/ . Embryol. exp. Morph. Vol. 53, pp. 353-365, 1979
Printed in Great Britain (p Company of Biologists Limited 1979
353
Responses of the early chick embryo
to external cAMP sources
By ALAN R. GINGLE 1 AND ANTHONY ROBERTSON 1
From the Department of Biophysics and Theoretical Biology,
University of Chicago
SUMMARY
Early chick embryos were stimulated with local sources of cAMP. Three major effects
were observed: bending of the embryonic axis, attraction of cells on the ventral surface of the
embryo, and disruption of the blastodisc. Each had a characteristic concentration dependence.
These results are compared with those from studies of cells disaggregated from similar
embryos.
INTRODUCTION
Soon after the discovery of the Amphibian organizer by Mangold & Spemann
(1927), reviewed by Spemann (1938), Waddington and his collaborators showed
that bird embryos also possessed an organizing region which could induce the
development of a secondary embryonic axis when grafted into a host embryo
(reviewed by Waddington, 1952). Furthermore, Waddington (1937) demonstrated
that the bird organizer could be interchanged with that from a mammal, suggesting that both worked in a similar way. Waddington also believed that the direction in which the avian axis developed was initially determined by an inductive
interaction between the endoderm and overlying ectoderm and mesoderm. He
showed this by rotating the epiblast with respect to the underlying hypoblast
(Waddington, 1933). More recently Gallera (1971) has performed a series of
experiments in which the extent and duration of organizer action and of
susceptibility to an implanted organizer in the chick are delineated. Nothing is
known, however, about the chemical nature of the inductive signal believed to be
released by the organizer.
Nowadays a convenient way to test the inducing capacity of a chemical is to
use a microsource which releases it near the explanted embryo's surface. This
makes a direct comparison with results of the grafting experiments of classical
embryology easy. In many of those experiments, the inductive capacities of
organizing tissues and substances were studied by implanting small pieces of
tissue or agar blocks containing diffusible substances into host embryos (Huxley
& De Beer, 1934). In such experiments the effects on the host embryos can be
1
Authors' address: Biological Research Corporation, Lexington, Georgia 30648, U.S.A.
354
A. R. GINGLE AND A. ROBERTSON
related to the source or tissue position within the embryo and there is minimal
disruption of the host.
We have found that microsources releasing cAMP have several effects on the
development of early chick embryos (Robertson & Gingle, 1977). A source near
an explanted embryo's ventral surface could attract cells and divert the embryonic axis. We also observed effects of cAMP on populations of cells dissociated from early chick embryos. Reaggregation studies showed critical cell
densities (at which there is an abrupt decrease in aggregate density) which were
increased by phosphodiesterase (PDE), an enzyme which converts cAMP to
linear AMP (Gingle, 1977).
Critical densities are characteristic of cells capable of relaying a chemical
signal (Gingle, 1976). The critical cell densities increased with the addition of
PDE, indicating that cAMP, its substrate, was probably the relayed signal
molecule (Gingle, 1977). cAMP-relaying competent cells will release a pulse of
cAMP molecules when stimulated by exposure to cAMP. Populations of dissociated chick embryo cells do indeed release cAMP into the extracellular
medium, but only when stimulated by cAMP concentrations between 10~8 M
and 5 x 10"6 M (Robertson, Grutsch & Gingle, 1978).
All of these results implied the need for further quantitative study of the effects
of cAMP on early embryonic development. In particular, the cAMP concentration dependencies of cell attraction and diversion of the embryonic axis
needed to be determined for comparison with the results of experiments on
dissociated cell populations. The dynamics of these phenomena, uncovered by
time-lapse filming, are also crucial for determining the qualitative effects of
different cAMP concentrations on development of the intact embryo.
MATERIALS AND METHODS
Embryos
The technique of explanting, culturing and filming is derived from that of
Waddington (1932) with modifications due to De Haan(1967), as published by
New (1966), and has been described earlier (Robertson & Gingle, 1977). Except
for the youngest embryos (up to 10 h incubation) it was possible to explant and
culture 80 % of the embryos successfully in a series of about 500. Each embryo,
in its petri dish, was either covered and returned to the incubator for later
examination, or was placed within a cylindrical copper water jacket, maintained at 38 °C and mounted directly on the microscope stage. The age of each
embryo is expressed as its stage according to Hamburger & Hamilton (1951).
Culture media
Chick Ringer: one liter of chick Ringer, made up in deionized water and
autoclaved at 115 °C for 30 min, contained 9-0 g NaCl, 0-42 g KC1 and 0-25 g
CaCl 2 (New, 1966). The CaCl2 was added after autoclaving.
Responses of chick embryo to external cAMP sources
355
Agar substrate: a 3:2 mixture of thin albumen and chick Ringer was made at
room temperature. One Pasteur pipetteful of this medium was added, in the
bottom of the sterile 5 cm plastic petri dish, to one pipetteful of a 2 % solution of
DIFCO purified agar in Ringer at 55 °C, mixed, covered and allowed to gel,
forming a reasonably flat substrate 2 mm deep.
Filming
16 mm movie films were taken with transmitted light on a Bolex H-16 M
camera coupled to a Nikon CFMA camera drive using a Nikon SKE or Apophot microscope body or a Zeiss model GF body. Long working distance planachromat objectives (x 1-2, x 2, x 5, x 10 or x 20) were used, with a x 5 eyepiece and x \ camera relay lens, giving a range of total magnifications between
\\ and 25 times. A green filter was used to enhance contrast on the film, and a
heat-filter to prevent overheating of the embryo by the light source. Frame rates
were in general four or eight per minute. Higher rates were occasionally used.
Kodak 4xR reversal film, rated at 225 ASA, was processed commercially.
35 mm stills were taken with a Nikon F camera back coupled to a Nikon CFM
with a x % relay lens on either Ilford FP3 or Kodak Plus-X film rated at 125
ASA.
For time-lapse films the exposure was set either manually, or automatically by
the Nikon CFMA camera drive; for 35 mm photographs a coupled, throughthe-lens light meter was used to aid manual exposure setting.
Stimulation with cAMP
cAMP was applied locally to embryos in two ways. In the first a Pyrex microelectrode with an open internal tip diameter between 10 and 20 jtim was used. The
tip was sealed with molten agar to prevent loss of the electrolyte by passive flow.
The electrolyte was cAMP in Ringer; negative ions were driven out electrophoretically by currents between a chlorided silver wire in the electrolyte and a
similar wire grounding the agar substrate. Full details of technique and electrode
calibration have been published (Cohen, Drage & Robertson, 1975). The microelectrode was held in a micromanipulator mounted on the microscope stage; its
tip was advanced until it was within 50 ^m of the ventral surface of the embryo,
but not touching.
In the second method a 2 mm straight length of Bio-Rad 50 dialysis tubing was
cemented at both ends into L-shaped pieces of stainless steel tubing from syringe
needles. The overall diameter of the tubing was 300 jam, with a wall thickness of
20 /an and transverse pore diameter of approximately 20 nm, allowing the passage of cAMP. cAMP in Ringer was perfused through the tubing by a Harvard
Apparatus Co. Model 975 infusion pump at nominal flow rates between 0-02
and 0-08 ml/min. At these flow rates the concentration of cAMP attained just
outside the tubing is approximately 4 % of that in the perfusing fluid and is
356
A. R. GINGLE AND A. ROBERTSON
independent of the flow rates used, as we showed by measurements with tritiated
cAMP. The tubing was supported by a micromanipulator mounted on the
microscope and advanced, held parallel to the embryo surface, until it was
approximately 100 jam above the embryo, which it never touched.
Calibration
Calibration of the microelectrode has been described before (Cohen et a!.,
1975). The dialysis tube source was calibrated by perfusion, at 0-028 ml/min,
with [3H]cAMP in Howard's Ringer at concentrations of 10~8, 10~6, 10~4 and
10~2 M. Perfusion was for 1 h; the source was immersed in 6 ml of Ringer contained in a scintillation vial. The vials containing Ringer and an unknown
amount of cAMP were placed at 60 °C for evaporation, leaving only the cAMP
and Ringer salts. Scintillation fluid, a mixture of toluene, Triton x-100, and
PPO-POPOP, was added and the vials were 'counted'.
The measured count rates were compared with those for vials containing
known amounts of [3H]cAMP, and the efflux of cAMP molecules from the
dialysis tube source was determined from these experiments. It is (1-70 ±0-12)
x 108 x C1X molecules sec."1 /.tm~2, where C1N is the cAMP concentration
(molarity) of the solution flowing through the dialysis tube. The tolerance
(± 0-12) molecules sec.-1 /im~2 is the standard deviation of fluxes measured over
the range of C1X. From the flux the cAMP concentration at the tube's outer
surface can be calculated and is (4-4 ± 0-3) % of C lN .
Measurement of angles
The frame showing maximal bending on each time-lapse film was backprojected on to a translucent screen. Straight-line segments were fitted by eye to
the anterior and posterior parts of each embryonic axis. The external angle at
their intersection was measured. The measurements were made 'blind'. These
angles were called 'the angles of maximum bend'.
RESULTS
1. Concentration dependence
In these experiments we scored all the embryos treated with a microelectrode
source for three effects: diversion of the embryonic axis, local attraction of cells
to the electrode tip, and detachment of the blastodisc from the vitelline membrane. All three showed a strong concentration dependence, illustrated in Figs.
1 A, IB and 1C. The axis was considered diverted if it changed direction by 5°
or more from its original orientation and towards the electrode source. For these
experiments the electrode was placed lateral to Hensen's node of embryos between H-H stages 3-5, and close to the boundary between the area opaca and
areapellucida. Great care was taken to avoid touching the surface of the embryo.
In some instances so many cells accumulated beneath the electrode tip that
Responses of chick embryo to external cAMP sources
1
1
I
1
i
1
1
1
A
•
•
2 0-4
/
0
i
i
1
1
10
1
- 4
(,
log,,, [cAMl'l
i
i
10
I
I
-•8
i
i
i
- 6
log,,, [cAMP]
Fig. 1. Fraction of embryos responding to a cAMP electrode vs. log10[cAMP] by (A)
bending of the embryonic axis, (B) exhibiting cell attraction, and (C) detachment of
the blastodisc. All embryos were observed for at least 8 h beginning at Hamburger
and Hamilton stages 3-4. At least 10 embryos were used for each concentration
point. The total sample was 137 embryos.
357
358
A. R. GINGLE AND A. ROBERTSON
contact was eventually made, particularly at concentrations of cAMP in the
electrode between 10~6 and 10~3 M. This always took several hours to develop
and did not affect scoring of the results, except that, at these concentrations,
detachment of the blastodisc often followed the accumulation of a cellular
mass beneath the electrode tip.
In our earlier report (Robertson & Gingle, 1977) we showed that at most 11 %
of control embryos, treated with electrodes containing only Ringer's solution,
were scored (blind) as having bent axes. Up to 76% treated with 10~ 3 McAMP were scored as having bent axes, and 43 % as showing cellular attraction
towards the electrode. In those experiments the embryos were prepared in
batches often and observed only before and after the experiment; they were not
filmed. In the experiments reported here all the embryos were filmed throughout
the experiment and were treated individually and more carefully. The results
show distinct, and different, thresholds for each phenomenon scored. In addition
the films can still be used to analyze the dynamics of the responses, some aspects
of which will be mentioned below. The threshold concentrations for axis bending, cell attraction and disruption of the embryo are between 10~9 and 10~8 M,
between lO"10 M and 10~9 M, and between 10"6 M and 10~5 M respectively, when
the threshold is defined as that concentration of cAMP, in the electrode, which
evoked a half-maximal response.
2. Angles of maximum bend
Angles of maximum bend were measured for 82 embryos, at least six for each
stimulus concentration. The mean for each concentration is shown in Fig. 2.
When bending is measured as a continuous variable it shows a roughly linear dependence on the logarithm of the stimulus concentration; the effect becomes
significant for cAMP stimuli between 10~9 and 10~8 M. It should be noted that
the data shown in Figs. 1 and 2 give an internal control for the effects of cAMP,
since no significant responses are found at the lowest concentrations used.
This is illustrated by Fig. 3, in which these data are plotted as histograms.
Each histogram contains data from three electrode concentrations, grouped into
high, medium and low ranges. One concentration range (Fig. 3C) is below the
threshold concentration for the induction of axial bending inferred from Figs.
1B and 2. For the lowest range there are no bends above 10°; in both the higher
(supra-threshold) ranges a substantial proportion is above 10°. In the middle
range there is no significant difference between the distribution of the total
sample and that from only the 10~9 M electrodes. In the high range seven out of
nine bend angles equal to or above 25° were from embryos stimulated with 10~3
M-cAMP.
3. Time courses of responses
In this section we give a more detailed description of three experiments to
illustrate the dynamics of the responses observed. The embryo shown in Fig. 4
Responses of chick embryo to external cAMP sources
1
1
359
1
1
24 r—
1
-A
7°
.im angle
-3
-
lUIIXBl
-
-
cc
Average 11
_
n
-
-
1
T
10
1
1
1
8
•6
[cAMPI
log10
i
- 4
Fig. 2. Average maximum angle of bend plotted as a function of cAMP concentration in the electrode. Bars represent standard errors about the mean.
(stage 3) was stimulated with a continuous signal of 10~ 3 M-CAMP. The sketches
cover a period of about 20 h, during which development was greatly influenced
by the electrode. The stimulus was switched off between frames 1 and 2 and on
again between frames 4 and 5 (see figure caption for details).
The main responses observed are accumulation of cells towards the electrode
and bending of the primitive streak. Both show considerable inertia in that they
continue for some time after cessation of stimulation and take time to develop
once the electrode is switched on again.
In Fig. 5 we show a similar series of an embryo, beginning at stage 6 + and
ending, 11 h later, with seven pairs of somites (stage 9). In this case stimulation
was with a dialysis tubing source through which 10~3 M-cAMP was flowing. The
exposed portion of the tubing is shown in black. As node regression proceeded
the posterior end of the embryo bent towards the source until, as shown in
frames 5 and 6, the embryo was distinctly curved, with its head region also displaced towards the tube. After removal of the tube the embryo continued to
develop, forming five more somite pairs and a beating heart, after which development ceased. The photograph, taken just after removal of the tube, shows a
dense accumulation of cells between the head region and the tube's original
position, as well as the beginning of premature vascularization, which we often
observed under these conditions of stimulation. In this embryo, as in all which
have beenfilmedduring the appropriate stages, cells which took part in formation
of the first pair of somites dispersed and were incorporated in the spreading margins of the omphalo-mesenteric veins, suggesting that both somitic and vascular
360
A. R. GINGLE AND A. ROBERTSON
1
1
1
8 A
1
1
4 1
-
n
n
2
-
-
8 -
4
-
-
n
_
16
C
2 -
-
8 -
-
4 -
-
0
1
1
1
I
20
40
Angle of .maximum bend (deg.)
1
60
Fig. 3. Histograms derived from the data plotted in Fig. 2. The data were grouped
in three cAMP concentration ranges, (A) high (10~3, 10~4 and 10~5 M), (B) medium
(10-6, 10-7 and 10-8 M) and (C) low (10"9, 10-10 and 10"11 M).
tissues use similar mechanisms for cell accretion, presumably by chemotactic
attraction.
The last figure (Fig. 6) in this series illustrates this effect well. Here the stimulus
was a microelectrode releasing 10~7 M-cAMP continuously, leading both to a
dramatic bending of the axis during node regression and a large accumulation
of cells in an area beneath and around the electrode tip. While these cells did not
make contact with the electrode the cellular mass detached during fixation, as is
shown by the photograph of the embryo which was fixed immediately after the
embryo had developed to the stage shown in frame 5. Note that, while three
pairs of somites were visible at this stage, those on the electrode side were poorly
defined and hard to distinguish from the surrounding mesoderm.
Responses of chick embryo to external cAMP sources
Frame
361
1
Frame
10
Fig. 4. Sketches from the projected film image of a stage-3 embryo stimulated
with 10~3 M-cAMP. Frame 1 is 0-6 h after the beginning of the experiment; frames
2-10 are, respectively: 7-6 h, 10-2 h, 12-6 h, 13-6 h, 15-2, 16-7, 18-2, 20-1 and 20-9 h
from the beginning. Frame width is 2-4 mm.
DISCUSSION
1. General. Our results show the importance of filming in order to obtain a
continuous record of the experiments. Many of the phenomena reported involve
movement, which can only be inferred and not accurately described when
periodic observation or histological techniques are used alone. Some are transient and would be missed altogether, or misinterpreted if only seen at one point
in their evolution.
2. Controls. Developing chick embryos are sensitive to a variety of disturbances. Those germane to this study are: the direct action of cAMP, mechanical
tensions introduced during explanting and culturing, mechanical tensions and
stresses introduced by the microsource's presence, and the effects of the small
electrical current necessary for the microelectrode's operation. The mechanical
tensions arising during explanting and culturing have been controlled for
(Robertson & Gingle, 1977); however, it should be noted that the lack of blastodisc detachment and embryo damage below cAMP concentrations of 10~6 M
(see Fig. 1C) is very good evidence for the gentleness of the explanting and
culturing procedures.
The mechanical influence of the microsource was controlled for by using
different source geometries. Both microelectrodes, having a circular disc-like
A. R. GINGLE AND A. ROBERTSON
2
Frame
Fig. 5. Sketches from the projected film image (1-5) and (6), photograph of the
embryo after filming, of a stage-6 embryo stimulated with a tube source containing
10~3 M cAMP. 1-6 are 0-2, 2-3, 405, 5-7, 10-9 and 10-9 h after the beginning of the
experiment respectively. Frame widths are 2-4 mm except for 6 which is 1-8 mm.
geometry, and dialysis tube sources, having a cylindrical geometry, were used.
The sources were placed as far from the embryo's surface as possible, in order
to prevent direct contact. Here the limiting factor is determined by chemical
diffusion and source geometry, since for large distances the cAMP concentration
at the embryo's surface will fall far below its source value. Of course the microelectrode source requires a small electrical current for its operation, which
needed to be controlled for. This was done by varying the current and also by
employing the dialysis tube source which does not require an electrical current.
Responses of chick embryo to external cAMP sources
363
Frame
A
Frame
Fig. 6. Sketches from the projected film image (1-5) and (6) photograph of the
embryo after fixation, of a stage-6 embryo stimulated with an electrode containing
10- 8 M-CAMP. 1-6 are 0 1 , 4-7, 8-6, 13-2, 17-2 and 17-2 h from the beginning of the
experiment. Frame widths are 3-4 mm.
Both sources effectively attracted cells and bent the embryonic axis when charged
with the appropriate cAMP concentrations. These experiments, employing a
wide range of cAMP concentration, constitute a further control for influences
other than those of cAMP. The geometry, placement, and operating parameters
for each source type were held constant over the range of cAMP concentrations
studied. The results of these control experiments completely rule out the possibility of biases due to the microsource design or placement.
3. Concentration dependence. The phenomena of cell attraction and axial bending are characterized by low cAMP concentration thresholds in the 10~10 M10~7 M range. These are consistent with normal development as long as the
cAMP electrode concentration is below about 10~6 M-10~ 5 M, the threshold for
blastodisc detachment. By this, we mean that normal development occurs after
the electrode is removed and, though cell attraction and axial bending occur,
the embryo retains its integrity. cAMP electrode concentrations between 10~10 M
and 10~8 M only induce cell attraction, a short-range phenomenon, leaving
most of the embryo relatively undisturbed. For cAMP concentrations between
10~8 M and 10~6 M the effects are more global extending from the cAMP microsource to the embryo's axis. Above 10~6 M detachment of the entire blastodisc
often occurs. This effect, which is certainly pathological, causes long-range
364
A. R. GINGLE AND A. ROBERTSON
damage to the embryo, destroys its integrity, and prevents further development.
Data from dissociated cells, however, suggest that such high ambient cAMP
concentrations will not occur normally since cAMP-induced cAMP release is
reduced by stimuli greater than 10~6 M, and fully suppressed by a stimulus of
6 x 10-6 M (Robertson et al., 1978).
Finally, the very low thresholds for both cell attraction and axial bending
suggest both that the responses are specific to cAMP and that they are mediated
by extracellular cAMP receptors. A similar situation holds for D. discoideum,
where evidence for two classes of extracellular cAMP receptor (or for a single
receptor with negative cooperativity) has been found (Newell, 1977). Even the
quantities, especially the numerical values of threshold and signal size, are
remarkably similar. Further, under the same stimulating conditions both ceJl
types show a cAMP concentration above which cAMP relaying, or induced
synthesis and release, is sharply repressed (3 x IO~7 M for D. discoideum) (Grutsch
& Robertson, 1978). Similar quantities can be inferred from recent work on
other cell systems (see, e.g., Nakahara et al. (1978); Sonne, Berg & Christoffersen
(1978)).
Work supported by a grant-in-aid from the Alfred P. Sloan Foundation and by the Biological Research Corporation.
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{Received 19 July 1978, revised 28 April 1979)
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