An analysis of the spacing of aggregation centers in Polysphondylium pallidum

DEVELOPMENTAL

An Analysis

BIOLOGY

18,

149-162

(19SSj

of the Spacing Polysphondylium

of Aggregation pallidurn

ARNOLD Deportment

of Zoology,

J.

Syracuse

Accepted

in

KAHX Urlicersity,

April

Centers

36.

Syruczrse,

New

York

7968

INTRODUCTION

During the life cycle of cellular slime molds, myxamoebae collect in functionally integrated groups called pseudoplasmodia or aggregates. The focal point of an aggregate is termed a center. Bonner and Dodd (1962a) h ave shown that the average number of aggregates formed per unit area of substrate remains relatively constant despite rather wide variations in cell density. Furthermore, the fruiting bodies (sorocarps) that develop from these aggregates often are rather precisely positioned with respect to the substrate, neighboring sorocarps, and other objects (Bonner and Dodd, 196213). Bonner and Hoffman (1963) p resented evidence that the distribution of aggregates and the positioning of sorocarps may be governed by a common factor present in the gaseous phase of the environment called the spacing substance. This investigation was undertaken to determine whether a nonrandom (“spaced”) distribution of sorocarps occurs in the cellular slime mold PoZysphondylium pallidurn and to determine the factors that are instrumental in determining this distribution. To clarify the present analysis of aggregation, the following definitions of significant terms are offered: Aggregation: The convergence transition from a single-celled, of multicellularity.

of cells into a mass that marks the vegetative mode of existence to one

Aggregate ( pseudoplasmodium) : The initial, functionally integrated group of cells (myxamoebae) that results from aggregation. Center of aggregation (aggregation center; center): The morphologically distinct cell mass formed at the center of a pseudoplasmodium into which streams of amoebae converge. 149

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Center den&y:

The number

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of centers

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per unit

area of substrate.

Aggregation territory: The space (area of substrate) contributing myxamoebae to a single center of aggregation. The establishment of a second, independently operating center generally does not occur within a territory. Spacing: The spatial distribution of aggregation centers and ultimately sorocarps on the substrate. This distribution may be random or nonrandom. In the latter situation, the centers may be distributed either close together (clustered) or farther apart (spaced) than one would expect from chance. These three possible types of distribution are shown in Fig. I.

A.

B.

C.

FIG. 1. A diagram illustrating three possible types of center distribution; (A) spaced, (I?) random, and (C) clustered. Center density is the same in all three cases.

Spacing Substance: A factor responsible for the spaced distribution of aggregation centers. This definition differs from that of Bonner and Hoffman (1963) in that it does not encompass a substance(s) involved in the orientation and positioning of fruiting bodies. For this, the term orientation factor is suggested. Suppressor: A factor(s) present in the gaseous phase of the environment that reduces the number of centers of aggregation and the rate at which they form, and which is removed or has its effect mitigated by charcoal, mineral oil, and light, Certain parallels in behavior suggest that the suppressor and the orientation factor might be the same substance (Bonner and Hoffman, 1963; Kahn, 1964).

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METHODS

Stock cultures of Polysphondylium pallidurn, strain WS-320, were maintained in association with Escherichia coli B/r on either dilute hay-infusion agar or on agar containing 0.1% lactose and 0.1%peptone (Kaper, 1951). The myxamoebae used in experiments were grown on L;. coli iu a nutrient solution containing 0.5% peptone, 0.1% dextrose. 0.05%yeast extract in a phosphate buffer of pH 6.2. Four or 5 X IO” spores and a loopful of bacteria were inoculated into tubes containing $5ml of the nutrient solution, and the tubes were incubated in a rotar! shaker at 2Tj’C. Cells were harvested in either the logarithmic or stationary phase of growth, washed essentially free of bacteria by differential centrifugation in several changes ot chilled distilled water. and rcsuspcndcd in Bonner’s salt solution (Banner, 1947). The cells were then dispensed in alicluots (generally 0.01 ml) on a buffered nonnutrient agar substrate ( 17.5 gm Bacto-agar; Na,HPO,*7H,O, 0.622 gm; KH.,PO,, 1.5 gm; distilled water, 1.000 ml; final pH about 6.2). If thr surface was sufficientlv dry, the excess liquid entered the agar \tithin a few minutes lea&g a circle of uniformlv distributed myxamoebac (Sussman and Noel, 1952). The number of cells per aliquot was varied depending upon the demands of the particular experiment, but generally fell within the range of 1 x lo” to 7 X lo” per milliliter. Incubation was at 2O’C either under constant, fluorescent illumination or in darkness. Mineral oil, when employed, was prechilled to 20^ and poured over the agar surface after the absorption of the excess salt solution from the alicluots. /Then charcoal was used. it was placed in a shallow, aluminum cup opposite the agar surfacca of inverted culture dishes leaving a separation of se\reral millimeters bctwcen the cells and the charcoal. Counts of the number of aggregation centers were made, in all cases, after 22-26 hours of incubation. In those instances where the rate of center formation was determined, counts were also made at various times during incubation. The distance between sorocarps was measurcyl from the base of the stalk at 20 X magnification with an ocular micrometer mounted in a stereomicroscope. The spatial relationship between sorocarps was determined by the method of Clark and Evans (1954). This method consists of calculating the mean nearest-neighbor distance expected if the distribution of sorocarps is random (?K; FE r= 2~-‘,‘~; p _- number of sorocarps/

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mm)2 and comparing this value, r E, with the mean nearest-neighbor distance obtained by actual measurement (T*) . If the distribution of sorocarps is random, then the ratio, R, of observed distance (?*) to expected (oh) is unity. If the fruiting bodies are closer together than expected, R < 1 and the distribution is termed “clustered”; if the centers are further apart, R > 1 and the distribution is termed “spaced.” The predicted R value for a distribution showing optimum spacing, with each unit equidistant from each of its neighbors, is 2.1491. In practice, values were found to range from 0.7 to 1.5. RESULTS

A total of 37 aggregating populations were examined with respect to fruiting body density and distribution. Of these, 16 (37%) exhibited nonrandom distributions as determined by the method of Clark and Evans (1954). Two of those classified as nonrandom were “clustered” and the remainder (14) were “spaced.” Of those that were spaced, nine (64%) developed under conditions that are expected to limit or preclude the action of a spacing substance (suppressor) present in the gaseous phase of the environment, i.e., in the presence of charcoal, mineral oil, and light (Bonner and Hoffman, 1963; Kahn, 1964). The fact that spaced distributions occur under these limiting conditions lessens the likelihood that the determination of centersorocarp distribution results from the action of a gaseous spacing substance. In Fig. 2, mean nearest-neighbor distance is plotted as a function of sorocarp density. The relationship expected if the distribution is random is shown by the curved line. Filled circles falling to the right of the line indicate statistically significant (P < 5%) departures toward a spaced distribution (R > 1); filled circles to the left, a clustered distribution (R < 1). It is evident from Fig. 2 that random and nonrandom distributions coexist over a wide range of densities. Thus, there is no strict correlation between density and distribution. However, note that those cell populations showing spaced distributions are located almost entirely within the lower range of sorocarp densities, while those few that are clustered are in the upper range. This dichotomy in nonrandom distribution suggests that at least the “type” of distribution, clustered or spaced, is associated with fruiting body density. This as-

ANALYSIS

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AC,GRl?C,ATIOK

I

I

4

0.5 1.0 15 Mean Nearest Neighbor Distance (mm)

I

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FIG. 2. The relation between mean nearest-neighbor distance (in millimeters) and the density of sorocarps (number of sorocarps/mm’). The curved line represents mean nearest-neighbor values expected if the distribution of centers is Filled symbols indicate significant derandom. Circles signify observed values. partures from random at the 5% level. In most cases, each point represents the mean of 50 nearest-neighbor determinations.

sociation, in turn, suggests that in order to understand the mechanism( s) involved in determining distribution we must first understand the factors that determine center (aggregate) density. Center (aggregate) density appears to be directly related to the rate of center formation. The faster the rate of center formation (number of centers/mm2/hour) the higher the final density of soro-

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0

1.41.2-

0.2-

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I I I i 2 3 4 Final Density (No. Sorocarps

I 526 /mm )

1

FIG. 3. The relationship between sorocarp density and the rate of center formation (number of centers/mm2/hour), The line represents the best fit of the points as determined by the least squares method. There appears to be a linear relationship between these two factors.

carps (number of sorocarps/mm2) (Fig. 3). Thus, a slow rate of center formation might be said to favor a spaced distribution; a fast rate, randomization or clustering. Bonner and Hoffman (1963) and Kahn (1964) have shown that the addition of charcoal and mineral oil to the culture environment, or incubation in the light results in an increase in the density of aggregation centers and sorocarps. If the rate-density correlation is correct, then the addition of these factors to the environment should result in an increase in the rate of center formation. The results of certain experiments, like that shown in Fig. 4, indicate that an increase in rate does occur in the presence of charcoal, mineral oil, or light. K. B. Raper had as early as 1940 indicated a relationship between light and the onset of center formation and center density. The time (or stage) at which cells are harvested from liquid culture also plays a role in determining ultimate aggregate-sorocarp

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Hours in incubation 1;~;. 4. The influence of various environmental factors on the rate of center formation. Each point represents the mean of eight determinations. 2’he symbols 0, 0, C, n indicate, respectively, the following enviromnental conditions: I,ight + mineral oil, light + c 11‘d rcoal, light, and dark. While the conditions of light and dark were maintained throughout the experiment. charcoal and mineral oil w(w not nddrd until after 1 hour of incubation.

density. Stationary phase cells begin aggregation almost immediately after being dispensed on the agar surface and exhibit a fast rate of center formation (Fig. 5). Cells harvested in the logarithmic phase of growth show an initial lag of one to several hours before the onset of center formation, followed, usually, by a slow rate of center formation (Fig, S). The exceptions are those log-phase amoebae incubated in the presence of light, mineral oil, or charcoal. Here, log phase amoebae may aggregate with stationary phase kinetics (Fig. 5). These enhanced kinetics are explicabIe if we assume that suppressor accumulation is responsible for the diminished rate of center formation and that this suppressor is removed or has its effects mitigated

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0

0

P24 Hours in incubation FIG. 5.

The relation of time of harvest from liquid culture to rate of center formation. Note that stationary phase cells (filled symbols) begin aggregation immediately after the deposition of the drops, while cells harvested in the logarithmic phase (open symbols) show a delay in the onset of center formation. In one series [O], log phase cells were covered after I hour of incubation with mineral oil; in the control series [O], the cells were left untreated. Note that although mineral oil did not hasten the onset of aggregation in the log phase cells, it did enhance the rate of center formation and ultimate density of centers. Each point (used in determining the slope of the lines) represents the mean of 5-10 counts; 24hour values, in all three cases, represent the mean of 10 counts.

by charcoal, mineral oil, or light (Kahn and Raper, 1963; Kahn, 1964). Another common observation was that myxamoebae at high density form a greater number of centers than those at lower density (Fig. 6). Note that the increase in the number of centers is accompanied by, if not the result of, an increase in the rate of center formation. In experiments dealing with the control of cell density, the practice is to vary the number of cells within a constant volume to be delivered on the substrate. Since, in this procedure, both density and cell number are varied simultaneously it is impossible to distinguish between the possible effects of these two factors. Some preliminary experiments have been done in which the number of cells was held constant while density was varied. The results corroborate those of

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125 I

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r

/ /

Y? 0,

0

g 75

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g 6 50 2 .~

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25 0

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X

c

0 * 2 3 4 Hours in incubation

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FIG. 6. The relation of cell density to rate of center formation. In this experiment, drops of the same volume (0.61 ml) but differing in cell concentration were dispensed onto the substrate. Counts of the number of centers were made at the hours indicated on the abscissa. Each point represents the mean of 10 counts; different symbols represent different cell concentrations. The symbols 0, 0, 0, X indicate, respectively, that the drops contained the following number of cells per 0.01 ml: 5 X lo’, 2.5 X lo”, 1.25 X 106, and 6.25 X 106.

and Raper (1961) and indicate that it is density, not number, that is important in the initiation of cell aggregation.

Konijn

DISCUSSION

A correlation has been drawn between the spacing of sorocarps and the density of aggregation centers. A low density of aggregation centers seemsto be associated with spaced distributions, while higher densities are related to clustering. Sorocarp density appears to be determined, at least in part, by the rate of center formation. The faster the rate, the higher the density of aggregation centers and sorocarps. A number of factors appear to influence the rate of center formation, among them whether the cells are in the logarithmic or stationary growth phase at the time that they are transferred to nonnutrient agar, the presence of absorbing materials, or light, and cell density. The combination of stationary phase cells, the presence of charcoal or

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mineral oil, and high cell density are expected to promote a fast rate of center formation, a high density of aggregation centers, and a random, if not clustered, distribution of centers. Log phase cells incubated in the dark and at low cell density will almost certainly favor the opposite; a slow rate of center formation, low center density, and a spaced distribution. A number of hypotheses may be constructed to explain the observed relationships between density, distribution, and the rate of center formation. Only three will be considered here. In the first hypothesis, sorocarp distribution is determined by the diffusion of a spacing substance from existing aggregates that prohibits other aggregates (centers) from forming in the immediate area. The second hypothesis states that part of the action of acrasin is to inhibit the formation of additional centers in the area where aggregation is already proceeding. The third hypothesis states that the distribution of centers and sorocarps is a function of cell depletion, i.e., as cells are drawn into the first-formed aggregates, they are removed from the population as potential founders and participants in other aggregations. All three hypotheses relate the rate of center formation to centersorocarp distribution in the following manner: The area constituting a territory is determined by the distribution of a spacing substance, acrasin or the removal of cells. Under conditions that promote a fast rate of center formation, first-formed centers can circumscribe only a small territory before other centers make their appearance. Since the existing territory is small, these other centers may occur at almost any distance from the first. This situation virtually assures a random if not clustered distribution. Under conditions that promote a slow rate of center formation, the delayed appearance of secondary centers allows for the creation of substantially larger territories before the appearance of a competitor. The center (aggregate)-free area, thus created, forms the basis for the spaced distribution of aggregation centers and resulting sorocarps. The data obtained in this study do not permit us to distinguish between these several hypotheses. The “cell-depletion” and “acrasin” models are favored since they satisfactorily account for the observations and do not require the postulation of an additional, unknown (spacing) substance. The acrasin hypothesis is further supported by the experiments of Francis (1965) which showed that in Polysphondy-

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lium pallidurn the action of founder cells (amoebae that initiate aggregation) may be masked or inhibited by participation in aggregation. No discussion of center density and distribution would be complete without a consideration of the role of cell pool size, the number of cells available for aggregation. Consider the following possibilities: If a sufficiently large number of cells are available after the initial wave of aggregation, secondary centers can form in the gaps between the preexisting ones. This means that a center can form at almost any distance from its neighbor, an event that virtually assures randomization. A large pool of cells also allows the formation of larger aggregates, a situation which favors the splitting of pseudoplasmodia ancl the formation of more than one center at a single locus (cf. Hohl and Raper, 1964). The proximity of resultant secondary centers could bc responsible for the clustering seen in some populations at high center density. On the other hand, low pool size would mean that once an aggregate had freed the immediate area of cells, a “gap” would exist in which other centers could not form. Such a circumstance would obviously favor the spaced distribution of centers. There are many instances of spacing or “pattern formation” in developing biological systems (Waddington, 1962; Sondhi, 1963). For example, in the development of Triturzis (Turicha) pigment patterns. it is thought that the presence of one set of pigment cells in a given territory inhibits the invasion by another (Twitty and Niu, 19S4). Similarly, the spacing of sensory plaques in the epidermis of the insect Rhodnius, is explained by the inhibition, through the depletion of an essential “inductor,” of one such structure bv another (Wigglesworth, 1959). To draw a simple parallel, if the initiation of slime mold aggregation requires a special inductor, then those cells which become “initiators” or “founders” may deplete the supply within the immediate area so that other centers cannot form. The result of this is a spaced distribution. Other cases of spacing are more complrx. The spatial distribution of bristles in Drosophila not only shows a positioning specificity between one bristle and another, but also with respect to particular sites on the epidermis. Moreover, this distribution is under strict genetic control and appears to require the postulation of a “prepattern” to explain certain features of its development (Stern, 1956). Still, there are rather strong similarities between bristle formation and slime mold

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aggregation. The components of the bristle (bristle cell, socket cell, and sensory cell) all stem from the division of a single, previously unspecialized cell within the spidermis. Likewise, the “founder” cell which appears at the focal point of aggregation in some species of slime mold (Shaffer, 1961; Francis, reported in Bonner and Hoffman, 1963) also arises from a previously unspecialized “cell among cells.” Yet in neither case is the development of these particular cells at random. Why is it that a certain cell becomes active while others surrounding it do not? Does the presence of this cell prohibit others within the immediate area from undergoing a similar fate, and, if so, how? The continued study of slime mold aggregation may supply answers to these questions, and in doing so provide clues to the more complex spacing phenomena in higher organisms. SUMMARY

The primary purpose of this investigation was to determine whether or not a nonrandom distribution of sorocarps occurs in Polysphondylium pallidurn, and to determine the factors that are instrumental in determining distribution. While a nonrandom distribution of centers does occur, the determining factor is probably not a substance present in the gaseous phase of the environment. There is a correlation between the density of sorocarps on the substrate and their spatial distribution. The clustered condition (sorocarps closer together than expected on the basis of chance) is associated with a high density of fruiting bodies, while a spaced distribution (sorocarps further apart than expected) is often found at low densities. Random distribution apparently can occur at almost any density. Center density depends upon the rate of center formation. This rate is markedly enhanced if the cells are in the stationary phase at the time of harvest and deposition, and are exposed to charcoal, mineral oil, or light. The latter factors are thought to remove or act upon a center suppressing substance in the environment. Cells taken from the logarithmic phase, incubated in the dark, and in the absence of charcoal and mineral oil show a reduced rate of center formation and a low center density. Three hypotheses are presented to account for the apparent relationship between the rate of center formation and center density, and the spatial distribution of centers: (1) A spacing substance surrounding the center may prohibit other centers from forming in the immediate

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area; (2) spacing may result from the removal of cells through aggregation in the area immediately surrounding the center; (3) spatial distribution may be determined by the action of acrasin. In all cases, the longer the interval between the appearance of one center and another, the greater the area controlled by the first center and the more spaced the distribution. The “acrasin” and “cell-depletion” hypotheses are favored for their equal acceptability as an explanation and because the postulation of an additional, unknown factor is not required. The dependence of sorocarp density and distribution upon cell pool size is considered. It is suggested that high cell number favors both randomization, by leaving enough cells after the first burst of aggregation to form secondary centers, and clustering, because aggregates that contain a large number of cells often split into closely associated secondary centers. Low cell number results in areas virtually free of myxamoebae surrounding newly formed centers, a condition which strongly favors a spaced distribution. The possible relationship between spacing in cellular slime aggregation and spacing in other developing, biological systems is considered. This investigation was conductecl at the University of Wisconsin. I wish to thank Professor Kenneth B. Raper for providing the research facilities used during the course of this investigation and for his encouragement and help. This study was supported by a Training Grant, 5-Tl-GM-686-03, issued to the Department of Bacteriology, University of Wisconsin. by thca National Institutes of Health. REFERENCES BOSNER, J. T. ( 1947). Evidence for the formation of cell taxis in the development of the slime mold Dictyostelium

aggregates by chemodiscoitl~rrn~. J. Exptl.

Zool. 106, l-26. BONNER, J. T., and DODD, M. A. (1962a). Aggregation territories in the cellular slime molds. Biol. BUZZ. 122, 13-24. BONNER, J. T., and DODD, ?\I. A. ( 196213). Evidence for gas-induced orientation in the cellular slime molds. Develop. Biol. 5, 344-361. BONNER, J. T., and HOFFMAN, hl. E. (1963). E v1.d ence for a substrate responsible for the spacing pattern of aggregation and fruiting in the cellular slime molds. J. Embryol. Exptl. Morphol. 11, 571-589. CLARK, P. J., and EVASS, F. C. ( 19.54). Distance to nearest neighbor as a measure of spatial relationships in populations. Ecology 35, 445453. FRANCIS, D. ( 1965). Acrasin and the development of Polysphot~dy~ilon @idtrn~. Develop. Biol. 12, 329-346. HOHL, H. R., and RAPER, K. B. ( 1964). Control of sorocarp size in the ctllular
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KAHN, A. J. ( 1964). The influence of light on cell aggregation in Polysphondylium pauidum. Biol. Bull. 127, 85-96. KAHN, A. J., and RAPEX, K. B. ( 1963). The effect of light on cell aggregation in Polysphondylium pallidurn. J. CeU Biol. 19, 37A. KONIJN, T. M., and RIPER, K. B. (1961). Cell aggregation in Dictyostelium discoideum. Develop. Biol. 3, 725-756. RAPER, K. B. ( 1940). Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Sci. Sot. 56, 241-282. RAPER, K. B. ( 1951). Isolation, cultivation, and conservation of simple slime molds. Quart. Rev. Biol. 26, 169-190. SHAFFER, B. M. ( 1961). The cells founding aggregation centers in the slime mold Polysphondylium uioluceum. J. Exptl. Biol. 38,, 833-849. SONDHI, K. C. ( 1963). The biological foundations of animal patterns. Quart. Reo. Biol. 38, 289-327. STERN, C. ( 1956). Genetic mechanisms in the localized initiation of differentiation Cold. Spring Harbor Symp. Quant. Biol. 21, 375-382. SUSSMAN, M., and NOEL, E. (1952). An analysis of the aggregation stage in the development of the slime molds, Dictyosteliaceae. I. The population distribution of the capacity to initiate centers. Biol. BuU. 103, 259-268. TWITTY, V. C., and NIIJ, M. C. (1954). The motivation of cell migration, studied by isolation of embryonic pigment cells singly and in small groups in vitro. J. Exptl. 2001. 125, 541-574. WADDINGTON, C. H. ( 1962). “New Patterns in Genetics and Development.” Columbia Univ. Press, New York. WIGGLESWORTH, V. B. (1959). “The Control of Growth and Form: A Study of the Epidermal Cell in an Insect.” Cornell Univ. Press, New York.