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Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum
DEVELOPMENTAL
BIOLOGY
81,
139-144 (1981)
Cell Patterning in Branched and Unbranched Fruiting Bodies of the Cellular Slime Mold Po/ysphondylium pallidum FAY 0. STENHOUSE AND KEITH L. WILLIAMS’ Department
of Genetics, Research School of Biological Sciences, The Australian P.O. Box 475, Ca.nberra, ACT 2601, Australia Received March $1, 1980; accepted in revised
National
University,
f&m June 19, 1980
Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidurn. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidurn. INTRODUCTION
A fundamental problem of embryogenesis is how cells are counted so that different types are formed in the correct proportions in the adult organism. Only in simple organisms such as the cellular slime molds is the direct measurement of cell patterning, the proportions of different cell types, feasible. Different species of cellular slime molds have different levels of complexity. Some species don’t exhibit cell patterning at all, since the fruiting body is comprised of only one cell type (e.g., Acytostelium has only spores and an acellular stalk (Raper and Quinlan, 19X3)), while some species form a simple fruiting body comprising a cellular stalk with spores on the top (e.g., Dictyostelium species; D. discoideum also has a cellular basal disk (Raper, 1935)). Polysphondylium species and related slime molds (Harper, 1932; Cavender et al., 1979) form either Dictyostelium-like unbranched fruiting bodies or more complex structures involving a main stalk and spore head plus whorls of small fruiting bodies along the main stalk. The cell patterning of Dictyostelium species has been examined and shown to be similar in small and large fruiting bodies in several studies (Bonner and Slifkin, 1949; Bonner, 1957; Bonner and Dodd, 1962). Recently ’ To whom requests for reprints and all correspondence should be addressed: Max Planck Institut fiir Biochemie, D-8033 Martinsried bei Miinchen, Federal Republic of Germany.
we showed that while cell patterning of D. discoideum is close to being size invariant, small and large fruiting bodies have an increased percentage of stalk and basal disk cells compared to average-sized fruiting bodies (Stenhouse and Williams, 19’77). There has been no study on the cell patterning (percentage stalk and spore cells) of species forming branched fruiting bodies except for a brief report of small unbranched fruiting bodies of Polysphondylium pallidum (Bonner and Dodd, 1962). We report here a study on branched and unbranched fruiting bodies of Polysphondylium pallidum strain PP28S. Overall cell patterning in this strain of P. pallidum is probably the same in unbranched and branched fruiting bodies over a large size range. The cell patterning of individual branches of the whorls is imprecise and it is possible that cell patterning is decided not at the level of the branch or the whorl, but at the level of the whole fruiting body. Finally, we show that while aggregation and fruiting body size is very light dependent in Polysphondylium species (Harper, 1932; Kahn, 1964), light barely alters the cell patterning of the P. pa.llidum fruiting body. METHODS
Growth. and harvesting of amoebas. Amoebas of the homothallic P. pallidum strain PP28S (Francis, 1975) were grown in dim light on SM agar, a rich growth 139 0012-1606/81/010139-06$02.00/O Copyright All rights
0 1991 hy Academic Press. Inc. of reproduction in any form reserved.
140
DEVELOPMENTALBIOLOGY
medium and harvested in sterile Bonner’s salt solution (SS = 0.6 g of NaCl, 0.75 g of KCl, 0.3 g of CaClz in 1 liter of distilled water) in exactly the same way as previously described for D. discoideum (Stenhouse and Williams, 1977). Conditions of differen.tiation. Dihydrostreptomycin sulfate (Sigma)-containing water agar plates were prepared as described previously (Stenhouse and Williams, 19’77). Four separate 20-&l drops, each containing 3 X lo5 P. pallidurn amoebas suspended in SS, were placed on each water agar plate. This gave a standard density of 4 X lo5 amoebas/cm’ and this density was used for most experiments, although for the experiments reported in Fig. 1, amoeba1 density was varied between 7.5 X 10” and 1.2 X lo6 amoebas/20-cl1 drop. The P. pallidum. amoebas were allowed to differentiate in either the light (incubator lit with Phillips TL 8W/33 lamp 30 cm from the plates) or the dark (foil-wrapped plates in the same incubator) at 22.0 f 0.5”C. In some experiments all manipulations, including harvesting and depositing amoebas on water agar plates, were carried out in a dark room lit with an Ilford safe light 902 S. It was found that exposure to light during harvesting did not affect morphogenesis of PP28S amoebas in the light or the dark and therefore in most experiments amoebas were harvested in the light. Spore and stalk cell counts in P. pallidum. A segment 14 “E ”
r
12 I
‘0’
i2
4 Amoebae
I 8
I 12 per
I 16
;O
cm2 (x10-‘)
FIG. 1. The density of aggregates (fruiting bodies) of P. pallidurn strain PP28S formed on water agar plates at 22 f 05°C after differentiation in the light (solid circles) or the dark (open squares) from amoebas at different initial densities. The number of aggregates equaled the number of fruiting bodies formed, so there was no breakup of aggregates under the conditions used. Bars represent standard error of the mean (n >‘ 5).
VOLUME 81, 1981
of water agar, with fruiting bodies attached, was cut out with a scalpel and placed upside down on a microscope slide. Individual fruiting bodies were then examined with an Olympus CK inverted microscope using a 40X objective. Several features of each fruiting body were noted, including the length of the branches and the main stalk, number of whorls, and number of branches per whorl. Then the numbers of stalk cells and spores on each branch, the number of stalk cells in the main stalk, and the number of spores on top of the main stalk were counted directly. This procedure was relatively uncomplicated because even the main stalk in strain PP28S is usually only one cell thick and each spore head contains only a few hundred spores. RESULTS
Fruiting Body Formation in P. pallidurn PP.%% in the Light versus the Dark
Strain.
The aim of this study was to examine cell patterning in branched fruiting bodies of P. pallidurn and also to determine the effect of light on this process for comparison with similar studies on D. discoideum (Stenhouse and Williams, 1977). Most strains of P. pallidum aggregate poorly, if at all, in the dark. The homothallic strain PP28S is a robust strain which fruits in both the light and the dark, so it was chosen for this work. Up to three times as many fruiting bodies were formed in the light as in the dark and in both treatments the density of fruiting bodies increased with higher initial concentrations of amoebas (Fig. 1). Hence this strain probably did not have a constant aggregation territory size (see Bonner and Dodd, 1962) although some macrocysts were formed which were not counted. As will be described shortly, the greater number of fruiting bodies formed in the light compared to the dark meant that light-developed fruiting bodies were small, most were unbranched, and no fruiting bodies with more than one whorl were examined. In the dark, unbranched fruiting bodies were uncommon, and the largest fruiting bodies examined had five whorls. The number of branches per fruiting body increased directly in proportion to the total cell number (Fig. 2). There was some effect of light on the transition size between the unbranched and branched fruiting body modes, with darkincubated fruiting bodies becoming branched at a smaller size than light-incubated fruiting bodies (Fig. 3). Conversely, there was a tendency for unbranched fruiting bodies in the light to be larger than those in the dark (Fig. 3). Thus it appears that light decreased both the territory size (or light increased the number of fruiting bodies/unit area) and the propensity to form branches.
STENHOUSE AND WILLIAMS
141
Cell Patterning in P. pallidum
Cell Pcltterlziny in Branched Fruiti.ny Bodies
. . . .
2
I.4
OL 0
1 Total
Cell
2 Number
3
4 (XlCrq
FIG. 2. The number of branches produced by P. pallidurn strain PP28S fruiting bodies of different size formed on water agar at 22 2 0.5”C in the dark. Bars represent standard error of the mean (n 3 5).
In the previous section all parts of the branched fruiting bodies were combined, i.e., stalk cells from both branches and the main stalk were added, while spores from the terminal head and branches were combined. Here the cell patterning of 33 branched fruiting bodies formed in the dark is examined in more detail. Figure 4 shows the cell patterning of (A) the whole fruiting bodies, (B) the whorls, and (C) the branches. The striking feature of these results is that the spore:stalk relationship in individual branches is highly variable (coefficient of determination for Fig. 4C = 0.03). That the organism ca~zpattern accurately in such small fruiting bodies is shown by examining unbranched fruiting bodies (Fig. 3) which are of similar size to the individual branches. The coefficient of determination for unbranched fruiting bodies in the dark (Fig. 3, inset) is 0.79. The spore:stalk relationship of whorls (i.e., spores of all branches and stalk cells of all branches in each whorl summed) is improved (coefficient of determination for Fig. 4B = 0.47), while the spore:stalk relationship of the whole fruiting bodies is even better (coefficient of determination for Fig. 4A = 0.89). The other point to be noted here is that the whorls
Overa. Cell Patterni,rzy in Branched and Unbra.nched Fruiting Bod,ies Formed in the Light or the Dark There was a linear relationship between the total numbers of spores and stalk cells in the four categories of fruiting bodies of P. pa.llidum. PP28S shown in Fig. 3, i.e., branched and unbranched fruiting bodies differentiated in the light or the dark. Examination of the residuals from regressions constructed from these results showed that linear equations fit the data better than exponential equations or semilogarithmic equations, in contrast to our findings for D. discoideu,m fruiting bodies (see Stenhouse and Williams, 1977). The spore:stalk relationships for the four categories (unbranched, dark; branched, dark; unbranched, light; branched, light) were all similar [test for equality of regression (Neter and Wasserman, 1974); 0.10 < P < 0.51. Therefore overall cell patterning in P. pa.llidwm fruiting bodies is statistically similar for branched and unbranched fruiting bodies differentiated in the light or the dark. However these results can also be analyzed by including the origin since the intercept in each of the four regressions is statistically close to zero. If this is done, the relationship between spores and stalk cells in the light compared to the dark, is close to being statistically different (probability that the equations are the same is greater than 0.05 but less than 0.10). There is, therefore, some hint of a change in cell patterning in the light, and the direction of that change is for fruiting bodies to be more stalky in the light.
3
l
I
- I
l
.&’ le * 0
0
1 1
I 2
I 3
I 4
Stalk Cell Number
I S
00
I 6
50
100
7
I 0
(~10~~)
FIG. 3. The relationship between total spore and stalk cells of individual fruiting bodies of P. pallidurn strain PP28S differentiated on water agar at 22 * 0.5”C. The main diagram shows the data obtained from branched fruiting bodies differentiated in the light (open squares) or in the dark (solid circles). The inset shows the spore:stalk cell relationship in unbranched fruiting bodies differentiated in the light (open squares) or in the dark (solid circles).
142 had much more “spory” than the whole fruiting
DEVELOPMENTALBIOLOGY
spore:stalk ratios (90% spores) bodies (78% spores).
Relatiolzsh ip betwee,n Fkting and Cell Patterning
VOLUME81, 1981 4000
A.
Whole
Fruiting
Bodies
Body Height
The number of stalk cells in the main stalk of branched fruiting bodies was directly proportional to the total cell number in the fruiting body and the relationship was Nstm = 0.14 ( 20.01) N,, where Nstm = number of cells in the main stalk and Nt equals the total number of cells in the fruiting body. The correlation coefficient was 0.93 and the equation was the same in fruiting bodies differentiated in the light or the dark (test for equality of regressions 0.75 > P > 0.50). Although the diameter of cells in the main stalk varied, being thin at the apex and thicker at the base, the main stalk was usually composed of a single column of cells over the entire length. Since the number of cells in the main stalk and the total cell number were tightly correlated (see above) it was not surprising that stalk height, stalk cell number, and total cell number were tightly correlated (e.g., correlation coefficient of stalk height to total cell number = 0.96). This finding contrasts with our studies on D. discoideum in which the stalk is several cells thick and the stalk length is not linearly correlated to stalk cell numbers.
0-i 0
400
200
Stalk 1200
r
Stalk 6oor
I
I
800
1000
Number
6. Whorls
DISCUSSION
This first study on cell patterning in branched fruiting bodies of P. pallidurn has produced some surprising results in relation to the effect of light and dark on cell patterning and to the nature of the pattern determining mechanism in the complex branched fruiting bodies. Since both aggregation and fruiting body size in P. pallidurn (and other Polysphondylium species, e.g., P. violaceum; Harper, 1932) are particularly light sensitive, much more so than D. discoideum (Raper, 1935), it was of interest to examine the effect of light on cell patterning. It is known in D. discoideum that light substantially increases the percentage of stalk and basal disk cells (Bonner and Slifkin, 1949; Stenhouse and Williams, 1977). Surprisingly, while light had a major effect on decreasing the size of P. pallidurn PP28S fruiting bodies (approximately three times more fruiting bodies were formed in the light compared to the dark), there was a barely detectable effect of light on cell patterning. This has relevance to the complexity of light responses at the molecular level in different slime mold genera. There are also some surprises in the cell patterning (percentage spores and stalk cells) in branched fruiting bodies of Polysph.ondy1iu.m pallidum. The first observation is that patterning may be the same in un-
Cell
I
600
c.
Cell
Number
Branches
. 20I
301 Stalk
Cell
40 I
.I
I
50
60
Number
FIG. 4. Spore-to-stalk-cell relationships of individual branched fruiting bodies differentiated in the dark at 22 f 0.5”C on water agar. Spore-to-stalk-cell relationship of: (A) whole fruiting bodies; (B) whorls; and (C) individual branches. The regression lines are included on each graph.
branched and branched fruiting bodies over a size range of 140 to 4142 cells. This is different from the cell patterning of D. discoideum in which small and large fruiting bodies have a greater percentage of stalk and basal disk cells than average-sized fruiting bodies (Stenhouse
STENHOUSEAND WILLIAMS
and Williams, 1977). We have no explanation for this difference between cell patterning in P. paMum and D. d,iscoidewm, although we note that D. discoidewm fruiting bodies are considerably larger than those of P. pa.llidwm. The range of fruiting body sizes examined in the study on D. discoideum. by Stenhouse and Williams (1977) was 620 to 18,000 cells. The above comments take a global view and do not consider the individual components of the branched fruiting bodies of P. pal1idu.m. A branched fruiting body can also be considered in terms of its components: a main stalk, terminal spore head, and a series of whorls, with between one and five branches, each of which can be considered to be a miniature fruiting body. We have shown here that the cell patterning in the branches, i.e., spore:stalk ratio of individual miniature fruiting bodies along the main stalk, is so poorly correlated that it seems to be barely regulated at all (Fig. 4C j, while small unbranched fruiting bodies of similar size have tightly correlated spore:stalk ratios (Fig. 3). If the cell patterning of whorls is considered (i.e., summing the spore and stalk cells of branches in each whorl), a correlation between the spore:stalk ratio is found (Fig. 4Bj, although it is still relatively poor. The cell patterning of the whorls (90% spores) is different from that of the whole fruiting bodies (78% spore) which include the large main stalk. The observation that the cell patterning of the whole branched fruiting body is tightly correlated (Fig. 4A) and is the same as that of unbranched fruiting bodies (Fig. 3), can be interpreted to indicate that cell patterning is decided at the whole aggregate level rather than at the level of the whorl or individual branch. Initially we thought this unlikely since P. pal1idu.m aggregates do not show prepatterning in the way D. discoideum aggregates do. In D. discoideum, prespore cells are identified by the presence of prespore vacuoles or prespore antigen (Takeuchi et al., 1977) and prestalk cells are identified by histochemical stains or neutral red staining (MacWilliams and Bonner, 1979). Similar experiments in P. pallidum detect almost entirely prespore cells in the aggregate on the basis of the presence of prespore vacuoles (Hohl et al., 1977) and the prespore antigen (O’Day, 1979). However, prestalk cells, which are mainly found at the front of the aggregate, have been found in a histochemical study (O’Day and Francis, 1973) and using neutral red (O’Day, 1979). As pointed out by O’Day (1979), this suggests that prespore vacuoles and the prespore antigen may not define the pattern in P. padlidum, since presumptive prestalk cells also contain these “spore specific” vacuoles and antigen. It has been shown recently that under some conditions D. discoideum amoebas destined to become stalk cells form some prespore vacuoles and then lose them (Kay
Cell Pa.tterning
in P. pallidurn
143
et al., 1978). While it is important to determine whether the percentage of cells stained with neutral red coincides with the stalk cell percentage, the findings of this study support the notion that the basic cell pattern is established in the whole P. pallidum aggregate. We suggest further that our finding that branched and unbranched fruiting bodies have the same cell patterning is consistent with the P. pall,idum. cell patterning being stable over a considerable time span. If the aggregate reestablished the cell pattern after each group of cells is lost from the main aggregate to form a whorl, the patterning of branched and unbranched fruiting bodies would be different. The high percentage of spores in the whorls is consistent with the formation of fruiting bodies in the branches of whorls from cells most of which are already predetermined to be spores (since most prestalk cells are at the front of the aggregate and the cells making up the whorl are lost from the rear). If this hypothesis is correct, the tips formed in the whorl which organize the individual branches have no cell patterning function but organize the morphogenetic movements of cells already committed to be stalk cells or spores. The poor correlation between spore:stalk ratios of whorls could be explained by variable proportions of prespore and prestalk cells dropped from the rear of the aggregate, while the essential absence of patterning in branches may reflect asynchrony in the morphogenetic movements (and hence capture) of cells by individual branch tips in each whorl.
We thank Robyn Smith for doing some of the data analysis, and Dr. Elizabeth Smith and Paul Fisher for helpful comments. Dr. D. Francis kindly provided P. pallidum strain PP28S.
REFERENCES BONNER,J. T. (1957). A theory of the control of differentiation in the cellular slime molds. Quart. Rev. Biol. 32, 232-246. BONNER,J. T., and DODD, M. R. (1962). Aggregation territories in the cellular slime molds. Biol. Bull. 122, 13-24. BONNER,J. T., and SLIFKIN, M. K. (1949). A study of the control of differentiation: The proportions of stalk and spore cells in the slime mold, Dictyostelium discoideum. Amer. J. Bat. 36, 727-734. CAVENDER,J. C., RAPER, K. B., and NORBERG,A. M. (1979). Dictyotenue: New species of the stelium aureo-stipes and Dictyostelium Dictyosteliaceae. Amer. J. Bot. 66, 207-217. FRANCIS,D. (1975). Macrocyst genetics in Polysphondy1iu.m pallidurn, a cellular slime mould. J. Gen. Microbial. 89, 310-318. HARPER, R. A. (1932). Organisation and light relations in Polysphondylium.
Bull. Torrey
Bot. Club 69,49-&L
HOHL, H. R., HONEGGER,R., TRAUB, F., and MARKWALDER,M. (1977). Influence of CAMP on cell differentiation and morphogenesis in Polysphondylium. In “Development and Differentiation in the Cellular Slime Moulds” (P. Cappuccinelli and J. M. Ashworth, eds.), pp. 149-172. Elsevier/North-Holland, New York.
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DEVELOPMENTALBIOLOGY
KAHN, A. J. (1964). The influence of light on cell aggregation in Polgsphondylium
pallidum.
Biol. Buti
127, 85-96.
KAY, R. R., GARROD,D., and TILLY, R. (1978). Requirements for cell differentiation in Dictyostelium discoideum. Nature (London) 271, 58-60. KONIJN, T. M., and RAPER, K. B. (1965). The influence of light on the time of cell aggregation in the Dictyosteliaceae. Biol. Bull. 128, 392-400. MACWILLIAMS,H. K., and BONNER,J. T. (1979). The prestalk-prespore pattern in cellular slime molds. Differentiation 14, l-22. NETER, J., and WASSERMAN,W. (1974). “Applied Linear Statistical Models.” Richard D. Irwin Inc., Illinois. O’DAY, D. H. (1979). Cell differentiation during fruiting body formation in Polysphondylium pal1idu.m. J. Cell Sci. 35, 203-215. O’DAY, D. H., and FRANCIS,D. W. (1973). Patterns of alkaline phosphatase activity during alternative developmental pathways in the
VOLUME81, 1981
cellular slime mold. Polysphondylium pallidum. Canad. J. Zool. 51, 301-310. RAPER,K. B. (1935). Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agr. Res. (Washington) 50, 135-147. RAPER, K. B., and QUINLAN, M. S. (1958). Acytostelium leptosmum: A unique cellular slime mold with an acellular stalk. J. Gen. Microbiol. 18, 16-32. STENHOUSE,F. O., and WILLIAMS, K. L. (1977). Patterning in Dictyostelium discoideum: The proportions of the three differentiated cell types (spore, stalk and basal disk) in the fruiting body. Develop. Biol. 59, 140-152. TAKEUCHI, I., HAYASHI, M., and TASAKA, M. (1977). Cell differentiation and pattern formation in Dictyostelium. In “Development and Differentiation in the Cellular Slime Moulds” (P. Cappuccinelli and J. M. Ashworth, eds.), pp. 1-16. Elsevier/North-Holland, New York.
BIOLOGY
81,
139-144 (1981)
Cell Patterning in Branched and Unbranched Fruiting Bodies of the Cellular Slime Mold Po/ysphondylium pallidum FAY 0. STENHOUSE AND KEITH L. WILLIAMS’ Department
of Genetics, Research School of Biological Sciences, The Australian P.O. Box 475, Ca.nberra, ACT 2601, Australia Received March $1, 1980; accepted in revised
National
University,
f&m June 19, 1980
Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidurn. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidurn. INTRODUCTION
A fundamental problem of embryogenesis is how cells are counted so that different types are formed in the correct proportions in the adult organism. Only in simple organisms such as the cellular slime molds is the direct measurement of cell patterning, the proportions of different cell types, feasible. Different species of cellular slime molds have different levels of complexity. Some species don’t exhibit cell patterning at all, since the fruiting body is comprised of only one cell type (e.g., Acytostelium has only spores and an acellular stalk (Raper and Quinlan, 19X3)), while some species form a simple fruiting body comprising a cellular stalk with spores on the top (e.g., Dictyostelium species; D. discoideum also has a cellular basal disk (Raper, 1935)). Polysphondylium species and related slime molds (Harper, 1932; Cavender et al., 1979) form either Dictyostelium-like unbranched fruiting bodies or more complex structures involving a main stalk and spore head plus whorls of small fruiting bodies along the main stalk. The cell patterning of Dictyostelium species has been examined and shown to be similar in small and large fruiting bodies in several studies (Bonner and Slifkin, 1949; Bonner, 1957; Bonner and Dodd, 1962). Recently ’ To whom requests for reprints and all correspondence should be addressed: Max Planck Institut fiir Biochemie, D-8033 Martinsried bei Miinchen, Federal Republic of Germany.
we showed that while cell patterning of D. discoideum is close to being size invariant, small and large fruiting bodies have an increased percentage of stalk and basal disk cells compared to average-sized fruiting bodies (Stenhouse and Williams, 19’77). There has been no study on the cell patterning (percentage stalk and spore cells) of species forming branched fruiting bodies except for a brief report of small unbranched fruiting bodies of Polysphondylium pallidum (Bonner and Dodd, 1962). We report here a study on branched and unbranched fruiting bodies of Polysphondylium pallidum strain PP28S. Overall cell patterning in this strain of P. pallidum is probably the same in unbranched and branched fruiting bodies over a large size range. The cell patterning of individual branches of the whorls is imprecise and it is possible that cell patterning is decided not at the level of the branch or the whorl, but at the level of the whole fruiting body. Finally, we show that while aggregation and fruiting body size is very light dependent in Polysphondylium species (Harper, 1932; Kahn, 1964), light barely alters the cell patterning of the P. pa.llidum fruiting body. METHODS
Growth. and harvesting of amoebas. Amoebas of the homothallic P. pallidum strain PP28S (Francis, 1975) were grown in dim light on SM agar, a rich growth 139 0012-1606/81/010139-06$02.00/O Copyright All rights
0 1991 hy Academic Press. Inc. of reproduction in any form reserved.
140
DEVELOPMENTALBIOLOGY
medium and harvested in sterile Bonner’s salt solution (SS = 0.6 g of NaCl, 0.75 g of KCl, 0.3 g of CaClz in 1 liter of distilled water) in exactly the same way as previously described for D. discoideum (Stenhouse and Williams, 1977). Conditions of differen.tiation. Dihydrostreptomycin sulfate (Sigma)-containing water agar plates were prepared as described previously (Stenhouse and Williams, 19’77). Four separate 20-&l drops, each containing 3 X lo5 P. pallidurn amoebas suspended in SS, were placed on each water agar plate. This gave a standard density of 4 X lo5 amoebas/cm’ and this density was used for most experiments, although for the experiments reported in Fig. 1, amoeba1 density was varied between 7.5 X 10” and 1.2 X lo6 amoebas/20-cl1 drop. The P. pallidum. amoebas were allowed to differentiate in either the light (incubator lit with Phillips TL 8W/33 lamp 30 cm from the plates) or the dark (foil-wrapped plates in the same incubator) at 22.0 f 0.5”C. In some experiments all manipulations, including harvesting and depositing amoebas on water agar plates, were carried out in a dark room lit with an Ilford safe light 902 S. It was found that exposure to light during harvesting did not affect morphogenesis of PP28S amoebas in the light or the dark and therefore in most experiments amoebas were harvested in the light. Spore and stalk cell counts in P. pallidum. A segment 14 “E ”
r
12 I
‘0’
i2
4 Amoebae
I 8
I 12 per
I 16
;O
cm2 (x10-‘)
FIG. 1. The density of aggregates (fruiting bodies) of P. pallidurn strain PP28S formed on water agar plates at 22 f 05°C after differentiation in the light (solid circles) or the dark (open squares) from amoebas at different initial densities. The number of aggregates equaled the number of fruiting bodies formed, so there was no breakup of aggregates under the conditions used. Bars represent standard error of the mean (n >‘ 5).
VOLUME 81, 1981
of water agar, with fruiting bodies attached, was cut out with a scalpel and placed upside down on a microscope slide. Individual fruiting bodies were then examined with an Olympus CK inverted microscope using a 40X objective. Several features of each fruiting body were noted, including the length of the branches and the main stalk, number of whorls, and number of branches per whorl. Then the numbers of stalk cells and spores on each branch, the number of stalk cells in the main stalk, and the number of spores on top of the main stalk were counted directly. This procedure was relatively uncomplicated because even the main stalk in strain PP28S is usually only one cell thick and each spore head contains only a few hundred spores. RESULTS
Fruiting Body Formation in P. pallidurn PP.%% in the Light versus the Dark
Strain.
The aim of this study was to examine cell patterning in branched fruiting bodies of P. pallidurn and also to determine the effect of light on this process for comparison with similar studies on D. discoideum (Stenhouse and Williams, 1977). Most strains of P. pallidum aggregate poorly, if at all, in the dark. The homothallic strain PP28S is a robust strain which fruits in both the light and the dark, so it was chosen for this work. Up to three times as many fruiting bodies were formed in the light as in the dark and in both treatments the density of fruiting bodies increased with higher initial concentrations of amoebas (Fig. 1). Hence this strain probably did not have a constant aggregation territory size (see Bonner and Dodd, 1962) although some macrocysts were formed which were not counted. As will be described shortly, the greater number of fruiting bodies formed in the light compared to the dark meant that light-developed fruiting bodies were small, most were unbranched, and no fruiting bodies with more than one whorl were examined. In the dark, unbranched fruiting bodies were uncommon, and the largest fruiting bodies examined had five whorls. The number of branches per fruiting body increased directly in proportion to the total cell number (Fig. 2). There was some effect of light on the transition size between the unbranched and branched fruiting body modes, with darkincubated fruiting bodies becoming branched at a smaller size than light-incubated fruiting bodies (Fig. 3). Conversely, there was a tendency for unbranched fruiting bodies in the light to be larger than those in the dark (Fig. 3). Thus it appears that light decreased both the territory size (or light increased the number of fruiting bodies/unit area) and the propensity to form branches.
STENHOUSE AND WILLIAMS
141
Cell Patterning in P. pallidum
Cell Pcltterlziny in Branched Fruiti.ny Bodies
. . . .
2
I.4
OL 0
1 Total
Cell
2 Number
3
4 (XlCrq
FIG. 2. The number of branches produced by P. pallidurn strain PP28S fruiting bodies of different size formed on water agar at 22 2 0.5”C in the dark. Bars represent standard error of the mean (n 3 5).
In the previous section all parts of the branched fruiting bodies were combined, i.e., stalk cells from both branches and the main stalk were added, while spores from the terminal head and branches were combined. Here the cell patterning of 33 branched fruiting bodies formed in the dark is examined in more detail. Figure 4 shows the cell patterning of (A) the whole fruiting bodies, (B) the whorls, and (C) the branches. The striking feature of these results is that the spore:stalk relationship in individual branches is highly variable (coefficient of determination for Fig. 4C = 0.03). That the organism ca~zpattern accurately in such small fruiting bodies is shown by examining unbranched fruiting bodies (Fig. 3) which are of similar size to the individual branches. The coefficient of determination for unbranched fruiting bodies in the dark (Fig. 3, inset) is 0.79. The spore:stalk relationship of whorls (i.e., spores of all branches and stalk cells of all branches in each whorl summed) is improved (coefficient of determination for Fig. 4B = 0.47), while the spore:stalk relationship of the whole fruiting bodies is even better (coefficient of determination for Fig. 4A = 0.89). The other point to be noted here is that the whorls
Overa. Cell Patterni,rzy in Branched and Unbra.nched Fruiting Bod,ies Formed in the Light or the Dark There was a linear relationship between the total numbers of spores and stalk cells in the four categories of fruiting bodies of P. pa.llidum. PP28S shown in Fig. 3, i.e., branched and unbranched fruiting bodies differentiated in the light or the dark. Examination of the residuals from regressions constructed from these results showed that linear equations fit the data better than exponential equations or semilogarithmic equations, in contrast to our findings for D. discoideu,m fruiting bodies (see Stenhouse and Williams, 1977). The spore:stalk relationships for the four categories (unbranched, dark; branched, dark; unbranched, light; branched, light) were all similar [test for equality of regression (Neter and Wasserman, 1974); 0.10 < P < 0.51. Therefore overall cell patterning in P. pa.llidwm fruiting bodies is statistically similar for branched and unbranched fruiting bodies differentiated in the light or the dark. However these results can also be analyzed by including the origin since the intercept in each of the four regressions is statistically close to zero. If this is done, the relationship between spores and stalk cells in the light compared to the dark, is close to being statistically different (probability that the equations are the same is greater than 0.05 but less than 0.10). There is, therefore, some hint of a change in cell patterning in the light, and the direction of that change is for fruiting bodies to be more stalky in the light.
3
l
I
- I
l
.&’ le * 0
0
1 1
I 2
I 3
I 4
Stalk Cell Number
I S
00
I 6
50
100
7
I 0
(~10~~)
FIG. 3. The relationship between total spore and stalk cells of individual fruiting bodies of P. pallidurn strain PP28S differentiated on water agar at 22 * 0.5”C. The main diagram shows the data obtained from branched fruiting bodies differentiated in the light (open squares) or in the dark (solid circles). The inset shows the spore:stalk cell relationship in unbranched fruiting bodies differentiated in the light (open squares) or in the dark (solid circles).
142 had much more “spory” than the whole fruiting
DEVELOPMENTALBIOLOGY
spore:stalk ratios (90% spores) bodies (78% spores).
Relatiolzsh ip betwee,n Fkting and Cell Patterning
VOLUME81, 1981 4000
A.
Whole
Fruiting
Bodies
Body Height
The number of stalk cells in the main stalk of branched fruiting bodies was directly proportional to the total cell number in the fruiting body and the relationship was Nstm = 0.14 ( 20.01) N,, where Nstm = number of cells in the main stalk and Nt equals the total number of cells in the fruiting body. The correlation coefficient was 0.93 and the equation was the same in fruiting bodies differentiated in the light or the dark (test for equality of regressions 0.75 > P > 0.50). Although the diameter of cells in the main stalk varied, being thin at the apex and thicker at the base, the main stalk was usually composed of a single column of cells over the entire length. Since the number of cells in the main stalk and the total cell number were tightly correlated (see above) it was not surprising that stalk height, stalk cell number, and total cell number were tightly correlated (e.g., correlation coefficient of stalk height to total cell number = 0.96). This finding contrasts with our studies on D. discoideum in which the stalk is several cells thick and the stalk length is not linearly correlated to stalk cell numbers.
0-i 0
400
200
Stalk 1200
r
Stalk 6oor
I
I
800
1000
Number
6. Whorls
DISCUSSION
This first study on cell patterning in branched fruiting bodies of P. pallidurn has produced some surprising results in relation to the effect of light and dark on cell patterning and to the nature of the pattern determining mechanism in the complex branched fruiting bodies. Since both aggregation and fruiting body size in P. pallidurn (and other Polysphondylium species, e.g., P. violaceum; Harper, 1932) are particularly light sensitive, much more so than D. discoideum (Raper, 1935), it was of interest to examine the effect of light on cell patterning. It is known in D. discoideum that light substantially increases the percentage of stalk and basal disk cells (Bonner and Slifkin, 1949; Stenhouse and Williams, 1977). Surprisingly, while light had a major effect on decreasing the size of P. pallidurn PP28S fruiting bodies (approximately three times more fruiting bodies were formed in the light compared to the dark), there was a barely detectable effect of light on cell patterning. This has relevance to the complexity of light responses at the molecular level in different slime mold genera. There are also some surprises in the cell patterning (percentage spores and stalk cells) in branched fruiting bodies of Polysph.ondy1iu.m pallidum. The first observation is that patterning may be the same in un-
Cell
I
600
c.
Cell
Number
Branches
. 20I
301 Stalk
Cell
40 I
.I
I
50
60
Number
FIG. 4. Spore-to-stalk-cell relationships of individual branched fruiting bodies differentiated in the dark at 22 f 0.5”C on water agar. Spore-to-stalk-cell relationship of: (A) whole fruiting bodies; (B) whorls; and (C) individual branches. The regression lines are included on each graph.
branched and branched fruiting bodies over a size range of 140 to 4142 cells. This is different from the cell patterning of D. discoideum in which small and large fruiting bodies have a greater percentage of stalk and basal disk cells than average-sized fruiting bodies (Stenhouse
STENHOUSEAND WILLIAMS
and Williams, 1977). We have no explanation for this difference between cell patterning in P. paMum and D. d,iscoidewm, although we note that D. discoidewm fruiting bodies are considerably larger than those of P. pa.llidwm. The range of fruiting body sizes examined in the study on D. discoideum. by Stenhouse and Williams (1977) was 620 to 18,000 cells. The above comments take a global view and do not consider the individual components of the branched fruiting bodies of P. pal1idu.m. A branched fruiting body can also be considered in terms of its components: a main stalk, terminal spore head, and a series of whorls, with between one and five branches, each of which can be considered to be a miniature fruiting body. We have shown here that the cell patterning in the branches, i.e., spore:stalk ratio of individual miniature fruiting bodies along the main stalk, is so poorly correlated that it seems to be barely regulated at all (Fig. 4C j, while small unbranched fruiting bodies of similar size have tightly correlated spore:stalk ratios (Fig. 3). If the cell patterning of whorls is considered (i.e., summing the spore and stalk cells of branches in each whorl), a correlation between the spore:stalk ratio is found (Fig. 4Bj, although it is still relatively poor. The cell patterning of the whorls (90% spores) is different from that of the whole fruiting bodies (78% spore) which include the large main stalk. The observation that the cell patterning of the whole branched fruiting body is tightly correlated (Fig. 4A) and is the same as that of unbranched fruiting bodies (Fig. 3), can be interpreted to indicate that cell patterning is decided at the whole aggregate level rather than at the level of the whorl or individual branch. Initially we thought this unlikely since P. pal1idu.m aggregates do not show prepatterning in the way D. discoideum aggregates do. In D. discoideum, prespore cells are identified by the presence of prespore vacuoles or prespore antigen (Takeuchi et al., 1977) and prestalk cells are identified by histochemical stains or neutral red staining (MacWilliams and Bonner, 1979). Similar experiments in P. pallidum detect almost entirely prespore cells in the aggregate on the basis of the presence of prespore vacuoles (Hohl et al., 1977) and the prespore antigen (O’Day, 1979). However, prestalk cells, which are mainly found at the front of the aggregate, have been found in a histochemical study (O’Day and Francis, 1973) and using neutral red (O’Day, 1979). As pointed out by O’Day (1979), this suggests that prespore vacuoles and the prespore antigen may not define the pattern in P. padlidum, since presumptive prestalk cells also contain these “spore specific” vacuoles and antigen. It has been shown recently that under some conditions D. discoideum amoebas destined to become stalk cells form some prespore vacuoles and then lose them (Kay
Cell Pa.tterning
in P. pallidurn
143
et al., 1978). While it is important to determine whether the percentage of cells stained with neutral red coincides with the stalk cell percentage, the findings of this study support the notion that the basic cell pattern is established in the whole P. pallidum aggregate. We suggest further that our finding that branched and unbranched fruiting bodies have the same cell patterning is consistent with the P. pall,idum. cell patterning being stable over a considerable time span. If the aggregate reestablished the cell pattern after each group of cells is lost from the main aggregate to form a whorl, the patterning of branched and unbranched fruiting bodies would be different. The high percentage of spores in the whorls is consistent with the formation of fruiting bodies in the branches of whorls from cells most of which are already predetermined to be spores (since most prestalk cells are at the front of the aggregate and the cells making up the whorl are lost from the rear). If this hypothesis is correct, the tips formed in the whorl which organize the individual branches have no cell patterning function but organize the morphogenetic movements of cells already committed to be stalk cells or spores. The poor correlation between spore:stalk ratios of whorls could be explained by variable proportions of prespore and prestalk cells dropped from the rear of the aggregate, while the essential absence of patterning in branches may reflect asynchrony in the morphogenetic movements (and hence capture) of cells by individual branch tips in each whorl.
We thank Robyn Smith for doing some of the data analysis, and Dr. Elizabeth Smith and Paul Fisher for helpful comments. Dr. D. Francis kindly provided P. pallidum strain PP28S.
REFERENCES BONNER,J. T. (1957). A theory of the control of differentiation in the cellular slime molds. Quart. Rev. Biol. 32, 232-246. BONNER,J. T., and DODD, M. R. (1962). Aggregation territories in the cellular slime molds. Biol. Bull. 122, 13-24. BONNER,J. T., and SLIFKIN, M. K. (1949). A study of the control of differentiation: The proportions of stalk and spore cells in the slime mold, Dictyostelium discoideum. Amer. J. Bat. 36, 727-734. CAVENDER,J. C., RAPER, K. B., and NORBERG,A. M. (1979). Dictyotenue: New species of the stelium aureo-stipes and Dictyostelium Dictyosteliaceae. Amer. J. Bot. 66, 207-217. FRANCIS,D. (1975). Macrocyst genetics in Polysphondy1iu.m pallidurn, a cellular slime mould. J. Gen. Microbial. 89, 310-318. HARPER, R. A. (1932). Organisation and light relations in Polysphondylium.
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Bot. Club 69,49-&L
HOHL, H. R., HONEGGER,R., TRAUB, F., and MARKWALDER,M. (1977). Influence of CAMP on cell differentiation and morphogenesis in Polysphondylium. In “Development and Differentiation in the Cellular Slime Moulds” (P. Cappuccinelli and J. M. Ashworth, eds.), pp. 149-172. Elsevier/North-Holland, New York.
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KAHN, A. J. (1964). The influence of light on cell aggregation in Polgsphondylium
pallidum.
Biol. Buti
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KAY, R. R., GARROD,D., and TILLY, R. (1978). Requirements for cell differentiation in Dictyostelium discoideum. Nature (London) 271, 58-60. KONIJN, T. M., and RAPER, K. B. (1965). The influence of light on the time of cell aggregation in the Dictyosteliaceae. Biol. Bull. 128, 392-400. MACWILLIAMS,H. K., and BONNER,J. T. (1979). The prestalk-prespore pattern in cellular slime molds. Differentiation 14, l-22. NETER, J., and WASSERMAN,W. (1974). “Applied Linear Statistical Models.” Richard D. Irwin Inc., Illinois. O’DAY, D. H. (1979). Cell differentiation during fruiting body formation in Polysphondylium pal1idu.m. J. Cell Sci. 35, 203-215. O’DAY, D. H., and FRANCIS,D. W. (1973). Patterns of alkaline phosphatase activity during alternative developmental pathways in the
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cellular slime mold. Polysphondylium pallidum. Canad. J. Zool. 51, 301-310. RAPER,K. B. (1935). Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agr. Res. (Washington) 50, 135-147. RAPER, K. B., and QUINLAN, M. S. (1958). Acytostelium leptosmum: A unique cellular slime mold with an acellular stalk. J. Gen. Microbiol. 18, 16-32. STENHOUSE,F. O., and WILLIAMS, K. L. (1977). Patterning in Dictyostelium discoideum: The proportions of the three differentiated cell types (spore, stalk and basal disk) in the fruiting body. Develop. Biol. 59, 140-152. TAKEUCHI, I., HAYASHI, M., and TASAKA, M. (1977). Cell differentiation and pattern formation in Dictyostelium. In “Development and Differentiation in the Cellular Slime Moulds” (P. Cappuccinelli and J. M. Ashworth, eds.), pp. 1-16. Elsevier/North-Holland, New York.