Diffusible signal molecules controlling cell differentiation and ...

Development Supplement I, 1991, 131-139 Printed in Great Britain © The Company of Biologists Limited 1991

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Diffusible signal molecules controlling cell differentiation and patterning in Dictyostelium

MARY BERKS, DAVID TRAYNOR, INES CARRIN, ROBERT H. 1NSALL and ROBERT R. KAY* MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England

* Author for correspondence

Summary 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 cell 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.

Introduction

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.

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

Key words: Dictyostelium discoideum, DIF-1, cyclic AMP, cell differentiation, DIF-1 receptor, patterning.

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Signalling and regulation 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. 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

Fig. 1. Tipped mound and mature fruiting body of Diciyosielium 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.

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 u C-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. 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

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# 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.

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75min 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. appropriate levels, and what do they do to the receiving cells? The signals controlling Dictyostelium development

Extracellular cyclic AMP has already been mentioned as the chemoattractant during aggregation (Konijn etal. 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 etal. 1989; Klein etal. 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 etal. 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

240 480 0 240 480 Distance from tip of final slug (urn)

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.

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

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Fig. 4. DIF structures. The structures were determined from quantities of less than 100//g of purified material in each case, mainly by mass spectroscopy, and confirmed by chemical synthesis.

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. 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 et al. 1989; Williams et 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). The patterning process in Dictvostelium 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). 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

Diffusible signal molecules in Dictyostelium 1h shaken suspension

ih shaken E suspension E O

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63 (pst A) 56 (pst B)

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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 DJ9 mRNA is still quite abundant after 1 h incubation with DIF-1, as might be expected if DIF-1 blocked transcription but did not cause a rapid turnover of the pre-existing mRNA and substantial pDdst (culminates)

none (prolonged migration)

The active concentrations (generally half-maximal effects) in cell differentiation assays are compared with the in vivo concentration averaged over the whole slug volume. The effects on slug patterning of depletion or addition of the effectors are also included: psp. prespore cells: pst. prestalk cells; st. stalk. References Cyclic AMP: PNAS 83 8624. 1986; PNAS 83, 48L9, 1986; Devi Biol. 64 210, 1978: JBC 254 9703, 1979; Cell Diff. 14 257. 1984: Devi Biol. 95 200. 1983; PNAS 85 8076. 1988: Development 103 611. 1988. DIF-1: Development 110 1990: Development 100 191. 1987: Development Supplement 81. 1989. Adenosine: JEEM 86 19. 1985; Cell 45 137, 1986. Ammonia: JEEM 52 171. 1979: Nature 303 244. 1983: J. cell. Phys. 94 37, 1978: JMB 116 161. 1977: Development 105 569. 1989.

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. The DIF-1 signalling 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. 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-l 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-l metabolism. Purified DM1 has about 7% of the specific activity of DIF-l in the stalk cell induction bioassay and the other metabolites even less activity: metabolism therefore inactivates DIF-X, 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

Diffusible signal molecules in Dictyostelium

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•DM101-DM1 1I'.DM121 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. 50 100 DIF-1 bound (fmol)

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 VJ2M2 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.

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. Conclusion 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 el 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. References AMACAI. A. (1984). Induction by ethylene of macrocyst formation

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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-offunction 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 cellautonomous marker for clonal analysis. Transposition occurs independently in epidermal and subepidermal lineages of the plant

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

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DM4—

'DM10i'DM11i'DM12i 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. 50 100 DIF-1 bound (fmol)

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]DLF-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.

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