susceptibility in amphibian development

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Susceptibility in Amphibian Development Author(s): A. W. Bellamy and C. M. Child Source: Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character, Vol. 96, No. 673 (Mar. 1, 1924), pp. 132-145 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/81064 . Accessed: 07/05/2014 11:28 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character. http://www.jstor.org This content downloaded from 169.229.32.136 on Wed, 7 May 2014 11:28:53 AM All use subject to JSTOR Terms and Conditions

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Page 1: Susceptibility in Amphibian Development

Susceptibility in Amphibian DevelopmentAuthor(s): A. W. Bellamy and C. M. ChildSource: Proceedings of the Royal Society of London. Series B, Containing Papers of aBiological Character, Vol. 96, No. 673 (Mar. 1, 1924), pp. 132-145Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/81064 .

Accessed: 07/05/2014 11:28

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of theRoyal Society of London. Series B, Containing Papers of a Biological Character.

http://www.jstor.org

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 11:28:53 AMAll use subject to JSTOR Terms and Conditions

Page 2: Susceptibility in Amphibian Development

132

Susceptibility in Amphibian Development.

By A. W. BELLAMY, Asst. Prof. of Zoology, and C. M. CHILD, Prof. of

Zoology, University of Chicago.

(Communicated by Prof. E. W. MacBride, F.R.S. Received November 7, 1923.)

In a recent paper Mr. H. Graham Cannon (1) has presented data on sus-

ceptibility in amphibian development, some of which are in disagreement with Bellamy's results (2, 3), and has also criticised the application of the

physiological gradient conception to the higher animals. In response to

inquiry whether the 'Proceedings of the Royal Society' were open to us if re-examination of contested points should make further discussion desirable, Dr. E. W. MacBride has very kindly consented to communicate to the Society a statement of our results and our position. We wish to express our appre- ciation of this courtesy and of the courtesy of the Royal Society in admitting this communication.

During the amphibian breeding season of the present year (1923) we have worked with four genera, Amblystoma tigrinum, Bufo americanus, Corophilus nigritus, and Rana pipiens, with reference to the points disputed by Mr. Cannon. The work was in part repetition of earlier work and in part new. The present paper includes results of this work so far as they concern points at issue: we have also taken the opportunity to clear up certain obscurities and to

supply certain omissions to which Mr. Cannon has kindly called attention. And finally, in the hope of avoiding to some extent future misunderstanding of our views we have attempted a brief statement of the chief results of the

susceptibility method and of the conclusions to which it has led.

Concerning Experimental Methods.

In the original report (2) it was not stated specifically that all solutions were made up in well water* although it was implied in a footnote, page 344. The experiments reported here were carried out in parallel series involving solutions made up in well water and once distilled water (from a tinned copper still with block tin condenser). In two series twice distilled (conductivity) water from a quartz still was used in addition. This was done to determine

* The composition of the well water used in this laboratory is indicated in the following analysis made in 1921. The amounts given are in parts per million :-Fe, 0 2 ; Mn, 0 1; SiO2, 7-8; Ca, 49-2; Mg, 16 7; NHI4, 0-03; Na, 3-0; K, 1-1; SO4 17.7; Cl, 6-0; Al103, 1-6; Non-volatile, 0-7.

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whether the disagreement between certain of Cannon's results and ours might be due, in part, to the fact that he used distilled water exclusively in making up solutions.

Having in mind the toxic effect of distilled water on many organisms, and

wishing to run controls in water similar to that in which development normally takes place, we naturally used well water both for controls and for making up solutions. Certainly in working with marine organisms one would not, in attempting the modification of development, make up solutions in anything but sea water. The same principle we believe applies here, and hence we have rather taken it for granted that the method of making up solutions would be clear. As a matter of fact one of us in earlier publications has stated

specifically that in studying the effects on development of external agents well water solutions were always used for fresh-water forms and were, at best,

approximations. Regardless of what happens to potassium cyanide, or any other chemical, when dissolved in well water or in sea water, we believe, for

comparative results, the method is essentially sound, whatever violence it

may do to the instincts of the biochemist.

Discussion of New Data.

Our experiments this spring on eggs of the four species mentioned above not only repeat some of the observations recorded in the original report (2) and certain'of Cannon's experiments, but also include some additional data on the susceptibility of these eggs to NH40OH. However, since the results as originally published are confirmed in all essential respects and since we

expect later to publish the results of further work, we do not feel justified in

taking space for detailed descriptions of experiments here.

Modification of Development in KCN.

New experiments with this agent confirm previous findings in all particulars. Experiments were run parallel in well, once distilled, and twice distilled water solutions. The solutions were always freshly made up and placed with the

eggs in Erlenmeyer flasks filled full and stoppered Since our previous findings were confirmed not only for R. pipiens, but similar results also were obtained with eggs of the other three species mentioned, the only experiment that

requires description is one repeating an experiment of Cannon's (p. 237 et seq., and his fig. 2).* The concentrations used were (distilled water solutions)

* According to the text the two eggs shown in Cannon's fig. 1 are upside down and the designations A and B reversed. In the course of personal correspondence, also, Mr. Cannon has admitted that this is the case.

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m/100, m/300, m/600, m/1000, m/1500, m/2000, m/5000, n/10,000, m/20,000 and m/50,000. Fig. 1 indicates our findings, viz., a marked differential

FIG. 1.-Inhibition in KCN. The eggs were introduced into the solution in 2- to 4-cell stage and exposed 41 hours. A, m/100; B, m/300; C, m/600; D, m/1000; E, m/1500; F, m/5000. Solutions made up in distilled water. Temperature 15? to 18? C. Decreasing inhibition, which is differential, with increasing dilution is clearly shown.

inhibition that decreases with decreasing concentration. We found not the

slightest evidence of inhibition increasing with increasing dilution, such as Cannon describes. Since, in all of the extensive literature on the action of

cyanides on living matter, we have found no other case of increasing toxicity with increasing dilution such as Cannon describes, we are at a loss for any explanation of his results, unless there is some most extraordinary difference between the eggs of R. pipiens and R. temporaria or some error in presentation. Under the circumstances we feel that his results require confirmation before further comment is justified.

Attention is called particularly to the fact, illustrated in fig. 1, that the inhibition just mentioned is markedly differential, i.e., is greatest in apical regions and diminishes basipetally. Work with Amblystoma eggs gave results similar to those obtained with eggs of R. pipiens.

Susceptibility to Lethal Concentrations of External Agents.

Eggs in cleavage stages were studied mainly in mercuric chloride in indicated concentrations ranging from mn/10,000 to m/30,000,000 made up in well and

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once distilled water, and ammonium hydroxide, 1 drop (specific gravity 0.90) to 100 cc. distilled water.

Parallel experiments in well and distilled water (HgC12 experiments) showed no significant difference, except that the eggs or embryos were affected a little

earlier, i.e., were slightly more susceptible in the distilled water solutions. This slight difference may be due to the loss of some mercuric chloride by combination with organic or other matter in the well water and egg membranes or to the action of distilled water, or both. Distilled water alone, while it accelerates slightly the early developmental stages, proves to be somewhat toxic to later stages, as indicated by the fact that embryos showed a slight differential inhibition and, in our experiments, hatched from four to six hours later than embryos from the same female, developing in well water. The gill plates and eyes were less conspicuous, the tails dorsoventrally narrowed

(unexpanded), and after several days the embryos showed considerable oedema.

In cleavage stages of about 64 cells, disintegration n NH40H begins at the apical pole and spreads toward the equatorial region in all directions over the black hemisphere, but somewhat more rapidly along a broad meridian. This meridional path of disintegration usually bisects the gray crescent region. In the late cleavage stages that immediately precede the appearance of the

blastopore disintegration usually begins first in the dorsal lip region and spreads apically and equatorially. The change proceeds apically most rapidly to

join an area of disintegration that has just begun at the apical pole. Finally, the entire apical hemisphere is involved before the yolk cells show any indication of disintegration.

In late yolk plug stages the disintegration pattern is substantially like that just described, i.e., a double disintegration gradient is revealed. Dis- solution begins first in the dorsal lip region of the circular blastopore and has

extended, usually, entirely around the margins of the yolk plug (not involving the yolk, however) by the time disintegration has begun at the anterior end. These two areas of disintegration then approach each other along the dorsal side and meet usually about one-third the distance from the apical region to the small yolk plug.

This double disintegration gradient persists up to the latest embryonic stage we have examined-late neural fold. In this stage, however, disintegra- tion begins almost simultaneously at the posterior and anterior ends of the dorsal, surface. These two areas then extend rapidly along the medullary plate to meet in the region where the neural folds first meet to form the neural

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tube. In some cases the anterior end leads slightly; in others the posterior end. The entire nervous area is destroyed before the lateral and ventral surfaces are attacked. Following the complete destruction of the nervous

area, disintegration spreads toward the ventral side and the last bit of ectoderm to undergo dissolution is in the mid-ventral region.

As regards mercuric chloride the areas affected and the sequence of dis-

integration changes are much the same as those seen in NH4OH or any of the other agents used, except for certain slight differences to be mentioned

presently. However, the appearance of the disintegrated areas following the action of mercuric chloride is more or less distinctive. Following the action of NH40H, e.g., with the rupture of the cell membranes the protoplasm literally flows out into the water as a soft whitish mass and fluffs up over the disin-

tegrated areas as a more or less coherent cloud. In mercuric chloride, on the other hand, the first apparent change is a graying of the surface followed in

many cases by the retreat of pigment to the centre of the cells. But with the breakdown of the cell membranes there is very little actual flowing out of protoplasm and the disintegrated areas do not have the downy or fluffy aspect mentioned above. The affected areas have, rather, a more or less

mouldy, granular appearance, and in certain regions become deeply eroded

after continued exposure in suitable concentrations.

Our observations on susceptibility to HgC12 may be summed up as follows:- 1. When suitable concentrations are found and precautions taken to insure

equal exposure of the entire egg surface, the disintegration pattern is sub-

stantially like that seen in any of the other agents used. On the other hand,

unequal exposure resulting from partial removal or local tearing of the jelly or local contact with other eggs, et cetera, may be followed by a disintegration

pattern determined by the differential exposure rather than by axial sus-

ceptibility relations. 2. Indicated concentrations of m/10,000 to m/25,000, and perhaps even

lower concentrations seem to produce some fixation, as indicated by the fact

that a varying number of eggs neither continue development nor undergo

disintegration. Our observations on this point are incomplete but we suggest

provisionally that some of the irregularities in disintegration noted by Cannon

may be associated with partial fixation or differential exposure, or both.

3. In late cleavage stages the disintegration pattern in HgCl2 differs some-

what from that seen in a number of other agents in that the surface changes indicative of beginning disintegration usually appear earlier, relative to the

apical region, in the equatorial zone associated with the future dorsal lip

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region. The surface changes then sweep apicalward to involve the apical pole region and typically the entire black hemipshere is affected either before the yolk cells have shown any change, or after at most, very slight changes in the yolk cells. It is, of course, obvious that these surface changes, whether they result from the action of mercuric chloride or some other agent, are somewhat more conspicuous in cleavage stages on the animal pole than on the vegetative pole, where the cells are white. But with suitable illumination and magni- fication, errors of observation are not particularly difficult to avoid.

4. In neural fold and later embryonic stages, while the pattern of early surface changes seen following the action of mercuric chloride differs most from that seen in other agents, the deeper disintegration and more extensive corrosion or "excavation" seen after continued exposure in suitable con- ,centrations of this agent reveals a disintegration pattern substantially similar to that seen in any of the agents we have used.

In a mid-neurula stage, for example, the graying with, here and there, slight rupture of cells, usually begins at the posterior end and sweeps forward over the embryo. The entire surface is involved. In some embryos we saw this

change proceed with the ventral side a little in advance of the dorsal, in others with the dorsal region a little in advance; and in still others at the same rate on all sides. It is essentially a wave of superficial change moving forward over the embryo, with the front sometimes oblique, sometimes perpendicular to the sagittal plane. However, the later and deeper disintegration that follows is restricted to the neural plate area and is deepest and most apparent at the anterior end, and later also in the tail bud region. Even with the

complete destruction of the dorsal or nervous area the lateral and ventral surfaces have shown no further change beyond the original graying, and slight loosening of cells that here and there have ruptured.

5. When concentrations and length of exposure of the eggs are such that

development is inhibited to some extent, but with no disintegration, the inhibition is differential.

6. Finally, we have not, and do not now offer the results previously pub- lished on the susceptibility relations of mercuric chloride, or for that matter

any other agent, or the brief comments on recent experiments, as final, and we welcome competent criticism. Further work is of course necessary with respect to many aspects of susceptibility.

Beyond calling attention to the early origin of tissue specificity in the higher animals, as revealed, e.g., by the numerous transplantation experiments on amphibia, and in other ways, it does not seem worth while at this time to

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comment at length on the apparent dissimilarity in the susceptibility relations to HgCl2 and other agents.

It would be surprising indeed if a vertebrate embryo did not begin early in

development to react in a specific manner to chemical differences in its environment and we are inclined to suggest that specificity is at the basis of differences in the reaction of later stages of the frog embryo to different

agents. It is not known at just what point in differentiation specificity of a particular part or organ in reaction to external or internal agents or factors is reached, but the evidence available indicates its appearance for some parts as early as gastrulation stages. The variations and apparent anomalies in

susceptibility to HgCl2 in the more advanced stages, seem to be largely or

wholly limited to the superficial ectoderm. The possibility may be noted that individual and regional differences in ciliary activity may be concerned in these variations. At any rate it appears to be true that these variations are associated with progressive differentiation of the ectoderm. But as regards the time of appearance and localisation of specific or apparently specific reactions to particular agents or groups of agents and as regards degree of

specificity much further investigation is necessary. It is to be emphasised, however, that the appearance of specificity at a certain

stage in development in no way invalidates the conclusions already reached and abundantly supported by experimental evidence, that the early stages of

development of higher forms and all stages of the life-history of many lower forms show a complete lack of specificity in their developmental response to certain ranges of concentration of a great variety of chemical and physical agents.

Value and Limitations of the Susceptibility Method.

In the hope of clearing up past, and avoiding future, misconception an

attempt is made in the following paragraphs to state the most important points in our views concerning susceptibility, its significance and its relation to the gradient hypothesis.*

1. It has been found experimentally that differences in susceptibility to

many different external agents, both chemical and physical, exist at different levels of the physiological axes of individuals or parts and in different individuals

* In order to avoid loading this paper with a long bibliography, references for each statement made are omitted. For the evidence on which the statements in this paper are based, so far as published before 1921, see (4), (5), Chaps. II, III, V, and the references there given.

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of the same species. These differences in susceptibility are evidently cor- related with differences of some sort in physiological condition.

2. It has been shown further that in the earlier stages of development of

many organisms and in many of the simpler animals and plants throughout life, these differences in susceptibility are similar for certain experimentally determined ranges of concentration or degree of action of different chemical and physical agents, so far as present knowledge goes.

3. This similarity as regards the differences in susceptibility, i.e., the sus-

ceptibility relations, cannot possibly be interpreted as indicating that all toxic agents act alike. Cannon's assertion (1, p. 240) that Bellamy makes such interpretation, even as regards different chemical agents, is in error. It was stated specifically by Bellamy (2, p. 354) that different chemical sub- stances do not act on protoplasm in exactly the same way, and similar state- ments have been made repeatedly by Child. Certainly we have never believed nor stated, and it is difficult to see how anyone can believe, that, for example, KCN, ethyl alcohol, HC1, NaOH, LiCI, neutral red or other vital dyes, lack of oxygen, accumulation of CO2, low temperature and various other agents all act alike on any living protoplasm. Nevertheless, it has been shown

experimentally for many organisms that susceptibility varies along an axis of an individual, or in different individuals of a species in the same way, i.e., in the same direction, though not necessarily in the same degree, for many or all of these and for various other agents in certain ranges of concentration or

degree of action. In short, this work on susceptibility shows that within certain ranges of concentration or degree of action all agents used act as general protoplasmic poisons or interfere radically in some way with general protoplasmic activity, and that within the limits of such action a high degree of uniformity between susceptibility relations as determined by the different agents and certain factors in physiological condition exists, at least in the earlier stages of development and in many of the simpler organisms throughout life.

4. It is also found in many cases that the relative susceptibility of a particular region or part differs widely for different agents or groups of agents. For example, in certain eggs or embryos with a considerable amount of localised yolk, the yolk-bearing region is more susceptible than other parts to certain agents or groups of agents and less susceptible than other parts to certain other agents or groups. In such cases the yolk-bearing region shows a specific, or apparently specific, susceptibility to certain agents or groups. In the more advanced stages of development of the higher animals and man such apparently specific differences in the susceptibilities of particular organs or parts to

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particular agents or groups of agents are the rule, rather than the exception. In fact, the mammalian physiologist and the pharmacologist are accustomed to regard the actions of drugs and other external agents as more or less specific for particular organs or parts, in most or all cases.

5. In the light of all the experimental data, including those of mammalian

physiology and pharmacology, as well as our own, and inclu'ding also what we

know of the physico-chemical character of the action of different external

agents, it seems probable that, in so far as the differences in susceptibility at different levels of an axis, or in different individuals of a species are similar

for many different agents or groups of agents of different constitution and

method of action, the physiological differences which underlie these differences

in susceptibility are not specific in character. If they were specific we should

expect to find some evidence of such specificity in the differences in suscep-

tibility to different kinds of agents. 6. So far as such physiological differences show no indications of specificity

in their relative susceptibility to different agents, the only possible conclusion

from the evidence seems to be that so far as the susceptibility relations are

concerned, they are essentially quantitative. If specific regional or individual

differences exist in the cases under discussion they are not such as to affect

the susceptibility relations.

7. Various other lines of experimental evidence, differences in rate of cellular

respiration, differences in rate of penetration of certain agents, differences in

rate of certain oxidation-reduction reactions, as indicated by rate and amount

of reduction of KMnO4 and of methylene blue and by the indo-phenol reaction, and differences in electric potential, demonstrate the existence of graded

quantitative differences along physiological axes, and these differences run in

general parallel to the observed differences in susceptibility. Differences in

protoplasmic structure in many cases and in rate of cell division, differentiation

and other processes, add further evidence. In the case of the cyanides, which

inhibit most protoplasmic oxidations and act as powerful protoplasmic poisons, even in very low concentrations, the parallelism between susceptibility and

rate of respiration is in general so close as to justify the belief that, within

certain limits and with proper precautions to insure equality of exposure, etc.,

susceptibility to cyanide can be used widely as an indicator of relative rate

of oxidation. 8. It has been shown for many cases that conditions which bring about

what are believed to be primarily quantitative changes in physiological con-

dition alter susceptibility in the same direction. In certain other cases, in

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Susceptibility in Amphibian Development. 141

which the altering condition and the agent used to determine susceptibility are additive, wholly or in part, in their physiological action, susceptibility may be altered in the direction opposite to that of the physiological change. If, for example, respiration is decreased by lack of oxygen, susceptibility to cyanide is increased.

9. The only possible conclusion from the data available seems to be that the physiological differences indicated by the susceptibility method within the limits stated above are primarily quantitative in character, and that they are associated with fundamentally quantitative differences in the processes which constitute life. The axial gradients have often been called metabolic

gradients, because differences in rate of metabolism, or more specifically of oxidative metabolism, as indicated by various experimental methods,

appear to be characteristic and conspicuous features of them. It is believed, however, that all differences in protoplasm, whatever their nature, which are

physiologically associated with differences in rate of metabolism are, or may be, characteristic features of these gradients, and the evidence at hand, though, of course, as yet only a beginning, indicates that this is true.

10. The study of several hundred species of animals and plants, including representatives of most of the larger groups of animals and many of the simpler plants, has shown that physiological gradients are characteristic features of

physiological axes during the earlier stages and that in the course of develop- ment various complications and modifications may occur: these latter, however, have been followed only in very small part. Moreover, it has been

possible by obliteration of previously existing gradients, to obliterate polarity and symmetry and by determining new gradients through the differential action of external factors on different regions, to determine new axes in the earlier developmental stages and in the reconstitution of pieces of various

species among the simpler organisms. The evidence indicates then that in a protoplasm of specific hereditary constitution, such a gradient is adequate as the initiating factor in the axial differentiation characteristic of that

species. 11. The apparent relation between susceptibility and quantitative factors

in physiological condition seems at present to be a special case of a general relation between dynamic systems and disturbances. For present purposes this relation may be stated in the following terms: The rate of change charac- teristic of a dynamic system of a particular kind, e.g., a protoplasm of a parti- cular constitution, a dynamo, a flowing stream, is a factor affecting the rate of changes occurring in the system as the result of disturbance. The higher

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the rate characteristic of the system the higher the rate of change resulting from disturbance. It follows from this relation that to a certain range of

extreme disturbance leading to disruption or profound alteration of the system the more active system is more susceptible than the less active. And it also

follows that to a certain range of slight or temporary disturbances, which

permit some degree of approach toward the pre-existing condition, i.e., some degree of equilibration (acclimation, acquirement of tolerance, recovery), the changes constituting the process of equilibration will take place more

rapidly in the more active than in the less active system. It may be

emphasised that the magnitude or degree of the disturbance of the system rather than the particular nature of the disturbing factors is concerned in this

relation, consequently disturbances of many different kinds but of a certain

order of magnitude may be similar in effect, so far as the susceptibility of a

particular kind of system, e.g., a particular kind of protoplasm, is concerned.

As regards the experimentil work on protoplasmic susceptibility, it is entirely

unnecessary to assume that all agents act upon any protoplasm in the same

way; in fact experimental evidence demonstrates that they do not. Each

of them, however, must affect some essential factor or factors of the system and the more rapid the complex of changes characteristic of the system, the more rapidly do the changes leading to disruption or alteration, or, on

the other hand, the changes constituting equilibration toward the pre-existing condition, i.e., acclimation or recovery occur.

In the experiments on modification of development a chronological factor

in relation to the period of development concerned is always involved. If

we disturb the processes of development at a particular stage by means of an

inhibiting agent, the result as regards form and proportion of a particular later stage may be a differential inhibition, if the primary effect of the agent

persists long enough. On the other hand, if acclimation or recovery occurs

rapidly in the course of development, the later stage in question may show

the secondary modifications in form and proportion characteristic of differential

acclimation or recovery. In the case of the sea-urchin, for example, acclima-

tion to or recovery from the action of cyanide is so slow that cyanide larvae

usually represent differential inhibition with little or no differential acclimation

or recovery as regards form and proportion, while with ethyl alcohol and acids

acclimation and recovery occur so rapidly, except in very high concentrations, that the resulting larva' usually show some degree of differential acclimation

or recovery (6). 12. Whatever the results of future investigation may be as regards the

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gradient hypothesis and the value of the susceptibility method it may be stated

emnphatically that both are based on experimental evidence. Experimental verification in the strict sense is of course often impossible, since the evidence, like a large part of scientific evidence, is indicative rather than demonstrative.

Consequently, many of the inferences drawn are provisional, or nothing more than suggestions. It would seem that criticisms of such inferences and sugges- tions ought to be based on some better foundation than the argument that we do not know all that is going on in living protoplasm or in development (cf. Cannon 1, pp. 247-248).

Certain of Mr. Cannon's criticisms require further brief consideration. As

regards the supposedly highly poisonous character of the disintegrating tissue

(Cannon, 1, p. 241), it may be pointed out that with relatively high concen- tration or intensity, disintegration may sweep over the whole length of an axis in a few seconds or a few minutes, that in embryos, larvae and adult animals in which more or less of the body is disintegrated, return to the normal medium results in cessation of disintegration, recovery, and, in the case of

embryos, further development of remaining parts, provided the action of the

agent has not been too severe. These facts have been stated in various papers. The possibility of poisonous action of the disintegrating tissue occurred to one of us long ago, but thus far no evidence of such action has been found

except possibly in cases in which the disintegration is slow enough to permit putrefactive changes in the dead parts.

As regards differences in permeability (Cannon, 1, p. 241), it has been stated repeatedly that differences in permeability, at least to certain agents, undoubtedly exist in certain, if not in all, axes. Furthermore, it has been

pointed out that these differences in. permeability cannot be the chief factors in susceptibility in general; first, because they are not concerned in the case of certain physical agents; second, because susceptibility relations along the axes in early stages and in many of the simpler organisms throughout life are the same for agents to which living membranes are highly permeable and to those which do not penetrate living unaltered membranes in appreciable amounts; and third, because differential acclimation and differential recovery occur most rapidly or most completely in those regions which, according to the criticism, must be most permeable.

It has also been pointed out that cuticular coverings and other differences of

exposure present difficulties to the susceptibility method when chemical agents are used. It may be granted that some of the earlier evidence presented was not sufficiently safeguarded against criticism in this respect, but evidence

VOL. XCVI.--B. L

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from early stages and comparative evidence from different forms, as well as the facts of differential acclimation and recovery and the susceptibility relations with various physical agents, indicate clearly enough that cuticular differences

along an axis are not in general primary factors in determining gradients in

susceptibility. As regards the extended criticism of Bellamy's classification of develop-

mental modifications and of various other points (Cannon, 1, pp. 244-248), it need only be pointed out that, aside from some actual mis-statements, the

argument seems to be misdirected. No attempt has been made by the writers to follow through, or to develop hypotheses concerning all the complex modi- fications from the primary gradient or gradients which occur in the course oi development. It is granted at once that this is at present impossible. All that was attempted in the case of the frog (2, 3), or in any other case, was to point out the changes in form and proportion in relation to the chief axes and to interpret them. Since they do not show, as regards their larger features, any indications of specificity with respect to different agents or groups of agents, during the early developmental stages, the inference was drawn from the evidence that they are based on factors that are primarily quantitative.

Cases in which certain regions of the embryo are primarily inhibited and remain inhibited more than other regions are certainly cases of differential

inhibition, whatever the factors involved. Cases in which the regions primarily inhibited become relatively accelerated in later stages while still exposed to the agent, certainly appear to be differential acclimations. And when it is

found, as it has been found, that the modifications are in general similar with

respect to the chief body axes for many different agents, the evidence indicates that the underlying physiological differences along the axes concerned in these modifications are not specific for the different agents. Specific differences

may exist at different levels of the axes, but they do not appear in the more

general features of these modifications. The only possible conclusion in the

light of the evidence seems to be that the physiological differences concerned

in the modifications are essentially quantitative, and this conclusion is supported by many other lines of evidence, both from the frog and from various other forms. Bellamy's classification of modifications is based on experimental evidence throughout, and is itself additional evidence for the interpreta- tion given.* Mr. Cannon's criticism apparently involves a fundamental

* The particular frog embryos from Bellamy's paper which are discussed by Mr. Cannon

on p. 245, as differential acclimations, are presented by Bellamy as differential inhibitions

(figs. 10, 11 "illustrate one of the less extreme embryos of this type "--(2), p. 493),

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Page 15: Susceptibility in Amphibian Development

Susceptibility in Amphibian Development.

misconception of the whole susceptibility method. To us the basis of this

misconception seems to be his mistaken belief that Bellamy regards all toxic

chemicals as acting " alike," their action differing only in degree. With this belief as a starting point, such misconception appears to be inevitable.

With respect to the modification called "meroblastic cleavage" Cannon

objects because it is discussed apart from other modifications of cleavage. The author (Bellamy) is accused of dismissing the event as "rare at best."

Such is not the case. The conditions under which such modifications appeared in his experiments are given in full on page 335 (2), and the reasons for the

"special treat,ment " are clear from the discussion there given. It was stated that: "So far as my observations go, this type of cleavage, rare at

best, is realised only when the eggs are exposed to the inhibiting conditions at

the time of, or immediately preceding, the appearance of the first cleavage plane." (Italics do not appear in the original paper.) From the fact that this modi- fication was obtained only under the conditions just stated, it was suggested first, that the inhibiting agent did not penetrate quickly enough to stop instantly or completely a process already well under way, but which did become effective before the first and perhaps the second cleavage planes had extended through the yolk. Second, in the case of the alcohol experiments in which a few of these cases were seen, it was pointed out that some modifica- tion of susceptibility relations is not surprising when a lipoid solvent such as alcohol is used. It is to be expected that the localisation of a large amount of yolk in the basal hemisphere Will determine a more or less specific reaction to certain ranges of higher concentrations of particular groups of agents. Nevertheless, the susceptibility relations of the egg and embryo are the same for these agents in certain ranges of lower concentrations as for other agents.

LITERATURE CITED.

(1) Cannon, H. Graham, ' Roy. Soc. Proc.,' B, vol. 94 (1923). (2) Bellamy, A. W., ' Biol. Bull.,' vol. 37 (1919). (3) Bellamy, A. W.,

' Amer. Journ. Anat.,' vol. 30 (1922). (4) Child, C. M., ' Biol. Bull.,' vol. 39 (1920). (5) Child, C. M., 'The Origin and Development of the Nervous System,' The University

of Chicago Press (1921). (6) Child, C. M., ' Journ. Morph.,' vol. 28 (1916).

consequently Mr. Cannon's argument does not seem to apply. There seems also to be a further misunderstanding as regards this case, for Bellamy agrees with embryologists in general in regarding the primordia of the central nervous system as originating in large part from the blastopore lips, but Mr. Cannon's argument seems to assume that neural tube and blastopore lips are regarded by Bellamy as wholly distinct.

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