/. Embryol. exp. Morph. Vol. 33, 1, pp. 227-241, 1975
227
Printed in Great Britain
The tip of the Dictyostelium discoideum
pseudoplasmodium as an organizer
By JONATHAN RUBIN 1 AND ANTHONY ROBERTSON 1
From Department of Biophysics and Theoretical Biology,
University of Chicago
SUMMARY
We have extended Raper's original work on the organizing ability of the tip of the Dictyostelium discoideum slug.
Our new results are that tips from all multicellular (pseudoplasmodial) stages act as
organizers; that the structure organized by a tip depends on the developmental stage of the
cells responding to the tip's signal; that tips from all stages release a qualitatively similar
signal which is continuous, most probably a gradient of c-AMP; and that the signal from
fruiting-body tips appears stronger than that from conus tips.
We discuss these results with reference to the control of morphogenetic movement and
patterned differentiation and point out that the D. discoideum tip is analagous to a classical
organizer, whose signal is interpreted according to the state of determination of cells in its
field of influence.
INTRODUCTION
Very little is known about the systems controlling cellular behaviour during
development. One of the most important concepts is that of the organizer
(Spemann, 1938), a group of cells in an embryo which can define a developmental
axis and can behave autonomously in grafting experiments. Although much of
the phenomenology of organizer behaviour has been described, the mechanism
of organizer action is not understood at all. However, Raper (1940) pointed
out that the tip of the Dictyostelium discoideum slug, the migratory phase of the
pseudoplasmodium, behaved like a classical organizer. More recently Farnsworth (1973) has measured the time for the determination of a new tip when
the original one has been removed from a pseudoplasmodium. (Full reviews of
D. discoideum biology are to be found in Raper (1940), Shaffer (1962), Bonner
(1967), Gerisch (1968), Ashworth (1971), Robertson & Cohen (1972).)
As D. discoideum provides a multicellular system sharing many of the properties, in particular differentiation and regulation (see Wolpert (1969) for a review),
of Metazoan embryos, we decided to investigate tip function further and thereby
examine the mode of action of a primitive organizer. A fortunate feature of
D. discoideum is that the aggregative signal is now quite well understood and is
1
Authors' address: Department of Biophysics and Theoretical Biology, University of
Chicago, 920 East 58th Street, Chicago, Illinois 60637, U.S.A.
15-2
228
J. RUBIN AND A. ROBERTSON
apparently retained to control at least some aspects of development throughout
the morphogenetic cycle. We now provide a brief review of those features of
the aggregative signalling system relevant to the experiments reported in this
paper.
There is good, albeit circumstantial, evidence that the molecule used as an
aggregative signal (acrasin) is cyclic adenosine monophosphate (c-AMP)
(Konijn, van de Meene, Bonner & Barkley, 1967; Konijn, Barkley, Chang &
Bonner, 1968). D. discoideum amoebae release c-AMP during aggregation, as
well as a phosphodiesterase (PDE) (Chang, 1968) which destroys the c-AMP.
High c-AMP levels can inhibit cell movement and aggregation (Konijn, 1972),
as can high extracellular beef heart PDE activity (Robertson, 1974). c-AMP
causes chemotaxis of aggregative amoebae and aggregation can be controlled
by an artificial source of c-AMP (Robertson, Drage & Cohen, 1972). However,
the experiments and conclusions in this paper do not depend on the identification
of acrasin with c-AMP.
During interphase, the period between the end of feeding and the beginning
of aggregation, amoebae successively become chemotactically sensitive to
c-AMP, capable of relaying a c-AMP signal and then, for a few amoebae,
capable of releasing acrasin signals autonomously (Cohen & Robertson, 1972).
By the time autonomous signalling cells have emerged all the amoebae in a field
entering interphase at the same time are responsive both chemotactically and
by being able to relay a c-AMP signal. There are well-defined, different, threshold
c-AMP concentrations for chemotaxis and relaying (Konijn et ah 1968; Cohen
& Robertson, 1971 a, b; Robertson, 1974; Robertson & Cohen, 1974). As the
times of emergence of the chemotactic and relaying competences are well defined
it is possible to use a field of amoebae in interphase as a sensitive monitor of
the waveform and amplitude of the signal released by a suspected c-AMP source.
For example, a periodic artificial signal source, in a field of amoebae 4 h into
interphase, evokes periodic chemotactic movements towards the signal source
(Robertson, et ah 1972), while a continuous signal evokes continuous movement
(Robertson, 1974). In a field approximately 6 h old both periodic and continuous
signals evoke periodic signal relaying if the relaying threshold concentration is
exceeded. Amoebae close to the source move either periodically or continuously,
while amoebae receiving a relayed signal move periodically.
In this paper we show that the D. discoideum tip acts as an organizer throughout
morphogenesis from the late aggregate stage, and that the signal it releases is
continuous.
MATERIALS AND METHODS
Amoebae
A stock of D. discoideum, NC-4, was obtained from Professor K. B. Raper.
The food bacteria were Aerobacter aerogenes.
Tip ofDictyostoliumpseudopIasmodium as organizer
229
Plating, growth, harvesting and standardization
Spores from culminated sorocarps were collected in a drop of distilled water
held in a bacterial inoculating loop. The spores were then transferred to a test
tube containing 3 ml of A. aerogenes suspension in growth broth, made up as
follows: yeast extract, 0-5 g/1.; peptone, 5 g/1.; dextrose, 5 g/1.; KH2PO4,2-25 g/1.;
K 2 HPO 4 .12H 2 O, 1-5 g/1.; MgSO4.7H2O, 0-5 g/1.
Four drops of spore-bacteria medium were transferred to growth plates containing 2 ° 0 agar with 0-5 % peptone and 0-5 % dextrose made up in KK 2 buffer.
The amoebae were allowed to grow vegetatively on the bacteria for 48 h at a
constant temperature of 23 °C. While the amoebae were still in the vegetative
phase, they were harvested in chilled KK 2 buffer, made up as follows: KH 2 PO 4 ,
2-31 g/1.; K 2 HPO 4 .12H 2 O, 1-3 g/1.; MgSO4.7H2O, 0-5 g/1.
The amoebae were washed free of bacteria by suspending them in buffer and
centrifuging at 650 g for 2 min. This process was repeated three times. The final
pellet of amoebae was then suspended in 5 ml of KK 2 buffer, and the concentration of amoebae was determined with a haemocytometer. It was adjusted to
5 x 107 cells/ml, and 0-5 ml of this suspension was plated out on a non-nutrient
agar plate. The amoebae were then synchronized according to Bonner (1967)
by returning the plates of harvested amoebae to an incubator at 23 °C for 2 h
and then refrigerating them at 4 °C until needed. The plates were then placed in
the 23 °C incubator for the desired incubation period.
Transplantation
Amoebae on some growth plates were not harvested but were allowed to
continue morphogenesis beyond the vegetative phase to provide material for
the transplant experiments. Glass micropipettes drawn into sharp points were
used to effect tip transfers. The removal of tips was accomplished by placing the
pipette at the base of the tip when there was a clear morphological difference
between the tip and the remainder of the structure (i.e. early fruiting body or
stage 17 or 18 of Farnsworth) (Farnsworth, 1973). If there was no clear morphological distinction between tip and body of the structure, as in a slug, the
removal was done as near to the front end of the structure as possible. The
transfer was then made to the recipient structure. In the later stages of the life-cycle
(Farnsworth stages 8-20) the tip was pushed into the recipient until it broke the
surrounding slime sheath, if present, and was securely attached. Care was taken
to deform the receiving structure as little as possible in the process. When transferring a tip into a field of interphase amoebae, the tip was placed into the field
as gently as possible in order to avoid injury to the tip. All of the transplants
were done under a Nikon Dissecting Microscope.
230
J. RUBIN AND A. ROBERTSON
Filming
The filming was done in a constant-temperature room (21 + 1 °C.) with a
Bolex 16 mm movie camera and a Nikon CFMA camera drive. The filming
microscope was a Nikon Apophot using transmitted light. The exposure was
set automatically. Frame rates varied from 2 to 6 per minute. The most common
magnification used was \% (5 x eyepiece, 1-2 x plan objective and -| x relay
lens). Experiments were recorded on Kodak 16 mm 4X reversal film.
Some transplants of tips into fields of interphase amoebae were recorded with
a Concord video-tape recorder, VTR-648, at a rate of 1\ frames/sec and played
back at the normal rate of 60 frames/sec. The images were displayed on a
Concord Mr-900 monitor.
Statistical experiments
One set of transfers was done to provide large numbers of results for statistical
purposes. The transplants were performed as previously described. However,
after each tip transplant was completed the recipient and transplant were transferred to a plain agar plate, allowed to develop, and observed to see whether
two fruiting bodies had formed. If one fruiting body was less than one-quarter
the size of the other the experiment was scored as a failure, as we had observed
that tips which fell off and regulated to make independent fruiting bodies never
attained that size.
Timing and period measurement
Time and periods were determined by projecting the films with a Traid
Selecta-frame projector, Model 16N/LS, equipped with an automatic frame
counter. Times were also measured with a stop-watch.
RESULTS
A. Grafts of tips into pseudoplasmodia
(i) Tips from all multicellular stages were transplanted to each multicellular
stage of the life-cycle of D. discoideum. Each tip was taken from the donor
structure and placed on the recipient. Each experiment was filmed by time-lapse
microphotography. Line drawings from films of each category of experiment
are shown in Fig. 1, and a typical graft is shown in the photograph.
The dynamics of a transplant were independent of the source of the tip or of
the stage of the recipient structure. In a successful transplant the tip began to
move within 15 min. It appeared to be orienting itself with respect to the recipient.
If the recipient was an upright structure, such as a conus, the tip often began
to slide downwards. However, within 30 min the tip stopped its movement and
began to set up its own field of influence in the recipient. This event was manifested by a ring or indentation which formed between the tip plus the field of
cells it had pulled in and the field of cells still under the control of the original
structure's tip. The average time for the indentation to appear was 42 + 16 min
Tip of Dictyostelium pseudoplasmodium as organizer
Transplant
Time after transplant (h)
231
Result
at 4 h
Conns
onto
(plan)
con us
Slug
onto
(plan)
slug
Fruiting body
onto
(plan)
slug
Slug
onto
(elevation)
con us
Slug
(elevation)
onlo
fruit inn bodv
Fruiting body
onto
(elevation)
fruiting bodv
Fruiting body
onto
(plan)
con us
COIHIS
onto
(elevation)
fruit inn bodv
Conus
onto
(plan)
slue
Two slugs
Two slugs
Two slugs
Two slugs
Two fruiting
bodies
Two fruiting
bodies
Two slugs
rn
Two fruiting
bodies
Two slugs
Fig. 1. Line drawings, taken directly from single frames of our films, showing pseudoplasmodial organization by grafted tips, which are shaded.
from the time of grafting. This corresponds well to the time of field formation,
approximately 34 min, measured by Farnsworth (1973). The indentation completely demarcated the structure under the control of the recipient's tip from
the newly formed structure under control of the transplanted tip (see the
photograph). Once it had formed the two structures acted independently of each
other. The recipient continued its developmental cycle independently of the
grafted tip and the cells which it had lost. The transplanted tip and the new cells
which it had attracted also proceeded through the standard developmental
cycle. In all cases in this group of experiments, the new structure produced by
the grafted tip was at the same stage as the recipient structure. If, for example,
a slug tip was transplanted on to a fruiting body, the slug tip and the cells it
had pulled in immediately culminated and fruited without going through the
normal migratory phase. In contrast, if a fruiting-body tip was transplanted
on to a conus, the two structures formed were slugs, both of which migrated,
culminated and fruited. The slug formed by the fruiting-body tip was morphologically indistinguishable from any other slug. Its migration was normal, and
232
J. RUBIN AND A. ROBERTSON
Table 1. Results of transplants of conus tips to fruiting bodies, fruit ing-body tips
to conuses, conus tips to conuses, and fruit ing-body tips to fruiting bodies
Each set of transplants is compared to a control consisting of a sham operation.
In each case, the difference between the control and transplant was highly significant
by the x2 test {P < 10~6). The table also shows the results of transplanting conus
and fruiting-body tips into fields of amoebae scored as their ability to cause directed
movement for 30 min, compared with the control transplantation of fruiting-body
bases. The difference is significant. (S = success, F = failure as % of total.)
Conus
Recipient
Conus
F.B.
Controls
F.B.
S
F
S
F
S
F
59
50
41
50
75
63
25
37
27
17
73
83
it fruited as a normal slug would. Several examples of all of the transplants in
the above experiments were recorded on film.
(ii) A subset of experiments (Table 1) was then selected in order that large
numbers of transplants could be performed to determine the proportion of
experiments in which there was successful field organization by grafted tips and
to see whether there were any quantitative differences between tips. The subset
chosen included conus to conus, conus to fruiting body, fruiting body to conus,
and fruiting body to fruiting body. As a control, sham operations were performed. In these, all manipulations were performed as in a normal transplant,
except that no tip was transferred. x* tests showed that in every case in Table 1
the difference between the experiments and controls was highly significant
(P < 10"6).
Table 1 shows that tips transplanted in these experiments always had a significant success rate in organizing fields when compared with the controls. It must
be remembered that the criterion for success that we adopted gives us a lower
limit for organizing ability and that when the experiments were filmed throughout
we could always see some evidence of organizing activity by grafted tips, in
particular the formation of an indentation between the grafted and host tips.
The controls, however, show that interference with a pseudoplasmodium often
leads to regulation and the formation of an extra tip, after a delay of at least
B. Transplantation of tips into fields of amoebae
Tips of conuses, slugs and fruiting bodies were transplanted into fields of
interphase amoebae. The tips were placed into fields of amoebae that were 4, 6
and 8 h past refrigeration - that is, 6, 8 and 10 h into interphase if one includes
the 2 h incubation period before refrigeration. As controls, fruiting-body bases
and the middle portions of slugs were also transplanted into fields. A summary
of the results is shown in Table 2.
Tip ofDictyostelium
pseudoplasmodium
as organizer
233
Table 2. Summary of results of transplants into fields of amoebae
(F.B. = fruiting body. A dash indicates that the category is not applicable to a
particular graft.)
Graft
No. of experiments
Attraction (%)
Initial continuous movement (%)
Periodic relaying (%)
Time to periodic relaying (min)
Time to pseudoplasmodium formation
(min)
Direct culmination (%)
F.B.
tip
Conus tip F.B. base
Slug
middle
93
94
94
24
88
150
93
84
84
5
—
87
61
8
0
8
—
—
41
100
0
100
30
290
4
26
—
0
(i) Fruit ing-body tips into fields
Ninety-three transplants were performed. In 87 of these chemotactic responses persisted for more than 30 min from the time of transplantation. The
responses were independent of the age of the field, demonstrating that all fields
had achieved both chemotactic and relaying competences.
When a tip was placed into an interphase field, amoebae were immediately
attracted toward the tip, moving continuously, and in straight lines. No pulsations were observed during this initial period. This implies that the attracting
signal was continuous. The initiation of periodic movements occurred from
40 min to 4 h after transplantation. This happened in 26 % (23/87) of those
experiments in which tips attracted amoebae for more than 30 min. The average
time for the initiation of periodic movements was 87-8 ± 39-5 min. The number
of periodic movements associated with each tip was quite variable, ranging up
to 12. After the initial pulse, the pulse period quickly decreased to about 10 min
(see Fig. 2). Once pulsations began and the field of amoebae showed periodic
movements towards the tip, streams formed. The streams either began at the
boundary of the tip or up to 50 /im away from the boundary. Thus the tips
caused periodic signal relaying, even though their own signal was continuous,
and the threshold for signal relaying may be exceeded at up to 50 jum from
the tip.
Approximately 2\ h after transplantation the tip and the cells it had attracted
formed a pseudoplasmodium (Fig. 3). In 96 % of the 48 cases which were observed
throughout this period, this was a conus which then transformed into a slug.
In two cases out of 48, however, a fruiting body formed directly, as can happen
in normal morphogenesis (Newell, Telser & Sussman, 1969).
234
J. RUBIN AND A. ROBERTSON
160 i—
120
80
40
J
I
12
10
Pulse number
Fig. 2. Periods of waves propagated from fruiting-body tips transplanted into
fields of amoebae.
40 -
.
,
30
20
10
r-L
4
6
10
Hours
Fig. 3. Histogram of times to pseudoplasmodium formation after transplantation
of fruiting-body tips into fields of amoebae.
(ii) Conus tips into fields
Eighty-four per cent (78/93) of conus tips attracted cells. The amoebae first
responded with continuous movement toward the tip and occasionally (5/78)
with later pulsations and stream formation. Twenty-six per cent (20/78) of the
transplants culminated without slug formation; the difference in times to
Tip ofDictyostelium pseudoplasmodium as organizer
40
235
-
20
10
2
Hours
Fig. 4. Histogram of times for pseudoplasmodium formation after transplantation
of conus tips into fields of amoebae.
20
15
10
LI H l i n i i n
0
10
20
30
m
40
50
60
70
Interval length (min)
m
80
90
100
Fig. 5. Periods of waves propagated from slug middles transplanted into
fields of amoebae.
culmination between the conus tips and the fruiting-body tips was significant
(P < 0-05). The mean time to slug formation was 87 min (Fig. 4) - much
shorter than that for fruiting-body tips (P < 0-05).
(iii) Base of fruiting bodies
To check that the tip behaves differently from an inert object placed into the
field of interphase amoebae, the ability of bases of fruiting bodies to attract
cells was investigated. Using the same criterion of continuous directed movement
toward the object for 30 min only 5 cases out of 61 base transplants were
236
J. RUBIN AND A. ROBERTSON
40
30
20
10
10
6
Hours
Fig. 6. Histogram of times for pseudoplasmodium formation after transplantation
of slug centres into fields of amoebae.
4
successful. In all of these five cases, an independent autonomous centre had
formed close to the base. This is close to the proportion ofconus tip transplants,
but significantly (P <^ 0-001) smaller than that of fruiting-body tip transplants,
showing periodic signalling. We therefore conclude that fruiting-body tips tend
to cause periodic signal relaying, but that conus tips do not. This implies that
conus tips secrete less attractant than do fruiting-body tips, as the threshold
for signal relaying is higher than that for chemotaxis (see Introduction).
(iv) Mid-portion of slugs
We placed 41 mid-portions of slugs into fields of amoebae 8 h into interphase.
The mid-portions were approximately 100 jumm diameter. All attracted amoebae
periodically. The first propagated waves occurred as early as 10 min from the
beginning of filming - that is, approximately 20 min after transplantation. The
distribution of recorded periods is shown in Fig. 5. After a mean time of 4 h
50 min (Fig. 6) all slug centres had regulated to produce conuses which then
proceeded through the normal developmental cycle to become fruiting bodies
containing some amoebae attracted from the field. The differences in mean times
between the production of slugs from fruiting-body tips (P < 0-01) or conus
tips (P <t 0-001) and slug middles placed in interphasefieldsare highly significant.
They are partially accounted for by the extra time required for regulation by
the slug middles to produce new tips - about 1£ h (Farnsworth, 1973).
Tip o/Dictyostelium pseudoplasmodium as organizer
237
Fig. 7. Typical result of grafting a fruiting-body tip into an early fruiting body, 1 h
after grafting. Two structures are beginning to separate; the indentations demarcating
the fields of the two tips are clearly visible. The frame width is 0-5 mm. The grafted
tip is on the right.
DISCUSSION
1. General remarks
Although there has been no complete review, it is now well established in the
literature that tips have special properties. We shall mention these briefly and
then discuss the relevance and significance of our own results.
Raper showed that the tip controls slug migration (Raper, 1940) and that
extra tips grafted into the side of a slug would take over cells posterior to the
graft site provided that the original polarity of the grafted tips was maintained.
He concluded that the tips acted as though they were organizers, controlling
cell movement and defining the developmental axis of the slug. Farnsworth has
shown that the presence of a tip on a pseudoplasmodium inhibits further tip
formation. The inhibition is released within approximately \ h - the tip determination time - when a tip is removed. Both Raper's and Farnsworth's results
support the analogy between tips and classical organizers.
Several authors have shown that tip cells have histochemical and ultrastructural properties not shared by other cells in the pseudoplasmodium (Bonner,
238
J. RUBIN AND A. ROBERTSON
Chiquoine & Kolderie, 1955; Hohl & Hamamoto, 1969; Maeda & Takeuchi,
1969; Gregg & Badman, 1970; Miiller & Hohl, 1973; Maeda & Maeda, 1974),
and that the cells that first form the tip remain in it until the end of morphogenesis (Takeuchi, 1969; Farnsworth, 1973). The tip forms the anterior 10%
or even less, of the pseudoplasmodium. Recently Maeda & Maeda (1974) have
found that the anterior (light) cells in D. discoideum also contain up to ten
times more c-AMP than the posterior (heavy) cells, though their light fraction
certainly includes pre-stalk cells posterior to the tip proper. Bonner (1949) had
demonstrated that for both slugs and fruiting bodies, the front released more
acrasin than the remainder of the pseudoplasmodium.
Finally, it has been shown that cells in isolated slug fragments de-differentiate,
and then re-differentiate (Gregg, 1965; Sakai, 1973) but only after there has
been sufficient time for regulation to produce a new tip.
2. The equivalence of tips
In Section A of the results we showed that tips from all pseudoplasmodial
stages act as organizers when grafted into all pseudoplasmodial stages and into
fields of amoebae before aggregation. The structures organized depend on the
developmental stage of cells in the recipient pseudoplasmodium. Therefore, tips
from all stages have qualitatively similar organizing abilities, and we can infer
that their organizing signals are qualitatively similar. This is analogous to the
interpretation by cells of the signal from a classical organizer such as the dorsal
lip of the amphibian gastrula (cf. Spemann, 1938) or the hypostome of hydra
(Wolpert, Hicklin & Hornbruch, 1971). It appears that organizers in general
produce a stage-independent signal which is interpreted by responding cells
according to their capacities or state of determination (Wolpert, 1969; Robertson
& Cohen, 1972).
3. Quantitative differences between tips
Our experimental results in Section B show that conus tips are less likely to
initiate signal relaying and periodic cell movement, when grafted into fields of
sensitive amoebae, than are fruiting-body tips, although both kinds of tip cause
chemotaxis. As the threshold for chemotaxis is lower than that for signal relaying
(Robertson & Cohen, 1974) we may conclude that the signal from a conus tip
is weaker than that from a fruiting-body tip. These results are therefore consistent with the possibility that tips release c-AMP. However, conus tips organize
new slugs more quickly than do fruiting-body tips. We do not know why. It is
possible that the conus tip secretes slime but that the fruiting-body tip does not.
The secretion of slime may expedite slug formation (Shaffer, 1962).
4. The tips' signal
The continuous movements of interphase amoebae towards tips show that
the tips release continuous signals. In experiments to be published in the next
Tip of Dictyostelium pseudoplasmodium as organizer
239
paper in this series we have found that beef heart PDE, when added to the
culture medium, inhibits the signal. These results support the postulate that tips
release c-AMP continuously, which in turn is consistent with the observed high
c-AMP concentration in the anterior cells of slugs. If the signal is a c-AMP
gradient then it can exceed a concentration, for fruiting-body tips, of 10~7 M,
the approximate threshold for relaying, at a distance of at least 50 jum from the
tip border. For conus tips the relaying threshold is not exceeded outside the tip,
although it may well be within the tip. In pseudoplasmodia cells are joined
closely by intercellular contacts. The amount of signal a tip must release to
cause relaying in a pseudoplasmodium will therefore be less than that required
to cause relaying by separated cells.
5. Pseudoplasmodial organization
The tip forms during late aggregation. Durston (1974) has shown that from
this time all autonomously signalling regions close to a tip are brought under
its control. As the autonomous sources are periodic we can understand this
observation if the tip releases a continuous signal that has a range greater than
that of the signal of the most stable autonomous pacemaker, that which propagates spiral waves at the refractory period of the field. The continuous signal
from the tip is also consistent with Durston's observation that the period of
signals propagated from an aggregate decreases monotonically to 2\ min, once
the tip has formed. This happens because the tip supplies a signal continuously
above the threshold for signal relaying, and because the refractory period for
signal relaying decreases monotonically to 2\ min. This property of the tip
may be fundamental to its ability to control pseudoplasmodial organization and
movement throughout the rest of the developmental cycle. In this model tips
dominate because they can initiate signal relaying at a distance and at the
refractory period of the medium (Durston, 1973, 1974).
The properties of slug mid-portions are illuminating in this respect. The
signal from middles placed in interphase fields is always pulsatile although the
period is ill-defined. It is consistent with the idea that cells in the middle, which
had been entrained by the refractory period signal caused by the slug tip, can
now exhibit autonomous signalling as they are no longer under the tip's influence.
The signals begin when the inhibition from the tip has disappeared (Farnsworth,
1973). A further possibility is that the signal gradient formed by the tip controls
the differentiation of pre-stalk cells to stalk cells, a view supported by Bonner's
observation that c-AMP can cause stalk cell differentiation (Bonner, 1970). As
we have shown that a tip can be produced, by regulation, from any portion of
a pseudoplasmodium, it is probable that its special signalling properties depend
on a geometrical feature of its organization rather than on an inherent capacity
of its constituent cells.
240
J. RUBIN AND A. ROBERTSON
We are very grateful for the assistance of David Drage and Diane Wonio, who performed
some of the first grafting experiments, and to Morrel Cohen and Susan Weiter for their
critical reading of the manuscript.
This work was supported by grant number GB-30784 from the National Science Foundation
to the University of Chicago. A.R. is an Alfred P. Sloan Fellow (1973-5).
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RAPER,
{Received 2 July 1974, revised 5 August 1974)
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