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Introduction

THE HISTORY OF CHEMICAL EMBRYOLOGY

J. BRACHET

Laboratoire de Morphologie animale Faculté des Sciences Université libre de Bruxelles Brussels, Belgium

Embryology, the science of development, lias, like ail other sciences, its own embryology—how and when it was conceived remains unknown; its first steps were as hésitant as those of a baby. Those very early days have been well described by the founder of chemical embryology, Jo­seph Needham, a gifted historian as well as an embryologist. After man discovered artificial incubation which was used even in the oldest civili­zations for very practical purposes, he must have had sufficient curiosity to break the shell immediately and look at the developing chick embryo. Without knowing it, he tlius became a "descriptive embryologist." When he made the same kind of observation on a duck embryo also, lie became a "comparative embryologist." Descriptive and comparative embryology remain at the root of modem embryology. Thèse two branches of em­bryology, which grew so successfully in the eighteenth and nineteenth centuries, may now look old; but they are by no means dcad, especially in view of the continuons progress made in the optical means of ob­servation. First, only the naked eye was used, then the primitive lens, and then the compound microscope; today the électron microscope is used in many laboratories. Numerous and valuable indeed are the papers which today describe the ultrafine structure of normal sea urchin, frog, or chicken embryos. This is the modem form of descriptive and com­parative embryology.

However, after man had a good look at the developmental steps which lead to the formation of the adult, lie became more curious than ever—"how" does this thing develop? After discussing at lengtli—as we still do—"preformation" and "epigenesis," he decided to play with em­bryos, as a child does with watches. So, eggs were partially destroyed

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by pricking; blastomeres were killed; embryos were centrifuged. Thus, "expérimental embryology" was born, culminating in discoveries, such as the organizer by Spemann. New theoretical concepts, such as poten-tialities, détermination, régulation, morphogenetic fields, and gradients, progressively emerged. Since thèse concepts remain as obscure today as ever, expérimental embryology, even though it is showing signs of sé­nescence, is still fruitful.

Since chemical embryology is nothing more than the biochemical analysis of embryonic development, it naturally followed the same trends as embryology itself and its development has been necessarily linked to that of embryology. As we shall see, chemical embryology began as a descriptive and comparative science and was deeply influenced by the progress of expérimental embryology. It is now being exposed to the impact of bacterial genetics and molecular biology, which will long prevent it from sénescence. In fact, it is shifting more and more toward "molecular embryology."

The first important landmark in the history of chemical embryology was Joseph Needham's "Chemical Embryology" (1931). Not only did Needham coin the name of the new science, but he assembled in three large volumes everything that was known at the time about the chemical composition of embryos at ail stages of their development. This was the opus magnum of a great modem humanist and a bible for the small group of chemical embryologists just beginning research at that time. In hundreds of tables data concerning the protein, glycogen, lipid, and water content of ail sorts of embryos were summarized from multitudi-nous papers. This compilation, which was so valuable at the time of its publication, has lost most of its usefulness now, since both problems and methods have undergone considérable changes; but "Chemical Em­bryology" remains a monument of the early days (antiquity and the middle âges, perhaps), when descriptive and comparative embryology were the only guides for the biochemist interested in embryonic de­velopment.

Yet, many papers published in those days still deserve mention and remain as models of good scientifîc analysis—for instance, Fauré-Fre-miet's important monographs on the biochemistry of Ascaris and Sabel-laria eggs or Needham's own contributions to comparative embryology. Needham was interested in the "succession of energy sources" during de­velopment (carbohydrates being utilized before fats and proteins) in varions animal forms, in nitrogen excrétion in "cleidoic" and "acleidoic" eggs, in the possible dissociation, by expérimental means, of growth and difîerentiation, in Child's axial gradients, and in many other problems. Some of thèse questions are of interest to this day. For example, the de-

THE HISTORY OF CHEMICAL EMBRYOLOGY 3

crease in arginase acti\'ity in chick embryo when it shifts from a "ureo-telic" to a "uricotelic" metabolism remains puzzling; but this problem is now attacked from a very différent angle. Is there inhibition of the enzyme, disappearance of an activator, repression by reaction end products, négative feedback control, or loss of inducibility? Today papers are being published on thèse problems which have great theo-retical importance and are well integrated in the realm of molecular biology.

Récent progress made in biochemistry and in physical chemistry, of course, had as much influence on the orientation of the more modem work in 1930, as thèse subjects do now. While reinvestigating Warburg's former observations on the increase of oxygen consumption during ferti-lization in sea urchin eggs, Runnstrom introduced for the fîrst time in thèse studies some really important factors: cytochrome oxidase (pre-viously considered the Atmungsferment), cytochromes, dehydrogenases, and adenosine triphosphate. Interesting work, such as that in Monroy's laboratory, is still going on in exactly the same fîeld. It was in the 1930s too that Needham, Chambers, Rapkine, Ephnissi and Wurmser, and Reiss tried to measure the pH and the redox potential of thèse eggs. Since the ultrastructure of eggs is now known to be so complicated that we can hardly consider them (even as a fîrst approximation) as a water solution surrounded by a lipid membrane, this type of research is no longer being performed. However, it was .shown by Chambers that the germinal vesicle of ovocytes neither reduces nor oxidizes injected dyes, and more récent studies have shown that the cell nucleus is remarkably inadéquate in oxidizing and reducing enzymes.

So much for the "pre-Needhamian" era. We now corne to the "rinas-cimento" and the "modem âges" of chemical embryology. In 1932, a révolution occurred, namely the discovery by Bautzmann et al. that a killed organizer is still capable of inducing a neural System. Biochemists discovered the existence of expérimental embryology, and the whole course of chemical embryology changed. This shift in interests is obvions when one compares Needham's "Chemical Embryology" ( 1931 ) with his "Biochemistry and Morphogenesis" ( 1942 ) or the author's own "Em­bryologie chimique" (Brachet, 1944). Because of wartime conditions, there was no possible contact between the two authors; nevertheless, the gênerai spirit of the two books is remarkably the same.

As soon as Holtfreter (1935) and Wehmeier (1934) found that the "inducing substance" ( or "evocating substance" as the Cambridge group, following Waddington's suggestion, preferred to call it) was of very widespread distribution in Nature, the quest for its identification began. Since an alcohol-treated fragment of horse or ox liver, as a killed or-

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ganizer, also "induces" the formation of neural structures in compétent ectoblasts, why net take the whole liver and fractionate it, following the biochemical methods which led to the isolation and purification of vita-mins and hormones? This was done in several laboratories and the field was almost as "hot" as the field of molecular genetics is right now. Pre-liminary notes, with stern remarks about priority, were shooting from ail sides. The contest became worse when it clearly appeared that every leading laboratory had its own "active substance," différent from those found in the others—Glycogen is the active substance! No, it is a sterol, and your glycogen is contaminâted with sterols! No, ail acids are indue-tors, and your sterols must contain fatty acids! No, the substance must be spécifie in order to explain why an embryo possesses a head and a tail: it must be a protein!

Suddenly, the setback came and the excitement ceased. In Cam­bridge we ( Waddington, Needham, and the author) were studying the respiratory metabolism of the organizer as compared to other parts of the gastrula (a warm field toc, but not really hot). Since méthylène blue was known to increase the respiratory rate of many cells, it was decided to implant pièces of agar containing méthylène blue in gastru-lae. Neural inductions were obtained using an "inductor" of nonbio-logical origin, which could not have been contaminated with sterols, fatty acids, or glycogen. Still worse, neutral red, which has no effect on oxygen consumption, was also active. So, ail thèse théories were both right and wrong—ail the substances tested were more or less active; but is was impossible to décide which was the right one, since the ecto-blast (like the unfertilized egg treated with parthenogenetic agents) could reaet in the same way (neural induction) to a large variety of chemical substances. This led Waddington et al. (1936) to suggest that the neuralizing factor was already présent in the ectoblast, but in a bound form; any agent which could "unmask" the inducing substance would induce the transformation of ectoderm into neural tissue. Later work by Barth and Graff (1938) led Waddington to the sad conclusion that both heterogeneous inductors and chemical substances easily pro­duce cytolysis of part of the very fragile ectoderm cells. The "true" ac­tive substance would be liberated by the dying cells, and the hope of isolating and identifying it became still more remote. The final blow came when Holtfreter (1947) demonstrated that even sublethal cytol­ysis is sufficient to induce the neuralization of ectodermal explants; a short acid or alkaline shock, which is insufficient to kill the cells, is sufficient to obtain a high percentage of nervous structures in such ex­plants. Now, one could and did despair.

However, things are never as bad as they look. Discussions about the

THE HISTORY OF CHEMICAL EMBRYOLOGY 5

mechanisms of cephalic (neural) and caudal (mesodermal) inductions led to a resumption of work in a fîeld which looked most unpromising from the time of Holtfreter's observations. Were thèse différences due to quantitative or qualitative factors? Were there distinct neuralizing and mesodermalizing substances? It has been to the crédit of men like Toivonen, Yamada, and Tiedemann to résume the quest for spécifie cephalic and caudal inducing substances this time (see Volume II, Chapter I) . Refîned methods of protein chemistry (ultracentrifugation, electrophoresis, column chromatography ) were used which led to the isolation from varions tissues (liver, bone marrow, chick embryos) of purified active neural and mesodermal inducing substances. Cephalic induction can be obtained with ribonucleoproteins (the protein part apparently being the active one), whereas pure proteins from bone mar­row and chick embryos act as caudal (mesodermal) inductors. Many problems, of course, remain to be solved. Are the mesodermal inducing proteins of bone marrow and chick embryo identical, similar, or en-tirely différent? Have they common antigenic sites? Have they some enzymatic activity? There are other, perhaps still more important, questions to answer. Is it certain that thèse spécifie substances act di-rectly (and not by a relay mechanism, the former unmasking of the true inducing substance)? The doubts we had 25 years ago are not yet com-pletely dissipated. Are the isolated proteins présent in a normal gastrula or neurula? If so, where is their localization? Thèse important questions can and will be solved. Yamada, in particular, is using a whole array of modem techniques, such as électron microscopy, autoradiography, and détection of the protein with labeled antibodies, according to the method of Coons. Immunological techniques, in particular, should be powerful tools for the solution of thèse basic problems.

But the topic of this introduction is the history of chemical embry-ology, not its présent state. We have moved, in the case of inducing sub­stances, from the 1930-1945 problems to the présent ones almost with-out noticing a transition. Is there a fundamental différence between the "modem history" of chemical embryology and its "contemporary his­tory"? The author should know the answer, since he wrote books on this same subject both in 1944 and in 1960. The main change between the modem and the contemporary history of chemical embryology is that we have undergone during the past 15 years an équivalent of the industrial révolution in the nineteenth century. It has been a révolution in the "techniques," rather than in the ideas, of chemical embryologists. We have now much more powerful weapons at hand, but the great problems to be solved remain the same.

Yet, there is another révolution occurring at this moment, a révolu-

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tion in oiir theoretical approaches stemming from the tremendous de-velopment of molecular genetics. It is often said that molecular biolo-gists, after having solved the riddles of heredity, will then enter the field of embryology and solve the problems of cell difEerentiation. We hope that this will really happen; a massive injection of first-class brains in our fîeld would undoubtedly lead to spectacular progress. However, un-til thèse welcome reinforcements arrive, chemical embryologists will carry the fight alone, using the concepts and methods which have been so successfully employed in genetics.

This new trend can be exemplifîed in a field which bas been the main battleground for molecular biologists, the control of spécifie pro-tein synthesis by nucleic acids. In the pre-Needhamian period, around 1930, there were no such things as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Thanks to the Feulgen reaction, it was known, however, that "thymonucleic acid" (our DNA) is présent in the nuclei of ail cells, hence, no longer deserving its name of "animal" nucleic acid. Pentose nucleic acids (RNA) were supposed to be "plant" nucleic acids, althougli one of thèse acids had already been isolated from the pan­créas. No reliable method for the estimation of the two nucleic acids was available, since it was still believed that they could not coexist in the same cell, one being spécifie for animal cells, the other for plant cells.

In the case of ovocytes, the very présence of thymonucleic acid in lampbrush chromosomes was still hotly disputed; discussions between the holders of the "migration" theory and those of the "net synthesis" theory were intense. According to the former, the germinal vesicle con-tained a reserve of nucleic acid which moved into the cytoplasm at maturation and migrated back into the nuclei during cleavage. Their opponents, of course, denied the existence of such a reserve and claimed that thymonucleic acid was formed de novo in the nuclei after fertiliza-tion. The fact that the very unreliable biochemical methods available for nucleic acid détermination indicated that there was no change during development confused the situation even more. This constancy of the nucleic acid content during development was, of course, the main argu­ment in favor of the migration theory; but the fact that the Feulgen reaction was négative in the ovocyte and strongly positive in the nuclei of blastulae and gastrulae apparently demonstrated the correctness of the net synthesis hypothesis.

As usual, it was the development of a new and more spécifie tech­nique (the diphenylamine method for DNA estimation of Dische) and of new hypothèses which clarified the situation. In 1933, it could be demonstrated that the unfertilized eggs contained only traces of DNA

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as compared to the high DNA content of gastriilae or neurulae. The reaHty of a net synthesis of DNA during development thus became an estabhshed fact. It was also proven at the same time that eggs contain large amounts of RNA; therefore, the latter is not a plant nucleic acid at ail, but is présent in ail cells. It was concluded on the basis of pentose estimations made with still very crude methods that there is a conversion of RNA (possibly after its dégradation to the nucleotide level) into DNA. That such a conversion occurs on a large scale was disproven when about 15 years later radioactive phosphate became a new tool for the study of nucleic acid synthesis.

A new question arose—what is the intracellular localization of RNA? In 1940 studies with the UV-microscope by Caspersson and the utiliza-tion of a simple staining procédure (combined with a digestion of the tissue sections with ribonuclease in order to ensure greater specificity) brought the desired answer: RNA is an ubiquitous constituent of ail cells; it is localized in the nucleolus and the cytoplasm; there is a close corréla­tion between the RNA content of a given cell and its ability to synthesize proteins.

In the eggs of the vertebrates ( those of the amphibians, in partic-ular) RNA is distributed along a primary polarity ( animal-vegetal ) gradient already présent in the ovocyte and the unfertilized egg. At gastrulation a new gradient, which is more dynamic and results from synthesis of fresh RNA, spreads from the dorsal to the ventral side. As a resuit of mutual interaction, dorsoventral and cephalocaudal RNA gradients become apparent in the late gastrula and the early neurula stages. Thèse gradients are parallel to or identical with the morpho-genetic gradients of the expérimental embryologists. Disorganization of the gradients by chemical or physical means and abnormalities in development go hand in hand.

This was the situation in 1940 during "rinascimento" and the "mod­em âges" of chemical embryology. How much has it changed during the "contemporary period" of this science? Once again, new and pow-erful methods ( autoradiography, for example ) have been put into opéra­tion; but still, the problems have remained the same, and many observa­tions are still disputed vigorously.

For instance, the migration versus net synthesis controversy is not yet extinct. New, even more sensitive and spécifie methods for DNA estimation have been developed; they have confirmed that the cyto­plasm of unfertilized eggs contains some kind of DNA. But, at the same time, autoradiography has shown that a net synthesis of DNA at the expense of simple precursors, such as thymidine and uridine, also occurs during cleavage. The fact that uridine, a ribonucleoside, can be utilized

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for DNA synthesis in the cleaving egg shows that, after ail, conversion of ribose derivatives into DNA actually occurs, even if only on a small scale. Again it looks as if both théories are correct. It is very likely now ( but it has not yet been proved ) that during cleavage DNA partly orig­inales from migration of a cytoplasmic DNA reserve and partly from a net synthesis at the expansé of simple deoxyribose and ribose precur-sors. The récent finding of Agrell and Bergqvist (1963) that during cleavage in frog eggs nuclei contain two différent types of DNA, un-equally résistant to acid hydrolysis, speaks strongly in favor of such a conclusion.

The signifîcance of the RNA gradients in amphibian eggs is also be-coming somewhat clearer. Autoradiography has now shown that RNA synthesis, which leads to the formation of the dorsoventral gradient at the gastrula stage, begins in the nucleus. On the other hand, électron microscopy has shown that the primary ( polarity ) gradient is essentially a ribosomal gradient. Combining thèse two fîndings, we can arrive at the following explanation: The inert polarity gradient of ribosomes be-comes activated toward gastrulation by messenger RNAs formed in the nuclei along the dorsoventral gradient. The resuit would be that protein synthesis will be more active in the anterior (cephalic) part of the embryo than in its posterior (ventral) part; protein synthesis would also decrease progressively from dorsal to ventral. Very récent experi-ments with spécifie inhibitors of messenger RNA synthesis (actinomy-cin D) and of protein synthesis (puromycin) entirely confirm thèse dé­ductions.

This is certainly progress; but the real problem, that of morphogen-esis, remains unsolved. To understand how production of spécifie mes­senger RNAs, controlled by localized gene activation leading to the synthesis of presumably spécifie proteins, ultimately leads to the dif-ferentiation of nervous System, chorda, or muscle cells is a task for the future; it is a task for "molecular embryology," whose birth we are now watching. It is hoped that this new science, based on solid theoretical foundations, will not be groping in darkness, as chemical embryology did initially. Hypothèses which "predict" (it is very exceptional now that the prédictions are not fulfilled by the experiments designed to test them) will replace our présent "guesses" and "hunches." One guess (almost a prédiction) is that électron microscopes with still higher and higher resolution will be built and will show in the developing egg the most important macromolecules changing their shapes or accumulating at stratégie spots. On that day "molecular embryology" and "descriptive embryology" will be synonymous.

Let us step back from the future for a final remark about the présent.

THE HISTORY OF CHEMICAL EMBRYOLOGY 9

Joseph Needham's opus magnum was a one-man work; the présent treatise is the resuit of the coopération of many scientists. This diflFer-ence shows what great progress has been made during the last 30 years. Those who took part in this battle against ignorance can be justifiably proud of what their génération has achieved in the field of chemical embryology. We hope that the présent treatise will be, as Needham's book was, a new milestone on the road and a new source of inspiration for the next génération of chemical embryologists. If so, it will have ful-fîlled a great purpose.

R E F E R E N C E S

Agrell, I., and Bergqvist, H . -Â . ( 1 9 6 3 ) . / . Cell Biol. 15, 604. Barth, D . G., and Graff, S. ( 1 9 3 8 ) . Cold Spring Harbor Sijmp. Quant. Biol. 6, 385. Bautzmann, H., Holtfreter, J., Spemann, H. , and Mangold, O. ( 1 9 3 2 ) . Naturwiss-

enschaften 20, 971. Brachet, J. ( 1 9 3 3 ) . Arch. Biol. (Liège) 44, 519 . Brachet, J. ( 1 9 4 4 ) . "Embryologie chimique." Desoer, L iège (Mas.son, Pari.s, 1945 ) . Brachet, J. ( 1 9 6 0 ) . "The Biochemi.stry of Deve lopment ." Pergamon, N e w York. Holtfreter, J. ( 1935 ). Wilhelm Roux' Arch. Entwicklungsmech. Organ. 133, 367. Holtfreter, J. ( 1 9 4 7 ) . / . Exptl. ïool. 106, 197. Needham, J. ( 1 9 3 1 ) . "Chemical Embryology." Cambridge Univ. Press, London and

N e w York. Needham, J. ( 1 9 4 2 ) . "Biochemistry and Morphogenesis ." Cambridge Univ. Press,

London and N e w York. Waddington , C. H., Needham, J., and Brachet, J. ( 1 9 3 6 ) . ?roc. Roy. Soc. B120, 173. Wehmeier , E. ( 1 9 3 4 ) . Wilhelm Roux' Arch. Entwicklungsmech. Organ. 132, 384.