Advances in Morphogenesis: Volume 10

By M. Abercrombie, Jean Brachet and Thomas J. King

Advances in Morphogenesis, Volume 10, provides an overview of the state of knowledge in morphogenesis. The book contains six chapters and opens with a discussion of the "organization center" of the amphibian embryo. This is followed by separate chapters on physiological gradients in development; molecular embryology of the sea urchin, mollusks, and other invertebrates; and changes in DNA, RNA, and protein during successive phases of embryonic development in vertebrates. Subsequent chapters deal with the control of growth in the filamentous prothalli and the causal factors promoting the transition to the two-dimensional gametophyte; and the development, inheritance, and origin of the plastid in Euglena.

• Language: English
• Publisher: Academic Press
• Release date: Oct 22, 2013
• ISBN: 9781483215617

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Advances in Morphogenesis

M. Abercrombie

Strangeways Research Laboratory, Cambridge, England

Jean Brachet

Faculté des Sciences, Université Libre de Bruxelles, Rhode-St-Genèse, Belgium

Thomas J. King

National Cancer Institute, Bethesda, Maryland

ISSN  0065-2962

Volume 10 • Number Suppl. (C) • 1973

Table of Contents

Cover image

Title page

Contributors to this Volume

Copyright page

Contributors to Volume 10

The Organization Center of the Amphibian Embryo: Its Origin, Spatial Organization, and Morphogenetic Action*

Publisher Summary

I Justification

II The Discovery of the Organization Center, and a Description of its Main Properties

III The State of Determination and Capacity for Differentiation of the Various Regions of the Early Gastrula

IV The Induction of the Mesoderm

V The Origin of the Regional Organization of the Mesoderm; Its Dorsoventral and Craniocaudal Polarity

VI Concluding Remarks

Acknowledgments

Physiological Gradients in Development — A Possible Role for Messenger Ribonucleoprotein

Publisher Summary

I Introduction

II Gradients and Determination in Early Embryos

III Some Other Gradient Systems in Development

IV Some Further Possible Analogies with Embryonic Animalizations

V Discussion

VI Summary

Appendix

Molecular Embryology of Invertebrates

Publisher Summary

I Introduction

II Molecular Embryology of the Sea Urchin

III Molecular Embryology of Other Invertebrates

IV Molecular Embryology of Molluscs

Addendum

Acknowledgments

Note Added in Proof

Biochemical Aspects of Early Differentiation in Vertebrates

Publisher Summary

I Introduction: The Problems in Analyzing Differentiation

II Differentiation in Early Embryonic Cells

III Primary Tissue Interactions

IV Protein and Nucleic Acid Changes during Organogenesis: Comparative Studies

V Biochemical and Functional Differentiation in Individual Organ Systems

VI Conclusions

Acknowledgments

Photomorphogenesis and Nucleic Acid Metabolism in Fern Gametophytes

Publisher Summary

I Introduction

II Normal Growth of the Gametophyte

III Growth of Filamentous Prothalli

IV Transition of Filamentous Prothalli to Biplanar Gametophytes

V Protein and Nucleic Acid Metabolism in the Induction of Biplanar Growth

VI Hypothetical Control Mechanisms

Acknowledgments

The Development, Inheritance, and Origin of the Plastid in Euglena

Publisher Summary

I Introduction

II The Development of the Proplastid into the Chloroplast in Euglena

III How Did the Plastid Originate?

IV Conclusions

Acknowledgment

Author Index

Topical Index

Contributors to this Volume

R.L. Brahmachary

P.D. Nieuwkoop

A.E. Demaggio

V. Raghavan

Elizabeth M. Deuchar

Jerome A. Schiff

Robert Wall

Copyright page

COPYRIGHT © 1973, BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED.

NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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PRINTED IN THE UNITED STATES OF AMERICA

Contributors to Volume 10

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

R.L. Brahmachary,     Indian Statistical Institute, Calcutta, India (115)

A.E. Demaggio,     Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire (227)

Elizabeth M. Deuchar,     Department of Anatomy, The Medical School, University of Bristol, Bristol, England (175)

P.D. Nieuwkoop,     Hubrecht Laboratory, Utrecht, The Netherlands (1)

V. Raghavan,     Department of Botany, The Ohio State University, Columbus, Ohio (227)

Jerome A. Schiff,     Biology Department, Brandeis University, Waltham, Massachusetts (265)

Robert Wall*,     Institute of Animal Genetics, Edinburgh, Scotland (41)

---

*Present address: 1A, Carlton Terrace, Edinburgh EH7 5DD, Scotland.

The Organization Center of the Amphibian Embryo: Its Origin, Spatial Organization, and Morphogenetic Action*

P.D. Nieuwkoop,     Hubrecht Laboratory, Utrecht, The Netherlands

Publisher Summary

This chapter discusses the origin, organization, and morphogenetic action of the amphibian embryo. The amphibian gastrula is dynamically determined in its constituent parts. Transplantation of upper blastoporal lips taken from gastrulae of different ages demonstrated that the head inductor is located in the dorsal lip of the early gastrula, the trunk inductor in that of the advanced, and finally the tail inductor in that of the completed gastrula. The morphogenetic movements do not take the form of a wandering of individual cells but are the expression of a supracellular phenomenon. Dorsal marginal zone plays a leading role in embryo formation and the region is called the organization center of the embryo. The phases of cleavage and blastocoel formation were considered as relatively unimportant though indispensable as preparations for the true morphogenetic events of gastrulation and embryo formation. The chapter discusses the several important morphogenetic events that happen prior to gastrulation, which are of utmost significance for embryogenesis.

I Justification 2

II The Discovery of the Organization Center, and a Description of its Main Properties 2

III The State of Determination and Capacity for Differentiation of the Various Regions of the Early Gastrula 5

A Determination and Differentiation Tendencies of the Presum ptive Endoderm 6

B Determination and Differentiation Tendencies of the Presumptive Mesoderm 8

C Determination and Differentiation Tendencies of the Presumptive Ectoneuroderm 9

D Differentiation Tendencies and Inductive Capacity of the Presumptive Prechordal Endomesoderm before and after Its Invagination 12

IV The Induction of the Mesoderm 13

A Endomesodermal Differentiation of Gastrula Ectoderm under the Influence of Heterogeneous Inductors 13

B Additional Mesoderm Formation in Centrifuged Embryos 14

C The Epigenetic Development of the Marginal Zone of the Amphibian Egg 15

D The in Situ Formation of the Mesoderm 17

E The Heterogeneity of the Cell Population in the Ectodermal Part of the Embryo 21

F The Vegetalizing Action of the Li Ion 23

G General Remarks 25

V The Origin of the Regional Organization of the Mesoderm; Its Dorsoventral and Craniocaudal Polarity 25

A The Formation of the Gray Crescent 25

B The Dorsoventral Polarization of the Endoderm and the Formation of the Dorsoventral and Craniocaudal Polarity of the Mesoderm 27

C The Invagination of the Endoderm and Mesoderm 31

D The Capacity of the Mesoderm for Self-Organization and Histodifferentiation 32

E The Self-Organization of the Entire Embryonic Anlage 33

VI Concluding Remarks 35

References 36

I Justification

Although several aspects of the subject to be reviewed here have been discussed by Holtfreter and Hamburger in their chapter on amphibian embryogenesis in Willier, Weiss, and Hamburger’s book Analysis of Development (1955), so many new data have accumulated in the last two decades that an up-to-date review of the origin, spatial organization, and morphogenetic action of the organization center of the amphibian embryo seems fully justified, the more so since the epigenetic character of its development has become more and more evident.

II The Discovery of the Organization Center, and a Description of its Main Properties

In his classical and highly comprehensive book Experimentelle Beiträge zu einer Theorie der Entwicklung * published in 1936, Spemann mentions the leading role of the dorsal marginal zone of the early embryo for the first time when describing the gastrulation process on p. 63.

Und nun setzen die Massen sich in Bewegung, erst langsam und kaum merklich, dann rascher und mit augenfälligem Erfolg. Es ist als ob von einem Punkte des Keims aus ein Zug ausgeübt würde, dessen Wirkung mit Zunehmen der Entfernung abnimmt, aber doch im ganzen Keim fühlbar bleibt. Das Zentrum, zu dem die Massen hinstreben, liegt median im Dotterfeld, nicht weit vom vegetativen Pol des Eies entfernt; da wo bald darauf die eigentliche Gastrulation mit der Bildung einer kleinen grube beginnt.

The morphogenetic movements apparently do not take the form of a wandering of individual cells but are the expression of a supracellular phenomenon (Vogt, 1923). It is therefore all the more surprising that the experimental analysis has brought to light that the single part processes, as described by Vogt (1929), are able to express themselves likewise in the corresponding, isolated parts of the embryo. This means that the amphibian gastrula is already dynamically determined in its constituent parts (Vogt, 1923). According to Vogt (1922) the most important of these part processes is the stretching of the marginal zone as a whole and particularly of its dorsal region. This led Spemann to the conclusion that the gastrula stage is a mosaic of regions with definite formative tendencies.

Apart from this dynamic determination in the young gastrula there also exists already a material determination.

Die drei Hauptregionen der jungen Gastrula, die präsumptiven Keimblätter, sind schon zu den normalerweise aus ihnen hervorgehenden ektodermalen, mesodermalen und entodermalen Organen bestimmt, letztere beide sogar schon im einzelnen. Das Material für die beiden ektodermalen Organe, Nervenrohr und Epidermis, scheint noch indifferent oder jedenfalls in äuszerst labilem Determinationszustand zu sein. [Spemann, 1936, p. 80.]

Although Spemann apparently first states that this material determination is already quite firm, he later concludes from experiments showing that in the early gastrula mesoderm and perhaps even endoderm can still be replaced by ectoderm, that the material determination in the early gastrula must still be labile.

In the early twenties there follows the sensational discovery of the leading role of the dorsal marginal zone in embryo formation in the classical experiment by Hilde Mangold. She grafted a dorsal blastoporal lip of an early gastrula of Triturus cristatus into the ventral or lateral side of a gastrula of approximately the same age of Triturus taeniatus or alpestris (Spemann and Mangold, 1924). The implant maintained its tendency to invaginate and continued to develop into mesodermal axial organs, i.e., notochord and somites. The neural plate, however, which arose above it, was, for the greater part, furnished by the host (see Fig. 1). Host and graft had formed an harmonious secondary embryonic anlage.

Fig. 1 Neurula of Triturus taeniatus. (a) Dorsal side with primary neural plate; (b) ventral side with secondary neural plate induced by grafted dorsal blastoporal lip of Triturus cristatus. (After Spemann and Mangold, 1924.)

Subsequently, Spemann (1936) analyzes two phenomena in more detail: (1) the capacity of the invaginated dorsal marginal zone to induce a neural plate in the overlying host ectoderm, and (2) the capacity of the implanted mesoderm to form a bilaterally symmetrical axial system.

(1) Bautzmann (1926) tested all regions of the gastrula as to their capacity to induce a neural plate. The regions endowed with this property evidently are the presumptive dorsal and lateral marginal zone, which after invagination will form the archenteron roof. Before invagination the entire presumptive axial mesoderm possesses the capacity to induce a medullary plate in presumptive gastrula ectoderm (see Fig. 2). By exchanging parts of the marginal zone, Bautzmann (1933) found that the presumptive notochordal material is superior to the other regions of the marginal zone both in fixity of determination and in inductive capacity.

Fig. 2 Spatial extension of organization center of the early urodelan gastrula. (a) Dorsal; (b) lateral aspect. (After Bautzmann, 1926.)

(2) Spemann and Mangold (1924) observed for the first time that in their heteroplastic grafts, beside the neural plate also parts of the axial mesoderm consisted of host tissue.

Aus einem Bruchstück des präsumptiven Mesoderms entsteht ein Achsensystem, eine Chorda, von zwei Reihen von Urwirbeln flankiert; es entsteht also ein Ganzes, oft unter Heranziehung eines Teils der neuen Umgebung. Dem musz eine Regulation vorhergehen, bei welcher sich das induzierende Bruchstück entweder rein in sich zur Ganzheit umbildet oder aber sich durch Angliederung der Umgebung erganzt. [Spemann, 1936, p. 94.]

When lateral halves of the dorsal blastoporal lip of gastrulae of different ages were grafted into the region of the lower lip (Mayer, 1935), it became evident that one half of a young blastoporal lip was able in itself to regulate into a bilaterally symmetrical axial system, whereas an older lip, apparently no longer being able to do so, completed itself from the material of the host. This latter process Spemann called assimilatory induction.

Die dort entstandene sekundäre Embryonalanlage enthielt immer ein bilateral-symmetrisches Achsensystem, an dessen Aufbau jedoch das Implantat je nach seinem Alter in verschiedenem Masze beteiligt war. Stammte das Implantat aus der begin-nenden Gastrula, so lag die Chorda nachher in der Mitte des vom Implantat gelieferten Materials, war also auch auf der innenständigen, dem Schnittrand entsprechenden Seite von einer Reihe von Implantat gebildeter Urwirbel flankiert. War das Implantat dagegen einer vorgeschrittenen Gastrula entnommen worden, so nahm die Chorda seinen inneren Rand ein; nur die äuszere Reihe der Urwirbel war vom Implantat gebildet, die der anderen Seite dagegen vom Mesoderm des Wirts. [Spemann, 1936, p. 106.]

In contrast to the mediolateral wholeness of the secondary embryonic analgen, their craniocaudal organization showed pronounced variations. This led Spemann to investigate the regional determination and inductive capacity of the mesoderm. Transplantation of upper blastoporal lips taken from gastrulae of different ages demonstrated that the head inductor is located in the dorsal lip of the early gastrula, the trunk inductor in that of the advanced, and finally the tail inductor in that of the completed gastrula (Spemann, 1931).

On the basis of these arguments, Spemann (1936) concluded that the dorsal marginal zone plays a leading role in embryo formation and called this region the organization center of the embryo. This terminology placed great emphasis on one particular part of the embryo. As a consequence, Spemann’s pioneering work led to a canalization of research toward the period of embryogenesis which starts with the process of gastrulation.

Spemann himself was, however, very well aware of the possible epigenetic origin of the organization center, which is apparent from his words:

Wann dieses Muster entsteht, ob es zurückgeht auf ein entsprechendes Muster in der Blastula oder gar im befruchteten Ei, läszt sich bis jetzt nicht sagen. Aber da es nach Verminderung des Ausgangsmaterials, also nach Stöning, sich wieder herstellen kann, so ist es durchaus wahrscheinlich, dasz es sich auf einem der Gastrula vorangehenden Stadium neu gebildet hat; natürlich nicht aus einem strukturlosen Ausgangsstadium, wohl aber aus einem solchen mit einer andersartigen, einfacheren Struktur. [Spemann, 1936, p. 80.]

However, his work unintentionally drew the attention away from the phases of development preceding gastrulation. The phases of cleavage and blastocoel formation were merely considered as relatively unimportant, though of course indispensable as preparations for the true morphogenetic events of gastrulation and embryo formation. This is clearly reflected in the literature of the next three decades. One of the purposes of this review is to show that several important morphogenetic events happen prior to gastrulation, which are of the utmost significance for embryogenesis.

III The State of Determination and Capacity for Differentiation of the Various Regions of the Early Gastrula

From numerous isolation experiments Holtfreter (1938a,b) has concluded that the early amphibian gastrula can be subdivided into three regions with different states of material determination, viz., the animal cap, the equatorial region, and the vegetative yolk mass regions which largely coincide with the areas of the future three germ layers (Vogt, 1929) (compare Figs. 3a and b with Figs. 3c and d). He states that the amphibian gastrula consists of parts of markedly different material determination and emphasizes that the mesodermal regions endowed with inductive capacities are not those that are the most firmly determined.

Fig. 3a and b Anlage map of the early urodelan gastrula. (a) Lateral; (b) vegetal pole aspect. A.P., animal pole; ep., epidermis; end., endoderm; l.p., lateral plate; n., notochord; n.t., neural tissue; ph., pharynx; s., somites; t.m., tail mesoderm; V.P., vegetal pole. (After , nutritive yolk. (After Holtfreter, 1938a.)

A Determination and Differentiation Tendencies*of the Presumptive Endoderm

†

According to Holtfreter (1938a,b) at the early gastrula stage, the isolated material of the entire presumptive endoderm, representing the various sections of the digestive tract as well as the lungs, liver, pancreas, etc., already show an almost complete material determination as well as regionally specific potencies for characteristic histological differentiation (see Figs. 3c and d).

However, for the following reasons, Holtfreter’s rather extreme conclusion turns out to be at least partially incorrect. In his experiments the isolated peripheral endoderm showed strong regional differentiation tendencies, but was nearly always accompanied by adjacent mesodern. Although he assumed that the mesoderm only served as a neutral substrate, the possibility of regionally specific interactions between mesoderm and endoderm cannot be excluded. This assumption may have given the impression of a much firmer determination for regionally specific differentiation of the endoderm than is actually present at the time of explanation (see below). In 1939, Holtfreter (1939a) stated that endoderm without substrate is not able to differentiate and soon undergoes cytolysis. Stableford (1948), investigating the vegetative hemisphere of Ambystoma blastulae, came to the same conclusion, stating that the endoderm fails to go through any organogenesis in the absence of a mesodermal substrate, except for blastoporal groove formation, while cell division also continues normally.

Balinsky (1948) was the first to demonstrate that even in the neurula, stomach and liver differentiation are still interconvertible under the influence of the corresponding lateral plate mesoderm. In an extensive series of publications, Okada (1953, 1954a,b, 1955a,b, 1957, 1960) showed that a specific mesenchymal substrate is a prerequisite for anterior, middle, as well as posterior endodermal differentiation. The axial mesoderm (notochord and somites) exerts no direct regional influence upon the differentiation of the endoderm. The isolated presumptive anterior endoderm of an early gastrula, in combination with different mesenchymes, always forms pharynx and the posterior endoderm always intestine, but, in addition, both stomach and intestine can be formed by the presumptive anterior endoderm, and stomach and pharynx by the posterior endoderm, provided they are associated with the proper kind of paraxial or head mesenchyme. In vivo mesoderm replacements lead to similar results. The presumptive gastric endoderm can be shifted anteriorly more easily than the presumptive intestinal endoderm, while the cephalic mesectoderm is the most effective for the anterior shift. These conclusions have been confirmed by Takata (1960a,b).

All these experiments lead to the general conclusion that the regional differentiation of the endoderm depends upon two sets of factors, one residing in the mesoderm, particularly the mesenchyme, and the other in the endoderm itself. The regional determination of the endoderm gradually becomes firmer during gastrulation and neurulation. The actual state of determination of the endoderm at the beginning of gastrulation, however, is still largely unkown.

B Determination and Differentiation Tendencies of the Presumptive Mesoderm

*

According to Holtfreter (1938a,b), the presumptive ventral and ventrolateral mesoderm shows pronounced regionally specific differentiation tendencies for blood island, lateral plates, gonads, mesonephros, heart, and limbs (see also Nieuwkoop, 1947). The dorsal and dorsolateral marginal zone also shows pronounced differentiation tendencies, viz., for notochord and somites, but with hardly any regional determination. These regions, moreover, show differentiation tendencies for neural tube and epidermis, which, however, decline toward the blastopore and are absent from the presumptive head mesoderm (see Figs. 3c and d).

In an extensive series of transplantation and recombination experiments, Yamada (1937, 1939a,b,c, 1940) demonstrated the dependence of various mesodermal differentiations upon the presence of the notochord. He showed unequivocally that likewise the normal dorsoventral sequence of differentiation in the mesodermal mantle, i.e., notochord, somites, pronephros, and lateral plate, and blood islands, is built up and maintained by a morphogenetic factor emanating from the notochord and declining with distance. These observations and conclusions were confirmed by Muratori (1939a,b,c).

Although Muchmore (1951) could confirm the main results of Yamada, he concludes that a single morphogenetic factor, as proposed by Yamada, is insufficient to explain the regional differentiation of the entire mesodermal mantle, since it cannot account for the characteristic craniocaudal location of e.g. heart, pronephros, and blood islands. Yamada (1950a) had already extended his theory by postulating the existence of a cephalocaudal factor as well. Regional differentiation of the mesodermal mantle would depend upon a double set of morphogenetic potentials (pdv and pcc). These would also be responsible for the regional differentiation of the overlying nervous system. Muchmore (1951) found in extensive defect experiments that in ovo the notochord is not indispensable for muscle and pronephros differentiation. He concludes that the differentiation of each anlage in the mesodermal mantle depends upon the entire complex of surrounding ecto-, meso-, and endodermal organ anlagen. He postulates the presence of separate but overlapping fields of organ-specific morphogenetic activity in the mesodermal mantle. During neurulation, each field will gradually be stabilized and finally become restricted to the area of the presumptive organ anlage concerned.†

Muchmore (1957) demonstrated the effect of tissue mass upon muscle differentiation and concluded that the factors for muscle differentiation are already present in the early neurula. He states, however, that the self-differentiation of the somites requires a favorable environment. Chuang and Tseng (1956) called attention to the fact that the differentiation of the mesodermal mantle is subject to inducing influences emanating from particular regions of the underlying endoderm, e.g., for the differentiation of blood islands, heart and splanchnic muscle, as well as of the overlying ectoderm, particularly from the neural plate. They conclude that each organ anlage requires its specific morphogenetic stimuli from other parts of the embryo. Takaya (1956) again emphasizes the influence of the notochord upon differentiation and segmentation of the somitic mesoderm. Lanot (1971) infers an early determination of the somitic mesoderm under the influence of both notochord and paraxial mesoderm.

Conclusion

Although local inductive influences from the underlying endodermal and overlying ectoneurodermal germ layers affect the regional differentiation of the mesodermal mantle, its overall regional organization, particularly in dorsoventral direction, seems to be governed by the notochordal anlage.

C Determination and Differentiation Tendencies of the Presumptive Ectoneuroderm

*

According to Holtfreter (1938a), the presumptive ectoneuroderm of the urodelan embryo represents an embryonic region characterized by the absence of any material determination at the beginning of gastrulation (see Figs. 3c and d). The only differentiation tendencies of the presumptive ectoneuroderm of the anuran embryo are those for sucker formation, which is an expression of epidermal differentiation (Holtfreter, 1938b).

In contrast to Holtfreter’s conclusion, Barth (1941) reported that the presumptive neurectoderm of the Ambystoma punctatum gastrula could be shown to possess neural differentiation tendencies in the complete absence of an inductor, if it were allowed to curl upon itself in a favorable, i.e., the natural direction. Holtfreter (1944c, 1945, 1947), however, showed that in explants neural differentiation is due to a cytolyzing effect of the medium to which Ambystoma ectoderm is very susceptible in contrast to that of Triturus, the direction of curling being of no significance. Although he originally attributed the neuralizing effect to influences liberated from the cytolyzed cells, he later inferred a direct, sublethal effect of the external medium upon the subcellular structures of the ectoderm, leading to a release of a neuralizing factor. High and low pH of the medium, as well as, e.g., the absence of Ca ions had pronounced neuralizing effects. According to him, however, neural differentiation of the ectoderm in ovo always depends upon an inductive action from the underlying archenteron roof.

Chuang (1955) used the neuralizing effect of a culture medium without Ca ions in order to analyze the time course of neural competence in Cynops orientalis. In this species, neural competence arises at the mid-blastula stage (24 hours before the beginning of gastrulation), has its maximum during the period between the initial and the sickle-shaped blastopore stage, and terminates at the middle yolk plug stage (see Fig. 4).

Fig. 4 Temporal course of successive competences in the animal, ectodermal half of the urodelan embryo. Mesodermal competence (—); neural competence (—); competence of transformation (—).

By means of the attachment of folds of competent ectoderm to the neurectoderm of gastrulae or neurulae, Niewkoop et al, 1952)) could demonstrate that the regional determination of the neural plate depends upon two successive inductive actions emanating from the underlying substrate; the first or activating (neuralizing) action determines the spatial extension of the neural anlage and subsequently leads to prosencephalic differentiation; the second or transforming action acts upon activated ectoderm and, depending upon its intensity, leads to mes- or rhombencephalic, or spinal cord differentiation. Both inductive actions are predominantly exerted by the mid-dorsal regions of the archenteron roof (see also Leussink, 1970). Sala (1955, 1956) could show that the two inductive actions have a different craniocaudal distribution in the archenteron roof, the activating action having its maximum in the prechordal and anterior chordal mesoderm, and the transforming action in the posterior chordal mesoderm. Hoessels (1957) found that the prechordal endoderm also exerts an activating effect. Toivonen and his co-workers were originally inclined to deny the existence of a separate transforming action and to ascribe the regional organization of the central nervous system to a specific mesoderm-inducing action* along, with a general neuralizing one, as the sole inductive principles acting in primary induction (see Toivonen et al, 1961; Saxén and Toivonen, 1962). Nieukoop et al, 1952)), however, distinguished a separate third inductive action, viz., the transforming action exerted by the differentiating mesoderm. Recent experiments of Toivonen and Saxén (1967), 1968), in which anterior neural plate and trunk mesoderm material were combined in varying proportions, have elegantly confirmed the role of a separate transforming inductive influence of the differentiating mesoderm in the regional organization of the central nervous system. Their experiments also demonstrate that the competence for transformation, which is supposed to arise immediately after activation (Nieuwkoop et al, 1952), declines rapidly around stage 14, the early neural plate stage (see Fig. 4).

Here a few words must be said about the tail mesoderm, which develops from the posterior part of the neural plate (Bijtel, 1931, 1936; Nakamura, 1942). The tail mesoderm acquires its final determination when the neural folds are closing as a result of an inductive action from the posterior part of the archenteron roof (Spofford, 1948). The observation of Chuang (1947) that prior to this final determination the presumptive tail mesoderm, when isolated, differentiates into epidermis and spinal cord, strongly supports Nieuwkoop’s (1966) suggestion that tail mesoderm formation may be regarded as the furthest step in the transformation process.

Conclusion

The presumptive ectoneuroderm of the early gastrula possesses no differentiation tendencies for neural development and only very weak tendencies for epidermal differentiation, both differentiations depending upon the presence of the underlying substrate. In epidermal differentiation the mesoderm, together with the mesectoderm, functions primarily as a necessary substrate, but for neural development, the endomesoderm† constitutes an indispensable inductor system, whose action emanates predominantly from the mid-dorsal region. Regional neural differentiation is accomplished by two successive inductive actions with a different spatial distribution in the archenteron roof.

All these studies demonstrate the leading role of the mesoderm, and particularly of the notochordal anlage, in the spatial organization of the axial system of the embryo.

D Differentiation Tendencies and Inductive Capacity of the Presumptive Prechordal Endomesoderm before and after its Invagination

The endo- and mesodermal portion of the archenteron roof situated in front of the notochordal anlage plays an important role in the spatial organization of the embryo, in that it is responsible for the formation of the head.

Okada and Takaya (1942a,b), Okada and Hama (1943, 1944, 1945), and Hama (1949) demonstrated that the isolated uninvaginated dorsal blastoporal lip of a young gastrula of Triturus (representing presumptive prechordal endomesoderm) forms trunk-mesodermal structures and induces corresponding sections of the nervous system in competent ectoderm, whereas the same material after invagination differentiates into head mesenchyme and foregut endoderm and induces forebrain structures. (The foregut structures comprise the pharynx, the gill pouches, and sometimes also stomach and liver.) In contrast, the presumptive notochordal region of the archenteron roof has nearly the same differentiation and inductive capacity before and after the invagination. The above-mentioned change in differentiation tendencies and inductive capacity of the presumptive prechordal endomesoderm can also be affected when the material, before bringing it into contact with competent ectoderm, is first cultured in Holtfreter solution for 10 hours at 21°C, a period during which the material would have invaginated if left in situ. This was also achieved when the uninvaginated material was combined with noncompetent neurula ectoderm (Kato and Okada, 1956). If brought directly into contact with neural plate it again formed trunk axial structures. Masui (1960a) showed that preculture in Li solution prevented the change in differentiation tendencies and inductive power.

This phenomenon was further studied in detail by Takaya (1953a,b), Kato and Okada (1956), Hoessels (1957), Kato (1958, 1959, 1963a,b) and Masui (1960a). The highest notochord-forming capacity is found in the dorsal blastoporal lip at the beginning of gastrulation (the presumptive prechordal endomesoderm) and shifts caudally toward the presumptive anterior notochordal portion of the archenteron roof during invagination. This means that the presumptive prechordal endomesoderm gradually loses its notochord-forming capacity. Hoessels (1957) could show that its transforming inductive action is likewise gradually lost, leaving only its activating action to express itself in the induction of forebrain structures. Slightly later the activating action also decreases markedly.

Conclusion

A drastic change in differentiation tendencies and inductive capacity evidently occurs in the presumptive prechordal endomesoderm during invagination and subsequent contact with the neurectoderm, its initial, purely chordomesodermal differentiation tendencies partly changing into those for head mesenchyme and partly into those for pharyngeal endoderm, and its inductive capacity changing simultaneously from that for rhombencephalon and spinal cord into that for forebrain structures.

IV The Induction of the Mesoderm

A Endomesodermal Differentiation of Gastrula Ectoderm under the Influence of Heterogeneous Inductors

When studying the regional inductive capacity of various adult tissues as expressed in their action upon early gastrula ectoderm (see Toivonen, 1940, 1949, 1950), Toivonen (1953) found that in contrast to a so-called spinocaudal inductor, such as guinea pig kidney, which leads to the formation of spinal cord accompanied by caudal axial mesoderm, bone marrow of the guinea pig almost exclusively induces mesodermal structures, such as notochord, somites, pronephros, limb rudiments, and mesenchyme. Takata and Yamada (1960) observed that bone marrow induced endodermal as well as mesodermal structures in early gastrula ectoderm. The endodermal structures (comprising pharynx, esophagus, lung, stomach, and intestine) differentiated after long cultivation only. Katoh (1962) showed that a purified protein extract prepared from guinea pig bone marrow induced axial mesoderm in explants of gastrula ectoderm. Kocher-Becker et al, 1965)) achieved an extensive mesodermization of the ectodermal portion of an early gastrula by injection into the blastocoelic cavity of a highly purified mesoderm-inducing factor isolated from chick embryos, the mesodermization leading to pronounced exogastrulation. Recently Kocher-Becker and Tiedemann (1971) reported that the same factor induces mesodermal as well as endodermal structures in isolated Triturus gastrula ectoderm. Among the mesodermal structures of the 3- to 7-week-old explants, a considerable number of primordial germ cells were found,* while the endodermal structures were represented in most cases by intestine, and less frequently by liver, pancreas, stomach, esophagus, and pharynx.

Leikola (1963) studied the autonomous loss of mesodermal and neural competence in isolated Triturus gastrula ectoderm by culturing it in Holtfreter solution for varying lengths of time before bringing it into contact with bone marrow or kidney tissue. He could show that there are two different periods of competence, a first one for mesodermal induction, which is lost earlier than the second one for neural induction (see Fig. 4). Leikola also showed (1965) that the loss of competence in isolated ectoderm cultured in vitro and in ectoderm aged in ovo follows the same pattern, but that the changes occur more rapidly in ovo. The fact that the loss of mesodermal differentiation always coincided with a pronounced increase in neural differentiation shows that the two competences are partially antagonistic. We shall return to this aspect later (see Sections IV,E and V,E).

When studying the effect of Li ions upon neural induction, Masui (1956) confirmed the original observations of Lehmann (1937) concerning the notochord-suppressing action of the Li ion. Further experiments showed that the Li ion exerts a caudalizing (transforming) effect upon the neural induction evoked by anterior archenteron roof (Masui, 1959) as well as upon isolated neuralized ectoderm itself (Masui, 1960c). He subsequently (1960b) found that gastrula ectoderm treated with Li partially undergoes mesodermal differentiation. He later observed (1961) that early gastrula ectoderm of Triturus, when exposed to Li formed both meso- and endodermal structures along with regionally corresponding neural structures. Ogi (1961) for the first time called this shift from ectodermal to meso- and endodermal differentiation tendencies in amphibian embryos vegetalization. Gebhardt and Nieuwkoop (1964) could show that blastula ectoderm is even more sensitive to Li than gastrula ectoderm. The formation of meso- and endodermal structures is probably the result of sublethal cytolysis of the exposed cells similar to the sublethal neuralizing effect of high or low pH found by Holtfreter (see Section III, C). Masui (1961, 1966) found that pronounced changes in pH and Ca or Mg ions sensitize the ectoderm to the meso- and endodermizing action of Li ions. However, the Li ion seems to exert an opposite, inhibitory, effect upon the neuralizing action of acidic solutions (Okano and Kawakami, 1959).

Conclusion

Craniocaudally arranged mesodermal structures of axial character accompanied by regionally corresponding endodermal structures can be induced in blastula and early gastrula ectoderm by quite different means, such as the heterogeneous inductor bone marrow or the Li ion.

B Additional Mesoderm Formation in Centrifuged Embryos

Pasteels (1947a,b) observed that centrifugation of amphibian blastulae and gastrulae at 460 g resulted in the production of additional complex ectoneurodermal and mesodermal formations in the ectoneurodermal region of the embryo. These complexes could have the character of very abnormal, disharmonic secondary embryonic anlagen. Their frequency, composition, and location varied greatly depending upon the stage of centrifugation. According to Pasteels they are due to cellular trauma as a result of the intracellular stratification of cell inclusions. The traumatic effect is followed by a local thickening of the blastocoelic roof and subsequent strong increase in RNA content of the affected area. He explains these meso- and ectoneurodermal formations by postulating an increase in morphogenetic potential according to Dalcq and Pasteels’ (1937, 1938) general morphogenetic theory.

Pasteels (1953a,b, 1954) observed that centrifugation always leads to a collapse of the blastocoelic cavity. Upon centrifugation at a nonsensitive stage, the blastocoel is reestablished, but at sensitive stages the collapsed blastocoelic roof forms a compact cell mass and morphogenesis sets in. From experiments involving explantation of the blastocoelic roof directly after centrifugation, Pasteels infers a direct sensitization of the ectodermal cells due to cellular trauma. He states, however, that complexes formed in explants usually remain amorphous, and that the regional organization occurring in situ apparently depends upon directing influences from the primary embryo. In a systematical study (Pasteels, 1953b), he described the various syndromes that occurred. Apart from the disharmonic embryonic axes already mentioned, extracerebral vesicles or additional sense organs may develop, while intracerebral notochordal nodules are also observed. The extraneural or placodal structures, according to Pasteels, are due to a subliminal sensitization of the ectoderm, resulting in an enhanced effect of the normal inductive action of the archenteron roof. Finally, different anuran and urodelan species show differences in susceptibility to the treatment and different sensitive phases for the various syndromes.

Karasaki and Yamada (1955) confirmed Pasteels’ observations on Rana and Bufo, but found that the isolated blastocoelic roof centrifuged by itself did not form any mesodermal or neural structures, while sublethal cytolysis of the isolated ectoderm evoked by ammonia yielded only prosencephalic or unorganized neural formations.

Conclusion

Although Pasteels’ experiments furnish good evidence that centrifugation causes general sensitization of the ectoderm due to cellular trauma, specific inductive influences from the rest of the embryo seem to be responsible for the extramesodermal and neural differentiations formed. We will return to these findings in Section IV,D.

C The Epigenetic Development of the Marginal Zone of the Amphibian Egg

Nakamura and Matsuzawa (1967) isolated dorsal, lateral, and ventral portions of the presumptive marginal zone of Triturus eggs at successive stages of development and reared them as explants wrapped in neurula ectoderm. Whereas the explants taken from stage 9 (Okada and Ichikawa, 1947), which corresponds to about stage 8 (Harrison, 1969), differentiated in a high percentage of cases into meso- and ectoneurodermal structures, those taken from (O & I) stage 7 (= H stage 6) did not differentiate and only formed an amorphous mass of yolk-laden cells. Explants taken from (O & I) stage 8 (= H stage 7) behaved much like those of stage 9, but their capacity for differentiation was somewhat lower. Explants taken from the different regions of the