/. Embryol exp. Morph. Vol. 35, 2, pp. 335-343, 1976
Printed in Great Britain
335
Cell specific events occurring during development
By CHARLES L.RUTHERFORD 1
From the Department of Biology, Virginia Polytechnic Institute and
State University, Blacksburg
SUMMARY
Ultra-microfluorometric techniques were adapted to follow the time sequence of glycogen
degradation during the differentiation of two cell types in Dictyostelium discoideum. Glycogen
content, glycogen phosphorylase activity, and inorganic phosphate accumulation were
localized in specific cell types during stalk and spore development.
Glycogen levels in pre-stalk cells remained constant during the pseudoplasmodium and
early culmination stages of development. However, as pre-stalk cells migrated into the position of stalk formation, a cell specific degradation of glycogen was observed. The loss of
glycogen from pre-stalk cells was accompanied by an increase in the activity of glycogen
phosphorylase. This increase in activity from 004 to 014moles/h/kg dry wt. occurred as
pre-stalk cells entered the position of stalk formation. An inverse relationship was found
between glycogen levels and inorganic phosphate (Pj) levels in the developing stalk. During
the process of stalk construction, a gradient of Pj levels occurred from the apex to the base
of the developing stalk. Glycogen degradation from pre-spore cells lagged behind that of
pre-stalk cells. No change in pre-spore cell glycogen levels was observed until stalk construction was nearly completed. The results emphasize the importance of the physical position of
a cell with respect to its composition and fate during development.
INTRODUCTION
This report describes the application of an ultra-microfluorometric technique,
to assess the extent to which cell specific events are correlated with differentiation in the cellular slime mold, Dictyostelium discoideum. With the development
of ultra-microchemical techniques by Lowry & Passonneau (1972), a method is
now available which is sensitive enough to make such a study possible. By
utilizing these techniques, glycogen, glycogen phosphorylase (EC 2.4.1.1), and
inorganic phosphate were all found to exhibit, not only time dependent changes,
but also changes in cell specific levels during differentiation. The results of these
studies emphasize the necessity of obtaining information on cell specific biochemical events, in attempting to understand the mechanisms underlying
differentiation in multicellular organisms.
The cellular slime mold D. discoideum, because of its unique life-cycle, offers
an ideal model system in which to assess the role of cell specific events. During
the development of D. discoideum, vegetative myxamoebae aggregate and form
1
Author's address: Department of Biology, College of Arts and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061, U.S.A.
336
C. L. RUTHERFORD
a multicellular pseudoplasmodium. After a period of migration, the pseudoplasmodium becomes organized so that presumptive stalk cells are located in
the anterior section and presumptive spore cells are located in the posterior
section. The pseudoplasmodium is eventually transformed into a fruiting body,
consisting of a stalk, with a spore-containing sorus at its tip. This localization
of presumptive stalk and spore-containing cells into distinct positions greatly
facilitates a study of the biochemical events occurring in each cell type.
MATERIALS AND METHODS
Growth and preparation of cells
Cells of D. discoideum, strain NC-4, were grown on a rich agar medium in the
presence of Escherichia coli as described previously (Rutherford & Wright,
1971). Amoebae were spread onto Whatman number 50 filter-paper discs and
were placed over a non-nutrient agar surface. At the desired stage of differentiation the filter paper was removed from the agar and was placed on dry ice.
The frozen tissue was then lyophilized overnight. The dry tissue was stored at
- 3 0 °C, under vacuum. On the day the assay was to be performed, the lyophilization flask was allowed to reach room temperature, the vacuum was
released, and the tissue was removed.
Description of the microtechnique
Individuals from various stages of development were dissected and assayed,
by adaptations of microtechniques as described by Lowry & Passonneau (1972).
Sections from pre-stalk or pre-spore cells were weighted on a quartz fiber
balance, capable of measuring 0-02 /tg dry weight. With the aid of hair points,
the dry samples were transferred to wells drilled into Teflon racks. Reaction
mixtures were added from hand-made pipettes, in volumes of from 0-1 to 5 (A.
The wells were then filled with mineral oil in order to prevent evaporation of the
small reaction volumes. In order to measure low substrate and enzyme activities
occurring in the microgram quantities of tissue, enzymic cycling was used to
increase the sensitivity of the method (Lowry & Passonneau, 1972).
Glycogen assay procedure
The assay was initiated by addition of 1-25 fi\ of 0-02 N-NaOH to the bottom
of a well in the Teflon rack. Tissue was then added, covered with mineral oil and
heated for 10 min at 60 °C. Appropriate blanks and standards were carried
through the entire procedure. To the NaOH was added 5-32 /i\ of a glycogen
reaction mixture, then incubated for 40 min at 37 °C. The glycogen reaction
mixture was made in 0-05 M imidazole-HQ buffer, pH 7-0, and contains 0-5 mM
magnesium acetate, 1-0 mM EDTA, 0-04 mM NADP+, 0-1 mM 5'AMP, 5 HIMK 2 HPO 4 , 0-02 % bovine plasma albumin, 0-5 mM dithiothreitol, 1-0 /*M glucose1,6-diphosphate, 3/*g/ml P-glucomutase, 0-5^g/ml glucose-6-P dehydrogenase
Cell specific events during development
337
and 60 /*g/ml glycogen phosphorylase a. After the incubation period, 5 /d of
0*1 N-NaOH was added to the reaction mixture and then heated at 60 °C for
15 min. The complete reaction mixture was then transferred to 100 /d of cycling
reaction mixture contained in a 3 ml test tube (Lowry & Passonneau, 1972).
Glycogen phosphorylase assay
The glycogen phosphorylase reaction mixture was made in 0-05 M potassium
phosphate buffer, pH 7-0, which contained 25 mM glycogen (glucosyl units),
0-5 mM magnesium chloride, 0-5 mM NADP+, 1-0 mM dithiothreitol, 1-0 /AM
glucose- 1,6-diphosphate, 0-1 mM 5-AMP, 10/*g/ml P-glucomutase and 2/*g/ml
glucose-6-P dehydrogenase. Dry tissue samples were added to 4-43 /d of cold
phosphorylase reaction mixture contained in the wells. The reaction mixture
was covered with oil and then incubated for 60 min at 37 °C. The reaction was
then stopped by adding 5-11 /d of 0-1 N-NaOH and heating for 10 min at 60 °C.
The complete reaction mixture was then added to 100 /d of cycling reaction mixture for amplification.
Inorganic phosphate assay
The reagent consisted of 0-05 M imidazole-HCl buffer, pH 7-0, containing
0-08 % glycogen, 0-03 mM NADP+, 0-01 mM 5'AMP, 0-02 % bovine plasma
albumin, 1 mM EDTA, 0-5 mM magnesium acetate, 0-5 mM dithiothreitol,
2-5 jLtglml glucose-6-P dehydrogenase, 3-0 /tg/ml P-glucomutase, and 60 /«g/ml
glycogen phosphorylase a. The assay procedure for inorganic phosphate was the
same as that for glycogen except for the following differences in volumes of
reagents added: 0-107/d 0-02N-NaOH, 0-102/d reaction mixture and 0-517/d
0lN-NaOH.
Chemicals
Agar, yeast extract, and peptone were obtained from Difco Laboratories
(Detroit, Michigan). All reagents were purchased from Sigma Chemical Company (St Louis, Missouri). Reagent stock solutions were kept frozen or refrigerated until used. The enzymes used in the cycling reaction mixtures were
Norite treated to remove nucleotide contaminants (Lowry & Passonneau, 1972).
RESULTS
Validation of the microtechniques
The metabolite levels and enzyme activities obtained with the microtechniques
are comparable to literature values obtained with less-sensitive procedures. For
example, glycogen levels at the pseudoplasmodium stage of development have
been reported at 0-05-0-25 moles/kg dry weight (Wright & Dahlberg, 1967;
Hames, Weeks & Ashworth, 1972; Wright, Rosness, Jones & Marshall, 1973).
22
EMB 35
338
C. L. RUTHERFORD
Table 1. Levels of glycogen, inorganic phosphate, and glycogen phosphorylase
during differentiation of stalk and spore cells
(The position of tissue samples is indicated by letters placed on the figures. The
intact stalk was dissected free of spore cells, before sectioning. Each individual is
approximately 2 mm in length. The dry weight of sections of an individual ranged
from 0-05 to 0-10 fig. Glycogen concentration is expressed in terms of glucosyl units.
Ten individuals were assayed at each stage of development shown. The values
shown are typical for each stage.)
Location of
tissue sample
1
1F
IG
/
\
i
/ K
L
\ M /
\N/
Pseudoplasmodium
(12 h)
/D
Glycogen level
(10~2 moles/kg
dry wt.)
Glycogen
phosphorylase
Inorganic
specific activity phosphate level
(10- 2 moles/h/kg (10~2 moles/kg
dry wt.)
dry wt.)
11-2
14-9
144
15-5
130
134
13-6
15-9
140
130
13-2
14-7
12-7
12-3
4-2
3-8
3-7
4-3
4-2
3-6
3-5
3-8
3-6
4-3
34
4-5
3-6
4-3
13-2
12-6
104
11-9
18-6
11-2
15-7
14-8
13-1
149
13-6
151
154
150
A 14-7
5-2
60
140
151
140
161
94
8-7
5-1
60
161
18-2
14-3
12-9
21-6
48-6
57-5
16-5
13-5
42-0
58-0
55-9
64-7
64-3
62-2
60-2
40-0
A
B
C
D
E
F
G
H
I
J
K
L
M
N
I
B 14-6
C 130
D 150
E 84
F 4-9
G 1-2
H 01
0-5
J
I
J
A
1-3
1-3
11-9
1-3
1-3
I
G
H
I
1-7
0-8
9-2
100
30
11
24
10
01
J
J
10
14
// F
F
I
\°/
11A
\ u
'M
1
40
760
744
604
•"••••
Culmination
(22 h)
a
\
3 Bj
E /
F
W
Sorocarp
(24 h)
B
C
D
E
F
1-6
1-5
14
1-2
Cell specific events during development
339
Table 1 shows that glycogen levels obtained with the microtechniques average
0-14 moles/kg dry weight. Glycogen phosphorylase activities (maximum activity)
of 3-3 x 10~9 moles/min/mg dry weight and 4-0 x 10~9 moles/min/mg dry weight
have been reported (Jones & Wright, 1970; Firtel & Bonner, 1972). Maximum
glycogen phosphorylase activity determined with the microtechniques was
3-1 x 10~9 moles/min/mg dry weight. The enzyme activity and substrate levels
obtained from microgram quantities of lyophilized tissue were also compared
to levels in tissue homogenates and were found to be similar when expressed on
a dry weight basis. Thus the preparation of extracts by freezing followed by
lyophilization, was sufficient to release enzymes and substrates to the assay
conditions. The glycogen and Pi levels and phosphorylase activities from
extracts of frozen-dried tissue (both pseudoplasmodium and sorocarp) were
also compared with extracts prepared with the french pressure cell, or by sonic
treatment. Results from all methods of extraction were not significantly different
(unpublished data).
The quartz fiber balances used for obtaining dry tissue weights were linear
over the range of sections assayed. In assays utilizing amplification by enzymic
cycling, the rate of cycling was linear over the concentration range of the
standards and tissue levels. The concentrations of stock reagents used as
standards, were determined by enzymatic assay. Glycogen and inorganic
phosphate assays were shown to go to completion during a 1-h incubation.
Internal controls of both substrates were included in the assays giving over 90 %
recovery in both cases. The glycogen phosphorylase assay was linear to at least
4 mg dry tissue/ml of reaction mixture. Likewise the rate was linear for at least
120 min at 37 °C. The coupling enzymes phosphoglucomutase and glucose6-phosphate dehydrogenase were not limiting in the assay. Appropriate controls
without substrates were carried through all steps. Endogenous levels of glycogen
or Pi were not sufficient to support significant phosphorylase activity.
Cell specific levels of glycogen, glycogen phosphorylase and inorganic phosphate
Glycogen levels have been shown previously to decrease during differentiation
with a concomitant increase in new carbohydrate end products, primarily
trehalose and cellulose (White & Sussman, 1961; Ceccarini & Filosa, 1965;
Sargent & Wright, 1971). Table 1 shows glycogen levels in each of the two cell
types during the time course of differentiation. At the pseudoplasmodium stage
of development (12 h), glycogen levels were 0-14 moles/kg dry weight (expressed
as glucose units). No localization of glycogen was found at this stage between
pre-stalk cells and pre-spore cells.
The differences in glycogen levels between cell samples observed at the
pseudoplasmodium stage of development, shows the experimental variation in the
method. Since these differences between sections were not localized in the same
position from one individual to another, the variations observed must represent
340
C. L. RUTHERFORD
the technical error. The standard error was near 10 % for both substrate and
enzyme assays.
At the culmination stage of development (22 h), a net degradation of glycogen
was observed in those cells near the base of the stalk. Glycogen levels in prestalk cells near the apex of the stalk, as well as glycogen levels in pre-spore cells,
remained constant. A gradient of glycogen levels was found between the apex
of the stalk and the base of the stalk. By 24 h of differentiation, a loss of glycogen
had occurred in the spore cells, but had significantly lagged behind the loss
observed in stalk cells. Finally, at the completed sorocarp stage of development,
both cell types had reached similar glycogen levels.
Glycogen phosphorylase activity was also found to exhibit cell specific
changes during differentiation of the two cell types. Activity associated with
pre-spore cells remained constant through the early stages of development at
near 40 mmoles/h/kg. At the final sorocarp stage, the activity dropped to
10 mmoles/h/kg. During culmination, the activity associated with cells at the
upper portion of the stalk, where stalk formation is initiated, showed an approximate fourfold increase over pre-stalk cells which had not yet begun the process
of stalk formation. This increase in glycogen phosphorylase activity, from 40
mmoles/h/kg to near 160 mmoles/h/kg, occurred as pre-stalk cells migrated into
the apical part of the stalk.
The increase in inorganic phosphate, known to occur during differentiation
(Gezelius & Wright, 1965, Jones & Wright, 1970) was found to be associated
with the base of the developing stalk. A gradient in phosphate level occurred
from the apex of the stalk to the base. Phosphate levels ranged from near 0-10
moles/kg dry weight at the apex of the stalk to 0-70 moles/kg at the base of the
stalk. Phosphate levels in the developing pre-spore cells showed no significant
change during development.
DISCUSSION
This report has described an attempt to study biochemical events in two
distinct cell types during their ultimate differentiation. By utilizing ultramicrotechniques cell specific events were shown to occur between pre-stalk and
pre-spore cells. These events occurred not only with respect to time, but in
addition to the position of the cell within a developing individual. Glycogen
degradation occurred in the stalk soon after pre-stalk cells had migrated into the
position of stalk formation. During this migration phase of the pre-stalk cells,
biochemical events were found to occur, creating a state favorable for glycogen
breakdown. The activity of the degradative enzyme glycogen phosphorylase
increased fourfold over the activity in prestalk cells. Likewise the substrate for
glycogen degradation, Pi? increased to saturation levels for phosphorylase. The
accumulation of Pj in stalk cells is sufficient to significantly inhibit glycogen
synthesis. Thus it must be concluded that during pre-stalk cell migration a
Cell specific events during development
341
kinetic situation is created favorable for glycogen degradation in those same
cells where loss of glycogen was actually shown to occur.
The results of this study show not only that cell specific events occur during
stalk and spore differentiation, but, in addition, may provide information as to
the rate limiting steps of this process. Wright (1973) has emphasized the necessity of determining which of the events, occurring during differentiation, are not
only essential but are also 'critical variables, i.e. limit the rate of differentiation
at particular points in time'. Table 1 suggests that the activity of glycogen
phosphorylase is limiting with respect to glycogen degradation, but only in
specific locations within the developing stalk. The rate of glycogen degradation
in stalk cells can be estimated from the time required for completion of stalk
construction. Although a 3 h period was required for total stalk construction,
total glycogen degradation per stalk cell was complete after about 1 h. As
shown in Table 1, pre-stalk cells begin glycogen degradation soon after entering
the apex of the developing stalk. These pre-stalk cells show complete loss of
glycogen over a time period required for completion of approximately onethird of the stalk. Studies of glycogen levels in specific areas of the stalk, in 23
individuals (247 determinations) showed that about 1 h was required for total
glycogen breakdown. Thus, the rate of net glycogen degradation in vivo, during
cell specific stalk formation, is approximately 0-14 moles/h/kg dry weight. In the
pre-stalk cells undergoing this rate of glycogen degradation, glycogen phosphorylase activity, as measured at maximum activity in vitro, was 0-14 moles/h/
kg dry weight. Although these values may not reflect the exact rates in vivo, the
similarity in the rate of glycogen degradation and the maximum activity of
glycogen phosphorylase suggests that the enzyme is 'limiting' the rate of glycogen degradation during stalk development. Thus, changes in the amount or
activity of this enzyme would significantly affect the rate of this initial step in the
differentiation of pre-stalk cells.
The inverse correlation between phosphate level and glycogen concentration
should also be emphasized. The Pi level found at the pseudoplasmodium stage
(0-13 moles/kg) is less than saturation concentration for glycogen phosphorylase
(Jones & Wright, 1970). As pre-stalk cells migrate into the position for stalk
construction, Pi levels begin to increase reaching levels near 0-60 moles/kg at
the base of the stalk. Table 1 shows that the initiation of glycogen degradation
in stalk cells at culmination (sections D and E) coincides with a twofold increase
of Pi levels in these same cells. It cannot be determined from the present data
whether the increase in PA levels preceded the loss of glycogen degradation, or
conversely if the initiation of glycogen degradation resulted in P t accumulation.
In either case the observed increase in Pi would result in higher levels of substrate for the degradative enzyme. Thus, in cells near the apex of the stalk, both
the increase of substrate availability as well as the elevated activity of glycogen
phosphorylase would result in a kinetic situation favorable for glycogen
degradation.
342
C. L. RUTHERFORD
The source of the P t accumulation in stalk cells cannot be determined from
this study alone. Autodegradation of stalk cells is known to occur, resulting
presumably in the release of large quantities of Pi from RNA breakdown
(Hames & Ashworth, 1974). Yet the inverse relationship between glycogen
degradation and Pi accumulation is worthy of consideration in this regard. For
each glucose unit released by glycogen degradation one molecule of Pi is used in
the phosphorylase reaction. Likewise, two molecules of Pi are produced in the
UDPG pyrophosphorylase reaction. Thus, a net yield of 1 mole of P t results
from each mole of glucose released. From the total degradation of glycogen in
stalk cells 0-14 moles/kg of P t would be expected to accumulate. The observed
increase in Pi during stalk construction is about 0-5 moles/kg. Thus, the close
correspondence of glycogen degradation and accumulation of Pi in specific
stalk cells suggests that at least part of the Pi accumulation in stalk cells could
be accounted for by the end products of glycogen degradation.
Furthermore, the accumulation of phosphate in stalk cells may result in
inhibition of glycogen synthesis. The enzyme which catalyzes the synthesis of
soluble glycogen would be inhibited by over 80 % at 0-60 moles/kg P1# This level
of Pi would also result in over 50 % inhibition of cell wall bound glycogen
synthetase (Wright, 1973).
Although this report shows that cell specific events occur during spore and
stalk differentiation, it does not distinguish between alternative mechanisms
for the regulation of this process. The events observed during cell migration
may result from an ordered time sequence of transcription or translation. In
this case migration of pre-stalk cells to the area of stalk construction is programmed to occur at the same point in time as is expression of glycogen phosphorylase activity. An alternative mechanism for the expression of the events
observed during pre-stalk and pre-spore differentiation involves 'positional
information' (Goodwin & Cohen, 1969; Wolpert, 1969; Crick, 1970). According
to this concept, cells migrating into the area of future stalk formation would
obtain information due to their actual physical location within the cell mass or
from adjacent cells. Farnsworth (1973) has suggested that the tip of the slime
mold cell mass acts as such an organizing region. The migration, and subsequent
differentiation of presumptive cell types would presumably be under regulatory
control by cells found in the tip. The cell specific events leading to a kinetic
situation favorable for glycogen degradation as described in this report could be
a result of positional information or alternatively simply programmed to occur
at a given time. In any case the results of this study emphasizes the necessity of
evaluating the transient biochemical composition of cells with respect to their
physical position within a developing organism.
This research was supported by the Brown-Hazen Program of Research Corporation. The
technical assistance of L. Kooshian is greatly appreciated.
Cell specific events during development
343
REFERENCES
C. & FILOSA, M. (1965). Carbohydrate content during development of the slime
mold, Dictyostelium discoideum. J. cell. comp. Physiol. 66, 135-140.
CRICK, F. (1970). Diffusion in embryogenesis. Nature, Lond. 225, 420-422.
FARNSWORTH, P. (1973). Morphogenesis in the cellular slime mold Dictyostelium discoideum:
the formation and regulation of aggregate tips and the specification of developmental axes.
J. Embryol. exp. Morph. 29, 253-266.
FIRTEL, R. A. & BONNER, J. (1972). Developmental control of a-1-4 glucan phosphorylase in
the cellular slime mold Dictyostelium discoideum. Devi Biol. 29, 85-103.
GEZELIUS, K. & WRIGHT, B. E. (1965). Alkaline phosphatase in Dictyostelium discoideum.
J. gen. Microbiol. 38, 309-327.
GOODWIN, B. C & COHEN, M. H. (1969). A phase-shift model for the spatial and temporal
organization of developing systems. /. theor. Biol. 25, 49-107.
HAMES, B. D., WEEKS, G. & ASHWORTH, J. M. (1972). Glycogen synthetase and the glycogen
synthesis in the cellular slime mold Dictyostelium discoideum during cell differentiation.
Biochem. J. 126, 627-633.
HAMES, B. D. & ASHWORTH, J. M. (1974). The metabolism of macromolecules during the
differentiation of myxamoebae of the cellular slime mold Dictyostelium discoideum containing different amounts of glycogen. Biochem. J. 142, 301-315.
JONES, T. H. D. & WRIGHT, B. E. (1970). Partial purification and characterization of glycogen
phosphorylase from Dictyostelium discoideum. J. Bact. 104, 754-761.
LOWRY, O. H. & PASSONNEAU, J. V. (1972). A Flexible System of Enzymatic Analysis, pp. 253255. New York: Academic Press.
RUTHERFORD, C. L. & WRIGHT, B. E. (1971). Nucleotide metabolism during differentiation
in Dictyostelium discoideum. J. Bact. 108, 269-275.
SARGENT, D. & WRIGHT, B. E. (1971). Trehalose synthesis during differentiation in Dictyostelium discoideum. II. In vivo flux determinations. J. biol. Chem. 246, 5340-5344.
WHITE, G. J. & SUSSMAN, M. (1961). Metabolism of major cell components during slime
mold morphogenesis. Biochim. biophys Acta 53, 285-293.
WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation.
/. theor. Biol. 25, 1-47.
WRIGHT, B. E., ROSNESS, P., JONES, T. H. D. & MARSHALL, R. (1973). Glycogen metabolism
during differentiation in Dictyostelium discoideum. Ann. N.Y. Acad. Sci. 210, 51-63.
WRIGHT, B. E. & DAHLBERG, D. (1967). Cell wall synthesis in Dictyostelium discoideum. II.
Synthesis of soluble glycogen by a cytoplasmic enzyme. Biochemistry, N. Y. 6, 2074-2079.
WRIGHT, B. E. (1973). Critical Variables in Differentiation, pp. 53-93. Englewood Cliffs,
New Jersey: Prentice-Hall.
CECCARINI,
(Received 19 September 1975, revised 8 December 1975)