Slime moulds, such as Dictyostelium discoideum, have biochemical, physiological and probably developmental features in common with both plants and animals. During development separate Dictyostelium amoebae first aggregate into collecting centers to form small multicellular organisms which, in their slug form, can migrate over the substratum toward light. Eventually a slug culminates to form a fruiting body consisting of a cellular stalk supporting a mass of spores. Development is highly regulative, indicating that it is controlled by signalling between the cells. A number of diffusible signal molecules have been discovered, including cyclic AMP, the chemoattractant in aggregation, and DIF-1, a novel chlorinated phenyl alkanone, which acts as a specific inducer of stalk cell differentiation.

The migrating slug contains three types of precursor cell: prespore, prestalk A and prestalk B cells. Differentiation of these cells from uncommitted amoebae can be brought about in ceil cultures by cyclic AMP and DIF-1 acting in combination: cyclic AMP alone favours prespore, DIF-1 alone favours prestalk B, cyclic AMP and DIF-1 together favour prestalk A cell differentiation. There is evidence suggesting that these signals act in the same way in the intact aggregate.

A cytoplasmic DIF-1 binding protein has been discovered, whose level increases as cells become sensitive to DIF-1 and which binds DIF-1 with an affinity and specificity suggestive of a receptor. At the same time, cells are able to inactivate DIF-1 by a metabolic pathway involving at least 12 metabolites. Metabolism may also serve to produce gradients of DIF-1 in the aggregate or other signal molecules from DIF-1. Manipulation of the DIF-1 signalling system will be an important means of further elucidating its role in development.

The cellular slime moulds have long been regarded as a bridge between unicellular and multicellular life-forms, but they might also be thought of as a bridge between the plant and animal kingdoms (or at least between the fungi and the protozoa). It is as if they grow as unicellular animals, but develop into multicellular plants. In the most studied species, Dictyostelium discoideum, the cells grow as separate amoebae, which phagocytose and feed on bacteria. These cells appear typically protozoan. But when they run out of their bacterial food they enter a developmental program, in which they form a multicellular fruiting body consisting of a stalk supporting a mass of spores. The fruiting body resembles a fungus, with vacuolated stalk cells and refractile spores; typical plant products are made:,the stalk and spores contain sporopollenins (Maeda, 1984) and cellulose (Gezelius, 1959), while high levels of IP6 (phytic acid) are found in both developing and growing cells (Martin et al. 1987). In the alternative sexual development, the plant hormone ethylene is used as a mating hormone (Amagai, 1984).

Development occurs without growth and with little cell division. In the first stage the separate amoebae come together in a unique aggregation process, which involves the propagation of waves of the chemoattractant, cyclic AMP, out from aggregation centers (Devreotes, 1983; Gerisch, 1987). The amoebae move into the aggregation centers, become cohesive and pile up in hemispherical mounds, which produce a primitive extracellular matrix, the slime sheath. At this stage the precursors of the stalk and spore cells (prestalk and prespore cells) first start to differentiate. A little later, a protruding tip appears on top of the mound (Fig. 1) and it elongates upwards and falls on its side to make the well-known migrating slug. The slug contains up to 105 cells and moves slowly over the substratum, toward light and up or down heat gradients, before it is triggered by suitable environmental conditions (overhead light, low humidity, low ammonia concentration) to transform into the mature fruiting body.

Fig. 1.

Tipped mound and mature fruiting body of Dictyostelium discoideum. Development is triggered by starvation and after a few hours the amoebae aggregate together by chemotaxis to cyclic AMP signals relayed out from aggregation centers. The resulting mounds start to secrete an extracellular matrix, the slime sheath, and eventually a protruding tip forms (shown just emerging, left), which thereafter leads the morphogenetic movements. The mound elongates upwards to form the standing slug and this can fall on its side to form the migrating slug which is both photo- and thermo-tactic. In suitable environmental conditions the slug culminates, resulting in the mature fruiting body (right) in which a basal disc and stalk support a droplet of spores. The whole process takes about 24 h in standard laboratory conditions and the fruiting body can contain up to 105 cells and be 5 mm tall. Photograph reproduced by permission of The Company of Biologists.

Fig. 1.

Tipped mound and mature fruiting body of Dictyostelium discoideum. Development is triggered by starvation and after a few hours the amoebae aggregate together by chemotaxis to cyclic AMP signals relayed out from aggregation centers. The resulting mounds start to secrete an extracellular matrix, the slime sheath, and eventually a protruding tip forms (shown just emerging, left), which thereafter leads the morphogenetic movements. The mound elongates upwards to form the standing slug and this can fall on its side to form the migrating slug which is both photo- and thermo-tactic. In suitable environmental conditions the slug culminates, resulting in the mature fruiting body (right) in which a basal disc and stalk support a droplet of spores. The whole process takes about 24 h in standard laboratory conditions and the fruiting body can contain up to 105 cells and be 5 mm tall. Photograph reproduced by permission of The Company of Biologists.

As in most other organisms, development in Dictyostelium is coordinated by signalling between the cells. This is already obvious during aggregation, which is brought about by cyclic AMP signalling. Signalling of some sort during multicellular development is indicated by regulation experiments. In the classic example, Raper (1940), having demonstrated that the front of the slug was destined to become the stalk and the rear the spores of the fruiting body, showed that isolated fronts or rears were capable of forming properly proportioned fruiting bodies. The very tip of the slug seems to have a special organizing role and can define a developmental axis: when a tip from one slug is grafted onto the flank of another, it captures part of the host and leads it off to form a separate small slug, and eventually a separate fruiting body (Fig. 2). These experiments show that the parts of the slug must somehow be in communication, so that the rear is ‘aware’ of the loss of the front, or of the grafting in of another tip. This communication system must be able to override the state of differentiation of individual amoebae and direct them to new fates and must also be able to control their organization within the aggregate. The suggestion that amoebae may be committed to stalk or spore differentiation at the start of development by their position in the cell cycle (Gomer and Firtel, 1987) seems impossible in the light of these regulation experiments.

Fig. 2.

An illustration of tip dominance and slug regulation. When additional tips are grafted onto the flanks of a host slug, they each capture part of the host tissue and lead it off to form a separate slug and eventually a separate fruiting body. Later work showed that this property (of causing the host slug to split) was graded in the donor slug, being greatest at the tip. It was also shown by Raper that isolated anterior and posterior fragments of a slug, though normally destined to form the stalk and spores respectively of the fruiting body, could regulate to form normal fruiting bodies. Modified from Raper (1940), with permission.

Fig. 2.

An illustration of tip dominance and slug regulation. When additional tips are grafted onto the flanks of a host slug, they each capture part of the host tissue and lead it off to form a separate slug and eventually a separate fruiting body. Later work showed that this property (of causing the host slug to split) was graded in the donor slug, being greatest at the tip. It was also shown by Raper that isolated anterior and posterior fragments of a slug, though normally destined to form the stalk and spores respectively of the fruiting body, could regulate to form normal fruiting bodies. Modified from Raper (1940), with permission.

There is, however, a continuing controversy about the mechanism by which isolated prestalk and prespore fragments regulate to allow normal fruiting body formation. At one extreme, it is proposed that the pattern of prestalk and prespore cells reforms, due to positional signals in the isolate inducing localized redifferentiation of the appropriate cell type; at the other extreme it is proposed that the missing cell type first differentiates at random locations in the isolate and then sorts to form the final pattern (Leach et al. 1973; Sternfeld and David, 1981; Tasaka and Takeuchi, 1979). In the case of the prestalk isolate, the first signs of prespore cell differentiation are strictly localised (Gregg and Karp, 1978), suggesting that positional cues are at work. The case of the prespore isolate is more complicated because this tissue also contains scattered prestalk cells (the anterior-like cells), which can be recognised by vital staining with dyes, such as neutral red (Sternfeld and David, 1981; Sternfeld and David, 1982). It is proposed that, during regulation, these cells sort out to form the regenerated anterior prestalk zone. However, in the experiments that led to this suggestion, the polarity of the aggregates was lost; for instance in one case they were shaped into small globs and covered in dilute agar during regulation.

We have performed a series of grafting experiments, in which polarity was maintained, and which indicate a position-dependent mechanism of regulation (Poggevon Strandmann and Kay, 1990). Host and graft tissue were distinguished by labelling the cells during vegetative growth with [14C]- or [3H]leucine and their distribution in the manipulated slugs determined by serial sectioning, followed by dual channel scintillation counting of the sections. In chimeric slugs with their anterior and posterior prespore zones differently labelled, the anterior cells contributed disproportionately to the regenerated prestalk zone following amputation of the original prestalk zone. Further, when a few hundred cells from the rear of a 14C-labelled slug were injected into the front of a regulating prespore fragment, a proportion of them were recruited into the new prestalk zone. When these same cells were injected into the rear of the regulating fragment, they remained in place and essentially none of them contributed to the new prestalk zone (Fig. 3). Recruitment does not take place if the cells are injected into the front of an intact slug, or into the front of a fragment where the injection is delayed for one hour after amputation of the prestalk zone. These results suggest that a specific signal which recruits prestalk cells transiently exists in the anterior of the regulating fragment and that this signal is not operative in an undisturbed slug.

Fig. 3.

Positionally dependent recruitment of cells into the regenerated prestalk zone (anterior) during regulation of a prespore isolate. A few hundred 14C-labelled cells from the rear of a slug were injected either into the rear (A) or front (B) of a regulating prespore isolate and their distribution determined when regulation was complete. None of the cells injected into the rear were recruited into the regenerated prestalk zone (taken as the front 30 % of the slug) (A), whereas about 20% of similar cells injected into the front were recruited (arrowed) (B); evidently for these cells, recruitment into the prestalk zone depends on their position in the isolate. As further controls to show that prestalk cells are capable of sorting in these conditions, prestalk cells from the front of a slug were injected either into the front (C) or the rear (D) of a regulating prespore mass. In both cases they sort to the front. In all cases the distribution of labelled cells was determined in the slugs 6h after injection by counting serial sections for both isotopes in a dual channel scintillation counter.

Fig. 3.

Positionally dependent recruitment of cells into the regenerated prestalk zone (anterior) during regulation of a prespore isolate. A few hundred 14C-labelled cells from the rear of a slug were injected either into the rear (A) or front (B) of a regulating prespore isolate and their distribution determined when regulation was complete. None of the cells injected into the rear were recruited into the regenerated prestalk zone (taken as the front 30 % of the slug) (A), whereas about 20% of similar cells injected into the front were recruited (arrowed) (B); evidently for these cells, recruitment into the prestalk zone depends on their position in the isolate. As further controls to show that prestalk cells are capable of sorting in these conditions, prestalk cells from the front of a slug were injected either into the front (C) or the rear (D) of a regulating prespore mass. In both cases they sort to the front. In all cases the distribution of labelled cells was determined in the slugs 6h after injection by counting serial sections for both isotopes in a dual channel scintillation counter.

Although cell-sorting seems not to be the primary mechanism of pattern regulation in this case, it probably is important in the formation of pattern within the prestalk zone during normal development (see below). Positional cues are also important during culmination. The stalk forms in a reverse fountain process, in which prestalk cells climb up the elongating stalk and are added to it from the top. As the amoeboid prestalk cells enter the top of the stalk tube they suddenly start to vacuolate, strongly suggesting that they experience a localized inductive signal (Bonner, 1944; George et al. 1972).

Communication between cells must also be involved in the complicated morphological movements of the aggregate, perhaps most clearly seen in the movement of the slug over the substratum and in its phototaxis, which is controlled by differential illumination across the tip (Poff and Loomis, 1973).

One of the major tasks in the field now is to understand the communication systems operating in the multicellular stages of development: what are the signal substances, how are they detected and maintained at appropriate levels, and what do they do to the receiving cells?

Extracellular cyclic AMP has already been mentioned as the chemoattractant during aggregation (Konijn et al. 1967). It is detected by a cell surface receptor linked, via G proteins, to the intracellular second messengers cyclic AMP, cyclic GMP and lnsP3 (Europe-Finner and Newell, 1985; Firtel et al. 1989; Klein et al. 1988). Levels of cyclic AMP in the environment are carefully controlled by cell-surface and released cyclic AMP phosphodiesterases and by the released inhibitor of cyclic AMP phosphodiesterase, so that they are in the range where changes are readily detectable by the cyclic AMP receptor (Gerisch et al. 1972; Podgorski et al. 1988; Yeh et al. 1978) Cyclic AMP signals guide the direction of cell movement during chemotaxis, stimulate the release of more cyclic AMP during signal relay and induce the expression of developmental genes, both during aggregation (Darmon et al. 1975; Gerisch et al. 1975) and during later development (Town and Gross, 1978). Adenosine is amongst the breakdown products of cyclic AMP, and it has been proposed that adenosine acts as an antagonistic signal to cyclic AMP, thus providing an intrinsic negative feedback loop (Schaap and Wang, 1986; Weijer and Durston, 1985).

Ammonia may seem to be an unlikely candidate for a signal substance, but there is good evidence that it plays an important role in regulating Dictyostelium development. It can reach millimolar concentrations in the immediate environment of a developing aggregate, as a result of protein and nucleic acid catabolism, and has been shown to be one of the factors which prolongs slug migration (Schindler and Sussman, 1977). The enzymatic removal of ammonia from a slug triggers its prompt transformation into a fruiting body and the maturation of prestalk cells into vacuolated stalk cells (Wang et al. 1990). It has also been proposed that ammonia acts during the initial establishment of the prestalk/prespore pattern to favour prespore cell differentiation (Gross et al. 1983). Although it has been widely confirmed that ammonia can stimulate spore formation during monolayer incubation of sporogenous amoebae (Town, 1984), the basis for this phenomenon is still unexplained, because ammonia does not appear to favour directly prespore over prestalk specific gene expression at this stage of development (Berks and Kay, 1990). The effect might therefore be indirect, by for instance affecting DIF-1 or cyclic AMP production.

The DIFs (Differentiation Inducing Factors) are a family of three closely related chlorinated alkyl phenones (Fig. 4), discovered as secreted factors able to induce isolated amoebae to differentiate into stalk cells (Morris et al. 1987; Town et al. 1976). A bioassay based on this observation has been used in all measurements of DIF levels to date, though the discovery of a high affinity binding protein (see later) should form the basis of a more convenient isotope dilution assay. DIF-1 accounts for about 95% of the bio-activity released into the medium by developing cells, and for this reason it has received most attention (Kay et al. 1983). DIF levels are low during aggregation but rise during multicellular development, as the prestalk/prespore pattern is established in the aggregate (Brookman et al. 1982; Sobolewski et al. 1983). In what follows we shall summarize work suggesting a central role for DIF-1 in cell differentiation and pattern formation and describe progress in understanding the DIF-signalling system.

Fig. 4.

DIF structures. The structures were determined from quantities of less than 100μg of purified material in each case, mainly by mass spectroscopy, and confirmed by chemical synthesis.

Fig. 4.

DIF structures. The structures were determined from quantities of less than 100μg of purified material in each case, mainly by mass spectroscopy, and confirmed by chemical synthesis.

DIF and a new morphology of the slug

For many years it was believed that the Dictyostelium slug contains just two major cell types: prespore and prestalk cells, though the existence of scattered nonprespore cells in the prespore zone had also been noted. Since unique markers for these anterior-like cells are lacking, it is not clear whether they are a distinct cell type or not (Bonner, 1957; Sternfeld and David, 1981). Distinct sub-types of prestalk cells have been recognised following work on DIF-induced gene expression. First, a number of mutants were isolated which were defective in the accumulation of DIF, but could still respond efficiently to added DIF (Kopachik et al. 1983). Second, genes whose expression is induced by DIF-1 were isolated by differential screening of a cDNA library with probes from DIF-treated and control mutant cells (Williams et al. 1987).

Two genes, pDd63 and pDd56, which encode proteins of the extracellular matrix, have been studied intensively, largely by J.G. Williams, K. Jermyn and collaborators. Promoter fusions to reporter genes show that the genes are expressed in (and define) sub-types of prestalk cell (Fig. 5) (Jermyn el al. 1989; Williams el al. 1989). Prestalk A cells express pDd63 mRNA and form the anterior cortex of the slug, whereas prestalk B cells express pDd56 mRNA and form an anterior core of the slug. Prestalk and prespore cells first appear in the mound that results from aggregation, perhaps two hours before the tip forms. Prestalk B cells first appear in a basal layer and later, as the tip is extending, in the core of the tip; prestalk A cells first appear in scattered positions in the mound and rapidly migrate to the tip. Prespore cells first appear in the central regions of the mound and maintain this position behind the prestalk cells as the mound elongates into a first finger (Krefft et al. 1983; Takeuchi et al. 1978).

Fig. 5.

Origins and patterning of prestalk cells. Prestalk A cells are first detectable at scattered positions in the mound resulting from aggregation (A) and later populate the tip of the slug (B), presumably as a result of migration within the aggregate. Prestalk B cells are first detectable in the base of the mound (C) and a second group appear in the core of the tip (D), as it elongates upwards to form a standing slug. Prestalk A and B cells were detected in strains transformed with reporter genes driven by the appropriate promoter. Modified from Williams et al. (1989), with permission.

Fig. 5.

Origins and patterning of prestalk cells. Prestalk A cells are first detectable at scattered positions in the mound resulting from aggregation (A) and later populate the tip of the slug (B), presumably as a result of migration within the aggregate. Prestalk B cells are first detectable in the base of the mound (C) and a second group appear in the core of the tip (D), as it elongates upwards to form a standing slug. Prestalk A and B cells were detected in strains transformed with reporter genes driven by the appropriate promoter. Modified from Williams et al. (1989), with permission.

The patterning process in Dictyostelium therefore appears to involve both positional differentiation (of prespore and prestalk B cells) and an element of cell sorting (of prestalk A cells). If prestalk A cells truly arise in scattered positions in the aggregate, then the likeliest mechanism may be a local interaction that promotes one cell to a particular fate, while inhibiting adjacent cells from adopting this fate. Other examples may be the differentiation of Drosophila neuroblasts from the neuro-ectoderm (Simpson, 1990), of the chick mesoderm and endoderm from the epiblast (Stern and Canning, 1990) and the generation of certain specialized cells in the nematode Caenorhabditas elegans (Sulston and White, 1980).

Control of prestalk and prespore cell differentiation

Regulation experiments show that amoebae can be directed toward either prestalk or prespore differentiation, up until at least the migrating slug stage of development, irrespective of their initial differentiation tendency. What directs the amoebae to follow a particular pathway of differentiation? This question has been investigated by studying the differentiation of cells in vitro, either in shaken suspension or in submerged monolayers. It is generally agreed that amoebae can be induced to differentiate into prespore cells by cyclic AMP. For instance, cyclic AMP stimulates the formation of prespore vesicles and expression of the prespore specific enzyme UDP-galactose polysaccharide transferase (Kay, 1979; Kay et al. 1978); more recently, it has been shown to induce the expression of a number of mRNA markers for prespore cells (Barklis and Lodish, 1983; Mehdy et al. 1983) (see Fig. 6).

Fig. 6.

Prestalk and prespore cell differentiation can be controlled by cyclic AMP and DIF-1 acting in combination. This is most clearly seen with mound stage cells taken at 8h of development, when prestalk and prespore differentiation is just starting. Expression of each marker is favoured by a different regime of effector molecules: prespore by cyclic AMP, no DIF-1; prestalk B by DIF-1, no cyclic AMP; and prestalk A by cyclic AMP with DIF-1. First finger stage cells, where prestalk and prespore differentiation is well under way, give similar results. However, in this case D19 mRNA is still quite abundant after 1 h incubation with DIF-1, as might be expected if D1F-J blocked transcription but did not cause a rapid turnover of the pre-existing mRNA and substantial pDd63 expression occurred in the absence of added DIF-1, perhaps due to the accumulation of endogenous DJF-1. After 8h (mounds) or 12 h (standing slugs) of development on agar, aggregates of strain V12M2 were partially disaggregated and transferred to shaken suspension with 1 mM cyclic AMP and 100nM DIF-1 as indicated. After 1h of incubation, mRNA was extracted for Northern analysis, using the pDd63, pDd56 and D19 mRNAs as markers for prestalk A, prestalk B and prespore cells respectively. See Berks and Kay (1990) for further details.

Fig. 6.

Prestalk and prespore cell differentiation can be controlled by cyclic AMP and DIF-1 acting in combination. This is most clearly seen with mound stage cells taken at 8h of development, when prestalk and prespore differentiation is just starting. Expression of each marker is favoured by a different regime of effector molecules: prespore by cyclic AMP, no DIF-1; prestalk B by DIF-1, no cyclic AMP; and prestalk A by cyclic AMP with DIF-1. First finger stage cells, where prestalk and prespore differentiation is well under way, give similar results. However, in this case D19 mRNA is still quite abundant after 1 h incubation with DIF-1, as might be expected if D1F-J blocked transcription but did not cause a rapid turnover of the pre-existing mRNA and substantial pDd63 expression occurred in the absence of added DIF-1, perhaps due to the accumulation of endogenous DJF-1. After 8h (mounds) or 12 h (standing slugs) of development on agar, aggregates of strain V12M2 were partially disaggregated and transferred to shaken suspension with 1 mM cyclic AMP and 100nM DIF-1 as indicated. After 1h of incubation, mRNA was extracted for Northern analysis, using the pDd63, pDd56 and D19 mRNAs as markers for prestalk A, prestalk B and prespore cells respectively. See Berks and Kay (1990) for further details.

The basic subdivision between spore and stalk cell differentiation can be brought about by DIF-1. Spore differentiation in monolayers is inhibited by DIF-1 and the cells become stalk cells instead (Kay and Jermyn, 1983); expression of all prespore markers tested can also be inhibited by DIF-1 (Early and Williams, 1988; Kopachik et al. 1985). DIF-1 induces both types of prestalk cell differentiation, but recent work suggests that cyclic AMP may bring about the division into A and B subtypes (Berks and Kay, 1990): prestalk A differentiation is stimulated by cyclic AMP, whereas prestalk B differentiation is inhibited (Fig. 6). Cells lacking both effectors appear to de-differentiate.

In summary it appears that cyclic AMP and DIF-1 act in a combinatorial manner to define four distinct patterns of gene expression: prespore=cyclic AMP, no DIF-1; prestalk A=cyclic AMP, plus DIF-1; prestalk B = no cyclic AMP, plus DIF-1; de-differentiation = no cyclic AMP, no DIF-1. We also have genetic evidence supporting this combinatorial model. Mutants have been isolated in which a presumed single lesion simultaneously blocks 2 pathways of differentiation: one class blocks prespore and prestalk A (both requiring cyclic AMP) but not prestalk B differentiation, and another blocks both types of prestalk differentiation (both requiring DIF-1) but not prespore differentiation (I. Carrin, unpublished observations).

Patterning is the supreme problem in Dictyostelium development, because it encompasses so many other problems. To what extent can the emerging knowledge about signalling within the aggregate explain the patterning and regulation phenomena described earlier? The short answer is that many fundamental aspects remain mysterious. The main difficulty is in linking experiments performed with cells in vitro, that show how cell differentiation can be controlled, to the intact organism. In particular, one would wish to understand the dynamics of the cyclic AMP and DIF signalling systems in the aggregate, so as to know the effective concentrations of these effectors experienced by cells in different places and at different times. However, the work to date does reinforce the evidence for central roles for cyclic AMP and DIF-1. Three basic types of observation have been made: (1) measurements of effector molecule concentrations in the aggregate; (2) effects of addition of effector molecules; (3) effects of depletion of effector molecules. The results summarized in Table 1 include work with adenosine and ammonia for completeness and generally refer to slugs as the most convenient multicellular stage to study. It should be remembered, though, that the important patterning events occur somewhat earlier in development. The mean concentrations of cyclic AMP (Brenner, 1978; Merkle et al. 1984), DIF-1 and ammonia (Schindler and Sussman, 1977; Wilson and Rutherford, 1978) in the aggregate all seem adequate to bring about the effects observed in vitro. In fact the concentration of DIF in the slug as a whole and in the prespore zone in particular (Brookman et al. 1987), may be 10 times higher than the concentration giving half-maximal inhibition of prespore cell differentiation in vitro (Berks and Káy, 1990). This raises the obvious problem of how prespore cell differentiation is possible at all in these circumstances. The resolution to this paradox may be the key to our understanding of the whole patterning problem, but it is not in sight yet. Exogenous cyclic AMP disrupts the aggregate (George, 1977; Nestle and Sussman, 1972; Wang and Schaap, 1985), presumably by swamping out the endogenous signals, whereas a localized depletion (caused by the insertion of a bead carrying cyclic AMP phosphodiesterase) causes prespore characters to be lost (Wang et al. 1988). Generalized depletion of cyclic AMP, resulting from over-production of the cyclic AMP phosphodiesterase, blocks development at the mound stage and mature stalk and spore cells never differentiate (Faure et al. 1988). Addition of exogenous DIF-1 causes an expansion of the prestalk zone of a slug by up to 50% (Kay et al. 1989; Kay et al. 1988; Wang and Schaap, 1989), whereas lowered DIF levels (in the rather ill-defined ‘DIF-less’ mutants) result in development blocked at the mound stage and allows differentiation of prespore but not prestalk cells (Kopachik et al. 1983). The addition of adenosine does not have any dramatic effect on slug morphology, whereas depletion using an enzyme cocktail causes prespore differentiation in the prestalk zone of a submerged slug (Schaap and Wang, 1986). Ammonia addition prolongs slug migration, whereas depletion, again with an enzyme cocktail, causes immediate fruiting (Schindler and Sussman, 1977) and in some circumstances the maturation of stalk cells in slugs without normal culmination (Inouye, 1988; Wang and Schaap, 1989).

Table 1.

Signal molecules from Dictyostelium

Signal molecules from Dictyostelium
Signal molecules from Dictyostelium

One route to understanding the roles of cyclic AMP and DIF-1 in development is to identify, and eventually manipulate the activity of, various components of the signalling systems. Here we concentrate on our recent progress with the DIF-1 system.

A receptor for DIF-1?

There are parallels between the DIFs and steroid hormones: both are small, lipophilic molecules which can rapidly affect gene expression. Extending this parallel, we searched for a cytoplasmic DIF-binding protein, analogous to a steroid hormone receptor. Using tritiated DIF-1 in conventional ligand binding assays, we discovered a DIF binding protein which has the properties expected of a DIF receptor: it has a Kd of 1-2 nM (Fig. 7), is present in nucleus and cytoplasmic fractions at a low abundance of 1000-2000 binding sites per cell, and analogues of DIF-1 compete for binding in the same potency series as they induce stalk cell differentiation in the DIF bioassay (Insall and Kay, 1990). The receptor is developmentally regulated, with the major rise in binding activity occuring just as cells become responsive to DIF-1 for prestalk specific gene expression. Efforts to purify and further characterise this binding protein are under way.

Fig. 7.

A specific binding protein for DIF-1. (A) Saturation curve. (B) Linear subtraction plot. (C) Scatchard plot with background correction of the same data. Equilibrium binding of [3H]D1F-1 to lysates of V12M2 cells taken at 12 h of development is shown, with bound and free DIF-1 separated using spun columns of Sephadex G15. Reproduced from Insall and Kay (1990) with permission.

Fig. 7.

A specific binding protein for DIF-1. (A) Saturation curve. (B) Linear subtraction plot. (C) Scatchard plot with background correction of the same data. Equilibrium binding of [3H]D1F-1 to lysates of V12M2 cells taken at 12 h of development is shown, with bound and free DIF-1 separated using spun columns of Sephadex G15. Reproduced from Insall and Kay (1990) with permission.

Metabolism and inactivation of DIF-1

If cells are to be able to respond to changes in the production of a signal molecule such as DIF-1, it is essential that there be some way of inactivating it as a signal. This is most likely to be by metabolism, which may also be important in creating gradients of DIF-1 (assuming these exist) and possibly further signal substances from DIF-1. We searched for DIF metabolism by incubating living cells with radioactive DIF-1 and then recovering the labelled compounds from cells and medium, after which they were analysed by HPLC and TLC (D. Traynor, unpublished work). DIF-1 is rapidly converted by living cells to a series of more polar metabolites which appear in the medium. Some of these metabolites are transient and disappear on prolonged incubation, suggesting that they are intermediates in a metabolic pathway. In all we have distinguished at least 12 metabolites and have been able to deduce the early steps in the metabolic pathway, from kinetic experiments and by determining the fate of purified metabolites re-fed to cells (Fig. 8). The first metabolite, DM1, is largely cell-associated, whereas the more distal ones are found mainly in the medium. DM2 and DM3 appear next and are also transient species, which rapidly disappear once all the DIF-1 is consumed. These are followed by DMs 4, 5 and 9, which are considerably more stable species. The remaining metabolites accumulate only slowly and are stable for prolonged periods in the medium; they appear to be the terminal products of DIF-1 metabolism. Purified DM1 has about 7% of the specific activity of DIF-1 in the stalk cell induction bioassay and the other metabolites even less activity: metabolism therefore inactivates DIF-1, at least as far as this assay is concerned. Metabolism is developmentally regulated; levels rise at the end of aggregation to reach a peak at the tipped aggregate stage, very similar to the regulation of the putative receptor. Even by the slug stage, we calculate that the metabolic capacity of the cells is sufficient to turn over the endogenous DIF-1 with a half-life of a few minutes. DIF-1 is therefore likely to be a very dynamic molecule and it is certainly conceivable that localized DIF-1 destruction could produce DIF-1 gradients in the aggregate. The production of so many metabolites is a puzzle: we are attracted to the idea that some of them may have a role of their own, but as yet there is no evidence as to what this might be.

Fig. 8.

Proposed pathway of DIF-1 metabolism by living Dictyostelium cells. Radioactively labelled DIF-1 is converted to at least 12 metabolites (DMs 1–12), which can be recovered from the cells and medium and separated by HPLC and TLC. The first metabolite in the pathway, DM1, is largely cell-associated whereas the other metabolites are mainly found in the medium. The pathway of metabolism was deduced by following the flow of counts from one metabolite to the next in kinetic experiments and confirmed by determining the fate of purified metabolites fed back to cells.

Fig. 8.

Proposed pathway of DIF-1 metabolism by living Dictyostelium cells. Radioactively labelled DIF-1 is converted to at least 12 metabolites (DMs 1–12), which can be recovered from the cells and medium and separated by HPLC and TLC. The first metabolite in the pathway, DM1, is largely cell-associated whereas the other metabolites are mainly found in the medium. The pathway of metabolism was deduced by following the flow of counts from one metabolite to the next in kinetic experiments and confirmed by determining the fate of purified metabolites fed back to cells.

Development in Dictyostelium is a highly regulative process, in which signalling between the cells seems to control both their differentiation into stalk or spore cells and the overall morphogenesis of the aggregate. The study of this morphogenesis promises to be interesting, as it will mark a convergence of the exciting work on single cell motility (Andre et al. 1989; De Lozanne and Spudich, 1987) with studies on their behaviour in multicellular conglomerates. Signalling between cells is mediated by cyclic AMP, DIF-1 and other molecules which include ammonia and possibly adenosine. The biochemical machinery for DIF-1 signalling appears at the end of aggregation in response to cyclic AMP signalling. DIF-1 controls the basic diversification of developing amoebae into stalk or spore cells; the recently recognized subtypes of prestalk cells may be brought into being by cyclic AMP, which represses differentiation of one type and stimulates differentiation of the other. Dictyostelium offers a unique set of experimental opportunities for studying short-range signalling by diffusible compounds: it is possible to study the components of the signalling machinery biochemically, mutate various of them using either classical or reverse genetics and finally observe the behaviour of cells in the living aggregate of both control and mutant cells. It is likely that this entire armament will be necessary to understand patterning and morphogenesis in this organism.

analysis is the technique of marking cells using transposition events. This technique has advantages and disadvantages over more conventional methods. The largest disadvantage of transposition is the lack of experimental control over the timing of transposition events. Transpositions can occur anytime during sporophyte development giving rise to a mixed population of sectors. While posing some disadvantages, this wide temporal range can also be an advantage when sectors of variable size are desirable. Moreover, irradiation levels during cell marking may also affect the growth of the plant. Secondary effects due to irradiation-induced damage can influence the behavior of cells or perhaps individual sectors. Besides uncovering a particular marker, cells may sustain multiple lesions during irradiation. The major assumption behind such studies is that the behavior of irradiated cells does not differ substantially from that of unirradiated meristematic cells, although estimates of such effects suggest this is minimal (McDaniel and Poethig, 1988).

While somatic impairment of marked cells may be minimal, irradiation techniques usually rely on loss of chromosome segments to uncover recessive markers. These losses usually prevent meiotic transmission. These potential problems are eliminated with transposon-induced sectors. Each lineage is marked by a single excision event and marked cells differ from unmarked cells by this single genetic change. More importantly, transposon marking usually takes place by a gain-of-function somatic mutation (e.g. expression of a color gene allowed by excision of transposon). This serves as a dominant genetic marker of cell lineages, as opposed to recessively marked sectors generated by gene loss during irradiation. As demonstrated below, dominantly marked sectors often allow a much finer resolution of lineages than do more commonly employed irradiation methods.

We have employed the excision of the maize transposable element Activator (Ac) to mark somatic and germinal lineages in the maize plant. The transposable element system, Activator-Dissociation (Ac-Ds), was first characterized and genetically studied by McClintock (McClintock. 1950, 1951). Ac elements are capable of catalyzing their own transposition; Dissociation elements are transposition-deficient but can transpose in the presence of an active Ac element. Several Ac and Ds elements have been cloned and analyzed at the molecular level. So far, all of the Ac elements studied are structurally identical; the Ds elements, however, are structurally quite diverse (reviewed by Fedoroff, 1989). Both Ac and Ds elements have similar inverted terminal repeats and both cause an eight base-pair (bp) duplication of host DNA upon insertion. Excision of these elements is imprecise, often leaving a modified version of the eight base-pair duplication at the site of excision.

Transposition events mark cell lineages of the pistillate flower

Perhaps the most studied Ac element at the genetic level is that inserted at the Pericarp locus (P) on chromosome IS. An unstable allele of this locus, which we designate P-VV:E, was first described by R. A. Emerson (Emerson, 1914). This unstable allele represents an insertion of Ac (previously referred to as Modulator or Mp) into the P-RR allele (Barclay and Brink, 1954). The P-RR allele conditions full red pericarp and glumes of the cob. The P-VV.E allele conditions variegated pericarp and cob phenotypes. The variegation results from somatic mutations from colorless to red pericarp caused by the excision of Ac from P-W.E, restoring P-RR function. These sectors are found in the glumes of the cob and in the pericarp. They range in size from small stripes nearly invisible to the naked eye to large patches of red pericarp that can cover multikernel sectors or even the entire ear inflorescence (Fig. 2A). The boundaries of sectors are always sharply defined, rarely is there any evidence that the pigment diffuses from red to colorless cells. This feature of P-RR expression makes it an ideal cell-autonomous marker for clonal analysis.

In variegated pericarp ears, two discrete types of pericarp stripes are discernable. One sector is dark red and usually does not enter the silk attachment region (crown), while the second type of sector usually originates in the crown at the silk attachment region and conditions a light red or pink stripe in the upper gown portion of the pericarp. When these sectors are large, covering many kernels, this distinction becomes even more evident. The large sectors result in two discrete pericarp phenotypes previously described as light crown and dark crown (Emerson, 1914; Randolf, 1926; Van Schaik and Brink, 1959). Dark crown sectors result in light red pigmentation that covers the gown (sides of the kernel) which becomes dark red at crown (Fig. 2B). Light crown sectors result in dark red pigmentation of the gown and a silk attachment region that is still variegated or colorless (Fig. 2C). Although the details of transposition were unclear at the time. Emerson (1914) was the first to point out the differential transmissibility of dark and light crown sectors. Subsequent studies, however, failed to support his assertion that these sectors represented mutations in independent layers of the pericarp (Randolf, 1926). Our evidence that these two patterns result from the independent excision of Ac in epidermal (dark crown) and subepidermal (light crown) lineages of the pericarp is given below.

The pattern of pericarp pigmentation in light crown and dark crown sectors suggests complexity in both cellular and lineage organization of pericarp tissue. We examined each type of sector in cross-section by light microscopy to determine which cells were pigmented in different regions of the kernel. Results of this analysis are shown in Fig. 3. Examination of the dark red gown of light crown kernels shows a red subepidermal layer and colorless epidermal layer (Fig. 3A). In the light red

Amagai
,
A.
(
1984
).
Induction by ethylene of macrocyst formation in the cellular slime mold Dictyostelium mucorotdes
.
J. gen. Microbiol
130
.
2961
2965
.
Andre
,
E.
,
Brink
.
M.
,
Gerisch
.
G.
,
Isenberg
,
G.
,
Noegel
.
A.
,
Schleicher
.
M.
,
Segall
.
J. E.
and
Wallraff
,
E.
(
1989
).
A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis
.
J. Cell Biol
.
108
,
985
995
Barklis
,
E.
and
Lodish
.
H F.
(
1983
).
Regulation of Dictvostehum discoideum mRNAs specific for prespore or prestalk cells
.
Cell
32
.
1139
1148
.
Berks
,
M.
and
Kay
.
R. R.
(
1990
).
Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum
.
Development
110
,
977
984
.
Bonner
,
J. T.
(
1944
).
A descriptive study of the development of the slime mold Dictyostelium discoideum
Am. J. Bot
31
,
175
182
.
Bonner
,
J. T.
(
1957
).
A theory of the control of differentiation in the cellular slime molds
.
Q. Rev Biol
.
32
,
232
246
.
Brenner
,
M.
(
1978
).
Cyclic AMP levels and turnover during development of the cellular slime mold Dictyostelium discoideum
.
Dev Biol
.
64
,
210
223
.
Brookman
,
J. J.
,
Jermyn
,
K. A.
and
Kay
,
R. R.
(
1987
).
Nature and distribution of the morphogen DIF in the Dictyostelium slug
.
Development
100
.
119
124
.
Brookman
,
J. J.
,
Town
.
C. D.
,
Jermyn
.
K. A.
and
Kay
.
R. R.
(
1982
).
Developmental regulation of a stalk cell differentiationinducing factor in Dictvostelium discoideum
.
Devi Biol
91
.
191
196
.
Darmon
,
M.
Bracket
.
P
and
Pereira Da Silva
,
L. M.
(
1975
).
Chemotactic signals induce cell differentiation in Dictyostelium discoideum
.
Proc. natn. Acad. Sci U.S.A
.
72
,
3163
3166
.
De Lozanne
.
A.
and
Spudich
.
J. A
(
1987
).
Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination
.
Science
236
,
1086
1091
.
Devreotes
,
P. N.
(
1983
).
Cyclic nucleotides and cell-cell communication in Dictyostelium discoideum
.
Adv. Cyclic Nucleotide Res
15
,
55
96
Early
,
A. E.
and
Williams
.
J. G.
(
1988
).
A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF tn vitro
.
Development
103
.
519
524
.
Europe-Finner
.
G. N.
and
Newell
.
P. C.
(
1985
).
Inositol 1.4.5-trisphosphate induces cyclic GMP formation in Dictyostelium discoideum
.
Biochem Biophys. Res Commun
.
130
,
1115
1122
.
Faure
,
M.
,
Podgorski
.
G. J.
,
Franke
,
J.
and
Kessin
.
R. H
(
1988
).
Disruption of Dictvostelium discoideum morphogenesis by overproduction of cAMP phosphodiesterase
.
Proc natn Acad. Sci. U.S.A
85
,
8076
8080
.
Firtel
,
R. A.
,
Van Haastert
.
P. J. M.
,
Kimmel
.
A. R.
and
Devreotes
.
P. N.
(
1989
).
G protein linked signal transduction pathways in development: Dictyostelium as an expenmental system
.
Cell
58
.
235
239
.
George
,
R. P.
(
1977
).
Disruption of multicellular organisation in the cellular slime molds by cyclic AMP
Cell Differ
.
5
.
293
300
.
George
,
R. P.
,
Hohl
.
H. R.
and
Raper
.
K. B
(
1972
).
Ultrastructural development of stalk producing cells in Dictyostelium discoideum. a cellular slime mould
.
J. gen. Microbiol
.
70
.
477
489
.
Gerisch
,
G.
(
1987
).
Cyclic AMP and other signals controlling cell development and differentiation in Dictvostelium A Rev
.
Biochem
56
.
853
879
.
Gerisch
,
G.
,
Fromm
.
H.
,
Huesgen
.
A.
and
Wick
.
U
(
1975
).
Control of cell-contact sites by cyclic AMP pulses in differentiating Dictyostelium cells
.
Nature
255
.
547
549
.
Gerisch
,
G.
,
Malchow
.
D.
,
Riedel
.
V.
,
Muller
.
E.
and
Every
.
M.
(
1972
).
Cyclic AMP phosphodiesterase and its inhibitor in slime mould development
Nature, New Biol
235
,
90
92
Gezelius
.
K.
(
1959
).
The ultrastructure of cells and cellulose membranes in Acrasiae
.
Expl Cell Res
.
18
,
425
453
.
Gomer
,
R. H
and
Firtel
.
R A.
(
1987
).
Cell-autonomous determination of cell-type choice in Dictyostelium development by cell-cycle phase
Science
237
.
758
762
.
Gregg
,
J. H.
and
Karp
.
G. C.
(
1978
).
Patterns of cell differentiation revealed by L-[3H]fucose incorporation in Dictyostelium
.
Expl Cell Res
112
.
31
46
.
Gross
,
J. D.
,
Bradbury
.
J.
,
Kay
.
R. R.
and
Peacey
.
M. J
(
1983
)
Intracellular pH and the control of cell-differentiation in Dictyostelium discoideum
.
Nature
303
.
244
245
.
Inouye
,
K.
(
1988
).
Induction by acid load of the maturation of prestalk cells in Dictvostelium discoideum
.
Development
104
,
669
681
Insall
,
R. H
and
Kay
.
R. R
(
1990
).
A specific DIF binding protein in Dictyostelium
.
EMBO J
.
9
,
3323
3328
.
Jermyn
,
K. A.
Duffy
.
K. T. I.
and
Williams
.
J G.
(
1989
).
A new anatomy of the prestalk zone in Dictvostelium
.
Nature
340
,
144
146
.
Kay
,
R. R.
(
1979
)
Gene expression in Dictyostelium discoideum: mutually antagonistic roles of cyclic AMP and ammonia
.
J Embryol. exp. Morph
.
52
.
171
182
.
Kay
,
R. R.
,
Berks
,
M
and
Traynor
.
D.
(
1989
).
Morphogen hunting in Dictyostelium
.
Development
107
.
Supplement
.
81
90
.
Kay
,
R R.
,
Berks
,
M.
,
Traynor
.
D.
,
Taylor
.
G. W.
,
Masento
.
M. S.
and
Morris
.
H R
(
1988
).
Signals controlling celldifferentiation and pattern-formation in Dictvostelium
.
Devi Genet
.
9
.
579
587
.
Kay
,
R. R.
,
Dhokia
.
B.
and
Jermyn
.
K. A.
(
1983
).
Purification of stalk-cell-inducing morphogens from Dictyostelium discoideum
.
Eur. J. Biochem
.
136
.
51
56
.
Kay
,
R R.
,
Garrod
,
D.
and
Tilly
.
R.
(
1978
)
Requirements for cell differentiation in Dictvostelium discoideum
.
Nature
271
.
58
60
.
Kay
,
R. R.
and
Jermyn
.
K. A.
(
1983
).
A possible morphogen controlling differentiation in Dictyostelium
.
Nature
303
.
242
244
.
Klein
,
P. S.
,
Sun
,
T. J.
,
Saxe
,
C. L.
,
Kimmel
.
A. R.
,
Johnson
.
R. L.
and
Devreotes
,
P. N.
(
1988
).
A chemoattractant receptor controls development in Dictvostelium discoideum
.
Science
241
.
1467
1472
Konijn
,
T. M.
,
Van De Meene
.
J. G C.
,
Bonner
.
J. T.
and
Barkley
,
D. S.
(
1967
).
The acrasin activity of adenosine-3’,5’-cychc phosphate
.
Proc. natn. Acad Set U.S.A
.
58
.
1152
1154
.
Kopachik
,
W.
,
Oohata
,
A.
,
Dhokia
.
B.
,
Brookman
,
J. J.
and
Kay. R
R
. (
1983
).
Dictyostelium mutants lacking DIF. a putative morphogen
.
Cell
33
.
397
403
.
Kopachik
,
W. J.
,
Dhokia
.
B
and
Kay
,
R. R.
(
1985
).
Selective induction of stalk cell specific proteins in Dictyostelium
.
Differentiation
28
,
209
216
.
Krefft
,
M.
,
Voet
.
L.
,
Mairhofer
.
H.
and
Williams
.
K.
(
1983
).
Analysis of proportion regulation in slugs of Dictyostelium discoideum using a monoclonal-antibody and a FACS IV
.
Expl Cell Res
.
147
.
235
239
.
Leach
,
C. K.
,
Ashworth
.
J. M.
and
Garrod
.
D. R.
(
1973
).
Cell sorting out during the differentiation of mixtures of metabolically distinct populations of Dictyostelium discoideum
J. Embryol exp Morph
.
29
.
647
661
.
Maeda
,
Y.
(
1984
).
The presence and location of sporopollenin in fruiting bodies of the cellular slime molds
.
J. Cell Sci
66
,
297
308
.
Martin
,
J. B.
,
Foray
.
M. F.
,
Klein
.
G.
and
Satre
.
M.
(
1987
).
Identification of inositol hexaphosphate in 31-P-NMR spectra of Dictyostelium discoideum amoebae. Relevance to intracellular pH determination
.
Biochim. biophys Acta
931
.
16
25
.
Mehdy
,
M. C.
,
Ratner
.
D.
and
Firtel
.
R. A.
(
1983
).
Induction and modulation of cell type specific gene expression in Dictyostelium
.
Cell
32
.
763
771
.
Merkle
,
R K.
,
Cooper
.
K. K.
and
Rutherford
.
C. L.
(
1984
).
Localization and levels of cyclic AMP during development of Dictyostelium discoideum
.
Cell Differ
.
14
,
257
266
.
Morris
,
H. R.
,
Taylor
,
G W.
,
Masento
.
M. S.
,
Jermyn
.
K. A.
and
Kay
,
R. R.
(
1987
)
Chemical structure of the morphogen differentiation inducing factor from Dictvostehum discoideum
.
Nature
328
,
811
814
.
Nestle
,
M.
and
Sussman
,
M
(
1972
).
The effect of cyclic AMP on morphogenesis and enzyme accumulation in Dictyostelium discoideum
.
Devi Biol
.
28
.
545
554
.
Podgorski
,
G. J.
,
Faure
.
M.
,
Franke
,
J
and
Kessin
,
R. H.
(
1988
).
The cychc-nucleotide phosphodiesterase of Dictvostehum discoideum: The structure of the gene and its regulation and role in development
.
Dev Genet
.
9
.
267
278
.
Poff
,
K. L.
and
Loomis
.
W. F
(
1973
).
Control of phototactic migration in Dictyostelium discoideum
.
Expl Cell Res
.
82
,
236
240
.
Pogge-Von Strandmann
,
R.
and
Kay
,
R. R.
(
1990
).
Positiondependent regulation of the prestalk-prespore pattern in Dictyostelium slugs
.
Dev. Genet
.
11
,
447
452
.
Raper
,
K. B.
(
1940
).
Pseudoplasmodium formation and organisation in Dictyostelium discoideum
.
J. Elisha Mitchell Sci. Soc
.
56
,
241
282
.
Schaap
,
P.
and
Wang
,
M.
(
1986
).
Interactions between adenosine and oscillatory cAMP signaling regulate size and pattern in Dictyostelium
.
Cell
45
,
137
144
.
Schindler
,
J.
and
Sussman
,
M.
(
1977
).
Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum
.
J. molec. Biol
.
116
,
161
169
.
Simpson
,
P.
(
1990
).
Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila
.
Development
109
,
509
520
.
Sobolewski
,
A.
,
Neave
,
N.
and
Weeks
,
G.
(
1983
).
The induction of stalk cell-differentiation in submerged monolayers of Dictyostelium discoideum - characterization of the temporal sequence for the molecular requirements
.
Differentiation
25
,
93
100
.
Stern
,
C. D.
and
Canning
,
D. R.
(
1990
).
Origin of cells giving rise to mesoderm and endoderm in chick embryo
.
Nature
343
,
273
275
.
Sternfeld
,
J.
and
David
,
C. N.
(
1981
).
Cell sorting during pattern-formation in Dictyostelium
.
Differentiation
20
,
10
21
.
Sternfeld
,
J.
and
David
,
C. N.
(
1982
).
Fate and regulation of anterior-hke cells in Dictyostelium slugs
.
Devl Biol
.
93
,
111
118
.
Sulston
,
J. E.
and
White
,
J. G.
(
1980
).
Regulation and cell autonomy during post embryonic development of Caenorhabditis elegans
.
Devl Biol
.
78
,
577
597
.
Takeuchi
,
I.
,
Okamoto
,
K.
,
Tasaka
,
M.
and
Takemoto
,
S.
(
1978
).
Regulation of cell differentiation in slime mold development
.
Bot. Mag. Tokyo Special Issue
47
60
.
Tasaka
,
M.
and
Takeuchi
,
I.
(
1979
).
Sorting out behavior of disaggregated cells in the absence of morphogenesis in Dictyostelium discoideum
.
J. Embryol. exp. Morph
.
49
,
89
102
.
Town
,
C. D.
(
1984
).
Differentiation of Dictyostelium discoideum in monolayer cultures and its modification by ionic conditions
.
Differentiation
27
,
29
35
.
Town
,
C.
and
Gross
,
J.
(
1978
).
The role of cyclic nucleotides and cell agglomeration in postaggregative enzyme synthesis in Dictyostelium discoideum
.
Devl Biol
.
63
,
412
420
.
Town
,
C. D.
,
Gross
,
J. D.
and
Kay
,
R. R.
(
1976
).
Cell differentiation without morphogenesis in Dictyostelium discoideum
.
Nature
262
,
717
719
.
Wang
,
M.
,
Roelfsema
,
J. H.
,
Williams
,
J. G.
and
Schaap
,
P.
(
1990
).
Cytoplasmic acidification facilitates but does not mediate DIF-induced prestalk gene expression in Dictyostelium discoideum
.
Devl Biol
.
140
,
182
188
.
Wang
,
M.
and
Schaap
,
P.
(
1985
).
Correlations between tip dominance, prestalk prespore pattern, and cAMP-relay efficiency in slugs of Dictyostelium discoideum
.
Differentiation
30
,
7
14
.
Wang
,
M.
and
Schaap
,
P.
(
1989
).
Ammonia depletion and DIF trigger stalk cell differentiation in intact Dictyostelium discoideum slugs
.
Development
105
,
569
574
.
Wang
,
M.
,
Van Drjel
,
R.
and
Schaap
,
P.
(
1988
).
Cyclic AMPphosphodiesterase induces dedifferentiation of prespore cells in Dictyostelium discoideum slugs: Evidence that cyclic AMP is the morphogenetic signal for prespore differentiation
.
Development
103
,
611
618
.
Welier
,
C. J.
and
Durston
,
A. J.
(
1985
).
Influence of cyclic AMP and hydrolysis products on cell type regulation in Dictyostelium discoideum
.
J. Embryol. exp. Morph
.
86
,
19
37
.
Williams
,
J. G.
,
Ceccarelli
,
A.
,
Mcrobbie
,
S.
,
Mahbubani
,
H.
,
Kay
,
R. R.
,
Early
,
A.
,
Berks
,
M.
and
Jermyn
,
K. A.
(
1987
).
Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen
.
Cell
49
,
185
192
.
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
,
1157
1163
.
Wilson
,
J. B.
and
Rutherford
,
C. L.
(
1978
).
ATP. trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum
.
J. cell. Physiol
.
94
,
37
46
.
Yeh
,
R.
,
Chan
,
F.
and
Coukell
,
M.
(
1978
).
Independent regulation of the extracellular cyclic AMP phosphodiesteraseinhibitor system and membrane differentiation by exogenous cyclic AMP in Dictvostelium discoideum
.
Devl Biol
.
66
,
361
374
.