Pattern Formation in Dictyostelium discoideum: Temporal and Spatial Distribution of Prespore Vacuoles Ursula Muller and H. R. Hohl Cytological Laboratory, Institute of General Botany, University of Zurich, Birchstr. 95, 8050 Zurich, Switzerland

Cell differentiation, cell determination and pattern formation in the pseudoplasmodium of Dictyosfelium discoideum was investigated using the prespore specific vacuole (PV) a s a morphological marker. Concomitantly, measurements of cell movement within the pseudo- plasmodium were made by tracing radioactively labelled cells. The main results indicate that 1) prespore cells appear first during late aggregation and occur randomly throughout the pseudoplasmodium with the exception of the very tip which stays free of prespore cells throughout development; 2) after late aggregation the number of prespore cells increases over a period of several hours; 3) each prespore cell takes on a progressively more prespore- like character a s judged by the increase in number of PVs it contains; 4) establishment of the distribution pattern of prespore and prestalk cells takes place within the first 2 h , mainly by a sorting out mechanism; 5) presumptive spore areas are likely to contain a small proportion of cells lacking PVs (prestalk-cells?) while presumptive stalk cell areas are homogeneous throughout; 6) maintenance of the pattern during migration may be facilitated by a circulation at low level of prestalk cells between prestalk and prespore areas; and 7) during the development of t h i s organism the events of cell determination, cell differentiation and pattern formation overlap substantially in time.

Introduction

In the migrating pseudoplasmodium of the cellular slime mold Dicfyostelium discoideum the presumptive stalk cells occupy the anterior region and the presumptive spore cells the posterior region of the cell mass. The proportion between the number of the two cell types remains remark- ably constant up to culmination and is restored even after parts of the cell mass are renioved.1

Two main mechanisms have been proposed to account for the establishment and maintenance of this characteristic pattern. According to the first, which is supported by the majority of the evidence, the two cell types sort out by moving to the anterior or posterior region of the pseudo- plasmodium. The second mechanism favours the idea that the fate of the cells is determined by their respective position within the cell mass.

One of the difficulties encountered in dealing with this question is that it has proved very difficult to distinguish accurately individual prespore cells from individual cells of the pre- stalk area. The various methods employed such

as dyes,2 radioactive markers,3 or immuno- fluorescence4 suffer from two main disadvantages : that the intensity of the reaction is normally insufficient for identification of individual cells, and that it is questionable whether the property of the cell which is used as a marker is relevant to the process of differentiation.

The two difficulties may be overcome to a large extent by using the prespore vacuole (PV) as a structural marker. These vacuoles have been shown to be an exclusive property of the prespore cells536 and to play a significant and indispensable role during sporogenesis.5 At present this marker can be identified only with the electron micro- scope, a drawback that renders any quantitative investigation on its distribution within the cell mass an exceedingly time consuming task.

Gregg and Badman’ have used the tight association of an electron-transparent vacuole with a cisterna of the rough endoplasmic reti- culum, the combination being termed spore vesicle (SV), as an additional criterion for

Diferentiation 1, 1973 267

Ursula Miiller and H. R. Hohl

identifying prespore cells. We have not included this character in our study because it does not occur frequently enough during the stages of most interest to us, and because the two con- stituents are found unassociated in both cell types. Thus, different degrees of association may be found and in addition cisternae of the endoplasmic reticulum may also be observed ensheathing other cell structures or wrapped around not one but several electron-transparent vacuoles. From our experience SVs are mainly found in prespore cells showing signs of cyto- plasmic dehydration characteristic of the terminal stages of spore differentiation.

The problem of pattern formation in Dictyo- steli14m has three major aspects, namely the establishment of the pattern, its maintenance and its restoration after an extensive disturbance. Though one might tacitly imply that all three aspects are governed by the same control mechanism there is u priori no reason for making this assumption.

In this report we describe the temporal and spatial distribution of the PVs in the pseudo- plasmodium during the different developmental stages. In addition we will try to correlate the distribution patterns observed with the rate of cell movement derived from measurements of the displacement of radioactively labelled myxamoebae within migrating pseudoplasmodia. The main emphasis is placed on the events that lead to the initial establishment and maintenance of the pattern, while the problem of restoration and regeneration after a major disturbance, such as cell mixing or dissection of the cell mass,3,4,8 will not be considered to any appreciable extent.

Methods

Dictyostclium discoidcum V-12 was grown at 22°C on Bonner’s standard medium (1.0% peptone, 1.0 % glucose, 2.0 % Difco agar in 0.16 M Soren- sen’s phosphate buffer at pH 7.0) with Eschcrichia coli Bjr as food organism. After 42-44 h the myxamoebae were harvested with cold buffer solution and washed by repeated centrifugation at 1700 rpm on a small table centrifuge. The concentration of myxamoebae was adjusted to 2x108 cells/ml and 0.5 ml of this suspension was plated on a Millipore filter placed in a 55 mm plastic petri dish. The filter rested on an absorbing pad saturated with 1.4 ml of Sorensen’s phosphate buffer, pH 6.0, containing 0.67 mg/ml of strepto- mycine sulphate.9 When incubated at 22°C the cultures pass through aggregation 9 h (tg) and

through culmination approximately 18 h (tl8) after the cells were deposited on the filter.

For electron microscopy the cells were fixed in a mixture of 6.25% glutaraldehyde, 2.0% osmium tetroxide and 1.0% phosphotungstate.10 The material was then dehydrated in ethanol and propylene oxide and embedded in Durcupan (Fluka). The thin sections were contrasted with Reynold’s lead citrate for 5-10 min and with uranyl acetate for 30 min and viewed in a Hitachi HU-11E electron microscope at 50 kV.

In order to prepare radioactively labelled myxdmoebae for the transplantations, 0.3 ml of a solution containing 1 mCi/ml of 3H-thymidine together with the inoculum of bacteria and slime mold spores was spread on Bonner’s standard medium and grown according to the method outlined above. The myxamcebae were harvested and washed several times with buffer before being deposited on plain 2 0 % agar for aggre- gation and pseudoplasniodium formation. The labelled pseudoplasmodia were then used for the experiments.

Results

First appearance and numerical change of PVs in relation to the developmental cycle Preliminary experiments indicated a total absence of PVs during the vegetative stage and interphase (to+). We have therefore analyzed more closely the interval between aggregation and incipient culmination (ts-tl5). Cells %ere harvested from Millipore cultures at 1 h intervals and analyzed for the presence of PVs. Five separate experiments were performed with a total of 12,568 cell profiles counted. The percentages of cell profiles with one or more PVs are plotted against time in Fig. 1.

Although there is a considerable degree of variation, it is found that the first PVs are formed 10 h after the cells were plated out, i.e. at a time when most of them have entered the aggregating mass. Subsequently the number of profiles with PVs increases steadily over the entire period of observation.

To indicate the general trend of the curve up to culmination we have added two more points in Fig. 1 . The first is added at mid-culmination when all the prespore cells contain large numbers of PVs (about 40-80) and up to 99% of all the sections through prespore cells contain PVs. Including the sections through the prestalk area this would amount to an overall of 85% of all sections with PVs, because approximately 15 7; of the cells are prestalk cells.11 The second point

Diflerentiution 1, 1973 268

Pattern Formation in Dictyosteliunz

I I I

9 11 13 15 23 t

Fig. 1 Percentage of cell profiles containing PVs (x) from pseudoplasmodia taken at various times (t, in hours) after the cells were placed on Millipore filters. The approximate developmental stages are indicated at the bottom.

is added at the final stage of culmination when all PVs have disappeared.5.6 Including these two points the overall picture is that the number of PVs increases over the entire period from late aggregation to mid-culmination and drops sharply to zero at the time the spore coat is formed.

Spatial distribution of PVs within the pseudoplasmodium Distribution of PVs along the main axis of the pseudoplasmodium - In this section we describe the distribution pattern of PVs within the pseudo- plasmodium at late aggregation, beginning of migration and late migration. Two main questions were asked: Do the first PVs appear in a discernible relationship to the shape of the pseudoplasmodium or is their occurrence apparently random? At what stage is the final differentiation between prespore and prestalk cells established?

Eight pseudoplasmodia were analyzed to establish the distribution of PVs at late aggre- gation (tlo). If the total length of the pseudo- plasmodium is taken as loo%, the average distance from the tip where no PVs could be found was 9 % (with values ranging from

Diferentiation 1, 1973

5-12.5%). In the remaining 91 % of the pseudo- plasmodium the PVs were found in small numbers and apparently randomly distributed (Fig. 2 4 .

The distribution pattern of PVs in early migrating pseudoplasmodia was studied in 6 slugs, aged tll-tlz. With the values ranging from 5-14X it was found that the average tip region totally free of PVs was 9.3 x, i.e. practically the same as in late aggregation. The distribution in the remaining portion of the pseudoplasmodium was again random, but larger numbers of PVs were encountered (compare Fig. 1). It also seemed that PVs were absent from the very end of the slug although this was difficult to ascertain because of frequent damage to the cells in that area (Fig. 2b).

Five slugs were taken to study the situation at late migration (tls). The average region at the tip free of PVs was 26% of the total length of the pseudoplasmodium with values ranging from 25530%. Again, the distribution of PVs within the remainder of the slug appeared to be random and the impression that there are no PVs at the very end of the slug could be confirmed (Fig. 2c).

In summary, the results indicate that at the very early stages of PV formation these organelles are

269

Ursula Miiller and H. R. Hohl

% I

30

20

a

I

-

-

Fig. 2 Diagrammatic representation of distri- bution of prespore cells within the pseudoplas- modium at late aggregation (a), early migration (b), and late migration (c). Black circles indicate cells with PVs, white circles cells without PVs and crossed circles the tip cells devoid of PVs through- out development.

randomly distributed over the pseudoplas- modium except in the very tip where PVs are lack- ing over the entire period of observation. At the culmination, PVs are observed in all cells of the prespore region. Hence, while cells lacking PVs may be capable of moving into the prestalk region during migration of the pseudoplasmodium, those still present in the prespore region during culmination apparently form PVs. The tip zone devoid of PVs is enlarged from approximately 10% to approximately 25% within the first two hours of migration and corresponds to the prestalk area described from light microscope investiga- tions.

Quantitative distribution of P Vs in cells of pseudo- plasmodia before and after migration - In this section we will try to establish first whether the overall increase in the percentage of cell profiles containing PVs is due to an increase in PVs per cell, an increase in the number of cells with PVs, or both, and secondly whether after a period of migration there is still a significant number of cells within the posterior region of the slug that are devoid of PVs, i.e. morphologically indistin- guishable from prestalk cells. The major obstacle for answering these questions is that with the electron microscope we can only determine the number of PVs per section and not per cell, unless we perform the practically impossible task of obtaining hundreds of serial sections from a large number of cells. The evidence presented here is, therefore, derived from counts of PVs in

individual sections taken at random through the posterior region of the pseudoplasmodium and the results obtained were gained indirectly from these counts as outlined below.

About 2,000 cell profiles, each from posterior regions of pseudoplasmodia before and after a period of migration, were analyzed. The migration lasted for about 12 h during which time the slugs moved towards a light on non-nutrient agar. The results are summarized in Table 1.

Of 1802 cell profiles from the beginning of migration, 1141 or 43.4% contained PVs, whereas of 1909 cell profiles from late migration

0 5 10 15 20 PV Fig. 3 Distribution of PVs among cell profiles from pseudoplasmodia at early (white circles) and late (black triangles) migration. Each curve represents total counts from about 2,000 cell profiles. The ordinate indicates percentage of cell profiles (%) containing the number of PVs indicated on the abscissa (PV).

270 Diferentiation 1, 1973

Pattern Formation in Dictyostelium

Table 1 (12 h) of migration.

Distribution of PVs among cell profiles from pseudoplasmodia before and after a period

No. of PVslcell profile

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21

Total

Total cell profiles counted

0

Pseudoplasmodia before migration No. of ”/, of cell Total no.

Pseudoplasmodia after migration No. of of cell Total no.

ce// profiles

290 205 192 125 114 74 58 27 16 22

8 3 5

1 1 1

-

- - - -

1141 661

1802

, 1

protiles of PVS

25.4 290 17.9 41 0 16.8 576 10.9 600 10.0 570 6.5 444 5.1 406 2.4 21 6 1.4 144 2.9 220 0.8 88 0.3 36 0.4 65

0.1 15 0.1 16 0.1 17

- -

- -

100 41 03 36.6 -

cell profiles

21 9 164 197 193 167 141 163 95 86 55 47 26 15 12 11 6 2

2 2 1

1606 303

1909

-

profiles

13.6 10.2 12.3 12.0 1055 8.8

10.1 5.9 5.4 3.4 2.9 1.6 0.9 0.8 0.8 0.4 0.1

0.1 0.1 0.1

-

100 15.9

of PVS

21 9 328 591 772 835 846

1141 760 774 550 51 7 31 2 195 168 165 96 34

38 40 21

-

a402 -

1606 or 84.1 % contained PVs. The increase from 63.4% to 84.1 % represents an increase of 33% of total sections containing PVs. During the same period of time the average number of PVs per section rose from 3.59 (4103 divided by 1141) to 5.24 (8402 divided by 1606) or by 46%, while the total number of PVs counted increased by 93 %.

We can now use these values to answer the first question, i.e. whether cells containing PVs acquired more PVs or whether cells without PVs at late aggregation subsequently developed PVs, or whether both events were taking place. The 46% increase in the average number of PVs per section provides clear evidence that after a period of migration the average number of PVs per cell must have substantially increased, particularly as we have only considered sections containing PVs and omitted all ‘zero’ sections from this calculation. This result is corroborated by the histogram in Fig. 3, which indicates that during migration the percentage of cell profiles with few PVs decreases with a corresponding increase

Diferentiation 1, 1973

of sections containing a larger number of PVs. The crucial question now arises if the observed

increase in the average number of PVs per cell can by itself account for the 93 % increase in the total PVs counted and the 33% increase of sections containing PVs. If the answer is no we would have to conclude that cells previously lacking PVs have acquired PVs during migration, i.e. have become prespore cells. We have tackled the problem from two angles, and both indicate that indeed more cells must acquire PVs.

The first approach simply compares the 46 % increase in the average number of PVs with the 93% increase of total PVs counted. If only cells already having PVs acquire new PVs the total number of PVs counted should not exceed 46 %, which it does, however, by a factor of 2. The second approach tries to establish that a doubling (more accurately, an increase by 93%) of the number of PVs in prespore cells would only lead to an increase in sections containing PVs of about 12 % instead of the observed 33 %, which is again incompatible with the assumption that only

271

Ursula Muller and H. R. Hohl

existing prespore cells acquire more PVs. The crucial relationship between the number of PVs per cell and the number of sections from such cells carrying PVs is given in Fig. 4. It represents a first approximation and the rationale for it is given in the Appendix at the end of the paper. Both approaches together are taken to indicate that during migration both the number of PVs per cell and the number of cells containing PVs increases substantially.

I I lo' lo2 2X1O2 PV

Fig. 4 Graph representing a first approximation of the relationship between the number of PVs per cell (PV) and the percentage of cell sections cut from such cells (:d) carrying PVs. For further explanations refer to text and Appendix.

At the end of the period of migration there are still about 16% of cell profiles in the posterior section of the slug devoid of PVs. To assess whether this means that there are cells without PVs (i.e. prestalk cells) even at this late stage in the posterior section of the slug, or whether this percentage simply represents the proportion of empty sections from cells that all contain PVs we employed the following approach. PVs and other cell organelles, such as nuclei and mitochondria, occur preferentially in the more central regions of the cell12 rather than in the

peripheral regions and thin pseudopodia. In order to eliminate these 'empty' regions of the cells we have re-evaluated all the cell profiles from late migration and considered only those that pass through central cell portions. Any section containing 4 or more mitochondria was arbitrarily considered a centrally located section. The histogram of distribution of PVs in sections with 4 or more mitochondria is given in Fig. 5 . It is essentially similar to that of Fig. 3, demonstrating

%

30

20

10

0 5 10 15 20 PV Fig. 5 Distribution of PVs among cell profiles from pseudoplasmodia at early (white circles) and late (black triangles) migration. In this figure are included only those cell profiles which contain at least 4 mitochondria, thus indicating that they originate from a more centrally located section through the cell. Note the close resemblance with Fig. 3.

272 Difereritiation 1, 1973

Pattern Formation in Dictyosteliurn

Fig. 6 Scheme of experimental set-up used for results presented in Table 2. Basically cell groups from radioactively labelled pseudoplasmodia are transferred and inserted into unlabelled pseudo- plasmodia. After a period of migration the pseudoplasmodium was cut into three equal parts and the radioactivity was recorded for each part separately.

that even after eliminating all a priori 'empty' sections the same fraction (approximately 16 %) of sections from the prespore area are devoid of PVs. This is taken as an indication that there remains within the prespore area a small but measurable fraction of cells lacking PVs.

As a last point we have investigated the pre- spore area during culmination, just before the prespores mature by laying down the spore wall layers. From about 140 cell profiles it was determined that the surface of the PVs of a cell exceeds the total surface of the cell by at least 50 %. Because the surface membrane of the PVs is covered with the initial spore coat layer, we conclude that there is more of the initial spore coat layer formed than would be necessary to cover the maturing spore surface evenly.

Rearrangement of "-thymidine labelled cells within the pseudoplasmodium If the major burst of pattern formation that

occurs within the first 2 h of migration was due to sorting out one would expcct a higher rate of cell migration during this period than during later stages of migration. To clarify this point we have established the rate of cell migration using 3H-thymidine labelled cells. The experiments were set up as follows: pieces from labelled slugs taken either from the front or back portion were inserted into unlabelled pseudoplasmodia. The slugs were then allowed to migrate from 4-7 h when each slug was cut transversely into three pieces of approximately the same size. Each section was then counted separately in a scintil- lation counter and the activity expressed as a percentage of the total activity recovered from the three parts. Figure 6 provides an illustration of the basic procedure. In one set of experiments (Table 2, experiment 4.4) the radioactive pieces were not inserted into an intact slug but were exchanged for the corresponding region of the recipient pseudoplasmodium.13 The result ob-

Table 2 Results of transplantation experiments whereby groups of radioactively labelled cells were inserted into unlabelled recipient pseudoplasmodia as outlined in Fig. 6.

Expt. Origin of Place of in- Developmental No. Distribution of label Migrafion rate

recipient slug and recipient cases Back Middle Front no. donor cells sertion in stage of donor of as % of total activity (% of popu/afion/h)

4.1 t ip back late aggregation 18 56(100) 19(0) 25*(0) 5.0 4.2 t ip back migration 20 74(100) 14(0) 12*(0) 2.1 4.3 back back migration 20 77(100) 18(0) 5YO) 1 .o

4.5 t ip t ip migration 10 12+(0) 29(0) 59(100) 2.1 4.4** back back migration 8 71(100) 2 7 ~ ) 3*@) 0.6

The distribution of the label in the three parts of the pseudoplasmodium was determined after a period of migration lasting 4-7 h.

The values given in parentheses are those at the start of the experiment. * These are important values, which were used for the determination of the rate of movement of the cells within the pseudoplasmodium. ** In this experiment the radioactively labelled cells were not inserted into an intact slug but, instead, were exchanged for the corresponding back region of the recipient pseudo- plasmodium.

Diferentiation 1, 1973 273

Ursula Miiller and H. R. Hohl

tained was not qualitatively different from the procedure normally employed.

The results of the various transplantations are summarized in Table 2. The averages recorded include all measurements made, even though up to 50% of the transplants did not seem to be integrated successfully because all the activity remained at the point of implantation. However, the results remain the same with and without the ‘failures’ except for the rate of exchange which in most cases would be up to twice as much if the successes only had been considered.

The results indicate that the migration rate (expressed as % of population/h that has mi- grated from front to back or vice versa) in old migrating slugs is less than half that measured in late aggregation stages. In addition, the rate of migration of cells from the slug tip is more than twice as high as that of cells from the prespore region but, nevertheless, a definite percentage of cells from the posterior section of an old slug migrated to the tip region within the time of observation, Finally, it was noted that in an old pseudoplasmodium the same percentage of cells cross the border between the prestalk and prespore areas from either side.

Discussion

The first electron microscope evidence of diff- erential cell determination during the develop- ment of D. discoideum was found at late aggre- gation, in accordance with Gregg and Badman.7 The first cells containing PVs appear at random over the entire pseudoplasmodium, with the exception of the very tip which throughout development remains unchanged. This tip should be considered as a specialized region within the prestalk area. It is quite likely that this most anterior 5-10% of the slug carries out functions of organizing and guiding the pseudoplasmodium which have been generally attributed more to the prestalk area as a whole, even though various authors have already indicated the possibly unique character of this small group of cells.1314-16

The random distribution of PVs within the early pseudoplasmodium agrees well with the results of other researchersl7.1* who also observed a random distribution of markers used to distinguish cell types. Because any theory based primarily on a preformed gradient as the major factor of pattern establishment would predict a non-random appearance of PVs, this result is in strong support of a sorting out theory.

Normal sorting out implies that the cell types

arise first (in our case prestalk cells and pre- spores) and then rearrange themselves. The situation in D. discoideum seems to be somewhat more complex in two ways: for one, the number of PVs per cell is increasing over a considerable period of time, indicating that the cells gradually take on a more prespore-like character during a period when sorting out is already taking place. Secondly, the cells acquire not only a more pronounced prespore character but, during the same period, more cells form PVs, i.e. switch from vegetative to prespore stage. This would indicate that cell determination and cell diff- erentiation are gradual events which considerably overlap in time with the establishment of the overall morphogenetic pattern.

Sorting out is likely to take place over an extended period of time, possibly over the entire period up to culmination.13 In addition, both our electronmicroscope observations and labelling experiments indicate that there is a major burst of activity within the first 2 h of migration which leads to the establishment of the borderline between prestalk and prespore cells. Subsequently cell movements within the slug occur on a much reduced scale. In view of these findings caution should be applied when working with prespore cell masses, because the pseudoplasmodium apparently contains a significant if variable and small proportion of cells which lack PVs. The assumption of Gregg and Badman7 that from the end of aggregation the posterior fraction of the slug is homogeneously composed of prespore cells (see their Fig. 8) must in consequence be qualified.

We have confirmed the observation of Francis and O’Dayl3 that even at late stages of migration there is still a considerable movement of cells from the tip to the posterior section which, it i? interesting to note, just equals the rate of forward movement observed during the same period of time. Although one might postulate that some cells die within the aggregate and that their remains will fall behind as the slug moves, the relative high rate observed can hardly be explained by an artifact alone. We rather think that some prestalk cells move back while others move forward within the slug, leading to a circulation at low level of prestalk cells. A similar idea has been entertained before 1,1920 but is novel in that only prestalk cells move. Thus we do not have to postulate that each time a cell crosses the border- line it has to reverse its determination. In fact our concept has the advantage of explaining the curious phenomenon that while cells are constantly crossing the borderline there are prestalk cells in

274 Dirermtiation 1, 1973

Pattern Formation in Dictyostelium

the prespore area but never prespore cells in the prestalk area. It is highly unlikely that prespore cells enter the prestalk area and suddenly lose their PVs in view of the observation by @egg* that prespore cells not in the process of division retain their PVs for hours. In addition, it might be possible to explain the faster regeneration of isolated prespore masses as compared to prestalk 'masses1 by their contents of prestalk cells.

While the majority of evidence favours sorting out, Farnsworth and Wolpertzl suggest a gradient theory as the best explanation of pattern for- mation. Their argument is primarily based on their failure to observe cell movement within the aggregate. We have unsuccessfully tried to repeat their experiments which include the use of a cell marker consisting of acridine orange tightly coupled to DEAE-cellulose particles. Our diffi- culties were twofold: first the slugs exhibited considerable autofluorescence, and secondly the stained particles were almost quantitatively eliminated from the cells during aggregation, most probably because they are located in the food vacuoles which are emptied during this stage of development.'* In this way the cells are rendered free of label. While their observations

References

1 . Raper, K. B., J. Elisha Mitckell scient. Soc., 56, 241, 1940.

2. Bonner, J. T., Amev. Naturalist, 86, 79, 1952. 3. Takeuchi, I., in: Nucleic Acid Metabolism,

Cell Diferer7tiation and Cancer Growth, eds. Cowdry, E. V. & Seno, S., pp. 297-304, Pergamon Press, 1969.

4. Gregg, J. H., Devl Biol., 12, 377, 1965. 5. Hohl, H. R. & Hamamoto, S . T., J. Ultra-

6. Maeda, Y . & Takeuchi, I., Develop., Growth

7. Gregg, J. H. & Badman, W. S . , Devl Biol.,

8. Gregg, J. H., Devl Biot., 26,478, 1971. 9. Sussman, M. & Lovgren, N., Expl. Cell Res.,

10. Schafer-Danneel, S. , Zeitschr. Zellfomch.

1 I . Bonner, J. T. & Slifkin, M. K., Am. J.Bot.,

struct. Res., 26,442, 1969.

and Diferentiation, 11, 232, 1970.

22, 96, 1970.

38, 97, 1965.

mikrosk, Anat., 78,441, 1967.

36, 727, 1949.

do, therefore, not refute sorting out it is never- theless quite possible that other mechanisms do participate, particularly after the initial pattern has been established. Furthermore, it is not yet understood by what mechanism the cells sort out.

The more quantitative approach attempted during this investigation has also pointed more closely to a situation noticed earlier5 that there are obviously more PVs produced than are necessary to provide sufficient material for the initial spore wall layer. The surplus material available possibly ensures that the apparent random drifting of PVs to the cell surface still leads to the formation of a coherent wall layer.

The work reported here has been supported by grant 3.242.69 from the 'Schweizerischer National- fonds zur Forderung der wissenschaftlichen Forschung' to one of us (H.R.H.). W e would like to thank Dr. Bonner for critical comments and Dr. Francis for various helpful suggestions during the course of this investigation.

Revised: July, 1973

12. George, R. P., Hohl, H. R. &Raper, K. B.,

13. Francis, D. & O'Day, D. H., J. exp. Zool.,

14. Bonner, J. T., J. exp. Zool., 110, 259, 1949. 15. Krivanek, J. 0. & Krivanek, R. C., J. exp.

16. Loomis Jr. W. F., Nature New Biol., 240,

17. Takeuchi, I., Devel Biol., 8, 1, 1963. 18. Bonner, J. T. & Adams, M. S., J. Embryol.

19. Bonner, J. T., Quart. Rev. Biol., 32,232, 1957. 20. Francis, D., Pseudoplasmodial movement in

Diclyostelium discoideum, M.S. thesis, University of Wisconsin.

21. Farnsworth, P. A. & Wolpert, L., Nature, 231,329, 1971.

J. gen. Microbiol., 70, 477, 1972.

176,265, 1971.

Zool., 137,89, 1958.

6, 1972.

exp. Morph., 6,346, 1958.

Diflrrentiation 1, 1973 215

Ursula Miiller and H. R. Hohl

Appendix

To solve the question whether an increase of 93 % of PVs in a population of prespore cells can lead to an increase of 33 % of sections containing PVs we have to know the relationship between the number of PVs per cell and the percentage of sections containing PVs taken from such cells, as well as the approximate number of PVs per cell at the migration stage.

At late migration an average of about 10% of a section (repersenting a slice through a cell) are taken up by PVs so that we can reasonably conclude that about 10% of the cell volume is occupied by PVs. Calculating the average volume of a PV from a series of measurements and dividing it into the total volume of the cell taken up by PVs we arrive at a tentative value of about 30-40 PVs per cell at late migration.

To approximate the relationship between number of PVs per cell and percentage of sections

through such a cell that will contain FVs we consider the following : the more PVs there are in a given cell the smaller will be the increment of sections with PVs after adding one more PV to the cell, mainly because of overlapping. In other words, by adding a PV to a cell with one PV we increase the percentage of sections containing PVs through such a cell much more than by adding one PV to a cell with, say, 50 PVs. To represent this situation at first approximation we use a log-function. Plotted semilogarithmically this relationship would be represented by a straight line as indicated in Fig. 4. The line was set to intersect the 1 PV/cell line at a few per cent (5-10) of sections with PVs and the 100%- sections-with-PVs line at about 200 PVsjcell, a value arrived at using various methods to ensure that every section would contain at least one PV (one method was based on calculating the number of PVs that could be closely packed into a cell, which amounted to about 300).

216 Differentiation 1, 1973