the ventral surface. (1) Indirect evidence derives from their eventual fate. Very late in culmination pstb cells are mostly confined to a cup just above the prespore mass (which we will term the upper cup), a cup just below it (which we will term the lower cup), the basal disc and the stalk tube (Figs 5K, 6B). The spore mass, and the psta cells which have yet to enter the stalk tube, remain unstained. Direct, visual observation of vitally stained cells has shown that this upper cup derives from ALC that accumulate at the prestalk-prespore boundary and the lower cup derives from ALC that accumulate at the base (Sternfeld and David, 1982). (2) By grafting the front of a non-transformant slug on to the back of a slug transformed with the ecmb gal fusion gene, we have confirmed that the upper and lower cups derive from posterior cells. In the mexican hat stage aggregates, obtained from such a grafted structure, staining cells are scattered through the cell mass, with a concentration of cells at the base and at the prestalk-prespore boundary and with no staining in the

10 784 K. A. Jermyn and J. G. Williams New PstB Subset of ALC which express ecmb PstA pper "Cup" Lower "Cup" "Old" PslB Anterlor-LIke > Cell -Derived Rearguard + Anterlor-LIke Cell-Derived? below the upper cup where the ecma gene is expressed at a high level but ecmb gene expression is much reduced. There is, therefore, either a difference in sorting behaviour between pstb cells, expressing the ecmb gene at different levels, or a difference in signalling, such that only those ALC proximal to the original prestalk-prespore boundary are induced to express the ecmb gene maximally. One point that remains unclear is the precise origin and fate of the basal cells. These seem to derive in part from ALC that migrate downwards and in part from rearguard cells of the migratory slug (although this distinction becomes somewhat arbitrary if the rearguard cells derive from ALC that accumulate at the back of the slug). The simplest possibility, and the one presented in Fig. 6, is that they intermingle and are split into two by extension of the stalk tube; the fraction that remain at the base constituting the outer part of the basal disc and the fraction that move up the stalk forming the lower cup. Alternatively, there may be separate populations of basal cells in the mid-culminant; one derived from rearguard cells and giving rise to the basal disc and one derived from those ALC that migrate to the base and give rise to the lower cup. It will be necessary to perform more precise grafting or transplantation experiments to clarify this point. Fig. 6. Diagramatic summary of the changes in ecmb gene expression and in the location of pstb cells during culmination. stalk tube (Fig. 7A). At a later stage in culmination, staining is almost totally confined to the upper cup, the lower cup and the basal disc (Fig. 7B. N.B. The example shown was stained for a relatively short period of time with X-gal and this presumably accounts for the apparent absence of expression in the outer part of the basal disc). In the reciprocal experiment, we obtain the opposite result, staining being essentially confined to the stalk tube (Fig. 7C). The scale of the increase in the level of ecmb gene expression in ALC at culmination can be seen in Fig. 8, which shows a slug and a late culminant that were stained together. At least part of the increase in ecmb gene expression occurs prior to the movement of ALC to surround the spore mass. It is not feasible to detemine whether there is a further elevation in the level of ecmb gene expression after sorting has occurred because individual cells cannot then be discriminated. At culmination ecma gene expression in ALC rises in parallel with ecmb gene expression, with strong staining in the upper and lower cups and in those ALC that are in process of sorting (Fig. 5C). In order to determine the degree of overlap in their expression, double staining, using co-transformants expressing the ecma gal and ecmb T-Ag CAT constructs, was performed. The two fusion gene products are colocalized in cells within the upper and lower cups and within the stalk (Fig. 9). Interestingly, there is a region Discussion Analysis using /3-galactosidase gene fusions has refined the view of slug structure that we obtained with an immunologicauy detectable reporter gene. It is now clear that, even in dark-migrated slugs, there is no entirely distinct class of pst 'O' cells, i.e. cells that completely fail to express either the ecma or the ecmb marker. Rather, there is a markedly discontinuous gradient of ecma gene expression, with cells in the front one-tenth of the slug expressing the gene most strongly. A previous study using a prespore-specific marker showed that, in some Dictyostelium strains, there is an increase in the ratio of prespore to prestalk cells in slugs migrating in the dark (Krefft et al. 1983). In agreement with this, we show that light stimulates prestalk cell differentiation, by causing a large elevation in the level of ecma expression in the pst 'O' zone. The ecma protein is produced by psta cells, the major prestalk species in the migratory slug, and is present in the slime trail and most probably also in the slime sheath. We believe that it probably plays an important role at this time as a structural component of the sheath. An elevation in the amount of the protein as the slug approaches the surface may be a preparatory step for culmination. Even after very long times of staining with X-gal, psta cells show no expression of the ecmb gene, indicating there to be a very clear cut distinction between psta and pstb cells. We have shown that some ALC express the ecma and some the ecmb gene. We cannot exclude the existence of ALC that are not

11 An /s-o/dictyostelium culmination 785 B Fig. 7. Whole mounts of preculminants and culminants derived from grafting experiments between ecmb reporter gene transformants and non-transformants. (A) Whole mount of a mexican hat derived from grafting the anterior region of a non-transformant slug on to the posterior region of an ecmb transformant slug. (B) As for A except that the structure resulting from the grafts was fixed at the preculminant stage. (C) Whole mount of a preculminant derived from grafting the anterior region of an ecmb transformant slug onto the posterior region of a nontransformant slug. detectably expressing either gene and there are definitely cells expressing one or other gene which are not detectably stained with neutral red. Thus there is a very considerable overlap between the ALC and psta and B cell populations but not a complete congruity. At culmination, the pstb cells originally present in the slug anterior form the vanguard of the stalk tube, eventually fusing with rearguard cells to form the inner part of the basal disc. The 'reverse fountain' directs psta cells into the entrance to the stalk tube and it is precisely at this point that expression of the ecmb gene is activated. By the end of culmination, all cells within the stalk tube are expressing, or at some time have expressed, both the ecma and ecmb fusion genes. Thus

12 786 K. A. Jermyn and J. G. Williams 8A Fig. 8. Elevation of the level of ecmb gene epression in ALC at culmination. This is a whole mount of a slug (A) and a mid-culminant (B) which came to lie together during fixation. Since the two structures were exposed to identical conditions it is meaningful to compare their intensities of staining. There is clearly a dramatic increase in the level of ecmb gene expression in ALC during culmination. At least part of this increase occurs prior to their migration to surround the spore head. We cannot rule out the possibility of a further elevation in ecmb expression after sorting because we cannot discern the level of expression in individual cells once the ALC have coalesced at the prestalk-prespore boundary and in the lower cup. B there is a temporal and spatial progression, with psta cells differentiating into pstb cells (i.e. activating the ecmb gene) as they enter the stalk tube. This is in some ways analogous of the situation in species such as D. mucoroides, which produce stalk continually during migration by the rapid trans-differentiation of 'prespore' cells at this region (Gregg and Davis, 1982). It would appear that the pstb cells in the tip of the migratory D. discoideum slug constitute a stalk tube primordium, with further progression being blocked until culmination is triggered. Consistent with this interpretation, we have recently shown that the pstb cells within the tip of the migratory slug contain both the ecma and ecmb fusion gene products (Jermyn and Williams, unpublished results). A similar conclusion was reached by Gregg and Karp (1978) who showed, by tissue autoradiography, that the majority of D. discoideum prestalk cells incorporate little, if any, fucose. However, they observed a region of relatively high fucose incorporation in the tip of the migrating slug, in precisely the position occupied by pstb cells. At culmination, cells within the stalk tube showed a similarly high level of labelling. Thus the entrance to the stalk tube appears to act as a site of positional signalling, causing psta cells to express the ecmb gene. A higher level of DIF is necessary for the initial induction of the ecmb gene than is required for the ecma gene (Berks and Kay, 1990). Hence the trigger for its induction could be a localized increase in DIF concentration or a drop in the concentration of an antagonist. While there is evidence to suggest that pstb cells in the migratory slug accumulate very high levels of DIF (Kwong et al. 1990), we favour the latter explanation. There are two potential DEF antagonists, ammonia and camp. If an enzymatic mixture that depletes ammonia is dropped on to migrating slugs they culminate immediately (Schindler and Sussman, 1977) and mutants that are hyper-sensitive to ammonia remain as slugs under conditions favouring culmination (Newell and Ross, 1982). Thus culmination is believed to be triggered by a drop in ammonia concentration, possibly caused by metabolic changes or an increased rate of diffusion as the slug perceives and orients towards overhead light (Bonner et al. 1982). The reduction in ammonia concentration may act to trigger culmination by decreasing intracellular ph (phi). If phi is artificially decreased, by treatment with CO 2, stalk cell differentiation is induced within the migrating slug (Inouye, 1988). Ammonia, and other weak bases, act as inhibitors of the induction, by DIF, of stalk cell differentiation and ecma and ecmb gene expression (Gross et al. 1983; Wang and Schaap, 1989). Cyclic AMP is an alternative candidate for the repressor of psta to pstb cell differentiation. The ecma and ecmb genes are dependent upon DIF for their expression (Jermyn et al. 1987) but cells must first be rendered competent to respond to DIF by a period of exposure to extracellular camp (Berks and Kay, 1988). The two genes differ in that, after DIFresponsiveness is achieved, expression of the ecma gene is stimulated by the addition of camp while expression of the ecmb gene is greatly repressed (Berks and Kay, 1988, 1990). The trigger for induction of pstb cell differentiation could therefore be a drop in the level of intracellular camp. These two candidates for the role of DIF antagonist need not be mutually exclusive. Exogenous ammonia stimulates the intracellular accumulation of camp (Riley and Barclay, 1990). Hence a drop in ammonia concentration might trigger a parallel drop in the intracellular concentration of camp. In addition to this positionally localized inductive signal we posit the existence of a non-localized signal responsible for elevating expression of the ecma and ecmb genes in ALC during culmination. This increase occurs both in cells that have migrated to surround the spore mass and in cells that have yet to do so (Fig. 8).

13 An analysis of Dictyostelium culmination 787 This indicates that, at least for the latter population of cells, the inductive signal must act throughout the aggregate. Induction of the ecma and ecmb genes in ALC during culmination was a somewhat unexpected result because, histologically, they give the appearance of undifferentiated cells (Sternfeld and David, 1982). This led to the suggestion that they might retain their motility and play an active role in lifting the spore mass up the stalk tube (Sternfeld and David, 1982). Consideration of the other site at which the ecmb protein accumulates leads us to believe that they may act in a structural role. The ecma protein is present in most, if not all prestalk cells, and we believe that it may help to maintain the integrity of the slime sheath. The ecmb gene is activated as psta cells enter the stalk tube, suggesting that its primary role is in the stalk tube and stalk cell walls. The stalk is a more rigid structure than the slime sheath and the fact that the ALC surrounding the prespore mass express the ecmb gene suggests that they may also be playing a supporting role, perhaps in maintaining the integrity of the spore head as it moves up the stalk tube. This may even be their primary role and their movement forward, to reconstitute the prestalk zone when the integrity of the slug is disrupted, may be a secondary function. We would like to thank Rob Kay and David Ratner for their constructive suggestions on this manuscript. References BERKS, M. AND KAY, R. R. (1988). Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum. Devi Biol. 125, BERKS, M. AND KAY, R. R. (1990). Combinatorial control of cell differentiation by camp and DLF-1 during development of Dictyostelium discoideum. Development 110, BONNER, J. (1957). A theory of the control of differentiation in the cellular slime molds. Q. Rev. Biol. 32, BONNER, J., CHIQUOINE, A. AND KOLDEJUE, M. (1955). A histochemical study of differentiation in the cellular slime molds. /. exp. Zool. 130, BONNER, J., DAVIDOWSKJ, T., HSU, W., LAPEYROLERIE, D. AND SUTHERS, H. (1982). The role of surface water and light on differentiation in the cellular slime molds. Differentiation 21, BORTH, W. AND RATNER, D. (1983). Different synthetic profiles and developmental fates of prespore versus prestalk proteins of Dictyostelium. Differentiation 24, CECCARELU, A., MCROBBIE, S. J., JERMYN, K. A., DUFFY, K., EARLY, A. AND WILLIAMS, J. G. (1987). Structural and functional characterization of a Dictyostelium gene encoding a DIP inducible, prestalk-enriched mrna sequence. Nuc. Acids Res. 15, DEVINE, K. AND LOOMIS, W. (1985). Molecular characterization of anterior-like cells in Dictyostelium discoideum. Devi Biol. 107, DlNGERMANN, T., REINDL, N., WERNER, H., HlLDEBRANDT, M., NELLEN, W., HARWOOD, A., WILLIAMS, J. AND NERKE, K. (1989). Optimization and in situ detection of Escherichia Coli B- galactosidase gene expression in Dictyostelium discoideum. Gene 85, GREGG, J. AND DAVIS, R. (1982). Dynamics of cell redifferentiation in Dictyostelium mucoroides. Differentiation 21, GREGG, J. AND KARP, G. (1978). Patterns of cell differentiation revealed by L-(3H)fucose incorporation in Dictyostelium. Expl Cell Res. 112, GROSS, J., BRADBURY, J., KAY, R. AND PEACEY, M. (1983). Intracellular ph and the control of cell-differentiation in Dictyostelium discoideum. Nature 303, HAYASHI, M. AND TAKEUCHI, I. (1981). Differentiation of various cell-types during fruiting body formation of Dictyostelium discoideum. Dev. Growth Differ. 23, INOUYE, K. (1988). Induction by acid load of the maturation of prestalk cells in Dictyostelium discoideum. Development 104, JERMYN, K. A., BERKS, M., KAY, R. R. AND WILLIAMS, J. G. (1987). Two distinct classes of prestalk-enriched mrna sequences in Dictyostelium discoideum. Development 100, JERMYN, K. A., DUFFY, K. AND WILLIAMS, J. G. (1989). A new anatomy of the prestalk zone of Dictyostelium. Nature 340, KREFFT, M., VOET, L., GREGG, J. H., MAIRHOFER, H. AND WILLIAMS, K. L. (1984). Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates. EMBO J. 3, KREFFT, M., VOET, L., MAIRHOFER, H. AND 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, KWONG, L., YINJUAN, X., DANIEL, S. M. AND WEEKS, G. (1990). A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells. Development 110, MCROBBIE, S., JERMYN, K., DUFFY, K., BLIGHT, K. AND WILLIAMS, J. (1988a). Two DIF inducible, prestalk specific messenger RNAs of Dictyostelium encode extracellular matrix proteins of the slug. Development 104, MCROBBIE, S., TILLY, R., BUGHT, K., CECCARELU, A. AND WILLIAMS, J. (19886). Identification and localization of proteins encoded by two DIF inducible genes of Dictyostelium. Devi Biol. 125, MORRISSEY, J., DEVINE, K. AND LOOMIS, W. (1984). The timing of cell-type-specific differentiation in Dictyostelium discoideum. Devi Biol. 103, 414-^24. NEWELL, P. AND ROSS, F. (1982). Genetic analysis of the slug stage of Dictyostelium discoideum. J. gen. Microbiol. 128, RAPER, K. (1940). Pseudoplasmodium formation and organisation in Dictyostelium discoideum. J. Elisha Mitchell Sci. Soc. 59, 241-2B2. RAPER, K. AND FENNELL, D. (1952). Stalk formation in Dictyostelium. Bull. Torrey Bot. Club 79, RJLEY, B. B. AND BARCLAY, S. L. (1990). Ammonia promotes accumulation of intracellular camp in differentiating amoebae of Dictyostelium discoideum. Development 109, SCHINDLER, J. AND SUSSMAN, M. (1977). Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum. J. molec. Biol. 116, STERNFELD, J. AND DAVID, C. N. (1981). Cell sorting during pattern formation in Dictyostelium. Differentiation 20, STERNFELD, J. AND DAVID, C. N. (1982). Fate and regulation of anterior-like cells in Dictyostelium slugs. Devi Biol. 93, VOET, L., KREFFT, M., BRUDERLEIN, M. AND WILLIAMS, K. (1985). Flow cytometer study of anterior-like cells in Dictyostelium discoideum. J. Cell Sci. 75, WANG, M. AND SCHAAP, P. (1989). Ammonia depletion and DIF trigger stalk cell differentiation in intact Dictyostelium discoideum slugs. Development 105, WILLIAMS, J., CECCARELU, A., MCROBBIE, S., MAHBUBANI, H., KAY, R., EARLY, A., BERKS, M. AND JERMYN, K. (1987). Direct induction of Dictyostelium prestalk gene expression by DLF provides evidence that DIF is a morphogen. Cell 49, WILLIAMS, J. G., DUFFY, K. T., LANE, D. P., MCROBBIE, S. J., HARWOOD, A. J., TRAYNOR, D., KAY, R. R. AND JERMYN, K. A. (1989). Origins of the prestalk-prespore pattern in Dictyostelium development. Cell 59, (Accepted 13 December 1990)