Young slugs of the cellular slime mould Dictyostelium discoideum drop small numbers of individual amoebae (∼ 10/mm) in the slime trail. With increased time of migration, slugs develop trailing tails and leave clumps of cells in their slime trails. Using reciprocal transplants between tips of young and old slugs and between a wild-type strain and an ‘aged” mutant it was shown that this age-dependent cell loss is due to changes in the bulk of cells comprising the slug, rather than to changes in the effectiveness of the tip (organizer region). Another property of the slug, the decision to continue migrating or form a fruiting body which is controlled by the tip, was less affected by age. This raises the possibility that cell autonomous properties of the slug are more subject to ageing than is the tip.

The cellular slime mould, Dictyostelium discoideum, is a simple eukaryote whose asexual life cycle can be divided into two separate phases, i.e. a growth phase and a differentiation phase. The organism is a soil amoebae which, during the vegetative phase, feeds on bacteria. When the food supply is depleted the amoebae aggregate to form a multicellular organism which, after a non-obligatory period of migration (slug stage), differentiates into three cell types, viz. spores, stalk cells and basal disc cells. During the migratory stage of the life cycle the slug moves towards light or heat. It is an elongated mass of cells enclosed by a layer of slime, the slime sheath, through which the mass moves leaving behind the collapsed sheath, or slime trail (Raper, 1940). Under conditions of high humidity and low ionic strength, the migratory stage lasts for several days in wild-type strains of D. discoideum. Both biochemical studies on developmentally regulated enzymes (Newell & Sussman, 1970) and studies of cell patterning of slugs (Takeuchi, Hayashi & Tasaka, 1977; Sampson, 1976) suggest that, although there are some age-related changes, development reaches a steady state in the slug and there are no major differences between young and aged slugs. By contrast, Bonner (1957) reported that the number of cells left in the slime trail increases with time of migration, suggesting that there is an age-related component to the integrity of the slug. In this report we investigate the loss of cells from the rear of the slugs of D. discoideum using young and aged slugs and a mutant strain, and we show that while the loss of small numbers of single cells occurs independently of age, the loss of clumps of cells is age-dependent. By the use of transplant experiments we show that the age-dependent characteristic is a property of the bulk cells in the slug rather than the organiser region in the tip. We also demonstrate that a slug property controlled by the tip, the ‘slug/fruit’ switch (Smith & Williams, 1980) is not very different in young and aged slugs.

Preparation of young and aged slugs of D. discoideum

Slugs of D. discoideum of strains NP84 (North & Williams, 1978) and AX3 (Loomis, 1971) were prepared as described previously and migrated towards light on non-nutrient agar plates (Smith & Williams, 1979). Aged slugs were prepared by allowing slugs to migrate for 5 days after the vegetative amoebae were put on water agar. During this time the light source was moved twice so that after 5 days the slugs were moving across a fresh area of agar. Because a slug which lies across the slime trail of another slug cannot be lifted without breaking at the point of contact with that slime trail, most of the aged slugs used were from the front of the field.

Transplant experiments

Transplant experiments were performed on slugs approximately 24 h after washed vegetative amoebae had been put on to non-nutrient agar, except when aged (5-day-old) slugs were used. The experiments involved the transfer of matched pairs of slugs to fresh non-nutrient agar plates, followed by reciprocal transplants of the intact rear portions (70–80 %) between two slugs, as previously described (Smith & Williams, 1980). The chimeras were replaced in standard slugging conditions (Smith & Williams, 1980).

Observation of slugs

The degree of contamination of slime trails with cells and the morphologies of the rears of slugs were noted, and in some cases slugs were drawn using a camera-lucida attachment on a Leitz dissecting microscope so that their morphologies could be easily compared. Numbers of cells left in slime trails were determined by marking the position of slugs at intervals and counting the number of cells in the slime trails between these points. This was possible because we have developed a technique (to be described elsewhere, Fisher & Williams [in preparation]) for attaching slime trails to transparent PVC discs and staining them with Coomassie Brilliant Blue (Sigma Chemical Co.), which stains both the slime trails and individual cells.

Morphology of slugs and contamination of trails

We have previously reported that slugs of strain NP84 leave only a few single cells in their slime trails over the first 4 to 5 days after vegetative amoebae are put under conditions which induce slug formation (Smith & Williams, 1979). Over this early period, the rear of the slug is well-defined and there are few cells in the slime trail (Fig. 1 A), but as the time of migration increases the rear becomes more trailing and clumps of cells, as well as slightly increased numbers of single cells, are left in the slime trail. The numbers of cells left during different periods of migration are given in Table 1 for five individual trails, and these results are consistent with the less detailed observations we have made on a large number of slime trails of strain NP84. It is clear that the numbers are fairly constant (∼ 10 cells/mm) over the first 70–80 mm (52 h) of migration and increase slightly over the next 24 h (∼ 15cells/mm). These results are consistent with those of Bonner (1957), who observed 10 cells/mm dropped by young slugs. After 76 h this level of cell loss is markedly increased, with large numbers of cells (> 100/mm) being left in clumps in the slime trail and the rear of the slug trailing (Fig. 1B), suggesting that the integrity of the NP84 slug is breaking down on prolonged migration.

Table 1.

Numbers of single cells and clumps of cells left in slime trails of migrating slugs of strain NP84

Numbers of single cells and clumps of cells left in slime trails of migrating slugs of strain NP84
Numbers of single cells and clumps of cells left in slime trails of migrating slugs of strain NP84
Fig. 1.

Camera-lucida drawings of slugs at various times after commencing migration. (A) NP84, 5 h; compact rear, few cells dropped in trail. (B) NP84, 76 h. (C) AX3 < 5 h and (D) AX3, 76 h; trailing rears, large numbers of cells dropped in trails. Slugs are approximately 1 mm long.

Fig. 1.

Camera-lucida drawings of slugs at various times after commencing migration. (A) NP84, 5 h; compact rear, few cells dropped in trail. (B) NP84, 76 h. (C) AX3 < 5 h and (D) AX3, 76 h; trailing rears, large numbers of cells dropped in trails. Slugs are approximately 1 mm long.

Young slugs of strains AX3 have a phenotype which is similar to that of aged NP84 slugs, but much exaggerated. The rears of AX3 slugs have a trailing appearance (Fig. 1C) and there is an increasingly high level of cell loss into the slime trail throughout the period of slug migration (Fig. 1D), which lasts only for up to 2, or occasionally 3, days before fruiting bodies are formed. As well as many single cells there are compact clumps of cells, which sometimes form fruiting bodies, in the trails of AX3 slugs. Based on these observations, we regard AX3 as showing premature and exaggerated ageing. The increased trailing with age could result from changes in cell speed (Inouye & Takeuchi, 1979) such that slower cells drop back despite end-to-end adhesion, resulting in a trail of cells behind the slug.

‘Slug/fruit’ developmental switch

Slugs of strain NP84 have a high propensity to continue migrating under conditions of high humidity, low ionic strength and unilateral light. We have observed slugs which have migrated for up to 11 days, and at this time are leaving a continuous stream of cells in the slime trail, so that eventually no fruiting body is formed. This suggests that the switch from continued migration to fruiting body construction is not age-related. However, there is an age-related component since we have noted that disturbance of NP84 slugs by subjecting them to overhead light and dry air for a few minutes is more likely to cause fruiting in aged slugs than in young slugs.

Slugs of strain AX3 do not need to be disturbed but have a high incidence of spontaneous fruiting body formation over the first two days of migration. Thus the ‘slug/fruit’ switch may be age-related, but this is convincingly demonstrated only in strain AX3 which we regard as an ‘aged’ mutant.

Use of transplant experiments to determine the location within the slug of the age-dependent characteristics

Heterotypic transplant experiments were carried out to determine whether the loss of cells in the slime trail and trailing rear described here are determined by the tip or by the cells in the bulk of the slug.

Morphology of slugs and contamination of trails

Since 1-day-old AX3 slugs have similar but exaggerated slugging characteristics to those of aged (5-day-old) NP84 slugs, we carried out tip transplants between young slugs of these two strains to determine whether the increased dropping of cells in the slime trail is a tip-controlled or a cell autonomous function. Control transplants between slugs of the same strain resulted in slugs with the characteristics of the original slugs. Reciprocal transplants between young NP84 and AX3 slugs, however, yielded slugs in which the shape of the rear and the degree of contamination of the trail were characteristic of the slug cells comprising the back portion of the chimeric slug (Fig. 2 A and 2B). Slugs composed of NP84 front/AX3 rear have trailing rears and many cells left in the slime trails (Fig. 2B). Slugs were rarely formed from transplants involving AX3 tips and NP84 rears, since the chimera almost always fruited immediately (Smith & Williams, 1980). However, in seven exceptional chimeras which migrated (Smith & Williams, 1980), the rear was characteristic of strain NP84, i.e. compact with little cell loss (Fig. 2 A). Thus the cells in the tip do not control the trailing of cells at the rear. Similar, but less extreme, results were obtained from transplants between young and aged NP84 slugs, in which the shape of the rear of the chimeric slug and the level of cell loss in the slime trail were characteristic of the slug cells comprising the back portion; i.e. young rear, few cells in trail; old rear, larger number of cells in trail.

Fig. 2.

Chimeric slugs 24 h after transplanting rear portions. (A) AX3 front/NP84 rear, showing compact rear and few cells dropped. (B) NP84 front/AX3 rear, showing a continuous stream of cells dropped in the trail. The tip of this slug is raised off the agar surface. Slugs are approximately 1 mm long.

Fig. 2.

Chimeric slugs 24 h after transplanting rear portions. (A) AX3 front/NP84 rear, showing compact rear and few cells dropped. (B) NP84 front/AX3 rear, showing a continuous stream of cells dropped in the trail. The tip of this slug is raised off the agar surface. Slugs are approximately 1 mm long.

The tip of the D. discoideum slug secretes cAMP (Rubin, 1976) and this may be involved in maintaining polarity and cohesion in the slug (Durston & Vork, 1979). While a tip is necessary for the cell mass to remain organised (Raper, 1940), our experiments show that it does not entirely control the integrity of the slug; an NP84 tip will not maintain the integrity of an NP84 front/AX3 rear chimera, nor will a young NP84 tip maintain the integrity of an aged NP84 rear. Also an aged NP84 tip which comes from a slug that has a trailing rear does not prevent the young NP84 rear portion of a chimeric slug from being well defined. This is consistent with the hypothesis that the integrity of the slug is maintained, at least in part, by cell autonomous factors such as cell surface components.

Understanding of cell loss from the rear of the D. discoideum aggregate may provide insight into the mechanism of branching in the more complex fruiting bodies of some species of cellular slime moulds. If D. discoideum strain AX3 formed stalk continuously during migration, as do most species of cellular slime moulds (e.g. D. mucoroides), the fruiting bodies formed from clumps of cells left in the slime trail would be branches along the stalk. There are now known to be species of cellular slime moulds which have irregularly spaced fruiting bodies along the stalk caused by leaving behind clumps of cells from the rear of the aggregate (e.g. D. aureostipes;Cavender, Raper & Norberg, 1979), as well as those in which loss of cells is more regular and evenly proportioned branches are formed (e.g. P. violaceum, P. pallidum;Harper, 1932; Bonner, 1967).

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