DEVELOPMENTAL
BIOLOGY,
Induction
44,239-t&%?(1975)
of Stalk Cell Differentiation
Susceptible
Variant
by Cyclic-AMP
of Dictyostelium
in a
Discoideum
WOON KIM CHIA’ Department
of
Biology, Princeton University, Princeton, New Jersey 08540 Accepted January 22, 1975
The P-4 variant of Dictyostelium discoideum (DdH) was found to produce a great excess of stalk cells compared to the wild type DdH. If the vegetative cells of P-4 were repeatedly washed, the variant changed back to the wild type phenotype, and if cyclic-AMP was added to the washed P-4 cells, the variant character was restored. Furthermore, if the concentration of added cyclic-AMP was increased, it was possible to induce 100% stalk cells in P-4. Phosphodiesterase would cause the variant to change to the wild type, while 5 -AMP and cyclic-nucleotides other than cyclic-AMP have no effect at all. Therefore it was concluded that cyclic-AMP plays a key role in stalk cell differentiation. A comparison between wild type DdH and the variant P-4 showed that DdH is ten times less sensitive to cyclic-AMP induction. They both produce the same amount of cyclic-AMP and extracellular phosphodiesterase, but the specific activity of P-4 cell-bound phosphodiesterase during development is significantly less than that in the DdH. One hypothesis that accounts for the P-4-DdH difference is that because of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence more stalk cell differentiation.
properties of the variant, an investigation was made to determine in what way it differed from the wild type.
INTRODUCTION
After a period of growth, the separate amoebae of the cellular slime mold Dictyostelium discoideum aggregate to form communal cell masses. These pseudoplasmodia migrate for varying periods of time during which the anterior cells slowly begin to differentiate into stalk cells, and the posterior cells into spores. It is known that in this species cyclic-AMP acts as an attractant during aggregation (Konijn et al., 1967, 1969; Barkley, 1969) and that it also can induce stalk cell differentiation if added in high concentrations to preaggregation cells (Bonner, 1970). This is a study of a variant of D. discoideum that is shown to be especially susceptible to stalk cell induction by cyclic-AMP. Furthermore, it can be made to change back to the wild type phenotype if different methods are used to reduce the extracellular cyclic-AMP. Because of these 1 Present address: Singapore Science 42, Friend Hill, Depot Road, Singapore Singapore.
Centre, Block 5, Republic of
MATERIALS
METHODS
Two strains of cellular slime molds were used; the wild type of Dictyostelium discoideum, a haploid strain of NC-4 (DdH) and a strain derived from Petite-4, a variant of DdH produced by uv irradiation (Hohl and Raper, 1964). Since it no longer resembles the original Petite-4, but somehow had become altered after prolonged storage, we shall henceforth call the modified form P-4. These strains were kindly provided by Professors Raper and Hohl in their original form some years ago. The amoebae of P-4 were grown with Escherichia coli B/r on buffered 0.1% lactose-peptone agar (lactose, 1 g; peptone, 1 g; KH2P0,, 0.272 g; Na,HPO, .7Hz0, 0.536 g; agar, 15 g; H,O, 1000 ml). The wild type DdH was grown on 1% dextrose-peptone agar (peptone, 10 g; dextrose, 10 g; Na,HP0,.7H,O, 0.719 g; KH,PO,, 1.45 g; 239
Copyright 0 1975 by Academic Press,Inc. All rights of reproduction in any form reserved.
AND
240
DEVELOPMENTAL
BIOLOGY
agar, 20 g; H20, 1000 ml). After a period of from 36 to 40 hr incubation at 21 * l”C, the E. coli were almost completely consumed by the growing amoebae. To obtain an amoebae suspension relatively free of bacteria, the amoebae were washed off the plates with 1% Bonner’s salt solution: 6 mg of NaCl, 7.5 mg of KC1 and 3 mg of CaCl, in 1 liter of distilled water (Bonner, 1947). The amoebae were washed by centrifuging three times at 75g for 5 min, adding fresh salt solution after each washing. After washing, the yield of P-4 amoebae for one 0.1% lactose-peptone agar plate (100 x 15 mm petri dish) was approximately lx lo7 cells and one 1% dextrose-peptone agar plate of DdH gave in the order of 8 x lo7 cells. Growing vegetative amoebae in liquid culture. P-4 and DdH were also grown in liquid culture on autoclaved E. coli B/r using Hohl and Raper’s (1963) modification of the technique of Gerisch (1959). The E. coli B/r was first grown for three days in nutrient broth (tryptose, 5 g; yeast extract, 5 g; glucose, 1 g; K2HPOI, 1 g and 1000 ml of distilled water). The bacterial cells were harvested by centrifuging the culture at 408Og for 20 minutes and then washed twice in pH 6.1 Sdrensen’s phosphate buffer (l/60 M) by centrifugation as above. After the washes, the concentration of E. coli in pH 6.1 phosphate buffer was adjusted to give a suspension with 4 absorbance units at a wavelength of 550 nm. The suspension was then autoclaved 20 min at 25 lb/in*, cooled, and inoculated with slime mold spores. It was found that a 500 ml Erlenmeyer flask containing 200 ml of E. coli B/r suspension provided the best amoeba growth. When growing P-4, the inoculum size was 5 x lo5 spores in each of the flasks, and for DdH, 2.5 x lo5 spores. The flasks were then placed on a gyrotory shaker (160 rpm) for three days, by which time the amoebae had grown and almost consumed all the bacteria in the suspension. The amoebae were harvested by centrifuging at 75g for 5 min and resuspended
VOLUME
44, 1975
in fresh pH 6.1 Sgrensen’s phosphate buffer solution. Washing experiments. Amoebae of P-4 were harvested from growth plates, washed three times, resuspended in 1% Bonner’s salt solution and then divided into two aliquots: (a) In order to follow their development, three drops were placed onto a small petri dish (50 x 12 mm) containing 2%’ agar; (b) the remainder was spread evenly on large petri dishes (100 x 15 mm) also containing 2% agar. After 2 hr, the amoebae on the large plates were again washed off for another treatment of three washes by centrifugation, and the whole procedure was repeated a second and third time. By observing the small plates put aside after each wash it was possible to see the cumulative effects of the washing at each stage (Fig. 1). Drum experiment to measure cyclicAMPproduction. The acrasin produced by amoebae at different stages of development was collected using the drum technique (Bonner et al., 1969). A drum consists of two close-fitting plastic cylinders, 8 cm in diameter, placed in a 5 x 9 cm crystallizing dish. Between these two cylinders is a dialysis cellophane membrane (size, 1% in. Cat. No. 3787-D-52, Arthur H. Thomas Co., Philadelphia), pretreated by boiling 5 min in 10m3 M EDTA and then soaked in distilled water for two days. The amoebae were harvested from culture plates and placed on the upper side of the dialysis cellophane membrane. The dish was filled with 1% Bonner’s salt solution to the level of the under surface of the dialysis cellophane membrane. The acrasin, or cyclicAMP produced by the amoebae during their various stages of development can be collected by removing the solution below the membrane at one or two hour intervals. After removal it is boiled for 5 min, and evaporated to dryness at 45°C. The residue was resuspended in 2 ml of distilled water and then tested for cyclic-AMP activity, using the cellophane square chemotaxis test.
WOON
KIM CHIA
O.ILPAgar Amoebae washed off, centrifuged and resuspended 3 times
Induction
of Stalk
241
Cell Differentiation
Plate
[
’ --2
% Agar
b:] b -2 I
% Agar
+ Cyclic-AMP
1 :.> ::
-__----
____
f
if
Bacteria-free
---_---
amoebae
2 % Agar
’ -,
~i-Ep4BoodTi~sterose
First Treatment of Washing Plate 2 % Agar
Amoebae &shed off. centrifuged and ’ resuspended 3 times
i -------------------j @W Second Treatment of Washing If 2 % Agar + Cyclic-
:;:s: >. ::.
2 % Agar 2 hour: later Amoebae washed off, centrifuged and resuspended 3 times
AMP
Plate [ ’ 7,
2 % Agar
b:-% J
-------------_---_---
‘l
2%
Agar
Cyclic
AMP
Third Treatment of Washing FIG. 1. Diagrammatic treatments are shown.
representation of the treatment The results of these experiments
The cellophane square chemotaxis test. Acrasin activity was assayed by the method of Bonner et al., (1966). In brief, a cellophane square, covered with amoebae at the aggregation stage, is placed on agar containing the substance to be tested. The rate at which amoebae move out is proportional to the chemotactic activity of the substance. By simultaneously running a series of tests of amoebae with different concentrations of commercial cyclic-AMP, the rates can be used as a rough estimation
of the cells washed by centrifugation. are presented in Fig. 5.
Only
three
of the amount of cyclic-AMP secreted by the amoebae. Assay of phosphodiesterase. The activity of soluble and cell-bound phosphodiesterase in a liquid culture of amoebae was assayed with a method in which the breakdown of cyclic-AMP to adenosine is measured (Brooker et al., 1968; Monard et al., 1969). The principle of this assay is that the phosphodiesterase from amoebae will degrade 3H-3’,5’-cylcic-AMP to 3H-5’AMP and 5’-nucleotidase will in turn con-
242
DEVELOPMENTAL
BIOLOGY
vert the 3H-5’-AMP to 3H-adenosine. The anion exchange resin binds and quenches the radioactivity of 3H-labeled cyclic-AMP but not of 3H-adenosine. Thus radioactivity of 3H-adenosine measured is proportional to the activity of phosphodiesterase. To separate the soluble phosphodiesterase from cell-bound phosphodiesterase, 10 ml of well mixed liquid culture of amoebae from the culture flask was centrifuged at 75g for 7 min, followed by 30 set at ll,OOOg. The supernatant (supernatant I) which contains all the soluble phosphodiesterase was separated and the amoebae in the pellet were washed in fresh Sgrensen’s phosphate buffer solution (l/60 M, pH 6.1) once, centrifuged again, and resuspended in fresh buffer solution. Cyclic-AMP (Calbiochem) and radioactive 3H-cyclic-AMP (Schwarz/Mann, Orangeburg, New York; Specific Activity = 28 C/nmole, Concentration = 0.5 mC/ml) were added to each of the 10 ml of supernatant I and 10 ml of amoebae buffer suspension to give a final cyclic-AMP concentration of 1O-4 M and 5 PC 3H (17.8 nn/r). The samples were incubated with shaking for 1 hr at 21 * 1°C and the reaction stopped by heating the reaction mixture to 100°C for 5 min. The amoebae suspension was immediately centrifuged (ll,OOOg for 30 set), and the supernatant (supernatant II) kept for assay. After cooling, supernatants I and II were adjusted to pH 7.8 with 0.1 N NaOH and brought to 4 x lo-’ it4 MgCl,. One mg of 5’-nucleotidase (snake venom, Ross Allen’s Reptile Institute, Inc., Silver Springs, FL) was added to 0.5 ml of the adjusted supernatant I and II and incubated for 30 min at 37°C. The hydrolysis was terminated by the addition of 2 ml of absolute ethanol containing 300 mg of Dowex-l-Cl (l-x8, 200-400 mesh, Calbiothem). After 10 min, another 2 ml of absolute ethanol was added, followed by 5 ml of the toluene scintillation cocktail containing 12 g of 2,5-diphenyloxazole (PPO) and 0.4 g of 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) per liter of tolu-
VOLUME
44,
1975
ene. The samples were counted in a liquid scintillation counter. The following controls for the assay were run in parallel: 3H-cyclic-AMP, 3H-5’-AMP and 3H-adenosine with and without Dowex, and also in combination with and without snake venom 5’-nucleotidase. Preparation of samples for microscopy. Most of the observations on the morphology of development were done directly with a binocular microscope and a compound phase microscope. When sectioned material was required, a method of plastic embedding of Feder and O’Brian (1968) was used. The cell masses were fixed for two hours at 4’C in 0.4% paraformaldehyde solution buffered at pH 7 (S$rensen’s buffer, l/60 M). Samples were dehydrated by sequential treatment for 12 hr at 4’C in ethylene glycol monoethyl ether, ethanol, propanol and butanol (all loo%), and then transferred to a monomer mixture which consisted mainly of glycol methacrylate (Hartung Associates, Camden, New Jersey) in which were dissolved 0.6% (w/v) of 2,2-azobis (2-methylpropionitrile) (Eastman Kodak Co., Rochester, New York) and 6% (v/v) polyethylene glycol 400 (Fisher). To ensure complete infiltration, the preparations were kept in the monomer mixture for a week during which the monomer mixture was changed twice. After infiltration they were transferred to No. 00 gelatin capsules and a freshly prepared glycol methacrylate mixture added to fill the capsule. These capsules were then polymerized by heating in an oven at 40°C for two days. Sections of 2 pm thickness were cut with a MT-l Sorvall “Porter-Blum” ultramicrotome and stained with toluidine blue (0.025 g/liter of distilled water) for 20 min. RESULTS
The development of P-4 The original strain of the P-4 variant of Dictyostelium discoideum (DdH) produced morphologically normal but smaller fruit-
WOON KIM
CHIA
Znductio
ing bodies than the wild type (Hohl and Raper, 1964). This strain, which has been kept for 8 yr in our laboratory, has acquired an additional character: it undergoes three types of development. When P-4 is grown on 0.1% lactose-peptone agar, three types of fruiting bodies are always produced (Fig.
Vegetative
n
of Stalk
243
Cell Differentiation
2). Type (1) is the normal but smaller fruiting body described by Hohl and Paper (1964) in the original strain, type (2) has a thickened stalk, and type (3) is what we call “stalk cell bumps”, which consist of a sphere of stalk cells with no spores at all. This was confirmed by inoculating these
Amoeboe A Primary
Aggregate
An Aggregate
Secondary
. P-F&
.___.._
Aggregates
Reudoplkmodia
A Pseudoplasmodium
A Mature
Dictvostelium
Fruiting
Body
discoideum
3 Types
of
Fruiting
Bodies
Petite - 4
FIG. 2. Pattern of pseudoplasmodium and fruiting body formation in DdH and P-4. (1) Normal body of P-4. (2) Fruiting body with a thick stalk of P-4. (3) Stalk cell bumps of P-4, and (4) Normal body of DdH.
fruiting fruiting
244
DEVELOPMENTAL
BIOLOGY
bumps with E. coli B/r on 0.1% lactosepeptone agar to see whether there are any spores which will germinate and give rise to a population of amoebae. Twenty bumps have been tested and no growth of amoebae has been detected. A fixed thin section of a typical stalk cell bump is shown in Fig. 3. Another difference between DdH and P-4 should be mentioned here. In DdH, at certain cell densities, each aggregate will form one pseudoplasmodium which in turn develops into one fruiting body. In P-4, in spite of the fact that the territory size of each aggregate is much larger than that of DdH, the P-4 aggregate subdivides into
VOLUME
44, 1975
secondary centers (Fig. 2). Hence in P-4, the primary aggregate always ends up with many smaller aggregates, each of which forms one pseudoplasmodium. Each of these pseudoplasmodia will then give rise to one of the three types of fruiting bodies. Under normal growing conditions on 0.1% lactose-peptone agar, each aggregate, depending on its size, gives roughly 50 to 200 fruiting bodies on a 50 x 12 mm petri dish. On the average 48% of these pseudoplasmodia will form stalk cell bumps, 13%) give rise to thick stalk fruiting bodies, and 39% develop into smaller, but normal fruiting bodies.
FIG. 3. A thin section of a stalk cell bump. (See materials and methods.) phase contrast. The vacuoles show as flat opaque areas separated by thick
The section is shown unstained, cellulose walls (x 117).
in
WOON KIM
CHIA
hduction
One aggregate of P-4 will produce from 50 to over 100 pseudoplasmodia by the breaking up of the aggregating streams, and it can be clearly seen that there is a larger number of normal fruiting bodies near the center of the large aggregate than at the edge. In order to study this phenomenon in greater detail, the aggregation territory was cut into various patterns, and the agar blocks were isolated (Fig. 4). The blocks were then observed to see how this affects the relative proportion of the three types of fruiting bodies in the different areas. In one such experiment an entire aggregate was cut out from the petri dish and further subdivided into 3 concentric rings (Fig. 4A). Cells on these three rings were allowed to develop separately and the number of each of the three types of fruiting bodies were scored. Nine such experiments gave consistent results; as can be seen from the table in Fig. 4, the central region of an aggregate produces more normal fruiting bodies, and their number decreases as one moves peripherally. In contrast, this unequal distribution disappeared if the agar was cut into rectangular shapes, each of which supported portions of
of Stalk
245
CellDifferentiation
just one stream of the aggregate (Fig. 4B). In this case, all the fruiting bodies from each block are normal. Still another difference between P-4 and DdH is that the amoebae and the spores of P-4 are larger than DdH. When amoebae are placed in a low concentration of Bonner’s salt solution (l%), the amoebae round up and thus their diameter can be measured accurately. It was found that the diameter of P-4 amoebae is 15.15 * 1.6 pm (SD) (52 samples) while the diameter of DdH amoebae is 8.8 i 1.23 pm (SD) (48 samples). The mean spore length of P-4 is 11.2 * 1.86 pm (SD) and in DdH 6.75 i 0.99 lrn (SD) (60 samples each). Therefore each P-4 amoeba has approximately 3 times more surface area and five times more volume than a DdH amoeba (this becomes important when we later compare the cell-bound phosphodiesterase of the mutant and the wild type). In order to see if P-4 was a homogenous cell line or a mixture of two or more genotypes, the amoebae were cloned. It was found that every P-4 clone (40 isolates) isolated from a single spore produced the three types of fruiting body characteristic of P-4. B
A
Normal
Ring-
3
Fruiting Bodies
P
Thick Stalk Fruiting Bodies Stalk Cell Bumps
/a
36 %
62
P
IO %
I0 %
23 %
_L
57%
52 %
15 %
33
-a
FIG. 4. The isolated agar block experiment: A. The P-4 aggregate one stream of the P-4 aggregate was cut into rectangular blocks.
%
100%
100%
was cut into three
100%
concentric
100%
rings,
and B.
246
DEVELOPMENTAL
BIOLOGY
Attempts to influence the proportion of normal fruiting bodies in P-4 (a) The effect of washing. The amoebae of P-4 were given successive washings by the centrifugation technique previously described (Fig. 1) during a period of 10 hr. After each treatment a few cells were allowed to develop on 2% agar. The result was striking: there was a progressive increase in the proportion of normal fruiting bodies (this was a qualitative observation, but the result was clear-cut). This was repeated numerous times with similar results: by the fourth and fifth washing, over 90% of fruiting bodies were normal (Fig. 5). This experiment suggests that a substance which induces stalk cells is removed by the washing procedure. In order to determine if the substance is dialyzable, the drum technique (used for cyclic-AMP collection as previously described) was used. The P-4 amoebae were placed on the side of a dialysis membrane upper stretched on a drum over a large reservoir of salt solution. It was assumed that if an inducer of small molecular weight was secreted by the amoebae which could diffuse through the dialysis membrane into the salt solution in the dish, then frequent removal of the salt solution means removal of the inducer. The result was that if the solution under the membrane was replaced
FIG.
5. Results
of various
experiments
on P-4 after
VOLUME
44, 1975
at repeated intervals (five times, once every 2 hr), then more than 90% of the fruiting bodies were normal (repeated three times with the same result). As a control, the salt solution was not replaced and the fruiting bodies were largely of the abnormal character. Clearly the stalk cell inducer can be effectively removed through a dialysis membrane. (b) Effect of cyclic-AMP on development of washed P-4 amoebae. The purpose of this experiment was to find out whether or not externally applied cyclic-AMP would reverse the effect of washing. Washed P-4 amoebae were placed on petri dishes containing agar supplemented with 10m5, 10e6 or 10m7 M cyclic-AMP. Whereas washed controls showed an increase of normal fruiting bodies through repeated washings, there was a significant increase in proportion (80 to 85%) of bumps in response to cyclic-AMP (Fig. 5). (c) Effect of cyclic-AMPphosphodiesterase on development of P-4 amoebae. Since the proportion of stalk cell bumps in P-4 can be increased by cyclic-AMP and 5’-AMP is ineffective, and phosphodiesterase degrades 3’,5’-cyclic-AMP to 5’-AMP, the induction of bumps was also examined with phosphodiesterase in the agar. P-4 amoebae were harvested and placed on a petri dish containing 0.2 units of phosphodiesterase (1 unit converts 1 /Imole of
successive
washings
and placed
on 2% agar plates.
WOON KIM
CHIA
Induction
3’,5’-cylcic-AMP to 5’-AMP; Sigma Company) in 2% agar. Although the P-4 amoebae had not been washed except during harvesting (which had little effect), their development on the phosphodiesterase agar was similar to those that had been extensively washed for 5 treatments: in both cases over 90% of the fruiting bodies were normal (Fig. 5). Attempts to increase the proportion of stalk cell bu’mps using the drum technique
(a) Effect of high concentrations of cyclic-AMP on development of P-4 amoehue. In this series of drum experiments, P-4 amoebae were permitted to develop on dialysis membrane in contact with 1% salt solution containing various concentrations of cyclic-AMP. Again, the cyclic-AMP in the salt solution affected the P-4 development by increasing stalk cell bumps and inhibiting normal fruiting body formation (Fig. 6). At concentrations of 10e4 and lo- 5 M all the cells turn into small stalk cell bumps (Fig. 7). As the concentration of cyclic-AMP was decreased from 1O-5 M to 10e6 M, the number of normal fruiting bodies progressively increased as the concentration was further lowered to 1O-7 M. A concentration of 10eB M cyclic-AMP had little effect on P-4 development: all
Cyclic-
,o-3M
,o-4w
AMP
P-4
Inhibited
of Stalk
247
Cell Differentiation
three types of fruiting bodies were formed. (b) Effect of high concentrations of cyclic-AMP amoebae. discoideum
on
development
The wild type (DdH), was treated identically for comparison. Cyclic-AMP also induces DdH amoebae to differentiate into stalk cell bumps on the drum, but the minimum concentration of cyclic-AMP necessary to produce 100% stalk cell bumps was lo-‘M, ten times more than needed for P-4 (Fig. 6). The concentration of lo-$ M cyclicAMP also delays the development of DdH for 12 hr and the amoebae develop into smaller but normal fruiting bodies. Another difference between the induced DdH bumps and P-4 bumps is that, unlike P-4, the DdH do not consist entirely of stalk cells. The center of the DdH bumps always consist of undifferentiated amoebae. (c) Effect of nucleotides other than cyclic-AMP on stalk cell induction. Adeno-
sine, 5’-AMP and some other cyclicnucleotides were tried on the development of P-4 and DdH using the drum technique. It is clear that only cyclic-AMP induced stalk cell differentiation (Fig. 8). It is interesting to note that 3’,5’-cyclic-CMP does reduce the territory size in DdH and smaller fruiting bodies are produced, while 3’,5’-cyclic-UMP does the same for both in
10-5M
100%
100%
Bumps
Bumps
I
Increase
1Increase
Norma
FIG.
6. Effect
of cyclic-AMP
on the development
of DdH of P-4, D.
of P-4 and DdH
on drums.
I
248
DEVELOPMENTAL
BIOLOGY
VOLUME
44, 1975
P-4 and in DdH. Finally, 3’,5’-cyclic-GMP has no effect on DdH but it slightly increased the proportion of the normal fruiting bodies and repressed the formation of stalk cell bumps in P-4.
concentration of extracellular cyclic-AMP increases during early aggregation and reaches its peak at late aggregation, decreasing rapidly at the end of the aggregation.
Cyclic-AMP production of P-4 development
Specific activities of phosphodiesterase in liquid culture of P-4 and DdH
during
the course
The cyclic-AMP produced by P-4 was collected by removing the salt solution from under the drum cellophane membrane at regular intervals while the amoebae developed on the upper surface. Since cyclic-AMP is heat stable, the large quantity of salt solution (400 ml from five drums) collected every 2 hours was evaporated to dryness at 45°C and resuspended in 2 ml of distilled water. It was then assayed for cyclic-AMP using the cellophane square test for chemotaxis. The
Since previous results show that (a) P-4 produces an excess of stalk cells and (b) P-4 is 10 times more sensitive than DdH to cyclic-AMP induction of stalk cells, it is important to find out whether there are differences between DdH and P-4 in cyclicAMP production or specific activity of phosphodiesterase. Comparison of the cyclic-AMP production in P-4 with the cyclic-AMP production in DdH (Bonner et al., 1969; Bonner, unpublished results) shows that P-4 and DdH produce the same
FIG. 7. A. Stalk cell bumps induced by lo-’ M cyclic-AMP on the dialysis machine Three stalk cell bumps and some individual stalk cells induced by lo-’ M cyclic-AMP under phase contrast (x 200).
in a drum (x 22). B. in the drum as seen
WOON KIM
Control I % Solt Solution Normal 3 Types of Fruitin< Bodies
$-AMP lO-4 Normal
CHIA
Induction
lO-4 lormal
-I
Cyclic Cyclic 3; 5’ - AMP ;, 5’- GMF
Idenosinc M
of Stalk
M
lO-4
M
lO-4
tvl
Cyclic Cyclic Cyclic 3-, 5’- UM P: i, 5’- CMF i, 5’- TMF 10-4M
lo-4
Cyclic j,dIMF
ld4M
lO-4
Normal
Normal
u
Lk!
M
Fewer Normal Stalk Cel I
but Smolle Fruiting Bodies
P-4
249
Cell Differentiation
M
k%fe of Norm< II but $nallt 9r F;;fFsg
? u Normal 1 Type of Frultint Body
Vormol
Jormal
lJormol IUormal Vormal but Smalle Sl ti Smaller Fruiting IFruiting 3odies I3odies
Normal
DdH
FIG.
8. The effect of 5’-AMP,
adenosine
and other
cyclic-nucleotides
amount of cyclic-AMP. A time course study was made of the phosphodiesterase specific activity in DdH and P-4 grown in liquid culture (on autoclaved E. coli B/r). The reason for using liquid culture amoebae are: (a) it provides a large quantity of amoebae, (b) the amoebae are better synchronized in their development, and (c) it is easier to separate and determine the two kinds of phosphodiesterase. It has been shown by Malchow et al. (1972) that DdH produces two kinds of phosphodiesterase: a soluble extracellular phosphodiesterase (Chang, 1968) and a membrane-bound phosphodiesterase. We measured the specific activity of these two phosphodiesterases in DdH and P-4 in liquid culture. The assay was started 8 hr before the amoebae had consumed all the bacteria and for 20 hr more, well past the time when aggregation would have occurred had they been growing on an agar surface. Both DdH and P-4 have two forms of phosphodiesterase. If one compares the
on P-4 and DdH
developing
on a drum.
specific activity of extracellular phosphodiesterase per unit area of cell surface over the 28-hr period in the two strains, they are remarkably close and show no significant difference (Fig. 9). However, the changes of specific activity of cell-bound phosphodiesterase per unit area of cell surface is significantly different in P-4 and DdH. The DdH cell-bound phosphodiesterase begins to rise early (2 hr) and peaks late (23 hr), while P-4 rises late (15 hr) and peaks almost immediately (18 hr). Furthermore, the amount of activity is consistently higher for DdH than P-4 (Fig. 9). These results for the wild type are similar to the results of previous workers for both the extracellular phosphodiesterase (Riedel and Gerisch, 1971; Bonner et al., 1972) and the cell bound phosphodiesterase (Malchow, et al., 1972). There are such major differences in techniques involved (e.g., different strains of D. discoideum) that it is not surprising that the absolute values do not exactly correspond. The surface area of the P-4 amoeba is
DEVELOPMENTAL 1.0
BIOLOGY
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r
Culture 0
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Cleared J! 8
of E.d IO
14 TIME
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30
HOURS
FIG. 9. Phosphodiesterase specific activities of DdH and P-4 in liquid culture. Cell-bound phosphodiesterase of DdH, (A); of P-4 (0). Soluble phosphodiesterase of DdH, (A); of P-4, (0). Since P-4 cells are larger than DdH cells the specific activity of the enzyme is calculated on the basis of the equivalent surface area for the two types. In other words, 1 x 10s DdH cells has the equivalent surface area of a third as many P-4 cells.
three times larger than the DdH amoeba. This means that 3.33 x lo5 P-4 cells is equal in total cell surface area to 10” DdH cells. To correct for this, the specific activity of phosphodiesterase is defined as enzyme activity per lo6 DdH cells, and 3.33 x lo5 P-4 cells. If cell volume is used instead of total cell surface area, then the relative values of both phosphodiesterases are lower for P-4. Therefore, calculating the phosphodiesterase specific activity either way shows that the cell-bound form is invariably lower in the variant than in the wild type. DISCUSSION
One of the key problems of development is the proportional differentiation of different cell types in a tissue, organ or in a whole organism. From the early work in the cellular slime molds it is known that a stable proportionality exists between stalk cells and spores over a wide range of
fruiting body sizes (Harper, 1932; Raper, 1935; Bonner, 1967). One way to approach the problem of proportionality is to treat the slime molds with different chemicals to see if one can shift the balance towards more spores, or more stalk cells. Another approach is to look for mutants (variants) with altered proportionality. In this study both of these methods have been employed. The P-4 variant of D. discoideum (DdH) normally has a great excess of stalk cells in a population of fruiting bodies. It is interesting to note that the spore-stalk cell ratio is not shifted equally in all pseudoplasmodia, but some are normal in their proportions, some have an abnormally high percent of stalk cells, and some are all stalk cells. I have found that if cells of this variant are washed, either by repeated centrifugations or by placing the amoebae on a cellophane drum so the water below can be replaced at intervals, then the
WOON
KIM
CHIA
Induction
variant changes phenotypically to the wild type. Since it has been shown that cyclic-AMP will induce DdH to form an excess of stalk cells (Bonner, 1970), this substance was added to washed P-4 cells. We found that that variant character was restored. Moreover, with increasing cyclic-AMP concentration, it was possible to obtain 100% stalk cells on the surface of the drum. This and the results obtained with 5’-AMP, adenosine and phosphodiesterase suggest that cyclic-AMP is the stalk cell inducer. Since both DdH and P-4 amoebae secrete cyclicAMP and phosphodiesterase, it is likely that cyclic-AMP is the natural inducer of stalk cells in cellular slime molds, and that the balance of phosphodiesterase to cyclicAMP production is crucial for the production of any particular phenotypic condition. Further evidence for this comes from the following observations: In the experiments of isolating agar blocks bearing P-4 aggregation streams (Fig. 4A), the central ring has a higher proportion of wild type phenotype because of the higher cell density and therefore the higher phosphodiesterase concentration. In the outer ring with a lower cell density, and thus less phosphodiesterase, there is an increase in the percent of stalk cell bumps. The cutting of agar into small rectangular blocks (Fig. 4B) is another way of increasing cell density and phosphodiesterase production; the result is an increase of normal fruiting bodies to 100%. Since millipore filters placed on pads in petri dishes are used in other laboratories (Sussman, 1966) as a means of supporting differentiating cellular slime molds, this method (using millipores PHWP 04700, average pore size 0.3 pm, Millipore Corporation) was also tried in the induction of P-4 stalk cells using cyclic-AMP. A range of concentrations of amoebae and of cyclicAMP were used, and they included concentrations known to produce stalk cell differentiation both on drums and on agar
of Stalk
Cell Differentiation
251
plates, but stalk cell induction never occurred under these conditions. It is presumed that since the millipore filter is permeable to macromolecules, the phosphodiesterase quickly converts any cyclicAMP to the inactive 5’-AMP. Comparing the experimental conditions of millipore filter, the agar plate, and the drum, one can only conclude that the total reservoir of liquid, and the distance over which diffusion can occur, is very much less in the millipore culture dishes, and as a result the cyclic-AMP is more quickly and effectively eliminated. The variant and wild type were compared with respect to their sensitivity to cyclic-AMP, cyclic-AMP production and the specific activity of extracellular and cell-bound phosphodiesterase. The P-4 variant is approximately 10 times more sensitive to induction of stalk cells by cyclic-AMP than the wild type. The cyclicAMP and the extracellular phosphodiesterase was the same for both, but the variant had significantly less cell-bound phosphodiesterase. One hypothesis that accounts for the P-4 - DdH difference is that as a result of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence the excess stalk cell production. This hypothesis would also account for the increased effectiveness of low concentrations of cyclicAMP in the induction of stalk cells in the variant as compared to the wild type. The study reported here is part of my Ph.D. thesis work. I express my gratitude and appreciation to Professor John Tyler Bonner for his advice. This work was supported by the National Science Foundation Grant GM 33439, and National Institutes of Health Grant GM 17856. I also benefitted from the central equipment facilities in the Department of Biology, Princeton University, supported by the Whitehall Foundation. REFERENCES D. Identification slime mold.
BARKLEY,
S.
1969. Adenosine-3’,5’-phosphate: as acrasin in a species of cellular Science 165, 1133-1134.
252
DEVELOPMENTAL
BIOLOGY
BONNER, J. T. 1947. Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. 2001. 106, 1-26. BONNER, J. T. 1967. “The Cellular Slime Molds,” Second edition. Princeton University Press, Princeton, NJ. BONNER, J. T. 1970. Induction of stalk cell differentiation by cyclic AMP in the cellular slime mold Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 65, 110-113. BONNER, J. T., BARKLEY, D. S., HALL, E. M., KONIJN, T. M., MASON, J. W., O’KEEFE III, G., and WOLFE, P. B. 1969. Acrasin, acrasinase, and the sensitivity to acrasin in Dictyostelium discoideum. Deuelop. Biol. 20, 72-87. BONNER, J. T., HALL, E. M., NOLLER, S., OLESON, JR., F. B., and ROBERTS, A. B. 1972. Synthesis of cyclic AMP and phosphodiesterase in various species of cellular slime molds and its bearing on chemotaxis and differentiation. Deuelop. Biol. 29, 402-409. BONNER, J. T., KELSO, A. P., and GILLMOR, R. G. 1966. A new approach to the problem of aggregation in the cellular slime molds. Biol. Bull. 130, 28-42. BROOKER, G., THOMAS, L. J., JR., and APPLEMAN, M. M. 1968. The assay of adenosine 3’,5’-cyclic-monophosphate and guanosine 3’,5’-cyclic monophosphate in biological material by enzymatic radioisotopic displacement. Biochem. 7, 4177-4181. CHANG, Y. Y. 1968. Cyclic 3’,5’-adenosine monophosphate phosphodiesterase produced by the slime mold Dictyostelium discoideum. Science 160, 57-59. FEDER, N. and O’BRIAN, T. P. 1968. Plant microtechnique: some principles and new methods. Amer. J. Dot. 55, 123-142. GERISCH, G. 1959. Ein Submerskulturverfahren fur entwicklungsphysiologische Untersuchungen an Dictyostelium discoideum. Naturwis. 46, 654-656. HARPER, H. A. 1932. Organization and light relations
VOLUME
44, 1975
in Polysphondylium. Bull. Torrey Bot. Club 59, 49-84. HOHL, H. R. and RAPER, K. B. 1963. Nutrition of cellular slime molds, I. Growth on living and dead bacteria. J. Bact. 85, 191-198. HOHL, H. R. and RAPER, K. B. 1964. Control of sorocarp size in the cellular slime mold Dictyostelium discoideum. Deuelop. Biol. 9,137-153. KONIJN, T. M., CHANG, Y. Y., and BONNER, J. T. 1969. of cyclic-AMP in Dictyostelium Synthesis discoideum and Polysphondylium pallidum. Nature 224, 1211-1212. KONIJN, T. M., VAN DE MEENE, J. G. C., BONNER, J. T., and BARKLEY, D. S. 1967. The acrasin activity of adenosine-3’,5’-cyclic phosphate. Proc. Natl. Acad. Sci. U.S.A. 58, 1152-1154. MALCHOW, D., NXGELE, B., SCHWARZ, H., and GERISCH, G. 1972. Membrane-bound cyclic-AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur. J. Biochem. 28, 136-142. MONARD, D., JANECEK, J., and RICKENBERG, H. V. 1969. The enzymatic degradation of 3’,5’ cyclic AMP in strains of E. coli sensitive and resistant to catabolic repression. Biochem. Biophys. Res. Comm. 35, 584-591. RAPER, K. B. 1935. Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agric. Res. 50, 135-147. RIEDEL V., GERISCH, G., MULLER, E., and BEUG, H. 1973. Defective cyclic adenosine-3’,5’-phosphatephosphodiesterase regulation in morphogenetic mutants of Dictyostelium discoideum. J. Mol. Biol. 74, 573-585. SUSSMAN, M. 1966. Biochemical and Genetic Methods in the Study of Cellular Slime Mold Development. “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. II, pp. 397-410. Academic Press, New York and London.
BIOLOGY,
Induction
44,239-t&%?(1975)
of Stalk Cell Differentiation
Susceptible
Variant
by Cyclic-AMP
of Dictyostelium
in a
Discoideum
WOON KIM CHIA’ Department
of
Biology, Princeton University, Princeton, New Jersey 08540 Accepted January 22, 1975
The P-4 variant of Dictyostelium discoideum (DdH) was found to produce a great excess of stalk cells compared to the wild type DdH. If the vegetative cells of P-4 were repeatedly washed, the variant changed back to the wild type phenotype, and if cyclic-AMP was added to the washed P-4 cells, the variant character was restored. Furthermore, if the concentration of added cyclic-AMP was increased, it was possible to induce 100% stalk cells in P-4. Phosphodiesterase would cause the variant to change to the wild type, while 5 -AMP and cyclic-nucleotides other than cyclic-AMP have no effect at all. Therefore it was concluded that cyclic-AMP plays a key role in stalk cell differentiation. A comparison between wild type DdH and the variant P-4 showed that DdH is ten times less sensitive to cyclic-AMP induction. They both produce the same amount of cyclic-AMP and extracellular phosphodiesterase, but the specific activity of P-4 cell-bound phosphodiesterase during development is significantly less than that in the DdH. One hypothesis that accounts for the P-4-DdH difference is that because of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence more stalk cell differentiation.
properties of the variant, an investigation was made to determine in what way it differed from the wild type.
INTRODUCTION
After a period of growth, the separate amoebae of the cellular slime mold Dictyostelium discoideum aggregate to form communal cell masses. These pseudoplasmodia migrate for varying periods of time during which the anterior cells slowly begin to differentiate into stalk cells, and the posterior cells into spores. It is known that in this species cyclic-AMP acts as an attractant during aggregation (Konijn et al., 1967, 1969; Barkley, 1969) and that it also can induce stalk cell differentiation if added in high concentrations to preaggregation cells (Bonner, 1970). This is a study of a variant of D. discoideum that is shown to be especially susceptible to stalk cell induction by cyclic-AMP. Furthermore, it can be made to change back to the wild type phenotype if different methods are used to reduce the extracellular cyclic-AMP. Because of these 1 Present address: Singapore Science 42, Friend Hill, Depot Road, Singapore Singapore.
Centre, Block 5, Republic of
MATERIALS
METHODS
Two strains of cellular slime molds were used; the wild type of Dictyostelium discoideum, a haploid strain of NC-4 (DdH) and a strain derived from Petite-4, a variant of DdH produced by uv irradiation (Hohl and Raper, 1964). Since it no longer resembles the original Petite-4, but somehow had become altered after prolonged storage, we shall henceforth call the modified form P-4. These strains were kindly provided by Professors Raper and Hohl in their original form some years ago. The amoebae of P-4 were grown with Escherichia coli B/r on buffered 0.1% lactose-peptone agar (lactose, 1 g; peptone, 1 g; KH2P0,, 0.272 g; Na,HPO, .7Hz0, 0.536 g; agar, 15 g; H,O, 1000 ml). The wild type DdH was grown on 1% dextrose-peptone agar (peptone, 10 g; dextrose, 10 g; Na,HP0,.7H,O, 0.719 g; KH,PO,, 1.45 g; 239
Copyright 0 1975 by Academic Press,Inc. All rights of reproduction in any form reserved.
AND
240
DEVELOPMENTAL
BIOLOGY
agar, 20 g; H20, 1000 ml). After a period of from 36 to 40 hr incubation at 21 * l”C, the E. coli were almost completely consumed by the growing amoebae. To obtain an amoebae suspension relatively free of bacteria, the amoebae were washed off the plates with 1% Bonner’s salt solution: 6 mg of NaCl, 7.5 mg of KC1 and 3 mg of CaCl, in 1 liter of distilled water (Bonner, 1947). The amoebae were washed by centrifuging three times at 75g for 5 min, adding fresh salt solution after each washing. After washing, the yield of P-4 amoebae for one 0.1% lactose-peptone agar plate (100 x 15 mm petri dish) was approximately lx lo7 cells and one 1% dextrose-peptone agar plate of DdH gave in the order of 8 x lo7 cells. Growing vegetative amoebae in liquid culture. P-4 and DdH were also grown in liquid culture on autoclaved E. coli B/r using Hohl and Raper’s (1963) modification of the technique of Gerisch (1959). The E. coli B/r was first grown for three days in nutrient broth (tryptose, 5 g; yeast extract, 5 g; glucose, 1 g; K2HPOI, 1 g and 1000 ml of distilled water). The bacterial cells were harvested by centrifuging the culture at 408Og for 20 minutes and then washed twice in pH 6.1 Sdrensen’s phosphate buffer (l/60 M) by centrifugation as above. After the washes, the concentration of E. coli in pH 6.1 phosphate buffer was adjusted to give a suspension with 4 absorbance units at a wavelength of 550 nm. The suspension was then autoclaved 20 min at 25 lb/in*, cooled, and inoculated with slime mold spores. It was found that a 500 ml Erlenmeyer flask containing 200 ml of E. coli B/r suspension provided the best amoeba growth. When growing P-4, the inoculum size was 5 x lo5 spores in each of the flasks, and for DdH, 2.5 x lo5 spores. The flasks were then placed on a gyrotory shaker (160 rpm) for three days, by which time the amoebae had grown and almost consumed all the bacteria in the suspension. The amoebae were harvested by centrifuging at 75g for 5 min and resuspended
VOLUME
44, 1975
in fresh pH 6.1 Sgrensen’s phosphate buffer solution. Washing experiments. Amoebae of P-4 were harvested from growth plates, washed three times, resuspended in 1% Bonner’s salt solution and then divided into two aliquots: (a) In order to follow their development, three drops were placed onto a small petri dish (50 x 12 mm) containing 2%’ agar; (b) the remainder was spread evenly on large petri dishes (100 x 15 mm) also containing 2% agar. After 2 hr, the amoebae on the large plates were again washed off for another treatment of three washes by centrifugation, and the whole procedure was repeated a second and third time. By observing the small plates put aside after each wash it was possible to see the cumulative effects of the washing at each stage (Fig. 1). Drum experiment to measure cyclicAMPproduction. The acrasin produced by amoebae at different stages of development was collected using the drum technique (Bonner et al., 1969). A drum consists of two close-fitting plastic cylinders, 8 cm in diameter, placed in a 5 x 9 cm crystallizing dish. Between these two cylinders is a dialysis cellophane membrane (size, 1% in. Cat. No. 3787-D-52, Arthur H. Thomas Co., Philadelphia), pretreated by boiling 5 min in 10m3 M EDTA and then soaked in distilled water for two days. The amoebae were harvested from culture plates and placed on the upper side of the dialysis cellophane membrane. The dish was filled with 1% Bonner’s salt solution to the level of the under surface of the dialysis cellophane membrane. The acrasin, or cyclicAMP produced by the amoebae during their various stages of development can be collected by removing the solution below the membrane at one or two hour intervals. After removal it is boiled for 5 min, and evaporated to dryness at 45°C. The residue was resuspended in 2 ml of distilled water and then tested for cyclic-AMP activity, using the cellophane square chemotaxis test.
WOON
KIM CHIA
O.ILPAgar Amoebae washed off, centrifuged and resuspended 3 times
Induction
of Stalk
241
Cell Differentiation
Plate
[
’ --2
% Agar
b:] b -2 I
% Agar
+ Cyclic-AMP
1 :.> ::
-__----
____
f
if
Bacteria-free
---_---
amoebae
2 % Agar
’ -,
~i-Ep4BoodTi~sterose
First Treatment of Washing Plate 2 % Agar
Amoebae &shed off. centrifuged and ’ resuspended 3 times
i -------------------j @W Second Treatment of Washing If 2 % Agar + Cyclic-
:;:s: >. ::.
2 % Agar 2 hour: later Amoebae washed off, centrifuged and resuspended 3 times
AMP
Plate [ ’ 7,
2 % Agar
b:-% J
-------------_---_---
‘l
2%
Agar
Cyclic
AMP
Third Treatment of Washing FIG. 1. Diagrammatic treatments are shown.
representation of the treatment The results of these experiments
The cellophane square chemotaxis test. Acrasin activity was assayed by the method of Bonner et al., (1966). In brief, a cellophane square, covered with amoebae at the aggregation stage, is placed on agar containing the substance to be tested. The rate at which amoebae move out is proportional to the chemotactic activity of the substance. By simultaneously running a series of tests of amoebae with different concentrations of commercial cyclic-AMP, the rates can be used as a rough estimation
of the cells washed by centrifugation. are presented in Fig. 5.
Only
three
of the amount of cyclic-AMP secreted by the amoebae. Assay of phosphodiesterase. The activity of soluble and cell-bound phosphodiesterase in a liquid culture of amoebae was assayed with a method in which the breakdown of cyclic-AMP to adenosine is measured (Brooker et al., 1968; Monard et al., 1969). The principle of this assay is that the phosphodiesterase from amoebae will degrade 3H-3’,5’-cylcic-AMP to 3H-5’AMP and 5’-nucleotidase will in turn con-
242
DEVELOPMENTAL
BIOLOGY
vert the 3H-5’-AMP to 3H-adenosine. The anion exchange resin binds and quenches the radioactivity of 3H-labeled cyclic-AMP but not of 3H-adenosine. Thus radioactivity of 3H-adenosine measured is proportional to the activity of phosphodiesterase. To separate the soluble phosphodiesterase from cell-bound phosphodiesterase, 10 ml of well mixed liquid culture of amoebae from the culture flask was centrifuged at 75g for 7 min, followed by 30 set at ll,OOOg. The supernatant (supernatant I) which contains all the soluble phosphodiesterase was separated and the amoebae in the pellet were washed in fresh Sgrensen’s phosphate buffer solution (l/60 M, pH 6.1) once, centrifuged again, and resuspended in fresh buffer solution. Cyclic-AMP (Calbiochem) and radioactive 3H-cyclic-AMP (Schwarz/Mann, Orangeburg, New York; Specific Activity = 28 C/nmole, Concentration = 0.5 mC/ml) were added to each of the 10 ml of supernatant I and 10 ml of amoebae buffer suspension to give a final cyclic-AMP concentration of 1O-4 M and 5 PC 3H (17.8 nn/r). The samples were incubated with shaking for 1 hr at 21 * 1°C and the reaction stopped by heating the reaction mixture to 100°C for 5 min. The amoebae suspension was immediately centrifuged (ll,OOOg for 30 set), and the supernatant (supernatant II) kept for assay. After cooling, supernatants I and II were adjusted to pH 7.8 with 0.1 N NaOH and brought to 4 x lo-’ it4 MgCl,. One mg of 5’-nucleotidase (snake venom, Ross Allen’s Reptile Institute, Inc., Silver Springs, FL) was added to 0.5 ml of the adjusted supernatant I and II and incubated for 30 min at 37°C. The hydrolysis was terminated by the addition of 2 ml of absolute ethanol containing 300 mg of Dowex-l-Cl (l-x8, 200-400 mesh, Calbiothem). After 10 min, another 2 ml of absolute ethanol was added, followed by 5 ml of the toluene scintillation cocktail containing 12 g of 2,5-diphenyloxazole (PPO) and 0.4 g of 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) per liter of tolu-
VOLUME
44,
1975
ene. The samples were counted in a liquid scintillation counter. The following controls for the assay were run in parallel: 3H-cyclic-AMP, 3H-5’-AMP and 3H-adenosine with and without Dowex, and also in combination with and without snake venom 5’-nucleotidase. Preparation of samples for microscopy. Most of the observations on the morphology of development were done directly with a binocular microscope and a compound phase microscope. When sectioned material was required, a method of plastic embedding of Feder and O’Brian (1968) was used. The cell masses were fixed for two hours at 4’C in 0.4% paraformaldehyde solution buffered at pH 7 (S$rensen’s buffer, l/60 M). Samples were dehydrated by sequential treatment for 12 hr at 4’C in ethylene glycol monoethyl ether, ethanol, propanol and butanol (all loo%), and then transferred to a monomer mixture which consisted mainly of glycol methacrylate (Hartung Associates, Camden, New Jersey) in which were dissolved 0.6% (w/v) of 2,2-azobis (2-methylpropionitrile) (Eastman Kodak Co., Rochester, New York) and 6% (v/v) polyethylene glycol 400 (Fisher). To ensure complete infiltration, the preparations were kept in the monomer mixture for a week during which the monomer mixture was changed twice. After infiltration they were transferred to No. 00 gelatin capsules and a freshly prepared glycol methacrylate mixture added to fill the capsule. These capsules were then polymerized by heating in an oven at 40°C for two days. Sections of 2 pm thickness were cut with a MT-l Sorvall “Porter-Blum” ultramicrotome and stained with toluidine blue (0.025 g/liter of distilled water) for 20 min. RESULTS
The development of P-4 The original strain of the P-4 variant of Dictyostelium discoideum (DdH) produced morphologically normal but smaller fruit-
WOON KIM
CHIA
Znductio
ing bodies than the wild type (Hohl and Raper, 1964). This strain, which has been kept for 8 yr in our laboratory, has acquired an additional character: it undergoes three types of development. When P-4 is grown on 0.1% lactose-peptone agar, three types of fruiting bodies are always produced (Fig.
Vegetative
n
of Stalk
243
Cell Differentiation
2). Type (1) is the normal but smaller fruiting body described by Hohl and Paper (1964) in the original strain, type (2) has a thickened stalk, and type (3) is what we call “stalk cell bumps”, which consist of a sphere of stalk cells with no spores at all. This was confirmed by inoculating these
Amoeboe A Primary
Aggregate
An Aggregate
Secondary
. P-F&
.___.._
Aggregates
Reudoplkmodia
A Pseudoplasmodium
A Mature
Dictvostelium
Fruiting
Body
discoideum
3 Types
of
Fruiting
Bodies
Petite - 4
FIG. 2. Pattern of pseudoplasmodium and fruiting body formation in DdH and P-4. (1) Normal body of P-4. (2) Fruiting body with a thick stalk of P-4. (3) Stalk cell bumps of P-4, and (4) Normal body of DdH.
fruiting fruiting
244
DEVELOPMENTAL
BIOLOGY
bumps with E. coli B/r on 0.1% lactosepeptone agar to see whether there are any spores which will germinate and give rise to a population of amoebae. Twenty bumps have been tested and no growth of amoebae has been detected. A fixed thin section of a typical stalk cell bump is shown in Fig. 3. Another difference between DdH and P-4 should be mentioned here. In DdH, at certain cell densities, each aggregate will form one pseudoplasmodium which in turn develops into one fruiting body. In P-4, in spite of the fact that the territory size of each aggregate is much larger than that of DdH, the P-4 aggregate subdivides into
VOLUME
44, 1975
secondary centers (Fig. 2). Hence in P-4, the primary aggregate always ends up with many smaller aggregates, each of which forms one pseudoplasmodium. Each of these pseudoplasmodia will then give rise to one of the three types of fruiting bodies. Under normal growing conditions on 0.1% lactose-peptone agar, each aggregate, depending on its size, gives roughly 50 to 200 fruiting bodies on a 50 x 12 mm petri dish. On the average 48% of these pseudoplasmodia will form stalk cell bumps, 13%) give rise to thick stalk fruiting bodies, and 39% develop into smaller, but normal fruiting bodies.
FIG. 3. A thin section of a stalk cell bump. (See materials and methods.) phase contrast. The vacuoles show as flat opaque areas separated by thick
The section is shown unstained, cellulose walls (x 117).
in
WOON KIM
CHIA
hduction
One aggregate of P-4 will produce from 50 to over 100 pseudoplasmodia by the breaking up of the aggregating streams, and it can be clearly seen that there is a larger number of normal fruiting bodies near the center of the large aggregate than at the edge. In order to study this phenomenon in greater detail, the aggregation territory was cut into various patterns, and the agar blocks were isolated (Fig. 4). The blocks were then observed to see how this affects the relative proportion of the three types of fruiting bodies in the different areas. In one such experiment an entire aggregate was cut out from the petri dish and further subdivided into 3 concentric rings (Fig. 4A). Cells on these three rings were allowed to develop separately and the number of each of the three types of fruiting bodies were scored. Nine such experiments gave consistent results; as can be seen from the table in Fig. 4, the central region of an aggregate produces more normal fruiting bodies, and their number decreases as one moves peripherally. In contrast, this unequal distribution disappeared if the agar was cut into rectangular shapes, each of which supported portions of
of Stalk
245
CellDifferentiation
just one stream of the aggregate (Fig. 4B). In this case, all the fruiting bodies from each block are normal. Still another difference between P-4 and DdH is that the amoebae and the spores of P-4 are larger than DdH. When amoebae are placed in a low concentration of Bonner’s salt solution (l%), the amoebae round up and thus their diameter can be measured accurately. It was found that the diameter of P-4 amoebae is 15.15 * 1.6 pm (SD) (52 samples) while the diameter of DdH amoebae is 8.8 i 1.23 pm (SD) (48 samples). The mean spore length of P-4 is 11.2 * 1.86 pm (SD) and in DdH 6.75 i 0.99 lrn (SD) (60 samples each). Therefore each P-4 amoeba has approximately 3 times more surface area and five times more volume than a DdH amoeba (this becomes important when we later compare the cell-bound phosphodiesterase of the mutant and the wild type). In order to see if P-4 was a homogenous cell line or a mixture of two or more genotypes, the amoebae were cloned. It was found that every P-4 clone (40 isolates) isolated from a single spore produced the three types of fruiting body characteristic of P-4. B
A
Normal
Ring-
3
Fruiting Bodies
P
Thick Stalk Fruiting Bodies Stalk Cell Bumps
/a
36 %
62
P
IO %
I0 %
23 %
_L
57%
52 %
15 %
33
-a
FIG. 4. The isolated agar block experiment: A. The P-4 aggregate one stream of the P-4 aggregate was cut into rectangular blocks.
%
100%
100%
was cut into three
100%
concentric
100%
rings,
and B.
246
DEVELOPMENTAL
BIOLOGY
Attempts to influence the proportion of normal fruiting bodies in P-4 (a) The effect of washing. The amoebae of P-4 were given successive washings by the centrifugation technique previously described (Fig. 1) during a period of 10 hr. After each treatment a few cells were allowed to develop on 2% agar. The result was striking: there was a progressive increase in the proportion of normal fruiting bodies (this was a qualitative observation, but the result was clear-cut). This was repeated numerous times with similar results: by the fourth and fifth washing, over 90% of fruiting bodies were normal (Fig. 5). This experiment suggests that a substance which induces stalk cells is removed by the washing procedure. In order to determine if the substance is dialyzable, the drum technique (used for cyclic-AMP collection as previously described) was used. The P-4 amoebae were placed on the side of a dialysis membrane upper stretched on a drum over a large reservoir of salt solution. It was assumed that if an inducer of small molecular weight was secreted by the amoebae which could diffuse through the dialysis membrane into the salt solution in the dish, then frequent removal of the salt solution means removal of the inducer. The result was that if the solution under the membrane was replaced
FIG.
5. Results
of various
experiments
on P-4 after
VOLUME
44, 1975
at repeated intervals (five times, once every 2 hr), then more than 90% of the fruiting bodies were normal (repeated three times with the same result). As a control, the salt solution was not replaced and the fruiting bodies were largely of the abnormal character. Clearly the stalk cell inducer can be effectively removed through a dialysis membrane. (b) Effect of cyclic-AMP on development of washed P-4 amoebae. The purpose of this experiment was to find out whether or not externally applied cyclic-AMP would reverse the effect of washing. Washed P-4 amoebae were placed on petri dishes containing agar supplemented with 10m5, 10e6 or 10m7 M cyclic-AMP. Whereas washed controls showed an increase of normal fruiting bodies through repeated washings, there was a significant increase in proportion (80 to 85%) of bumps in response to cyclic-AMP (Fig. 5). (c) Effect of cyclic-AMPphosphodiesterase on development of P-4 amoebae. Since the proportion of stalk cell bumps in P-4 can be increased by cyclic-AMP and 5’-AMP is ineffective, and phosphodiesterase degrades 3’,5’-cyclic-AMP to 5’-AMP, the induction of bumps was also examined with phosphodiesterase in the agar. P-4 amoebae were harvested and placed on a petri dish containing 0.2 units of phosphodiesterase (1 unit converts 1 /Imole of
successive
washings
and placed
on 2% agar plates.
WOON KIM
CHIA
Induction
3’,5’-cylcic-AMP to 5’-AMP; Sigma Company) in 2% agar. Although the P-4 amoebae had not been washed except during harvesting (which had little effect), their development on the phosphodiesterase agar was similar to those that had been extensively washed for 5 treatments: in both cases over 90% of the fruiting bodies were normal (Fig. 5). Attempts to increase the proportion of stalk cell bu’mps using the drum technique
(a) Effect of high concentrations of cyclic-AMP on development of P-4 amoehue. In this series of drum experiments, P-4 amoebae were permitted to develop on dialysis membrane in contact with 1% salt solution containing various concentrations of cyclic-AMP. Again, the cyclic-AMP in the salt solution affected the P-4 development by increasing stalk cell bumps and inhibiting normal fruiting body formation (Fig. 6). At concentrations of 10e4 and lo- 5 M all the cells turn into small stalk cell bumps (Fig. 7). As the concentration of cyclic-AMP was decreased from 1O-5 M to 10e6 M, the number of normal fruiting bodies progressively increased as the concentration was further lowered to 1O-7 M. A concentration of 10eB M cyclic-AMP had little effect on P-4 development: all
Cyclic-
,o-3M
,o-4w
AMP
P-4
Inhibited
of Stalk
247
Cell Differentiation
three types of fruiting bodies were formed. (b) Effect of high concentrations of cyclic-AMP amoebae. discoideum
on
development
The wild type (DdH), was treated identically for comparison. Cyclic-AMP also induces DdH amoebae to differentiate into stalk cell bumps on the drum, but the minimum concentration of cyclic-AMP necessary to produce 100% stalk cell bumps was lo-‘M, ten times more than needed for P-4 (Fig. 6). The concentration of lo-$ M cyclicAMP also delays the development of DdH for 12 hr and the amoebae develop into smaller but normal fruiting bodies. Another difference between the induced DdH bumps and P-4 bumps is that, unlike P-4, the DdH do not consist entirely of stalk cells. The center of the DdH bumps always consist of undifferentiated amoebae. (c) Effect of nucleotides other than cyclic-AMP on stalk cell induction. Adeno-
sine, 5’-AMP and some other cyclicnucleotides were tried on the development of P-4 and DdH using the drum technique. It is clear that only cyclic-AMP induced stalk cell differentiation (Fig. 8). It is interesting to note that 3’,5’-cyclic-CMP does reduce the territory size in DdH and smaller fruiting bodies are produced, while 3’,5’-cyclic-UMP does the same for both in
10-5M
100%
100%
Bumps
Bumps
I
Increase
1Increase
Norma
FIG.
6. Effect
of cyclic-AMP
on the development
of DdH of P-4, D.
of P-4 and DdH
on drums.
I
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P-4 and in DdH. Finally, 3’,5’-cyclic-GMP has no effect on DdH but it slightly increased the proportion of the normal fruiting bodies and repressed the formation of stalk cell bumps in P-4.
concentration of extracellular cyclic-AMP increases during early aggregation and reaches its peak at late aggregation, decreasing rapidly at the end of the aggregation.
Cyclic-AMP production of P-4 development
Specific activities of phosphodiesterase in liquid culture of P-4 and DdH
during
the course
The cyclic-AMP produced by P-4 was collected by removing the salt solution from under the drum cellophane membrane at regular intervals while the amoebae developed on the upper surface. Since cyclic-AMP is heat stable, the large quantity of salt solution (400 ml from five drums) collected every 2 hours was evaporated to dryness at 45°C and resuspended in 2 ml of distilled water. It was then assayed for cyclic-AMP using the cellophane square test for chemotaxis. The
Since previous results show that (a) P-4 produces an excess of stalk cells and (b) P-4 is 10 times more sensitive than DdH to cyclic-AMP induction of stalk cells, it is important to find out whether there are differences between DdH and P-4 in cyclicAMP production or specific activity of phosphodiesterase. Comparison of the cyclic-AMP production in P-4 with the cyclic-AMP production in DdH (Bonner et al., 1969; Bonner, unpublished results) shows that P-4 and DdH produce the same
FIG. 7. A. Stalk cell bumps induced by lo-’ M cyclic-AMP on the dialysis machine Three stalk cell bumps and some individual stalk cells induced by lo-’ M cyclic-AMP under phase contrast (x 200).
in a drum (x 22). B. in the drum as seen
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Control I % Solt Solution Normal 3 Types of Fruitin< Bodies
$-AMP lO-4 Normal
CHIA
Induction
lO-4 lormal
-I
Cyclic Cyclic 3; 5’ - AMP ;, 5’- GMF
Idenosinc M
of Stalk
M
lO-4
M
lO-4
tvl
Cyclic Cyclic Cyclic 3-, 5’- UM P: i, 5’- CMF i, 5’- TMF 10-4M
lo-4
Cyclic j,dIMF
ld4M
lO-4
Normal
Normal
u
Lk!
M
Fewer Normal Stalk Cel I
but Smolle Fruiting Bodies
P-4
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Cell Differentiation
M
k%fe of Norm< II but $nallt 9r F;;fFsg
? u Normal 1 Type of Frultint Body
Vormol
Jormal
lJormol IUormal Vormal but Smalle Sl ti Smaller Fruiting IFruiting 3odies I3odies
Normal
DdH
FIG.
8. The effect of 5’-AMP,
adenosine
and other
cyclic-nucleotides
amount of cyclic-AMP. A time course study was made of the phosphodiesterase specific activity in DdH and P-4 grown in liquid culture (on autoclaved E. coli B/r). The reason for using liquid culture amoebae are: (a) it provides a large quantity of amoebae, (b) the amoebae are better synchronized in their development, and (c) it is easier to separate and determine the two kinds of phosphodiesterase. It has been shown by Malchow et al. (1972) that DdH produces two kinds of phosphodiesterase: a soluble extracellular phosphodiesterase (Chang, 1968) and a membrane-bound phosphodiesterase. We measured the specific activity of these two phosphodiesterases in DdH and P-4 in liquid culture. The assay was started 8 hr before the amoebae had consumed all the bacteria and for 20 hr more, well past the time when aggregation would have occurred had they been growing on an agar surface. Both DdH and P-4 have two forms of phosphodiesterase. If one compares the
on P-4 and DdH
developing
on a drum.
specific activity of extracellular phosphodiesterase per unit area of cell surface over the 28-hr period in the two strains, they are remarkably close and show no significant difference (Fig. 9). However, the changes of specific activity of cell-bound phosphodiesterase per unit area of cell surface is significantly different in P-4 and DdH. The DdH cell-bound phosphodiesterase begins to rise early (2 hr) and peaks late (23 hr), while P-4 rises late (15 hr) and peaks almost immediately (18 hr). Furthermore, the amount of activity is consistently higher for DdH than P-4 (Fig. 9). These results for the wild type are similar to the results of previous workers for both the extracellular phosphodiesterase (Riedel and Gerisch, 1971; Bonner et al., 1972) and the cell bound phosphodiesterase (Malchow, et al., 1972). There are such major differences in techniques involved (e.g., different strains of D. discoideum) that it is not surprising that the absolute values do not exactly correspond. The surface area of the P-4 amoeba is
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FIG. 9. Phosphodiesterase specific activities of DdH and P-4 in liquid culture. Cell-bound phosphodiesterase of DdH, (A); of P-4 (0). Soluble phosphodiesterase of DdH, (A); of P-4, (0). Since P-4 cells are larger than DdH cells the specific activity of the enzyme is calculated on the basis of the equivalent surface area for the two types. In other words, 1 x 10s DdH cells has the equivalent surface area of a third as many P-4 cells.
three times larger than the DdH amoeba. This means that 3.33 x lo5 P-4 cells is equal in total cell surface area to 10” DdH cells. To correct for this, the specific activity of phosphodiesterase is defined as enzyme activity per lo6 DdH cells, and 3.33 x lo5 P-4 cells. If cell volume is used instead of total cell surface area, then the relative values of both phosphodiesterases are lower for P-4. Therefore, calculating the phosphodiesterase specific activity either way shows that the cell-bound form is invariably lower in the variant than in the wild type. DISCUSSION
One of the key problems of development is the proportional differentiation of different cell types in a tissue, organ or in a whole organism. From the early work in the cellular slime molds it is known that a stable proportionality exists between stalk cells and spores over a wide range of
fruiting body sizes (Harper, 1932; Raper, 1935; Bonner, 1967). One way to approach the problem of proportionality is to treat the slime molds with different chemicals to see if one can shift the balance towards more spores, or more stalk cells. Another approach is to look for mutants (variants) with altered proportionality. In this study both of these methods have been employed. The P-4 variant of D. discoideum (DdH) normally has a great excess of stalk cells in a population of fruiting bodies. It is interesting to note that the spore-stalk cell ratio is not shifted equally in all pseudoplasmodia, but some are normal in their proportions, some have an abnormally high percent of stalk cells, and some are all stalk cells. I have found that if cells of this variant are washed, either by repeated centrifugations or by placing the amoebae on a cellophane drum so the water below can be replaced at intervals, then the
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KIM
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Induction
variant changes phenotypically to the wild type. Since it has been shown that cyclic-AMP will induce DdH to form an excess of stalk cells (Bonner, 1970), this substance was added to washed P-4 cells. We found that that variant character was restored. Moreover, with increasing cyclic-AMP concentration, it was possible to obtain 100% stalk cells on the surface of the drum. This and the results obtained with 5’-AMP, adenosine and phosphodiesterase suggest that cyclic-AMP is the stalk cell inducer. Since both DdH and P-4 amoebae secrete cyclicAMP and phosphodiesterase, it is likely that cyclic-AMP is the natural inducer of stalk cells in cellular slime molds, and that the balance of phosphodiesterase to cyclicAMP production is crucial for the production of any particular phenotypic condition. Further evidence for this comes from the following observations: In the experiments of isolating agar blocks bearing P-4 aggregation streams (Fig. 4A), the central ring has a higher proportion of wild type phenotype because of the higher cell density and therefore the higher phosphodiesterase concentration. In the outer ring with a lower cell density, and thus less phosphodiesterase, there is an increase in the percent of stalk cell bumps. The cutting of agar into small rectangular blocks (Fig. 4B) is another way of increasing cell density and phosphodiesterase production; the result is an increase of normal fruiting bodies to 100%. Since millipore filters placed on pads in petri dishes are used in other laboratories (Sussman, 1966) as a means of supporting differentiating cellular slime molds, this method (using millipores PHWP 04700, average pore size 0.3 pm, Millipore Corporation) was also tried in the induction of P-4 stalk cells using cyclic-AMP. A range of concentrations of amoebae and of cyclicAMP were used, and they included concentrations known to produce stalk cell differentiation both on drums and on agar
of Stalk
Cell Differentiation
251
plates, but stalk cell induction never occurred under these conditions. It is presumed that since the millipore filter is permeable to macromolecules, the phosphodiesterase quickly converts any cyclicAMP to the inactive 5’-AMP. Comparing the experimental conditions of millipore filter, the agar plate, and the drum, one can only conclude that the total reservoir of liquid, and the distance over which diffusion can occur, is very much less in the millipore culture dishes, and as a result the cyclic-AMP is more quickly and effectively eliminated. The variant and wild type were compared with respect to their sensitivity to cyclic-AMP, cyclic-AMP production and the specific activity of extracellular and cell-bound phosphodiesterase. The P-4 variant is approximately 10 times more sensitive to induction of stalk cells by cyclic-AMP than the wild type. The cyclicAMP and the extracellular phosphodiesterase was the same for both, but the variant had significantly less cell-bound phosphodiesterase. One hypothesis that accounts for the P-4 - DdH difference is that as a result of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence the excess stalk cell production. This hypothesis would also account for the increased effectiveness of low concentrations of cyclicAMP in the induction of stalk cells in the variant as compared to the wild type. The study reported here is part of my Ph.D. thesis work. I express my gratitude and appreciation to Professor John Tyler Bonner for his advice. This work was supported by the National Science Foundation Grant GM 33439, and National Institutes of Health Grant GM 17856. I also benefitted from the central equipment facilities in the Department of Biology, Princeton University, supported by the Whitehall Foundation. REFERENCES D. Identification slime mold.
BARKLEY,
S.
1969. Adenosine-3’,5’-phosphate: as acrasin in a species of cellular Science 165, 1133-1134.
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BONNER, J. T. 1947. Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. 2001. 106, 1-26. BONNER, J. T. 1967. “The Cellular Slime Molds,” Second edition. Princeton University Press, Princeton, NJ. BONNER, J. T. 1970. Induction of stalk cell differentiation by cyclic AMP in the cellular slime mold Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 65, 110-113. BONNER, J. T., BARKLEY, D. S., HALL, E. M., KONIJN, T. M., MASON, J. W., O’KEEFE III, G., and WOLFE, P. B. 1969. Acrasin, acrasinase, and the sensitivity to acrasin in Dictyostelium discoideum. Deuelop. Biol. 20, 72-87. BONNER, J. T., HALL, E. M., NOLLER, S., OLESON, JR., F. B., and ROBERTS, A. B. 1972. Synthesis of cyclic AMP and phosphodiesterase in various species of cellular slime molds and its bearing on chemotaxis and differentiation. Deuelop. Biol. 29, 402-409. BONNER, J. T., KELSO, A. P., and GILLMOR, R. G. 1966. A new approach to the problem of aggregation in the cellular slime molds. Biol. Bull. 130, 28-42. BROOKER, G., THOMAS, L. J., JR., and APPLEMAN, M. M. 1968. The assay of adenosine 3’,5’-cyclic-monophosphate and guanosine 3’,5’-cyclic monophosphate in biological material by enzymatic radioisotopic displacement. Biochem. 7, 4177-4181. CHANG, Y. Y. 1968. Cyclic 3’,5’-adenosine monophosphate phosphodiesterase produced by the slime mold Dictyostelium discoideum. Science 160, 57-59. FEDER, N. and O’BRIAN, T. P. 1968. Plant microtechnique: some principles and new methods. Amer. J. Dot. 55, 123-142. GERISCH, G. 1959. Ein Submerskulturverfahren fur entwicklungsphysiologische Untersuchungen an Dictyostelium discoideum. Naturwis. 46, 654-656. HARPER, H. A. 1932. Organization and light relations
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in Polysphondylium. Bull. Torrey Bot. Club 59, 49-84. HOHL, H. R. and RAPER, K. B. 1963. Nutrition of cellular slime molds, I. Growth on living and dead bacteria. J. Bact. 85, 191-198. HOHL, H. R. and RAPER, K. B. 1964. Control of sorocarp size in the cellular slime mold Dictyostelium discoideum. Deuelop. Biol. 9,137-153. KONIJN, T. M., CHANG, Y. Y., and BONNER, J. T. 1969. of cyclic-AMP in Dictyostelium Synthesis discoideum and Polysphondylium pallidum. Nature 224, 1211-1212. KONIJN, T. M., VAN DE MEENE, J. G. C., BONNER, J. T., and BARKLEY, D. S. 1967. The acrasin activity of adenosine-3’,5’-cyclic phosphate. Proc. Natl. Acad. Sci. U.S.A. 58, 1152-1154. MALCHOW, D., NXGELE, B., SCHWARZ, H., and GERISCH, G. 1972. Membrane-bound cyclic-AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur. J. Biochem. 28, 136-142. MONARD, D., JANECEK, J., and RICKENBERG, H. V. 1969. The enzymatic degradation of 3’,5’ cyclic AMP in strains of E. coli sensitive and resistant to catabolic repression. Biochem. Biophys. Res. Comm. 35, 584-591. RAPER, K. B. 1935. Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agric. Res. 50, 135-147. RIEDEL V., GERISCH, G., MULLER, E., and BEUG, H. 1973. Defective cyclic adenosine-3’,5’-phosphatephosphodiesterase regulation in morphogenetic mutants of Dictyostelium discoideum. J. Mol. Biol. 74, 573-585. SUSSMAN, M. 1966. Biochemical and Genetic Methods in the Study of Cellular Slime Mold Development. “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. II, pp. 397-410. Academic Press, New York and London.
