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Development 101. 653-657 (1987) Printed in Great Britain © The Company of Biologists Limited 19S7 General interest paper 653 The epigenetic nature of vertebrate development: an interview of Pieter D. Nieuwkoop on the occasion of his 70th birthday J. C. GERHART Department of Molecular Biology, University of California. Berkeley. CA 94720, USA Interviewer's commentary Professor Pieter Nieuwkoop has made numerous contributions to amphibian embryology over the past 40 years. These include his formulation of the normal table of Xenopus development, the analysis of re- gional aspects of neural induction, the discovery and analysis of mesoderm induction, the discovery of the inductive formation of germ cells in urodeles, studies of the cytoplasmic organization of eggs, and the study of the evolution of land-adapted vertebrate eggs. He has sought an understanding of epigenesis in ver- tebrates through his observations of morphology and through the methods of experimental embryology. Since the body of information represented by his work is less and less familiar to contemporary re- searchers, I have provided him with a list of questions that might encourage him to summarize in writing some aspects of his purview. When you began your training in embryology, what were the important theoretical and experimental issues? My training in embryology began with the arrival of Dr Chr. P. Raven as Professor of Zoology at the State University of Utrecht in September, 1938, one year after I received my B.Sc. degree in Biology. Professor Raven was a pupil of Professor M. W. Woerdeman who had worked with Professor H. Spemann in Freiburg, Germany, in the 1920's. As I studied comparative anatomy and descriptive embryology, I was particularly fascinated with the formation of a new individual from the mere union of egg and sperm, because it represented for me 'the creation of new life'. In contrast, I found rather uncompelling the use of comparative anatomy and embryology as evidence for the course of evolution, a speculative scholarly endeavour that had been carried forward from the late 1800's. The main theoretical issues at the time were 'vitalism' versus 'causal analysis,' and 'holism' versus 'reductionism'. Various critics questioned seriously the value of experimental work at all since one must perturb the natural state of the embryo and must focus on less than the entirety of the developmental process. However, as is largely taken for granted today, one can only learn about developmental pro- cesses by disturbing them and then relating the observed abnormalities to the normal condition. Other biologists at the time assumed they could predict the integrated behaviour of the embryo from that of its cells or subcellular materials. The holism- reductionism controversy certainly continues and has been strongly aggravated by the advent of molecular biology. In experimental embryology, the main issue at the time was of course induction, not just primary (neural) induction as discovered and studied by Spemann and his colleagues, but a great variety of inductive interactions in vertebrates in general. Young experimental embryologists were strongly drawn to the phenomenon of induction, the analysis of which seemed to promise many answers to ques- tions about epigenesis. What changes of issues have stood out for you over the period of your career? I have witnessed the rapid development of molecular biology and its penetration into nearly all fields of biology, including embryology. The deification of the DNA/RNA/protein dogma has led to a substantial neglect of the role of the cytoplasm in cellular differentiation. Moreover, the significance of the whole organism as well as of its environment in the development of a new individual has been so thoroughly forgotten during the advance of molecular biology that by now there are hardly any all-around

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Page 1: The epigenetic nature of vertebrate development: …...Epigenetic nature of vertebrate development 655 the initial role of the primary mesenchyme in sea urchin development or of the

Development 101. 653-657 (1987)Printed in Great Britain © The Company of Biologists Limited 19S7

General interest paper 653

The epigenetic nature of vertebrate development: an interview of

Pieter D. Nieuwkoop on the occasion of his 70th birthday

J. C. GERHART

Department of Molecular Biology, University of California. Berkeley. CA 94720, USA

Interviewer's commentary

Professor Pieter Nieuwkoop has made numerouscontributions to amphibian embryology over the past40 years. These include his formulation of the normaltable of Xenopus development, the analysis of re-gional aspects of neural induction, the discovery andanalysis of mesoderm induction, the discovery of theinductive formation of germ cells in urodeles, studiesof the cytoplasmic organization of eggs, and the studyof the evolution of land-adapted vertebrate eggs. Hehas sought an understanding of epigenesis in ver-tebrates through his observations of morphology andthrough the methods of experimental embryology.Since the body of information represented by hiswork is less and less familiar to contemporary re-searchers, I have provided him with a list of questionsthat might encourage him to summarize in writingsome aspects of his purview.

When you began your training in embryology,what were the important theoretical andexperimental issues?

My training in embryology began with the arrival ofDr Chr. P. Raven as Professor of Zoology at the StateUniversity of Utrecht in September, 1938, one yearafter I received my B.Sc. degree in Biology. ProfessorRaven was a pupil of Professor M. W. Woerdemanwho had worked with Professor H. Spemann inFreiburg, Germany, in the 1920's.

As I studied comparative anatomy and descriptiveembryology, I was particularly fascinated with theformation of a new individual from the mere union ofegg and sperm, because it represented for me 'thecreation of new life'. In contrast, I found ratheruncompelling the use of comparative anatomy andembryology as evidence for the course of evolution, aspeculative scholarly endeavour that had been carriedforward from the late 1800's.

The main theoretical issues at the time were'vitalism' versus 'causal analysis,' and 'holism' versus'reductionism'. Various critics questioned seriouslythe value of experimental work at all since one mustperturb the natural state of the embryo and mustfocus on less than the entirety of the developmentalprocess. However, as is largely taken for grantedtoday, one can only learn about developmental pro-cesses by disturbing them and then relating theobserved abnormalities to the normal condition.Other biologists at the time assumed they couldpredict the integrated behaviour of the embryo fromthat of its cells or subcellular materials. The holism-reductionism controversy certainly continues and hasbeen strongly aggravated by the advent of molecularbiology.

In experimental embryology, the main issue at thetime was of course induction, not just primary(neural) induction as discovered and studied bySpemann and his colleagues, but a great variety ofinductive interactions in vertebrates in general.Young experimental embryologists were stronglydrawn to the phenomenon of induction, the analysisof which seemed to promise many answers to ques-tions about epigenesis.

What changes of issues have stood out for youover the period of your career?

I have witnessed the rapid development of molecularbiology and its penetration into nearly all fields ofbiology, including embryology. The deification of theDNA/RNA/protein dogma has led to a substantialneglect of the role of the cytoplasm in cellulardifferentiation. Moreover, the significance of thewhole organism as well as of its environment in thedevelopment of a new individual has been sothoroughly forgotten during the advance of molecularbiology that by now there are hardly any all-around

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654 J. C. Gerhart

morphologists left for proper instruction of the newgeneration of biologists.

What Is your current view of the process ofneural induction and of the patterning of theneural plate?

I must confess that at present I am revising some ofmy former views on neural induction in the amphib-ians, views in which diffusion gradients played animportant role. I still believe that neural inductionoccurs in at least two successive steps: first, theprimary neuralization of the ectoderm by the advanc-ing prechordal portion of the invaginating archen-teron roof, which, in the absence of further inductivestimuli, leads to prosencephalic (forebrain) develop-ment; and second, the subsequent transformation ofthe neurectoderm into more caudal structures of thecentral nervous system (CNS) by a secondary actionof the notochordal region of the archenteron roof.We now know that primary neural induction, which isinitiated only at the median, i.e. notochordal andprechordal, regions of the archenteron roof, whereintimate contact (adhesion) develops with the over-lying ectoderm, spreads mediolaterally and anteriorlyin the plane of the neurectoderm by means ofhomoiogenetic (i.e. between similar cells) induction.This spreading from cell to cell is a slow process,which continues as long as the ectoderm maintains itsneural competence and stops when neural com-petence is lost. The boundary of the neural plate isthus not set by a threshold in a diffusion gradient, as Ithought previously, but by a termination of com-petence. The second induction process, for which anew competence develops in the activated neurecto-derm, seems to follow the same principles as far as itsmediolateral spreading is concerned. In the cranio-caudal segregation of specific regions of the CNSmany more factors play a role, such as the relative ageof the inducing caudal mesoderm, the actual onset ofthe inductive action and the duration of competenceof the neurectoderm, while concentration differencesof the inducing factor or graded mechanical factorsmay also play a role. At present I am working on thisproblem.

In primary neural induction, the level of instruc-tiveness between the mesoderm and neurectodermmay be rather low, since at the time of contact theectoderm is thoroughly prepared for the switch intoneural development. The level of instructiveness maybe higher in the second induction process. However,even in the first induction process some specificitycannot be denied to the inducing factors, although aschemical substances these may comprise quite com-mon materials ranging from ionic imbalances to high-molecular-weight extracellular matrix components.

What is your view of the process of patterningof the mesoderm in vertebrates?

The origin of the embryonic endomesoderm (themesoderm and pharyngeal and dorsal endoderm) inthe amphibians is entirely epigenetic. Specifically, itarises by the inductive interaction of two cellularizedregions of the egg, namely, the vegetal endodermalyolk mass and the still totipotent animal ectodermalmoiety. Although the simplest explanation of theregional patterning of the mesoendoderm is still thatbased on dorsoventral differences in the onset andintensity of one and the same inductive interaction,there is recent evidence for a more complex type ofinduction involving different ventral and dorsal in-ductive actions by the endoderm. Mesoendoderminduction looks very much like a homoiogeneticprocess which spreads very slowly animalward fromthe boundary of the two regions of the amphibianblastula, starting at the 32- to 64-cell stage andreaching about halfway around the presumptive mar-ginal zone by the early gastrula stage. The process isaccelerated during gastrulation by a contact interac-tion between the invaginated (anterior) and thenoninvaginated (posterior) regions of the presump-tive marginal zone.

Mesoendoderm induction is, I think, a generalfeature of vertebrate development. It has beendemonstrated convincingly for amphibians and birds,and there is no convincing evidence that the sameprinciple does not hold for fish, reptiles and mam-mals. If the Cephalochordates also share this induc-tion process and if they are the descendants of thetrue ancestors of the vertebrates, mesoendoderminduction may then hold for the entire phylum ofchordates, an opinion which I still hope to verify bystudying further the early development of Amphi-oxus.

In what ways do you see the development ofthe vertebrates as different from that of theinvertebrates?

I do not believe that the principles of development ofvertebrates and invertebrates are essentially differ-ent. However, the timing of determinative eventsmay vary markedly, as shown by early and irrevers-ible determination in some organisms such as nema-todes, intermediate determination in other organismssuch as vertebrates, and late determination in yetothers such as coelenterates. Moreover, the leadingrole of a particular cell type (or cell layer) indevelopment may vary greatly: e.g. the initial crucialrole of the endoderm in vertebrate development,leading to mesoendoderm formation, the latter thendirecting further development; in contrast, there is

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the initial role of the primary mesenchyme in seaurchin development or of the ectoderm in insectdevelopment, the last mentioned establishing meso-derm by an inductive interaction.

At the morphological level, a unique aspect ofvertebrate development, of course, lies in the originof the CNS. The first step in neural induction leads tothe formation of the highest integrative centres ofneuronal organization, namely the forebrain orprosencephalon, while the second step of inductiveinteraction transforms some of this tissue into themore caudal, motor regions of the CNS.

What main advances would you draw attentionto in the evolution of vertebrate development?

The evolution of the vertebrates is characterized byan extension of the forebrain region and a relativereduction of the hindbrain-spinal cord region of theCNS. This is reflected in the development ofincreased amounts of prechordal mesoderm at theexpense of the chordamesoderm, as one comparesvertebrate classes. In vertebrate evolution, embry-onic development is, moreover, markedly extended,with the elimination of larval development, andgrowth is markedly enhanced during embryonic de-velopment. Vertebrate evolution is, moreover,characterized by the transition from aquatic to terres-trial life (and viviparous reproduction), with a con-comitant adaptation of egg structure from aquaticeggs in fishes and amphibians to land eggs in reptilesand birds and perhaps back to aquatic (uterine) eggsin mammals.

What aspects of development do you thinkdeserve more attention than they are presentlyreceiving?

We should pay much more attention to the spatial andtemporal aspects of developmental processes, es-pecially to epigenetic processes, which I define asthose by which the complexity of the embryo'sorganization is systematically increased through theinteraction of juxtaposed parts, leading to the gener-ation of new regions at their boundaries. Embryonicinduction is clearly an epigenetic process which playsa fundamental role in establishing the three-dimen-sional pattern of organ anlagen of the new individual.There is, of course, great value in the current gen-etic-molecular approach to development, but a cleardistinction should be made between the kinds ofcomplexity existing at successive levels of organiz-ation, i.e. the molecular, subcellular, cellular andorganismal levels. More attention should be given tothe relevance and validity of the proposed governing

principles at these higher levels of organization whereepigenesis is so apparent. I feel that moleculardevelopmental biology cannot advance successfullywithout constantly confronting the development ofthe whole organism.

I think a deeper insight into various multicellularphenomena is essential for a better understanding ofembryonic development. For example, groups ofisopotential cells of the embryo show a capacity for'self-organization,' as, for example, in the spatialorganization of the mesodermal mantle or of theCNS. We know little about selforganizing properties.Interactions with surrounding tissues certainly play arole, but local conditions inside an initially homo-geneous cell mass (e.g. peripheral versus centralposition) could also be of great importance.

Finally, we should also pay more attention to thephenomenon of cytoplasmic segregation as an earlystep in cytoplasmic diversification, leading eventuallyto the development of different cell types. In embry-onic development the expression of genes is governedby the composition of the cytoplasm, as nucleartransplantation experiments have convincinglydemonstrated. The spatial diversification of the cyto-plasm into cellular and subcellular units determinesthe temporal and regional activation of genes. In itsturn, the spatial diversification of the cytoplasm isestablished in oogenesis and early embryonic devel-opment by integrated epigenetic processes aboutwhich we know little. Cell membrane properties alsoseem to depend on the composition of the cytoplasm,so that the cytoplasm is actually the foremost com-ponent. This is, of course, too simplistic a picture,since there is a constant interaction between nucleusand cytoplasm and extra- and intracellular membranesystems, but I use it to emphasize the early dominantrole of the cytoplasm in embryonic development.

If you were now starting your career inresearch, yet having the knowledge you have,what lines of research would you pursue?

I would again direct my research at embryonicinduction as one of the most important developmen-tal events, but would concentrate on the properties ofthe reacting system, in particular on the appearance,full development, and decline of successive phases ofcompetence. In the preceding decades attention hasbeen, I think erroneously, nearly exclusively directedat the nature of the inducing factors. Happily, we seea recent renewed interest in these inductive aspects ofembryonic development among molecular develop-mental biologists. Inductive interaction causes achange in the properties of the responding cells,which leads to new morphogenetic events: e.g.mesoendoderm induction leads to gastrulation, and

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656 J. C. Gerhart

neural induction to neurulation. These phenomenaare multicellular-organismal phenomena in whichlarge numbers of cells are involved, behaving in acoordinated manner.

What were your general aspirations in bringingtogether young researchers, both visitors andstaff, at the Hubrecht Laboratory?

In organizing the International Research Groups ofthe Laboratory, I had two objectives in mind: first, togive young, promising researchers from other

countries a chance to go abroad and gain newexperience in attacking fundamental embryologicalproblems and, second, to let them participate inscientific cooperation on an international basis.Through this cooperation, they could perhaps learnto appreciate the value of different points of vieworiginating from different religious and philosophicalbackgrounds, and in this way help overcome inter-national misunderstandings.

With regard to the scientific staff of the Labora-tory, I was happy to encourage the association of awide variety of researchers emphasizing different

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Epigenetic nature of vertebrate development 657

approaches and problems in development. I con-sidered it valid that, as a research program, thetraditions of experimental embryology, as founded bySpemann and his school and transferred by Woerde-man and Raven to my generation, could be extendedwith new techniques such as EM analysis, organculture, biochemical and biophysical methods, butalways in a multilevel approach in which integratedepigenetic processes would remain foremost in ourview.

Biographical information

Pieter Dirk Nieuwkoop was born July 7, 1917 inEnschede (located in an eastern province of theNetherlands) as the second child of a secondaryschool teacher's family. He completed his secondaryeducation in Wageningen and entered the StateUniversity of Utrecht in September, 1934, for thestudy of Biology. After obtaining a B.Sc. degree in1937 and an M.Sc. in 1940, he worked for a Ph.D.degree during the German occupation of The Nether-lands in the Second World War. He was preventedfrom receiving a Ph.D. degree during wartime be-cause he chose to write his thesis in English. Thisdegree (with qualification cum laude) was granted inJuly, 1946 (supervisor Professor Chr. P. Raven).

He was employed as a scientific assistant from 1942to 1949 in the Department of Zoology in Utrecht. Heundertook the reorganization of the Hubrecht Lab-oratory (seat of the "Institut International d'Embryo-logie") as Deputy Director in September 1947 and

continued this endeavour as Director, a position heaccepted in January 1953, after a one year leave(1951-52) as a Rockefeller Fellow in Baltimore,Chicago and Woods Hole, USA. He was appointed toa special Professor's chair in Experimental Embry-ology at the State University of Utrecht in Sep-tember, 1959. Among his doctoral research studentshave been E. Boterenbrood, H. Eyal-Giladi, K.Hara, L. Sutasurya, A. Johnen, and B. Albers. Heretired from his Directorship of the Hubrecht Lab-oratory in July, 1980, and from his special Professor-ship in September, 1984. At present he is continuingresearch at the Hubrecht Laboratory (amphibians)and at the Institute of Technology in Bandung,Indonesia (reptiles).

His bibliography contains about 70 scientificarticles, mostly in international journals, as well asthe following four monographs: the Xenopus NormalTable (with J. Faber; North-Holland Publishing Cy,1956, 1st edition); Primordial Germ Cell Developmentin the Chordates (with L. A. Sutasurya, 1979); Primor-dial Germ Cell Development in the Invertebrates (withL. A. Sutasurya, 1981); and The Epigenetic Nature ofChordate Development (with A. G. Johnen and B.Albers, 1985), the last three published by CambridgeUniversity Press, Cambridge, UK. He has served onthe editorial boards of the Journal of Embryology andExperimental Morphology and of Wilhelm Roux'sArchives of Developmental Biology.

He was married in December 1942, to Miss H. C.M. Beukers, a primary school teacher. Their son,Wouter, was born in June 1952, in Chicago, Illinois,USA.