responses of the early chick embryo to external camp sources · responses of chick embryo to...

/ . Embryol. exp. Morph. Vol. 53, pp. 353-365, 1979 3 5 3Printed in Great Britain (p Company of Biologists Limited 1979

Responses of the early chick embryoto external cAMP sources

By ALAN R. GINGLE1 AND ANTHONY ROBERTSON1

From the Department of Biophysics and Theoretical Biology,University of Chicago

SUMMARYEarly 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 theembryo, and disruption of the blastodisc. Each had a characteristic concentration dependence.These results are compared with those from studies of cells disaggregated from similarembryos.

INTRODUCTION

Soon after the discovery of the Amphibian organizer by Mangold & Spemann(1927), reviewed by Spemann (1938), Waddington and his collaborators showedthat bird embryos also possessed an organizing region which could induce thedevelopment of a secondary embryonic axis when grafted into a host embryo(reviewed by Waddington, 1952). Furthermore, Waddington (1937) demonstratedthat the bird organizer could be interchanged with that from a mammal, suggest-ing that both worked in a similar way. Waddington also believed that the direc-tion in which the avian axis developed was initially determined by an inductiveinteraction between the endoderm and overlying ectoderm and mesoderm. Heshowed this by rotating the epiblast with respect to the underlying hypoblast(Waddington, 1933). More recently Gallera (1971) has performed a series ofexperiments in which the extent and duration of organizer action and ofsusceptibility to an implanted organizer in the chick are delineated. Nothing isknown, however, about the chemical nature of the inductive signal believed to bereleased by the organizer.

Nowadays a convenient way to test the inducing capacity of a chemical is touse a microsource which releases it near the explanted embryo's surface. Thismakes a direct comparison with results of the grafting experiments of classicalembryology easy. In many of those experiments, the inductive capacities oforganizing tissues and substances were studied by implanting small pieces oftissue 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 minimaldisruption of the host.

We have found that microsources releasing cAMP have several effects on thedevelopment of early chick embryos (Robertson & Gingle, 1977). A source nearan explanted embryo's ventral surface could attract cells and divert the em-bryonic axis. We also observed effects of cAMP on populations of cells disso-ciated from early chick embryos. Reaggregation studies showed critical celldensities (at which there is an abrupt decrease in aggregate density) which wereincreased by phosphodiesterase (PDE), an enzyme which converts cAMP tolinear AMP (Gingle, 1977).

Critical densities are characteristic of cells capable of relaying a chemicalsignal (Gingle, 1976). The critical cell densities increased with the addition ofPDE, indicating that cAMP, its substrate, was probably the relayed signalmolecule (Gingle, 1977). cAMP-relaying competent cells will release a pulse ofcAMP molecules when stimulated by exposure to cAMP. Populations of dis-sociated chick embryo cells do indeed release cAMP into the extracellularmedium, but only when stimulated by cAMP concentrations between 10~8 Mand 5 x 10"6 M (Robertson, Grutsch & Gingle, 1978).

All of these results implied the need for further quantitative study of the effectsof cAMP on early embryonic development. In particular, the cAMP con-centration dependencies of cell attraction and diversion of the embryonic axisneeded to be determined for comparison with the results of experiments ondissociated cell populations. The dynamics of these phenomena, uncovered bytime-lapse filming, are also crucial for determining the qualitative effects ofdifferent cAMP concentrations on development of the intact embryo.

MATERIALS AND METHODSEmbryos

The technique of explanting, culturing and filming is derived from that ofWaddington (1932) with modifications due to De Haan(1967), as published byNew (1966), and has been described earlier (Robertson & Gingle, 1977). Exceptfor the youngest embryos (up to 10 h incubation) it was possible to explant andculture 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 laterexamination, or was placed within a cylindrical copper water jacket, main-tained at 38 °C and mounted directly on the microscope stage. The age of eachembryo is expressed as its stage according to Hamburger & Hamilton (1951).

Culture media

Chick Ringer: one liter of chick Ringer, made up in deionized water andautoclaved at 115 °C for 30 min, contained 9-0 g NaCl, 0-42 g KC1 and 0-25 gCaCl2 (New, 1966). The CaCl2 was added after autoclaving.

Responses of chick embryo to external cAMP sources 355Agar 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 thebottom of the sterile 5 cm plastic petri dish, to one pipetteful of a 2 % solution ofDIFCO 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 Mcamera coupled to a Nikon CFMA camera drive using a Nikon SKE or Apop-hot microscope body or a Zeiss model GF body. Long working distance plan-achromat objectives (x 1-2, x 2, x 5, x 10 or x 20) were used, with a x 5 eye-piece 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 aheat-filter to prevent overheating of the embryo by the light source. Frame rateswere 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 CFMwith a x % relay lens on either Ilford FP3 or Kodak Plus-X film rated at 125ASA.

For time-lapse films the exposure was set either manually, or automatically bythe Nikon CFMA camera drive; for 35 mm photographs a coupled, through-the-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 micro-electrode with an open internal tip diameter between 10 and 20 jtim was used. Thetip 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 electro-phoretically by currents between a chlorided silver wire in the electrolyte and asimilar wire grounding the agar substrate. Full details of technique and electrodecalibration have been published (Cohen, Drage & Robertson, 1975). The micro-electrode was held in a micromanipulator mounted on the microscope stage; itstip 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 wascemented at both ends into L-shaped pieces of stainless steel tubing from syringeneedles. The overall diameter of the tubing was 300 jam, with a wall thickness of20 /an and transverse pore diameter of approximately 20 nm, allowing the pass-age of cAMP. cAMP in Ringer was perfused through the tubing by a HarvardApparatus Co. Model 975 infusion pump at nominal flow rates between 0-02and 0-08 ml/min. At these flow rates the concentration of cAMP attained justoutside the tubing is approximately 4 % of that in the perfusing fluid and is


independent of the flow rates used, as we showed by measurements with tritiatedcAMP. The tubing was supported by a micromanipulator mounted on themicroscope and advanced, held parallel to the embryo surface, until it wasapproximately 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 and10~2 M. Perfusion was for 1 h; the source was immersed in 6 ml of Ringer con-tained in a scintillation vial. The vials containing Ringer and an unknownamount of cAMP were placed at 60 °C for evaporation, leaving only the cAMPand Ringer salts. Scintillation fluid, a mixture of toluene, Triton x-100, andPPO-POPOP, was added and the vials were 'counted'.

The measured count rates were compared with those for vials containingknown amounts of [3H]cAMP, and the efflux of cAMP molecules from thedialysis 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 overthe range of C1X. From the flux the cAMP concentration at the tube's outersurface can be calculated and is (4-4 ± 0-3) % of ClN.

Measurement of angles

The frame showing maximal bending on each time-lapse film was back-projected on to a translucent screen. Straight-line segments were fitted by eye tothe anterior and posterior parts of each embryonic axis. The external angle attheir intersection was measured. The measurements were made 'blind'. Theseangles were called 'the angles of maximum bend'.

RESULTS1. Concentration dependence

In these experiments we scored all the embryos treated with a microelectrodesource for three effects: diversion of the embryonic axis, local attraction of cellsto the electrode tip, and detachment of the blastodisc from the vitelline mem-brane. 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 theseexperiments the electrode was placed lateral to Hensen's node of embryos be-tween H-H stages 3-5, and close to the boundary between the area opaca andareapellucida. 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 357

2 0-4

0

1

A

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1

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i

I

i

1 i

1 1

1

•

1

1 1

•

10 (, - 4

log,,, [cAMl'l

i i I I i i i

10 - • 8 - 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 ofthe blastodisc. All embryos were observed for at least 8 h beginning at Hamburgerand Hamilton stages 3-4. At least 10 embryos were used for each concentrationpoint. The total sample was 137 embryos.


contact was eventually made, particularly at concentrations of cAMP in theelectrode between 10~6 and 10~3 M. This always took several hours to developand did not affect scoring of the results, except that, at these concentrations,detachment of the blastodisc often followed the accumulation of a cellularmass 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 10~3M-

cAMP were scored as having bent axes, and 43 % as showing cellular attractiontowards the electrode. In those experiments the embryos were prepared inbatches often and observed only before and after the experiment; they were notfilmed. In the experiments reported here all the embryos were filmed throughoutthe experiment and were treated individually and more carefully. The resultsshow distinct, and different, thresholds for each phenomenon scored. In additionthe films can still be used to analyze the dynamics of the responses, some aspectsof which will be mentioned below. The threshold concentrations for axis bend-ing, 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, whenthe threshold is defined as that concentration of cAMP, in the electrode, whichevoked a half-maximal response.

2. Angles of maximum bend

Angles of maximum bend were measured for 82 embryos, at least six for eachstimulus concentration. The mean for each concentration is shown in Fig. 2.When bending is measured as a continuous variable it shows a roughly linear de-pendence on the logarithm of the stimulus concentration; the effect becomessignificant for cAMP stimuli between 10~9 and 10~8 M. It should be noted thatthe 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 intohigh, medium and low ranges. One concentration range (Fig. 3C) is below thethreshold 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 middlerange there is no significant difference between the distribution of the totalsample and that from only the 10~9 M electrodes. In the high range seven out ofnine 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 toillustrate the dynamics of the responses observed. The embryo shown in Fig. 4


24

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Bl

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11

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1 1 1 1

-

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- 4

Fig. 2. Average maximum angle of bend plotted as a function of cAMP concentra-tion in the electrode. Bars represent standard errors about the mean.

(stage 3) was stimulated with a continuous signal of 10~3M-CAMP. The sketchescover a period of about 20 h, during which development was greatly influencedby the electrode. The stimulus was switched off between frames 1 and 2 and onagain between frames 4 and 5 (see figure caption for details).

The main responses observed are accumulation of cells towards the electrodeand bending of the primitive streak. Both show considerable inertia in that theycontinue for some time after cessation of stimulation and take time to developonce the electrode is switched on again.

In Fig. 5 we show a similar series of an embryo, beginning at stage 6+ andending, 11 h later, with seven pairs of somites (stage 9). In this case stimulationwas with a dialysis tubing source through which 10~3 M-cAMP was flowing. Theexposed portion of the tubing is shown in black. As node regression proceededthe posterior end of the embryo bent towards the source until, as shown inframes 5 and 6, the embryo was distinctly curved, with its head region also dis-placed towards the tube. After removal of the tube the embryo continued todevelop, forming five more somite pairs and a beating heart, after which develop-ment ceased. The photograph, taken just after removal of the tube, shows adense accumulation of cells between the head region and the tube's originalposition, as well as the beginning of premature vascularization, which we oftenobserved under these conditions of stimulation. In this embryo, as in all whichhave been filmed during the appropriate stages, cells which took part in formationof the first pair of somites dispersed and were incorporated in the spreading mar-gins of the omphalo-mesenteric veins, suggesting that both somitic and vascular


8 -

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1 1

1 1

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16

2

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C

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0 20 40 60Angle of .maximum bend (deg.)

Fig. 3. Histograms derived from the data plotted in Fig. 2. The data were groupedin 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 chemotacticattraction.

The last figure (Fig. 6) in this series illustrates this effect well. Here the stimuluswas a microelectrode releasing 10~7 M-cAMP continuously, leading both to adramatic bending of the axis during node regression and a large accumulationof cells in an area beneath and around the electrode tip. While these cells did notmake contact with the electrode the cellular mass detached during fixation, as isshown by the photograph of the embryo which was fixed immediately after theembryo had developed to the stage shown in frame 5. Note that, while threepairs of somites were visible at this stage, those on the electrode side were poorlydefined and hard to distinguish from the surrounding mesoderm.


Frame 1

Frame 10

Fig. 4. Sketches from the projected film image of a stage-3 embryo stimulatedwith 10~3 M-cAMP. Frame 1 is 0-6 h after the beginning of the experiment; frames2-10 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 hfrom the beginning. Frame width is 2-4 mm.

DISCUSSION

1. General. Our results show the importance of filming in order to obtain acontinuous record of the experiments. Many of the phenomena reported involvemovement, which can only be inferred and not accurately described whenperiodic observation or histological techniques are used alone. Some are tran-sient and would be missed altogether, or misinterpreted if only seen at one pointin their evolution.

2. Controls. Developing chick embryos are sensitive to a variety of distur-bances. Those germane to this study are: the direct action of cAMP, mechanicaltensions introduced during explanting and culturing, mechanical tensions andstresses introduced by the microsource's presence, and the effects of the smallelectrical current necessary for the microelectrode's operation. The mechanicaltensions arising during explanting and culturing have been controlled for(Robertson & Gingle, 1977); however, it should be noted that the lack of blasto-disc detachment and embryo damage below cAMP concentrations of 10~6 M(see Fig. 1C) is very good evidence for the gentleness of the explanting andculturing procedures.

The mechanical influence of the microsource was controlled for by usingdifferent source geometries. Both microelectrodes, having a circular disc-like

A. R. GINGLE AND A. ROBERTSON2

Frame

Fig. 5. Sketches from the projected film image (1-5) and (6), photograph of theembryo after filming, of a stage-6 embryo stimulated with a tube source containing10~3 M cAMP. 1-6 are 0-2, 2-3, 405, 5-7, 10-9 and 10-9 h after the beginning of theexperiment 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 orderto prevent direct contact. Here the limiting factor is determined by chemicaldiffusion and source geometry, since for large distances the cAMP concentrationat the embryo's surface will fall far below its source value. Of course the micro-electrode source requires a small electrical current for its operation, whichneeded to be controlled for. This was done by varying the current and also byemploying the dialysis tube source which does not require an electrical current.


Frame

A

Frame

Fig. 6. Sketches from the projected film image (1-5) and (6) photograph of theembryo after fixation, of a stage-6 embryo stimulated with an electrode containing10-8 M-CAMP. 1-6 are 0 1 , 4-7, 8-6, 13-2, 17-2 and 17-2 h from the beginning of theexperiment. Frame widths are 3-4 mm.

Both sources effectively attracted cells and bent the embryonic axis when chargedwith the appropriate cAMP concentrations. These experiments, employing awide range of cAMP concentration, constitute a further control for influencesother than those of cAMP. The geometry, placement, and operating parametersfor each source type were held constant over the range of cAMP concentrationsstudied. The results of these control experiments completely rule out the possi-bility of biases due to the microsource design or placement.

3. Concentration dependence. The phenomena of cell attraction and axial bend-ing are characterized by low cAMP concentration thresholds in the 10~10 M-10~7 M range. These are consistent with normal development as long as thecAMP electrode concentration is below about 10~6

M-10~5 M, the threshold forblastodisc detachment. By this, we mean that normal development occurs afterthe electrode is removed and, though cell attraction and axial bending occur,the embryo retains its integrity. cAMP electrode concentrations between 10~10 Mand 10~8 M only induce cell attraction, a short-range phenomenon, leavingmost of the embryo relatively undisturbed. For cAMP concentrations between10~8 M and 10~6 M the effects are more global extending from the cAMP micro-source to the embryo's axis. Above 10~6 M detachment of the entire blastodiscoften occurs. This effect, which is certainly pathological, causes long-range


damage to the embryo, destroys its integrity, and prevents further develop-ment.

Data from dissociated cells, however, suggest that such high ambient cAMPconcentrations will not occur normally since cAMP-induced cAMP release isreduced by stimuli greater than 10~6 M, and fully suppressed by a stimulus of6 x 10-6 M (Robertson et al., 1978).

Finally, the very low thresholds for both cell attraction and axial bendingsuggest both that the responses are specific to cAMP and that they are mediatedby extracellular cAMP receptors. A similar situation holds for D. discoideum,where evidence for two classes of extracellular cAMP receptor (or for a singlereceptor with negative cooperativity) has been found (Newell, 1977). Even thequantities, especially the numerical values of threshold and signal size, areremarkably similar. Further, under the same stimulating conditions both ceJltypes show a cAMP concentration above which cAMP relaying, or inducedsynthesis and release, is sharply repressed (3 x IO~7 M for D. discoideum) (Grutsch& Robertson, 1978). Similar quantities can be inferred from recent work onother 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 Bio-logical Research Corporation.

REFERENCES

COHEN, M. H., DRAGE, D. J. & ROBERTSON, A. (1975). Iontophoresis of cAMP. Biophys. J.15, 753-764.

GALLERA, J. (1971). Primary induction in birds. Advances in Morphogenesis 9, 148-180.GINGLE, A. R. (1976). Critical density for relaying in Dictyostelium discoideum and its relation

to phosphodiesterase secretion into the extracellular medium. / . Cell Sci. 20, 1-20.GINGLE, A. R. (1977). cAMP enhanced aggregation of cells from early chick embryos. Devi

Biol. 58, 394-401.GRUTSCH, J. F. & ROBERTSON, A. (1978). The cAMP signal from D. discoideum amoebae.

Devi Biol. 66, 285-293.DE HAAN, R. L. (1967). Avian embryo culture. In Methods in Developmental Biology (ed.

F. H. Wilt & N. K. Wessels), pp. 401-412. New York: Crowell.HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of

the chick embryo. / . Morph. 88, 49-92.HUXLEY, J. S. &DEBEER, G. R. (1934). The Elements of Experimental Embryology. (Cambridge

University Press, London; reprinted in 1963 by Hafner, New York.)MANGOLD, O. & SPEMANN, H. (1927). Uber Induktion von Medullar durch Medullarplatte an

jiingeren Keim, ein Beispiel homoeogenetischer oder assimilatorischer Induktion, WilhelmRoux Arch. EntwMech. Org. Il l , 341-422.

NAKAHARA, Y., TESADA, S., PINCUS, J., FLOURET, G. & HECTOR, O. (1978). Neurohypophysealhormone-responsive adenylate cyclase. J. biol. Chem. 253, 3211-3218.

NEW, D. A. T. (1966). The Culture of Vertebrate Embryos, pp. 245. London: Logos.NEWELL, P. C. (1977). Aggregation and cell surface receptors in cellular slime molds. Micro-

biological Interactions, Receptors and Recognition, Series B, 3 (ed. B. Reissio), pp. 1-57.Chapman & Hall, London.

ROBERTSON, A. & GINGLE, A. R. (1977). Axial bending in the early chick embryo by a cyclicadenosine monophosphate source. Science 197, 1078-1079.

Responses of chick embryo to external cAMP sources 365ROBERTSON, A., GRUTSCH, J. F. & GINGLE, A. R. (1978). cAMP production by embryonic

chick cells. Science 199, 990-991.SONNE, O., BERG, T. & CHRISTOFFERSEN, Y. (1978). Binding of 125I-labeled glucagon and

glucagon stimulated accumulation of adenosine 3':5'-monophosphate in intact rat hepa-tocytes. J. biol. Chem. 253, 3203-3210.

SPEMANN, H. (1938). Embryonic Development and Induction, pp. 410. (Yale University Press,reprinted in 1967 by Hafner, New York.)

WADDINGTON, C. H. (1932). Experiments on the development of chick and duck embryoscultivated in vitro. Phil. Trans. R. Soc. B 221, 179-230.

WADDINGTON, C. H. (1933). Induction by the primitive streak and its derivatives in the chick./ . exp. Biol. 10, 38-46.

WADDINGTON, C. H. (1937). Experiments on determination in the rabbit embryo. Archs Biol.Paris 48, 273-290.

WADDINGTON, C. H. (1952). The Epigenetics of Birds, pp. 272. (London: Cambridge UniversityPress.)

{Received 19 July 1978, revised 28 April 1979)

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