Sorting out in Pseudoplasmodia of Dictyostelium discoideum

DAVID FRANCIS AND DANTON H. ODAY Department of Biological Sciences, University of Delaware, Newark, Delaware 1971 1

ABSTRACT During its period of migration the pseudoplasmodium of D. dis- coideum changes from a homogeneous mass of cells to one in which the cells of the front part stain more darkly with neutral red than those of the rear part. It has been asked whether this color change represents differentiation in situ of two cell types or sorting out of previously differentiated cell types.

Experiments are described which (1) show that the transition of the pseudo- plasmodia from one type to another is a long continued process, and (2) show by means of labeling parts of pseudoplasmodia with vital dyes or C14 thymidine, that exchange of cells across the color boundary separating tip and rear parts is continuous during the life of a pseudoplasmodium. From the results it appears that sorting out of cell types is probably a major cause of the color change.

When combined with previous work, our results suggest that sorting out and redifferentiation are active together both in maintaining and restoring the normal proportions of cell types in a slug. It may prove useful to compare these processes with the components of regeneration in metazoan organisms.

The cellular slime mold Dictyostelium discoideum forms aggregates of many amebae which exist as creeping, slug- like masses for a period before transform- ing to fruiting bodies composed of stalk cells and spores.

A young slug is uniformly colored when vitally stained with neutral red. Older slugs show a darkly stained front portion, consisting of prestalk amebae, and a lightly stained rear part, containing pre- spore amebae. The color boundary is usu- ally very sharp, and occurs about one- quarter of the slug's length back from the tip (Bonner, '52). Two explanations of this change from uniform to bicolor slugs have been proposed. Bonner ('52, p. 82) believing that the change occurred very quickly within a period of 15 minutes, suggested that i t represents the im- mediate differentiation of two cell types in a previously homogeneous cell popula- tion. The alternative explanation is that the color change is due to sorting out of previously differentiated pale and dark cells across the color boundary. Early evidence implied that the cells of a slug are indeed able to sort themselves out; cells taken from the front part would return there if displaced to the rear (Bon-

J. EXP. ZOOL., 176: 265-272.

ner, '52, '59). Recently Takeuchi ('69) has performed very elegant experiments along this line. By marking cells with radioactive label, he showed that cells from the front or rear return to their original place even when dispersed and allowed to reaggregate to form a new

Our experiments are designed to ex- amine further the role of sorting out dur- ing the blush of slugs.

slug.

MATERIALS AND METHODS The strain of D. discoideum that we

used was V-12, obtained from K. B. Raper. It produces large slugs which are partic- ularly easy to manipulate.

Vital staining. A drop of a con- centrated suspension of Escherichia coli was mixed with a much smaller drop of 0.1% aqueous neutral red chloride. The mixture was spread as a streak on a petri dish of 2% agar in tap water, and inoculated with D. discoideum. The col- ored slugs which are formed move away from the streak towards a source of light and are well separated from each other.

1 Research supported by a grant from the National Research Council of Canada, and by the University of Delaware. The writers are indebted to Dr. Nancy Col- burn for valuable advice on DNA digestion.

265

266 DAVID FRANCIS AND DANTON H. O'DAY

Microdensitometry. We modified a Zeiss GFL microscope for use as a den- sitometer, A diaphragm was placed below the condenser, which focused a circular spot of 83 p diameter on the microscope stage. The diaphragms in the light source and condenser were stopped down so that light passing through the slug on the microscope stage was a beam of nearly parallel rays. A high pressure mercury vapor lamp (Osram HBO 200) was used as the li ht source, and the light emitted

A Zeiss light meter on top of the mi- croscope was connected to a Rustrak recorder, The stage was driven at 14 p/ sec by a 1/15 rpm synchronous motor attached to one of the mechanical stage knobs.

Slugs were placed in special flat ob- servation chambers. To make a scan, the chamber was positioned so that the slug was just beside the spot of light on the stage. The motor was turned on and a recording made of the changing light intensity as the spot swept over the length of the slug.

Tracer technique. To provide radio- actively labeled slugs: 5 pC of thymidine- 2-Cl4 were added to 2 cm3 of nutrient agar in a small petri dish, which was then sparsely inoculated with E. coli. After one or two days the bacteria were scraped off and used to feed D. dis- coideum as described above. This proce- dure gave slugs with a total count of 20-50 cpm above background. As shown below, most of the label is in the frac- tion digested by DNAse.

After an experiment the parts of a slug were placed separately in the center of planchets and counted for ten minutes in a Nuclear Chicago model 1042 plan- chet counter.

The fol- lowing method was used to determine which molecular fraction was labeled with C14 thymidine:

Ten to twenty labeled slugs were ho- mogenized in 1.0 ml of distilled water in a hand homogenizer, then sonicated. The mixture was split into three parts, the first to be digested with DNAse (30 pg/ml Worthington DNAse; 0.01 M MgClZ), the second to be digested with RNAse (30 pg/ ml Worthington RNAse), the third to serve

at 5460 % was selected by a green filter.

Cellular localization of label.

as a control. All three fractions were incubated 30 minutes at 37"c, then mixed with equal volumes of ice cold 20% trichloroacetic acid (TCA) and kept at 0°C for 30 minutes. The precipitate was collected by centrifugation and washed in three changes of cold 10% TCA, one change of cold 70% ethanol, and one change of cold 2: l 100% ethanol/ether. All supernatants were collected for count- ing. The final pellet was dissolved in 0.3 ml of hyamine hydroxide, then counted in a Beckman scintillation counter.

EXPERIMENTS AND RESULTS

A . Scanning experiment. Our first experiment was intended to test Bonner's statement that the blushing of a slug occurs very quickly, sometime during the first 3 mm of a slug's migration (or the first 3 hours of its life) (Bonner, '52; Bonner and Frascella, '52). We used slugs stained with neutral red. Individual slugs were selected and the absorption along their length was measured at intervals. At the same time, the diameter of the slug was measured at a number of points so as to allow calculation of C X , the spe- cific absorbance per unit thickness, from the equation:

In 10 - In Ix X C X =

where 10 is the intensity of the incident light, IX is the intensity of the emergent light, and x is the thickness of the object.

Figure 1 shows a set of absorbance curves for a single slug from an early stage when it was nearly uniformly col- ored through to the time when it was plainly two-toned. It is very clear that the (Y of the tip region increases with time. Furthermore, it is evident that cx increases continuously. The average specific ab- sorbance of the tips of older slugs, which are already two-toned, also increases con- tinuously. Experiments with 18 slugs of different ages gave an average increase in tip 01 of 1,0/mm/3 hours (table 1). Figure 2 shows an example, from which it appears that the increase in (Y of the tip of these older slugs is nonuniform through the tip zone.

At the same time that the tip is becom- ing gradually darker the zone just behind the tip initially becomes somewhat

SORTING OUT IN SLIME MOLDS 267

TABLE 1 Changes in specific absorbance (a) during aging of slugs. Starred numbers are significantly

diflerent f r o m zero, a t the 95% confidence level, us ing student’s T test

Tip Middle Rear

Unstained ff

slug5 (Imm) Aff

(/mm/3 hours)

5.0 3.4 3.1 (std. dev. = 0.32; n = 17) -0.02 -0.02 f0 .16

(std. dev. = 0.5, 0.3, 0.1; n = 7)

Neutral red ff 6.9 - 19.9 7.2 7.5

Aff f1.0’: +0.3 +0.1 stained slugs i n all slugs (std. dev. -, 1.1, 1.3; n = 45)

(std. dev. = 1.0, 0.8, 0.6; n = 13)

(std. dev. = 0.8, 0.2, 0.3; n = 5)

i n slugs < 12 hours old

in slugs >24 hours old A a 4-1.0” +0.03 f 0 . 3

1240 PM.

DISTANCE (rnrn.)

Fig, 1 Changes of a in a young slug.

clearer. This decrease in a just behind the tip is visible in the young slug of figure 1. In the older slug of figure 2, the a of this zone changed very little. The results with 18 slugs are summarized in table 2.

These results might be easily explained by the sorting out of red amebae from pale ones in the area where the color boundary appears.

B. Labeling experiments. For further investigation of these problems it is neces- sary to find a way to distinguish amebae which originate in the rear of the slug from those which originate in the front. Then the movements of the two types could be followed separately. Therefore

\ - ,

‘0 front a 5 1.0 7.5 back ZO DISTANCE (mm.)

Fig. 2 Changes of a in an old slug.

we tried labeling front or rear amebae, using vital dyes and radioactive com- pounds.

In one experiment, some slugs were stained with neutral red, and others were left clear. Then the red tip of an old stained slug was grafted onto the rear part of an unstained slug. The redistribu- tion of the red color in the new composite

268 DAVID FRANCIS AND DANTON H. O’DAY

TABLE 2

Differences i n a between the clear area j u s t behind t h e t ip and t h e middle of t h e slug. T h e starred n u m b e r i s significantly di f ferent f r o m zero b y t h e criterion of table 1

m i d a - j b t a

Slugs < 12 hours old Slugs >24 hours old

+0.16 (std. dev. = 0.48, n = 16) +0,61:$ (std. dev. = 0.43, n = 10)

slug was followed using the microdensito- meter. In similar fashion, a clear tip was grafted onto a rear from a red-stained slug and the resulting slug was scanned at intervals.

The results with 29 slugs are shown in figure 3, and indicate very clearly that there is transfer of stained material in both directions across the color boundary separating tip and rear. These results are caused a t least in part by movements of cells carrying the dye, as Bonner (‘57) showed by direct microscopical observa- tion of the amebae in similar grafts. Nevertheless it might be argued that some of the stain moves out of the original part by mere physical diffusion through the slug material.

In order to investigate this possibility, a second labeling experiment was per- formed in which CI4 thymidine was used as the label. We expected the label to be localized in the nuclei of cells, so that any redistribution which might occur would be due to movement of cells carry- ing labeled nuclei.

This assumption is confirmed by the results of an experiment in which the amount of label in DNAse and RNAse digestible material was measured and compared with the total label. (The de- tailed procedure is given in the materials and methods.) Table 3 summarizes the averaged results of three replicate exper- iments. The results indicate that about 80% of the label is in DNA (defined as the fraction precipitable by 10% TCA and digestible by DNAse). The remainder

0 .... ..... ..... ... .. :. :

.:. - . . . :: *

.... . . a . ..*. . ... . . . ....

t = 3 HR

(SD.0.3, N.14)

t 0.2 (SD= 0.5, N* 14)

. . . .... -0.2

Fig. 3 The results of exchanging the tips of a neutral red stained slug and an unstained one. Aa is the change in specific absorbance i n a three- hour period, expressed as lmm13 hours. Starred figures are significantly different from zero by the criterion of table 1.

is in the small molecular fraction, and is perhaps free to diffuse between cells, Nevertheless this fraction is too small for our subsequent results to be explainable by diffusion of label.

The same grafts were used as with stained slugs. For accuracy in separating tips from rears, both C14 labeled and un- labeled slugs were stained with neutral red. The tips of an unlabeled and a C‘4

labeled slug were exchanged. The com- posite slugs so formed were allowed to migrate for 21-24 hours before being

TABLE 3

Localization of label

Total Digestion Washed TCA First 2nd-5th

precipitate supernatant supernatants Total

DNAse RNAse None

1 85 79

93 10 11

6 5

10

% 100 100 100

SORTING OUT IN SLIME MOLDS 269

separated at the color boundary into a tip and a rear which were counted sep- arately. Other grafted slugs were tran- sected and counted at one-half to three hours after the graft to serve as controls. Figure 4 shows the results: Eight per cent of the total label in a slug moves across the color boundary from front to rear in 21-24 hours, and 11% (or essentially the same proportion) crosses the boundary in the opposite direction. If we interpret these movements as solely due to move- ment of amebae, then on the average 1.2% of the total amebae in the slug cross the tip-rear boundary in three hours, in each direction.

DISCUSSION

(1) The data from the C14 experiments give directly the per cent of the cells of a slug that cross the tip-rear boundary in a unit of time.

It is possible to get similar information from the scanning data on grafted red slugs. The necessary assumption is that the total absorbance in one area of a slug is the sum of the absorbance due to red cells and to clear cells. That is:

a r c .Xrc = a r .Xr + a c (xrc - X r ) >

where x r and (xrC -x,) are the “thick- nesses” of red and white cells, corre- sponding to the proportions of red and white cells in that area; and a r and a C are the specific absorbances of slug material composed of pure red cells or pure clear cells. This equation can be solved to find the proportion of red cells:

We measured ac directly in an unstained slug. We measured the specific absorb- ance of the tip of an old red slug as an estimate of a,.. This gave a r = 14.5, a c = 5.15. Then we used the a’s shown in figure 3 as the x r c and calculated xr Ixrc for different parts of grafted slugs.

When interpreted in this way, the data of figure 3 show that an average of 1.6% of the cells in a slug cross the tip-rear boundary in each direction in three hours.

The same calculations can be made on the data for entire red slugs which are

Fig. 4 The results of exchanging the tips of a slug labeled with C14 thymidine and an unlabeled slug. The counts are expressed as percent of the total in the entire original labeled slug. Since both slugs were always of nearly the same size, the un- derlined figures show directly the percent of cells of the entire composite slug which cross the tip- rear boundary from front to rear (left) or from rear to front (right). The starred figures are sig- nificantly different from zero by the criterion of table 1.

aging, given in table 1. Here the average exchange rate is 2.1% per three hours. The near agreement of these two values indicates that exchange of red dye pro- ceeds in composite slugs at a rate that can account for the observed rate of blushing of the tips of normal red slugs.

Both of these values (1.6%, 2.1%) are near the rate of exchange of cells cal- culated on the basis of the C14 labeling experiment, which was 1.2% in three hours. This agreement reinforces the interpretation that the color change can be quantitatively accounted for by the

2 70 DAVID FRANCIS AND DANTON H. O D A Y

exchange of cells of two distinct types, red ones and clear ones.

(2) Evidence from other sources indi- cates that there are in fact two discrete cell types during the slug stage-prespore cells and prestalk cells. In D. mucoroides they contain particles which stain bright- ly with fluorescent antispore sera (Take- uchi, '63), and which may be the prespore vacuoles described in prespore cells of D. discoideum by Hohl and Hamamoto ('69). The prestalk cells possess no known distinctive morphological features at the slug stage, although they are recognizable histochemically (see Gregg, '66), and are less dense (Takeuchi, '69; Miller et al., '69).

In the old slug of D. discoideum, pre- spore cells are concentrated in the rear, and this is the part that stains lightly with neutral red. Similarly, prestalk cells are concentrated in the tip which stains deep red with neutral red. However, a separate study is needed to determine whether individual amebae taken from neutral red stained slugs do fall into two groups, red ones and clear ones. Pre- liminary observations on amebae of dis- sociated slugs indicate that the situation may not be simple. The red stain is con- centrated in intracellular particles and there seems to be a spectrum of cell types from those with many large red granules to those with very few or no small red granules (fig. 5) .

We have also made some preliminary observations on how the proportions of these types change in different parts of an aging red-stained slug. We removed the tip or middle third of a red-stained slug of known age and gently squashed it, then examined the dispersed amebae so obtained. The amebae were classified according to the eight types of figure 5 . The data from 11 slugs are summarized in table 4. Very plainly, the proportion of dark red amebae (classes F, G, H) in- creases in the tip of the slug at the same time that it is decreasing in the mid-section. This coincidence may have another explanation, but it agrees very well with the expectations of the sorting- out hypothesis.

(3) Our scans show that the tip of a slug continues to darken over many hours and so contradict Bonner's 1952 conclu- sion that the color change occurs in the course of a few minutes. Bonner was arguing from the appearance of slugs in a time-lapse movie. It may be that the sudden change in contrast which can be seen in his film when the color boundary appears is due to the nonlinear properties of the photographic emulsion. Alterna- tively, it may be that the darkening which we have shown to continue for many hours does start suddenly. Possibly also important is the fact that we used strain V12 and Bonner used another strain of D. discoideum.

0 A

E

0 B

0 F

c l

0 D

GI H Fig. 5 The classes of amebae observable in a slug stained with neutral red,

SORTING OUT IN SLIME MOLDS 271

TABLE 4

Changes in proportions of amebae

Class t = O t = 24 hours t = 48 hours

Tip one-third A + B 2% 2 3 C 35 11 4 D + E 54 51 42 F + G + H 9 36 51

Mid one-third A + B 1 2 14 C 21 65 67 D + E 68 26 11 F + G + H 10 7 8

(4) The experiments with neutral red and C*4 labels indicate that there is a long-continued exchange of cells across the front-rear boundary of the slug; and the observations on the changes in pro- portions of cell types indicate that the exchange consists of a sorting out of the two cell types across this boundary. If this is the only sorting out that occurs, and if the different classes of cell types that we observed are truly prespore and prestalk cells as seems likely, then one would expect that both the front and the rear part of the slug will be composed of a mixture of prestalk and prespore cells for many hours during migration. But, this expectation is in direct conflict with recent conclusions of Gregg and Badman (170) and Bonner, Sieja and Hall (171). Gregg and Badman used the prespore vacuole visible in the electron microscope as a marker to recognize prespore cells. Similarly, Bonner et al. made use of a particular strain which forms large spores and whose cells become segregated in the prespore area of a mixed slug in order to demonstrate the homogeneity of a pre- spore or prestalk fraction. Although they used these two entirely independent char- acters to identify the cells of a particular region, both laboratories found that sort- ing out of prespore and prestalk cells is completed by the time the slug has mi- grated one centimeter, or within 5-10 hours of its formation.

In the face of this result we must sup- pose that, either (1) our strain V12 of D. discoideum sorts out very slowly and differs in this respect from the three strains used by Bonner (which were all originally derived from NC4) and the one

used by Gregg; or (2) the primary quick sorting out defined by Bonner and Gregg is followed by a slower secondary sorting out which is correlated with the appear- ance of the color boundary between front and rear. Which alternative is correct remains to be decided.

(5) Our experiments do not indicate whether sorting out is the only process involved in causing the color change, how- ever, and in particular they say nothing about the role of redifferentiation of cells during the slug stage. The early experi- ments of Raper (’40) extended by the work of Bonner, Chiquoine and Kolderie (‘55) gave prima facie evidence for redif- ferentiation of prestalk cells to prespore cells, and of prespore cells to prestalk cells. Raper found that an isolated tip of a slug formed a fruiting body with a dis- proportionately large stalk if it fruited immediately, but it formed a normally proportioned fruiting body containing many more spores if allowed to migrate for some time.

Even these results might also be ex- plained by long-continued sorting out, however. Much stronger evidence for re- differentiation is provided by the recent work of Gregg (‘65, ’68). He used fluo- rescent antibodies as Takeuchi (‘63) did and concluded that after the tip is iso- lated, it first develops antigens of the young slug stage, then later develops pre- spore and prestalk antigens. Gregg sug- gests that the prespore cells revert to an earlier developmental stage, then differ- entiate again to one or the other cell type. So far he has only demonstrated these phenomena in slugs of drastically abnor- mal proportions which are produced by

272 DAVID FRANCIS AND DANTON H. O’DAY

transection, and it is not clear to what extent redifferentiation partakes in con- trol of prestalk-prespore proportions in un- disturbed normal slugs.

We might conclude tentatively that both processes are involved in creating a slug of proper proportions. These two processes (sorting out and redifferentiation) recall the two suggested mechanisms involved in regeneration of lower invertebrates, which are morphallaxis, or the rearrange- ment of pre-existing cells, and epimor- phosis and other processes, which involve renewed differentiation of cells. The cel- lular slime molds seem to be another case in which both mechanisms are operative. It will be of the greatest interest to work out this case in detail, to see to what degree the proportion regulating system in this protozoan resembles the compar- able systems of multicellular organisms.

LITERATURE CITED Bonner, J. T. 1952 The pattern of differentia-

tion in ameboid slime molds. Amer. Nat., 86:

1957 A theory of the control of dif- ferentiation in the cellular slime molds. Quart, Rev. Biol., 32: 232-246.

1959 Evidence for the sorting out of cells in the development of the cellular slime molds. Proc. Nat. Acad. Sci., 45: 379-384.

Bonner, J. T., A. D. Chiquoine and M. Q. Kol- derie 1955 A histochemical study of differ- entiation in the cellular slime molds. J. Exp.

79-89.

Zool., 130: 133.158.

Bonner, J. T., and E. B. Frascella 1952 Mi- totic activity in relation to differentiation in the slime mold Dictyostelium discoideum. J. Exp.

1971 Further evidence for the sorting out of cells in the differentiation of the cellular slime mold Dictyostelium discoideum. J. Embryol. Exp. Morph., in press.

Gregg, J. H. 1965 Regulation in the cellular slime molds. Devel. Biol., 12: 377-393.

Organization and synthesis in the cellular slime molds. In: The Fungi. Vol. 11. G. C. Ainsworth and A. S. Sussman, eds. Academic Press, New York, pp. 235-281.

1968 Prestalk cell isolates in Dicty- ostelium. Exp. Cell. Res., 51: 633-642.

1970 Morphogenesis and ultrastructure in Dictyostelium. Devel. Biol., 22: 96-111.

Hohl, H. R., and S. T. Hamamoto 1969 Ultra- structure of spore differentiation in Dictyostel- ium: the prespore vacuole. J. Ultrastructure Res., 26: 442-453.

Miller, Z. I. , J. Quance and J. M. Ashworth 1969 Biochemical and cytological heterogene- ity of the differentiating cells of the cellular slime mold, Dictyostelium discoideum. Bio- chem. J., 114: 815-818.

Raper, K. B. 1940 Pseudoplasmodiuiii loiiiia- tion and organization in Dictyostelium discoi- deum. J. Elisha Mitchell Sci. SOC., 56: 241-282.

Takeuchi, I. 1963 Immunochemical and im- munohistochemical studies on the development of the cellular slime mold Dictyostelium mu- coroides. Devel. Biol., 8: 1-26.

1969 Establishment of polar organiza- tion during slime mold development. In: Nu- cleic Acid Metabolism, Cell Differentiation and Cancer Growth. E. V. Cowdry and S. Seno, eds. Pergamon, N.Y.

Zool., 121: 561-571. Bonner, J. T., T. W. Sieja and E. M. Hall

1966