EXPERIMENTS ON THE EFFECT OF DYES ONINDUCTION AND RESPIRATION IN THE

AMPHIBIAN GASTRULA

BY R. A. BEATTY, S. DE JONG AND M. A. ZIELINSKIFrom the Zoological and Biochemical Laboratories, Cambridge

(Received 12 October 1938)

(With One Plate)

INTRODUCTION

FROM the work of experimental embryologists and biochemists on the gastrulationperiod in amphibian development, it appears that the cells of the blastula all containthe morphogenetic stimulating substance or substances which are later responsiblefor the neural induction. But they contain it in some inactive or masked form, fromwhich it is liberated only at the beginning of imagination at the dorsal lip of theblastopore. In other regions of the gastrula, it may indeed be liberated, but only byartificial treatment; such as the denaturation of the proteins of the ventral ectodermor yolk endoderm by heat or by treatment with organic solvents (Holtfreter, 1933).But it is not absolutely necessary to kill the inactive tissues in order to cause theappearance of the inductive power in them, for Waddington et al. (1936) showedthat after pieces of ventral ectoderm had remained isolated for some time in dilutesolutions of methylene blue, they would, when implanted into the blastocoelecavity of another embryo, perform neural inductions. These might be only on thepart of the host, but the graft also in many cases showed considerable neuralization.

In later experiments, which are reported in the present paper, it was found thatneuralization of the isolated explant would occur without any implantation intoanother embryo.

It was at first thought probable, on the basis of the classical experiments ofBarron and his collaborators (Barron, 1929, 1930; Barron & Harrop, 1928; Barren& Hoffman, 1930), that the effect of methylene blue was primarily on the meta-bolism, either respiration or glycolysis, leading to the liberation of the active sub-stance from its masked or inactive form. If this were so, it would be expected thatdyes such as Janus green or neutral red, of rather negative oxidation-reductionpotential, which were shown by Barron & Hoffman to have no effect at all on theoxygen consumption of echinoderm eggs, would also have no effect on the ventralectoderm of the gastrula. It will however be shown in the present paper that thesedyes seem to have a similar effect to that of methylene blue on the inductive proper-

Effect of Dyes on Induction and Respiration in Amphibian Gastrula 151

ties of the isolated ventral ectoderm.1 On the other hand, methylene blue in diluteconcentrations appears to have an acceleratory effect on the oxygen consumption ofgastrula tissue, although it is not so marked as in other cases such as sea-urchineggs.

Since the original discovery of the methylene blue effect by Waddington et al. [193 6]it has been generally assumed that the mechanism must be indirect, i.e. a liberationof the natural organizer substance contained in the ventral ectoderm; rather thandirect, i.e. the action of the dye itself as an organizer substance. The latter possi-bility cannot yet, however, be excluded, and it would be interesting to implant intothe blastocoele cavity of gastrulae graded doses of dyes, running down to verysmall concentrations, in order to determine the optimum concentration. In thisconnexion, it is interesting that Finkelstein & Schapiro (1937) implanted dinitro-phenol in agar into the blastocoele cavity, but obtained no neural differentiations.At the same time, there is evidence (Needham & Boell, 19396) that the anaerobicglycolysis of the ventral ectoderm is increased considerably by dinitro-o-cresol. Itis clear that a great deal more work is required on these and similar phases of theproblem.

METHODS

Isolation of the ventral ectoderm from over the blastocoele cavity was carriedout by the usual Spemann technique of watchmaker's forceps and glass needlesunder strictly sterile conditions.2 Early gastrulae of Triton alpestris were used. Thedye solutions were made up in sterile distilled water, but not themselves sterilizedafterwards. After a variable period (from 1 to 3 days) in the dye solutions, theexplants were removed, fixed in Michaelis solution, serially sectioned and stained.

Measurement of the respiratory rate of the isolated pieces of ventral ectoderm,etc., was carried out by the Cartesian Diver technique of Linderstrem-Lang (1937)as applied to this purpose by Boell & Needham (1939). Full descriptions of thismicro-manometric technique, which is about fifteen hundred times more sensitivethan the standard Warburg manometers, will be found in the latter paper. The onlymodification used by us was the employment of Holtfreter solution containingmethylene blue at the desired concentration, instead of ordinary Holtfreter solution.For obtaining the amounts of tissue used, the micro-Kjeldahl technique describedby Needham & Boell (1939a) was used. Gastrulae of Rana esculenta and Ambly-stoma mexicanum were used as material. The former were obtained by implantationof pituitary glands of the same species (July) as described by Rugh (1937), andartificially fertilized; the latter were obtained by injection of Antuitrin S, and natur-ally fertilized. The main batch of eggs was kept in the ice chest and samples used asrequired. We always allowed some eggs from these samples to develop in order tomake sure that their development was normal.

1 The experiments of Marston (1923) and Commoner (1938) on the formation of dye-proteincomplexes render the expectation that dyes could liberate the organizer substance from its inactiveprecursor by competitive complex-formation not without plausibility.

* In order to make sure that no piece of tissue approaching the limits of the organizer region hadbeen taken, each gastrula was carefully examined after the explant had been removed from it.

R. A. BEATTY, S. DE JONG and M. A. ZIELINSKI

With a batch of Discoglossus pinctus embryos we had the curious experience thatalthough their development seemed to be normal, their respiratory rate was extremelylow. Dissection of the gastrulae was accomplished with Spemann glass needles inthe ordinary way, but the stiff envelopes of Rana esculenta eggs made it necessary toopen them with iridectomy scissors as described by Needham et al. (1939). It isimportant that in every experiment the pieces should come from the same gastrulaand that they should be bilaterally symmetrical, i.e. left and right pieces, lookingat the yolk-plug with the dorsal blastopore lip upwards.

RESULTS

The results of the explantations into dye solutions, from the laying seasons of1937 and 1938, are given in Table I. Janus green and neutral red are the dyes whichare not known to have any effect on the respiratory rate of cells, yet, as can be seenfrom the table and the illustrations (PI. I), two possibly neural tubes were obtainedwith the former, and a number of lesser neuralizations with both. Pyocyanin gaveone tube, but phenazine methochloride, which Dickens (1936) found to increase therespiratory rate of tumour tissue 250%, gave no positive results.

Table

uyc

Methylene blue

Pyocyanin

Dinitro-o-cresol

Janus green

Neutral red

Phenazinemethochloride

Control

I. Explantattons of ventral ectoderm into

Molarconcentration

1-25 x io-»1-25 x io-*1-25 xio-"1-25 x io~*1-25 x io~*1-25 x io~°

I X IO~*I X IO~*

1-25 x io~*1-25 x io~5

1-25 x 10-*1-25 x io~*1-25 x io-1

0

No. ex-planted

1

343

19374

22

9255

13

3

No.cut

1

341

18334

179

15S6

3

dye solutions

Reaction

1t

np

—11

——————————

—

t

I

I

I

I?

All B

—1

—

3—1

—1

42

——

—

D

1211

1522

4154

1356

3

Note. In the above table, the classification is made according to the plan of Waddington et al.(I93S)> save that all types of B+ are included under B, and B— is included under D. np = neuralplate; t = neural tube. A, neural plate or tube inductions; B, all grades of palisade inductions;D, ectodermal proliferation.

The results of the manometric experiments are shown in Table II. It will benoted that at the lowest concentration of the dye there is an increase of about 45 %,while at the higher concentrations there is an inhibition in nearly all the cases. Thisagrees with many observations in the literature, e.g. those of Elliott & Baker (1935)who worked with mammalian tissue slices. The increase of 45 % is nothing like so

Effect of Dyes on Induction and Respiration in Amphibian Gastrula 153

great as that recorded for echinoderm eggs by Barron & Hoffman (1930), but morenearly resembles the increases found by Bodine & Boell (1936) on the grasshopperembryo. Here the increases were 2% on the pre-diapause embryos, 100% on thediapause embryos, and about 25 % on the post-diapause embryos. Whether there isany parallelism between the effects of methylene blue on respiratory rate hererecorded, and the effect on induction, must be left for further work to determine.

The only error which might be introduced into the experiments required for theresults of Table II would be that the nitrogen content of the dye in the diver isincluded in the Kjeldahl nitrogen, thus giving too low values for the experimentalvalues as opposed to the controls. However, it may be calculated that this error,even in the strongest concentrations of dye, cannot exceed 5 %.

Table II. Effect of methylene blue on oxygen uptake of gastrula tissues

Material

Rana esculenta

Amblystoma mexicattum

Molarconcentration

1-25 x io~*

1-25 x io~~*

1-25 x io"J

1-25 x io~*

Region

vevevedltie

dldldlventnt

Q'o,

Control

6-34-23-83-26-8

n o6-73 36-8

3'43-3

Dye

8 45 74'26 44 18 74 24 24'53O2'3

Ratio d y C

control

i '331 3 6i n2-OO0 6 00 7 90 6 31 2 70660 8 80 7 0

Note. J2o, = c.mm. oxygen uptake by tissue corresponding to i mg. nitrogen of Kjeldahl perhour. dl=donal lip region; u« = ventral ectoderm; nt=just closed neural tube.

SUMMARY1. It is shown that pieces of presumptive epidermis (ventral ectoderm of the

gastrula), when isolated into weak solutions of several dyes, will undergo neuraldifferentiation. Dyes such as Janus green and neutral red, which are not known toaccelerate cell respiration, appear to have this effect, as well as methylene blue, theaccelerating action of which on cell respiration is well known.

2. Measurements of the oxygen consumption of isolated pieces of the gastrulaby the Cartesian Diver method show that methylene blue, if in weak concentration,has an accelerating action of about 45 %. In stronger concentrations it is inhibitory.

ACKNOWLEDGEMENTS

One of us (S. de J.) held a grant from the Stokvis Reisfonds of AmsterdamUniversity during the course of the work, and one of us (M. A. Z.) was on leavefrom the University of Warsaw. The work forms part of a plan of investigationsproposed by Dr C. H. Waddington and Dr J. Needham, to both of whom we are

154 R- A. BEATTY, S. DE JONG arid M. A. ZIELINSKI

indebted for help and advice. The statistics of explantation include a few isolationsmade by the former in the summer of 1937. We also wish to thank Dr F. Dickensfor the gift of a specimen of phenazine methochloride, and the Government GrantCommittee of the Royal Society for a grant to Dr Needham which partially defrayedthe cost of the work.

REFERENCES

BARRON, E. S. G. (1929). J. biol. Chem. 81, 445.(1930). J. exp. Med. 52, 447.

BARRON, E. S. G. & HARROP, G. A. (1928). J. biol. Chem. 79, 65.(1928). J. exp. Med. 48, 207.

BARRON, E. S. G. & HOFFMAN, L. A. (1930). J. gen. Physiol. 13, 483.BODINE, J. H. & BOBLL, E. J. (1936). PTOC. SOC. Exp. Biol., N.Y., 34, 629.BOKLL, E. J. & NEEDHAM, J. (1939). PTOC. roy. Soc. B (in the Press).COMMONER, B. (1938). J. cell. comp. Physiol. 12, 171.DICKENS, F. (1936). Biochem.J. 30, 1064.ELLIOTT, K. A. C. & BAKER, Z. (1935). Biochem. J. 29, 2396.FINKELSTEIN, E. M. & SCHAPIRO, E. M. (1937). Exp. Med. {Ukraine), 3, 5.HOLTFRETER, J. (1933). Arch. EntwMech. Org. 128, 584.LINDERSTR0M-LANO, K. (1937). Nature, Lond., 140, 108.MARSTON, H. R. (1923). Biochem. J. 17, 851.NEEDHAM, J. & BOELL, E. J. (1939a). Biochem. J. (in the Press).

(i939fr). PTOC. roy. Soc. B (in the Press).NEEDHAM, J., BOELL, E. J. & ROGERS, V. (1939). Proc. roy. Soc. B (in the Press).RUGH, R. (1937). Science, 85, 588.WADDINGTON, C. H., NEEDHAM, J. & BRACHET, J. (1936). Proc. roy. Soc. B, 120, 173.WADDINGTON, C. H., NEEDHAM, J., NOWINSKI, W. W. & LEMBERG, R. (1935). Proc. roy. Soc. B, 117,

289.

EXPLANATION OF PLATE I

Fig. 1. RA3—e. Methylene blue, M 1-25 x io~5. A well-formed neural tube (A), x 235.Fig. 2. RA 3—e (another piece). Methylene blue, same concentration. A palisade of neural cells (B).

x 140.Fig. 3. RA5— d. Janus green, M 1-25 x io~*. A large cartwheel-shaped neural (?) induction (A).

x 100.Fig. 4. RA5— q. Janus green, M 1-25 x io~*. A neural plate of unusual form (B). x 100.Fig. 5. JG5 — 2. Janus green, M 1-25 x 1 o~6. Chaotic neuralization (B). xioo.Fig. 6. RA2 —h. Pyocyanin, M 1-25 x io~*. A palisade (B). x 100.

JOURNAL OF EXPERIMENTAL BIOLOGY, XVI, 2. PLATE I

Fig. 1. Fig. 2.

Fig. 5- Fig. 6.

BEATTY, DE JONG AND ZIELINSKI.—EXPERIMENTS ON THE EFFECT OF DYES ONINDUCTION AND RESPIRATION IN THE AMPHIBIAN GASTRULA (pp. 150-154).