PERGAMON SCIENCE SERIES

Editors for the Biological Sciences :

J. E. HARRIS and E. W. YEMM

AN OUTLINE OF
DEVELOPMENTAL PHYSIOLOGY

AN OUTLINE OF l^^^

DEVELOPMENTAL PHYSIOLOGY

by

Chr. p. raven

professor of zoology in the university
of utrecht

Translated by
L. DE RUITER, bioL drs.

NEW YORK: Mc GRAW-HILL BOOK CO., INC.
LONDON: PERGAMON PRESS LTD.

1954

First published in the Dutch language 1948
First English edition 1954

Published in Great Britain by Perganion Press Ltd.
Maxwell House, Marylebone Road, London N. W. I.

Published in U.S.A. by the Me Graw-Hill Book Co.. Inc..
330 West 42nd Street. New Yor/? 36, N.Y.

Printed in the Netherlands by J. Noorduijn en Zoon N.V.

CONTENTS

page

PREFACE VII

I INTRODUCTION 1
II THE INITIATION OF DEVELOPMENT: THE

FERTILISATION OF THE EGG 7

III THE STRUCTURE OF THE FERTILISED EGG 27

IV POLARITY AND SYMMETRY; GRADIENT-

FIELDS 39

V CHEMODIFFERENTIATION 50
VI THE REALISATION OF THE NUCLEAR

FACTORS 64

Vn THE TOPOGENESIS OF THE EMBRYO 91

VIII INDUCTION AND ORGANISATION :

I. NEURULATION 106

IX INDUCTION AND ORGANISATION:

II. THE PERIOD OF ORGAN DEVELOPMENT 128

X THE LATER STAGES OF DEVELOPMENT 151

XI REGENERATION 160

Xn SOME FINAL CONSIDERATIONS 182

Preface

This little book was written in 1942 but, as a consequence of
the war and its aftermath, the Dutch edition did not appear
until 1948. It was meant in the first place for readers who,
though interested in its subject and having some general know-
ledge of science, were not acquainted with more than the first
elements of biology. This determined the design of the whole
book. Highly technical digressions had to be avoided, and, as
far as possible, the construction of a well-rounded picture of
the phenomena of development had to be attempted. It will
be clear that this made it impossible sometimes to make a
sharp distinction between well-founded facts, and the more or
less hypothetical trains of thought which link them together.
However, all that was possible has been done to steer clear of
the major obstacle to all popularization of science: the danger
of telling half truths. As for relating the results of investiga-
tions, the author has tried to apply the same strict standards
as are used in a purely scientific publication. In one respect
only he has made a concession to the character of the work:
he is aware that the way in which the scientific terminology
has been used in this book will not satisfy rigorous norms.
This could not be remedied without confusing the reader by
introducing a still greater number of technical terms.

In the preparation of the English edition, no essential change
has been made in this design or its realization. An attempt
has been made, however, to bring the book up to date by
taking into account the literature up to about the middle of
1952. This is not a textbook, but a list of references has been
added in the interest of those who might want to refer to the
original publications on some special problem. This list, too,
has been brought up to date in the English edition. At the end
of the book, a glossary of scientific terms has been included
for the benefit of those readers who are not professional
biologists.

VII

The author wishes to acknowledge his gratitude to Mr. L. de
Ruiter, Groningen, who undertook the task of translating the
book and preparing the manuscript of the English edition. He
also sincerely thanks Prof. J. E. Harris, of Bristol University,
for reading the manuscript and for helpful suggestions and
constructive criticisms.

Chr. P. R.

Translator's Note

I am greatly indebted to Prof. J. E. Harris, who carefully
read the whole manuscript of the translation, and suggested
many improvements in style and choice of terms.

L. de R.

CHAPTER I

Introduction

"Developmental physiology" is the term that will be applied
here to the branch of biology that studies the causal relation-
ships in animal development. About 1880, Wilhelm Roux
founded this branch of science on the strength of theoretical
considerations and a small number of specially designed expe-
riments. He called it "developmental mechanics". This name,
however, might well give rise to misconceptions as to its
nature. Roux applied the term "mechanics" in the wide sense of
"theory of causal connections" but its use may easily lead to the
idea that an attempt is being made to explain the developmental
phenomena on the basis of the laws of mechanics. For that
reason we prefer the name "developmental physiology", which
has the same scope. This means that the word "physiology" is
used here in its widest sense, viz. that of "causal science of
living organisms", so that it does not imply that we shall study
only those processes during development that are normally
included in "physiology" in its narrower sense, such as meta-
bolism, excitability, etc. The term "development" is meant to
mclude all irreversible changes that the organism goes through
m the course of its existence, from the moment of its origin
until the death of the individual.

In broad outline, the development of all multicellular animals
proceeds along the same route. We shall give a short account
of this now, taking the fertilised egg as the starting point of
development. This arises from the fusion of a male and a female
germ cell (called sperm and egg respectively). (For the present
we shall disregard special cases such as that of vegetative
reproduction, in which the new individual originates from a cell
or cell group that separates itself from the body of an animal).
The first developmental process is a division of the fertilised

Raven - Outline Physiologie .

2 INTRODUCTION

egg into cells. This so-called cleavage results in the formation
of a small lump of more or less uniform cells, the blast omeres.
A cavity develops in the centre of this cell lump, and the
vesicular germ is then called a blastula. In the period following
cleavage the germ goes through important changes, consisting
of a series of movements of cell groups. The blastomeres are
thereby arranged in several layers which in their turn will
divide into more or less well defined masses of cells. Together
these processes may be called the topogenesis of the embryo;
they serve to divide the germ into a number of organ primordia,
each of which contains the material for one definite organ or
group of organs of the embryo. At first, these primordia consist
of cells which are all very similar, and of an almost undifferen-
tiated, embryonic character. This changes during the next phase
of development, when each cell of the primordia specializes in
a particular direction; in this way the various tissues of the
body are formed. This is called the period of tissue differentia-
tion (histogenesis) . Once development has proceeded so far, the
organs and tissues of the embryo begin to take up their re-
spective functions. Development has by no means ended yet,
but in the subsequent period the functions of the organs play
a decisive role in their differentiation. This is therefore called
the functional stage of development. Finally, two categories of
change which occur at a very late stage may be mentioned, viz.
those which lead to full maturity of the organism, and those
of senility. The latter are mainly of a disintegrative character,
and eventually result in the death of the individual. A further
category that must be specially mentioned is formed by de-
velopmental processes that do not manifest themselves during
the normal, undisturbed life of the individual, but may lead to a
more or less complete regeneration, a restitution of the struc-
ture of the organism when this has been damaged.

When we survey the outline of the course of development
just given, its most striking characteristic appears to be an
increase in spatial multiplicity. This is seen most clearly if we
compare the culminating point of development, the adult in-
dividual, with its starting point, the fertilised egg. On the one
hand we have a very complicated whole of organs, tissues and

INTRODUCTION 3

cells. Each of these has its own place in the organism, and its
own characteristic, orderly structure. Together they form an
integrated system of a very high degree of multiplicity. On the
other hand we see a small lump of protoplasm with a nucleus,
very simple in shape, and practically without a visible structure.
Clearly the "spatial multiplicity" of the organism has increased
greatly during its development. Yet this increase might be
apparent only, for the structure of the egg, though invisible
and therefore seemingly simple, could in reality be very com-
plicated. In past centuries, it has indeed been assumed that the
egg contained the future organism in its full complexity, al-
though not in an easily visible form. This is the theory of
Preformation (Evolutio). Later this concept was modified as
follows. It was assumed that the whole organism itself was not
present in the egg, but that each part of the egg was predestined
to form one definite part of the embryo, and that each part
contained the original factors for this process. This implied
that the multiplicity of the future embryo was, in a different
form, present in the egg already (W. Roux's theory of Neo-
Evolutio). We shall see in Chapter III that experimental in-
vestigations have shown that this view cannot be maintained.
It has been demonstrated that the egg has really a very simple
spatial structure of a low degree of multiplicity, and hence
that development involves an increase in orderly spatial multi-
plicity.

The word spatial must be stressed here, for there can be no
doubt that the protoplasm of the egg is a highly complicated
mixture of substances. From that point of view it has a great
multiplicity already. This, however, is not a spatial multiplicity
for the various components are not restricted to fixed places
within the system but are distributed more or less homo-
geneously over the egg instead. We may call this a non-spatial
or intensive multiplicity, as opposed to spatial or extensive
multiplicity in which the various parts are arranged side by
side in space ^). In this terminology, development may be

1) H. Driesch has used these terms in another sense. He attributes a
metaphysical meaning to the concept of "intensive multiplicity". It should be
stressed that this is not implied in the present use of the word.

4 INTRODUCTION

characterised as a transition from intensive to extensive
multiplicity.

At this point we can briefly indicate how we can imagine
such a development, in which multiplicity changes from in-
tensive to extensive, to take place. This will at the same time
illustrate the line of thought underlying this book. Before we
start, however, two points must be made clear. First, it must
be pointed out that development follows very different lines
in different animal groups. It is only in broad outline that all
these different modes of development are governed by the same
laws. Only these general features can be sketched here, and
naturally we shall often have to resort to broad generalization
in so doing. In the second place, it must be remembered that
developmental physiology is a young branch of biology, and
that it has by no means yet constructed a generally accepted
and balanced system of ideas. Therefore, the best attempt at
a well rounded picture of development we can now make will
still be of a very subjective and preliminary nature, and will
be open to many future changes and improvements. The need
for generalization entails the danger of partiality. Moreover,
it will be necessary now and then to draw conclusions from
factual data that are at present insufficient, and to bridge gaps
in our knowledge by means of hypothetical constructions. We
can base such opinions only upon the facts that have so far
become known to science. Therefore, the reader must take care
not to regard the picture of animal development given in this
book as definitive and irrefutable. We have only tried to place
the results of the several experiments in such a context that
a wider circle of readers may appreciate the importance of
the phenomena discovered.

The fusion of sperm and egg starts a number of processes
which inaugurate development (Ch. II). The structure of the
fertilised egg is still very simple; the various components of
the cytoplasm are more or less evenly distributed so that all
parts of the egg are still approximately equivalent (Ch. III).
Yet the main axes of the egg are already fixed; it has a
polarity, and often a bilateral symmetry. This polarity and

INTRODUCTION 0

symmetry are probably localized partly in the more solid outer
layer, or cortex of the egg, often in the form of gradient-fields
(Ch. IV). Next, certain components of the egg cytoplasm (so-
called determining substances) begin to accumulate locally
under the influence of these cortical factors. This leads to the
onset of heterogeneity in the egg, for now its parts begin to
vary in chemical composition. This process is called the chemo-
differentiation of the egg (Ch. V). The variation in chemical
composition within the egg influences other properties of the
protoplasm as well, such as permeability, metabolism, etc.
Moreover, the determining substances begin to interact with
one another. In this way the spatial multiplicity of the egg-
system increases rapidly. Now the genes, the carriers of the
hereditary properties which are localized in the nucleus, begin
to intervene. In the course of cleavage, the zygote nucleus
which arose from the fusion of the nuclei of egg and sperm
has divided into a great number of segmentation nuclei. These
come to be situated in parts of the cytoplasm which vary in
physicochemical composition as a consequence of chemodiffer-
entiation. Consequently in each segmentation nucleus certain
genes are "activated", whereas others remain inactive. The
activated genes begin to interact with the cytoplasm surround-
ing them. Part of the substances produced by this interaction
remains in the cells, and determines their further development.
Another part, however, diffuses from the cells, and exerts its
influence in other parts of the body. These latter substances
have been called gene hormones (Ch. VI). Under the influence
of the ever-increasing chemodifferentiation, divergent speciali-
zation takes place in the motility of the protoplasm of the
various cell groups. The different parts of the blastula vary in
this respect, and consequently a system of shifts of cell groups
occurs, which we have already named the topogenesis of the
embryo (Ch. VII). This in the first place moves the material
for the future organ primordia to the appropriate places in the
embryo. Secondly, however, it establishes direct contact between
cell groups of different physico-chemical composition which
were spatially separated at first. These cell groups thereby
influence each other (induction), and this interaction results

6 INTRODUCTION

in a rapid increase in the chemodifferentiation of the embryo.
Here a leading role may sometimes be played by particular
cell groups (organisers), distinguished perhaps by a higher
content of certain determining substances (Ch. VIII). In the
following period, a complicated system of topogenetic and in-
ductive processes transforms the embryo into a mosaic of
organ primordia. Each of these has different physicochemical
properties, and consequently different developmental potencies.
This stage is succeeded by the period of tissue differentia-
tion, during which the cells of the primordia develop into
definite cell- and tissue structures, as determined by their
previous chemodifferentiation (Ch. IX). Later the structure of
organs and tissues may be further perfected under the influence
of their functions (functional adaptation) (Ch. X). Finally,
some sort of an equilibrium is achieved. From then on, the
importance of developmental processes is very small only. In
many animals, however, a disturbance of this equilibrium by
the removal of part of the body leads to renewed developmental
processes which result in a more or less complete regeneration
of the lost part (Ch. XI).

CHAPTER II

The initiation of development:
The fertilisation of the egg

In sexual reproduction, which occurs in all multicellular
animals, two types of germ cells or gametes are produced by
the adult individuals. In some cases both types are produced
by the same individual, in other cases they are formed by
different animals. The two types are female germ cells, or eggs,
and male germ cells, or sperms (spermatozoa). Each egg fuses
with a sperm; this is the process of fertilisation. The fertilised
egg, or zygote, develops into a new individual.

As a rule, the egg is a very large cell, consisting of protoplasm
with a large nucleus, commonly called a germinal vesicle. In
the protoplasm, a great quantity of food, in the form of protein
and fat globules, is often accumulated. This is the so-called
yolk, which constitutes a store, given to the young individual
as it sets out upon its path of life. Its supplies the energy for
the first stages of development, during which the embryo can-
not yet take up food from its environment. The eggs originate
from small cells, the so-called oogonia, in the ovary of the
maternal animal. They swell strongly in the course of the
accumulation of the yolk substances, and grow into immature
eggs, or oocytes.

In contrast with the eggs, the sperms are very small, usually
thousands of times smaller than the egg; they do not contain
any yolk. Apart from slight changes in shape, eggs as a rule are
immobile ; sperms, on the other hand, have the power of locomo-
tion. This enables them to move actively towards the eggs,
and to penetrate into them, which results in fertilisation. The
shape of the sperms varies rather considerably among the differ-

8 THE INITIATION OF DEVELOPMENT.

exit groups of animals, but usually we can distinguish a head,
representing the nucleus of the cell, a middle piece, and a long,
motile tail. The undulating movements of the latter propel
the sperm.

In all organisms, the nucleus of each cell contains a number
of small bodies with a high affinity for stains, the chromo-
somes. They become visible each time the nucleus divides.
Within each species, their number is constant. We shall see later
that they are very important for the life of the organism
(Ch. VI). During fertilisation the nuclei of egg and sperm fuse
(amphimixis), so that a double number of chromosomes is
present in the nucleus of the fertilised egg, and likewise in that
of all the cells of the new individual, which arise from it by
cell division. This would lead to doubling of the chromosome
number in each successive sexually reproduced generation, but for
the fact that during the formation of the germ cells the number
is each time reduced again to the single value. This takes place
in the so-called reduction division, actually two divisions in
quick succession in the course of which the double (diploid)
set of chromosomes is reduced to a single (haploid) set. In the
formation of sperms, these reduction divisions take place in
the male gonad, or testis, one diploid spermatocyte giving rise
to four haploid spermatids which then change into spermatozoa.
In eggs, the reduction process takes a slightly different course.
Here it is known as maturation, and proceeds as follows. The
nucleus of the oocyte moves towards the surface; its nuclear
membrane disappears, and a mitotic spindle develops. Mean-
while each chromosome has duplicated itself, and the double
chromosomes now arrange themselves in pairs in the centre
of the spindle (Fig. la). The two members of each pair then
move apart, so that two equal groups of double chromosomes
are formed. One of these groups, together with a small quantity
of protoplasm, is expelled from the egg, and forms the first
polar body (Fig. lb). Immediately afterwards, a new spindle
forms in the egg, and the remaining double chromosomes
(dyads) arrange themselves in the centre of this spindle. The
two halves of each dyad separate (Fig. Ic). One group is ex-
pelled again, forming the second polar body. From the remain-

THE FERTILISATION OF THE EGG 9

ing chromosomes a new nucleus is formed, the so-called female
pronucleus (Fig. Id).

x^

\

Fig. 1. Maturation divisions in the egg of a starfish, (o) first

maturation division; (b) extrusion of the first polar body; (c)

second maturation division; (d) mature egg, (below: pronucleus,

at top: first and second polar body). After Buchner.

This maturation of the egg may occur at different times.
Sometimes both maturation divisions take place within the
ovary or in the genital ducts of the maternal animal. In other
cases, maturation begins in the ovary, but comes to a stop at a
certain stage, and is not completed until after fertilisation.
Finally, in many species the female lays unripe oocytes, and
the whole process of maturation takes place outside the mater-
nal body. In a number of instances of this last type, the nature
of the stimulus which causes the onset of maturation has been
investigated. Sometimes the maturation divisions do not begin
until after the sperm has penetrated into the egg. In other
cases, maturation occurs a few minutes after the oocytes have
been laid in sea water. It has been found that the calcium ions

10 THE INITIATION OF DEVELOPMENT:

present in sea water play an important role (Dalcq, Pasteels,
Heilbrunn). These ions, possibly in combination with potassium
ions, appear to provide the specific stimulus which causes the
membrane of the germinal vesicle to disappear, and which
thereby initiates maturation.

In normal development, the polar bodies are always formed
at one pole of the egg, the so-called animal pole (p. 39). At
the beginning of maturation, the germinal vesicle, or the
maturation spindle arising from it, moves towards this pole;
and the spindle there orients itself at right angles to the egg
surface (Fig. la). Apparently this part of the surface attracts
the spindle. In several cases it has been possible to weaken this
attraction by a special treatment of the egg, e.g. by means of an
excess of calcium ions in the medium in starfishes and in some
molluscs, or by means of a rise of temperature in sea urchins.
Dalcq (1924) has called this depolarisation. As a result the
maturation spindle does not come close enough to the egg surface,
and consequently abnormally large polar bodies are formed.
In the case of an even stronger depolarisation the. maturation
spindle does not even orient itself at right angles to the surface,
and the polar bodies fail to appear altogether.

The maturation of the egg has still another consequence.
Once the nuclear membrane has dissolved, the nucleoplasm
contained in the germinal vesicle mixes with the cytoplasm.
This causes a change in several of the physical and physiolo-
gical properties of the cytoplasm (e.g., its viscosity, and
permeability). Moreover, it appears that many eggs cannot be
fertilised until this has happened. No sperms can penetrate into
immature oocytes, or, if they can, they remain inactive in the
the egg cytoplasm for the time being, and do not "awake" until
maturation has begun. Costello (1940), working with fragment-
ed oocytes of the polychaete worm Nereis, showed that only
those parts that contained the germinal vesicle could be success-
fully fertilised. In other animals, e.g. starfishes and sea urchins,
it is equally impossible to fertilise non-nucleated egg fragments,
if the germinal vesicle was intact at the moment of fragmenta-
tion. However, if maturation had already set in at that time,
non-nucleated fragments can be successfully fertilised, and will

THE FERTILISATION OF THE EGG 11

go on developing (so-called merogony). This was found by
Delage as early as 1899. All this shows that the mixing of
nuclear sap and cytoplasm at the beginning of maturation is
of great importance for fertilisation.

In the fusion of e^g and sperm, substances secreted by each
of these cells play a major role (Hartmann, Schartau and
Wallenfels, 1940). In sea urchins, both gametes have been
shown to secrete two types of fertilisation substances, or
gamones. Gynogamone /, secreted by the eggs, accelerates the
movements of the sperms, and perhaps also attracts them so
that they reach the eggs more rapidly. Once the sperms have
reached the egg, they are "agglutinated" (made sticky) by
gynogamone II. This substance also paralyzes sperms of other
species, and thereby reduces the danger of hybrid fertilisation.
The sperms produce androgamone /, which slows down their
movements so that they are immobile in the male genital ducts,
and do not exhaust their energy too early. They also produce
androgamone II which dissolves the envelopes of the egg, and
counteracts the agglutinating action of gynogamone II, there-
by enabling the sperms to penetrate quickly into the egg. In
this way, the gamones, which are probably of common occur-
rence, ensure the normal course of the fertilisation process.
Investigations by Tyler (1948) have demonstrated that the
reaction between male and female gamones has a great similar-
ity to immunological reactions. In American literature both
gynogamones are grouped together under the name of ''ferti-
lizing, and both androgamones under the name of "anti-
fertilizin".

The situation in mammals deserves special mention. When
leaving the ovary, the eggs of this group are surrounded by a
layer of cells, the cumulus oophorus. These originate from the
follicle. They are kept together by a viscous substance, probably
consisting mainly of hyaluronic acid, or a related polysaccha-
ride. Now it has been proved that the sperms contain an enzyme,
hyaluronidase, which breaks down hyaluronic acid, and thereby
causes the disintegration of the cumulus oophorus so that the
sperms gain free access to the egg surface.

After the sperm has pierced the envelopes of the egg, and

12

THE INITIATION OF DEVELOPMENT;

reached its surface, it penetrates into the egg. In many species,
the egg forms a small surface projection in the region towards
which the sperm is making its way, and the latter is taken
up by this so-called fertilisation cone, seen in sea urchin eggs
by Fol as early as 1876 (Fig. 2). Therefore, the egg is by no
means entirely passive during fusion. According to Chambers
(1930), the egg of the starfish even emits fine filaments of

Fig. 2. Fertilisation of the egg of a starfish, (a) the egg in its en-
velopes, surrounded by spermatozoa; ib-e) a sperm pierces the
egg envelope and penetrates into the "fertilisation cone". The initial
stages of the formation of a perivitelline cavity between egg and
vitelline membrane. After Fol and Wilson.

protoplasm, one of which establishes contact with the sperm,
and, contracting rapidly, draws the latter inwards. The activity
of the outer layer of the protoplasm may also be responsible
for the fact that fragments of unfertilised eggs can only be
fertilised if at least part of this "ectoplasm" is intact.

The penetration of the sperm causes the egg to "awake" at
once. Within a few seconds, it reacts with an instantaneous
change of properties. Usually the formation of a fertilisation
membrane is the first visible effect. The unfertilised egg is
surrounded by a vitelline membrane, which is sometimes fairly
thick, sometimes however barely visible. Immediately after the
penetration of the sperm, this membrane begins to lift from
the egg surface (Fig. 2d-e, 3), because substances expelled by

THE FERTILISATION OF THE EGG

13

the egg accumulate in the space between egg surface and
membrane, the so-called perivitelline space. These substances,
part of which are colloids, take up water from the environment
by osmosis. Consequently, the perivitelline space increases

Fig. 3. Formation of the fertilisation membrane in the sea urchin
Strongylocentrotus purpuratus (the jelly layer around the egg has
been omitted), (a) sperm in contact with the egg, about 2 seconds
after insemination; (b-e) 30-50 seconds after insemination, formation
and coalescence of blebs at the egg surface; (/) 2-3 minutes after
insemination, the fertilisation membrane has been formed. After Chase.

rapidly. The extrusion of these substances often results in a
considerable decrease in size of the egg. A few minutes after
its elevation, the fertilisation membrane undergoes a process of
consolidation, after which it resists further stretching. In sea
urchins, this consolidation occurs only in the presence of calcium
ions, or other bivalent cations.

Usually the formation of the fertilisation membrane does not
set in simultaneously in all parts of the egg surface, but starts
from the spot where the sperm has penetrated (Fig. 3c), and
proceeds from there in all directions over the egg. Obviously,
the extrusion of the perivitelline substances, which causes the
elevation of the fertilisation membrane, must be due to a sudden
change in the outer layer of the egg, its so-called cortex. In
sea urchins, this cortical reaction is even visible, and has been

14 THE INITIATION OF DEVELOPMENT:

described by Moser (1939). Starting from the point where the
sperm has entered, certain granules which are present in the
cortex of the unfertilised egg, vanish suddenly. This process
spreads over the egg surface in a wave-like progression, and
reaches the opposite pole within ten seconds. This reaction, too,
depends upon the presence of calcium ions; it fails to appear
in a calcium free medium. Runnstrom and collaborators (1946)
have shown that the granules which are expelled in the cortical
reaction, coalesce with the inside of the vitelline membrane.
Chemically, they consist largely of polysaccharides. They react
with the proteins of the vitelline membrane, thereby causing
a structural change which probably involves both interlinking
by side chains, and stretching of the protein molecules. This gives
the fertilisation membrane its very tough, horny consistency.

In the marine polychaete worm Nereis, too, cortical granules
are extruded during fertilisation (Costello, 1949). Here, how-
ever, they coalesce into a substance, the greater part of which
passes through the vitelline membrane, and forms a thick
gelatinous mantle on the outside of the latter. This jelly is
again a polysaccharide.

Apart from bringing about the formation of the fertilisation
membrane, the cortical reaction is very important in another
respect as well. In the great majority of animals, normally
only one sperm finds its way into the egg; penetration of more
than one sperm into the egg, so-called polyspermy, causes ab-
normal development. In a few groups only, e.g. in birds, we
find physiological polyspermy, i.e. penetration of several sperms
into one egg, is normal and does not lead to disturbances in
development.

Where monospermy is normal, there must be a mechanism
that prevents the penetration of more than one sperm. Now it
has been shown that the cortical reaction which follows the
penetration of the first sperm, causes changes in the egg
surface which prevent the entrance of any further sperms.
Just, for instance, has demonstrated that in the sea urchin
Echinarachnius a "wave of negativity" spreads over the egg
surface from the spot where the sperm has entered, and that it
is impossible for other sperms to enter at any place which the

THE FERTILISATION OF THE EGG 15

wave has passed. In completely normal eggs, this happens so
rapidly that the egg cortex becomes impenetrable practically
instantaneously. In eggs under unfavourable conditions, how-
ever, the process is retarded, and its advance can be followed.
In the frog's egg, e.g., such a retardation of the cortical reaction
can be produced by treatment with a sodium chloride solution
(Bataillon, 1919). It increases the chance that more than one
sperm will penetrate, and may easily give rise to polyspermy.

We have seen above (p. 11) that one of the substances
secreted by the sea urchin egg paralyses sperms of other
species. This may be regarded as a barrier against hybrid
fertilisation, but it does not completely prevent it. Closely
related species can often be crossed easily. By means of such
artifices as high sperm concentrations, and modification of the
acidity of the medium, it is possible to combine eggs and
sperm of species which are very far apart taxonomically. In
this way, sea urchins' eggs have been successfully fertilised,
even with sperm of worms or molluscs (Kupelwieser, God-
lewski). We shall later return to these experiments.

The sperm penetrates head first into the egg, but soon after-
wards it rotates through 180°, so that its head points towards
the egg surface, and its middle piece towards the centre. In
those cases where the tail of the sperm has entered into the
egg as well, this now drops off and dissolves in the egg cyto-
plasm. The head of the sperm swells strongly, and, regaining
the appearance of a normal nucleus, becomes the male pro-
nucleus. Originating from the middle piece of the sperm, a
star-shaped radiation, the sperm aster, appears in the egg
cytoplasm (Fig. 4, ^-^). Chambers has shown by microdissection
that this is due to local gelation of the cytoplasm. This process
of gelation spreads like a wave in all directions through the
protoplasm. At the same time, the centre of the sperm aster
liquefies again, forming an area of liquid protoplasm around
the male pronucleus. Simultaneously also, the male and female
pronuclei move towards each other, and meet in the central
area of the sperm aster. They may lie side by side for some
time, or, alternately, they may fuse immediately (Fig. 4, '*).
Now two new radiations appear, one on each side of the pair

16

THE INITIATION OF DEVELOPMENT:

of pronuclei (Fig. 4, ^). They meet, forming a so-called amphi-
aster. Then the nuclear membranes of both pronuclei disappear,
thereby liberating their chromosomes. A mitotic spindle de-
velops between the poles of the amphiaster, and the chromosomes

Fig. 4. Cytology of fertilisation in a sea urchin, 1-4: formation of
the sperm aster, fusion of the nuclei of egg and sperm; 5-7: forma-
tion of the amphiaster, first cleavage division, u : female pronucleus;
sp: sperm nucleus. After Fry and Parks.

arrange themselves in the centre of the spindle (Fig. 4, ^■'^),
thereby completing the union of the two nuclei (amphimixis).
The next step will be a cell division, the first cleavage^ which
marks the beginning of the development of the germ.

A number of experiments have provided information as to
the factors governing the processes here described. First, it
appears that at least two conditions must be fulfilled for the
development of the sperm within the Qgg to take place: (1),
the maturation of the egg must have begun, so that the nuclear
sap of the germinal vesicle has mixed with the egg cytoplasm
(p. 10); (2), the sperm must have traversed the egg cortex.
Sperms that have been artificially injected into the egg remain
completely inactive (Kite). The same is true of sperms which
have penetrated into egg fragments without cortical plasm
(Chambers). Evidently, the sperms are activated during their

THE FERTIL,ISATION OF THE EGG 17

passage through the cortex, and it is this activation which
enables them to complete their further development.

The best study of the processes which bring about the fusion
of the pronuclei was made by Fankhauser (1925-41) in newts.
In this group, there is so-called facultative polyspermy, i.e.
more than one sperm may penetrate into one egg without
disturbing the development. One of the male pronuclei combines
with the nucleus of the egg. The other sperms which have
penetrated show some initial development, but then they dis-
integrate. Fankhauser constricted eggs shortly after fertilisa-
tion, by means of a hair ligature, dividing them either com-
pletely or partially into two halves. One half contained the
egg nucleus, with or without one or more sperm nuclei. The
other half contained only sperm nuclei.

Fankhauser observed that, if constriction was complete, a
number of sperms developed simultaneously, forming large
asters in the half without the egg nucleus. In the case of
incomplete constriction, what happened in this half of the now
dumb-bell shaped egg depended upon whether or not, in the
other half, the egg nucleus had meanwhile united with a sperm
nucleus. If this had taken place, development occurred mainly
in those sperms in the half without the egg nucleus that were
farthest removed from the peduncle which joined the two
halves (Fig. 5). If the egg nucleus had not fused (because there
were no sperms in its half of the egg), development took place
in the sperm closest to the ligature in the other half. This
sperm nucleus and the egg nucleus moved towards each other,
and met on, or close to, the bridge. Fusion of the pronuclei then
took place. Fankhauser concluded from these observations, (1)
that there is an attraction between male and female pronuclei,
(2) that the egg nucleus promotes the development of the
nearest sperm, and (3) that this "favoured" sperm, and later
the zygote nucleus originating from the fusion of the pronuclei,
inhibit the development of the remaining sperms. It seems
probable that these promoting and inhibiting influences are due
to substances secreted by the nuclei, and diffusing into the
protoplasm.

Some observations on the egg of the fresh water snail

Raven - Outline Physiologie 2

18 THE INITIATION OF DEVELOPMENT:

Limnaea suggest that the mutual attraction between egg and
sperm nuclei does not arise until both pronuclei are swollen.
This swelling itself depends upon a special condition of the
egg cytoplasm, a condition which normally is not realised until
after the second maturation division, but which may be pre-

Fig. 5. Egg of a newt, Triton palmatus, constricted before the be-
ginning of cleavage (cf. Fig. 13). In the half on the right, the egg
nucleus has fused with a sperm nucleus, and cleavage has set in.
In the left half, the sperm nuclei farthest removed from the constric-
tion are dividing, whereas those closer to it are inhibited. After

Fankhauser.

cipitated by external influences (Raven and Roborgh, 1949).

The penetration of the sperm into the egg thus starts a
series of processes in each of which the components of egg
and sperm interact, and which result in the initiation of the
development of the fertilised egg- Now it is a very remarkable
fact that the same result can be achieved along completely
different lines. In eggs of many animals, development has been
successfully provoked by means of a wide variety of artificial
treatments. This is called artificial parthenogenesis. Evidently,
the stimulus for development, normally given by the penetrating
sperm, is not very specific; very different external stimuli may
have the same result.

In the sea urchin egg, for instance, a method devised by
J. Loeb gives very good results. Unfertilised eggs are first
treated for a few minutes with a weak solution of butyric acid
in sea water. When returned to normal sea water, they im-

THE FERTILISATION OF THE ECxG

19

mediately form fertilisation membranes. After 15-20 minutes
they are then brought, for a period of 30-60 minutes, into sea
water made hypertonic by the addition of sodium chloride.
Finally they are returned to normal sea water. Many of the
eggs then cleave, and develop into normal larvae.

In the eggs of the frog satisfactory results are obtained with
a treatment discovered by Bataillon (1910-1913). When punctur-
ed with a fine needle, such eggs extrude their perivitelline fluid,
and develop a fertilisation membrane. Cleavage, however, occurs
only if the eggs have previously been moistened with blood. It
appears that, for normal development to occur, a live blood
corpuscule, containing a nucleus, must be brought into the egg.
In this way, cleavage can be provoked in great numbers of eggs,
and in a certain percentage of the cases normal further de-
velopment will take place. A number of parthenogenetic larvae
have even been raised successfully through metamorphosis.

The experiments on artificial parthenogenesis have provided
the opportunity for a further analysis of the processes that
initiate development. Two components of this phenomenon can
now be separated experimentally, which in normal fertilisation
are so interwoven that they can hardly be distinguished. For
instance, sea urchin eggs
have been treated with bu-
tyric acid, as in the first part
of Loeb's method, but then
kept in normal sea water.
When such eggs are brought
back into normal sea water,
the cortical reaction takes
place, with the result that
the fertilisation membrane is
elevated. However, cleavage
and further development fail
to occur. After some time,
the egg nucleus swells, its
nuclear membrane disap- f'^- ^- Activated egg of a star-
, . , fish. Egg nucleus in monaster. No

pears, and a single aster ap- fertilisation membrane has been
pears in the surrounding formed in this case. After Dalcq.

20 THE INITIATION OF DEVELOPMENT:

cytoplasm (Fig. 6). Each chromosome divides into two halves,
but the halves remain together, and finally all the chromosomes
reunite into one nucleus in the centre of the now very much
enlarged aster. This process may repeat itself several times,
but irregularities soon appear, and, finally, this so-called mon-
astral cycle terminates in the death of the eg^.

In frog eggs, which have been pricked, but not inoculated with
cell material, we again find a cortical reaction with elevation
of a fertilisation membrane, followed by a monastral cycle,
which in the end comes to a stop, culminating in the death of
the egg.

Evidently, certain stimuli release a chain of reactions in the
egg, called the activation of the egg. The most conspicuous
links of this chain are the elevation of the fertilisation mem-
brane, and the monastral cycle. These processes, however, do
not result in normal development, but lead into a blind alley,
ending with the death of the egg. For normal development to
occur, other factors must bring the egg cytoplasm into such a
condition that, instead of the monastral radiation, a dicentric
radiation, or amphiaster, can be formed. The latter leads to
normal division, and thereby to cleavage of the egg. Bataillon
has named this process ''regulation''. In Loeb's method, the
treatment with hypertonic sea water is the regulating factor;
in Bataillon's method the inoculation of cell material acts as
such.

Consideration of these two groups of processes shows that
widely divergent means can be used for the activation of the
egg in different species. Both mechanical stimulation (pricking)
and physical treatment (illumination, induction shock, increase
or decrease of temperature, or of osmotic pressure) will serve
the purpose. Chemical treatment especially, however, has been
applied on a large scale (various acids, salts, and alkalis, but also
non-electrolytes, such as urea, saponin, etc.). This variety of
methods tends to show that none of them constitutes the
specific, natural agent itself. The inherent properties of the egg
itself must be responsible for the fact that it reacts in the same
way to such entirely different stimuli. Now the results of
several investigations indicate that calcium ions play a special

THE FERTILISATION OF THE EGG 21

role in activation. In several species, the egg can be activated
only in the presence of these ions in sufficient quantity
(Pasteels, 1938; Moser, 1939). Heilbrunn (1937) holds the view
that the egg cortex consists of a protein-calcium compound,
which can be broken down by the action of various stimuli.
The liberated calcium is then taken up by the internal cyto-
plasm of the egg. This causes an increase in the viscosity of
this region, while the cortex simultaneously liquefies. These
processes are considered to be the real cause of activation.

Membrane formation and monastral cycle, the two processes
especially characteristic of activated eggs, are not linked to-
gether inseparably. In the starfish, the formation of a monaster
unaccompanied by a fertilisation membrane can be caused by
treatment of the egg with a mixture of certain chlorides
(Dalcq), (Fig. 6). On the other hand, in certain cases membrane
formation may take place without a monastral cycle. Nor is
the fertilisation membrane indispensable for further develop-
ment of the egg. It is possible to prevent membrane formation
in eggs, either after fertilisation, or during parthenogenetic
development. This does not always upset development.

Activation causes a marked change in the physiological
properties of the egg. A great increase in the permeability of the
egg surface is quite common. This is probably due to the cortical
reaction. In the sea urchin egg, many chemical reactions take
place in the first few minutes after activation. Large molecules
are first broken down into smaller ones, and, somewhat later,
this is followed by resynthesis. Finally, activation often results
in infertilisability of the egg. This, however, is only true in the
case of optimal activation ; if the activating agent has operated
for too short or too long a period, so that activation is in-
complete, the egg retains its fertilisability.

In normal fertilisation, the egg is activated by the penetrat-
ing sperm, as can be seen from the occurrence of the cortical
reaction. Apparently the sperm introduces a "regulating" factor
as well, preventing the occurrence of a monastral cycle, and
allowing the nuclei of egg and sperm to join in the formation
of a bipolar mitosis, which leads to cleavage of the egg. We
have seen above that the amphiaster usually arises in connect-

22 THE INITIATION OF DEVELOPMENT:

ion with the development of the sperm aster, viz. in the central
liquefaction area of the latter.

According to the classical idea of Boveri, the middle piece
of the sperm contained a specific cell organ for division, the
centrosome. This acted first as the starting point for the form-
ation of the sperm aster in the egg cytoplasm. Next it divided,
and an aster developed around each half; together these asters
constituted the amphiaster. In other words, development was
initiated by the activity of the centrosome carried by the sperm.

More recently this view has been questioned. It was stressed,
especially by Fry, that the centrosome may not exist, at least
as a separate plasmatic body. It cannot be denied that the
sperm, especially its middle piece, contains a centre which
readily gives rise to a gelation (aster) in the egg cytoplasm,
but similar asters, so-called cytasters, can be produced at other
places in the cytoplasm by a variety of stimuli. They are there-
fore certainly not dependent on the presence of a special
centrosome.

Two hypotheses have been advanced, concerning the factor
which, in normal fertilisation, is responsible for the occurrence
of the amphiaster with its bipolar mitotic spindle. Dalcq
suggested the following hypothesis. The sperm, especially its
nucleoplasm, is the carrier of the principle that causes "di-
centry". The formation, on the disappearance of the nuclear
membrane, of a bipolar spindle with its asters, is an inherent
property of this nucleoplasm. Dalcq's view is founded on the
following observations. (1) Sometimes, in the act of penetrat-
ing, the sperm remains stuck with its head in the cortex. In
such cases, normal development of the sperm nucleus does not
take place. The egg is activated only, and a monastral cycle
ensues. This occurs in some cases of heterogeneous fertilisation,
e.g. when eggs of the toad Bufo calamita are fertilised with
sperm of the newt Triton alpestris (Bataillon). It may also
occur when the sperm has been damaged, e.g. by trypaflavin
poisoning (Dalcq). (2) If the fusion of the pronuclei is prevent-
ed, each may, under certain circumstances, develop separately
in such a way that the male pronucleus undergoes a normal
mitosis, whereas the female pronucleus gives rise to a monaster.

THE FERTILISATION OF THE EGG

23

\

Fig. 7. Egg- of a sea urchin, treat-
ed with ether. The pronuclei have
not fused. The egg nucleus {^)
forms a monaster, the sperm
nucleus (d") an amphiaster. After
Wilson,

This was observed, e.g., by
Ziegler (1898) when, in his
experiments with fragmented
sea urchin eggs, the nuclei of
egg and sperm were isolated
in different fragments. Wil-
son (1901), by preventing
the fusion of the pronuclei in
sea urchin eggs by means of
an ether treatment, observed
the same phenomenon (Fig.
7). In Amphibia, the same
thing may occur in unripe or
overripe eggs, in which the
fusion of the pronuclei is
retarded by the abnormal

condition of the protoplasm (Bataillon and Tchou Su, 1934).
(3) The co-operation of the chromatin of the sperm nucleus
is not indispensable for a normal bipolar mitosis. This is proved
by some cases of heterogeneous fertilisation, in which the sperm
penetrates into the egg, and male and female pronuclei unite,
but the chromatin of the sperm nucleus remains entirely
compact. In the course of a mitosis which in all other respects
is normal, this chromatin does not divide into chromosomes, and
is soon afterwards extruded into the cytoplasm, and resorbed.
This occurs, e.g., in the case of fertilisation of sea urchin eggs
with sperm of the mussel Mytilus (Kupelwieser, 1908) (Fig. 8).
The same may happen after fertilisation with sperm damaged by
radium irradiation (O., G., and P. Hertwig) or trypaflavin treat-
ment (Dalcq). (4) Cytasters, produced e.g. in sea urchin eggs,
by treatment with hypertonic sea water, are usually unable to
divide, and therefore remain monocentric. If, however, they are
situated in the neighbourhood of a normal mitosis, they can
sometimes "capture" part of the nuclear material of the latter.
Thereupon they divide, and form an amphiaster (Fry, 1925).
Working with sperm treated with trypaflavin, Dalcq obtained
amphiasters which, as a consequence of irregularities in the
course of division, contained no chromosomes, but only part

24

THE INITIATION OF DEVELOPMENT:

of the nucleoplasm of the sperm nucleus. These were able to
divide repeatedly, thereby giving rise to new ampiasters (Fig .9).
Dalcq concluded from these experiments that the power of di-
vision, especially that of forming a bipolar mitosis, is an inherent
property of the sperm nucleus, in particular of its nucleoplasm.
Yet there is an objection to this conclusion. There are several

v^N. ^

ii//^.

r-i

'h.

^

\

Pig. 8. Egg of a sea urchin, fertilised with sperm of a mussel,

Mytilus. (a) fusion of the pronuclei; (b-c) first cleavage, the male

chromatin (cT) is eliminated; (d) four-cell stage, male chromatin

in one of the blastomeres. After Kupelwieser.

exceptions to the rule that co-operation of the male pronucleus
is necessary for the formation of a normal bipolar mitosis. In
Crepidula (Conklin, 1904) and Ciona (Duesberg, 1926), the
female pronucleus will form an amphiaster, even if fusion of
the pronuclei is prevented. On the other hand, in Fankhauser's
experiments already described (see above p. 17), the super-
fluous sperms in the half without the egg nucleus often form
a monaster, although, naturally, the nucleoplasm of the sperm
is present here. Further, Dalcq's hypothesis does not explain

THE FERTILISATION OF THE EGG

25

"regulation", such as is caused, e.g., by treatment of the sea
urchin egg with hypertonic sea water in Loeb's method. It is
true that in Bataillon's method for artificial parthenogenesis
in Amphibia regulation is caused by the introduction into the
egg of a blood corpuscle with its nucleus, but this nucleus does
not unite with the egg nucleus. Moreover, Einsele (1931) has

Fig. 9. Two sections of a frog's egg, fertilised with two trypaflavin
treated sperms.-. : haploid nuclei, originating from the egg nucleus;
(f: sperm chromatin has remained compact; a: mitotic spindles with-
out chromosomes. After Dalcq.

shown that the same result may be obtained with an extract
of blood or sperm. According to a recent investigation by
Shaver (1949), the activity of such extracts is mainly due to a
fraction consisting of cytoplasmic granules, so-called micro-
somes, which are very rich in ribonucleic acids.

For these reasons, Bataillon has attempted an entirely differ-
ent explanation of the origin of dicentry. In his opinion, the
occurrence of a monastral cycle in activated eggs is due to an
"anachronism" between the development of the nucleus and
that of the asters arising from the cytoplasm. It is as though
the nucleus has proceeded further in its development than has
the cytoplasm so that the nuclear membrane disappears before
the aster has yet had time to develop into an amphiaster. In
normal fertilisation, the penetrating sperm accelerates the
activity of the cytoplasm. This "regulates" the phase difference
between nucleus and cytoplasm. Bataillon points out that, in

26 THE INITIATION OF DEVELOPMENT:

the normal course of fertilisation, first a gelation of the proto-
plasm, the sperm aster, takes place, with the penetrating sperm
as its starting point. This spreads as an "onde de gelification"
in all directions through the egg cytoplasm. At the same time
the centre of the sperm aster solates (liquefies) again, and it
is only then that the amphiaster forms in this central area,
and that the nuclear membranes of the pronuclei, which have
in the meantime fused, disappear. In Bataillon's opinion, the
passing of this "onde de gelification" prepares the egg cyto-
plasm for the formation of an amphiaster, and thereby abolish-
es the anachronism between nucleus and protoplasm. In arti-
ficial parthenogenesis in Amphibia, the "onde de gelification"
starts from the cell fragments with which the egg has been
inoculated when it was punctured. Chambers (1921) has shown
that in Loeb's method the treatment of the sea urchin eggs
with hypertonic sea water causes an "onde de gelification" in
the cytoplasm around the nucleus. Therefore, Bataillon's hypo-
thesis, in contrast with that of Dalcq, is able to explain both
the phenomena of normal fertilisation, and those of artificial
parthenogenesis. In future, it may become possible to unite
both points of view into a single more general hypothesis, for
it must be admitted that in many cases the gelation of the
cytoplasm starts from the sperm nucleus.

CHAPTER m

The structure of the fertilised egg

The structure of the egg at the starting point of its development
seems to be quite simple. But, as we have seen in the Intro-
duction, it is conceivable that this simplicity is only apparent.
The whole complicated spatial structure of the later organism
might already be present, in a not easily recognisable form,
in the egg. According to the preformation theory, which had
many supporters in the 17th and 18th centuries, the complete
young animal was already contained in the fertilised egg, in
the same way as a complete stalk with leaves, flowers, etc.,
can be contained in the bud of a plant. The development of the
egg would be no more than the unfolding of this preformed
young germ. The opposite view was maintained by the theory
of epigenesis, which held that the embryo was not yet present,
as such, in the egg, but that it would arise as a new product
in the course of development. In the 19th century, a thorough
study of the developmental phenomena became possible, thanks
to the great progress in microscopic technique made in that
period. By this means, the dispute between preformation and
epigenesis was at that time settled in favour of the latter theory.
A few years after 1880, however, the issue was reopened, in a
different form, by Wilhelm Roux. It had indeed been shown that
the embryo as such is certainly not yet present in the fertilised
egg, but was it not possible that the "degree of multiplicity"
of the egg's spatial structure was almost, or quite as high as
that of the embryo ? The egg might contain, in an invisible form,
such a complex system of causal factors, that each of its parts
would be able, quite independently, to produce one definite
part of the embryo, and nothing else. This implied that each
part of the embryo would be preformed in one definite part of the

28 THE STRUCTURE OF THE FERTILISED EGG

egg. As a consequence of the structure of the egg, in which
each part had its fixed place, the various organs, though each
developing independently, would later fit together as parts of
a "mosaic". Therefore, development would not involve an "in-
crease of spatial multiplicity", but only the manifestation of
a previously invisible, preformed, spatial multiplicity. This is
the hypothesis of neo-yre formation. Its alternative, that of
neo-epigenesis, is the hypothesis that the spatial multiplicity
of the embryo is in no way preformed in the egg, but that it
arises during development.

W. Roux, the creator of the science of "developmental me-
chanics", considered the solution of this problem its first and
most important task. Since then, it has been the motive for a
great number of investigations in a wide variety of animal
groups. Such studies are characteristic of the first period of
research in developmental physiology. On the strength of these
results, we can now assert on good grounds that the spatial
structure of the egg is generally very simple, and that there-
fore the spatial multiplicity of the embryo increases strongly
during development, as is postulated by the neo-epigenetic
hypothesis. We shall now discuss a number of experiments
which have led to this conclusion.

We have already seen that the first step in development is
the cleavage of the fertilised egg into two cells. Each of these
cells divides into two again, and so on, until a great number of
cleavage cells, or blastomeres, has been formed. This process
does not involve cell growth, so that the resulting blastula is
still of about the same size as the original egg. In 1891, Driesch
made an experiment which was to prove of the greatest im-
portance for the development of our views in this field. He took
sea urchin eggs which had just begun developing, and were
in the two-cell stage. Driesch shook those eggs in a test tube
with sea water, and thereby separated the two blastomeres of one
egg in a number of cases. He then studied the further devel-
opment of these isolated halves, and observed that each of
them was able to develop into a harmoniously built young larva.
These larvae were only half the size of ordinary ones, but were
otherwise normal in form (Fig. 10). Moreover, Driesch found

THE STRUCTURE OF THE FERTILISED EGG

29

Fig, 10. Gastrula and pluteus of a sea urchin. Left: from a whole
egg; right: from a lo-blastomere. The latter are smaller, but har-
moniously built. After Morgan.

that even blastomeres isolated at the 4-cell stage could still
develop into harmoniously built larvae of reduced size.

The same phenomenon, the origin of normal embryos from
one half or one quarter of an egg, was found to occur in other
groups of animals as well. In several species of newts, for
example, the two blastomeres of the two-cell stage can be
separated by constricting the egg with a hair ligature in the
cleavage plane. Here, too, under certain circumstances both
halves will develop into harmoniously built embryos of half
the normal size, which lie together in one egg capsule (Spemann,
1903), (Plate I). Even if newt eggs are divided into two halves
at a much older stage, after the completion of cleavage, each
half may still develop into a complete, normal embryo.

From other eggs, large parts of the egg cytoplasm can be

30

THE STRUCTURE OF THE FERTILISED EGG

removed before the beginning of
cleavage, without any disturbance
of their further development.

In such cases, a normal embryo
originates from part of an egg.
However, the opposite, devel-
opment of a single embryo from
the fusion of two or more eggs,
has also been observed. Driesch
(1900) has done this experiment
in sea urchins, and it has been re-
peated and extended by Bierens
de Haan (1913). The envelopes of
two sea urchin eggs were removed,
and the eggs were gently pressed
together. They then often fused
into one germ of double size. Here
too, if certain conditions are ful-
filled, a single, harmoniously built
embryo will develop from the pro-
duct of such a fusion (Fig. 11).

Mangold and Seidel (1927) have
done the same experiment in newts.

Fig. 11. (a) pluteus of a
sea urchin, Paracentrotus
lividus, which has originated
from, the fusion of two eggs,
(b) the same pluteus, for
comparison with (c) a
normal pluteus drawn at the
same scale. After Bierens
de Haan.

After removal of the envelopes,

newt eggs at the two-cell stage often assume a dumb-bell shape.
When one was placed crosswise on top of the other, two of
these dumb-bells often fused into one single germ. Each pair
of opposite quadrants of such an embryo was derived from
one of the original eggs. Part of the products of these fusions
proved to be able to develop into single, more or less har-
moniously built embryos. This was possible even when the
fused eggs belonged to different species of newts so that the
resulting embryo was a "chimera", consisting of quadrants
which alternately belonged to either of the two species
(Plate 11).

The view that the egg has a fixed spatial structure, and that
this is responsible for the development of its parts into definite
organs, is rendered highly improbable by these experiments.

THE STRUCTURE OF THE FERTILISED EGG 31

It becomes even less tenable in the light of the following facts.
A profound disturbance of the structure of the egg cytoplasm
often does not result in aberrant development. Such a disturb-
ance can be caused by centrifuging the egg. Under the in-
fluence of the centrifugal force, the materials of the egg are
arranged according to their specific gravity. The lighter
substances (especially the fats) are accumulated at the centri-
petal pole, the heavier material moves toward the centrifugal
pole. In this way the contents of the egg are arranged in layers,
an arrangement very different from the normal structure of
the egg cytoplasm (Plate III). Nevertheless normal development
will often take place in such centrifuged eggs, e.g. in polychaete
worms and in molluscs. In the course of this development, the
stratification is lost to some extent, and the material of the
egg is redistributed more evenly. Finally, normal embryos will
be formed.

All these experiments give positive indications that the cyto-
plasm of the fertilised egg cannot contain a complex spatial
structure, in which each part represents one definite organ,
or part of the body, of the future embryo. For if this were true,
the division of such a structure into two halves (as in the
experiments with isolated blastomeres) would be bound to
cause defects in the embryos developing from each half.
Further, it is very difficult to imagine how fusion of two of
these systems could again result in a unity of entirely similar
properties. Finally, one cannot understand how such a struc-
ture, once it has been disturbed (as in the centrifuge experi-
ments), could return to its original state.

The conclusion obviously must be that the egg cytoplasm
has no such complicated structure, no "extensive multiplicity",
but that it is a more or less homogeneous system. Such a system
— a water drop is a convenient model — can be divided, and,
alternately, two such systems can fuse, without any change
of properties. A change which produces heterogeneity will be
undone as soon as the factors which caused it suspend their
activity, because the components of the system return to their
position of equilibrium, i.e. the homogeneous distribution.

32 THE STRUCTURE OF THE FERTILISED EGG

Therefore, the multiplicity of the egg cytoplasm must be mainly
intensive.

For the sake of completeness, an alternative explanation of
the experiments described above must be mentioned. One can
still maintain that the egg cytoplasm has a complicated struc-
ture, which is disturbed in these experiments. In that case, the
existence must be postulated of a more or less mysterious
"vital force" which sees to it that the normal structure of the
egg is restored after the disturbance. Such an explanation was
given by Driesch, who regarded the result of his experiments
as proof of the existence of an entelechy which, after a disturb-
ance, "regulates" the course of development again. Undoubtedly,
this is a possible explanation, but it is not the most obvious
one because it is founded on the introduction of a new, and
entirely hypothetical, factor. Now it is the task of science
always to look for the simplest possible explanation, and to
accept this as long as it has not been disproved. Therefore, we
must reject Driesch's hypothesis so long as no more compelling
arguments can be advanced in favour of it, and we must accept
the conclusion that, on the whole, the egg cytoplasm is a homo-
geneous system with intensive multiplicity only.

In the Introduction it was pointed out that the various animal
groups by no means behave identically in their development.
This applies also to the results of the experiments described
above. Often they show different results in other eggs than
those mentioned. Centrifuge experiments, for example, do in
fact cause disturbances in the development of the eggs of many
species. For some time past, therefore, two types of eggs were
distinguished, (1) "mosaic eggs" which possessed a complicated
spatial structure, and in which each disturbance of the system
resulted in disturbed development, and (2) "regulation eggs",
with a poorly developed, or highly plastic spatial structure,
in which disturbances of the system were easily "regulated".
This distinction, however, has proved unfounded. It has been
shown that the aberrant behaviour of „mosaic eggs" is not
due to fundamental differences in the structure of the egg, but
to a number of adventitious phenomena (cf. p. 63). Therefore,
it can be said of these eggs as well that, at the beginning of

PLATE I. Development of two embryos from a single egg. If a newt egg
IS divided into two halves by a ligature (cf. fig. 13), both halves may under
certam circumstances develop into embryos (A-B). In the case of incomplete
constriction, the resulting twins form a double monster (C-D). After Spemann,

\

a

6

?5^.#

d

PLATE II. Development of a single embryo from two fused eggs. Crosswise
fusion (a) of two newt eggs at the 2-cell stage results in the production of
a single embryo (b), the four quadrants of which arise fi'om one or the
other of the original eggs altei-nately. (c). Even eggs of different species
(Triton taeniatus and T. alpestris), fused in this way, may produce a single
embryo (d). After Mangold and Seidel.

THE STRUCTURE OF THE FERTILISED EGG 33

development, the egg cytoplasm has no, or at most very little,
spatial multiplicity.

The application of modern methods of investigation has
considerably increased our insight into the structure of the
cytoplasm in the last few years. In some respects, it was
shown to have the properties of a liquid. The viscosity of the
protoplasm of sea urchin eggs is only a few times higher than
that of water. In many eggs an obvious protoplasmic current
can be observed. Brownian movements of granular inclusions have
been seen in eggs, e.g. of nematode worms. Other observations,
however, show that cytoplasm is not a pure liquid, but that
it has, for instance, elastic properties. Iron particles taken up
by a cell can be moved by means of a magnetic field, but will
elastically return to their original position when the field is
removed.

At present Frey Wyssling's view is generally accepted that
protoplasm contains a network of long polypeptide chain-
molecules, interconnected by side-chains. These "contacts",
however, are continually being broken and re-connected. The
meshes of the network are filled with a watery solution of salts,
lipids, phosphatids, etc. These dissolved substances are located,
in variable arrangements, around the free side-chains of the
polypeptide molecules.

In some eggs, moreover, a coarser structure, of a microscopic
order of magnitude, is apparently superimposed upon this
"molecular" basic structure. This came to light especially in
Monne's studies (1946) of sea urchin eggs. Here the egg cyto-
plasm consists of a meshwork of 50-100 m^ thick fibrillae. The
interstices of this "spongioplasm" are filled with a more liquid
phase, the ''enchylema" which contains the mitochondria, yolk
granules, oil droplets, etc. The spongioplasm fibrillae are not
homogeneous, but consist alternately of granules with a strong
affinity for certain stains (the chromidia or microsomes), and
interchromidial parts. The chromidia are rich in ribonucleic
acid. Under the polarisation microscope, the spongioplasm
fibrillae proved to be negatively birefringent in longitudinal
direction; it was concluded that they consist of bundles of pro-
tein chain molecules, with lipid molecules at right angles to the

Raven - Outline Physiologie 3

34 THE STRUCTURE OF THE FERTILISED EGG

chains. The polypeptide chains are probably folded in the region
of the chromidia, but stretched in the intervening parts.

Several investigations, among which those of J. Brachet are
prominent, have demonstrated that microsomes can be isolated
from damaged cells by ultracentrifugation. These microsomes
may be identical with Monne's chromidia. Apart from ribo-
nucleic acid, they contain sulfhydryl compounds, calcium,
magnesium, phosphatids, and numerous enzymes. Probably
these microsomes are important centres of metabolism and
protein synthesis. It seems likely that they are able to multiply
by division.

At the egg surface, we find a layer of a more solid consist-
ency, the egg cortex. In the majority of cases, this is already
formed by a peripheral condensation process before fertilisation
takes place. It is a few /x thick, at most. Investigations by
Monroy (1947) and Monne have shown that the cortex of sea
urchin eggs probably consists of alternating protein layers and
lipid lamellae. The filamentous polypeptide chains are parallel
to the surface of the egg, whereas the rod-shaped lipid molecules
are at right angles to it. During fertilisation and the first
cleavages of the egg, rhythmical changes take place in the
structure of the cortex.

All this tends to show that the composition of the egg cyto-
plasm is very complicated indeed, but that its structures do not
adumbrate the structure of the future embryo. They are entirely
in the nature of an intensive multiplicity, as defined above
(p. 3).

Apart from the egg cytoplasm, however, the fertilised egg
also contains a nucleus, originating from the fusion of the
nuclei of egg and sperm. During cleavage this nucleus also
divides into a number of cleavage nuclei which find their way
into the various blastomeres. Could it not be that the egg has
a complicated spatial structure, localised, however, not in its
cytoplasm but in the nucleus, in which, after all, the spatial
multiplicity of the future embryo might be preformed in some
way or another?

Weismann advocated this hypothesis in 1892. In his opinion,
the nucleus of the fertilised egg contained the id, a three-

THE STRUCTURE OF THE FERTILISED EGG 35

dimensional structure, consisting of material particles, the
determinants. In the course of the nuclear divisions during
cleavage the id would be distributed over the various cleavage
nuclei. The nucleus of each blastomere would receive only part
of the determinants, and, eventually, there would be only one
determinant in each cell. This would then exert its influence
upon the cell, thereby unequivocally determining its different-
iation. In other words, the spatial multiplicity of the embryo
would be preformed in the architecture of the id; the rules
governing the distribution of the determinants during cleavage
would keep the process of development on its normal course.

Two deductions from Weismann's theory are open to ex-
perimental verification: (1) there must be qualitative differ-
ences among the nuclei formed during cleavage, and (2)
disturbances of the normal course of cleavage must result in
an abnormal distribution of determinants over the cells, and
therefore in abnormal development. If it can be shown ex-
perimentally that these conclusions do not correspond to reality,
it follows that the theory is not correct.

Now it can already be seen from the experiments on the
isolation of cleavage cells (see above p. 28) that the nuclei of
the first few blastomeres are not qualitatively different. We have
seen that a single blastomere from the two-cell stage of a newt
or the 4-cell stage of a sea urchin can still produce a complete,
normally built embryo. This proves that the nucleus of these
cells must still be equivalent to the original nucleus of the
fertilised egg. In other words, no division has occurred, up
to this stage of cleavage, that led to non-equivalent results.
This is demonstrated even more clearly by the following
experiments. Driesch (1893) kept developing sea urchin eggs
between two sheets of glass, so that they were slightly flatten-
ed. This changed the direction of the plane of the divisions, and
resulted in abnormal positions of the cleavage cells relative
to one another. Consequently, the cleavage nuclei became located
in the "wrong" cells (Fig. 12). In spite of this, the eggs devel-
oped into normal embryos. It follows that disturbance of the
course of cleavage does not influence further development.

Spemann (1928) constricted fertilised, but uncleaved newt

36

THE STRUCTURE OF THE FERTILISED EGG

Fig. 12. Diagram of the cleavage in a sea urchin egg under pressure.
(a-c) normal cleavage (4, 8, and 16-cell stages); id-i) cleavage in
an egg flattened by external pressure; (d-f) seen from the side;
ig-i) seen from above. Analogous nuclei marked in the same way
in all cases. After Diirken.

eggs with a hair ligature, giving them a dumb-bell shape. The
nucleus lay in one end of the dumb-bell shaped egg, and only
this part was able to start cleaving. The other, non-nucleated
half remained uncleaved (Fig. 13 a, b). After a certain number
of divisions, however, when the nucleated part had divided into
8 or 16 cells, one of the cleavage nuclei would pass from this
half, through the ligatured peduncle, into the other part
(so-called "retarded nucleation" of this half), (Fig. 13 c).
This half then began to cleave as well. If at this stage the
two halves were entirely separated, each would, under certain
circumstances, develop into a normal embryo (Fig. 13 d).
Therefore, the nucleus which passed through the peduncle, and
which represents only one eighth or one sixteenth of the
original fertilisation nucleus, is sufficient to bring about normal

THE STRUCTURE OF THE FERTILISED EGG

37

Fig. 13. "Retarded nucleation" in Triton. Zygote nucleus pushed to
one side by the constriction of the egg. (a) first cleavage in the
nucleated half; (&) further cleavages in this part, the other half
is still un cleaved; (c) passage of one nucleus from the nucleated
into the non-nucleated half, beginning of development in the latter;
id) the two embryos that developed from the constricted egg; the
one produced by the half in which nucleation was retarded is
considerably younger, but normally built. After Spemann and

Fankhauser.

development of the half in which nucleation was retarded. It
is evidently equivalent to the fertilisation nucleus. This proves
once again that, at least in the first stages of development,
there is no qualitative difference between the division products
of the nucleus.

These and similar experiments prove that Weismann's theory
cannot be maintained. Development is not due to a qualitatively

33 THE STRUCTURE OF THE FERTILISED EGG

unequal distribution of a spatial system of developmental
factors, localised in the nucleus of the fertilised egg.

The extensive multiplicity of the later organism is not pre-
formed, either in the cytoplasm or in the nucleus of the fertilised
egg. Therefore, development involves an increase in extensive
multiplicity.

CHAPTER IV

Polarity and symmetry;
Gradient-fields

In the previous chapter we have seen, that, in broad outlines,
the newly fertilised egg is to be regarded as a homogeneous
system, and that its multiplicity is only intensive, not extensive.
However, we shall now discuss a number of phenomena that
show that this is true only with certain restrictions.

Even a superficial examination shows that the egg can not
be so entirely homogeneous as is, for instance, a water drop,
because we can distinguish between egg cytoplasm and nucleus.
But, apart from this, a further organisation is demonstrable in
the egg; this is expressed in its polarity and its symmetry.

All animal eggs have a polar structure, i.e. two opposite
poles can be distinguished, called the animal and vegetative
poles respectively. They are connected by the main axis of the
egg. Considering the egg as a globe, we may call all planes
that contain the main axis meridian planes, whereas the plane
that bisects the main axis at right angles is the equatorial plane.
In some cases, e.g. in the oblong eggs of insects (Fig. 26) and
cuttlefish, the polarity is revealed in the shape itself of the
egg. In other cases, the egg is more or less spherical, but its
polarity is evident in other ways: the polar bodies are given
off at the animal pole of the egg (p. 10) ; after fertilisation,
the zygote nucleus often lies in an eccentric position, nearer
to the animal pole. The first two cleavages nearly always take
place in meridional planes so that they intersect in the main
axis of the egg.

Further the polarity is often apparent in the arrangement
of the inclusions of the egg cytoplasm. In many eggs, the food
substances which together constitute the yolk are not evenly

40

POLARITY AND SYMMETRY;

e/7

fe/7/r.

7b/-J'

L

Fig. 14. Egg of a frog,
Rana fusca, after the for-
mation of the grey crescent
igr.h.m.). an: animal side;
veg: vegetative side; dors'.
dorsal side; ventr: ventral
side. After Schleip.

distributed; often their density increases from the animal
toward the vegetative pole. There may also be differences in
pigmentation. In many amphibians, for example, the animal
region of the egg surface is darkly
pigmented, whereas the vegetative
region is unpigmented (Fig. 14).

In the great majority of animals,
the unripe eggs {oocytes) possess
a polar structure already on leav-
ing the ovary. Evidently, the
polarity originates during the
growth of the egg in the ovary.
In many cases a connection can
be observed between the polarity
of the oocyte, and the way in
which it is attached to the ovary.
The place of attachment, which is
also the place where the food
stream from the maternal tissues

enters the oocyte, is in some groups the future animal pole,
in others the future vegetative pole of the egg.

Apart from polarity, many eggs possess an obvious bilateral
symmetry. This, too, may be expressed in the shape itself of
the unfertilised egg, as in insects and cuttlefish. In other cases,
the symmetry is not visible until after fertilisation. In the eggs
of many amphibians, a zone of lighter pigmentation, the so-
called grey crescent, develops on one side of the egg along the
boundary of the dark animal half in the first few hours after
fertilisation. This marks the dorsal side of the egg, and later
of the embryo. The opposite side becomes the ventral side
(Fig. 14). Once the grey crescent has been formed, we can
divide the egg into symmetrical halves in only one way, viz.
through the meridional plane which bisects the grey crescent.
In normal development this plane, the median plane, will be-
come the plane of symmetry of the embryo. It contains the
main axis, and also the dorso-ventral axis which is at right
angles to the main axis, and connects two opposite points on
the equator of the egg.

GRADIENT-FIELDS 41

By making small lesions in the surface of frog eggs, Ancel
and Vintemberger (1935) were able to show that the formation
of the grey crescent is due to shifting of the egg cortex relative
to the deeper layers of the cytoplasm. On the dorsal side, the
egg cortex moves toward the animal pole, and also slightly
towards the median plane. Part of the superficial pigment gets
caught in this movement, and a less pigmented area, the grey
crescent, is left behind.

The ascidian egg is another example of an egg in which
bilateral symmetry does not become visible until after fertilisa-
tion (p. 58).

For frog eggs, Ancel and Vintemberger (1948) have also
solved the problem of what factors determine the position of
the plane of symmetry of the egg. Immediately after spawning,
the orientation in space of the amphibian egg is arbitrary.
When, however, some time after fertilisation the perivitelline
fluid is extruded, the eggs can turn freely in their capsules,
and are oriented by gravity. They rotate so that the main axis
is vertical, with the animal pole pointing upwards. This rotation
proved important for the determination of bilateral symmetry.
The meridional plane in which the rotation takes place will
later be the plane of symmetry of the egg. The grey crescent
will form on the side along which the vegetative pole has
descended. The point of entry of the sperm into the egg also
plays a part: the grey crescent is formed preferably at the
opposite side of the egg. The influence of the sperm, however,
is less marked than that of the orienting rotation. Normally,
the grey crescent is formed in accordance with the latter. The
influence of the sperm will take effect only in eggs whose
animal pole accidentally pointed upwards from the beginning,
and which therefore did not rotate. Both factors can be
eliminated experimentally by fixing unfertilised eggs so that
their animal poles point upwards, and activating them by means
of an electrical induction shock (p. 20). In this case, the grey
crescent may be formed anywhere. This shows that the origin
of bilateral symmetry, per se, does depend on the starting of
development by fertilisation or by activation, but not on the
one-sided penetration of a sperm or on the orienting rotation.

42 POLARITY AND SYMMETRY;

The direction of the plane of symmetry, however, may be
determined by the last two factors. According to Ancel and
Vintemberger, these factors cause a slight asymmetry in the
position of the cortex relative to the internal parts of the egg.
This would then direct the development of bilaterality.

We have already said that the phenomena of polarity and
symmetry complicate the very simple picture of the egg drawn
in the previous chapter. They show that it would not be correct
to regard the egg as a completely homogeneous system. Polarity
and symmetry can only be explained on the assumption that
there are local differences in composition within the egg, and
a closer examination of the egg proves the truth of this as-
sumption. Some of its physical and chemical properties appear
to be unevenly distributed. As a rule, these properties have the
character of gradient systems. Therefore we shall now briefly
consider the meaning of this "gradient" concept.

By "gradient- field'' is meant a spatial distribution of a
physical or chemical quantity, whereby the value of this quan-
tity gradually changes from point to point. The properties
concerned are always so-called "scalar" quantities, i.e. a
numerical value can be attributed to them (which might be
read on a scale), but no direction; e.g., temperature, pressure,
concentration of a substance, electrical potential, and the like.
In a gradient-field, we can distinguish planes of equal intensity.
At all points in such a plane the scalar quantity has the same
value. The rate of change of the quantity at any given point
in the field is called the gradient at that point. At each point
of the field, the gradient has one, and only one, definite value
and direction, since it is always measured at right angles to the
plane of equal intensity through the point.

Several different types of gradient-field can be distinguished.
The scalar quantity may have a maximum at one end of the
system under consideration, and a minimum at the opposite
end. In this case the gradients have the same direction through-
out the field. This is called a linear gradient system, or, if the
direction of the gradient coincides with one of the axes of the
system, an axial gradient system (example: the temperature
gradient along an iron bar which is heated at one end, and

GRADIENT-FIELDS 43

cooled at the other). On the other hand, a gradient system may
extend over a flat or curved surface, the quantity concerned
decreasing, for example, in all directions from a central maxi-
mum (for instance: atmospheric pressure gradients at the
surface of the earth). This is called a surface gradient. Finally,
a gradient may extend in all directions in space, e.g. the con-
centration gradient of a salt crystal dissolving in water. This
is called a spatial gradient system.

Now in many eggs gradients have been found which show
some connection with polarity or symmetry. We have already
mentioned that there is an increase in yolk concentration along
the main axis of many eggs (p. 39). This may be called a yolk
gradient. Further, Child and his collaborators have for many
years stressed the importance of local differences in the in-
tensity of metabolism, as expressed in differential sensitivity
to noxious influences in different regions. In animal eggs, as
a rule the sensitivity is greatest at the animal pole, and from
there it decreases gradually in the direction of the vegetative
pole. This points to a variation in metabolism of the egg along
its main axis. Similar results have been obtained in determin-
ations of the redox potential (rH) of the egg cytoplasm with
various dyes, such as Janus green and methylene blue (Ries
and Gersch, 1936).

In this context experiments by Spek (1930-1934) deserve
mention. He measured the degree of acidity (pH) of proto-
plasm by means of indicator dyes (substances whose colour
depends on the pH of their environment), e.g. neutral red, nile
blue hydrochloride and brilliant cresyl violet. Initially, most
eggs showed a more or less uniform coloration, but shortly
after the beginning of development a ''bipolar differentiation"
took place, i.e. the colours of animal and vegetative regions
began to differ. The colour of the animal cytoplasm indicated
an alkaline reaction, that of the vegetative part of the egg an
acid reaction. Spek explained this as follows. Originally colloid
particles with positive and negative electrical charges were
mixed throughout the egg. Later, the positive particles would
move to one pole, and the negative particles would migrate
to the other pole. The mixture would thereby be separated, and

44 POLARITY AND SYMMETRY;

particles of the same sign would be concentrated at each of
the poles. This migration of the particles was assumed to be
caused by an electric field, due to differential penetration of
certain ions (potassium ions playing a major role) from the
environment into the egg. A difference in permeability of the
egg surface near the animal pole would be responsible for this
penetration. Later investigations, however, have shown that
"bipolar differentiation" is due not to segregation of colloid
particles of opposite signs in the cytoplasm, but rather to
shifting of the yolk of the egg. This material, consisting of
fat- and protein globules, now assumes its final position with
regard to the egg axis (Raven, 1938b). The acid yolk proteins
accumulate at the vegetative pole ; their disappearance from the
animal side causes the pH there to shift in the direction of
alkalinity.

Gradient systems also play an important role in the devel-
opment of sea urchin eggs. Cleavage in these eggs is very
regular. It results in a blastula, a vesicle with a wall consisting
of a single layer of cells with cilia on their outer surfaces. In
the next stage, the wall of the blastula invaginates at the
vegetative pole, thereby forming the archenteron of an embryo
called a gastrula. At the animal pole the gastrula possesses a
long tuft of cilia (Fig. 15a). The further development of a
gastrula into a pluteus larva involves the growth of long
"arms", and the secretion of a skeleton of calcareous rods by
cells originating from the vegetative wall of the blastula. More-
over, the larva is now surrounded by a band of cilia which also
extends over part of the arms (Fig. 15b).

Driesch has shown, as mentioned above (p. 28), that sea
urchin blastomeres isolated at the 4-cell stage can still develop
into a complete larva. This stimulated several investigators, in
particular Horstadius (since 1931) to study the developmental
potencies of blastomeres and groups of blastomeres, isolated
at later stages. In these investigations, the potencies of the
egg material were shown to change gradually from the animal
towards the vegetative pole. Isolated animal halves of a germ
will produce a larva with a large apical tuft, but without gut
and skeleton; isolated vegetative halves will grow into a larva

GRADIENT-FIELDS

45

with a large gut, and an irregular skeleton, but without either
apical tuft or band of cilia (Fig. 15). Evidently, the potency
for the formation of a gut decreases from the vegetative to-
wards the animal pole, whereas that for the formation of an

Fig. 15. Sea urchin larvae, (a) normal gastrula and (b) normal
pluteus; (c) embryo with a large tuft of cOia, produced by a frag-
ment of the animal side of an egg; id) embryo containing gut and
skeleton formed by a fragment of the vegetative side. After

Horstadius.

apical tuft decreases in the opposite direction. When cell groups
from different parts of the germ are brought together, more
or less normal larvae will result even from highly abnormal
combinations of blastomeres, so long as cells with animal and
cells with vegetative potencies are present in balanced
proportions.

Runnstrom (1928) has suggested that there are two op-

46

POl^ARITY AND SYMMETRY;

positely directed gradients in this case, one animal-vegetative,
and one vegetative-animal gradient. The fate of each cell will
be determined by the interaction of the two. Addition of certain
chemicals to the sea water surrounding the germ may produce
an ascendancy of one of the two gradients over the other.
Lithium ions suppress the animal gradient so that the vegetative
differentiation of the embryo begins to preponderate at the
expense of the animal differentiation (Fig. 16). Other sub-
stances, such as sodium thiocj^anate, have the opposite effect;
they reinforce the animal gradient at the expense of the
vegetative gradient so that the animal differentiation of the
embryo is promoted, and its vegetative differentiation inhibited.
Lindahl's investigations (1936) indicated that these gradients

Fig. 16. Abnormal sea urchin embryos, produced by treatment with

lithium, (a) exogastrula; (b-e) increasing growth of the endoderm

at the expense of the ectoderm; ect.: ectoderm; ent.: endoderm; Tnes.:

mesenchyme cells. After Herbst.

GRADIENT-FIELDS 47

were associated with two different metabolic processes, viz. the
animal gradient with carbohydrate metabolism, and the vege-
tative gradient with protein metabolism. Lithium was found to
inhibit carbohydrate metabolism. This would explain its in-
fluence on the development of the embryo. However, attempts
at a direct demonstration of metabolic differences between the
animal and vegetative halves remained unsuccessful. Moreover,
recent investigations, mainly those of Horstadius and Gustaf-
son (1950), indicate that lithium may also influence the syn-
thesis of proteins. Certain cell enzymes may be the primary point
of attack, and its influence on metabolism may be secondary.

On the other hand, the work of Ranzi and his collaborators
has shown that substances which in vivo influence the de-
termination of the sea urchin's egg, i7i vitro modify the
structure of various proteins with long chain-molecules. Lithium
and other substances which reinforce the vegetative gradient
of sea urchins' eggs were shown to effect condensation of these
proteins. Substances such as thiocyanate, which favour the
animal trends in development, in vitro caused a loss of fibrillar
structure in the proteins. From these facts, Ranzi (1947) con-
cluded that these fibrous proteins play an important role in the
determination process.

The egg of the snail Limnaea stagnalis furnishes another
example of the influence of gradient-fields in development.
Here again, treatment with lithium chloride was found to cause
characteristic disturbances in development (Raven, 1948, 1949).
It results in embryos in which the middle part of the head is
reduced so that eyes and tentacles (normally paired) unite
on top of the head (so-called cyclopia). In normal development
the parts that are suppressed by lithium develop from the cells
lying around the animal pole of the egg. Here, just as in sea
urchins, lithium evidently inhibits an animal gradient-field
which plays a role in normal development. In accordance with
Ranzi's views, lithium proved to have a condensing influence
on the cytoplasm of the egg in this case as well. On the other
hand, it caused increased swelling of the nuclei.

If the polarity of the egg is determined by axial gradients,
disturbance of these gradient systems may be expected to

48 POLARITY AND SYMMETRY;

cause changes in polarity. We have mentioned above (p. 31)
that the structure of the egg can be profoundly modified by
centrifuging. As a rule, the protein yolk is heavier, and the
fatty yolk lighter than the clear cytoplasm. Therefore, they
will accumulate at opposite poles, with the cytoplasm occupying
the middle zone of the egg (Plate III). The direction of this
stratification need not coincide with the original polarity, but
may be at any angle to it. In this way, the normal yolk gradient
and the correlated gradients of metabolism are completely
destroyed. Nevertheless, the further development of such eggs
shows that in the majority of cases their polarity has remained
unchanged. The egg material, displaced by centrifuging, often
after a short time returns to its normal position with regard to
the original egg axis (Raven, 1938b; Raven and Bretschneider,
1942); polar bodies are given off at the original animal pole;
cleavage and development of the embryo are oriented with
regard to the original polarity, and are independent of the
direction in which the centrifugal force has been operating.
Evidently, the factors governing polarity have not been shifted
by the centrifuging; they must be inherent in a component
of the egg that is not moved by the centrifugal force. Presumably
this is the more solid outer layer of the egg, the cortex.
Polarity, then, depends on factors localised in the egg cortex;
the axial gradients arise only as a result of these factors. There
may be some exceptions to this rule. In amphibians, for example,
the yolk gradient seems to be of decisive importance for the
direction in which the embryo will later develop. If the eggs
of an amphibian are turned upside down, and fixed in this
position, the heavy yolk material will sink through the cyto-
plasm from the vegetative side toward the originally animal
side of the egg. According to Motomura (1935) and Pasteels
(1938-39), this inversion by gravity of the yolk gradient results
in an inversion of the polarity of the egg.

Just as, in general, cortical factors are of importance for
polarity, so they are for symmetry. Pasteels has made a study
of amphibian eggs in which the internal structure was dis-
turbed by gravity or centrifugal force. Their further development
proved that the localisation of the primordia of the future

% ^>

PLATE III. Disturbance of the structure of the egg by centri-
fuging. Egg of a snail, Limnaea stagnalis, centrifuged before the
beginning of cleavage, i : Cap of fat with large vacuoles at tne
centripetal pole. 2: clear cytoplasm with few inclusions. 3: zone
of mitochondria. 4: centrifugal cap containing the protein yolk.
The animal pole is at the right, with the egg nucleus (e.n.) and
the two polar bodies (l.p.b. and 2.p,b.).

PLATE IV. The effect of genetic factors in hybrids of Triton taeniatus and
T. cristatus. (a-d) four larvae of about the same age. (a) Triton taeniatus;
(b) T. cristatus; (c) T. cristatus i X T. taeniatus cT . (d) T. taeniatus V X
T. cristatus cj". Note the white pigment cells in the caudal fin. (e-g) the
shape of the limbs in larvae of the same age. (e) Triton taeniatus; (f) T.
taeniatus f^ X T. cristatus (f. (g) T. cristatus. After Hamburger.

GRADIENT-FIELDS 49

embryo, and the direction of its plane of symmetry depend on
the interaction of two factors: (1) the yolk gradient, localised
in the internal cytoplasm, and (2) a gradient-field in the cortex,
the so-called "cortical field". The variable quantity in this
field is a hypothetical "C-factor", whose nature is yet unknown.
In normal eggs, its maximum intensity coincides with the centre
of the grey crescent; its minimum lies at the opposite side.
All gradients in this field run therefore in a dorsoventral
direction, and the planes of equal intensity intersect the egg
surface in a series of concentric circles. This field originates
during the formation of the grey crescent, which, according to
the work of Ancel and Vintemberger, is accompanied by a
contraction of the egg cortex towards the dorsal side (see
above, p. 41).

In sea urchins, cortical factors apparently also play a role.
Pease (1939) studied eggs of Dendraster which had been
centrifuged at a very high speed. He found that the position
of the ventral side of the embryo was determined by the inter-
action of a cortical gradient system with certain substances
localized in the internal protoplasm, which accumulate at the
centripetal side during centrifuging. This case, therefore, is
completely analogous to that of the amphibians discussed above.

Summarizing these facts, we can state with some confidence
that the directional organisation of the egg is governed by the
interaction of axial and cortical gradient- fields. These fields
determine the polarity and symmetry of the egg, and thereby
simultaneously determine the main directions of the future
embryo. This directional organisation of the egg provides it
with what may be called a fixed system of coordinates, and
all further developmental processes are oriented with regard
to this system. There are fixed relationships, therefore, between
the polarity and symmetry of the egg, and the organisation
of the future embryo.

Raven - Outline Physiologie

CHAPTER V

GhemodifFerentiation

In the foregoing chapters we have reached the conclusion that,
at the beginning of development, the egg protoplasm is of a
practically homogeneous constitution, and has a very low degree
of multiplicity, though there is a polar and bilateral structure,
governed by the axial and cortical gradient systems. We shall
now see how the spatial multiplicity of the structure increases
in the course of development.

Several investigations have made it plain that in many cases
the first step in this process consists of a local accumulation
of substances that previously were evenly distributed over the
egg. These accumulations exert a definite influence on the
fate of those parts of the germ in which they occur. Originally
it was assumed that they supplied the material for certain
organs of the future embryo, and for this reason they have been
named ''organ- forming substances''. Later this view proved to
be far too schematic. The substances accumulated in certain
parts of the germ do exert a determining influence on the
further development of these parts; they cannot, however, be
regarded simply as building material for the organs concerned.
It is better, therefore, to call them ''determining substances" .

Penners' work (1922-25) on the development of the worm
Tubifex may be cited as a first example. Soon after fertilisation,
accumulations of a special protoplasmic material occur both
at the vegetative and at the animal pole of the eggs of this
species. These masses are called the vegetative and animal
pole-plasm (Fig. 17 a). Experiments by Lehmann (1940) have
shown that this accumulation is probably caused by attractions
exerted by certain parts of the egg cortex on the material
concerned. For even in centrifuged eggs, in which the distribu-

CHEMODIFFERENTIATIOX

51

tion of the protoplasmic material had been profoundly modified,
the pole-plasm still assembles under the cortex at the two
original poles of the egg (Fig. 18). The physico-chemical
composition of the pole-plasm is slightly different from that
of the rest of the protoplasm. For one thing, it is very rich in
certain enzymes (oxidases; Lehmann, 1948). During further
development, the two masses of pole-plasm unite in the centre
of the egg. In Tubifex, as in most worms and molluscs, cleavage
has a characteristic very regular pattern. First the egg divides
into four blastomeres. At the animal side each of these then
buds off a number of small cells, the micromeres. In this way

'/Js/r

Fig. 17. Development of Tubifex. (a) uncleaved egg, (an r=: animal,
veg =r vegetative pole-plasm); (h) 22-cell stage, first and second
somatoblast have been formed; (c) formation of the mesodermal
germ bands (nies.k.str.) from the primary mesoderm cells
ioermes.c), (tel = teloblasts); (d) older embryo, from the right
side, with right ectodermal germ band (ect.k.str.), and, at the hind
end of the latter, the teloblasts (tel.). After Penners.

.52

CHEMODIFFERENTIATION

a liumbc. of "micromere quartets" are formed, surrounding
!iie aiiimal pole in a spiral arrangement (so-called ''spiral
cleavage''). At first, the pole-plasm is situated in one of the
first four blastomeres. Later, it is distributed over two of the
micromeres produced by this cell; these micromeres are larger
than the others. They are called the first and second somato-
hlasts (Fig. 17b). In later development, the first somatoblast
soon divides into a right and a left half. Each of these halves

Fig. 18. Eggs of Tubifex. a: normal ^gg, with mitotic spindle (sp),
and animal {an) and vegetative {veg) pole plasm; b: centrifuged
egg (centrifugal force parallel to the axis of the egg), strong
accumulation of pole-plasm (p.p.) at the centripetal pole (above),
whereas only little pole-plasm is formed at the centrifugal pole;
c: egg centrifuged with the centrifugal force acting at right angles
to the egg axis, animal pole (an) at one side; d: as c, somewhat
later, centripetal pole above; the pole-plasm has accumulated at the
place of the original animal and vegetative poles. After Lehmann.

CHEMODIFFERENTIATION 53

then divides into a number of large cells, the teloblasts. The
latter remain at the surface of the germ, and, by budding off
small cells toward the anterior end of the embryo, form a left
and a right ectodermal germ-hand (Fig. 17d). The second
somatoblast also divides into two. Its two daughter-cells, the
primary mesoderm cells, move into the deeper layers where,
again by budding off cells unilaterally, they give rise to a left
and a right mesodermal gerhi-hand (Fig. 17c) lying under the
ectodermal germ-bands of the same side. During further devel-
opment, left and right germ-bands gradually unite, the process
beginning at the anterior end. Together they form the primor-
dium of the future embryo. The nervous system and the circular
musculature will arise from the ectodermal germ-bands,
whereas the longitudinal musculature, the segmental excretory
organs, etc., will be produced by the mesodermal germ-bands.
Skin and gut will originate from the remaining cells which
contain no pole-plasm.

Penners irradiated the eggs with a very narrow pencil of
ultraviolet light. In this way he was able to kill certain cleavage
cells, thereby excluding them from further development. He
proved that the pole-plasm was indispensable for the formation
of the embryo. If the cells containing the pole-plasm had been
killed, the remainder of the e^g was unable to produce an
embryo. If, on the other hand, a number of other cells, together
constituting a large part of the volume, were eliminated, then
an abnormally small, but harmoniously built embryo would be
produced, so long as the somatoblasts remained intact. Elimina-
tion of the first somatoblast led to absence of the ectodermal
germ-bands, whereas the mesodermal germ-bands would be
present. The killing of the second somatoblast produced the
opposite result.

Application of heat causes abnormalities in the cleavage of
these eggs. In this case, the pole-plasm is often not restricted
to one of the first two cleavage cells only, but distributed evenly
over both of them (Fig. 19a). In further development, each of
the blastomeres will form a first and a second somatoblast,
containing pole-plasm (Fig. 19b). Two embryo primordia will
develop in such a germ; together they form a double monster

54

CHEMODIFFERENTIATION

/sm. 1

Fig. 19. a: egg of Tubifex, divided into two equivalent cells (7 and
II). Animal (an) and vegetative {veg) pole-plasm are both halved;
&: a similar ^^% during cleavage, two first {l.soin.I, LsomJI) and
two second somatoblasts i2.soni.I^ 2.som.II) have been formed;
c: the result is a double monster (so-called duplicitas cruciata)
with two pairs of germ-bands (7 and 77). After Penners.

(Fig. 19c). This shows clearly that the presence of pole-plasm
really is the factor to which the cells owe the potency to form
an embryo, or, in other words, the factor that determines them
to form an embryo. Therefore, the pole-plasm is a real determin-
ing substance.

CHEMODIFFERENTIATION

55

In the eggs of many worms and molluscs, a special pole-plasm
is accumulated at the vegetative pole. This shows a character-
istic behaviour during the first few cleavages. As soon as the
first cleavage furrows the egg, the pole-plasm becomes to some
extent separated from the rest of the egg, and forms the polar
lobe. This remains connected with one of the two blastomeres,
and later fuses with it so that all the pole-plasm is taken up
by one cell. This process may be repeated several times in the

O

Fig. 20. Cleavage in Dentalium. (a) uncleaved egg, with animal
pole-plasm and polar bodies at the animal pole, and vegetative
pole-plasm at the vegetative pole; (b) first cleavage, the vegetative
pole-plasm, now concentrated in the polar lobe, almost completely
separated from the rest of the egg; (c) 2-cell stage, the polar lobe
has fused with one of the cleavage cells; (d) second cleavage, polar
lobe has been formed anew; (e) 4-cell stage, polar lobe fused with
one of the cells; (/) third cleavage, polar lobe formed once more;
ig) 8-cell stage, pole-plasm in one of the cleavage cells. After Wilson.

56

CHEMODIFFERENTIATION

subsequent cleavages (Fig. 20). Here again the entire pole-
plasm finally finds its way into the first and second somato-
blasts. Wilson (1904) has shown experimentally that, in these
cases too, the pole-plasm is of great importance for further
development. Complete removal of the polar lobe results in
defective embryos, lacking certain organs ; partial removal leads
to poor development of these organs (Fig. 21d-f). Wilson
isolated parts of the eggs at later stages as well. In such cases
the cells that contained the pole-plasm differentiated into small
larvae, which were complete, though not harmoniously built,

Fig. 21. Dentalium. (a) normal trochophore larva; (h) larva from
an isolated blastomere without pole-plasm; (c) larva from a cleavage
cell with pole-plasm; (d) larva from an egg, the first polar lobe of
which had been removed; (e) as d, but only half of the first polar
lobe resected; (/) larva developing from an egg after resection of
the second polar lobe. After Wilson.

CHEMODIFFERENTIATION 57

showing an overdevelopment of the organs concerned. An
isolated group of blastomeres without pole-plasm developed
into a defective embryo (Fig. 21a-c). Under abnormal cir-
cumstances the polar lobe may be distributed equally over the
first two blastomeres; this results in the formation of a double
monster, in the same way as in Tubifex. But if the two blast-
omeres of such eggs become completely separated, each will
develop into a complete embryo (Titlebaum, 1928). Wilson
(1929) studied the development of fragments of eggs which
had been halved before the beginning of cleavage. He found
that the pole-plasm was already present in the unfertilised egg.
At first, it is distributed homogeneously over the egg plasm;
later it assembles near the vegetative pole. This does not always
happen at the same time; in some species, it takes place in the
unfertilised egg, in others immediately after fertilisation, or
a short time before the first cleavage.

Interesting results were also obtained by Wilson in his work
on the egg of the mollusc Dentdlium. In this species, a polar
lobe is formed three times, viz. at the first, second, and third
cleavages (Fig. 20). Dentalium has a so-called trochophore
larva, i.e. a larva characterized by a ring of cilia all round the
body. This ring separates the anterior pretrochal region from
the posterior posttrochal region. The animal side of the larva
bears a group of cilia, the so-called apical organ. Removal of
the polar lobe at the first cleavage results in a larva without
an apical organ, and with a reduced posttrochal region (Fig.
21d). Removal of the polar lobe during the second cleavage
produces larvae in which the posttrochal region is still reduced,
but which usually possess an apical organ (Fig. 21f). Evidently,
the factor responsible for the development of an apical organ
is located in the first, but not in the second polar lobe. There-
fore, in the interval between first and second cleavages, a shift
of the determining substances concerned must take place in
the egg protoplasm. This shows that these substances are "pre-
formed" but not "prelocalised" in the egg; they can change
their position during development.

The development of the ascidian egg provides another good
example of the local accumulation of determining substances.

58

CHEMODIFFERENTIATION

In the protoplasm of newly laid eggs of this group, three differ-
ent substances can be distinguished because of their different
pigmentation. At the animal pole, we find the ectoplasm. This
has originated in the course of maturation from the nuclear
sap of the oocyte nucleus; on the disappearance of the nuclear
membrane, this sap mixes with the surrounding e^g proto-
plasm. A thin layer of mesoplasm lies at the surface of the egg,
and the rest of it comprises the endoplasm (Fig. 22a). Soon

Fig. 22. The egg of an ascidian, before (a) and after (b) fertilisa-
tion, ect: ectoplasm; end: endoplasm; mes: mesoplasm; ch.n: chor-
doneuroplasm. After Conklin.

after fertilisation, complicated streaming movements of these
substances can be observed. Ultimately the following distribu-
tion is reached: the ectoplasm occupies the animal side, and
the endoplasm the vegetative side of the egg; the mesoplasm
has accumulated at the ventral side, near the equator, forming
the so-called "yellow crescent". Meanwhile a fourth substance,
the chordoneuroplasm has appeared at the dorsal side of the
egg, also near the equator, where it forms the so-called "grey
crescent" (Fig. 22b). Consequently, the egg now has a clear-cut
bilateral symmetry. In the course of cleavage, the various
cytoplasmic substances become localized in different cells. In
further development they will give rise to different organs and
tissues of the embryo. The skin is produced by the cells contain-
ing the ectoplasm; those with the endoplasm will form the gut.
The cells of the yellow crescent, which contain the mesoplasm,
will supply musculature and connective tissue, and the chordo-
neuroplasmic cells of the grey crescent will develop partly into
notochord and partly into central nervous system.

CHEMODIFFERENTIATION

59

Several experiments have proved that the cells containing
the different types of protoplasm really do have different
developmental potencies. Isolated blastomeres produce almost
exclusively structures to which they would have given rise
in normal development (Conklin, 1905-11). If all the cells con-
taining a particular type of protoplasm are removed, the larva
will lack the organs concerned (Von Ubisch, 1939). Centrifuga-
tion modifies the relative positions of the protoplasmic sub-
stances in the uncleaved egg ; such treatment results in embryos
in which the various tissues and organs are present, but mixed

<?c^r. _

^'cjcV^^:

Fig. 23. (a) an ascidian larva, seen from the right, after Kowa-
lewsky; (b-c) abnormal embryos formed by centrifuged eggs, after
Conklin. ect =■ ectoderm, ent = endoderm, mes = mesoderm, tnch^=^
mesenchyme, zw = nervous tissue, ch = notochord, hh =^ brain
vesicle, st = statocyst, o ^ eye spot.

60 CHEMODIFFERENTIATION

up in a highly abnormal manner (Conklin, 1931) (Fig. 23).

From these and similar experiments it has been concluded
that the various protoplasmic substances may be regarded as
determining substances for the various organs of the embryo,
i.e. that they steer the development of the cells in which they
have become located in a certain direction. However, later
experiments by Dalcq (1938), Meryl Rose (1939), Reverberi
and Minganti (1946-49), and others, point to the fact that the
ascidian egg must not be regarded as merely a mosaic of
independently developing primordia. Complicated interactions
between the parts play a role in the development of these eggs
too. Yet, apart from these interactions, the localisation of the
protoplasmic substances undoubtedly is important for the devel-
opmental potencies of the cells. This applies in particular to
the mesoplasm, which does apparently determine the differ-
entiation of the cells in which it is present into muscle and
connective tissue, and is, therefore, a real determining sub-
stance. In this case, again, the localisation of the various cyto-
plasmic components after fertilisation is probably due to the
influence of factors in the egg cortex.

Using histochejnical methods, Ries (1939) has proved that
the mesoplasm of ascidians is characterized by the presence of
certain enzymes (oxidases and peroxidases), as is the pole-plasm
of Tuhifex. This enzyme-containing protoplasm can be displaced
by centrifugation. Now Ries has shown that in embryos devel-
oping from centrifuged eggs muscle tissue originates from those
cells that happen to harbour the enzyme-containing plasm
(Fig. 24). This is a strong argument in favour of the hypothesis
that these enzymes themselves are responsible for the determina-
tion of the cells to muscular tissue. In other words, they may
be regarded as the determining substances in question. These
enzj^m.es are known to be important for cell metabolism; it is
probable, therefore, that they exert their influence through the
metabolic processes. This modification in the metabolism, in
its turn, may be the cause of the differentiation.

We have seen above that the original conception of ^'organ-
forming substances'' supplying the building material for definite
organs proved to be an oversimplification. We have then re-

CHEMODIFFERENTIATION

61

placed it by the hypothesis of "determining substances" which
would determine the cells to a certain development. In recent
years, however, the view has gained ground that this conception
is still too schematic, and that the actual relationships are
probably even more complicated. Presumably the differentiation
of a cell is not determined by only one determining substance.
It is more likely that the direction taken by the development
of any cell depends on the mutual quantitative relations of a
great number of substances, which together govern the cell
metabolism, e.g. enzymes, vitamins, etc. Slight differences in the

Fig. 24. Abnormal embryos, formed by centrifuged eggs of
ascidians. Left: living larvae; the lines mark the places where
muscle contractions have been observed. Right: the same animals
after fixation and benzidine treatment for the demonstration of
peroxidases. Positive reaction (dotted areas) in the regions where
muscle cells occur. After Ries,

62 CHEMODIFFERENTIATION

amount of these substances will modify the ''histochemical equi-
librium" and thereby the direction of development. Peltrera
(1940) has given a clear statement of this view on the basis of his
work on the distribution of various physico-chemically important
substances in the egg of the marine snail Aplysia. What we
have so far called "determining substances" are probably only,
for some fortuitous reason, the more conspicuous components
of this complicated system of factors; they may themselves be
mixtures of a great number of substances.

We have discussed in this chapter how, after the beginning
of development, the originally more or less homogeneous egg
cytoplasm acquires a complicated spatial structure because of
the concentration of various substances in definite parts of the
egg. These substances may at first have been evenly distributed
throughout the cytoplasm, their accumulation now taking place
under the orienting influence of the egg's "coordinate system",
especially its cortex (see p. 49). On the other hand it is possible
that the substances were not originally present in the egg, but
that they arose de novo as a consequence of chemical reactions
taking place in the egg, e.g. of such an interaction between its
axial and cortical gradient systems, as was assumed by Dalcq
for the amphibian egg. At any rate, the various parts of the
egg, which were all alike before, now begin to show differences
in chemical composition. Following J. S. Huxley, we may call
this phenomenon "chemodifferentiation".

The differences in physico-chemical composition, now existing
between the parts of the germ, result in differences in their
developmental potencies. In broad outline, the egg at first was
an "equipotential system"; now, it is divided up into a mosaic
of parts with diverging potencies.

Chemodifferentiation may set in at earlier or later stages in
development. We have seen above (p. 58) that in ascidians the
uncleaved egg is already a mosaic of different plasmatic sub-
stances. In such a case, the developmental potencies of the
different parts of the germ are unequal from a very early
stage, and development has the character of a "mosaic devel-
opment". In other cases chemodifferentiation begins much later,
the various parts of the germ remaining equivalent for a long

CHEMODIFFERENTIATION 63

time, both physico-chemically and as regards their potencies.
One half of an egg, or two fused eggs, can develop into a single
harmoniously built embryo in this case. The one condition for
this to occur is, of course, that after the disturbance the co-
ordinate system of the egg can readjust itself by the formation
of a new equilibrium, essentially similar to the original one,
between the axial and cortical gradient-fields. Even in later
stages the addition or removal of material may still be regulated
in these cases. Such eggs have the character of "regulation
eggs". Therefore, the difference between mosaic eggs and
regulation eggs is due mainly to an earlier or later occurrence
of chemodifferentiation.

The accumulation, in definite places in the egg cytoplasm,
of preformed or newly built determining substances constitutes
the first beginning of spatial multiplicity in the egg. At first,
this spatial structure is still very simple, but we can imagine
how this situation will be the starting point of increasing com-
plexity. The local concentration of certain substances will start
chemical reactions which previously were unable, or almost
unable, to take place because of the dilution of the reagents,
or because of the presence of inhibiting substances. New sub-
stances will thereby be produced in the egg, which again may
be locally concentrated. These local accumulations will affect
the physical conditions (pH, rH, interfacial tension, electrical
relationships), and the intensity of metabolism in the areas
they occupy. They will also interact, e.g. by mutual attraction
or repulsion. All these factors give increasingly complex pro-
perties to this physico-chemical system, whose spatial multi-
plicity originally was characterised by polarity and symmetry
only. Once started, chemodifferentiation will steadily proceed,
and the egg's intensive multiplicity will be more and more
transformed into an extensive multiplicity.

So far we have considered the egg cytoplasm only. We shall
now discuss a new complication in the system of actions and
interactions in the egg, namely the activity of the complex of
factors localised in the nucleus.

CHAPTER VI

The realisation of the nuclear factors

We have seen in Chapter II how in fertilisation the nuclei of
egg and sperm unite to form a single nucleus, the zygote nucleus
or synkaryon. This contains the chromosomes of both nuclei,
i.e. it has a double set of chromosomes. Genetical and cyto-
logical research, which lies outside the scope of this book, has
shown that we must regard the material carriers of heritable
properties, the so-called genes, as localised in the chromosomes.
Therefore, the nucleus of the fertilised egg contains two sets
of genes, one paternal, and one maternal; these transmit the
heritable characters of both parents to the new individual.
These characters are thus present, in an undeveloped form, in
the fertilised egg, and must be realised in the course of the
embryo's development. We shall now discuss this process.

During cleavage, the synkaryon divides first into two, then
into four, and so on, until a great number of cleavage nuclei has
been formed, one in each blastomere.

We have seen above (p. 35) that, according to Weismann's
theory, there would be a qualitative difference between the
products of these nuclear divisions. The hereditary factors
(Weismann's "determinants") would thereby be distributed
over the various cleavage nuclei so that, in the end, each cell
would contain certain of these factors only. We have also dis-
cussed the experiments which refute this theory; there are in
fact no qualitative differences among the products of the
nuclear divisions during cleavage. At least in the earlier stages
of development, the cleavage nuclei are identical with each
other, and with the zygote nucleus. Each of them contains all
the genes, and there are good grounds for the assumption that
even in the course of further development only genotypically

THE REALISATION OF THE NUCLEAR FACTORS 65

equivalent nuclei are formed, so that all the nuclei of the
organism (possibly with a few exceptions) will contain the full
complement of genes typical for the species.

For this reason, Weismann's hypothesis that the whole of
development is governed by the nuclear factors cannot be
correct. On the contrary, nuclei and cytoplasm co-operate in
this process. We have discussed the complicated interplay of
the developmental processes in the cytoplasm. Evidently, the
nuclear factors must in some way or another exert their in-
fluence on these processes.

It has been known for a long time that the nucleus is
generally indispensable for the life of the cell. Enucleated cell
fragments or protists may survive for a while but they show
no further development, and finally they die. In addition, the
nucleus is often to be found in those parts of the cell where
growth or differentiation takes place. This observation sug-
gests that the nucleus plays a leading, or at least an important,
role in these processes. Many investigators claim to have proved
that substances or particles produced by the nucleus pass into
the cytoplasm, either migrating through the nuclear membrane,
or mixing with the cytoplasm when this membrane has dis-
appeared during nuclear division (cf. what has been said above,
p. 58, about the ectoplasm of the ascidians). In many eggs the
nucleoplasm of the germinal vesicle has been shown to be rich
in sulphydryl compounds. These are poured out into the cyto-
plasm when the nuclear membrane disappears. It has also been
demonstrated that the nuclei of many cells (e.g. oocytes and
neurones) produce ribonucleic acid. This substance diffuses
through the nuclear membrane into the cytoplasm (Caspersson,
Hyden), where it may be used in the construction of microsomes
and mitochondria. The microsomes are probably concerned
mainly with protein synthesis (cf. p. 34), the mitochondria with
the cell's energy metabolism. The mitochondria are the site of
energy-supplying oxidation processes, as well as of phosphor-
ylations, which fix the liberated energy in the form of energy-
rich phosphate bonds, especially as adenosine triphosphate.
Substances produced by the nucleus are supposed to ensure
the coupling between oxidations and phosphorylations (Brachet,

Raven - Outline Physiologie 5

66 THE REALISATION OF THE NUCLEAR FACTORS

1952). In this way the nucleus may govern the main processes
of metabolism and construction in the cell.

It is well known that the nucleus plays an important role in
cell division. It might be said that, at least in uni-nuclear cells,
each cell division is preceded by a nuclear division. Cell frag-
ments without a nucleus will not divide any more. This proves
that, generally speaking, there is a fixed causal relation between
nuclear and cellular division. This statement, however, can be
applied to the egg only with certain restrictions. In certain
cases, eggs that contain no nucleus at all are nevertheless able
to divide. E. B. Harvey (1938), for instance, treated enucleated
fragments of sea urchin eggs with parthenogenetic agents. This
led to cleavage, and even to developmental processes ("partheno-
genetic merogony") which went as far as to produce a more or
less normal looking blastula, consisting of cells without nuclei,
but then came to a stop. The same phenomenon was found in
amphibian eggs. This exception to the rule that non-nucleated
cells cannot divide can probably be explained as follows. During
the growth of the oocyte and after the disappearance of the
nuclear membrane of the germinal vesicle, a large quantity of
products of the nucleus has been accumulated in the cytoplasm
of those eggs. Divisions can go on in the absence of the nucleus,
therefore, until this store is consumed. It is significant, how-
ever, that in not even a single case has further differentiation
of non-nucleated cells been observed.

The following observation also suggests that there is a
certain measure of autonomous activity in the cytoplasm during
cleavage. If the polar lobe of certain mollusc eggs is removed
at the first cleavage, rhythmic contractions and changes in
shape can be observed in the non-nucleated, isolated polar lobe ;
these movements coincide with the subsequent cleavages of the
rest of the egg.

The correlation between the size of the cell and that of its
nucleus must also be mentioned here. This was studied, for
instance, by Boveri (1905) in sea urchin eggs. He compared
parthenogenetic or merogonic eggs (see p. 18 and 10), which
contained only half the normal number of chromosomes, with
normal eggs, and with eggs containing double the normal

THE REALISATION OF THE NUCLEAR FACTORS 67

number. The latter he obtained by shaking normal eggs a few
minutes after fertilisation; this caused irregularities in the
first nuclear division. Boveri found that the size of nuclei and
cells depended on the number of chromosomes they contained.
When the chromosome number was doubled, both the size of
the nucleus (according to Boveri its surface area), and the
volume of the cell were doubled too. However, the total number
of cells of which the embryo will eventually consist, is inversely
proportional to the number of chromosomes. This is due to
the fact that in embryos with higher chromosome numbers
(larger nuclei) the cell divisions are slower, and fewer in
number. Therefore, the final size of the embryo is more or less
constant and independent of the chromosome number.

The validity of these so-called "rules of Boveri" has repeat-
edly been confirmed in other animals, though it was not the
surface area but the volume of the nucleus which was, as a
rule, found to be proportional to the chromosome number.
Evidence was also obtained that the regulation of the body
size can go even farther. In amphibian larvae with chromosome
numbers varying from haploid to pentaploid, and accordingly
with cells of very different sizes, it was found that not only the
total body size, but also the thickness of the Vx^alls of the organs
was approximately constant. This implies that not only the
number of cells varies with the chromosome number, but their
shape as well (Fankhauser, 1945). However, this regulation
of the body size is not without exceptions. In plants and in
several invertebrate animals, polyploids often are larger than
normal diploid specimens, at least in the first few generations
after the production of polyploidy.

During cleavage the egg cytoplasm is distributed over a great
number of cells. In as far as chemodifferentiation has already
taken place, there will be differences in physical and chemical
properties of the cytoplasm among the various blastomeres.
Therefore, the genotypically identical cleavage nuclei will be-
come located in non-identical parts of the egg cytoplasm. The
interactions of these nuclei with the cytoplasm surrounding
them will result in differences among the reactions that are
set going in the various parts of the egg. The nuclear factors

68

THE REALISATION OF THE NUCLEAR FACTORS

will influence the composition of the cytoplasm, and, vice versa,
the cytoplasm will influence the nuclei and direct the course of
their development in divergent ways. We shall first discuss an
example of the latter effect.

In the parasitic nematode, Ascaris megalocephala, the nucleus
contains a very small number of chromosomes; in one race, the
so-called univalens race, there are only two. Consequently, the
division process can very easily be followed in these nuclei, and

Fig. 25. Chromatin diminution in Ascaris megalocephala. (a-b)
second cleavage, diminution in AB, normal division of the chromo-
somes in Pi; (c) 4-cell stage; (d) new arrangement of the cleavage
cells, diminished nuclei in A and B, diminution taking place now in
EMSt, and normal division in P2. After Boveri.

THE REALISATION OF THE NUCLEAR FACTORS 69

this animal constitutes an excellent subject for cytological
research. The fertilised egg of the worm contains two long,
curved chromosomes. At the first cleavage, the egg divides into
an animal and a vegetative blastomere. At the second cleavage,
each of these divides once more into two cells, and, during this
division, a peculiar process, known as chromatin diminution,
take place in the animal blastomeres. The ends of the chromo-
somes are thrown off into the cytoplasm, where they dis-
integrate, and the middle part of each chromosome divides into
a number of fragments (Fig. 25a). At this stage, no diminution
has yet occurred in the vegetative blastomere. In the next
cleavage, however, this will take place in one of its daughter-
cells (Fig. 25d). The process is repeated several times, and in
the end all cells contain diminished chromatin, except for a
single cell, Vv^hich still has two chromosomes of the original
shape. This cell contains cytoplasm that was originally located
at some distance from the vegetative pole of the egg, and
it is from this cell that the primary germ-cells will develop,
which supply the building material for the gonads of the new
individual.

Boveri (1910) investigated and experimented on both normal
and abnormal eggs of Ascaris. He put forward the hypothesis
that the nature of the cytoplasm into which the nucleus finds
its way at cleavage determines whether or not diminution will
occur. The cytoplasm originally located at the vegetative pole
has different properties from that of the rest of the egg. For
this reason, chromatin diminution would not take place in this
cytoplasm. Although later observations by Von Ubisch (1943)
and Pasteels (1948) have led to a modification of this view, the
fact remains that evidently the development of the nuclei is
influenced by local differences in the egg cytoplasm.

The dragon fly Platycnemis pennipes, investigated by Seidel
(1929-34), provides another clear-cut example of the interaction
of nucleus and cytoplasm. This insect has oblong eggs, the
hinder ends of which have special properties. Seidel named this
region the activating centre, because it is of decisive importance
for the further development of the egg. If at an early stage of
development this centre is killed with a hot needle, or separated

70

THZ; REALISATION OF THE NUCLEAR FACTORS

from the rest of the egg by means of a tight ligature, no
embryo will develop in the anterior part of the egg (Fig.
26 a-d). The activating centre does not begin its activity, how-
ever, until one of the cleavage nuclei has reached it. Cleavage
in insects differs from that in other animals in that the zygote
nucleus, which is located in the middle of the egg, divides
repeatedly without accompanying cell-divisions. Therefore, the
cleavage nuclei can spread freely throughout the egg. It is only

Fig. 26. The activating centre in Platycnemis pennipes, and its

effect, (a) constriction of a very small part of the caudal end of the
egg does not prevent the development of the primordium of an
embryo (K), from which a normal embryo will later develop (b);
(c) constriction of a larger part prevents the formation of an
embryo (d); (e) incomplete ligature prevents the cleavage nuclei
(black dots) from reaching the activating centre; again, no embryo
is formed in this case. After Seidel.

when a large number of these nuclei has been formed that they
migrate to the superficial layer of the egg cytoplasm. Then each
nucleus, with the surrounding cytoplasm, is partitioned off
from the remainder of the egg to form a separate cell (so-
called ''superficial cleavage''). Now Seidel constricted the eggs
just anteriorly to the activating centre so tightly that the
cleavage nuclei were unable to reach the centre through the
narrow passage that remained. In that case no further devel-
opment took place (Fig. 26e). But if one nucleus manages to
reach the activating centre, a substance is produced there
whieh diffuses into the anterior regions of the egg, and there

THE REALISATION OF THE NUCLEAR FACTORS 71

sets the development of the embryo going. The diffusion of this
substance is not impeded by an incomplete constriction of the
egg. Evidently, the production of this essential activating sub-
stance is due to an interaction between the cytoplasm of the
activating centre and a cleavage nucleus. All cleavage nuclei
are equivalent also in this respect. Seidel demonstrated this
as follows. He killed the hindmost nucleus, a descendant of
which would in the normal course of events move into the
activating centre, by irradiation with a narrow beam of ultra-
violet light. The place of this nucleus was then taken by one
of the other cleavage nuclei, and development proceeded in the
normal way.

These experiments give us a clue as to the way in which the
nuclear factors influence development. During cleavage, the
nuclei have become located in areas of different physico-
chemical properties. Once they begin to interact with their
environment, in certain parts of the egg chemical reactions
will occur between the nuclear factors and the cytoplasm of
those areas. Nuclear factors, which have so far remained in-
active, can begin to unfold their activity once they have found
a suitable substrate on which to act, or with which to react.
It may be said, therefore, that they are activated by the sur-
rounding cytoplasm. In other parts of the egg, where the cyto-
plasm is of a different composition, the reaction in question
will not occur, but other nuclear factors may be activated there.

We have not yet entered into the question of the nature of
these nuclear factors. It is natural in this connection to think
of the genes, which we may regard as localized in the chromo-
somes. An experiment by Boveri (1907) demonstrates that
the chromosomes do indeed play an important role in devel-
opment. If sea urchin eggs are fertilised with concentrated
semen, into many eggs two sperms will penetrate simultaneously
(so-called double fertilisation). As a rule both sperm nuclei
fuse with the egg nucleus in such eggs, forming a "triploid"
zygote nucleus, containing three sets of chromosomes, instead
of two as in normally fertilised, "diploid", eggs. At the first
cleavage the majority of such abnormal eggs divides at once
into three or four daughter-cells simultaneously. This, according

72

THE REALISATION OF THE NUCLEAR FACTORS

to Boveri, is due to the fact that each of the penetrating sperms
has introduced a centrosome into the egg. One, or more often
both, of these centrosomes will divide into two, giving rise
to a total of three or four centrosomes which then will constitute
the poles of a three- or four-polar mitosis. The haploid chromo-
some number of these sea urchins is 18. The triploid zygote nucleus
therefore contains 54 chromosomes. Each of them doubles it-
self, and the members of each pair move apart to the neigh-
bouring poles of the spindle (Fig. 27). As the distribution of
the chromosomes over the three or four spindles is entirely
random, it is a matter of chance how many chromosomes will
arrive at each of the poles, and combine to
form a daughter-nucleus. The average number
will be 108 : 4=27 in the case of a four-polar,
and 108 : 3=36 in that of a three-polar mitosis,
but the numbers may vary widely in individual
cells.

By two different methods Boveri made a care-
ful study of the further development of eggs
after double fertilisation. Sometimes he allowed
complete eggs to develop, but at other times
he separated the three or four simultaneously
formed daughter-cells by bringing them into
calcium-free sea water (this simple method
for the isolation of the blastomeres was dis-
covered by Herbst). Boveri found that, on
the whole, the isolated cells developed poorly,
but that there were great individual differ-
ences. Some cells died very soon, others devel-
oped better and produced fairly normal small
larvae. Experiments with whole eggs had the
same result. As a rule, cleavage was normal,
but then development came to a stop in certain
sectors of the egg, and the cells of those

Fig. 27. Diagram of a division of the tetraster type in a sea urchin
egg after double fertilisation; the numbers indicate the distribution
of the chromosomes over the various spindles and daughter nuclei.

After Stomps.

THE REALISATION OF THE NUCLEAR FACTORS

73

parts died. In general, eggs which had divided into three devel-
oped better than those which had divided according to the
tetraster type.

These facts give clear indications already as to the
importance of the chromosomes, because the difference be-
tween the three or four simultaneously formed cells lay in
their chromosome complements. But Boveri was able to do
more. He determined the size of the nuclei in the various parts

Fig. 28. Diagram of the distribution of the chromosomes in a
tetraster division, taking place in a sea urchin egg after double
fertilisation, taking 4 as the haploid chromosome number, (a) the
12 chromosomes of the triploid egg become distributed at random
over the 4 spindles; (b) consequently, only one of the cells (bottom
left) receives a complete set of chromosomes, a, b, c and d.

After Boveri.

of germs developing from an intact egg, and in germs develop-
ing from isolated blastomeres. As we have seen, this is pro-
portional to the chromosome number. This method enabled
Boveri to establish that the success of development did not
depend only on the number of chromosomes present in the
nucleus. Sometimes parts with a larger nucleus were less
successful than other parts with a smaller nucleus, i.e. with a
smaller chromosome number. Boveri concluded that the result
of development is determined by the combination of chromo-
somes. He suggested that there were qualitative differences
between the chromosomes, and that only those parts of the egg
could develop normally which had at least one complete
(haploid) set of chromosomes in their nucleus (Fig. 28).

74 THE REALISATION OF THE NUCLEAR FACTORS

Later investigations have shown, indeed, that absence of one
chromosome, or even of a small part of a chromosome, is
sufficient to bring development to a stop at an early stage.
Poulson (1940-45), for instance, studied the consequences of
"deficiencies" of varying extent in the X-chromosome of the
fruit fly, Drosophila. Complete absence of this chromosome
resulted in disturbances as early as the end of cleavage, at the
moment when the cleavage nuclei migrate to the surface of the
egg (cf- p. 70). In the case of absence of one particular half
of the X-chromosome the disturbances also became manifest
at the same stage, but the distribution of the cleavage nuclei
over the surface layer was slightly less abnormal. If the other
half of the chromosome was absent instead, the superficial
cell layer developed normally, but no embryo was formed.
Finally, absence of small parts of the chromosome led to certain
characteristic aberrations at later stages, e.g. in the develop-
ment of the nervous system or of the gut.

These investigations prove that the full complement of
chromosomes is necessary for normal development. Since the
absence of very small parts of a chromosome may result in the
disturbance of very definite developmental processes, it seems
probable that the normal course of these processes depends
on the presence of special genes, located in these parts.

We owe our insight into the way in which the genes work
in the first place to a number of investigations on so-called
lethal genes, i.e. genes which lead to the death of the embryo
at some given moment during its development. Sometimes these
genes take effect even when heterozygous, in other cases only
if homozygous. Each of them was found to exert its influence
at one definite stage of development, and often also at one defi-
nite place, by directing the course of development of some organs
or parts of the embryo into abnormal channels, leading to the
death of the embryo. In many cases, however, one gene proved
to influence several developmental processes, localised in differ-
ent parts of the germ, at the same time. Side by side with the
dying tissues, other organs on which the lethal factor has no
influence often occur in the same embryo. If such tissues are
removed in good time, and grafted into a healthy embryo, they

THE REALISATION OF THE NUCLEAR FACTORS 75

are able to develop normally in spite of the fact that the nucleus
of each of their cells contains the lethal factor that caused the
death of the other tissues. The importance of work of this kind
lies in the fact that from the mode of operation of these lethal
genes, the conclusion can be drawn that the corresponding
processes in normal development are governed by normal genes.
This can be seen as follows: lethal genes have originated from
normal ones by mutation. Consequently, the resulting disturb-
ance in development must be due to the disappearance, or
modification, of the normal action of the gene. Indirectly,
therefore, these observations give us information as to the time
and place at which the genes exert their influence in normal
development.

The genes co-operate with the cytoplasmic factors. We can-
not find out, simply by looking at the result, what has been
the part played by each in the origin of the embryo. In order to
assess the relative importance of cytoplasmic and nuclear
factors, we must combine them in such a way that the groups
can be told apart by their specific recognition marks. This can
be done by combining nuclear material of one species with egg
cytoplasm of another ; the properties of the embryo that results
from such a combination may give some information on the
problem under consideration.

The crossing of two related species is the simplest way to
achieve this. It results in eggs, the cytoplasm of which is almost
entirely maternal, whereas the nucleus contains genes of two
species, because it arises from the fusion of egg and sperm
nuclei. One might expect that it would be very easy to conclude,
from the development of such hybrids, when and how the action
of the paternal genes becomes manifest in development.

In practice, however, this type of crossing experiment is
fraught with certain difficulties which seriously reduce its
usefulness for the solution of our problem. Firstly, crosses of
somewhat distantly related species do not as a rule give viable
embryos. Broadly speaking, it can be said that the more remote
the two parent species are from each other, the earlier the
development of the hybrids will come to a stop. However, taxon-
omic affinity is not the only factor to be considered, as may be

76 THE REALISATION OF THE NUCLEAR FACTORS

seen from the fact that sometimes the result of the combination
of eggs of species A with sperm of species B is entirely different
from that of the opposite combination. The arrest of devel-
opment may be due to different factors. Sometimes disturbances
occur in the nuclear divisions so that the distribution of the
chromosomes among the cells is abnormal. In other cases, the
cells show pathological phenomena from a given moment on-
wards. These may be restricted to certain parts or organs of
the germ, whereas other parts are not affected, or, on the
other hand, they may be present more or less equally in all
cells. This in itself does prove that for normal development
nucleus and cytoplasm must be "attuned" to each other, but
it makes a further analysis of the role of each group of factors
impossible in these cases. Hybridisation of more closely related
species is attended with another difficulty. The characters in
which these species differ do not appear until a very late stage
in development. During the major part of the process, the
hybrid is indistinguishable from either parent, and if there are
any influences of the paternal genes, they cannot become visible.
Their activity becomes manifest only at the end of development,
when all the more essential steps in the process have long ago
been completed.

In spite of these obstacles hybridisation experiments have
in a few cases thrown some light on the problem under dis-
cussion. We shall now discuss the results of such work with
sea urchins, and with some species of amphibia.

In accordance with the foregoing remarks, the result of a
cross between two sea urchin species depends very much on the
species used. In the most favourable cases, the hybrids will
develop into normal pluteus larvae. This will occur in particular
if the hybridised species are closely related, e.g. in the cross
between Psammechinus microtuherculatus and Paracentrotus
lividus. The plutei of these species are very similar, and can
be distinguished only by slight details in the structure of the
calcareous rods. In a careful investigation, Horstadius (1936)
obtained proof that the hybrid larvae were intermediate between
the parent species on this point. The same result can be seen
more clearly in the case of the fertilisation of eggs of Sphaer-

THE REALISATION OF THE NUCLEAR FACTORS

77

echinus granularis with sperm of either Psammechinus or Para-
centrotus. The plutei of these forms (Fig. 29) show marked
differences. Those of Spkaerechinus are of a plumper shape;
the relative lengths of the arms are different, and the structure
of the calcareous rods is more complicated. One of the latter,
the anal rod, consists of 3-5 parallel rods with cross connections.
Another, the apical rod, has three prongs at its top. In Para-
centrotus, the anal rod is single, and the apical rod club-shaped,
but not branched (in Psammechinus the latter shows an
incipient branching; see below p. 83). Now it was found that
the hybrid larvae were intermediate, both in shape and in

Fig. 29. Pluteus larvae of (a) Sphaerechinus granularis, (h) Psamm-
echinus micro tuberculatus, and (c) the hybrid Sphaerech. gran. X
Psammech. microtuberc. After Boveri.

structure of the skeleton. Their anal rods consisted of 2-3 rods,
without cross connections; their apical rods were club-shaped,
but also showed a tendency to branch (Boveri, 1889-95). It is
a remarkable fact that, apart from intermediate larvae, other
larvae are sometimes found with a preponderance of paternal
characters ("patrocline" larvae). Conditions in the culture
seem to play a role here ; in particular the temperature appears
to influence the structure of the calcareous skeleton.

In this case, the influence of the paternal chromosomes on

78 THE REALISATION OF THE NUCLEAR FACTORS

development was unmistakable. Other crosses, however, had
different results. In many cases, the hybrid was entirely similar
to the species to which the eggs belonged (so-called matrocline
hybrids), e.g. in the cross Sphaerechinus Q X Arhacia ^. In
other cases, the incompatibility of the paternal chromosomes
with the surrounding "foreign" cytoplasm manifested itself in
a somewhat dramatic way. In the cross Paracentrotus Q X
Arbacia r^ , the paternal chromosomes take a normal part in
the nuclear divisions during cleavage, but at the blastula stage
they are suddenly expelled into the cytoplasm where they dis-
integrate. From then on development is disturbed. We have
already seen that the cross Sphaerechinus X Psammechinus (or
Paracentrotus) gives normal hybrids of intermediate structure.
The reciprocal cross, however, Psammechinus (Paracentrotus)
X Sphaerechinus has an entirely different result. The paternal
chromosomes do unite with the egg nucleus, but in the first
or second division, 16 of the 20 Sphaerechinus chromosomes are
eliminated, and such development as takes place is matrocline.

In the experiments here described, the activity of the paternal
chromosomes did not become visible until the pluteus stage.
Yet an investigation by Tennent (1922) has proved that even
before that stage they play a role in development. Tennent
crossed two sea urchins, belonging to the genera Cidaris and
Lytechinus. In Cidaris, the formation of the archenteron begins
20 hours after fertilisation, and the cells which will form the
skeleton disengage themselves from its wall a few hours after-
wards. In Lytechinus, on the other hand, the archenteron
invaginates after only 9 hours, and the skeleton-forming cells
arise from the vegetative wall of the blastula at an even earlier
time. In the hybrid Cidaris X Lytechinus, development first
takes a matrocline course, viz. until the end of the blastula
stage. However, the skeleton-forming cells originate from the
blastula wall, simultaneously with the beginning of the in-
vagination of the archenteron. On this point, therefore, the
influence of the paternal chromosomes expresses itself already.

In amphibians, too, the result of crossing experiments
depends very much upon the nature of the species crossed.
Good development of the hybrids occurs, e.g., in the cross

THE REALISATION OF THE NUCLEAR FACTORS 79

Bufo communis '^ ^ ^- viridis (f , whereas in the reciprocal
cross the hybrids develop poorly, and in the cross Rana escu-
lenta X R. fusca development ceases already at the gastrula
stage. Here we also find so-called "false hybrids", in which the
penetrating sperm has set development going, but does not
otherwise take part in the process (e.g. in Rana esculenta X
Bufo viridis).

J. A. Moore (1941) studied the development of a great
number of American frog hybrids. In many cases, these devel-
oped normally until the early gastrula stage, but then develop-
ment ceased; the rate of development was entirely similar to
that of the parent species which had produced the eggs. In
hybrids in which development proceeded further, its rate was
again at first determined exclusively by maternal factors. How-
ever, with the appearance of the first organ primordia after
the completion of the gastrula stage, the influence of the
paternal chromosomes on the rate of development became
noticeable.

Further it has been found in frog hybrids which stopped
developing at the gastrula stage, that simultaneous deviations
occurred in respiration and carbohydrate metabolism (Barth
and Jaeger), and the synthesis of ribonucleic acid which nor-
mally begins at this stage dit not set in (Brachet, 1952).
Evidently the presence of the foreign chrom.osomes inhibits
certain processes by which the nucleus influences the cell meta-
bolism (cf. p. 65). Curiously enough, parts of such "lethal
hybrids", grafted into normal embryos, proved able to continue
their development more or less normally. This is accompanied
by a distinct synthesis of ribonucleic acid in the grafts. This
"revitalisation" of the lethal tissues must probably be ascribed
to the fact that the substances indispensable to metabolism,
which the cells of the hybrid cannot themselves synthesise, are
now supplied by diffusion from the environment.

The influence of the paternal nuclear factors on further
development has been carefully studied in some newt crosses.
Hamburger (1936) crossed Triton taeniatus and T. cristatus.
The hybrid larvae initially were the same size as those of the
maternal species. Later, however, differences developed. T.

80 THE REALISATION OF THE NUCLEAR FACTORS

cristatus grows more rapidly than does taeniatus. The hybrids
are intermediate, though cristatus X taeniatus grows some-
what faster than the reciprocal hybrid. This difference between
the hybrids shows that growth does not depend only on the
nuclear factors, but on the cytoplasmic factors as well: the
composition of the nuclei is identical in the two hybrids, but
their cytoplasm is different, being almost exclusively maternal.
Another difference between the parent species is that the white
pigment cells on the caudal fin appear earlier in Triton cristatus
than in taeniatus, and have somewhat different patterns in the
two species. In both hybrids, the appearance of this character
coincides with that in cristatus, and at that time the pattern,
too, essentially tallies with that of cristatus (Pl.IVa-d) *. Later,
however, it begins to approach the taeniatus pattern. The
similarity in behaviour of the two hybrids on this point shows
that cytoplasmic factors do not play a role here, but that the
formation of these pigment cells is governed entirely by the
nuclear factors. Finally, there are marked differences between
taeniatus and cristatus larvae in the structure of the fore-limb,
viz. in shape and length of the fingers. The limbs of the hybrids
are intermediate between those of the parent species (PI. IV
e-g). There is no difference between reciprocal hybrids on this
point, so that here again only nuclear factors seem to play a role.

In a study of American species of the genus Triturus, Twitty
(1936) was able to carry the analysis still further by means of
a combination of transplantations and hybridisations. There are
characteristic differences in pigmentation between the species
studied by him. In Triturus torosus, the black pigment cells
form a well defined black band on each side along the base
of the dorsal fin. In T. rivularis, they are dispersed all over
the sides of the body. The pigmentation of hybrids between
these species is more or less intermediate between that of their
parents. Evidently the distribution of the pigment cells over
the sides is governed by the nuclear factors (PI. V a-c).

Twitty tried to find out how the nuclear factors influence the
distribution of the pigment cells. These cells originate dorsally,
from the embryonic neural folds (cf p. 98), and spread from
there over the sides of the body. In T, torosus, they accumulate

* Facing page 49.

THE REALISATION OF THE NUCLEAR FACTORS 81

in the dorsal regions of the sides, above the muscle segments,
whereas in T. rivularis they become evenly scattered over the
whole flank. The differences in pigmentation, therefore, are
due to differences in the migrations of the pigment cells. These
in their turn might depend either upon differences in the nature
of these cells, or upon differences in the orienting and attract-
ing influences from the environment to which they are exposed.
By means of transplantation experiments, Twitty and his
collaborators proved, indeed, that both the skin epithelium and
the muscle segments of various species have different properties
in this respect. But the specific differences in the pattern of
pigmentation proved to be due mainly to the properties of the
pigment cells themselves. In T. torosus, the latter are strongly
attracted by the muscle segments, and to a lesser degree by
the central nervous system. The pigment cells of T. rivularis
lack this property ; hybrids possess it to a smaller extent. Parts
of the pigment cell primordia in the neural fold of torosus
embryos were replaced by corresponding parts of rivularis
embryos, and vice versa. In this case, the pigment cells of the
torosus graft accumulated over the muscle segments of the
rivularis host, whereas the pigment cells of the rivularis graft
scattered evenly over the side of the torosus host. In other
words, the graft's own specific pattern will develop even in the
foreign host (PL V d-e) *. Twitty (1945) has shown in later
experiments that the dorsal accumulation of the torosus melano-
phores is a secondary phenomenon. As soon as they have reach-
ed a certain degree of differentiation, the cells withdraw their
processes, thereby moving closer together. In T. rivularis, the
melanophores always remain at a lower level of differentiation ;
they do not become so strongly pigmented, and do not show
secondary accumulation. The nuclear factors, therefore, are
directly responsible for a difference in the ultimate level of
differentiation of cells of this category; indirectly, this results
in a modification of the pattern of skin pigmentation.

In the cells of hybrids, the paternal nuclear material is always
combined with the maternal chromatin of the egg nucleus. This
means that the influence of the paternal genes on the foreign

* Facing page 96.

Raven - Outline Physiologie 6

82 THE REALISATION OF THE NUCLEAR FACTORS

cytoplasm will not be able to express itself unhampered. It is
always more or less masked by that of the maternal genes.
If a better picture of the mutual influence of nuclear and cyto-
plasmic factors is desired, a nucleus of species A should be
combined with cytoplasm of species B in the absence of nuclear
material from the latter. If this can be achieved, the foreign
nucleus will not be able to use the "mediation" of this material
when influencing the cytoplasm, but the two must interact dir-
ectly. Properties of the hybrid which are characteristic of A are
then undoubtedly caused by the nucleus, and B-characters equally
certainly by the cytoplasmic factors. Such combinations can
indeed be realised in a number of cases. Non-nucleated egg-
fragments or whole eggs, the nucleus of which has been re-
moved or killed, can under certain circumstances develop after
fertilisation with nuclear material derived only from the sperm
nucleus present in the cell. The general term for this pheno-
menon is merogony. If combined with hybrid fertilisation —
the sperm nucleus belonging to another species — it is called
heterospermic or hybrid merogony. The ideal case described
above is realised here. The heterospermic merogone consists of
cytoplasm of one species, and a nucleus of another.

In experiments on heterospermic merogony, the same dif-
ficulties are generally met with as in normal hybridisation:
hybrid merogones in which distantly related species are com-
bined, are of low viability, and as a rule they soon die. It is
interesting to note that, just as in the case of lethal genes
(p. 74), this "lethality" often expresses itself in the disturbance
of definite developmental processes at a definite stage, whereas
other processes, in other organs, may take a fairly normal
course. The combination of more closely related species gives
better results, which however are not quite so interesting from
our point of view, because the differences between the parents
do not become manifest till late in development. Yet these
experiments furnished some results, in particular again in sea
urchins and amphibians.

Horstadius (1936) used the following method for the pro-
duction of sea urchin merogones. With a sharp needle he re-
moved from the egg a small segment containing the nucleus.

THE REALISATION OF THE NUCLEAR FACTORS

83

The enucleated eggs were then fertilised. In this way he obtain-
ed hybrid merogones between Psammechinus microtuberculatus
and Paracentrotus lividus. These developed well, and produced
harmoniously built plutei. It is true that the plutei of the parent
species are very similar to each other, but after a very minute
investigation, Horstadius was able to demonstrate, by means
of slight differences found in the shape of the calcareous rods,
that the sperm nucleus had a clear-cut influence on these
organs. The tips of the apical rods are slightly more irregular

Fig. 30. Apical rods of the skeleton of the pluteus larva of (a)
Psammechinus microtuberculatus, (b) Paracentrotus lividus, and
(c) the hybrid merogone Parac. liv. (cytoplasm) X Psammech.
micro tub. (nucleus). In the latter, the shape of the apical rods is
more similair to that of Psammechinus. After Horstadius.

in Psammechinus than in Paracentrotus j, also they show a
slight tendency to branch. In this respect, the hybrid merogones
behaved in the same way as the species that supplied the sperm
nucleus (Fig. 30). Evidently this character is governed by the
nuclear factors. Some further information on their mode of
operation may be obtained from the combination of this result
with observations by von Ubisch (1937) on the way in which
the calcareous skeleton of the pluteus develops. It is built by
mesenchyme cells of the gastrula, which are grouped to the
left and right of the archenteron, and fuse into a so-called
syncytium. The material of the rods is then secreted within
this syncytium (Fig. 32). The viscosity of the cytoplasm of
these cells appears to be an important factor in the final

84 THE REALISATION OF THE NUCLEAR FACTORS

determination of the shape of the calcareous rods. Lower vis-
cosity, which may occur as a specific character in some species,
and may be caused by a rise in temperature in others, leads to
more complicated branching of the skeleton. Therefore, the
primarj^ influence of the nuclear factors in the merogones
mentioned above might consist in a modification of the vis-
cosity of the protoplasm of the skeleton-building cells. The
activity of the nuclear factors would in that case manifest
itself in an influence on the physico-chemical properties of
certain cells of the body.

In Amphibia, merogonic development may be brought about
by several means. G. and P. Hertwig killed the nucleus of the
egg by irradiation with mesothorium, and then fertilised with
normal sperms. Or, on the other hand, the nucleus may be
removed from the egg with a fine pipette. Finally, a method
often followed is to constrict the eggs with a hair soon after
fertilisation in such a way that one half contains the egg
nucleus, and the other a sperm nucleus. The latter half will
then develop merogonically. Homospermic merogones, i.e. mero-
gones in which the sperm nucleus belongs to the same species
as the egg cytoplasm, are often able to differentiate into a
harmoniously built embryo. In heterospermic merogones be-
tween newts of the genus Triton, development will cease at a
given stage, and this will be earlier, the more distant the parent
species. In any case, development will stop before the stage at
which the differential characters between the parent species
begin to show (Baltzer, 1920). For that reason, these mero-
gones themselves, again, do not help us much towards the
solution of our problem. Hadorn (1932) found, however, that
e.g. in merogones of Triton palmatus (cytoplasm) and T. cris-
tatus (nucleus) not all tissues are damaged to the same extent,
although the merogones die at an early stage. The mesenchyme
cells of the head die, and the muscles and nervous tissue are
also incapable of differentiation, but skin ectoderm, heart, noto-
chord, gut, and pronephros, however, are apparently unimpair-
ed. This shows that the antagonism between nucleus and
cytoplasm of different species is not manifested equally strongly
in all parts, but especially where the cells must achieve certain

THE REALISATION OF THE NUCLEAR FACTORS 85

differentiations. This is another indication that the "activation"
of the nuclear factors takes place at different times in the
different parts of the embryo. In related species of Triton these
nuclear factors may be partly identical, and partly different.
Normal differentiation will be possible in those cells in which
no nuclear factors are activated during differentiation, or only
such factors as occur in the maternal species too, and which
therefore "fit in" with the protoplasm. Disturbances, sometimes
resulting in the death of the cells, will only occur where factors
are activated which differ in the two species. Hadorn managed
to keep the unimpaired tissues of the merogones alive to much
later stages by grafting them into normal embryos. There they
differentiated normally. In a few cases, skin ectoderm of such
a merogone, when grafted into an embryo of Triton alpestris,
remained intact until after the metamorphosis of the host.
Meanwhile, the specific characters of this tissue had become
visible, and they were found to agree entirely with those of
the species which had supplied the cytoplasm, i.e. Triton
palmatus. No specific influence of the nucleus of T. cristatus
was observed.

Comparable experiments were made by Dalton (1946) with
American newts of the species Triturus torosus, T. similans,
and T. rivularis, the same species that were used by Twitty
(see p. 80). Here, too, the hybrid merogones die early, before
the development of specific differences in pigmentation. But
it was again possible to keep the primordia of the pigment cells
alive for a longer time by early transplantation into a normal
embryo. We have already seen that the pattern of pigmentation
depends upon the properties of the pigment cells. Dalton found
that it is determined mainly by the nuclear factors, though
the cytoplasm has some influence.

Finally, mention must be made of an experiment by Baltzer
(1947) on the pigmentation of merogonic hybrids between black
and white axolotls. The difference in pigmentation between
these races is caused by a single gene, black being dominant
over white. The character does not depend on the pigment cells
themselves, but on the epidermis covering them. In the black
axolotl, this is said to secrete a substance, probably an oxidase,

86 THE REALISATION OF THE NUCLEAR FACTORS

which is necessary for the production of melanin in the pigment
cells. In the white axolotl, the production of this . substance
would be reduced (Dushane, 1935). (This, at least, is the pre-
vailing opinion. Dalton gives a somewhat different explanation
of the phenomenon, into which we cannot now enter.) It appear-
ed from Baltzer's experiments that merogones consisting of
cytoplasm of the black axolotl, and of nuclear material of the
white race, had the same type of pigmentation as the white
race. Even after early transplantation into a black larva, skin
grafts of such a merogone developed a pigmentation of the
white type. In this case, the nature of the epidermis, on which
the pigmentation depends, appears to be determined entirely
by the nuclear factors.

Mutual comparison of these experiments on heterospermic
merogony shows that generalizations with respect to the role
of nucleus and cytoplasm in early differentiation are impossible.
Specific characters are determined sometimes by the cytoplasm,
sometimes by the nucleus alone, and in other cases again by a
combination of the two.

Little is known yet about the nature of the cytoplasmic
factors we are discussing here. Some investigators assume that
particles occur in the cytoplasm, which can multiply by division,
and are in some respects similar to the genes of the nucleus.
They are called plasmagenes. Production and multiplication of
these plasmagenes would be governed partly by the nuclear
genes, partly also by external influences. According to Brachet
(1952), they are probably identical with the microsomes
(see p. 34).

We arrive, therefore, at the following conclusion as regards
the way in which nuclear factors influence development. As a
result of earlier chemodifferentiation, the cytoplasm of certain
cells is of such a composition that it can '"activate" certain
genes in the nucleus. A reaction then takes place between these
genes and the cytoplasmic factors, resulting, for example, in
the production of substances which in their turn influence other
developmental processes. We must distinguish here between
two cases. Firstly, the substances produced as a consequence
of gene activity may be restricted to the cells in which they

THE REALISATION OF THE NUCLEAR FACTORS 87

have been formed, and may influence the chemical, physical,
morphological, or physiological properties of these cells only.
Probably the pigment cells of newts (see p. 81) and the
skeleton-building cells of sea urchins (see p. 84) are examples
of this kind. In other cases, the substances produced under the
influence of certain genes may diffuse from the cells where
they originated into the blood. They will then be transported
throughout the body, and will be able to influence other parts
of the embryo as well. Such substances have been called ''gene
hormones''. Their existence has been proved, and their activity
studied, in particular in insects (Plagge, 1939; Ephrussi, 1942).
Research on this problem has been carried out mainly on the
moth Ephestia kuhniella by German investigators (Caspari,
Plagge, Becker, and others) in Kiihn's laboratory, and on the
fruit fly Drosophila melanog aster by a great number of French
and American workers (Ephrussi, Beadle, Clancy, Tatum, and
others). Similar results, however, were also obtained in the
parasitic wasp Habrobracon juglandis (Whiting, 1932-34) and
in the moth Bombyx mori (Morohosi, Kikkawa, Kawaguchi,
1937-38). We shall take Drosophila as an example because the
greatest progress has been made in this species.

In Drosophila two mutations occur, known as "vermilion"
(v), and "cinnabar" (en), respectively. The eye colour of these
mutants is lighter than that of normal individuals. The eyes of
the latter are coloured dark red by the combination of a red
and a brown pigment. The brown pigment is absent both in v
and in en mutants. Both mutants are due to single gene muta-
tions. If grafted early into a normal animal, the eye of a 'U'-mutant
developed the same dark red colour as is found in the wild form.
The same was seen on transplantation of v-eyes into a cn-
animal. Eyes of a cn-larva also became dark red when trans-
planted into a normal animal. However, they remained light if
grafted into a v-animal. The result did not depend upon which
particular part of the body the eyes were grafted into. It can
be concluded from these observations that the whole body,
both of the normal animal, and of the cn-mutant contains a
factor, probably a substance, which gives rise to brown pigment
in the eyes of the -u-mutant, and which is evidently absent in

88

THE REALISATION OF THE NUCLEAR FACTORS

the v-mutant itself. Similarly, a factor necessary for the forma-
tion of brown pigment in cn-eyes is present in the wild form,
but not in either v- or cn-mutants. Apparently the cells of
certain organs (eyes, Malpighian tubes, fat-body) of the normal
animal produce an excess of these substances. Transplantation
of these organs from a normal animal into a v- or cn-mutant
resulted in normal, wild type coloration of the host's eyes.
Injection of lymph from a normal animal had the same effect.
Therefore, the substances concerned must be present in lymph
as well. Further experiments showed that in a normal, wild
type Drosophila a reaction-chain occurs which first leads to
the production, under the influence of a single gene, of a so-
called v-h substance. Under the influence of a second gene this
substance is transformed into a second substance, the cn-\-
substance, which in its turn is indispensable for the normal
pigmentation of the eyes. In the mutants, this catenary reaction
is interrupted in various places. In "vermilion", the gene that

ye/Z/cA

}/^C/7

Fig. 31. Drosophila. (a) the fat body of a normal animal {v^rcn-\-)
is transplanted into a vcn-\- animal, where it secretes v-\- substance;
(b) the host tissues transform this into cn-\- substance, so that both
the eyes of the host, and a grafted v-\-cn eye can now develop
normal pigmentation; (c) the same transplantation into a f en animal
has no effect, because there the v^r substance cannot be trans-
formed into cn-\- substance. After Ephrussi and Chevais.

THE REALISATION OF THE NUCLEAR FACTORS 89

takes care of the production of the v + substance has fallen out.
In "cinnabar", the v+ substance is produced, but it cannot be
transformed into the cn+ substance because of the absence of
the necessary gene (Fig. 31). As we have seen, these substances
are produced by certain cells of the body under the influence
of certain genes, and then spread throughout the body via blood
or lymph, so that they satisfy the definition of gene hormones.
A single gene hormone may influence several different devel-
opmental processes. In Ephestia, one substance influences the
speed of development, and the viability of the embryo, the
pigmentation of eyes and skin of the caterpillar, and the
pigmentation of eyes, brain, and sex organs of the imago.
Another remarkable discovery is that gene hormones are not
specific. The v+ and cn+ substances of Drosophila mela-
nogaster were also found in other flies, parasitic wasps, and
butterflies. The catenary reaction found in Drosophila appar-
ently occurs in insects in general, and the existence of muta-
tions entirely analogous to ''vermilion" and "cinnabar" in
Drosophila has been demonstrated in Ephestia and Habrobracon^
where, as a matter of fact, they had been known for a long
time under different names. On the other hand, the end point
of the reaction is not always the same. There is a wide variety
of eye pigments in different insects. Moreover, the pigments of
eyes and skin in Ephestia are not identical, though the pro-
duction of both is governed by the same gene. Evidently, the
gene substances do not influence the last stages of pigment
formation, but instead they constitute a link in earlier processes.
Later work has thrown some light on the nature of these processes.
The v-\- substance was found to be identical with kynurenin,
a derivative of tryptophane. The cn+ substance is a derivative
of kynurenin. Apparently, the genes exert their influence
through the production of enzymes which first transform
tryptophane — itself a common intermediate in protein meta-
bolism — into kynurenin, and subsequently kynurenin into
other substances which are the building stones for skin and eye
pigments. In this case, therefore, the name gene hormone may
not be entirely appropriate, because the substances concerned
are not themselves catalysts, but building material that is

90 THE REALISATION OF THE NUCLEAR FACTORS

used up in the course of development. For that reason, the
neutral term "gene substance" is nowadays preferred.

Recent experiments by Beadle and coworkers on mutants of
the fungus Neurospora give strong support to the hypothesis
that the primary action of genes consists of the production of
certain enzymes. Their results indicate that each gene gives
rise to one definite enzyme, so that mutation of the gene causes
a link in the metabolic processes to fall out. It is conceivable
that genes generally exert their influence on development in
this way. However, we must await further data on this point
before a final conclusion can be drawn.

CHAPTER VII

The topogenesis of the embryo

In the foregoing chapters we have seen how a series of processes
transforms the initially fairly homogeneous structure of the
egg into a system with a considerable degree of spatial multi-
plicity. First, the primary co-ordinate system of the egg,
expressed in its polarity and symmetry, gave rise to the
localisation of determining substances. This was followed by
the production and local accumulation of new components of
the cytoplasm, resulting from the chemical and physical pro-
cesses now started in the egg. Differences began to occur, both
chemically and physically, among the various parts of the egg.
Finally, reactions between nuclei and cytoplasm took place as
a consequence of the activation of the genes under the influence
of the local cytoplasm. Taken together, all these processes
break up the originally fairly homogeneous system into a mosaic
of physically and chemically different areas. We have called
this the chemodifferentiation of the egg.

As a rule, chemodifferentiation does not manifest itself very
clearly in the egg's external appearance. In exceptional cases,
some of the determining substances may be visible externally
because they have pigments of different colours; if so, their
movements can be followed easily. In general, however, only
experiments can inform us of the changes that have taken place
in the egg.

Another process, however, also subsequent to fertilisation,
and usually more or less simultaneous with the primary chemo-
differentiation, can be easily observed. This is the cleavage
of the egg, whereby the originally continuous mass of egg
cytoplasm is divided into a number of cells. The zygote nucleus
also divides a great number of times so that, in the end, the

92 THE TOPOGENESIS OF THE EMBRYO

egg consists of a number of cleavage cells, or blastomeres, each
containing one cleavage nucleus. A close correlation nearly
always exists between the direction of the first cleavages, and
the polarity and symmetry of the egg, as determined by its co-'
ordinate system. As a rule, the first two cleavages are in a
meridional plane, and at right angles to each other, whereas
the third cleavage lies in the equatorial plane ^). From this
point onwards, cleavage may follow several different courses.
Sometimes it remains very regular so that the cleavage planes
always cut through the egg in a definite direction, and two eggs
of the same species in the same stage have identical structures.
Evidently, the direction of cleavage is rigidly determined in
such cases. The eggs of most molluscs and annelid worms may
serve as examples (see above, p. 51). In other cases, cleavage
soon becomes more or less irregular. The direction of the
cleavage planes does not follow any general rule, and is ap-
parently more or less independent of the polarity and symmetry
of the egg. These differences are not essential for the further
course of affairs, and they can be neglected here.

During cleavage the egg cytoplasm is distributed between
the blastomeres. As a consequence of the previous chemo-
differentiation, the cytoplasm of the various blastomeres will
be physically and chemically different. We have already seen
how this influences the realisation of the nuclear factors by
the activation of different genes in different cells. We have also
discussed (p. 62) how differences in the developmental potencies
of the cells arise thereby. In other words, the germ now becomes
a complex of cell groups with divergent potencies. The necessary
conditions are thereby provided for the processes which now
follow, in the course of which the embryo is formed (embryo-
genesis). This consists of two phases: the development of form,
or topogenesis, and the differentiation of the tissues, or
histogenesis.

Topogenesis is a series of movements, resulting in cell
migrations, which bring the prospective tissues of the body

1) Ascaris is an exception; here, the first cleavage is equatorial (Fig. 25).
See also p. 70 on cleavage in insects.

THE TOPOGENESIS OF THE EMBRYO 93

to their appropriate places in the structural plan. These move-
ments may take many different forms, such as folding, in-
vagination, or shifting, either of cell groups, or of isolated cells.

In the eggs of most animals, a period sets in, directly after
the completion of cleavage, in which these movements dominate
the whole picture of development. So far, the egg has been
rather inert, cleavage being its only manifestation of activity.
Now, however, it suddenly seems to wake up, and to begin its
real task, the formation of an embryo. The whole set of topo-
genetic phenomena immediately following cleavage is called
gastrulation. Cleavage had already produced a vesicular germ,
the blastula. During gastrulation this enters upon a phase in
which some of the main components of the future embryo can
already be recognized. We shall now discuss the process of
gastrulation in two animal groups which have often been used
for experiments in developmental physiology, viz. in sea urchins
and amphibians.

The sea urchin germ at the end of cleavage is a vesicle with
a wall consisting of a single layer of cells (Fig. 32a). The cells
of the vegetative side are slightly higher than those of the
animal pole. The latter carry a long tuft of stiff cilia, whereas
the other cells have short, motile cilia. The beginning of gastrul-
ation is marked by an indentation at the vegetative pole of the
blastula. At first, this is shallow, but it penetrates gradually into
the interior, thereby forming the archenteron (Fig. 32 d, e).
The opening which connects the archenteron with the exterior
is called the blastopore. The cells which form the wall of the
archenteron are called the endoderm; to the cells remaining at
the surface of the germ the term ectoderm is applied. Further,
a small number of cells, originally occupying the vegetative pole
of the blastula, now disengage themselves from the epithelium,
and move into the space between ectoderm and endoderm. They
constitute the so-called primary mesenchyme (Fig. 32b, c). We
have already seen (p. 78) that the formation of the primary
mesenchyme may take place at different stages in development.
If this occurs at an early stage, the cells become free from
the blastula wall even before the invagination of the archen-
teron; if at a later stage, they are first carried along by the

94

THE TOPOGENESIS OF THE EMBRYO

Fig. 32. The normal development of Psammechinus micro tuber-

culatus. (a) early blastula; (b-c) formation of the primary mesen-
chyme (p.m.); id) beginning invagination of the archenteron; (e)
gastrula, from the left; formation of the secondary mesenchyme
(s.m.); (/) older gastrula, dorsal view; formation of the skeleton;
(g) the archenteron bends towards the ventral side; (h) formation
of the mouth (m). After Schmidt.

invagination, and later become disengaged from the tip of the
archenteron. Originally, the archenteron is straight, but later
its terminal part bends, and comes into contact with the ecto-
derm (Fig. 32 g, h). At this point, an invagination, called the
stomodaeum, is formed by the ectoderm. This breaks through
into the archenteron, thereby forming the definitive mouth.
The archenteron becomes the gut, the blastopore becomes the
anus. The symmetry of the egg which, though preformed, was
not yet visible, manifests itself in these processes. The embryo
is now bilaterally symmetrical. The mouth has formed at its
ventral side ; the opposite side is dorsal. The cells of the primary

THE TOPOGENESIS OF THE EMBRYO 95

mesenchyme, later reinforced by a second generation of cells
which have become free from the endoderm (Fig. 32 e), ac-
cumulate in two groups, to the left and right of the gut. We
have described above (p. 83) how the skeleton develops within
these two cell masses (Fig. 32 f). The calcareous rods of the
skeleton show a rapid increase in length, and where their ends
come into contact with the ectoderm, the latter bulges out,
forming the so-called arms- In this way the blastula develops,
via the gastrula stage, into a pluteus larva which already
possesses the primordia of several organ systems characteristic
of the sea urchins (Fig. 10, 11, 15, and 29). This development
is achieved by a series of folding, invaginating and shifting
processes.

In amphibians, gastrulation is somewhat more complicated.
Cleavage here results in a blastula with a multi-layered wall.
Its cavity (hlastocoel) is eccentric, lying nearer to the animal
pole of the egg. At this end, it is bordered by several layers of
small cells, whereas the floor of the cavity is formed by several
layers of large cells which are rich in yolk (Fig. 34a).

The size of the cells increases gradually from the animal pole
towards the vegetative pole. The same statement applies to
their yolk content. In many amphibians, the original, bilateral
structure of the egg can be discerned in the blastula, because
the grey crescent, which was formed soon after fertilisation, is
still present. This marks the dorsal side of the egg.

Here, again, a complicated system of cell movements begins
after the completion of cleavage, which shifts the cell material
of the prospective organs towards their appropriate places.
The best way to elucidate this complicated process is to consider
the surface of the blastula to be divided into three parts by
two imaginary lines. These parts are: (1) a marginal zone
which surrounds the egg in the vegetative half, and is more
or less parallel to the equator; it is wider at the dorsal side,
and here it extends into the animal half; (2) the animal fields
consisting of small, usually darkly pigmented cells; (3) the
vegetative field, consisting of large, unpigmented cells, con-
taining much yolk (Fig. 33). The boundaries of these areas
cannot be distinguished in the egg by their outward appear-

96

THE TOPOGENESIS OF THE EMBRYO

ye^ye/c/ I ^er/77^ff/'.

Fig. 33. Diagram of
an amphibian blastu-
la. Anxynale veld =
animal field; oerm.gr.
=z blastopore furrow;
randzone = marginal
zone; veg. veld =
vegetative field.

ance ; nevertheless, their importance will
appear in the later development.

The beginning of gastrulation is mark-
ed by a small indentation at the dorsal
side of the blastula, in the vegetative
field, just under the grey crescent which
here lies in the marginal zone. This in-
dentation is called the blastopore furrow.
It is produced by an active change in
shape of the cells of that area, which
become club-shaped and thereby move
inwards. The depth of the indentation
soon increases, and in this way the
archenteron extends into the egg, along
its dorsal side, and in the direction of
the animal pole (Fig. 34 a-e). In the
course of this invagination, material
which was originally on the surface is
carried into the interior, so that the wall of the archenteron
consists of cells which initially lay at the surface. Material of
the vegetative field as well as material of the neighbouring
dorsal part of the marginal zone is rolled in over the lips of
the blastopore. The vegetative material will form the ventral
wall of the archenteron, its so-called floor, whereas the material
from the marginal zone will form its roof. The invagination
then begins to spread laterally, more or less along the boundary
between marginal zone and vegetative field. All along this
boundary, marginal zone material begins to roll in, forming
a protruding edge, called the Up of the blastopore (Fig. 37 a, b).
The invaginating archenteron gradually compresses the blasto-
coel. In the end, gastrulation leads to an almost complete dis-
appearance of the material of the vegetative field into the
interior. Only a small part of this material still protrudes from
the blastopore as the yolk-plug (Fig. 34 c, d). Finally this, too,
disappears, and the blastopore lips, approaching each other
from all sides, close the blastopore. The material of the marginal
zone also disappears from the surface by rolling-in over the
lips of the blastopore. At the same time, the material of the

%.

?f*SF-iBF^

»*'^" V ' '*

-^

•»-«,-- -^-

PLATE V. The effect of genetic factors on the pigmentation of newts,
(a) larvae of Triturus rivularis; (b) larvae of T. torosus; (c) hybrids of
T. rivularis X T. torosus. (d) transplantation of pigment cell primordium
from T. torosus into T. rivularis; (e) ditto from T. rivularis into T. torosus.
The pigmentation in the area occupied by the graft is the same as that in
the species to which the graft belongs. After Twitty.

a

c

a.

6

p.S,

PLATE VI. The organiser and its activity, (a) Gastrula of Triton with im-
planted organiser (dark pigmentation) opposite the blastopore lip. (b) The
same embryo at the neurula stage, (ventral view) ; a secondary neural plate
has been induced, (c) The same embryo a few days later. A secondary embryo
primordium (right), with head and tail-bud has been formed at the side
opposite to the primary primordium. Both embryos have a common anus (a),
situated between the primary (p.s.) and secondary (s.s.) tail-buds. After

Spemann.

THE TOPOGENESIS OF THE EMBRYO

97

Fig. 34. Diagram of gastrulation in amphibians, (a) blastula, halved
in the median plane, with thin roof and thick floor (coarse hatching:
animal field; fine hatching: marginal zone, the outline of cells is
indicated in the vegetative material), blastopore furrow below at
right; (a-e) invagination of the archenteron, starting from the
blastopore furrow; inward movements of the vegetative material;
rolling in of the marginal zone material over the edge of the blasto-
pore; (/) cross section through the same stage as in (e), the trough
shaped endoderm forms the floor and the sides of the archenteron,
the prospective notochord and mesoderm its roof, whereas the
ectoderm covers the outer surface of the germ; (g) dorsal view of
neurula, showing the neural plate, surrounded by a raised fold
which separates it from the rest of the ectoderm; (h) cross section
through the same stage as in (g); the endoderm has closed dorsally,
forming the gut, the archenteron roof has differentiated into noto-
chord and mesoderm, the ectoderm into neural plate and skin ecto-
derm. After Hamburger and Mayer.

Raven - Outline Physiologie

98 THE TOPOGENESIS OF THE EMBRYO

animal field expands strongly, and finally covers the whole
surface of the germ. It also becomes thinner, and the multi-
layered arrangement of the cells is replaced by a single-layered
one. At the end of gastrulation, therefore, the embryo consists
of (1) an external ectoderm, or outer germ-layer, originating
from the animal field, (2) an internal endoderm, or inner germ-
layer, forming the floor and the sides of the archenteron, and
arising from the original vegetative field, and (3) the roof of
the archenteron, formed by the invaginated marginal zone
material, and constituting the middle germ-layer, or chorda-
mesoderm. The archenteron roof, consisting of the prospective
notochord and mesoderm sheets, extends ventrally between
ectoderm and endoderm (Fig. 34f). The main axes of the embryo
have now become visible: The blind end of the archenteron
marks the anterior end of the embryo; here the mouth will
break through. The blastopore lies at the posterior end, and
will become the anus. The back of the embryo will be formed
at the dorsal side of the germ, and its belly at the opposite side.
At the conclusion of gastrulation, topogenesis has not yet
come to an end. The next phase, known as neurulation, follows
immediately. During this period, new movements result in the
formation of the first organ systems of the embryo from the
material brought into place by gastrulation. The dorsal ecto-
derm thickens and becomes the neural plate (Fig. 34 g). The
edges of this plate fold upwards (neural folds), and finally
fuse, forming the neural tube. This tube is the primordium of the
embryonic central nervous system. The rest of the ectoderm
is the prospective epidermis. The mid-dorsal cells of the
archenteron roof, under the middle of the neural plate, become
free from the rest of the roof, and form a longitudinal cylin-
drical rod, the notochord. The adjoining strips, to the right
and left of the notochord, first thicken, and then divide into
a number of segments, the so-called somites, lying in a longi-
tudinal row. These will give rise to the musculature of the
trunk, etc. The remaining, lateral and ventral parts of the
mesodermal sheets supply the lateral mesoderm in which the
body cavity of the embryo will develop. Initially, the endoderm
was trough-shaped, but now its edges approach each other

THE TOPOGENESIS OF THE EMBRYO 99

under the notochord, and fuse so that the archenteron is now
surrounded by endoderm on all sides. From then on it becomes
the gut of the embryo (Fig. 34 h).

In this way, the processes of gastrulation and neurulation,
which follow each other without delay, transform the simple
structure of the blastula into the complicated architecture of
the embryo, in which the primordia of several organ systems
can already be recognized.

Only a small part of the movements here described can be
seen by direct observation of the germ. The development and
closing of the neural plate can be easily seen, but the shifting
processes during gastrulation, as in the infolding of marginal
zone material, can be made visible only by means of special
techniques. Our insight into these processes has been greatly
deepened by the method of colour marking intra vitam, invented
by Walter Vogt (1925-29). In this method, small marks are
made on the surface of the germ with so-called "vital" stains,
i.e. dyes which do no damage to living cells. In this way, we
can investigate the particular organs of the embryo into which
the cells of the various areas of the germ will eventually find
their way, and these organs of the embryo can, so to speak, be
projected back onto the surface of the blastula (Fig. 35).

All movements involved in gastrulation and neurulation are
due to active changes in shape of the cells of which the germ
consists. Motility is one of the elementary properties of all
cells. Most cells are capable of active changes in shape, although
not all cells show this so clearly as the elements of the
musculature. Many animal eggs, too, are motile to a lesser or
greater extent. In some lower animals, such as the sponges,
the eggs can crawl around freely in the maternal tissues. These
eggs are very similar to amoebae. In other animals, the eggs
show amoeboid movements only at certain times, e.g. in the
snail Limnaea immediately after the expulsion of the polar
bodies. We can imagine that this power of locomotion becomes
specialized in divergent directions in the various cell groups
of the germ, due to the modifications in the physico-chemical
condition of the parts of the egg, caused by chemodifferentia-
tion. The cells of one area will show a tendency to expand

100

THE TOPOGENESIS OF THE EMBRYO

c/orsffd/

/'^/^//Vc?/

Fig. 35. Diagrammatic projection of the organ primordia of a
urodele embryo onto the blastula surface, seen (a) from the vege-
tative pole, and (b) from the left side. Coarse hatching := epidermis,
fine hatching rzi neural plate, closely dotted = notochord, sparsely
dotted -=■ mesoderm, white = endoderm; future somites numbered;
g\ boundary of the invaginated area; oe: future blastopore lip;
oerm.gr.: blastopore furrow; St.: tail area, z.y.'. lateral plate meso-
derm. After Vogt.

THE TOPOGENESIS OF THE EMBRYO 101

parallel to the germ surface, whereas in another area they
tend to extend at right angles to it. In yet another area they
may assume a club-shape by enlargement of the part pointing
towards the centre of the germ, and narrowing of the other
end. This will cause the cell to move into the interior, and
thereby give rise to an invagination of the superficial material.
It has indeed been proved experimentally that different parts
of the early gastrula of amphibians show tendencies for differ-
ent movements. Part of the marginal zone tissue of one gastrula
can be grafted into the ventral side, or
into the animal field, of another early
gastrula. A blastopore will then be
formed by invagination in the graft, all
of which is rolled into the interior, often
together with a number of cells of the
host. This shows that marginal zone

material must have the potency to roll „^ ^-r , , ^

... ' -li. 1 ^ J- ^ Fig. 36. "Hyperblastu-

m actively, simultaneously extending to ^^,r ^^ Triton formed

form an archenteron. On the other hand, ^y the animal half of

the material of the animal field shows an egg. Strong ex-

a tendency to expand parallel to the pansion of the aninial

n o /ir\n-i\ J. J. 1-1 J. material leads to fold-

surface. Spemann (1931) cut twoblastu- ^ After Ruud

lae in halves along the equator, and

grafted the two animal halves together. This resulted in a
germ with an excessively expanded ectoderm, forming many
loose folds and flaps (Fig. 36). This can be explained as follows.
In normal development, the expanding ectoderm takes the place
of the marginal zone material that is rolled in during gastrula-
tion. But, in the germ resulting from the fusion of two animal
halves, no rolling in takes place, and there is therefore no room
for the expanding ectoderm, which is obliged to fold. The
material of the vegetative field shows hardly any capacity for
active movement. It seems to be more or less passive in gastrul-
ation; only the very first beginnings of the blastopore furrow
in this area appear to be due to an active power of invagination
of the cells, which assume a club shape, as mentioned above
(Vogt; 1922, 1929).
This power of active movement, or topogenetic potency of

102

THE TOPOGENESIS OF THE EMBRYO

Fig. 37. (a-b) normal gastrulation, seen a: from the vegetative side,
and b: fro'm the left side; (c-d) exogastrulation. The arrows indicate
the directions in which the material moves. After Vogt and

Holtfreter.

the cells, is very clearly shown if the normal co-operation of
the migratory tendencies in gastrulation is disturbed in some
way or other. Holtfreter (1933) obtained such a disturbance
in newt eggs by taking them out of their capsules at an early
stage, and transferring them into certain salt solutions. The
resulting disturbance of gastrulation is known as "exogastrul-
ation". No invagination of the archenteron, accompanied by
rolling in of the material of marginal zone and vegetative field
takes place. On the contrary, an evagination occurs at the edge of
the blastopore, because marginal zone material moves through
the blastopore furrow to overlie the vegetative field. The result is
a germ in which endoderm and chordamesoderm are not sur-
rounded by, but lie altogether outside the ectoderm. The latter
forms an empty, folded pouch which may eventually become
completely separated from the rest of the germ at the blasto-
pore edge (Fig. 37). Now it is a remarkable fact that all the
other movements of gastrulation, and also part of the sub-
sequent movements of neurulation, take place in the normal

THE TOPOGENESIS OF THE EMBRYO 103

way, even though invagination has been suppressed, and turned
into an evagination. Notochord and somites develop from the
chordamesoderm, although the latter is not surrounded by-
ectoderm. There is a beginning of differentiation into head,
trunk, and tail, and even the first primordia of branchial pouches
are formed in the endoderm of the head-region. This shows that
the various topogenetic processes are more or less mutually
independent. It is essential for normal gastrulation, however,
that these processes are linked together in an orderly fashion.
Schechtman (1942) has demonstrated that normal rolling in of
prospective notochord material, and closure of the blastopore,
will only occur if the connections between the parts of the
marginal zone are uninterrupted.

Much of our insight into the nature of the moving forces
in gastrulation is due to experiments by Holtf refer (1943-44).
He found that the expansive tendencies of the animal field
depend on the presence of a tough, semi-elastic surface "coat"
which unites all cells into one mechanical unit, and governs
their mass-movement. This "coat" shows a tendency to expand,
and for that reason the region tends to occupy the largest
possible area. The formation of this surface layer is probably
due to the so-called Gibbs phenomenon, i.e. accumulation at
phase boundaries of substances that lower the interfacial
tension. In so far as it is provided with a similar layer, the
vegetative field also shows expansive tendencies, but to a lesser
extent than the animal field.

Certain specific attractions and repulsions existing between
different types of cells also result in topogenetic processes.
Holtfreter (1939), who studied these phenomena, has sum-
marized them under the term "tissue affinity''. These forces
manifested themselves clearly in experiments in which different
tissue elements were cultivated together. Holtfreter found that
under their influence parts of a cell mass may become separated,
or, conversely, cell groups of different types may fuse and
interpenetrate, so that they come into close contact. Such
processes play a very important role, particularly in later devel-
opment when the organs of the embryo begin to differentiate.

The following investigations have thrown some light on the

104

THE TOPOGENESIS OF THE EMBRYO

problem of the causation of the different topogenetic potencies
in different parts of the blastula. It has been known for a long
time that the gastrulation of amphibian eggs is often disturbed
if, soon after fertilisation, the eggs are fixed with the animal
pole downwards (Schultze, 1894), (of. p. 48). Schleip and
Penners (1925-28), and Motomura (1935) repeated this ex-

Fig. 38. Diagram of the co-operation of the gradients in the
development of amphibians. The cortical factor C decreases from
dorsal side (left in the figure) to ventral side (on the right), accord-
ing to line a . The yolk factor V decreases from the vegetative
pole (below) to the animal pole (above), according to line Q-
The product (7 X V is maximal at F (centre of the dorsal marginal
zone), and decreases gradually from there. The lines a, b, c, d, and
e indicate planes in which the product has the same value. The lines
HI and KL are the boundaries of areas in which the ratio C/V has
the same value. After Dalcq and Pasteels.

periment, and finally Pasteels gave a satisfactory analysis of
the phenomenon (1938-39). It appeared that in such eggs a
blastopore may be formed by invagination at any point where
yolk material is in direct contact with the egg surface, but

THE TOPOGENESIS OF THE EMBRYO 105

preferably at the side directed toward the original centre of
the grey crescent. From these observations, Dalcq and Pasteels
concluded that the occurrence of the potencies for gastrulation
in normal eggs is governed by (1) the yolk gradient, and (2)
the cortical field (see above p. 49). At any point, both the
"yolk factor" V, and the ''cortical factor'^ C, have a definite
value. Dalcq and Pasteels thought it probable that the product
of these factors, C X V, which also has a definite value at each
point of the egg surface, was a measure of the differences in
topogenetic potency. The first thought that comes to mind here
is that we are dealing with a chemical reaction between two
substances, which obeys the law of mass action. Where the
product C X V exceeds a certain value, the cells will acquire
a tendency to roll in. Where it remains below the liminal value,
there is a tendency for expansion in all directions. Where the
yolk concentration rises above a certain absolute value, the
cells remain more or less inert. Dalcq and Pasteels succeeded
in giving an elegant derivation, based on their hypothesis, of the
distribution of the topogenetic potencies in the normal blastula
(Fig. 38).

We can summarise the above as follows. The chemodiffer-
entiation which occurs under the influence of the gradient-
systems governing the egg's polarity and symmetry, gives rise
to different topogenetic potencies in the various parts of the
egg. At a given moment, these lead to the occurrence of cell
migrations which result in gastrulation, and further topogenesis.

CHAPTER Vm

Induction and organisation
L Neurulation

The cell migrations during gastrulation, and the further topo-
genetic processes to which the cells are subjected, bring the
materials for the embryo's future organs into those places
where they belong according to its structural plan. But they
also have another consequence. Cell groups which originally
were separated by considerable distances, and which, as a
result of chemodifferentiation, differ in physical and chemical
condition, now become immediate neighbours. It may be ex-
pected, therefore, that they will now influence one another, and
that the new topographical relations between the cell groups
will in this way initiate new processes of chemodifferentiation.
The outcome of these topogenetic processes will thus be a
considerable increase in the spatial multiplicity of the embryo.
There are, indeed, some observations, which give clear
evidence of changes in the physico-chemical constitution of the
cells during gastrulation. Woerdeman (1933) was the first to
show that a sudden fall occurs in the glycogen content of the
glycogen-rich marginal zone cells of amphibians, at the moment
when these cells are rolled in over the lip of the blastopore.
The following experiments proved that there was a causal
connection between the processes of invagination and of
glycogen decomposition. First of all, dorsal marginal zone
material of an early gastrula was grafted into the ventral margi-
nal zone of another germ. It then became rolled in, either over the
ventral lip of the host blastopore, or over the edge of a second-
ary blastopore (see p. 101). The moment of disappearance of
the glycogen always coincided with that of rolling in. Secondly.

I. NEURULATION 107

cells of the animal field were grafted into the dorsal marginal
zone. In this material, too, glycogen decomposition took place
when it was rolled in over the lip of the blastopore, even though
in normal development it would have stayed at the surface,
and retained its glycogen. Thirdly, the same material did not
lose its glycogen if brought directly into the interior of the
germ through a slit cut in the roof of the blastula (Raven,
1933-35). Finally, marginal zone material does not lose its
glycogen when cultured in isolation from the rest of the embryo
(Jaeger, 1945). Hence, the disappearance of the glycogen pre-
sumably is a direct consequence of the rolling in of the cells
during gastrulation.

In this context mention must also be made of observations
on the distribution of ribonucleic acid and sulphydryl com-
pounds (Brachet, 1940-42), and of alkaline phosphatase (Kruge-
lis, 1947) in amphibian embryos. In each case, a more homo-
geneous distribution, obtaining in the younger stages, was seen
to be replaced during gastrulation by a distribution in which
there were distinct differences in concentration among the
various parts of the germ.

However, such observations on changes in the physico-
chemical constitution of the cells are rare. In most cases, the
occurrence of chemodifferentiation cannot be demonstrated
directly; it appears only indirectly from changes in the
potencies of the cells during gastrulation. We possess more
accurate knowledge on this point, particularly in amphibians.

The histogenetic potencies, or potentiality for differentiation,
of the cells can be studied by removing a small group of cells
from the germ, and rearing it in isolation in an appropriate salt
solution. This method is called explantation. TroMsplantation,
the grafting of the removed cell group into another embryo,
is another useful method. In the case of explantation, we may
generally assume that the cells are not exposed to any specific
modifying influences on the differentiation processes. Such
experiments inform us what the cells concerned can do "on
their own", and therefore give us an insight into their auto-
nomous powers of differentiation. Cells transplanted into an-
other embryo will there be subjected to the action of the

108 INDUCTION AND ORGANISATION

surrounding tissues of the host. This will vary according to the
place and time of grafting, so that divergences in different-
iation will occur. By grafting similar cell groups into various
parts of hosts of different nature and age, the range of possible
differentiations of the material can be studied.

The differentiation potencies of various parts of young
amphibian gastrulae have been studied by these methods. The
following results were thereby obtained. The material of the
vegetative field has a very strong capacity for self-differentia-
tion already. Holtf refer (1931) cultured this material in a salt
solution, and found that it differentiated into vesicles with an
epithelial wall. The structure of this epithelium varied in accord-
ance with the place of origin of the explant. Sometimes it was
similar to the epithelium of the branchial part of the gut, at
other times it had the character of stomach or mid-gut epi-
thelium. This shows that there are qualitative differences in
differentiation potency even within the vegetative field. On the
other hand, the range of possible differentiations of this
material is limited. If transplanted it produces only endo-
dermal tissues- This proves that the cells' inherent ''differentia-
tion tendency'' can maintain itself, in spite of environmental
influences that work in another direction.

The marginal zone material also appears to have an intrinsic
capacity for differentiation. In Holtfreter's explantation ex-
periments, it differentiated into notochord, muscle, kidney
epithelium, etc. Within the marginal zone, too, there are
qualitative differences in differentiation potencies. The tendency
to form a notochord preponderates in the dorsal part, that to
form muscular tissue in other places, etc., as proved for
example by Bautzmann's (1933) transplantation experiments.
But the mode of differentiation of the various parts of the
marginal zone is not yet irrevocably fixed, or "determined".
In Holtfreter's experiments, notochord and muscle nearly al-
ways arose simultaneously from the same material. If the
explant is large, it may even produce ectodermal tissues, as
was first noted by Lopashov (1935). Evidently, the marginal
zone material has a wider range of possible differentiation
than is manifest in normal development.

I. NEURULATION IQQ

Hence, the cells of the vegetative field and the mai'ginal
zone, at the beginning of gastrulation, possess inherent capaci-
ties for self-differentiation, which even show local qualitative
differences. The animal field, on the other hand, gives an
entirely different picture. When isolated in a salt solution,
material from this area produces only irregular masses of cells,
in which the individual cells have an atypical structure. Only
if mesenchyme cells are present as well, vesicles will be formed,
the walls of which consist of an epithelium that is completely
similar to the epidermis of normal embryos. This shows that
here the capacity for self-differentiation is still very poor. On
the other hand, the range of possible differentiations is still
practically unlimited. A wide variety of transplantation ex-
periments has shown that the cells of the animal field can
differentiate either into muscle, or into nervous tissue, into
notochord, or kidney, gut or liver. In other words, practically
all the tissues of the body can be formed by this material. It
is still "omnipotent" (Holtfreter, 1933; Raven, 1935). There
are no local differences within the animal field in this respect.
Cells from the dorsal half, i.e. material of the prospective
neural plate, behave in entirely or practically the same way as
cells from the ventral half, i.e. prospective epidermis (cf. Fig.
35). If parts from these two regions of the animal field are
exchanged, they fit harmoniously into their new environments,
and can entirely substitute for each other (Spemann, 1918),
(Fig. 39). Evidently the cells of the animal field are of a
highly indifferent character; their chemodifferentiation is still
very slight.

Now it is in this material that the progress of chemo-
differentiation during gastrulation, and the modification of
differentiation potencies involved, are manifested most clearly.
For a completely different situation is found if after gastrula-
tion, at the neurula stage, the potencies of the ectoderm are
again investigated by means of transplantations and explant-
ations. At this stage the ectoderm cells are no longer alike in
their differentiation potencies. A part of the neural plate will
produce nervous tissue, even if grafted into skin ectoderm
(Fig. 48), and, conversely, a piece of skin ectoderm, grafted

110

INDUCTION AND ORGANISATION

into the foreign environment of the neural plate, will still
produce epidermis (Spemann, 1918). Also in the case of ex-
plantation, neural plate material will differentiate into nervous
tissue, and skin ectoderm (in the presence of mesenchyme cells)
into epidermis (Holtfreter, 1931). Evidently, the two regions
of the ectoderm have now acquired different differentiation
potencies. At the same time, the range of possible different-
iations has been cut down. After transplantation into a foreign

Fig, 39. Heteroplastic transplantation of presumptive ectoderm at
an early gastrula stage, (a) gastrula of Triton taeniatus with graft
from T. cristatus; (b) gastrula of T. cristatus with taeniatus graft;

(c) the taeniatus embryo of fig. (a), at a later stage; the graft
(light pigmentation) occupies the front part of the neural plate;

(d) cross section of this taeniatus embryo; part of the brain wall
(between the crosses) has developed from the light cristatus graft,
which has produced brain tissue in accordance with its new environ-
ment. After Spemann.

I. NEURULATION 111

environment, ectodermal grafts will produce only ectodermal
tissues, and no muscle tissue, notochord, or gut any more
(Mangold, 1923). This shows that chemodifferentiation has
progressed in the animal material, the parts of which were still
equivalent at the beginning of gastrulation. The cells have
been fixed in one definite direction; they have become de-
termined to one definite differentiation.

How does this determination take place? Experiments by
Spemann (1918) have given a clear answer to this question. It
appears that the material of the dorsal part of the marginal
zone is provided with special properties. We have seen that
during gastrulation this material is rolled in over the dorsal
lip of the blastopore, and subsequently it forms the roof
of the archenteron, from which the notochord and somites
originate. Spemann removed part of this material from
an early gastrula, and grafted it into the ventral side of an-
other germ of the same age. In this region the primordium
of a complete embryo was formed, consisting of neural tube,
notochord, somites, and gut. Spemann's pupil Hilde Mangold
(1924) repeated this experiment, but combined two different
species, in a so-called heteroplastic transplantation. The graft
was taken from the dorsal lip of the blastopore of Triton
cristatus, and it was grafted into the ventral side of T. taeniatus.
It became clear in this case that only part of the secondary
embryo originated from the graft itself. The latter was, indeed,
rolled in under the influence of its special topogenetic potency,
and it produced notochord and somites. The neural primordium
of the secondary embryo, however, did not arise from the graft,
but entirely or for the greater part from the overlying ectoderm
of the host. The secondary gut primordium also developed from
host material (Fig. 40). This means that the graft enforced
a definite course of development on the cells of the host. It
''induced" them to undergo a cert^.ln differentiation. This,
however, did not lead to a chaotic aggregate of tissues, but
the various organs produced by the co-operation of graft and
host showed normal topographical interrelations so that to-
gether they constituted an organised embryo. In very favour-
able cases, this may go so far that the anterior end of the

112

INDUCTION AND ORGANISATION

Fig-. 40. Organiser activity in amphibians, (a) neurula of Triton
taeniatus, with (b) a secondary neural plate at the ventral side,
induced by a piece of dorsal marginal zone material from T. crista-
tus, part of which is still visible as a narrow white band in the
neural plate; (c) embryo of T. taeniatus with a secondary embryo
primordium (with neural tube, somites, ear-vesicles, and protruding
tail-bud) in its left side; (d) cross section of the embryo of fig. (c),
showing primary embryo primordium on the left, secondary pri-
mordium on the right; the latter consists partly of the light tissues
of the organiser, partly of the dark material of the host. After

Spemann and H. Mangold.

a

, m^zW'^ ♦ » * ifg,~

^^.

PLATE VII. Head- and trunk organiser, (a) Part of the blastopore lip
of an older gastrula has induced a secondary primordium, of a trunk-like
nature, in the trunk of the host. The two embryos are parallel. In both, the
notochord is sectioned lengthwise (pr.ch. and s.ch.), whereas the neural tube
is sectioned at two places (pr.n. and s.n.). The somites of the two embryos
may be seen on both sides of each notochord. (b) Part of the archenteron
roof of an older gastrula has induced a piece of fore-brain, with eye-vesicles
(S.O.), in the trunk area of the host, (cf. Fig. 47a). After Spemann.

s./?.

S

O

♦ ?•«

^ y *'

^ * ♦ - /

*\' * * ■ , t

^ ^ • * -- ^ .. ' - /

* ^ ^

PLATE VIII. The co-operation between organiser and host tissues, (a)
Secondary embryo primordium, organised by one half of the blastopore lip
of an early gastrula. The graft (light pigmentation) produced the notochord,
flanked on either side by somite material, and the floor of the secondary
neural tube (s.n.). The host produced all the remainder, (b) Secondary
embryo primordium, organised by one half of the blastopore lip of a late
gastrula. The graft has formed only the notochord, and the somites of one
side. The host has produced all the rest. p.n. = primary neural tube; s.n. =
secondary neural tube; d. = lumen of the common gut. After Mayer.

I. NEURULATION

ii:

Uo*

secondary embryo forms a distinct head with brain, eyes, ear-
vesicles, etc., while its posterior end develops into a tail bud.
The secondary embryo is then distinguishable from the primary
embryo only by its slightly smaller size (PI. VI) *. For these
reasons, Spemann called such a graft an ''organiser", and the
dorsal marginal zone from which it originated the ''organisa-
tio7i centre'' of the embryo. In this way he voiced his conviction
that this area played a role in
normal development as well. Bautz-
mann (1926) determined the exact
limits of the organisation centre.
At the beginning of gastrulation,
the organising power, or, more
accurately, the power to induce a
neural plate, was found to be limit-
ed to the dorsal half of the margin-
al zone, i.e. the area which in
normal development provides the
notochord and somites (Fig. 41).
The first experiments of Spe-
mann and Hilde Mangold had al-
ready shown that in secondary
embryos the organ boundaries did
not necessarily coincide with those
between graft and host. In some

cases, part of the graft was not rolled in, but remained at the
surface, and associated with the ectoderm. This was then
incorporated into the neural plate of the secondary embryo,
so that this organ was a "chimaera", consisting partly of graft
tissue, and partly of host tissue (Fig. 40 b, d). A similar
chimaerical composition was sometimes encountered in the
notochord and somites of the secondary embryo, where cells of
the host's ventral mesoderm, bordering on the graft, took part
in the formation of these organs (Fig. 40 d). This means that
the inductive activity of the graft did not only manifest itself
in the overlying ectoderm, and the underlying endoderm; it
had evidently also extended from cell to cell within the meso-
derm, and forced a development in an aberrant direction on

* Facing page 97.

Rav^en - Outline Physiologie 8

Fig. 41. Extent of the orga-
nisation centre (dotted) in
the initial stage of gastru-
lation of Triton taeniatus.
Germ seen from the left;
the wavy line indicates the
blastopore furrow. After
Bautzmann.

114

INDUCTION AND ORGANISATIOM

the mesoderm of the host. It is important to make a clear
distinction between these two types of embryonic induction,
and to discuss them separately. They are: (1) induction by
contact; here the "inductor'' and the material that reacts to
it, the so-called ''reaction system", are in immediate contact, and

Fig. 42. Diagram of a cross section through the dorsal part of an
embryo, illustrating the difference between contact induction and
progressive induction. The organiser (hatched) influences the over-
lying ectoderm and the underlying endoderm by contact induction
(arrows drawn in full), but it influences the surrounding mesoderm
by progressive induction (interrupted arrows).

(2) progressive induction, which extends from cell to cell in
a coherent layer of cells (Fig. 42).

In normal development, the material of the dorsal marginal
zone, or organisation centre, becomes invaginated during gastrul-
ation, and forms the archenteron roof. It becomes located under
that precise part of the ectoderm which will later differentiate
into the neural plate. The hypothesis is obvious that here,
again, "contact induction" plays a role, and that the neural
plate is determined under the influence of the underlying
archenteron roof. Marx (1925) demonstrated that the latter
material does indeed possess inductive powers. He cut out
part of the archenteron roof of an older gastrula, and brought
it into the blastocoel of a blastula. The slit in the blastula wall
through which it was inserted soon healed again (Fig. 43 a).
During the gastrulation of the host, the blastocoel was obliter-
ated by the archenteron, and the implant then lay immediately
under the ectoderm. Therefore, it was in a position to exert
such inductive influence as it possessed (Fig. 43 b). This
"insertion method" has proved very effective, and it has been

I. NEURULATION

115

Fig. 43. Diagram of the "insertion method"; (a) through a small

incision, the implant is inserted into the blastocoel; (b) during

gastrulation it is pushed against the ventral ectoderm of the host

by the invaginating archenteron.

employed in a great number of other investigations on the in-
ductive powers of certain implants. In Marx's experiments,
small but distinct neural plates developed in the ectoderm
overlying the grafts consisting of archenteron roof, proving that
this material possessed inductive powers.

In this way the mechanism of the determination of the neural
primordium in normal development was elucidated. Position
and size of the neural plate evidently depend on position and
size of the underlying archenteron roof. Moreover, Spemann
(1931) demonstrated the existence of qualitative differences
within the organisation centre, which are responsible for the
regional differentiations of the neural plate. The first in-
vaginated part of the marginal zone, which becomes located at
the anterior end of the archenteron roof, induces especially the
cranial parts of the neural primordium, i.e. the brain, with eyes
and ear-vesicles. The material that invaginates later, and forms
the caudal parts of the archenteron roof, induces the caudal
regions of the neural primordium, i. e. the spinal cord (PI. VII) *.
It appeared that the influence of the archenteron roof extended
to the further development of the neural primordium as well.
Topogenesis and differentiation of the central nervous system
proceed in immediate dependence on the underlying notochord

* Facing page 112.

116 INDUCTION AND ORGANISATION

and somites. Many experiments by Lehmann (1926-35), Holt-
freter (1933), and others have demonstrated that defects in
the archenteron roof cause characteristic abnormalities in the
brain and spinal cord. Further, it was shown that the chorda-
mesoderm retains its inductive powers for a long time. Pieces
of notochord or somites from older embryos (neurula or tail-
bud stage) have been investigated by the "insertion method".
They proved still to be able to induce a neural plate in the
ectoderm of the gastrula (Bautzmann, 1928; Holtfreter, 1933).
In contrast, the reactivity of the ectoderm to stimuli inducing
a neural plate was found to be restricted to a certain stage
only^). Once the germ has reached the neurula stage, the
reactivity has disappeared. This is the reason why the secondary
neural plate nearly always becomes visible simultaneously with
the primary neural plate, even though the inducing stimulus
has had an opportunity to exert its influence long before that.

The inductive activity is not species-specific. In the original
work of Spemann and H. Mangold an organiser of Triton
cristatus was already found to be active after heteroplastic
transplantation into T. taeniatus. Moreover, induction was
found to take place when two more distant species were com-
bined (so-called xenoplastic transplantation). In Geinitz' (1925)
experiments, marginal zone material not only from axolotl and
Pleurodeles, but even from frogs (Rana fusca and R. esculenta),
and toads (Bomhinator) induced neural plates in the ectoderm
of Triton.

Even greater interest was aroused by the discovery that the
power of inducing a neural plate was possessed, not only by
the roof of the archenteron, but also by other tissues, which
are not in normal development involved in the formation of
the neural plate. This was demonstrated by bringing such ab-
normal inductors into immediate contact with overlying gastrula-
ectoderm by means of the "insertion method" mentioned above.
Inductive powers were found not only in the neural plate itself,
and in brain tissue of older larvae (Mangold and Spemann,
1927), but in material of the larval limb primordia as well

1) Nowadays such a reactivity is often called "competence".

I. NEURULATION 117

(Mangold, 1928). Next, Holtfreter (1934) showed that many
tissues of a wide variety of animals, both vertebrate and in-
vertebrate, have the power of induction. He studied not only
living tissues, but also dried, boiled, and minced material, and
these, too, appeared to have inductive capacities in many cases.
Moreover, not only neural plates, but also other organs, such
as olfactory pits, eyes, ear-vesicles, limbs, etc., were formed
by the host ectoderm under the influence of the inductors. It
was concluded from these experiments that the inducing agent,
or possibly agents, are very widespread in animal tissues, and
that the processes concerned are of a very general character,
and of low specificity.

All interest now centred on the problem of the nature of
induction. Marx (1931) found that narcotized organisers did
not lose their power of induction. Spemann (1931) found the
same in ground organisers. Even dead tissue proved still to
possess inductive powers. Organisers that had previously been
dried, frozen, boiled, or killed with alcohol, still caused the
formation of neural plates in the overlying ectoderm (Holt-
freter, 1933). The remarkable discovery was made that even
embryonic tissues which had no power of induction when alive,
acquired this capacity after death. This was found, for example,
in the prospective ectoderm and endoderm of gastrulae.

It was an obvious hypothesis at this stage to ascribe the
induction to a substance, given off by the inductor, which
causes the ectoderm cells to form neural tissue. This inducing
substance was called "evocator'' by Waddington, and "organ-
isiwe" by Dalcq. In normal development it would be given off
only by the archenteron roof. It might be present in other
parts of the embryo, but there it would be prevented from taking
effect in some way or other. It was assumed that many other
animal tissues also contained the substance. This was confirmed
by the successful preparation of extracts from animal tissues
which, absorbed by an agar-agar carrier, induced a neural
plate in the ectoderm of an amphibian gastrula. In principle,
the determination of the nature of these evocators had thereby
become possible. On this point, however, research has not led
to satisfactory results. It was possible, indeed, to determine the

1,18 INDUCTION AND ORGANISATION

chemical nature of certain of the extracted products, and even
to obtain inductions with synthetic preparations of such sub-
stances. But a wide variety of organic compounds, such as fatty
acids, nucleotides and sterols, had the same effect. The con-
clusion was inevitable that no specific chemical actions are
involved, but that the ectoderm will react to the most diverse
influences with the same reaction, namely, the formation of
a neural plate. Recent experiments by Holtfreter (1945-47)
have made it likely that an injury to the host tissues is the
primary effect in many cases of so-called induction by means
of chemical substances, or of dead, or foreign tissue. The injury
is supposed to increase the water permeability of the ecto-
dermal cell walls, and this, in turn, to lead to enzymatic
decomposition of certain components of the cytoplasm.
Presumably, substances are thereby liberated, which direct the
differentiation of the cells into a course leading to a neural
plate. This makes it unlikely that experiments with such
abnormal agents can be used to deepen our insight into the
normal process.

In later years, therefore, another course has been followed,
which took V/oerdeman's results (p. 106) as its starting point.
Woerdeman found a very rapid decomposition of glycogen in
cells of the marginal zone that were being rolled in. Heatley
and Lindahl (1937) confirmed this, using quantitative micro-
analytical methods. Although it was found that glycogen itself
could not be regarded as the evocator, yet attention was now
called to the metabolic processes in the marginal zone material.
Several investigations, in particular those of J. Brachet, showed
that the cell metabolism in the organisation centre differs from
that in the rest of the germ. Both the COo production, and the
respiratory quotient have higher values. Further research in-
dicated that the main role is played here by protein metabolism,
and not by carbohydrate metabolism. Attention was drawn to
the sulphydryl compounds which are highly important in the
oxidation and reduction processes in the cell, and later also
to the ribonucleic acids, with which the sulphydryl compounds
are closely linked. J. Brachet (1938, 1942) proved that in amphi-
bian embryos these compounds are specially accumulated at

I. NEURULATION 119

places where the processes of development are most active.
Originally, they are to be found in the nucleus of the oocyte;
later, they scatter over the egg cortex, especially at the dorsal
side. During gastrulation, a considerable accumulation of
ribonucleic acid and sulphydryl compounds occurs in the lips of
the blastopore, and afterwards in the archenteron roof, in
particular in those parts of the cells that face the overlying
ectoderm. The inductive powers of dead inductors are pro-
portional to their ribonucleic acid contents, and disappear on
decomposition of these substances, e.g. by boiling or by enzyme
treatment (Brachet, 1945) • All this suggests that these substances
play a major role in development, and that they are important
for the contact induction of the neural plate as well. It has not
yet been ascertained whether or not the latter process is
produced by diffusion, from the archenteron roof into the
overlying ectoderm, of ribonucleic acid, or of oligonucleotides
formed by its hydrolysis. Another view was in recent years
put forward by Brachet (1952) ; according to him the micro-
somes, which contain the major part of the ribonucleic acid
contents of the cell, would pass as such from the cells of the
archenteron roof into those of the ectoderm. Finally, mention
must be made of experiments by Kuusi (1951) which suggest
that different substances play a role in the induction of the
rostral and caudal parts of the neural plate (cf. what was said
above, p. 115, on the qualitative differences in the inductive
powers of the archenteron roof). Ribonucleic acid may be
specially important for the induction of the anterior parts of
the brain, whereas the evocator for the caudal part of the
neural plate may have the properties of a protein. Recent
experiments by Nieuwkoop (1952), however, have led to the
assumption that the determination of the neural plate takes
place in two phases. The first of these, "activation", would be
the same in all regions of the neural plate. Possibly it is due
to the ribonucleoproteins of the whole archenteron roof. Activ-
ation would be followed by a second phase, during which the
posterior parts of the central nervous system acquire their
regional character. It is conceivable that Kuusi's spinal evocator
is the agent causing the second phase.

120 INDUCTION AND ORGANISATION

Whatever their nature may in the end prove to be, we must
in any case assume that the evocators originate in the cellular
metabolism of the archenteron roof (possibly also in that of many
other animal tissues), and that they are taken up by the over-
lying ectoderm, and influence its chemodifferentiation so that
it becomes able to form a neural plate. For this purpose, the
ectoderm cells must be sensitive to the activity of these partic-
ular substances. We have seen that this is the case only during
a short period. Therefore, the production of the evocator by
the organiser, and the specific sensitivity of the reaction system
must CQincide. Presumably the sensitivity is due to the fact
that at exactly the right moment the evocator fits the cellular
mechanism of the reaction system, as a key fits the lock. It
may be, for instance, that the evocator can saturate unsaturated
bonds which would not be present at any other stage, or that
it can catalyse certain reactions which take place at this stage
in the cell's development, and which are impossible at all
other times. It is the mutual adjustment of the chemical activity
of the cells of organiser and reaction system, that ensures the
normal course of development.

We have already mentioned that the activity of a trans-
planted organiser is not restricted to the ectoderm of the host,
but that the adjacent mesoderm is influenced as well. Evidently,
the induction spreads from cell to cell in the latter (Fig. 42).
This causes the cells of the host's mesoderm to differentiate
into notochord and somite tissue. The inductor, therefore, makes
this tissue similar to itself, hence the name "assimilative in-
duction'' for this process. As there are a number of differences
between this type of induction and contact induction, we shall
now discuss progressive induction.

We have already seen that the grafted organiser does not
always form the same components of the secondary embryo.
Often the materials of graft and host co-operate in the forma-
tion of its organs, giving them a "chimaerical" composition,
which varies from case to case, depending upon the experi-
mental conditions. This is clearly illustrated by a number of
experiments by B. Mayer (1935). He performed heteroplastic

I. NEURULATION 121

transplantations of a lateral half of the organization centre of
one embryo into the ventral side of another. One edge of the
graft, therefore, consisted of mid-dorsal material which would
normally have formed a notochord, and the rest consisted of
prospective somites (cf. Fig. 35). The result of the experiment
depended on the age of the gastrula from which the graft was
taken. If it was taken from an older gastrula, its originally
median part formed a notochord, whereas the remainder pro-
duced a single series of somites. Both areas, therefore, differ-
entiated according to their original potencies (PL VIII, b) *.
Grafts taken from an early gastrula, however, produced a noto-
chord flanked on both sides by somites (PL Villa) *. This shows
that "regulation" took place here, which made possible the
development of a symmetrical archenteron roof from one half
of the organiser, and led to a deviation in the differentiation of
certain regions from their normal fate. In both cases, the host's
mesoderm supplied the missing parts of the archenteron roof.
The latter effect was also seen in another experiment, in which
the graft formed only a neural primordium, whereas the host
itself produced notochord and somites (Raven, 1938). The most
important aspect of these facts is that, whatever the shares
of graft and host in the composition of the organs may be, yet
in many cases a more or less complete secondary embryo is
formed. The tissues of graft and host supplement each other;
one produces what the other cannot supply. There is endless
variety, therefore, in the ways in which the secondary embryo
may arise from the co-operation of graft and host. The use
to which the cell material is put varies from case to case, and
its origin from graft or host does not determine its fate. The
organising process is not restricted to the organiser. From
there, it extends into the surrounding tissues as well, and
unites graft and host tissues into one integrated system. The
differentiation of each cell depends only on its position in this
system, and not on its origin. The best way to formulate these
facts seems to be the statement that, starting from the or-
ganiser, an organisation-field arises.

The organisation-field is a system within which a number of
developmental factors are distributed in such a way that the

Facing page 113.

122 INDUCTION AND ORGANISATION

formation of certain organ primordia in the field is thereby
determined. The fate of each element depends on its position in
the field. The field possesses the power of regulation. This
expresses itself in two phenomena. Firstly, it is not tied to
definite material elements, but instead it displays a certain
freedom as regards them. It can extend over the material, and
readjust itself; the field can be ''transposed". Secondly, its
size may vary according to circumstances. After the removal,
or addition, of material, it can adjust itself to the new con-
ditions. We shall now discuss some examples of these regulative
capacities.

Early gastrulae of Triton can be halved, by transection or
constriction, in such a way that each part contains one half
of the organisation centre. Two complete embryos will be form-
ed in this case. Both may be slightly less well developed on the
side of the transection, but in all other respects they are
harmoniously built (Ruud and Spemann, 1922). Evidently an
entirely new organisation-field has arisen in each half. The
original field has been divided into two, and has, on a smaller
scale, reconstructed itself in each half. However, if the early
gastrula is halved frontally, so that its dorsal and ventral
halves are separated, the ventral half, which has no organisation
centre, will often produce only a "belly piece" lacking such
axial organs as neural tube, notochord and somites^). The
dorsal half, which contains the whole organisation centre, will
nearly always produce a harmoniously built embryo of half
the normal size (Fig. 44). Here, again, regulation takes place,
for in spite of the fact that the whole organisation centre is
present, the organisation-field is formed on a smaller scale,
adapted to the lesser proportions of the halved embryo. Dalcq
and Huang (1948) have repeated these experiments with a more
accurate technique. They have shown that the processes under-
lying this regulation are of a far more radical nature than
Ruud and Spemann suspected. An entirely new organisation
takes place in the halved germs, which disposes of the available

1) According to recent work by Dalcq and Huang, howev^er, such a ventral
half will, if the ligature was made at the blastula stage, often form an embryo.

NEURULATION

123

material in a more or less "arbitrary" way, varying from case
to case.

This power of regulation was also encountered in experiments
in which material of the organisation centre was explanted,
and cultured in a salt solution. In this case, it produced not

\M.y

Fig. 44. Above: Closing neurula of Triton taeniatus, seen from
dorsal, from ventral, and from the right side. Below: embryo de-
veloping from the dorsal half (after frontal transection of an early
gastrula) at the same stage of development. The latter embryo is
quite harmoniously built, as may be seen on comparison with the
normal embryo. After Ruud and Spemann.

only notochord and muscular tissue, but, if sufficient material
was present (e.g. because several blastopore lips were cultured
together), ectoderm would be formed as well (Lopashov, 1935).
This ectoderm surrounded the other tissues, and here, too, in
part of it the formation of nervous tissue was induced. In this
way organ complexes were formed that had some faint re-
semblance to embryos.

The organisation-field can also extend over indifferent

124

INDUCTION AND ORGANISATION

material that comes within its reach. Such material then
acquires the power of organisation, and can thenceforward act
as an organiser itself (Spemann and Geinitz, 1927). A piece
of prospective ectoderm from one early gastrula, grafted into
the dorsal lip of the blastopore of another, will in 24 hours

Fig. 45. Diagram of operation, (a) part of the presumptive ecto-
derm, of an early gastrula is (b) grafted into the dorsal lip of the
blastopore of another germ of the same age. After 24 hours, (c),
part of the graft has already been rolled in over the edge of the
host's blastopore. The part that is not yet rolled in is now removed,
and (d) grafted into the ventral side of a third gastrula.

acquire all the properties of blastopore lip material. If removed
again, and grafted into the ventral side of a third embryo
(Fig. 45), it will first of all prove to have acquired the topo-
genetic capacity of independent invagination, followed by
differentiation into notochord and somites. Moreover, it has
become an organiser which gives rise to the primordium of
a secondary embryo at the place of implantation (Raven,

NEURULATION

125

\

1938 a), (Fig. 46). This means that here we have another case
of assimilative induction; the piece of ectoderm has acquired
organiser properties by its stay in the organisation-field; this
is the reason why it is able to give rise to a new organisation-
field after transplantation into an indifferent area. It is possible,
in other words, to transfer the organ-
ising power to indifferent material. This
is fresh evidence of the freedom of the
field with regard to the material ele-
ments of the germ.

At first sight, it may seem that the
concept "organisation-field" does not
help us much to acquire a real under-
standing of the phenomenon of organ-
isation. It does not reduce the great
problem, how qualitatively different
organs can arise side by side from one
common material. The concept, however,
has shown its fecundity by giving rise
to working hypotheses for the solution
of this problem. Dalcq's hypothesis may
be taken as an example. Its main features

i

are that the organisation-field is sup-

Fig. 46. The ecto-
derm graft has ac-
quired the properties
of an organiser be-
cause of its stay in
the blastopore lip. It
has organised the
primordium of a se-
condary embryo in the
right flank of the
host, o : secondary
blastopore.

posed to be a gradient-field (cf. p. 42),
due to local differences in the concentra-
tion of a substance, ''organisine", iden-
tical with that which is responsible for
contact induction. Its concentration
would be highest in the centre of the
dorsal marginal zone, and from there it would decrease in all
directions, though perhaps not everywhere at the same rate.
This chemical gradient-field, in turn, is supposed to owe its
origin to the interaction of the yolk gradient and the cortical
gradient of the egg (cf. pp. 49 and 105). Here, however, it
would be the ratio, and not the product, of the factors C and V
that would be important (cf. Fig. 38). The origin of qualitatively
different organs in an orderly spatial arrangement would be
due to the dependence of cell differentiation upon the "organ-

126 INDUCTION AND ORGANISATION

isine" concentration. At very high concentrations, a notochord
would be formed; at a lower concentration, somites, and so on.
Which differentiation occurs, would depend on whether or not
the concentration exceeds a certain liminal value. The regulation
phenomena can easily be explained on this basis. After disturb-
ances in the germ, the organisine would be redistributed. Simple
diffusion would lead to an equilibrium in which its concentration
decreased again in all directions from a central maximum. The
expansion of the organisation-field from a grafted organiser
into the surrounding tissues can also be explained simply by
diffusion of organisine, and the same applies to the trans-
ference of organising activity to indifferent material grafted
into the organisation centre.

Later, Dalcq has extended his hypothesis by attributing
great importance to the ''physiological competition'' between
parts of the germ. This concept was introduced by Spiegelman
(1945), who used it in a theoretical discussion of the phenomena
of regeneration (cf. Ch. XI). Neighbouring groups of developing
cells are supposed to compete by monopolizing food substances,
and by flooding each other with the waste products of their
metabolism. A cell group favoured in some way, would thus
be able to suppress the differentiation of other groups in its
environment. This is the so-called phenomenon of dominance.
Dalcq (1947) applied this idea to the relationships in the
organisation-field, and assumed that the cells compete for the
limited quantity of "organisine" that is available. He succeeded,
indeed, in giving simple explanations of several observed
phenomena on this basis.

Dalcq's expositions form a fertile working hypothesis which
deserves to be tested by further experiments. Several recent
studies have already given it strong support, e.g. the work
of Raven and Kloos (1945) who investigated the inductive
powers of the median and lateral parts of the archenteron
roof. In any case, the hypothesis shows that it is possible, in
principle, to tackle the problem of organisation, however
mysterious it may seem at first sight, and to find a simple
explanation for the origin of the orderly spatial multiplicity
of the germ.

I. NEURULATION 127

If Dalcq's hypothesis proves correct, new problems will arise
at once. The phenomenon that the material of the germ can
react to quantitatively different organisine concentrations by
qualitatively divergent differentiation awaits further analysis.
In this way new riddles, setting new tasks to science, are
brought to light with each new problem solved.

CHAPTER IX

Induction and organisation
II. The period of organ development

Neurulation is immediately followed by a rapid increase in the
spatial multiplicity of the embryo, caused by topogenetic pro-
cesses which now begin at various places in the body. During
gastrulation and neurulation, the topogenetic processes in the
germ formed a single coherent system, but now the processes
of folding, the cell migrations, and the invaginations and ex-
trusions, are of a more local nature. They result in the almost
simultaneous demarcation of a great number of organ primordia.
We shall now discuss this period of organ formation or
organogenesis.

There is conclusive evidence that here, again, a major role
is played by the interactions of the parts of the germ which,
under the name of induction and organisation, we have studied
in the previous chapter. The development of the eyes in amphi-
bians may serve as a first example for the discussion of these
phenomena, because these organs were among the first to be
studied from the point of view of developmental mechanics,
and because analysis has already made very good progress here.

The vertebrate eye is a complicated organ. It arises by the
combination of parts from different origins. After the neural
plate has closed, thereby forming the neural tube, both lateral
walls of the latter form a vesicular protrusion, the primary
eye-vesicle, in the region of the prospective fore-brain (Fig.
47 a). These protrusions at first have a wide communication
with the lumen of the neural tube, but this soon becomes
constricted so that the eye-vesicles are connected with the brain
only through the eye-stalks. The outer wall of the eye-vesicles
soon establishes contact with the skin ectoderm covering the

II. THE PERIOD OF ORGAN DEVELOPMENT

129

Fig. 47. Diagram of the development of a vertebrate eye, (a) pro-
trusion of the eye-vesicles (o) from the brain (h); Z = lens primor-
dium; (b) invagination of the eye-vesicle, resulting in the formation
of the eye-cup; formation of the lens-furrow, (Z); (c) eye-cup still
connected with the brain through the narrow eye-stalk (o.s.);
differentiation of the wall of the eye-cup into retina (r) and pigment
epithelium (p); lens (l) has become free from the epidermis (ep).

After Kiihn.

sides of the head. At this point, the ectoderm thickens, thereby
forming the lens-placode which then becomes invaginated, and,
as a lens-vesicle, disengages itself from the ectoderm. At the
same time, the eye-vesicle also invaginates, thereby forming
the double walled eye-cup (Fig. 47 b). The inner wall of the cup
becomes thicker, and develops into the retina; the outer wall
remains thin and forms the pigment epithelium surrounding
the retina (Fig. 47 c). Later the iris will arise from the edges
of the eye-cup, and the eye-stalk will develop into the optic
nerve which connects the eye with the brain. The transparent
cornea is formed by the skin overlying the eye. Finally, the
mesenchyme surrounding the eye also contributes its share. It
penetrates into the eye-cup and takes part in the formation

Raven - Outline Physiologie 9

130

INDUCTION AND ORGANISATION

of the vitreous hody^ and it also forms several tough membranes
around the eye.

One of the first problems arising in connection with the
development of the eye is: What causes the protrusion of the
eye- vesicles from the wall of the brain? Experiments in which

Fig. 48. (a) the right eye primordium is removed from the rostral
part of the neural plate of one embryo of Bombinator, and (b) is
grafted into the right side of another embryo; (c) cross section
of the larva developed from the embryo pictured in (b); an eye-cup
(o) has developed from the graft. After Spemann.

II. THE PERIOD OF ORGAN DEVELOPMENT 131

parts of the rostral end of the neural plate were transplanted
into other areas of the germ have shown that such grafts
could develop into normal eyes (Fig. 48). Evidently, the potency
for the formation of an ej^e-vesicle is already present in the
anterior parts of the newly formed neural plate. Mangold
(1928-29) has even shown that the material concerned possess-
es this eye-forming potency as early as the late gastrula stage,
before there is any neural plate at all. The ectoderm acquires
this potency under the influence of induction by the anterior
part of the archenteron roof. Experiments by Spemann (1931)
have shown that, after transplantation into the ventral side
of another embryo, this part of the organisation centre induces
not only brain tissue, but eyes as well (PL VII b). It is note-
worthy that the eye-forming potency is at first evenly dis-
tributed throughout the anterior region of the neural plate,
so that any part of it will form an eye after transplantation.
It is not until later that the eye-forming power becomes con-
centrated in two lateral centres, and lost in the median region.
Experiments by Adelmann (1929-37) have shown that this,
too, is due to the activity of the archenteron roof. From a
certain stage onwards, a narrow median strip of the archenteron
roof begins to exert an induction which suppresses the eye-
forming potency in the overlying ectoderm. This observation
supplies the explanation of a common deformity: chemical or
mechanical injury to the median part of the archenteron roof
in the head region destroys its inhibitory influence, and there-
fore, instead of two lateral eyes, a single median eye now
arises from the bottom of the neural tube (so-called cyclopia).
The next problem is whether, simultaneously with the pro-
trusion of the eye-vesicle, its later differentiation into retina,
pigment epithelium, and eye-stalk is also unequivocally deter-
mined. A grafted eye-primordium invaginates in the normal
manner, and forms an eye-cup. The differentiation of retina
and pigment epithelium also takes its normal course. This
shows that the determining influence of the archenteron roof
has also to some extent fixed the later phases of the develop-
ment of the eye-cup. Experiments by Dragomirow (1932-37),
however, have shown that, at the eye-vesicle stage, parts of the

132 INDUCTION AND ORGANISATION

vesicle wall can still replace each other. The final determination
of the lateral wall to form a retina does not take place until
the beginning of the invagination of the vesicle. These experi-
ments make it plausible that induction from the invaginating
lens-vesicle co-operates in this determination.

Let us now turn to the development of the lens. Transplant-
ation experiments have proved that in some amphibians the
ectoderm that will later form the lens has a weak lens-forming
potency already at the late neurula stage. If it is grafted, e.g.
into the ventral side, it will produce more or less well developed
small lenses (Harrison, 1920; Woerdeman, 1934). This potency
becomes much stronger at the time when the protruding eye-
vesicle comes into contact with the ectoderm. The assumption
was obvious that an inductive action from the eye-vesicle on
the overlying ectoderm plays a role here. The following experi-
ments proved that this hypothesis was correct. (1) In many
amphibians, no lens is formed if the eye-vesicle is removed
before it establishes contact with the ectoderm ; in other species,
only a slight thickening of the epidermis will occur, and in
only a few (e.g., Rana esculenta) a well developed lens is
formed, apparently independently of the eye-vesicle (Spemann,
1912), (Fig. 49). (2) If the lens ectoderm is replaced by other
ectoderm, originating, e.g., from head or belly, a lens will under
certain circumstances be formed by this foreign ectoderm.
(3) The same thing happens if an eye- vesicle is cut out, and
grafted under the ectoderm of another part of the body (Lewis,
1904), (PI. IX a, facing p. 136).

However, in the last two experiments, it appeared that the
reaction of the ectoderm is not always the same in this respect.
At early stages, it is true, the whole ectoderm still has the
power to react to induction from the eye-vesicle with the
formation of a lens. But at least in some amphibians, after
neurulation this capacity becomes restricted to the more or
less immediate neighbourhood of the eye. The area of the "re-
action-field" decreases in the course of development. The
capacity to react, the competence, remains strongest at the
place where the lens is normally formed, and decreases
gradually from this centre toward the periphery.

II. THE PERIOD OF ORGAN DEVELOPMENT

133

Fig. 49. Independent differentiation of the lens in Rana esculenta.
(a) extirpation of the right eye primordium at the neural plate
stage; (d) cross section of the resulting larva, 14 days after the
operation; the right eye (left in this figure) is absent; nevertheless,
a lens (Z) has been formed, which, however, (b) is smaller than that
of (c) the normal left eye. After Spemann,

Some details of this induction process may be mentioned.
First of all, it appears that there is a correlation between the
size of the induced lens and that of the inducing eye-vesicle.
The size of the latter can be reduced or increased experiment-
ally, by extirpation of material or fusion of two eye-primordia,
respectively. Such modified eye-vesicles always induce a lens
which approximately fits the eye-cup (Fig. 50). But this is
only true if eye-vesicle and lens ectoderm belong to the same
species, and not in the case of heteroplastic combinations.
Rotmann (1939) has shown that under the influence of the
(larger) eye-vesicle of Triton cristatus the ectoderm of T.
taeniatus forms a lens of the normal taeniatus size, and there-
fore too small in comparison with the eye. In the reverse

134

INDUCTION AND ORGANISATION

combination, the cristatus lens is too big for the taeniatus eye-
cup (PI. IX b-c). No continuous activity of the eye-cup is
necessary for the final differentiation of the lens. If an eye-
vesicle is removed after it has influenced
the ectoderm for 24 hours, the lens
primordium, as yet consisting only of a
slight thickening of the ectoderm, will go
on differentiating independently (Fila-
tow, 1934). The internal wall of the lens-
vesicle always supplies the lens fibres,
and its external wall the lens epithelium.
This polarity of the lens, too, is de-
termined by the eye-cup. However, other
organs than the eye-cup can exert the
same influence. If the lens-vesicle lies
in an abnormal part of the head, and
is in contact with an ear-vesicle, an
olfactory pit, or with the brain, lens
fibres are formed at the place of contact
(Balinsky, 1930; Dragomirow). Apart
from its polarity, the lens also has a
bilateral structure. Woerdeman (1939-
41) has shown that the various process-
es which play a part in the normal de-
velopment of the lens, such as the
formation of the lens-placode, the in-
vagination of the lens-vesicle, fibre
differentiation, and the development of
polarity and bilateral symmetry are
determined not simultaneously, but
successively.

The induction which gives rise to a
lens is not species-specific. Not only in
heteroplastic, but even in "xenoplastic" transplantations a lens
may be induced, e.g., by an anuran eye-vesicle in the ectoderm
of a urodele, and vice versa (Holtfreter, 1935).

It appears from an investigation on chick embryos by
McKeehan (1951) that direct contact of the eye-vesicle with

Pig. 50. The size of
the lens depends on
that of the eye-cup.
Left: small eye-cups
of Bombinator, ob-
tained by the removal
of varying quantities
of material. Right:
the normal eye-cups
of the other half of
the body. After Spe-
mann.

II. THE PERIOD OF ORGAN DEVELOPMENT 135

the ectoderm is necessary for the induction of a lens to take
place. If the contact is locally interrupted by the insertion
of a strip of cellophane, no lens placode is formed at that
place. This has led P. Weiss to the conclusion that lens in-
duction is not due simply to the diffusion of an "evocator", but
rather to a contact phenomenon, e.g. a stereochemical reaction
in the interface between inductor and reaction-system. However,
recent data from Woerdeman's laboratory cannot be brought
into full agreement with this view. Evidence was obtained there
by means of serological methods that specific lens proteins
are already present in the lens-primordium at the lens-groove
and lens-vesicle stages, i.e. before the beginning of actual fibre
differentiation (Ten Gate and Van Doorenmalen, 1950). Further-
more, Woerdeman (1950) demonstrated that such lens proteins
are also formed in mixtures of extracts of eye-vesicles and of
head-ectoderm from early embryos in which no lens formation
has yet begun. Here the reaction concerned evidently takes
place quite independently of the normal structure of the cells.

A further group of experiments was made on the origin of
the cornea. Both the eye-cup and the lens may induce the
formation of a cornea. In normal development, therefore, this
tissue is apparently formed under the combined influence of
both factors (Lewis, 1905; Fischel, 1915-19). The whole skin
ectoderm of the larva has the capacity to differentiate into a
cornea under the influence of these inductions. The presence
of the eye is necessary, not only for the development of a
cornea, but for its maintenance as well. After the extirpation
of eye-cup and lens, de-differentiation of the cornea takes place,
and it alters into normal skin. Here, again, the induction is not
species-specific.

It is clear, therefore, that the normal development of the
eye is based on an interplay of inductions, whereby each part
influences the development of all others, and also reacts to the
influences from all others, all parts having acquired the ne-
cessary reactivity in their previous chemodifferentiation.

Similar situations obtain in other organs. The olfactory organ
of the amphibians originates from a pair of thickenings of the
ectoderm in the anterior region of the head. Here, in front of

136 INDUCTION AND ORGANISATION

the eye-vesicles, the lateral walls of the neural tube come into
very close contact with the ectoderm. Later this part of the
brain will form a paired protusion, the cerebral hemispheres.
The ectoderm which is in contact with these parts of the brain
wall thickens into a pair of olfactory placodes, which later, by
invagination, form the olfactory pits. Several experiments
have shown that this differentiation of the ectoderm is also
caused by inductions from the brain wall (Raven, 1933). If
the rostral end of the brain is double, either partly, or com-
pletely, an olfactory pit is formed in contact with each of the
cerebral hemispheres. If one hemisphere is absent, the corres-
ponding olfactory pit is absent, too. In cyclopic embryos, the
fore-brain is poorly developed because of deficiencies in the
anterior part of the archenteron roof. In such cases, there is
not only a single median eye, but also a single olfactory pit,
situated in front of the anterior end of the brain. This, again,
is a non-specific induction. Holtfreter (1935) found that, in the
xenoplastic combination of anuran with urodele cell material,
induction of olfactory pits was still possible.

In the case of the olfactory organs, the stage at which the
definitive determination by contact induction takes place is
probably preceded by a phase during which there is a slight,
labile "predetermination" already, just as we have seen in the
case of the lens of the eye (above, p. 132). In particular in some
anurans, this early phase of determination may be very im-
portant, and can replace induction by the fore-brain completely,
or nearly so (Zwilling, 1940) ; it probably originates under the
influence of the archenteron roof.

The auditory organ, too, arises from an ectodermal thicken-
ing, the ear-placodCj which invaginates and becomes separated
from the ectoderm, forming the ear-vesicle. This gives rise
to the various parts of the internal ear by a series of folding
processes. In urodeles the area concerned generally acquires
an autonomous capacity to form ear-vesicle during the closing
of the neural tube. In anurans this may happen a little earlier.
Yntema (1939), however, has shown that in Amblystoma
punctatum a first, weak determination takes place at a much
earlier stage, viz. at the late gastrula stage.

"t

^4

PLATE IX. Induction of a lens, (a) Lens induction (L,. ind.) in the belly
epidermis of Triton taeniatus by an implanted eye-cup (Au. impl.) of T.
alpestris, after Mangold, (b) The eye-cup of T. taeniatus has induced a lens
in transplanted ectoderm from T. cristatus (left). This lens is larger than
a normal taeniatus lens (right), (c) The eye-cup of T. cristatus has induced
a lens in transplanted ectoderm from T. taeniatus (left). This lens is smaller
than a normal cristatus lens (right), (b-c) after Rotmann.

A.

V?

a

h

S^V-^hT^ ^-f ^^^ specificity of the reaction to the stimulus of induction Bellv
HPr? rT n""? f ^^°^ ^^^ FvT^"^^ ^^to the mouth area of a newt embryo^
Here (b, d) it fornis a mouth with horny teeth and a pair of suckers (Hn)
Both organs are characteristic of frog larvae, and do not occur in new"s

After Spemann and Schotte.

II. THE PERIOD OF ORGAN DEVELOPMENT 137

These phenomena were further investigated by Harrison
(1936). Initially, the potency to form ear-vesicle is not strictly
limited to the material that will later actually produce it. It
extends also over the surrounding ectoderm, though there is a
gradual decrease in intensity. Any cell group from this zone
can produce a complete ear-vesicle after transplantation, e.g.
into the ventral side. The size of such an ear-vesicle depends
upon that of the graft, but its structure may be entirely normal.
Several ear-vesicles can originate from one "prospective ear
region" in this way, and, conversely, two such regions can fuse
if grafted side by side. In the latter case, they produce one
ear-vesicle of double size. This proves that here, too, we find an
organisation-field possessing a certain freedom with respect to
the cell material.

Harrison has also investigated how the symmetry of the
auditory organ is determined. Each of these organs is in itself
asymmetrical, for it has different shapes in the three dimen-
sions. But one auditory organ is symmetrical to that of the
other side of the body. It is an important problem how the
determination into a right or left-side organ takes place. As
we shall discuss this more fully in connection with the limbs,
the following remarks will suffice here. Initially, the ear
primordium is still unpolarized, so that after transplantation it
develops into a left or a right ear in accordance with its new
environment. But soon the rostrocaudal axis, and then also the
dorsoventral axis, become fixed. Once this has happened, these
axes cannot be reversed any more, and when inverted the
primordium will go on developing in accordance with its original
polarity. From then on, a left primordium will always become
a left auditory organ, even if transplanted into the right side.

At a still later stage the determination of the component
parts within the "ear-field" takes place. After this, a grafted
part of the field no longer produces a complete auditory organ,
but only certain parts of one. Exchange of cell groups now
results in ear-vesicles of abnormal composition (Kaan, 1926).
This shows that the ear-field is no longer a unity, but that it is
broken up into a mosaic of parts with different potencies.
Moreover, the ear-forming potency is now restricted to those

138 INDUCTION AND ORGANISATION

cells that will really take part in the formation of the ear-
vesicle; the peripheral parts of the ear-field have lost this
potency.

For a long time it remained doubtful what, in normal devel-
opment, induces the ear-vesicle. On the one hand, Dalcq was
able to show that in Discoglossus there is a centre of ear-
vesicle induction in the lateral parts of the archenteron roof.
On the other hand, Trampusch (1941) made it seem likely that
an important role is played in the induction of the auditory
organ by a cell group lying in the neural folds during the
neural plate stage. Later, this group, the neural crests migrates
in a ventral direction under the ectoderm at the sides of the
head. Extensive experiments by Yntema (1950) have now
elucidated this problem, at least for Amblystoma 'punctatum.
According to his results, the determination of the auditory
organ takes place in two phases. At the late gastrula or early
neurula stage, a first, weak determination is effected by the
archenteron roof. From the late neurula stage onwards, a second
induction takes place, this time by the brain or the neural
crest. This gives the determination of the ear-vesicle a more
final character. There are qualitative differences between these
two inductions, and in the ectoderm two qualitatively different
phases of competence correspond to them. In other words, each
induction attains its maximum at about the same time at which
the ectoderm's ability to react also reaches its correspond-
ing peak.

Finally we must mention that xenoplastic induction of the
auditory organ between anurans and urodeles is possible
(Holtf refer, 1935). Such a xenoplastic labyrinth develops
normally for a long time. The speed of its development, and
its size and proportions are entirely analogous to those in the
species to which the ear-forming ectoderm belongs. It may even
function normally in the foreign larva. At a certain stage in
larval life, however, an incompatibility reaction suddenly sets
in, and the xenoplastic labyrinth rapidly disintegrates (Andres,
1949).

The anterior end of the archenteron, which was the first to
invaginate during gastrulation, comes into close contact with

II. THE PERIOD OF ORGAN DEVELOPMENT 139

the rostral ectoderm. A mouth plate, consisting of ectoderm
and endoderm is thereby formed. Later this breaks through,
and a mouth is formed. This process is due to induction by the
endoderm of the archenteron wall. If, at the neural plate stage,
this endoderm is grafted somewhere under the ectoderm of
the flank, a mouth cavity will be formed at this point (Stroer,
1933). Balinsky (1948) has demonstrated that here, too, a
weak predetermination precedes the final determination of the
mouth. Probably this predetermination takes place during
gastrulation. The results of xenoplastic transplantations are
highly interesting. In urodeles (newts), the larvae have real
teeth, consisting of dentine. In the anurans (tail-less amphi-
bians), however, they have horny teeth, formed by local
proliferation and cornification of the mouth epithelium, and
the mouth edges are covered with horny jaws. Spemann and
Schotte (1932) grafted belly ectoderm of anurans into the ventral
side of the head of Triton. This ectoderm reacted to the inductive
activity of the Triton mouth endoderm, with which it came
into contact, by forming a mouth cavity. This, however, had
the character of an anuran mouth, and was provided with horny
jaws and teeth. Moreover, immediately behind the mouth, a
pair of suckers, consisting of glandular epithelium, developed
from the grafted anuran ectoderm. These organs are always
found in the corresponding place in anuran larvae, but the
urodeles do not have them (PI. X). The tissue of the Triton
host, therefore, had induced organs in the graft which the host
itself did not possess. Holtf refer (1935) made the reverse
experiment, which had the expected result. Belly ectoderm
of Triton, grafted into the head of an anuran embryo, produced
a mouth with dentine teeth. These experiments give a very
clear illustration of the nature of induction. The cells of the
graft react to an "order" given by the inductor, but they do
it in their own way, by virtue of their own species- and organ-
specific reactive powers, which are due to their genetic con-
stitution, and their previous chemodifferentiation. Induction
does nothing but activate a definite developmental capacity,
present in the cell, and dependent on the nature of the latter.
Somewhat more caudally, a row of lateral extrusions, the

140 INDUCTION AND ORGANISATION

branchial pouches, is formed on each side by the walls of the
gut. These endodermal pouches come into contact with the
ectoderm, and at these points branchial grooves are formed by
the ectoderm. Later the grooves break through into the pouches,
thereby forming the gill slits. The branchial arches separating
the slits are lined with endoderm on the inner side. Externally,
they are initially covered only by the ectoderm, though later
the endodermal epithelium grows out under this ectoderm.
Within each branchial arch there is a band of mesoderm, which
produces the muscles, and some mesenchyme originating from
the neural crest, which forms the cartilaginous skeleton of the
branchial arches. Branched external gills grow out from the
outer surfaces of the hindmost three pairs of branchial arches.
They consist of epithelium, filled with mesenchyme.

The formation of these gills was found to be due to induction
by the endoderm, the former archenteron wall. If gill region
endoderm was grafted under the ectoderm of the flank, gills
were formed there (Soveringhaus, 1930). Here, too, it appears
that the gill-forming potency initially extends over a large area,
its intensity decreasing from a central maximum toward the
periphery. As long as the graft is not too small, a normal
set of gills develops from any part of this "gill- field" on trans-
plantation. Two gill-fields grafted side by side fuse into one,
and produce one normal set of gills, provided that both were
oriented in the same way (Harrison, 1921). The gill endoderm
is not only responsible for the origin of the gills, but it also
determines their polarity. This was proved by inverting the
endoderm, when the resulting gills also were inverted. More-
over, the endoderm determines the specific form, size and
pigmentation of the outgrowing gills. This was proved by
heteroplastic transplantations between species of the genus
Ambly stoma (Harrison, 1927). The ectoderm, however, is not
altogether without influence on gill development. Rotmann
(1931-35) exchanged the gill-region ectoderm between Triton
taeniatus and cristatus, and between axolotl and Triton. He
found that the gill-ectoderm determines especially the speed
of gill development during the earlier stages (Fig. 51). It was
also shown that it influences the size of the gills.

II. THE PERIOD OF ORGAN DEVELOPMENT

141

-?^f%-.

x>.

Fig. 51. (a) Embryo of Triton cristatus, into which a piece of pro-
spective ectoderm of T. taeniatus has been grafted at the gastrula
stage (cf. Pig. 39b). The graft occupies the prospective gill area
of the right side, (b) Dorsal view of the same embryo at a later
stage; development has proceeded farther in the gills on the right,
formed by the taeniatus ectoderm, than in those on the left (formed
by the host), (c) Cross section through the gill region of the embryo
(b) (the gill primordium formed by the graft is on the left in this

figure). After Spemann.

Very many experiments have been made in connection with
the limbs of amphibians. Their first visible primordia are
formed by the local accumulation, under the ectoderm of the
flank, of mesenchyme originating from the lateral mesoderm.
First, a half-spherical limb-bud is formed. This then assumes a
conical shape, and subsequently begins to expand at its tip,
thereby forming the hand- (or foot-) plate. The digits are
formed by indentation of the edge of this plate, and become
elongated in a certain sequence. Still later the joints are form-
ed, and the limb is rotated so that it assumes the normal
position relative to the body. Meanwhile, the skeleton and the
musculature of the limb have differentiated from the mesen-

142 INDUCTION AND ORGANISATION

chyme, and the girdle skeleton with its muscles has developed
in the flank. In urodeles, the fore-limbs are formed at an
early stage, whereas the hind-limbs develop much later. In
anurans, on the other hand, both pairs are formed at about
the same time. Most of the experiments have been made on the
fore-limbs of urodeles.

In this group, the limb-forming potency can be demonstrated
as early as the gastrula stage. If grafted into an abnormal
place, the material concerned (ectoderm and mesoderm to-
gether) will still develop into a limb (Detwiler, 1929-33). Here,
too, the potency initially extends over a larger area, its in-
tensity decreasing in all directions from a central maximum
(Harrison, 1915-18). Probably the potency is originally located
exclusively in the mesoderm. At somewhat later stages, how-
ever, when the limb-bud has become visible, the ectoderm
covering the bud has also acquired this potency. If at this
stage this ectoderm alone is grafted into another place, it will
produce a limb in co-operation with the local flank mesenchyme
(Filatow, 1930). Rotmann (1931-33) has effected separate
heteroplastic transplantations of limb mesoderm alone, and
limb ectoderm alone, between Triton taeniatus and T. cristatus.
His results prove that the mesoderm has the main influence
on the limb's specific form and size. It is not until later larval
stages that a slight influence of the ectoderm on the size of
the limb and the shape of the digits becomes manifest.

At first, the limb rudiment has all the properties of an
organisation-field: transplanted parts of the primordium can
produce a complete, harmoniously built limb, so that one
primordium can give rise to as many as four limbs. On the
other hand, two fused primordia can produce a single harmo-
nious limb. All the cells of the rudiment, therefore, are still
able to produce any part of the limb. The field has not yet
become tied to certain cells, but it can be displaced, can divide
into a number of equivalent fields, or can fuse with another
field into one unity. The field can be ''transposed" with regard
to the material.

Harrison (1915-25) carried out a large number of experi-
ments on the symmetry relationships of the limbs. Left and

II. THE PERIOD OF ORGAN DEVELOPMENT

143

right limbs are mutually symmetrical. Here, again, we face the
problem of how the development of a primordium into either
a right or a left limb is determined. In order to solve this,
Harrison transplanted limb rudiments of Ambly stoma punctatum
in various stages, rotating them 180° on any one of their three
axes (viz. rostro-caudal, dorso-ventral, and medio-lateral), or
on two or three axes simulta-
neously. If these experiments
were made at an early stage,
a short time after neurula-
tion, Harrison found that the
dorso-ventral and the medio-
lateral organisation of the
growing limb were always
in harmony with that of its
new environment. If, how-
ever, the limb had been
rotated on its rostro-caudal
axis, it grew out obliquely
forwards, instead of back-
wards, so that the symmetry
was reversed (Fig. 52). This

Fig. 52. Amblystoma larva, into
which a right Umb bud, rotated
on its rostrocaudal and dorso-
ventral axes, was grafted in the
flank. The graft (tr) has produc-
ed a limb with normal dorso-
ventral structure, in which, how-
ever, the rostrocaudal organisation
is inverted so that the graft is
the mirror image of the normal
right fore-limb. After Harrison.

shows that, at the time of the operation, the rostro-caudal
axis was already irrevocably fixed in the material, but that
the other axes had not yet been determined. Further exper-
iments showed that the determination of the rostro-caudal axis
takes place at the gastrula stage already, whereas that of the
other axes does not occur till later stages, irreversible determ-
ination taking place first in the dorso-ventral, and then in the
medio-lateral axis. Moreover, it was found that the different
species of urodeles did not behave identically in this respect.
In Triton taeniatus, for example, the direction of the dorso-
ventral axis is determined much earlier than in Amblystoma
(Brandt, 1922-28). The sequence, however, in which the three
axes become determined, is probably the same everywhere.
Presumably, the limb mesoderm is solely responsible for this
polarity, and the ectoderm does not play a role on this point
(Balinsky, 1931).

144 INDUCTION AND ORGANISATION

Interesting results were also obtained in successful attempts
to induce limbs in abnormal places by grafting material into
the side of the body. Balinsky (1925-27) discovered that in
Triton taeniatus grafting of an ear-vesicle under the ectoderm
of the flank often results in the outgrowth of a limb in this
place. That this is not a specific induction is shown clearly by
the fact that Balinsky obtained the same result by grafting
an olfactory pit, or even a piece of celloidin, under the flank
ectoderm. He made an extensive analysis of this phenomenon
in a series of further experiments (1929-37). He found that a
limb can be induced anywhere in the flank. The percentage of
successful inductions decreases from the fore-limb region in
a caudal direction, but increases again in the vicinity of the
hind-limb. In the anterior regions of the body, the induced
limbs have the character of fore-limbs, farther caudally more
that of hind-limbs. The more caudal the induced limb, the later
it will develop, irrespective of the moment of implantation.
At a certain stage, however, the capacity to react to this in-
duction disappears. Probably the whole of the flank of a urodele
has a latent limb-forming potency in such a way that there is
some sort of an equilibrium between the tendencies to form
"body-wall" and "limb". Presumably, a non-specific agent may
lead to preponderance of the latter tendency by causing a local
increase in the intensity of tissue metabolism. Where this
intensity is low, however, the tendency to form body-wall
predominates.

A large number of experiments might be added to those
mentioned above. These, however, will suffice to give an im-
pression of the very complicated interplay of inductions that
is at work in the embryo during this phase of development.
It is hardly an overstatement to say that all parts of the
embryo which are in contact, or which in the course of topo-
genesis come into contact, influence each other by contact
induction. Each part, therefore, is at the same time an inductor,
emitting certain influences, and a reaction system, reacting
to the influences received. The physicochemical constitution,
and therefore the potency, of each cell or cell group is modified
according to the inductions it encounters, either simultaneously
or successively.

II. THE PERIOD OF ORGAN DEVELOPMENT 145

It is highly probable that this contact induction is brought
about by material influences, i.e. that the cells secrete sub-
stances which diffuse into the environment, and which are
taken up by, or at least act on, the neighbouring cells. It must
be admitted that for most inductions this has not been proved
with the same certainty as for neural plate induction. In many
cases, however, there are plenty of indications which support
this assumption. The fact that dead tissues, and tissue extracts,
often can induce not only neural tissue but many other organs
as well, is one strong argument in its favour.

We must now turn our attention to the problem of how far
the specificity of the effect depends on the nature of the in-
duction, i.e. whether each organ is induced by its "own"
evocator. Several possibilities must be considered here. One
and the same evocator might be operating in all cases. The
divergent development of the cell groups, e.g. into organs so
different as an eye-lens, an olfactory pit, or an auditory organ,
would then be due to differences in the reactivity of these cell
groups themselves. The influence of induction would then be
of a purely activating nature; it would not cause the intrinsic
differences between the parts of the germ, but only make them
manifest. We must not forget here that such a specific re-
activity of the cell groups must owe its origin to previous
chemodifferentiation. This, in its turn, would require an
explanation.

Another attempt at an explanation also takes one evocator
as its starting point, but attributes great importance to varia-
tions in the concentration of this substance. The occurrence
of specifically different organ-forming potencies would, in this
view, be a consequence of concentration differences, to which
the reaction system would be extremely sensitive. Which of two
possible courses development would take, would depend on
whether or not the evocator concentration exceeded a certain
"threshold". We have already seen (p. 126) how Dalcq ex-
plained the properties of the primary organisation-field of the
germ by means of this hypothesis, and it may well be asked
how far such an explanation might apply to the phenomena
of organogenesis as well.

Raven - Outline Physiologie 10

146 INDUCTION AND ORGANISATION

It is true that in certain cases differences in the concentration
of an evocator could give rise to specifically different patterns
of development of cell groups, in the way indicated by Dalcq.
It is probable, therefore, that such differences may play a role
in the normal induction processes. Yet it is not very likely that
this alone could account for the strong increase in specificity
of the parts of the germ occurring in this phase of development,
which transforms the embryo into a system with a very high
degree of multiplicity. Moreover, several investigations, in
particular those of Chuang (1938-40) and Toivonen (1940-45)
give strong support to the view that there are several
qualitatively different evocators. Both investigators proved that
living and dead tissues of different origins induce organ
complexes of different composition, if grafted into the blasto-
coel of Triton. In some cases, the rostral organs of the head
are formed, such as fore-brain, eyes, and olfactory pits. In
other cases, organs of the posterior region of the head are
produced, such as the caudal parts of the brain and ear-vesicles,
or organs of the trunk, such as spinal cord, muscle tissue,
kidney, etc. Heat treatment of the inductors sometimes results
in a change in the nature of the induced organs. All this tends
to show that these "abnormal inductors" contain chemically
different substances, and that this is the reason why the germ
reacts with the formation of different organs. Hence, it is
very likely that qualitatively different evocators play a role
in normal development as well. In that case the specificity of
the induced organs can be ascribed at least in part to differ-
ences in the evocators.

Taking all the investigations in this field as a whole, it
seems probable that at early stages (gastrulation and early
neurulation), a quantitative difference in evocator concentration
plays the main role, whereas in the course of further develop-
ment more and more qualitative differences arise in the
chemical constitution of the evocators for the various organs.

Special attention must in this connection be paid to the
situation in the head-region. We have seen above th-^t we know,
for several organs of the head, e.g. lens, olfactory pits, auditory
organs, mouth, that their determination takes place in two

II. THE PERIOD OF ORGAN DEVELOPMENT 147

phases: (1) an early phase, at the end of gastrulation, during
which a weak tendency to form these organs arises in the areas
concerned, and (2) a later phase, during which specific contact
inductors, e.g. retina, hemispheres of the brain, neural crest,
mouth endoderm, bring about the final determination of the
organs. The first phase, which we have called "predetermina-
tion", apparently takes place under the influence of the
archenteron roof, but it is probable that an even earlier
gradient-field in the ectoderm also plays a role (Nieuwkoop,
1947; Lombard, 1952). In any case, there are strong indications
that this predetermination of the organs of the head takes
place, in a common "head organisation-field", in the way dis-
cussed above (p. 125). This view is supported, for instance, by
the fact that treatment of the germ with lithium ions modifies
the pattern of the head as a whole. Probably only quantitative
differences in a "head evocator" play a role here. In the second
phase, the place and character of the organs would be irre-
vocably determined by qualitatively different contact inductions.

The evocators probably originate as products of the cell
metabolism of the inductors, and diffuse into the environment.
If they are to exert an effect on the reaction system, the
cells of the latter must be sensitive to the action of just these
substances. This is often the case only during a very limited
period. Here, again, the chemical processes in the cells of
inductor and reaction system must be accurately adjusted to
each other for normal development to be possible. If the
evocator is not produced at the right moment, or if the reaction
system has not yet acquired its sensitivity by that time, or
has lost it again, development will be upset. Whether or not
these developmental processes will be interlinked in the correct
way, depends on the course of events in the previous phases
of development. This explains why a slight disturbance at a
certain moment may give rise to serious abnormalities in later
stages.

The inductions cause the formation of the primordia of the
various organs. These primordia are groups of cells which all
possess the potency to form the organ in question. Initially, this
organ-forming potency is not equally strong at all points, but

148 INDUCTION AND ORGANISATION

from a central maximum it shows a gradual decrease towards
the periphery. Therefore, a peripheral zone, in which the
potency is weak, gradually merges into material that lacks it
altogether. Moreover, the area in which the potency occurs is
initially much larger than the area from which the organ will
eventually develop. In the beginning, therefore, the organ
primordia occupy fairly large areas with vague boundaries. At
this stage, these areas may overlap at the edges, the cells in
the intermediate zones possessing two or more organ-forming
potencies simultaneously. In the course of development, the
potency becomes concentrated in the centre of the primordium.
The peripheral parts, in which the potency was weaker already,
now lose it altogether. Probably the ''physiological competition"
between the cells (p. 126) plays a role here. The result is a clear
demarcation of the organ primordia from their environment.

The organ primordia, again, have the character of organ-
isation-fields. This is proved by the fact that they possess the
power of regulation, and that the organ-forming potency can
be transposed with respect to the material of the germ. In early
primordia, parts of the material of the field can be exchanged,
e.g. by inverting part of the primordium, without causing
disturbances in later development. A smaller, but harmoniously
built organ will develop from an isolated part of a primordium,
so that in experiments one primordium may give rise to several
organs (cf. our discussion of the limb primordium, p. 142).
Conversely, two or more equivalent primordia can fuse so
that they produce a single harmoniously built organ of double
size. In other words, the organisation-field may be divided into
several fields, and, on the other hand, two equivalent fields may
be combined into one unity. In each organ-field, therefore, we
find, on a smaller scale, a repetition of the regularities shown
by the organisation-field of the whole embryo at an earlier
stage in development (cf. p. 122).

Initially, all cells of the field possess the same organ-forming
potency, though not all to the same degree. Later, however,
a further subdivision of the field takes place : the determination
of the parts of the organ. Under the influence of the field-
factors, chemodifferentiation takes place within the field, so

II. THE PERIOD OF ORGAN DEVELOPMENT 149

that the originally equivalent cells begin to differ in potencies.
Once this has happened, each part of the primordium can
produce one part of the organ only, e.g. only a retina, instead
of a whole eye. At this stage the parts can no longer replace
one another; disturbances and displacements of material within
the primordium are no longer regulated. The organ-forming
potency can no longer be transposed, but is tied to definite cell
groups. Following Paul Weiss, (1926), we may call these
happenings ''autonomisation" because they make the parts of
the organ autonomous so that from now on they behave as
mutually independent units. In the primary organisation-field
of the embryo, too, such a process of autonomisation takes
place. We have seen that it was this process that led to the
formation of the various organ primordia. Viewed in this light,
development may be regarded as a stepwise subdivision of the
primary field. The first step leads to autonomisation of the
organ primordia within the field, breaking it up into a number
of more or less independent organ-fields. The second step is the
division of the organ-fields into their parts. These parts, in
their turn, behave at first as organisation-fields. Experiments,
e.g. on the chick embryo, have shown that the field of the leg
becomes divided into the sub-fields "upper leg", "lower leg",
and "foot". On transplantation of any given part of one of
these sub-fields, the graft no longer forms a whole leg, as in
the previous stage, but only the appropriate part of the limb
(Murray, 1926). It may be assumed that at a still later stage,
these sub-fields will again be subdivided by "autonomisation"
so that, e.g., the field of the foot will break up into tarsus,
mid-foot and toes. At present, however, no information is
available on this point.

The mutual contact inductions of the parts of the germ, and
the autonomisation of the parts within the organ-fields, result
in a further increase of the spatial multiplicity of the embryo.
More and more, the embryo is broken up into a mosaic of cell
groups which differ from each other in physico-chemical
constitution. This will soon express itself in the external
appearance of the cells. Starting from the more or less in-
different embryonic cell type, the cells now begin to specialise

150 INDUCTION AND ORGANISATION

for the particular functions which each of them will have to
fulfil in the whole of the organism. The period of organ-
formation, therefore, is followed immediately by a phase in
which the differentiation of the tissues, or histogenesis,
dominates the picture. During this period are formed the tissues
of which the embryo consists, such as muscle tissue, connective
tissue, nervous tissue, cartilage, etc. It is a direct consequence
of the preceding chemodif f erentiation ; it is the visible ex-
pression of the diversity of the cells which, in an invisible
form, was already present.

Histogenesis is the last important step in the realisation of
the structural plan. The egg has now given rise to an organized
whole, a complex of histologically different organs and tissues
with fixed topographical relations. In a word, an embryo has
been formed.

CHAPTER X

The later stages of development

In the previous chapter we have seen how, in the course of
development, the embryo is subdivided into ever smaller groups
of cells, which are more or less independent, and have
different potencies. In the earlier phases of development, the
whole embryo behaves as one unit. It reacts to disturbances
with regulation, so that a more or less harmoniously built
embryo is formed in spite of the interference. In the later
stages, however, the situation is different. The power of regula-
tion, it is true, still exists within the individual organisation-
fields, but major disturbances, such as the removal or dis-
placement of complete organ primordia, can no longer be
regulated, and result in aberrant development. At this stage,
the embryo can be said to consist of a mosaic of building
stones which cannot be removed or displaced without damage
to its development. Each of these building-stones, the organ
primordia, develops more or less autonomously, and it can
no longer be replaced by any of the others. This phase may
therefore be called the ''mosaic stage^' of development.

That, indeed, the organ primordia are irreplaceable at this
stage, is clearly proved by experiments in which the whole
tail-bud of young amphibian embryos was cut off. Such embryos
developed into tail-less larvae, nor was the missing part of the
body replaced at later stages (Schaxel, 1922), (Fig. 53). This
is the more remarkable because complete regulation would have
taken place if the material concerned had been removed at a
slightly earlier stage. Other material would then have formed
a tail. And if, on the other hand, the tail is amputated at later
stages, regeneration will occur, and a new tail will be formed
by a proliferation of cells (cf. Chapter XI). The mosaic stage,

152

THE LATER STAGES OP DEVELOPMENT

Fig. 53. Extirpation of the tail primor-

dium of Triton at an early stage (a)

results in an entirely tail-less larva (h).

After Sehaxel.

therefore, is a develop-
mental phase of limited
duration during which
regulation exists no
longer, and regenera-
tion not yet.

Results similar to
those described for the
tail primordium have
been obtained with
other organs as well at
this stage. Complete
extirpation of a limb
primordium, for in-
stance, results in last-
ing absence of the limb
concerned, both in am-
phibians, and in the
chick embryo (Fig. 54). The same applies to eyes, gills, etc.,
and even to the blood of amphibians : removal of the rudiment of
the blood cells leads to the development of larvae without blood
corpuscles (Frederici, 1926).

The high degree of independence of the organ primordia
is also demonstrated by their behaviour on transplantation.
In the previous chapter we have seen many examples of the fact
that, once they have been determined, organ primordia grafted
into a foreign environment can continue their development in
complete independence, and that they can in this way reach
a high degree of differentiation. This, moreover, is true not
only of amphibians, but of other animals at the same stage
as well. Organ primordia of the chick embryo, grafted into the
egg membranes of another, much older embryo, will often
continue their development, and differentiate into the organ
they were destined to form. The same applies to fishes. Here
organ primordia have been grafted onto the yolk sac of other
embryos. Organ rudiments of mammals, e.g. rabbits and rats,
can be grafted onto the omentum in the abdominal cavity of a
rabbit. In that case, and, what is more, even after transplantation

THE LATER STAGES OF DEVELOPMENT

153

Fig. 54. Extirpation of the fore-limb primordium of Amblystoma
at an early stage (left), leads to the production of a larva (right),
in which the limb concerned is completely absent. After Harrison.

onto the egg membranes of a chick embryo, they, too, were
found to continue their differentiation.

The case of the mesonephros primordium of the chick embryo
deserves special mention. In normal development, the meso-
nephros of the chick becomes reduced from the tenth day of
incubation onwards, its function being taken over by the meta-
nephros. If grafted onto the egg membranes of an older embryo,
the primordium first differentiates into a typical mesonephros.
Reduction, however, sets in at about the sam.e time at which
it would have happened in the normal embryo (Danchakoff,
1924). This shows that not only the nature of the progressive
differentiation, but its duration as well is determined in the
organ primordium. Holtfreter (1931) found a similar case in
amphibians. Here, explanted endoderm of the prospective mid-
gut remains compact for several days, but it becomes broken
up into loose cells at the time at which, in the normal embryo,
the yolk-rich endoderm cells would have been expelled into the
mid-gut lumen.

Apart from transplantations, explantations have also been
applied to the study of the properties of organ primordia. This
method proved that in many cases the primordia are capable
of independent differentiation outside the body, provided they
are cultured in a suitable medium. The heart rudiment of
amphibians, for instance, developed in vitro into a pulsating
heart in which the various parts of a normal heart were clearly

154 THE LATER STAGES OF DEVELOPMENT

distinguishable (Stohr, 1924). Explanted parts of cephalopod
(cuttle-fish) embryos also proved able to continue their differ-
entiation in the normal way (Ranzi, 1931).

A somewhat different situation obtains in tissue cultures.
Here the tissues are kept in a medium specially provided with
food and growth promoting substances. This stimulation of
growth inhibits differentiation. Therefore, embryonic tissues
are usually unable to differentiate in these cultures, and such
structure as may already be present in the tissues will even be
reduced so that the cells become less differentiated in their
external appearance. However, under certain circumstances,
differentiation is possible in tissue cultures. Gaillard (1931),
for instance, has shown that the differentiation of bone-forming
tissue is promoted by the successive addition to the culture of
a series of extracts from embryos of steadily increasing age.

In this context, it is important to note that the loss of visible
cell differentiation in tissue cultures does not imply that the
cells really relapse into an undifferentiated, embryonic con-
dition. It is true that cells determined into different directions
e.g. bone-forming cells, and cells of the heart-primordium, in
tissue cultures become indistinguishable in external appearance,
but even after a very prolonged stay, they still retain their
different properties, and as soon as circumstances permit they
return to their particular course of differentiation. There is no
real "de-differentiation'\ therefore, but only ''modulation" (P.
Weiss, 1950).

We have already seen that the beginning of the mosaic stage
is soon followed by visible tissue differentiation. After this,
growth becomes gradually more important. Admittedly the
embryo may have increased in size before this stage, but this
was mainly due to water uptake, and was more a swelling
process, than real growth. True growth cannot begin until
differentiation has proceeded so far that the embryo is able to
take up food independently, or at least, until its blood circula-
tion is well enough developed for the transport of food from
the yolk, or from the maternal tissues, toward the embryo to
be possible. In other words, the period of differentiation
generally precedes growth.

THE LATER STAGES OF DEVELOPMENT

155

Though growth is highly dependent upon such external
factors as temperature and food supply, yet each species has
its own rate of growth. This was clearly shown by experiments
in which the primordia of a limb were exchanged between

embryos of two newt species,
viz. Amblystoma punctatum
and A. tigrinum, which at their
later stages show different
growth rates. It was found that
the grafted limbs retained their
own growth rates, and did not
adapt themselves to the slower
or faster rate of the host
(Harrison, 1924), (Fig. 55).

Moreover, the various organs
also have their own character-
istic growth rates. This causes
changes in the body proportions
in the course of development.

Finally, various organs in-
fluence the growth of other
organs. This was demonstrated
by combining, by means of a
heteroplastic transplantation, a
lens of Amblystoma punctatum
with an eye-cup of A. tigrinum.
The more rapidly growing ti-
grinum eye-cup stimulated the
more slowly developing punc-

Fig. 55. Larvae of (a) : Am-
blystoma punctatum and (b):
A. triginum, between which
the left fore-limb primordia
have been exchanged at an
early stage. After 50 days, the
transplanted limbs are about
the same size as those of the
animal from which they origin-
ate. After Huxley and de Beer.

tatum-lens, to faster growth.
The same is true of a tigrinum lens when combined with a
punctatum eye-cup. A mutual adaptation in size and growth
rate occurs between the two parts (Harrison, 1929; Twitty and
Schwind, 1931). In Rotmann's experiments, too, the originally
maladjusted eye-cup and lens of the heteroplastic combinations
(see above p. 133) were later on found to show mutual
adaptation in size by differential growth.

This mutual influence is also clearly expressed in the central

156 THE LATER STAGES OF DEVELOPMENT

nervous system. The size of the spinal ganglia depends to a
great extent upon the size of the peripheral area with which
they are connected. Increase of this area, e.g. by the implanta-
tion of a supernumerary limb in the flank, causes an increase
in the number of sensory neurones in these ganglia, and there-
by an increase in their size (Detwiler, 1920). If, on the other
hand, no skin is present on one side of the body, as in the case
of two amphibian embryos fused side by side, the ganglia on
this side remain very small (Detwiler, 1926). The number of
cells in the ventral half of the spinal cord, that will differentiate
into motor neurones, also varies with the size of the innervated
peripheral area (Hamburger and Keefe, 1944). The nerve fibres
growing out from the cells seem to play a role here, but the
effect is not dependent on the normal conduction of nervous
impulses. If amphibians are kept under permanent anesthesia,
their development proceeds normally, including that of the
nervous system.

The growth processes in the brain are partly governed by
stimulation by the ingrowing nerves from the large sense organs
of the head. If these sense organs are extirpated at an early
stage, the brain centres with which they are normally connect-
ed do not attain their normal size. Conversely, the implantation
of a supernumerary eye or olfactory pit will cause an over-
development of certain parts of the brain, if the nerve coming
from the graft penetrates into the brain of the host.

The growth of peripheral nerves towards their end organs
seems to be governed by attractions exerted on the nerves by
strongly growing organs. A limb transplanted into the flank
will establish connections with any nerve that happens to be in
the neighbourhood, even though normally this nerve has no-
thing to do with limb innervation. The nerve fibres enter into
the limb and become connected with its muscles, so that in
many cases the latter will function more or less normally
(Detwiler, Weiss). An eye or an olfactory pit, transplanted
into the flank, also attracts the neighbouring spinal nerves
(Detwiler, 1927), though in this case no functional connections
between nerve and organ are formed.

Once the connections of the nervous system with the various

THE LATER STAGES OF DEVELOPMENT 157

organs have become established, the former begins to influence
the development of the latter. It is true that practically normal
differentiation can take place in amphibian limbs without
nerves, but such limbs remain smaller than normal ones (Ham-
burger, 1929). Evidently, the nervous system has a trophic
influence which intensifies growth. In other cases, however, the
connection with the nervous system is indispensable for the
maintenance of the structure of the organ. Marked atrophy
occurs, for instance, in muscles as soon as the motor nerves
are severed. This is even more true in the case of the lateral
line sense organs and the skin taste buds of fishes, which
degenerate entirely after transection of the afferent nerve
(Olmsted, 1920). This influence of the nervous system is
probably due to the secretion of a substance by the nerves.

The development of the blood circulation also has important
consequences for further development, as it supplies the various
organs with the food substances needed for their growth. The
distribution of these substances between the organs will depend
upon the total quantity that is available. The body proportions
of maximally fed animals may differ sharply from those of
starved specimens. In extreme cases, certain organs or parts of
the body may be completely reduced in consequence of want of
food.

The possibility of hormone transport is another important
consequence of the development of the vascular system. Hor-
mones are substances produced by certain glands, which become
distributed throughout the body by the blood so that they can
exert their influences on the development of other organs in
remote places. In another context, we have already mentioned
the "gene hormones" of insects (p. 87). Hormones play a very
important role, in particular in the later stages of development,
and in the adult animal. A more extensive discussion of these
phenomena lies outside the scope of this book.

Finally, in the later stages, the function itself of each organ
has an important influence on its development. Once differentia-
tion has proceeded so far that the organs begin to take over
their own specific functions, new processes are started under
the influence thereof. These processes influence the structure

158 THE LATER STAGES OF DEVELOPMENT

of the organs, and modify their growth and histological differ-
entiation, or the arrangement of their cells and tissues. We
shall now briefly review this 'Afunctional stage'' of development.
The best known phenomenon in this context is the hypertrophy
of an organ caused by excessive use of that organ. If one kidney
of a vertebrate animal is extirpated, the other kidney increases
in size. Another well known example is the great increase in
the size of striated muscle as a consequence of heavy muscular
work.

The influence of the functional relationships on the arrange-
ment of tissue elements is clearly illustrated by Weiss's (1929-
33) experiments. He exposed tissue cultures of connective tissue
cells (fibroblasts) to local tension. The cells then became
arranged in accordance with the direction of the lines of tension,
and multiplied more rapidly in this direction. In normal devel-
opment, such reactions play a role in the origin and regenerat'on
of tendons, etc., as was shown by Lewy's experiments as early
as 1904. The detailed architecture of bones obeys the same law.
The trabeculae (bone bars) in spongy bone are arranged in the
direction of the stresses normally acting on the bone. If the
direction of these forces is modified, e.g. by the extraction of
a molar from the jaw, the direction of the trabeculae is changed
accordingly. The growth of bones also depends upon their
function. A motionless limb which is not subjected to any stress
is retarded in growth, as compared with a normally functioning
limb.

Function is of paramount importance also for the final
structure of the vascular system. With the exception of the
very first blood vessels, which differentiate locally, all others
arise in the course of embryonic development by the branching
of existing vessels. Initially, massive "buds" of endothelium cells
are formed. These extend into the tissues and soon become
hollow. Manifold connections between the outgrowing blood
vessels are then formed, so that a network develops. At first,
all these vessels are of about the same calibre. The same
process takes place in regeneration. This primitive network
differentiates into the definitive vascular system in the follow-
ing way. Some of its meshes disappear, others become capil-

THE LATER STAGES OF DEVELOPMENT 159

laries, but the lumen of a few channels becomes widened, and
their walls thickened so that they become the major arteries and
veins. Clark and collaborators (1931, etc.) have studied this
process in living animals, viz. in the transparent caudal fins
of frog larvae, and in specially constructed transparent cells,
let into the ear of a rabbit. They found that the fate of each
part of the original network depends on the currents which
prevail there because of the pressure differences in the system.
Where the blood stagnates, the diameter of the vessels de-
creases, and finally they disappear. Strong perfusion, on the
other hand, causes a widening and thickening of the vessels
concerned, leading to their differentiation into major blood
vessels. In this way the blood stream itself models the eventual
pattern of the blood vessels.

The gills of larval newts may be taken as a last example. In
water with a low oxygen content, these organs are large and
strongly branched; the overlying skin is thin, and they are
well vascularized. In water with a high oxygen content, on the
other hand, where skin respiration can easily take place over
the whole body surface, the gills are poorly developed. Here,
again, the structure becomes adapted to the functional require-
ments under the influence of the function itself.

CHAPTER XI

Regeneration

Regeneration is the term applied to a process of restitution
which occurs after removal of a part of the body and which
results in complete or partial replacement of the lost part.

If an animal of simple structure, e.g. a planarian, is cut
into two transversely, a bud-like outgrowth will within a few
days develop on the cut surface in each half. This is the so-
called regeneration-bud. It grows steadily, and replaces the lost
part of the body, i.e. a new fore-part is formed on the caudal
half, and a new hind-part on the anterior half (Fig. 56). The
position of the cut is of no, or at most of secondary importance.
Whether it is made in the middle, or in the front or hind part
of the worm, the result is always the same. This shows already
that the quality of the regenerate is not determined exclusively
by the nature of the regenerating material. The situation is not
such that, e.g., in the front half of the
body "tail-forming" cells or substances
are present, and "head-forming" mate-
rial in the posterior half. At each level
in the body, both a head and a tail can
be produced. What will in a given case
develop from the regeneration-bud,
depends only on whether the cut at
which this bud is formed faces forwards
backwards. In other words, the

C3 CD

<:0 CZ3

or

original polarity of the worm determines
the result of the regeneration. Evidently,
this polarity has remained intact in both
pieces formed by the transection of the
worm. This is true even if the worm is

Fig. 56, Regeneration
of rostral and caudal
halves in the flat
worm Polycelis nigra,
after 0, 7, 9, and
14 days respectively.
After Dresden.

REGENERATION 161

divided into several parts by a series of transverse cuts. The
original polarity is retained by each of these parts, and each
of them will regenerate a head at its front edge, and a tail at
its hind edge. Only in exceptional cases can this inherent polarity
of a fragment be suppressed or reversed by certain treatments.

In some cases, however, the situation is more complicated.
In earthworms, such as Eisenia foetida, a number of regions
with different powers of regeneration can be distinguished
(Gates, 1950). In the foremost six segments of the body, a
head is regenerated at a front edge, but no regeneration takes
place at a hind edge. In the succeeding zone of about 11
segments, heads are formed by both front and hind edges.
From segment 21 to segment 34, a head or a tail may be formed
at either edge. Next comes a region, extending to segment 54,
in which only tails are regenerated at both edges. Finally, m
the zone behind segment 54, a tail is formed by a hind edge,
but no regeneration occurs at a front edge.

It is important to study the nature of the regeneration
process somewhat more closely. We have already mentioned
that the formation of a regeneration-bud is the first visible
consequence of the transection. This consists of cells of a very
indifferent character. The origin of this material is not in all
cases sufficiently well known. In some lower organisms, un-
differentiated cells are present at various places in the body.
After transection, these migrate into the wound area, and
accumulate there. Miss Dubois (1949), for instance, has demon-
strated this in the case of Planaria. Regeneration can be sup-
pressed here by irradiation with X-rays; the irradiated animals
succumb after a few weeks. But if only the foremost two fifths
part of the body is irradiated, and the head is then amputated,
regeneration does occur, though it begins a month later than
in non-irradiated animals. Miss Dubois has been able to prove
that this is due to the fact that the regeneration cells, or
neoblasts, migrate from the posterior, non-irradiated part of
the body into the wound area. In the course of this migration,
they must traverse the irradiated zone. The wider this zone is,
the more regeneration is retarded. This migration is not
spontaneous ; it takes place only after a wound has been made,

Raven - Outline Physiology 11

162 REGENERATION

and it is always directed towards the wound. The cells can move
both forwards and backwards, and sideways. The stimulus that
causes the onset of migration spreads throughout the body; it
disappears four to five days after the wound has been made.
Once started, the migration continues until a regeneration
blastema has formed in the wound area. Apart from the capacity
to form a regeneration-bud, the neoblasts possess the power
to repair other damage in the body. They are omnipotent, i.e.
they can form any other tissue.

It is possible that in other cases the regeneration cells are
formed by the de-differentiation of tissue cells which thereby
acquire a more embryonic character, and an increased power
of division.

Once the regeneration-bud has reached a certain size, an
outwardly visible differentiation begins to take place in this
material, and it becomes possible to see what will develop
from it.

Now it has been found that in many annelid worms the
regeneration-bud which develops at a front edge will always
differentiate into a fixed number of segments, which agree
with those found in the rostral end of the body, the "head" of
a normal worm. The number of segments in the regenerate is
entirely independent of the place of the cut. In the polychaete
worm Sabella, for instance, three segments are always formed,
the foremost of which carries a crown of branchial palps, even
if the regenerating fragment belongs to the caudal end of a
normal worm (Fig. 59). This shows that here regeneration
does not lead to restitution of the missing part of the body,
but produces only a new apical end by an autonomous differ-
entiation. In contrast, the type of differentiation in regeneration
buds at a caudal edge depends upon the nature of the tissues
of the fragment, and here the lost part of the body is completely
replaced.

The same applies to planarians. Here, again, regeneration at
a front edge, irrespective of its position, produces only a head,
whereas that at a hind edge leads to complete restitution of
the missing parts.

Similar phenomena are found in the restitution processes

REGENERATION

163

\^

Fig. 57. Reorganisation in the hydroid polyp Tubularia. A very small
part produces only (a): the apical part of a polyp; if more material
is present, the more basal parts are formed accordingly (b-d).

After Child.

that occur after the transection of hydroid polyps, though here
as a rule we find no regeneration, but only a "reorganisation"
of the old tissues, which are subjected to a morphological de-
differentiation, followed by new differentiation. If a stem
fragment of such a polyp is isolated, it will change into a new
hydranth provided the size of the fragment is sufficient. Smaller
parts produce only the apical parts of a polyp, but these apical
parts are of normal size^). Evidently, the process of re-
organisation begins with the formation of the apical parts,
and proceeds from there in the direction of the base, until
all the available material has been used (Fig. 57).

The size of the apical parts formed depends more or less
upon the external circumstances. In Planaria, for instance, only
a small head is produced by a regeneration-bud at low
temperatures, or under the influence of anesthetics. High
temperatures, on the other hand, result in the production of
very big heads (Child, 1915), (Fig. 58). In the hydroid
Tubularia, the size of the hydranths produced depends on the

1) In many cases, apical parts arise at both ends of the stem fragment.
We shall not enter into this complication.

164

REGENERATION

oxygen tension. Here, too,
narcotics cause a reduct-
ion in size, which how-
ever can be compensated
by increased oxygen
tension (Barth, 1944).
In normal regeneration,
the role played here by
anaesthetics is probably
duplicated by inhibiting
substances formed in the
tissues (waste products
of metabolism). After
transection, regeneration
is started by the better
oxygen supply to the
wound area, and by the
disappearance of inhibit-
ing substances by dif-
fusion.

In some cases, local
stimulation without re-
moval of material is suf-
ficient for a new head,
or, in hydroids, a new
hydranth, to be formed.
Goldsmith (1940), for in-
stance, caused the devel-
opment of supernumerary
heads in planarians by
making incisions or local
burns in the rostral part
of the body. In the earth-
worm a supernumerary

head may develop laterally in the front part of the body,
if a wound is made there, and a cut end of the ventral nerve
cord is brought into the wound (Avel, 1947). Evidently
the nerve cord has a stimulating or activating influence, for

Fig. 58. Correlation between the size
of the regenerated head and its or-
ganising power in Planaria. All fig-
ures illustrate the regeneration of a
caudal half of the original, cut as
in (a); (b) regeneration under normal
circumstances; (c-e) regeneration
under the influence of increasing con-
centrations of narcotics; reduction
in size and development of the re-
generated head, and corresponding
reduction in the size of the induced
pharynx (ph), and in the distance
between head and pharynx; (/) re-
generation at optimal temperature,
larger head and pharynx, and greater
distance between pharynx and head.
After Child.

REGENERATION

165

d

the wound itself does not cause head formation at this place.

It may be assumed that in the differentiation of organs from
a regeneration blastema similar mutual influences to those
we have encountered in the development of embryos play a
role. In fact, the origin of eyes in a regenerate in the flat-worm
Polycelis proved to depend on an influence from the cerebral
ganglion which spreads over a certain distance (Wolff and
Lender, 1950-51).

The head formed by regeneration at the front edge of a caudal
fragment of the body does not at first harmonise with the old
tissues of the fragment,
because certain intermediate
parts of the body and their
organs are absent. The
normal structure of the
organism is restored by a
second process, subsequent
to that of regeneration in the
proper sense. This process is
called morphallaxis. In its
course, regenerate and frag-
ment mutually adjust their
sizes (Fig. 56), and, more-
over, the newly formed
head influences the adjacent
tissues in such a way that
the missing parts are form-
ed here by "reorganisation"
of the tissues. If, for ex-
ample, the caudal part of a
planarian has regenerated a
new head, the pharynx which
in planarians occupies the
middle of the body, at first
is absent. By subsequent
morphallaxis, however, a
cavity is formed in the
old tissue of the original

A

Fig. 59. Four stages (a, b, c, d)
in the regeneration of the rostral
end in Sabella. As regeneration
proceeds, the foremost 4 segments
change from the abdominal into
the thoracic type, by the loss of
their old hooks and bristles and
the formation of new ones in an-
other position. This change pro-
ceeds in a rostro-caudal succession.
After Berrill and Mees.

166

REGENERATION

fragment. This develops into a new pharynx sheath, and breaks
through to the exterior, forming a new mouth. The pharynx
itself is formed by the regeneration blastema and grows into
the sheath from the rostral side (Van Asperen, 1946).

Morphallaxis is very clear in Sahella. In this worm, the
foremost 5 to 11 segments, the so-called "thoracic" segments,
are distinguished by a number of characters from the more
caudal "abdominal" segments. If the animal is transected in
the abdominal zone, the posterior part regenerates a head
consisting of three segments. This, however, is followed by
changes in a number of the foremost abdominal segments,
which thereby acquire thoracic characters (Berrill, 1931). This
reorganisation proceeds in a rostro-caudal direction (Fig. 59).

Fig. 60. Organising activity of a transplanted head in Planaria.
(a) a laterally implanted graft has induced a lateral outgrowth
and a secondary pharynx, (b-c) a subterminal graft has induced
an outgrowth (directed forwards), and two phar3niges; (d) a ter-
minal graft has caused a reversal of polarity in the caudal part of
the host, and a secondary pharynx; in this last case the hind part
was capable of autonomous movement (dotted outline), i.e. it be-
haved as an independent individual. After Santos.

REGENERATION 167

There are a number of experiments that leave no doubt that
morphallaxis is due to "organising" activities of the previously-
regenerated head. Santos (1929) transplanted parts of the
head of one planarian into the caudal region of another. The
graft regenerated the missing parts of the head, and induced
a pharynx some distance away in the host tissues. The direction
of this pharynx agreed with that of the implanted head. In
many cases the host tissue even supplied a complete new caudal
part, adjusted to the implanted head (Fig. 60). Similar effects
were obtained in the case of heteroplastic transplantation
between two species of Planaria.

In hydroids, too, the existence of such organising activities
has been demonstrated. Here they are exerted by the apical parts
of the polyp. In Hydra, the peristome (i.e. the area around the
mouth) of one polyp, implanted laterally into the body of an-
other, induces the formation of tentacles and the production
of a bud.

The comparison is obvious between these organising in-
fluences of regenerated heads or apical parts and the activity
of organisers in embryonic development. Here, again, the
situation can be described as an "organisation-field", originating
from the regenerate and spreading from there over the adjacent
areas of the original fragment; the role of organiser is here
played by the regenerate. Under the influence of the field, the
cell material undergoes certain changes characteristic of
morphallaxis, changes which adapt regenerate and fragment
to each other. Therefore, the study of regeneration may deepen
our insight into the properties of organisation-fields.

First of all, the field in certain cases appears to adapt its
size to the available material. In a fragment of the ascidian
Clavellina, for instance, complete de-differentiation of the
tissues into a compact mass of cells may occur. This is then
followed by fresh differentiation into a well-proportioned
Clavellina of smaller size (Huxley, 1926). Evidently the field
in this case harmoniously imposes itself on such material as
is present; its size is adjusted to the available quantity of cells.
However, that this is not always so, may be seen from the
case of hydroid polyps described above, in which fragments

168 REGENERATION

that were too small did not produce complete polyps on a
smaller scale, but only parts of hydranths of normal size.
On this point, therefore, the field may behave in either of two
different ways. As yet it is entirely unknown which factors are
responsible for the choice between the two.

Further, it was found that the area eventually occupied by
the field depends upon the "state of activity" of the regenerate
acting as organiser. This, in turn, can be influenced by external
factors. If, under the influence of cold or narcotics, the re-
generated head of a planarian has remained small, the pharynx
which arises by morphallaxis under the influence of the field
also remains smaller, and it is formed at a shorter distance
from the rostral end. There is a direct correlation between the
size of the head, that of the pharynx, and the distance between
the two (Child, 1915), (Fig. 58). Similar phenomena have been
found in hydroids. In Sabella, illumination of the regenerating
head causes a strong expansion of the field. In darkness, only
about four abdominal segments develop into thoracic segments
during morphallaxis, but this number may be increased to as
many as eighty by illumination (Berrill and Mees, 1936).

The organising power proved not to be a privilege of the
extreme rostral, or apical, part of the body. As a rule, each
part of the body has an organising influence on the more caudal
parts. A fragment from the middle of the body of a planarian
can regenerate a tail at its hind edge, even if no regeneration
of a head takes place at its rostral end. Even a pharynx may
be formed in this case, at least if the fragment originated from
the pre-pharyngeal part of the body, but not if it originated
from the post-pharyngeal region. This agrees with the observa-
tion by Okada and Sugino (1934) that not only transplanted
heads, but also more caudal parts of the body of a planarian
have inductive effects v/hen grafted into still more posterior
regions of another individual.

The same applies to the polyp Corymorpha. Here parts of
the stem, grafted laterally into the stem of another polyp, in a
number of cases induce a new hydranth. This happens more
often, according as the original position of the graft in the
stem was more apical (Child, 1929), (Fig. 61).

REGENERATION

169

The inductive power, therefore,
is not a property of one definite,
specific tissue only, but it is due
to a physiological condition the
intensity of which decreases as a
gradient from the rostral, or
apical, towards the caudal, or
basal, end of the body. In other
words: each part of the field in-
fluences the more caudal (basal)
parts, but is itself influenced by
the more rostral (apical) parts.

Little is known so far about the
physical nature of this gradient,
though it is obvious to think of
electro-physiological phenomena in
this connection. The recent work
of Moment (1946-49) is highly im-
portant in this respect. We have
seen above (p. 162) that a re-
generation-bud at a hind edge as
a rule completely replaces the lost
part of the body. Now Moment has
shown that during regeneration of
a caudal half in the earthworm

W^

Fig. 61. Organisation by
transplanted parts of the
stem in the hydroid polyp
Corymorpha. (a) an apical
part of the stem induced a
complete new hydranth in
48 hours; ib-c) more basal
parts of the stem induced
smaller (&) or abnormal
outgrowths (c) in the same
time. After Child.

Eisenia foetida the formation of
new segments stops when the total number of segments has
reached an approximately normal value. In a normal worm,
there is an electric potential difference between the rostral and
caudal ends of the body. This potential is destroyed by amputa-
tion, but during regeneration it is gradually built up again
until it has regained its original value, which happens at the
very same moment at which the formation of new segments
comes to a stop. On this basis. Moment concluded that the
production of new segments is brought to a close by an in-
hibitory action exerted by the electric field as soon as this has
attained a certain degree of intensity.

Two parts originally situated at different levels in the

170

REGENERATION

gradient-field can be
brought into contact by
transplantation. In this
case, a strong prolifera-
tion of tissue occurs at
the boundary (Okada and
Sugino). The greater
the discontinuity in the
gradient, the more marked
is this proliferation. This
was beautifully demon-
strated in an experiment
by Schewtschenko (1937),
who grafted heads of
planarians into different
regions of the hosts. Im-
planted caudally, the
heads caused a very
strong outgrowth, but in
grafts in more rostral
areas the effect became
gradually weaker. Trans-
plantation of caudal parts had exactly the opposite effect:
strong proliferation in rostral areas, much weaker proliferation
more caudally (Fig. 62).

One of the effects of the "dominant" area (Child) on the
subordinate parts of the field is of an inhibitory nature. Miller
(1938) transplanted the head of a planarian into the trunk
area, and then transected the host just rostral to the graft.
No head was then regenerated at the front edge, because the
graft repressed such regeneration. A head grafted into the
trunk sometimes moves in a forward direction because (1)
strong proliferation takes place at the caudal side of the graft,
and (2) the tissues in front of the graft are resorbed. This
movement goes on until the head has found its way to the
rostral end.

The apical "organiser", therefore, has a twofold influence.
It prevents the formation of new apical parts, and it forces

Fig. 62. The effect of discontinuities
in the gradient in Planaria. (a) the
growth caused by grafted heads is
the more marked when the head is
implanted more caudally; (b) on the
other hand, transplanted hind ends
produce strongest growth in the
rostral parts of the host. After
Schewtschenko.

REGENERATION 171

the other cells within the field to form subordinate organs in
accord with their place in the field.

If, therefore, a tissue is to form a new apical region, it is
necessary for it to be removed from the inhibitory influence
of the existing apex. This can be done by cutting the latter
away, but in normal development it may occur as a consequence
of growth. The field emanating from the "dominant" apex has
a limited extent, and consequently part of the tissue can be
removed from its sphere of influence by growth — so-called
''physiological isolation" (Child). This tissue will then form a
new apical region. This is the law governing the occurrence
of new buds in colony-building organisms, such as hydroid
polyps. We have seen that the extent of the field depends upon
the state of phj^siological activity of the tissues. The distance
between buds formed under favourable conditions of food,
temperature, etc., will therefore be greater than that between
buds formed under less favourable conditions. This may
completely modify the appearance of the colony as a whole
(Child, 1929). A similar phenomenon occurs in some planarians
which reproduce asexually by transverse division. Here, at a
certain distance from the original head of the individual, the
caudal part forms a new head. If, under unfavourable circum-
stances, the size of the old head is reduced (see p. 168), the
field emanating from it extends less far, and division takes
place at a much smaller size of the body.

Child has demonstrated in many experiments that the
dominant apical parts are often highly sensitive to noxious
influences, and that this sensitivity decreases in a caudal (or
basal, respectively) direction. He ascribed this to differences
in the "physiological (i.e. metabolic) activity" of the tissues,
which would be highest in the apical parts, and decrease
gradually from there. Similar results were obtained with an-
other method for the demonstration of differences in the in-
tensity of metabolism, viz. measurements of the velocity of
decolorisation of certain redox dyes, such as methylene blue,
at low oxygen pressures. From these differences in physio-
logical activity. Child explained such morphogenetic influences
as "dominance" and "organisation", exerted by the apical part.

172 REGENERATION

It is doubtful, however, whether in doing so he did not confuse
cause and effect. In other words, the morphogenetic dominance
of the apical region might be the primary phenomenon, and the
difference in physiological activity of the tissues its con-
sequence. So far it has not been possible to decide which of
the two hypotheses is correct.

On the basis of experiments on regeneration in hydroid
polyps, Spiegelman (1945) has developed a theory which re-
duces the dominance of a cell group over its neighbours to
"physiological competition" between the cells by the absorption
of food substances and the secretion of noxious waste products.
It is possible that several phenomena in this field can be
explained in this way. However, in an investigation on the
mutual influences of rostral and caudal regenerate in transverse
sections of Euplanaria luguhriSj Raven and Mighorst (1948)
showed that in this case the facts are not what one
would expect on the basis of the theory of "physiological
competition".

All examples discussed so far referred to so-called "total
regeneration", provoked by transection of the whole body.
However, the study of the regeneration phenomena which occur
after the removal of subordinate parts of the body has further
contributed to our insight into the mode of action of or-
ganisation fields. Interesting results were obtained in particular
in experiments on the regeneration of limbs and tail in
amphibians.

If a limb or the tail of a larval or adult newt is cut off, a
regeneration bud, consisting of more or less indifferent cells, is
formed in the wound area. The origin of this material and the
processes resulting in the formation of the regeneration blastema
have in recent years been the subject of many investigations. It
has been found that, immxediately after the amputation of a limb,
a marked de-differentiation takes place in the tissues of the
stump near the wound area. A partial disintegration of skeleton
and musculature takes place, and this process supplies great
numbers of free cells of a more or less indifferent appearance.
In the course of the next few days, a regeneration blastema
forms in the cut.

REGENERATION 173

It is practically certain that the cells of this blastema do
not, as, e.g., in planarians, originate from a store, somewhere
in the body, of cells which have retained embryonic properties,
and which migrate towards the wound. In contrast, they arise
locally by de-differentiation of the tissues near the wound.
The following type of experiment supports this view. The distal
part of a limb is replaced by a limb graft from another
individual, the cells of which can be distinguished from those
of the host, e.g. by their pigmentation, or haploidy. Thereupon,
the graft is amputated almost completely, but so that the cut
goes through the grafted tissue only. In this case, the cells of
the regenerate have in every respect the character of the grafted
tissue, even though the normal tissue of the host begins at a
short distance from the wound. Furthermore, it has been
demonstrated that the formation of the blastema begins even
before there is any considerable increase in the number of
cell divisions in the neighbourhood of the wound, so that it is
clearly not formed by multiplication of cells. It has not yet
been ascertained whether its cells originate mainly from the
de-differentiating tissue of muscles and skeleton, or from the
connective tissue, or whether, on the other hand, they are
supplied for the most part by the proliferating epidermis, as
was suggested by Rose (1948).

On the one hand, de-differentiation of the old tissues supplies
the cells for the regeneration blastema, but on the other hand
the blastema, once it has been formed, seems to bring the
process of de-differentiation to a stop. Butler concluded this
from experiments in which regeneration was studied after
irradiation of the limb with X-rays. After strong irradiation,
the power of regeneration had disappeared completely. Am-
putation of the limb led to marked de-differentiation of the
stump tissues, but no blastema was formed, and de-differ-
entiation continued until the whole stump had been resorbed.
The same thing happened after the amputation of a limb which
had previously been denervated by transection of its nerves
(Butler and Schotte, 1941). Apparently the presence of nerves
is indispensable for a normal equilibrium between de-differ-
entiation and blastema-formation. Singer (1942-49) has shown

174 REGENERATION

that both motor nerves and sensory nerves play a role here.
A certain minimum number of nerve fibres per unit area of the
wound is required for the normal progress of regeneration. It
also appears that a considerable invasion of nerve fibres into
the epidermis of the regenerate takes place in the early stages
of regeneration. Finally, experiments by Butler and Schotte
(1949) have proved that the presence of nerves is necessary
for the first phases of regeneration, during which the blastema
is formed and determined, but that the later morphogenesis,
differentiation, and growth of the regenerate are independent
of the nervous system.

The de-differentiation which supplies the material for the
regeneration blastema can be inhibited not only by the forma-
tion of this blastema, but also by a premature growth of skin
over the wound. This may be the explanation of the difference
in regenerative capacity between urodeles and anurans. In
urodeles, an amputated limb is completely regenerated, both in
larvae and in adults, whereas in anurans the capacity to re-
generate is permanently lost during metamorphosis. Now it
has been found in adult anurans that the skin soon overgrows
the amputation wound, and that the wound is covered by a
scar tissue consisting of coarse connective tissue fibers. No
regeneration blastema is then formed. It is of great practical
importance that de-differentiation has been successfully pro-
voked by mechanical (Polezajev, 1939-41) or chemical stimula-
tion (Rose, 1942-45; Polezajev, 1945-46). In adult anurans,
too, a certain degree of regeneration has been produced in this
way. Man and the mammals do not possess the capacity for
spontaneous regeneration, but these investigations have revealed
methods of stimulating the regeneration of lost parts of the
body which might bring regeneration in this group within the
realm of practical possibilities.

We shall now discuss some details of the later development
of these regeneration-buds. Here all the missing parts are re-
placed by the regenerate, in contrast to what we have seen
earlier in the case of total regeneration at a front edge. The
differentiation in the regenerate adapts itself completely to
the remaining organs of the stump. Moreover, we find no trace

REGENERATION 175

of the influence of the regenerate on the stump which was so
characteristic of total regeneration. In the present case, re-
generation is not followed by morphallaxis. Whereas in total
regeneration, all interest was focused on the influence of the
regenerate on the fragment, here, in contrast, we must study
the influence of the stump on the regenerate.

First of all, it appears that the differentiation of the re-
generate is governed by the stump of the amputated organ,
and not by influences originating in the body as a whole. Fore-
limbs have been grafted into the place of a hind-limb, and
amputated after some time. In such cases a fore-limb was
regenerated on the stump. If the limb had been rotated during
transplantation (e.g., implanted upside down), the orientation
of the regenerate agreed with that of the stump, and not with
that of the body (Weiss, 1924). We have already seen (p. 151)
what will happen if a limb or tail is extirpated completely and
no stump left at all: no regeneration takes place in this case.
This led Guyenot (1927) to the formulation of the concept of
"territoire de regeneration''. For each part of the body, a certain
region can be indicated within which regeneration of this part
can take place ; if this region is removed completely, no regenera-
tion occurs. Within the region, the formation of the part in ques-
tion can even be induced by local stimulation, e.g. by an out-
growing or regenerating nerve. If a nerve of the hind-leg (nervus
ischiadicus) in amphibians is diverted under the skin in the
vicinity of the fore-limb, a supernumerary fore-limb is formed
there. If diverted under the skin of the tail, it causes the forma-
tion of an extra tail (Guyenot, 1928).

The fact that the organs developing in the regenerate are in
direct connection with the remaining organs of the stump
might lead to the assumption that the former originate simply
as outgrowths of the latter. In other words, each tissue that is
exposed in the wound would grow out into the regenerate, and
in this way the replacement of the missing part would be
brought about by the co-operation of the organs. The following
experiments disprove this view. Weiss (1925) extirpated the
humerus from the fore-limb of a newt, and a little later
amputated this limb in the upper-arm region. No skeleton was

176

REGENERATION

present in the wound area, therefore. Nevertheless a normal
forearm and hand skeleton were formed in the regenerate,
whereas in the upper arm the extirpated humerus was not
regenerated (Fig. 63). Therefore, the skeleton in the regenerate
is certainly not formed by the outgrowing skeleton of the
wound; it must have differentiated on the spot from the re-
generation material. Weiss has made similar experiments on
skin. The same point was illustrated even more clearly in an
experiment by Umanski (1938). He irradiated a limb of a black

Fig. 63. Diagram of fore-limb regeneration in Triton, (a) normal
skeleton of the fore-limb; (b) extirpation of the humerus, followed
by the amputation of lower-arm and hand; (c) the skeleton of the
regenerated lower-arm and hand is normal, but the extirpated
humerus is not regenerated. After Przibram.

axolotl with X-rays, thereby destroying the regenerative
capacity of its tissues. The skin of this limb was then replaced
by that of a normal specimen of the white axolotl. The limb
was thereupon amputated, and regeneration took place. The
lack of pigmentation in the regenerate proved that it originated
from the grafted skin which had retained its power of prolifera-
tion. Similar experiments were made by Trampusch (1951).
Instead of skin, he grafted in other cases skeleton or muscle
tissue into the irradiated limb. It appeared that in these cases,
too, regeneration occurred, and the assumption was again in-
evitable that all or nearly all of the material for the regener-
ation originated from the implanted healthy tissues.

The following experiments supply some information on the

REGENERATION

177

degree of determination in the regeneration buds. Young buds,
which have just become visible as half-spherical outgrowths
are resorbed if transplanted into the flank of another animal
of the same age. Older buds, on the other hand, go on devel-
oping, and produce approximately the same parts as they would
have formed if they had not been transplanted. A young re-
generation bud of a fore-limb, grafted onto the stump of an
amputated hind-limb, develops into a hind-limb regenerate

Fig. 64. Transplantation of a regeneration bud. (a) amputation of
a fore-limb. If the regeneration bud alone is transplanted onto the
stump of an amputated hind-limb (d), it will form a hind-limb (e),
but if part of the original stump (hatched) is transplanted as well
(b), a fore-limb will be formed by the graft (c). After Przibram.

(Milojevic, 1924), (Fig. 64, d-e). A young tail regeneration
bud produces a fore-limb if grafted into the neighbourhood
of a fore-limb, though an older tail regeneration bud develops
into a tail (Weiss, 1927). Conversely, a young limb regeneration
bud forms a tail if transplanted onto the base of a tail (Guyenot,
1927). These experiments show that young regeneration buds
are still more or less indifferent, and not yet determined for a
definite course of development. After transplantation, they
develop in agreement with their new environment, evidently

Raven - Outline Physiology

12

178 REGENERATION

under the influence of this environment. An experiment by
Schotte and Hummel (1939) has demonstrated clearly that the
tissue of young buds really is of an entirely indifferent,
"embryonic" nature. They transplanted young regeneration
buds into the inner eye-chamber. Here the buds formed a lens
under the influence of the retina.

Older regeneration buds, on the other hand, have already
been determined for development in a definite direction. After
transplantation they produce the same material as they would
also have formed if they had not been displaced. The moment
of determination at which the buds lose their indifferent
character depends on various circumstances; on the average,
the indifferent stage of limb buds lasts about two weeks.

Some Russian authors have in later years raised objections
to these experiments and their explanation (e.g., Polezajev,
Liosner). They pointed out that in many of these experiments
the possibility was not excluded that the graft did not go on
developing, but that it was repressed and replaced by the
stump's own regeneration bud. This would then explain why
the properties of the regenerate are in accordance with those
of the stump, regardless of the origin of the graft. In reality,
early regeneration buds would not be indifferent, but possess
a certain degree of labile determination already. In support of
this view they described experiments the results of which
did not agree with those mentioned above. An early regeneration
bud of a tail, for instance, produced a tail-like appendix after
transplantation onto an amputated limb. So far, however, their
results are not very convincing.

In regeneration, just as in embryonic development, de-
termination seems to take place stepwise. First the regeneration
bud as a whole is determined to form a fore-limb, or hind-limb,
etc., but the fate of each individual cell is not yet fixed. Schaxel
(1922) has shown that part of an already determined regenera-
tion bud is still able to produce a harmoniously built limb after
transplantation. If a limb regeneration bud is split into two,
each half will produce a complete limb (Swett, 1928). On the
other hand, two buds which are already determined, may still
fuse so that they form a single, harmoniously built limb.

REGENERATION 179

At a somewhat older stage, such experiments have a different
result. The parts of the regenerate have then been determined,
so that defective limbs are formed after the extirpation of part
of the material, or splitting of the bud.

As regards the problem of how the determination of the
various organs within the regenerate takes place, it can be
stated on the basis of the experiments already described
(p. 176) that this process does not occur in such a way that
the determination of each tissue of the regenerate is governed
by the cells of similar tissues in the stump. The subdivision
of the regenerate into its organs is a process entirely similar
to the autonomisation of the organ-fields in embryonic devel-
opment. Each part of the regeneration material is as though
"imprinted" with a certain determination, depending on its
position in the system. The fate of each element is a function of
its position. Here again we are dealing with an organisation-field.

If we want to solve the problem of the origin of this field,
we must realise that the regenerate replaces the part that has
been removed by amputation, i.e. that its development varies
in accordance with the position of the cut. Therefore, the field
governing the development of the regenerate must also vary
from case to case. The simplest explanation seems to be that,
e.g. in the limb of the adult animal, the original limb-field is
retained intact. After amputation, it is still present in the
stump. From there, it extends into the indifferent material of
the regeneration bud, and determines the differentiation of
this material. The field in the regenerate, therefore, would be
the immediate continuation of the field in the stump, and this
would explain the complete harmony between the regenerated
organs and those in the stump.

Certain experimental results, however, conflict with this
explanation. We have seen that an early regeneration cone of
a fore-limb, grafted onto the stump of a hind-limb, differentiates
into a hind-limb, in accordance with its new environment. If
however a thin slice of the old tissue of the fore-limb is trans-
planted along with the regenerate, the latter will develop into
a fore-limb (Milojevic, 1924), (Fig. 64). The disc of fore-limb
tissue, therefore, prevents the influence of the hind-limb stump

180 REGENERATION

from taking effect, and gives rise to a fore-limb field in the
regenerate. In this case the field is certainly not the immediate
continuation of that of the stump. Weiss (1927) amputated the
hand of a fore-limb, halved the lower arm longitudinally, and
removed one of the halves. He then covered the lateral wound
with skin, so that regeneration could not take place in this
region, but only at the distal transverse cut. A complete hand
was then formed by the regenerate on the halved lower-arm.
Here, again, the field of the regenerate could not be simply the
continuation of that of the stump. The opposite experiment can
also be made: if two limbs are made to grow together longitu-
dinally with their axes pointing in the same direction, and then
amputated, a single regenerate will develop in the common
wound (Swett, 1924). Polezajev (1936) went even farther, by
completely mincing the internal organs of the stump of an
amputated limb. Yet a more or less complete regeneration of
the limb was found to take place. If, however, the stump was
filled with minced tail tissue, tail-like regenerates were formed.
It follows that it is not necessary for the formation of a normal
organisation-field in the regenerate that the organs of the
stump should be in their normal positions. The field is clearly
not an extension of a pre-existing field in the stump, but it is
formed de novo in the regenerating tissue by an autonomous
process. The composition of the field is such that the organs
differentiating under its influence fit exactly onto those of the
stump. The following experiment proves that the nature of the
field is determined only by the level of the cut, and not by the
composition of the rest of the stump. Part of a limb, e.g. a
forearm, is isolated by means of two transverse cuts, one at
the elbow, and one at the wrist. It is then put back onto the
stump in an inverted position, so that the elbow is at the distal
end. Now at this end, a regenerate is formed consisting of the
parts that normally lie distally to this cut, i.e. forearm as
well as hand; a new forearm is consequently formed in the
regenerate, in spite of the fact that an (inverted) forearm is
present already. This must be due to the "elbow" qualities of
the wound in which regeneration takes place.

Liosner and Woronzowa (e.g., 1937) have made a number of

REGENERATION 181

experiments in an attempt to analyse the complex of determ-
ining factors that originates in the wound. They transplanted
either only musculature or only skeleton from fore-limb into
hind-limb, from tail into limb, from upper arm into forearm,
and vice versa. After this, they amputated the part in question.
During regeneration they found that both muscle grafts and
skeleton grafts influenced the nature of the regenerate. They
concluded that the determination of the regenerate is brought
about by the co-operation of the various transected organs
in the wound.

Trampusch concluded on the basis of his experiments (p. 176)
that the skeleton is important in particular for the longitudinal
growth of the regenerating limb, and the muscle tissue for its
increase in girth, but that the skin is the most important
organiser for the shape of the regenerate.

Summarising the foregoing, we can say that in many respects
the phenomena of regeneration show a parallel to those of
embryonic development, but that in a number of respects we
have found new relationships here. These throw some light
from a fresh angle on the problem of the organisation-field.
We may expect a deeper insight into the laws governing devel-
opment from the further analysis of these phenomena.

CHAPTER XII

Some final considerations

How does the spatial multiplicity of the adult organism arise
in the course of its development? This is the central problem,
underlying the whole of the present discussion. We have seen
that the spatial multiplicity as such is not yet present in the
fertilised egg. The latter shows only a slight extensive multi-
plicity, expressed in its polarity and bilateral symmetry, and
probably located mainly in the egg cortex. Development, how-
ever, involves a steady increase in the complexity of the
structure of the embryo. This begins with the local accumulation
of preformed determining substances under the influence of
directive activities originating in the primary coordinate system.
The parts now begin to influence each other. The next step
is the formation and local concentration of new substances in
cytoplasm and nucleus, leading to a rapid increase in the
chemodifferentiation of the egg. This, in turn, starts certain
topogenetic processes which result in migrations of cell material,
and thereby create new topographical relationships which set
the scene for new mutual influences, called induction. Finally,
the physical and chemical variety of the cells caused by their
chemodifferentiation becomes outwardly visible. This marks the
beginning of tissue differentiation, which prepares the organs
for their definitive functions in the organism. Even after the
attainment of the functional stage, development does not come
to a stop, but it goes on under continuous interaction of the
parts. Last of all, the phenomena of regeneration show that
even in the adult animal the parts still interact ceaselessly:
removal of a part of the body causes new processes in the
remaining part which result in complete or partial restitution
of the missing part.

To a very great extent, therefore, development has the

SOME FINAL CONSIDERATIONS 183

character of an epigenesis. The spatial structure is not pre-
formed in the egg, but it arises de novo in the development of
each individual. We must not forget, however, that in another
form the multiplicity is already present in the egg, viz. as in-
tensive multiplicity. But for this intensive multiplicity of the
egg, its further development would be inconceivable. The spatial
multiplicity of the egg, therefore, does not arise from nothing
at all, but rather by the transformation of intensive into ex-
tensive multiplicity. In other words, the structural plan is
potentially present in the egg already, though not, it is true,
in a spatial form; it is actualised during development. Ultima-
tely, all developmental processes can be referred back to the
constitution of the egg, from which they follow according to
fixed laws. This is the cause of their harmonious interlinking,
which is so essential for the normal course of development. The
orderly character of development, therefore, is due to the
constant composition of the egg, which is given once and for all.
The secret of development lies in the composition of the
fertilised egg; from it, all the rest follows of necessity.

It can easily be understood that the consideration of the
phenomena of development, in which the marvellously perfect
structure of the organism seems to arise from nothing, has
given rise to questions regarding the nature of this great
mystery: Life. The very first investigations in the field of
"developmental mechanics" were designed to shed some light
on this problem. Roux, the "father of developmental mechanics",
himself adhered to the then dominant ''machine theory'* of life,
which regarded the living organism as nothing but a well
functioning machine. On this view, all biological phenomena
would be explicable entirely by means of the laws of physics
and chemistry. The egg, too, was regarded as such a complicated
machine; its development would be nothing but the setting
in motion of a wound-up clockwork. Weismann's theory, which
we have discussed above (p. 34), followed this train of thought
to its final logical conclusion.

A campaign against these conceptions was started by Driesch.
His experiments, in particular those on sea urchins, had con-
vinced him that the machine theory could not explain the

184 SOME FINAL CONSIDERATIONS

phenomena of development. The chief obstacle to this theory
was the explanation of the phenomena of regulation, whereby
normal embryos are formed in spite of disturbances during
development. The machine theory was not able to make this
comprehensible. According to Driesch it was not possible to
conceive a machine which can be divided into any number of
machines of the same structure, or which, after a disturbance
of its structure or functioning, returns to the normal situation.
Hence he concluded that, apart from the "machine" there was
another, non-mechanical factor, which he called ''entelechy".
It was this factor that, after disturbances, was able to repair
the machine and to make it return to the right track. He
opposed this theory, a form of "vitalism", to the "mechanism"
of Roux and Weismann. Because of the interference of the
"vital force", or entelechy, in the mechanical course of the life
phenomena, no purely physico-chemical explanation of these
phenomena would be possible. This was the basis of Driesch's
conviction that he had proved the "autonomy of life".

We have already seen (p. 32) that this "proof" by Driesch
it not valid. His conclusion that, apart from the machine, the
assumption of a special vital force is necessary to explain
regulation, is not the only possible conclusion, nor even the
first one that comes to mind. The phenomena of regulation can
be explained far more easily by the hypothesis that, at the
stage in question, there is no machine at all as yet, i.e. that no
system of spatial multiplicity has yet been formed. This dis-
poses of Driesch's arguments, and his vitalism thereby loses
its raison d'etre as an empirically founded theory.

Now does this mean that "mechanism" is the only correct
view on the nature of life, and that, therefore, the organism
is nothing else and nothing more than a physico-chemical
system that can be explained entirely by the laws of physics
and chemistry? In this discussion, we have always strongly
emphasised the physical and chemical processes in the develop-
ing organism. Hence it might be assumed that the author
supports this view. Such, however, is not the case ; and for that
reason we must devote our attention for a moment to the
relation between biological and physicochemical systems, and to

SOME "FINAL CONSIDERATIONS 185

the conclusion on the nature of life to which this leads. We
may start from the considerations developed by H. J. Jordan
(1941, and elsewhere).

In Jordan's opinion, the difference between physics and
chemistry, on the one hand, and biology, on the other, is a
difference in method rather than a difference in subject. It is
not really correct to oppose physics and chemistry, as the
sciences of lifeless nature, to biology, as that of living nature.
Physics and chemistry are sciences of the whole of nature, both
living and lifeless. Their task is the study of the modes of
operation occurring in nature. Therefore, being analytic and
experimental sciences, they attempt as far as possible to
isolate the factors acting on one another. They attempt to
become independent of the fortuitous circumstances of the ex-
periment by eliminating, one after the other, all those factors
which interfere with the analysis of the phenomenon that is
being studied. In this way, physics and chemistry arrive at
formulations of "laws of nature", which are expressions of the
modes of operation encountered. These sciences are not in-
terested in the fact that the circumstances postulated in these
"laws" do not "occur" in nature in this form, so that no falling
object behaves entirely according to the simple law of gravita-
tion, and no billiard ball is so perfect that it does not show
deviations from the basic laws of collision. What is more, they
are scarcely, if at all interested in the actual occurrence^ the
"here and now" of the phenomena. Physics studies the properties
of electrical discharges, but does not ask where the lightning
strikes. Chemistry states that A reacts with B if the two meet,
but it is not interested in knowing whether or not the two ever
do meet in nature without the mediation of the investigator,
and whether this occurs often or rarely.

In biology, the situation is entirely different. Here all in-
terest is focused on the "here'' and "now'\ It is not the fact
that A reacts with B which is the most important thing in
biology, but that this reaction takes place exactly here, and at
just this moment. All phenomena in living organisms have their
fixed place and time of occurrence; only by virtue of this fact
is the orderly progress of living phenomena possible. Interest

186 SOME FINAL CONSIDERATIONS

centres on this orderliness in biology, whereas physics and
chemistry consider only isolated modes of action and are blind
to their association in the real process.

It follows from these considerations that we must divide the
question of the relationship between biology, physics and
chemistry into two parts. We must consider, on the one hand,
the modes of action occurring in living organisms, as such.
But, on the other hand, we must take into account their spatial
and temporal connections.

It follows from the above that it is meaningless to ask
whether in biology modes of action will at any time be found
which for fundamental reasons cannot be reduced to, and ex-
plained by, the laws of physics and chemistry. For if the
biologist, in the course of his analyses of living organisms,
encounters modes of action that are not yet known to, or
analysed by, the physicist or chemist, it will be the task of the
latter to study these phenomena with their own methods, i.e.
by complete isolation, and to trace the laws on which they are
founded. This is not just a hypothetical case, as may be seen
from the number of times that physics and chemistry have
already borrowed their problems from biology. A well known
example is the importance of the role played in the development
of the theory of electricity byGalvani's observation that exposed
frog muscles twitch if they touch two different metal con-
ductors. In a later period, physical chemistry received an
important impetus from Pfeffer's observations on osmotic
phenomena in plant cells, which led to a closer analysis by
physicists and chemists of the laws on which these phenomena
are founded. It might be said, in general, that the sciences of
"here" and "now" (to which belong, apart from biology, such
other branches of science as geology, physical geography,
meteorology, and astronomy), supply the material that is
studied more closely by physics and chemistry. These latter
disciplines unravel this material into its elementary components,
in order to derive the natural laws that are valid for the whole
of the reality of nature. Once we have understood this, we see
that the assumption is unfounded that biology may some time
detect modes of action which, for fundamental reasons, do

SOME FINAL CONSIDERATIONS 187

not belong to the domain of physics and chemistry, and which
these sciences will be unable to analyse. On the other hand,
it follows that biology, wherever it employs causal analysis,
will greatly benefit by the use of the concepts coined by
physics and chemistry, and of the laws discovered by these
disciplines.

However, the matter takes on an entirely different aspect,
if we state the problem as follows: Is it possible to give a
complete explanation of the phenomena of life on the basis
of physics and chemistry? In other words, is biology nothing
but a part of physics and chemistry ? We have already seen that
what matters in the living organism are not only the modes of
action as such, but in particular also their localisation in space
and time. The modes of action as such are not different from
those studied by physics and chemistry; it is their mutual
connections, the "here" and "now", in a word, the orderliness
of the phenomena of life, that constitutes the typical subject
of biology. We need not consider here whether or not non-
living systems can have an "order", comparable to the
orderliness found in living organisms (Kohler's "physische
Gestalten''), The important fact is that physics and chemistry
are powerless to explain this "order" because it is outside their
domain. This "order" cannot be analysed further with the causal
method ; if we trace the phenomena backwards through time, we
find that the orderly progress of the phenomena of life always
proceeds from the preformed order of an earlier situation.
Without this order, life is not conceivable; biology, therefore,
will never be able to dispense with it without belying its most
essential nature.

If we apply these ideas to animal development, we see that
the phenomena as such, taken severally, can be described
simply as physical and chemical processes. Movements of
material particles, diffusion and osmosis, chemical reactions,
colloid-chemical processes — physical and chemical laws govern
the course of all these phenomena. But as soon as we focus
our attention on the orderly character of the developmental
phenomena, the way in which they are harmoniously inter-
linked, their well-arranged course in space and time — character-

188 SOME FINAL CONSIDERATIONS

istics which constitute the most essential trait of development,
because it is only by their virtue that the result is not a chaotic
aggregate, but an organism — as soon as we do this, physics
and chemistry fail us. We can reduce the orderly course of
development only to preformed order in the constitution of its
starting point, the egg, in which the future embryo is contained,
not, it is true, in an actually spatial form, but potentially, non-
spatially, as an intensive multiplicity.

If now we ask whence this order of the egg originated, we
can go still farther back in time, and trace the developmental
processes which gave rise to the egg, but always again we
encounter the order, given once and for all, which is the
characteristic of all life. In the phenomena of life, all order
is a consequence of previous order. Here we encounter the
great enigma of Life, which cannot be solved by means of a
causal approach. We must accept this order as an essential
characteristic of life, a basic phenomenon, of which no causal
explanation is possible.

GLOSSARY

The following list contains the explanations of a number of
biological technical terms used in this book.

Amoeboid movement: movement by means of pseudopodia ('false

feet', protoplasmic processes), similar to that of an Amoeba.
Amphiaster: double star, consisting of two asters connected by a

mitotic spindle. Cf. monaster.
Amphimtvis: fusion of two gametes (in particular: fusion of their

nuclei), formed by two different individuaJs.
Androgamone: fertilisation substance produced by the male germ

cells. Cf. gamone, gynogamone.
Animal pole: pole of the egg at which the polar bodies are formed.

Opp.: vegetative pole.
Assimilative induction: influence which causes other cells to develop

in the same way as those of the inductor.
Aster: star-shaped structure in the cytoplasm, formed by local

gelation.
Atrophy: reduction in size and functional activity. Cf. hypertrophy.
Autonomisation: process leading to independence of the parts of

a whole.
Bilateral symmetry: case of symmetry in which there is only one

symmetry plane, i.e. in which the body can be divided into

symmetrical halves in only one way.
Blastema: organ primordium consisting of undifferentiated cells.
Blastomere: cell formed by the cleavage of the egg.
Blastula: vesicular stage of cleavage, with an internal cavity.
Caudal: pertaining to or directed towards the tail. Opp.: cranial,

rostral.
Centrifuge: rapidly rotating apparatus for subjecting bodies to

strong centrifugal forces.
Centrosome: corpuscle forming the centre of an aster, or a pole of

a mitotic spindle.
Chemodifferentiation: division of a whole into parts differing in

their (physical and) chemical properties.
Chimaera: organism consisting of tissues belonging to different

species.
Chromatin: substance with a high affinity for stains, localised in

the nucleus.
Chromidium: cytoplasmic granule with high affinity for stains.
Chromosome: corpuscle containing the chromatin during nuclear

division.

190 GLOSSARY

Cilia: hair-like, motile cytoplasmic processes.

Cleavage: the division of the egg into a number of cells by a process

of cellular division, which is not accompanied by cell growth.
Cortex: outer layer.
Cortical: pertaining to the cortex.

Cranial: pertaining to or directed towards the head. Opp.: caudal.
Cumulus oophorus: the follicle cells surrounding the egg in the

follicle of the mammalian ovary.
Cyclopia: abnormality, in which only one median eye is present.
Cy taster: aster formed at an arbitrary place in the cytoplasm.
Cytology: the science of cells.
Cytoplasm: protoplasm of the cell, as opposed to that of the nucleus

(cf. nucleoplasm).
De-differentiation: reduction in the differentiation that has already

taken place.
Depolarlsation: loss or reduction of polarity.

Determinants: material particles, regarded as the carriers of in-
heritable properties. Cf. genes.
Determination: the fixation of the course of future development in

a part of the germ.
Differentiation: the division of a whole into recognisable parts; the

appearance of differences among originally identical parts.
Diploid: containing a double set of chromosomes. This is the usual

condition in the majority of the cells of the body. Cf. haploid,

polyploid.
Dispermy: Fertilisation by two sperms. Cf. monospermy, polyspermy.
Double-monster: organism formed by two individuals grown to-
gether.
Ectoderm: outer germ-layer, the outer cell -layer of the gastrula.

Cf. mesoderm, endoderm.
Ectoplasm: outer layer of the cytoplasm; also, protoplasm which

will later be located in the ectoderm cells. Opp.: endoplasm.
Embryogenesis : the development of the embryo from the egg.
Enchylema: the more liquid part of the protoplasm.
Endoderm: inner germ-layer, inner cell-layer of the gastrula. Cf.

ectoderm, mesoderm.
Endoplasm: the central part of the cytoplasm, or: the protoplasm

that will later be located in the endoderm cells. Opp.: ectoplasm.
Endothelium: the tissue lining cavities in the body, such as blood

and lymph vessels.
Entelechy: a hypothetical, purposive vital force.
Enzyme: organic substance with the properties of a catalyst; =■

ferment.
Epigenesis: the appearance of new structures in the course of

development.
Epithelium: tissue, consisting of a sheet of closely packed cells,

covering an internal or external surface of the body. Cf. endo-
thelium.

GLOSSARY 191

Equatorial plane: plane that bisects at right angles the line connect-
ing the poles.

Equipotential system: cell group, all parts of which have the same
potency.

Exogastrulation: abnormal gastrulation, in the course of which the
archenteron evaginates, instead of invaginating.

Explantation: the culturing of pieces of tissue outside the body.
Cf. implantation, transplantation.

Extensive multiplicity: spatial multiplicity: a complexity of struc-
ture which is recognisably different in form or nature in differ-
ent regions of an organism or cell. Opp. : intensive multiplicity.

False hybrid: hybrid formed by the fusion of an egg with a sperm
of another species, in which the nucleus of the latter does not
take part in development.

Ferment: = enzyme.

Fibroblast: young connective tissue cell.

Follicle: multicellular covering of the egg in the ovary.

Frontal: at right angles to the median plane, separating dorsal and
ventral halves.

Gamete: germ cell.

Gamone: fertilisation substance, formed by the gametes at fertilisa-
tion. Cf. androgamone, gynogamone.

Gastrula: developmental stage, consisting of 2 (or 3) germ-layers.

Gastrulation: development of a gastrula from a blastula.

Gene: unit carrier of inheritable properties. Cf. determinants.

Genotype: the inherited constitution of the individual.

Germ-band: thickening of one or several germ-layers, from which
certain organs will develop.

Germinal vesicle: the nucleus of the oocyte.

Germ-layer: one of the cell-layers of the gastrula, each of which
will later develop into definite organs.

Gradient: change of a scalar quantity from point to point.

Gynogamone: fertilisation substance formed by the female germ
cells. Cf. androgamone, gamone.

Haploid: containing a single set of chromosomes. This is the normal
condition in ripe germ cells. Cf. diploid, polyploid.

Heterogeneous fertilisation: fertilisation by a sperm of another,
usually distantly related, species.

Heteroplastic transplantation: transplantation into an individual of
another, closely related species. Cf. xenoplastic transplantation.

Heterospermic merogony: development of a non-nucleated egg
fragment after fertilisation by a sperm from another species.
Opp.: homospermic merogony.

Heterozygote : organism developing from a zygote formed by the
fusion of gametes that were dissimilar as regards the gene
under consideration. Opp.: homozygote.

Histochemistry: the determination of the chemical composition of
cells and tissues.

192 GLOSSARY

Histog-enesis : the origin of tissues.

Homospermic merogony: development of a non-nucleated egg
fragment after fertilisation by a sperm of the same species.
Opp.: heterospermic merogony.

Homozygote: organism arising from a zygote formed by the fusion
of gametes that were identical as regards the gene under
consideration. Opp.: heterozygote.

Hormones: substances secreted in definite parts of the body, under
the influence of which certain processes take place in other
parts of the body to which they are carried by the blood.

Hybrid: individual arising from a cross between two races or species.

Hypertrophy: excessive growth by the increase in size of tissue
elements.

Implantation: the grafting of a cell group or organ into an abnor-
mal place, in particular into one of the body cavities. Cf. ex-
plantation, transplantation.

Induction: the influence exerted by an inductor.

Inductor: cell group which causes other cells to develop into a
certain direction.

Intensive multiplicity: A complexity of structure which is not based
on recognisable regional differences in form or nature within
the organism or cell. Opp.: extensive multiplicity.

Malpighian tubes: excretory organs of insects, opening into the
hind gut.

Matrocline : in which the maternal characteristics dominate over
the paternal ones. Opp.: patrocline.

Median plane: plane of symmetry of a bilaterally symmetrical
organism.

Melanophore: black pigment cell.

Meridian plane: plane containing both poles.

Merogony: development of a fertilised fragment of an egg in which
no female pronucleus is present.

Mesenchyme: embryonic connective tissue.

Mesoderm: middle germ-layer, middle cell-layer of the gastrula.
Cf. ectoderm, endoderm.

Mesoplasm: cytoplasm that will later become located in the meso-
derm cells. Cf. ectoplasm, endoplasm.

Microdissection: operations on cells under the microscope.

Micromeres: small cleavage cells.

Microsomes: very small granules in the cytoplasm.

Mitochondria: larger, rod-shaped or granular corpuscles in the cell
cytoplasm.

Mitosis: indirect nuclear division.

Modulation: change in the external appearance of cells, caused by
external circumstances, and not accompanied by a simultaneous
change in potencies.

Monaster: simple radiation in the cytoplasm, in the centre of
which the chromosomes are located. Cf. amphiaster.

GLOSSARY 193

Monospermy: fertilisation by a single sperm. Cf. dispermy, poly-
spermy.

Morphallaxis : restitution by the transformation and shifting of
material, not by growth. Cf. reorganisation.

Morphogenesis: the origin of form.

Morphology: the science of form.

Mutant: individual or race arising by mutation.

Mutation: spontaneous change in an inheritable property.

Neoblast: undifferentiated cell in the body of an adult animal, which
plays a role in regeneration.

Neo-epigenesis: the hypothesis that the embryo develops from a
structureless egg. Cf. Neo-evolutio.

Neo-evolutio : the hypothesis that the egg already contains an in-
visible spatial structure, from which the embryo will later
develop. Cf. Neo-epigenesis.

Neural: pertaining to the nervous system.

Neurula: developmental stage, subsequent to the gastrula stage,
in which the primordium of the central nervous system (the
neural plate) has developed from the ectoderm.

Neurulation: the development of a neurula from a gastrula.

Notochord: axial rod, in the body of Tunicates and Vertebrates.

Nucleoplasm: non-chromatic content of the nucleus, nuclear sap.

Omentum: a fold of the peritoneum.

Omnipotent: capable of development into any organ or tissue.

Oocyte: unripe egg, before the completion of the maturation
divisions.

Oogonium: mother cell of the egg.

Organisation: arrangement of the parts of a whole into an orderly
system of organs.

Organisation centre: part of the germ, which plays a leading role in
the organisation of the embryo.

Organiser: cell group which incites tissues to form an organised
system of organs.

Organogenesis: the development of organs.

Ovary: female reproductive gland. Cf. testis.

Oxidase: enzyme promoting oxidation by oxygen. Cf. peroxidase.

Parthenogenesis: reproduction by means of gametes which develop
without fertilisation.

Patrocline: in which the paternal characteristics dominate over the
maternal ones. Opp.: matrocline.

Pentaploid: containing five sets of chromosomes. Cf. diploid, haploid,
triploid.

Peristome: mouth area.

Perivitelline space: the interspace between the egg and the fer-
tilisation membrane.

Peroxidase: enzyme which liberates oxygen from peroxides. Cf.
oxidase.

Raven - Outline Physiology 13

194 GLOSSARY

Pharynx: part of the intestine, immediately caudal to the mouth

cavity.
Physiology: the causal science of the phenomena of life.
Placode: thickening of the ectoderm.
Plasmagene: corpuscle in the cytoplasm which in certain respects

corresponds to the genes in the nucleus.
Pluteus: larva of a sea urchin or brittle star, with a calcareous

skeleton consisting of radially protruding rods.
Polar bodies: small cells expelled from the egg in the course of its

maturation divisions.
Polarity: condition of the body in which an axis connecting two

different poles can be distinguished.
Polyploid: containing more than two sets of chromosomes. Cf. tri-

ploid, pentaploid.
Polyspermy: the penetration of several sperms into a single egg.

Cf. dispermy, monospermy.
Postpharyngeal: caudal to the pharynx. Opp.: prepharyngeal.
Posttrochal: basal to the band of cilia in the trochophore larva.

Opp.: pretrochal.
Potency: power of development.
Predetermination: first phase in the process of determination, during

which the fixation of the fate of the cells is still labile.
Preformation: presence in the egg of the structure that will become

unfolded during the development of the embryo.
Prepharyngeal: rostral to the pharynx. Opp.: postpharyngeal.
Pretrochal: situated above the band of cilia in the trochophore larva.

Opp.: posttrochal.
Pronucleus: nucleus of a gamete.

Protoplasm: the substance of which living cells consist.
Reaction system: cell group reacting to an induction with a definite

developmental process.
Reductional division: nuclear division in the course of which the

chromosome number is halved.
Regenerate: cell group formed by regeneration for the restitution

of lost organs or parts of the body.
Regeneration: the reformation or restitution of lost organs of parts

of the body.
Regulation: in biology, this term is applied in particular to processes

that lead to a return to the normal situation after a disturbance.
Reorganisation: restitution by transformation or shifting of material.

Cf. Morphallaxis.
Rostral: pertaining to the front end.

Scalar quantity: quantity to which a numerical value can be attri-
buted, but which is not directional.
Somatoblast: cell from which an important part of the body of the

embryo will arise.
Sperm aster: aster in the egg cytoplasm arising from a sperm that

has penetrated into the egg.

GLOSSARY 195

Sperm (atozoon) : male germ cell.

Spong^ioplasm: the more solid, fibrous part of the protoplasm.

Stomodaeum : mouth cavity formed by the ectoderm.

Syncj'tiiim: multi-nuclear mass of protoplasm.

Synliarion: zygote nucleus, arising from the fusion of a male and

a female pronucleus.
Teloblast: end-cell of a row of cells.
Testis: male reproductive gland. Cf. ovary.
Tetraster: mitotic spindle with four poles.
Topogenesis: the development of the form of the body through the

shifting of material during development.
Transplantation: grafting of an organ or cell group into another

place. Cf. explantation, implantation.
Triploid: containing three sets of chromosomes. Cf. diploid, haploid.
Trochophore: larval stage, occurring in worms, etc., with crowns

of long cilia by means of which it moves.
Trophic: pertaining to the nutrition.

Ultracentrif uge : centrifuge working at very high speeds.
Vacuole: cavity in the cytoplasm, filled with a liquid.
Vegetative pole: one of the poles of the egg. Opp.: animal pole.
Vegetative reproduction: asexual reproduction.
Vitalism: theory according to which the phenomena of life cannot

be described entirely in terms of the laws of physics and

chemistry.
Vitamins: essential food substances, very small quantities of which

are sufficient.
Vitro, in — : "in glass", term applied to the culturing of tissues

outside the body.
X-chromosome : sex chromosome, playing a role in the inheritance

of sex.
Xenoplastic transplantation: transplantation into an individual of

another, very distant, species. Cf. heteroplastic transplantation.
Zygote: fertilised egg, cell formed by the fusion of two gametes.
Zygote nucleus: the nucleus formed by the fusion of the male and

female pronuclei.

REFERENCES

Extensive lists of literature may be found in the works of Schleip
(1929), Dalcq (1928 and 1941), Huxley and de Beer (1934), Spemann
(1936), Weiss (1939), Brachet (1944 and 1952), and Lehmann
(1945). Therefore, the following list contains in general only those
of the papers quoted in this book, which are not mentioned by, or
appeared after the publication of these works, to which the reader is
referred for all other references.

1948 ANCEL, P. and P. VINTEMBERGER: Recherches sur le de-

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1949 ANDRES, G.: Untersuchungen an Chimaeren von Triton und

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1946 ASPEREN, K. VAN: Pharynx regeneration in postpharyngeal

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1947 AVEL, M. : Les f acteurs de la regeneration chez les Annelides.

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1948 BALINSKY, B. I.: Korrelationen in der Entwicklung der

Mund- und Kiemenregion und des Darmkanals bei
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1947 BALTZER, F. : Weitere Beobachtungen an merogonischen
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1934 BATAILLON, E. and TCHOU SU: L'analyse experimentale
de la fecondation et sa definition par les processus
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1936a BERRILL, N. J. and D. MEES: Reorganization and regenera-
tion in Sabella. 1. Nature of gradient, summation, and
posterior reorganization. J. exp. Zool. 73, 67 (1936).

1936b Reorganization and regeneration in Sabella. 2. The in-
fluence of temperature. 3. The influence of light. J. exp.
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1944 BRACHET, J, Embryologie chimique, Paris — Li^ge, 1944.

1952 Le role des acides nucleiques dans la vie de la cellule

et de I'embryon. Actwalites biochim., Lidge ■ — Paris 1952.

1949 BUTLER, E. G. and O. E. SCHOTTE: Effects of delayed

denervation on regenerative activity in limbs of Urodele
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REFERENCES 197

1931 CLARK, E. R., W. J. HITSCHLER, H. T. KIRBY-SMITH,
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the ingrowth of new blood vessels into standardized
chambers in the rabbit's ear and the subsequent changes
in the newly grown vessels over a period of months.
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1940 COSTELLO, D. P.: The fertilizability of nucleated and non-

nucleated fragments of centrifuged Nereis eggs. J.
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1949 The relations of the plasma membrane, vitelline mem-
brane, and jelly in the egg of Nereis limbata. J. gen.
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1928 DALCQ, A. Les bases physiologiques de la fecondation et de
la parthenogenese. Paris 1928.

1941 L'oeuf et son dynamisme organisateur. Paris 1941.

1947 The concept of physiological competition (Spiegelman)

and the interpretation of vertebrate morphogenesis. Proc.
6th Intern. Congr. Exp. Cytol. 1947.

1946 DALTON, H. C: The role of nucleus and cytoplasm in de-
velopment of pigment patterns in Triturus. J. exp. Zool.
103, 169 (1946).

1936 DRAGOMIROW, N.: Ueber Induktion sekundarer Retina im
transplantierten Augenbecher bei Triton und Pelobates.
Roux' Archiv 134, 716 (1936).

1949 DUBOIS, F.: Contribution a I'etude de la migration des cellules

de regeneration chez les planaires dulcicoles. Bull. Biol.
France Belg. 83, 213 (1949).

1942 EPHRUSSI, B.: Chemistry of "eye color hormones" of Droso-

phila. Quart. Rev. Biol. 17, 327 (1942).
1945 FANKHAUSER, G. : Maintenance of normal structure in
heteroploid salamander larvae, through compensation of
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1950 GATES, G. E.: Regeneration in an earthworm, Eisenia foetida

(Savigny, 1826). III. Regeneration from simultaneous
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(1950).

1940 GOLDSMITH, E. D,: Regenerative and accessory growths in
Planarians. II. Initiation of the development of re-
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(1940).

1950 GUSTAFSON, T.: Survey of the morphogenetic action of the
lithium ion and the chemical basis of its action. Rev.
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1936 HADORN, E.: Uebertragung von Artmerkmalen durch das
entkemte Eiplasma beim merogonischen Triton bastard,

198 REFERENCES

palmatus -PlOisma, X cristatus-Kern. Verh. Deutsch. Zool.
Ges. 1936, 97.
1936 HAMBURGER, V.: The larval aevelopment of reciprocal
species hybrids of Triton taeniatus, Leyd (and Triton
palmatus, Duges) X Triton cristatus, Laur. J. exp. Zool.
73, 319 (1936).

1940 HARTMANN, M., A. SCHARTAU, and K. WALLENFELS:

Untersuchungen iiber die Befruchtungsstoffe der Seeigel.

II. Biol. Zentralbl. 60, 398 (1940).
1936 HARVEY, E. B.: Parthenogenetic merogony or cleavage with-
out nuclei in Arhacia punctulata. Biol. Bull. 71, 101

(1936).
1939 HOLTFRETER, J.: Gewebeaffinitat, ein Mittel der embryo-

nalen Formbildung. Arch. exp. Zellf. 23, 169 (1939).
1943-44 A study of the mechanics of gastrulation. I. J. exp.

Zool. 94, 261 (1943). — II. J. exp. Zool. 95, 171 (1944).
1945 Neuralization and epidermization of gastrula ectoderm.

J. exp. Zool. 98, 161 (1945).

1947 Neural induction in explants which have passed through

a sublethal cytolysis. J. exp. Zool. 106, 197 (1947).

1936 HORSTADIUS, S.: Studien iiber heterosperme Seeigelmero-
gone nebst Bemerkungen iiber einige Keimblattchimaren.
Verh. Kon. Natuurk. Mus. Belgie (II), 3, 801 (1936).

1934 HUXLEY, J. S. and G. R. DE BEER: The elements of ex-

perimental embryology. Cambridge 1934.

1941 JORDAN, H. J.: Die theoretischen Grundlagen der Tierphysio-

logie. Biblioth. Biotheor. 1, Leiden, 1941.

1951 KUUSI, T.: Ueber die chemische Natur der Induktionsstoffe

mat besonderer Beriicksichtigung der Rolle der Proteine
und der Nukleinsauren. Ann. Zool. Soc. Zool. Bot. Fenn.
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1945 LEHMANN, F. E.: Einfilhrung in die physiologische Embryo-
logie. Basel 1945.

1948 - — ■ Zur Entwicklungsphysiologie der Polplasmen des Eies

von Tubifex. Rev. Suisse Zool. 55, 1 (1948).

1936 LINDAHL, P. E.: Zur Kenntnis der physiologischen Grund-

lagen der Determination im Seeigelkeim. Acta Zool.
(Stockholm) 17, 179 (1936).

1937 LIOSNER, L. D. and M. A. WORONZOWA: Recherches sur

la determination de processus regeneratifs chez les
Amphibiens. Arch. Anat. micr. 33, 313 (1937).

1952 LOMBARD, G. L.: An experimental investigation on the

action of lithium on amphibian development. Thesis,
Utrecht 1952.

1935 LOPASHOV, G.: Die Entwicklungsleistungen des Gastrula-

mesoderms in Abhangigkeit von Veranderungen seiner
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REFERENCES 199

1951 McKEEHAN, M. S.: Cytolog-ical aspects of embryonic lens

induction in the chick. J. exp. Zool. 117, 31 (1951).

1938 MILLER, J. A.: Studies on heteroplastic transplantation in

Triclads. I. Cephalic grafts between Euplanaria doroto-
cephala and E. tigrina. Physiol. Zool. 11, 214 (1938).
1949 MOMENT, G. B.: On the relation between growth in length,
the formation of new segments, and electric potential
in an earthworm. J. exp. Zool. 112, 1 (1949).

1946 MONNE:, L.: Struktur- und Funktionszusammenhang desZyto-

plasmas. Experientia 2 (1946).

1947 MONROY, A.: Further observation on the fine structure of

the cortical layer of unfertilized and fertilized sea urchin
eggs. J. cell. comp. Physiol. 30, 105 (1947).
1941 MOORE, J. A.: Developmental rate of hybrid frogs. J. exp.
Zool. 86, 405 (1941).

1939 MOSER, F. : Studies on a cortical layer response to stimulating

agents in the Arbacia egg. I. Response to insemination.
J. exp. Zool. 80, 423 (1939).

1935 MOTOMURA, I.: Determination of the embryonic axis in the

eggs of Amphibia and Echinoderms. Sci. Rep. Tohoku
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1947 NIEUWKOOP, P. D. : Investigations on the regional determina-
tion of the central nervous system. J. exp. Biol. 24, 145
(1947).

1952 and others: Activation and organization of the central

nervous system in amphibians. J. exp. Zool. 120, 1 (1952).

1934 OKADA, Y. K. and H. SUGINO: Transplantation experiments

in Planaria gonocephala. Proc. Imp. Acad. Tokyo 10,
37 (1934).
1951 PASTEELS, J.: Centre organisateur et potentiel morphogene-
tique chez les Batraciens. Bull. Soc. Zool. Fi^ance 76,
231 (1951).

1940 PELTRERA, A.: La capacita regolative dell'uovo di Aplysia

limacina L. studiata con la centrifugazione e con le
reazioni vitali. Pubhl. Staz. Zool. Napoli 18, 20 (1940).
1939 PLAGGE, E.: Genabhangige Wirkstoffe bei Tieren. Ergebn.
d. Biol. 17, 105 (1939).

1936 POLEZAJEV, L.: La valeur de la structure de I'organe et les

capacites du blasteme regeneratif dans le processus de
la determination du regenerat. Bull. biol. France Belg.
70, 54 (1936).

1945 POULSON, D. F.: Chromosomal control of embryogenesis in
Drosophila. Anier. Natural. 79, 340 (1945).

1951 RANZI, S.: The proteins in the cells and in embryonic de-
velopment. Experientia 7, 169 (1951).

1935 RAVEN, CHR. P.: Zur Entwicklung der Ganglienleiste. IV.

Untersuchungen liber Zeitpunkt und Verlauf der ,,ma-

200 REFERENCES

teriellen Determination" des prasumptiven Kopf ganglien-

leistenmaterials der Urodelen. Roux' Archiv 132, 509

(1935).
1938a RAVEN, CHR. P. Ueber die Potenz von Gastrulaektoderm

nach 24-stiindigeni Verweilen im ausseren Blatt der

dorsalen Urmundlippe. Roux' Archiv 137, 661 (1938).
1938b Experimentelle Untersuchungen Uber die ,,bipolare Dif-

ferenzierung" des Polychaeten- und Molluskeneies. Acta

neerl. morphol. 1, 331 (1938).
1948 The chemical and experimental embryology of Limnaea.

Biol. Rev. 23, 333 (1948).
1952 Lithium as a tool in the analysis of morphogenesis in

Limnaea stagnalis. Experientia S, 252 (1952).
1942 RAVEN, CHR. P. and L. H. BRETSCHNEIDER: The effect

of centrifugal force upon the eggs of Limnaea stagnalis

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1945 RAVEN, CHR. P. and J. KLOOS: Induction by medial and

lateral pieces of the archenteron roof, with special refer-
ence to the determination of the neural crest. Acta neerl.
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1948 RAVEN, CHR. P. and J. C. A. MIGHORST: On the influence

of a posterior wound surface on anterior regeneration in
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Wetensch., Amsterdayn. 51, 434 (1948).

1949 REVERBERI, G. and A. MINGANTI: Ulteriori richerche sulla

formazione del cervello, degli organi di senso e dei palpi
nelle Ascidie. Riv. di Biol. 41, 125 (1949).
1936 RIES, E. and M. GERSCH: Die Zelldifferenzierung und Zell-
spezialisierung wahrend der Embryonalentwicklung von
Aplysia limacina L. Zugleich ein Beitrag zu Problemen
der vitalen Farbung. Puhbl. Staz. zool. Napoli 15, 223
(1936).

1946 RUNNSTROM, J., L. MONNfi and E. WICKLUND: Studies

on the surface layers and the formation of the fertiliza-
tion membrane in sea urchin eggs. J. Coll. Sci. 1, 421
(1946).

1942 SCHECHTMAN, A. M.: The mechanism of amphibian gastrula-
tion. I. Gastrulation-promoting interactions between
various regions of an Anuran egg {Hyla regilla). Univ.
Calif. Publ. Zool. 51, 1 (1942).

1929 SCHLEIP, W. : Die Determination der Primitiventwichlung.
Leipzig, 1929.

1939 SCHOTTfi, O. E. and K. P. HUMMEL: Lens induction at the
expense of regenerating tissues of Amphibians. J. exp.
Zool. 80, 131 (1939).

1936 SPEMANN, H.: Experimentelle Beitrdge zu einer Theorie der
Entwicklung. Berlin, 1936.

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1945 SPIEGELMAN, S.: Physiological competition as a regulatory
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(1945).

1951 TRAMPUSCH, H. A. L.: Regeneration inhibited by X-rays
and its recovery. Proc. Kon. Ned. Akad. Wetensch., Am-
sterdam, C 54 (1951).

1936 TWITTY, V. C: Correlated genetic and embryological ex-

periments on Triturus. I and II. J. exp. Zool. 74, 239
(1936).

1945 The developmental analysis of specific pigment patterns.

J. exp. Zool. 100, 141 (1945).

1948 TYLER, A.: Fertilization and immunity. Physiol. Rev. 28,

180 (1948).

1937 UBISCH, L. VON: Die normale Skelettbildung bei Echino-

cya^nus pusillns und Psamniechinus miliaris und die
Bedeutung dieser Vorgange fiir die Analyse der Skelette
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1938 UMANSKI, E.: The regeneration potencies of Axolotl skin

studied by means of exclusion of the regeneration capa-
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1947 WADDINGTON, C. H.: Organizers and genes. Cambridge,
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1926 WEISS, P.: Morphodynamik. Ahh. theor. Biol. 1926, H. 23.

1939 Principles of development. New York, 1939.

1950 Perspectives in the field of morphogenesis. Quart. Rev.

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1949 WOERDEMAN, M. W.: L'induction du cristallin chez les

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1950 YNTEMA, C. L.: An analysis of induction of the ear from

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113, 211 (1950).

Name Index

ADELMANN, H. B. 131
ANCEL, P. 41, 42, 49, 196
ANDRES, G. 138, 196
ASPEREN, K. VAN 166, 196
AVEL, M. 164, 196

BALINSKY, B. I. 134, 139,

143, 144 196
BALTZER, F. 84, 85, 86, 196
BARTH, L. G. 79, 164
BATAILLON, E. 15, 19, 20, 22,

23, 25, 26, 196
BAUTZMANN, H. 108, 113
BEADLE, G. W. 87, 90
BECKER, E. 87
BERRILL, N. J. 166, 168, 196
BIERENS DE HAAN, J. A. 30
BOVERI, TH. 22, 66, 67, 69,

71, 72, 73, 77
BRACKET, J. 34, 65, 79, 86,

107, 118, 119, 196
BRANDT, VV. 143
BRETSCHNEIDER, L. H. 48,

200
BUTLER, E. G. 173, 174, 196

CASPARI, E. W. 87
CASPERSSON, T. 65
GATE, G. TEN 135
CHAMBERS, R. 12, 15, 16, 26
CHILD, C. M. 43, 163, 168, 170,

171
CHUANG, H. H. 146
CLANCY. C. W. 87
CLARK, E. R. 159, 197
CONKLIN, E. G. 24, 59 60
COSTELLO, D. P. 10, 197

DALCQ, A. 10, 21, 22, 23, 24,
26, 60, 62, 104, 105, 107, 122,
125, 126, 127, 138, 145, 146,
197

DALTON, H. C. 85, 86, 197
DANCHAKOFF, V. 153
DEL AGE. Y. 11
DETWILER, S. R. 142, 156
DOORENMALEN, W. J. VAN

135
DRAGOMIROW, N. 131, 134,

197
DRIESCH, H. 3, 28, 30, 32, 35,

44, 183, 184
DUBOIS, F. 161, 197
DUESBERG, J. 24

EINSELE, W. 25
EPHRUSSI, B. 87, 197

FANKHAUSER, G. 17, 24, 67,

197
FILATOW, D. 134, 142
FISCHEL, A. 135
FOL, H. 12
FREDERICI, E. 152
FREY WYSSLING, A. 33
FRY, H. J. 22, 23

GAILLARD, P. J. 154
GALVANI 186
GATES, G. E. 161, 197
GEINITZ, B. 116, 124
GERSCH, M. 43. 200
GODLEWSKI, E. 15
GOLDSMITH, E. D. 164, 197
GUSTAFSON, T. 47, 197
GUYENOT, E. 175, 177

HADORN, E. 84, 85, 197
HAMBURGER, V. 79, 156, 157,

198
HARRISON, R. G. 132, 137,

140, 142, 143, 155
HARTMANN, M. 11, 198
HARVEY, E. B. 66, 198

204

NAME INDEX

HEATLEY, N. G. 118

HEILBRUNN, L. V. 10, 21

HERBST, C. 72

HERTWIG, G. 23, 84

HERTWIG, O. 23

HERTWIG, P. 23, 84

HOLTFRETER, J. 102, 103,
108, 109, 110, 116, 117, 118,
134, 136, 138, 139, 153, 198

HORSTADIUS, S. 44, 47, 76,
82, 83, 198

HUANG, A. C. 122

HUMMEL, K. P. 178, 200

HUXLEY, J. S. 62, 167, 198

HYDEN, H. 65

JAEGER, L. 79, 107
JORDAN, H. J. 185, 198
JUST, E. E. 14

KAAN, H. W. 137
KAWAGUCHI, E. 87
KEEFE, E. L. 156
KIKKAWA, H. 87
KITE, G. L. 16
KLOOS, J. 126, 200
KOHLER, W. 187
KRUGELIS, E. J. 107
KUHN, A. 87
KUPELWIESER, H. 15, 23
KUUSI, T. 119, 198

LEHMANN, F. E. 50, 51, 116,

198
LENDER, TH. 165
LEWIS, W. H. 132, 135
LEWY, O. 158
LINDAHL, P. E. 46, 118, 198
LIOSNER, L. D. 178, 180, 198
LOEB, J. 18, 19, 20, 25, 26
LOMBARD, G. L. 147, 198
LOPASHOV, G. 108, 123, 198

MANGOLD, H. Ill, 113, 116
MANGOLD, O. 30, 111, 116,

117, 131
MARX, A. 114, 115, 117
MAYER, B. 120

McKEEHAN, M. S. 134, 199
MEES, D. 168, 196
MIGHORST, J. C. A. 172, 200
MILLER, J. A. 170, 199
MILOJEVIC, B. D. 177, 179
MINGANTI, A. 60, 200
MOMENT, G. B. 169, 199
MONNfi, L. 33, 34, 199, 200
MONROY, A. 34, 199
MOORE, J. A. 79, 199
MOROHOSI, I. 87
MOSER, F. 14, 21, 199
MOTOMURA, I. 48, 104, 199
MURRAY, P. D. F. 149

NIEUWKOOP, P. D. 119, 147,
199

OKADA, Y. K. 168, 170, 199
OLMSTED, J. D. M. 157

PASTEELS, J. 10, 21, 48, 69,

104, 105, 199
PEASE, D. C. 49
PELTRERA, A. 62, 199
PENNERS, A. 50, 53, 104
PFEFFER, W. 186
PLAGGE, E. 87, 199
POLEZAJEV, L. 174, 178, 180,

199
POULSON, D. F. 74, 199

RANZI, S. 47, 154, 199
RAVEN, CHR. P. 18, 44, 47,

48, 107, 109, 121, 124, 126,

136, 172, 199, 200
REVERBERI, G. 60, 200
RIES, E. 43, 60, 200
ROBORGH, J. R. 18
ROSE, S. M. 60, 173, 174
ROTMANN, E. 133, 140, 142

155
ROUX, W. 1, 3, 27, 28, 183, 184
RUNNSTRGM, J. 14, 45, 200
RCJUD, G. 122

SANTOS, F. V. 167
SCHARTAU, A. 11, 198

NAME INDEX

205

SCHAXEL, J. 151, 178
SCHECHTMAN, A. M. 103, 200
SCHEWTSCHENKO, N. N. 170
SCHLEIP, W. 104, 200
SCHOTTfi, O. E. 139, 173, 174,

178. 196,' 200
SCHULTZE, O. 104
SCHWIND, J. L. 155
SEIDEL. F. 30, 69, 70, 71
SEVERINGHAUS, A. E. 140
SHAVER, J. R. 25
SINGER, M. 173
SPEK, J. 43
SPEMANN, H. 29, 35, 101,

109, 110, 111, 113, 115, 116,

117, 122, 124, 131, 132, 139,

200
SPIEGELMAN, S. 126, 172, 201
ST5HR, P. 154
STROER, W. F. H. 139
SUGINO, H. 168, 170, 199
SWETT, F. A. 178, 180

TATUM, E. L. 87
TCHOU SU 23, 196
TENNENT, D. H. 78
TITLEBAUM, A. 57
TOrVONEN S. 146
TRAMPUSCH, H. A. L. 138,
176, 181, 201

TWITTY, V. C. 80, 81, 85, 155,

201
TYLER, A. 11, 201

UBISCH, L. VON 59, 69, 83,

201
UMANSKI, E. 176, 201

VINTEMBERGER P. 41, 42,

49, 196
VOGT, W. 99, 101, 102

WADDINGTON, C. H. 117, 201
WALLENFELS, K. 11, 198
WEISMANN, A. 34, 35, 37, 64,

65, 183, 184
WEISS, P. 135, 149, 154, 156,

158, 175, 176, 177, 180, 201
WHITING, A. R. 87
WILSON, E. B. 23, 56, 57
WOERDEMAN, M. W. 106,

118, 132, 134, 135, 201
WOLFF, E. 165
WORONZOWA, M. A. 180, 198

YNTEMA, C. L. 136, 138, 201

ZIEGLER, H. E. 23
ZWILLING, E. 136

Subject Index

Abdominal segments 165 et seq.,

168
Acidity of protoplasm 43
Acids, fatty 118
Activating centre 69 et seq.
— substance 71
Activation, in induction 119,

139, 145
— , in regeneration 164
— , of egg 19 et seq., 25, 41
— , of nuclear factors 71, 85

et seq., 91 et seq.
Activity, metabolic 171
— , physiological 171 et seq.
— , state of 168, 171
Adaptation, functional 6, 155
Adenosine triphosphate 65
Affinity, tissue 103
Agglutination, sperm 11
AmUystoma 136, 138, 140,

143, 153, 155
Amoeboid movements 99, 189
Amphiaster 16, 20 et seq., 189
Amphibia, artificial partheno-
genesis 25 et seq.
— , bilateral symmetry 40 et

seq., 48 et seq.
— , cell size and body size 67
— , centrifugation 48
— , cleavage without nuclei 66
— , determination of neural plate

111 et seq.
— , development of eyes 128

et seq.
— , explantation of heart 153
— , fertilisation 23
— , gastrulation 95 et seq.,

101 et seq.
— , gradients 48, 62
— , heterospermic merogony

84 et seq.
— , hybridisation 78 et seq.

Amphibia, inversion of egg 48, 104
— , neurulation 98 et seq., 106

et seq.
— , polarity 40, 48
— , regeneration of limbs and

tail 172 et seq.
— , removal of organ primordia

151 et seq.
— , transplantation of organ

primordia 152 et seq.
Amphimixis 8, 16, 189
Androgamones 11, 189
Anesthesia 156
Anesthetics 163, 164
Annelids 92, 162
Antifertilizin 11
Anurans 134, 136, 138, 139,

142, 174
Apical organ 57

— region 162 et seq., 167 et seq,
Aplysia 62

Arbacia 78

Archenteron 44, 78, 83, 93 et
seq., 138, 139, 140

— roof 96 et seq., Ill et seq.,
131, 136, 138, 147

Architecture of bones 158

Arteries 159

Ascaris 68, 69, 92

Ascidians 167

— , bilateral symmetry 41, 58

— , centrifuging of eggs 59,

60, 61
— , cytoplasmic substances

57e^ seq., 62, 65
— , isolation of blastomeres 59
Aster, sperm 15, 16, 17, 22,

26, 194
Atrophy 157, 189
Attraction of nerve fibres 156
Auditory organ 136 et seq.,

145, 146

SUBJECT INDEX

207

Autonomisation 149, 179, 189

Autonomy of life 184

Axes, of ear 137

— , of limb 143

Axis, dorsoventral 40

— , main 4, 39, 40, 41, 43, 48

Axolotl 116, 140, 176

— , pigmentation 85, 86

Belly piece 122

Biolog-y, relation to physics and

chemistry 184 et seq.
Bipolar differentiation 43 et seq.
Birds 14
Birefringence 33
Blastema 162, 189
Blastocoel 95, 96, 114, 115, 146
Blastomeres 2, 24, 34, 35, 51,

64, 69, 92, 189
— , isolation of 28 et seq., 35,

44, 56, 59, 63, 72
Blastopore 93 et seq., 101
— , secondary 106, 125
— , furrow 96, 100, 101, 102
— , lip 98 et seq., 106, 107,

111 et seq., 119
Blastula 2, 5, 28, 44, 66, 78, 93,

95 et seq., 99 et seq., 104, 107,

114, 189
Blood 152, 157

— circulation 154, 157

— vessels 158
Bomhinator 116, 130, 134
Bombyx 87

Bone 154, 158

Brain 113, 115, 116, 119 129,

130, 131, 134, 135, 136, 138,

146, 156
Branchial arches 140

— grooves 140

— palps 162

— pouches 103, 140
Brilliant cresyl violet 43
Bufo 22, 79
Butterflies 89

Calcium 9, 10, 13, 14, 20, 21, 34, 72
CaDillaries 158

Carbohydrate metabolism 47,

79, 118
Catalysts 89
Cell fragments 65, 66
Centrifugation of eggs 31, 32,

48, 49, 50, 59, 60, 63
Centrosome 22, 72, 189
Cephalopods 154
Cerebral ganglion 165

— hemispheres 136, 147
Chain molecules 33, 47
Chemical treatment, acids 20

alkalis 20

butyric acid 18, 19

calcium 10

ether 23

lithium 46, 47, 147

saponin 20

salts 20

sodium chloride 15

sodium rhodanide 46, 47

trypaflavin 22, 23, 25

urea 20
Chemodifferentiation 5, 6, 50
et seq., 62, 63, 67, 86, 91, 92,
99, 105, 106 107, 109, 111,
120, 135, 139, 145, 148, 150,
182, 189
Chick 134, 149, 152, 153
Chimera 30, 113, 120, 189
Chordamesoderm 98 et seq., 116
Chordoneuroplasm 58
Chromatin 23, 24, 25, 81, 189

— diminution 68, 69
Chromidia 33, 34, 189
Chromosomes 8, 16, 20, 23, 25,

64, 66, 67, 68, 69, 71 et seq., 189
Cidaris 78
Cinnabar 87 et seq.
Ciona 24
Clavellina 167
Cleavage 2. 5, 16, 19, 20, 28,

29, 34, 35, 39, 64, 66, 67, 91,

92, 93, 190
— , centrifuged eggs 48
— , Dentalium 55
— , insects 70, 92
— . sea urchins 24. 35

208

SUBJECT INDEX

Cleavage, spiral 52

— , Triton 18, 36, 37

— , under compression 35, 36

Coat, surface 103

Colloid particles 43, 44

Competence 116, 132, 138

Competition, physiological 126,

148, 172
Concentration of evocator 145,

146
Connective tissue 158, 173, 174
Constriction of eggs, Amphibia

17, 18, 29, 35, 36, 37, 84
Contact phenomenon 135
Cornea 129, 135
Comification 139
Cortex 5, 22, 48, 49, 60, 62,

119, 182, 190
— , Amphibia 41, 42
— , attraction 10, 50
— , composition 21
— , in fertilisation 13 et seq.
— , structure 34
Cortical factor 105

— field 49, 105

— granules 14

— reaction 13 et seq., 19, 20, 21
Corymorpha 168, 169
Crepidula 24

Crescent, grey 40, 41, 49, 58,

95, 105
— , yellow 58
Crossing, species 75 et seq.
Cumulus oophorus 11, 190
Cuttlefish 39, 40, 154
Cyclopia 47, 131, 136, 190
Cytaster 22, 23, 190
Cytoplasm, acidity 43, 44
— , properties 10, 21, 69
— , structure 28 et seq.

De- differentiation 135, 154, 162,

163, 167, 172, 173, 174, 190
Dendmaster 49
Denervation 173
Dentalium, cleavage 55, 57
— , isolation of blastomeres 56

— , removal of polar lobe 56, 57
— , trochophore 56, 57
Dentine 139
Depolarisation 10, 190
Determinants 35, 64, 190
Determination 46, 47, 49, 50,
54, 60, 61, 84, 108, 111 et
seq., 131 et seq., 174, 177 et
seq., 190

— of bilateral symmetry 41
— , labile 178
Development, course of 1 et

seq., 4 et seq.

Dicentry 22, 25

Differentiation 2, 6, 35, 60, 65,
66, 81, 85, 103, 107 et seq.,
131 et seq., 152 et seq., 157,
162, 163, 165, 167, 174 et seq.,
179, 180, 190

Diffusion 79, 119, 126, 135, 145,
147, 164, 187

Digits 141, 142

Diploid 8, 67, 71, 190

Discoglossus 138

Disintegration 172

Division, cell 1, 8, 9, 35, 36, 66,
67, 173

— , nuclear 8, 23, 24, 35, 64,
66, 67, 76, 78

— , planarians 171

Dominance 126, 170, 171, 172

Dragon fly 69 et seq.

Drosophila, chromosome deficien-
cies 74

— , gene hormones 87 et seq.

Duplicitas cruciata 54

Dyads 8

Ear-field 137, 138
placode 136

— -vesicle 113, 115, 117, 134,
136 et seq., 144, 146

Earthworms, regeneration 161,

164, 169
Echinarachnius 14
Ectoderm 46, 93 et seq., 190
Ectoplasm 12, 58, 65, 190

SUBJECT INDEX

209

Eisenia 161, 169
Electric charge 43
— field 44, 169
Embryo 2, 3, 5, 6, 150
— , secondary 111 et seq.,

120 et seq.
Embryogenesis 92, 190
Enchylema 33, 190
Endo'derm 46, 93 et seq., 190
Endoplasm 58, 190
Endothelium 158, 190
Energy metabolism 65
Entelechy 32, 184, 190
Envelopes, egg 11, 12, 30
Enzymes 11, 34, 47, 51, 60, 61,

89, 90, 119, 190
Ephestia 87, 89
Epidermis 85, 86, 98, 100, 109,

173, 174
Epigenesis 27, 183, 190
Equatorial plane 39, 92, 191
Equilibrium, histochemical 62
Equipotential system 62, 191
Euplanaria 172
Evocator 117 et seq., 135, 145,

146, 147
Evolutio 3

Exogastrula, Amphibia 102, 191
— , sea urchins 46
Explantation 107, 108, 110,

123, 153 et seq., 191
Extracts 135, 154
— , induction with 117 et seq.,

145
Eye 113, 115, 117, 128 et seq.,

146, 149, 152, 156, 165

— colour genes 87 et seq.
cup 128 et seq., 155

— pigments 89
stalk 128 et seq.

vesicle, primary 128 et seq.,

136

Factors, nuclear 64 et seq.
False hybrids 79, 191
Fat 7, 31, 44, 48
Fertilisation 7 et seq., 34, 41,
57, 64

Fertilisation cone 12

— , heterogeneous 22, 23, 191

— membrane 12 et seq., 19,
20, 21

— substances 11
Fertilizin 11
Fibroblasts 158, 191
Fibrous proteins 47

Field, animal 95, 96, 98, 101,

103, 109
— , cortical 49, 105
— , ear- 137, 138
— , electric 44, 169
— , gill- 140
— , gradient- 5, 42 et seq., 63

125, 147, 170
— , organ- 148, 149, 179
— , organisation- 121 et seq.,
137, 142, 145, 147 et seq., 151,
167 et seq., 179 et seq.
— , reaction- 132
— , sub- 149

— , vegetative 95, 96, 101, 103, 108
Fishes 152, 157
Flat-worms 160, 165
Flies 89
Follicle 11, 191
Food substances 126, 154, 157,

172
Foot 149

plate 141

Fore-brain 128, 136, 146
Fore-limb 175, 176, 177, 179, 180,

181
Fragments, egg 10, 12, 16, 23,

57, 66, 82
— , in regeneration 162, 163,

165, 166, 167, 168, 175
Frog 25, 116, 159
— , artificial parthenogenesis

19 et seq.
— , bilateral symmetry 41
— , cortical reaction 15
— , grey crescent 40, 41
— , hybrids 79
Fruit fly 74, 87
Functional stage of development

2, 158 etseq., 182

Raven - Outline Physiology

14

210

SUBJECT INDEX

Fungus 90

Fusion of eggs 30 et seq., 63

— of organ primordia 133, 137,
140, 142, 148

— of regeneration buds 178

Gametes 7, 11, 191

Gamones 11, 191

Gastrula 29, 44, 45, 79, 108,

110 et seq., 191
Gastrulation 93 et seq., 106,

111, 114, 128, 138, 191
— , amphibians 95 et seq., 103
— , sea urchins 93 et seq.
Gelation of protoplasm 15, 22,

26
Gene 5, 64, 65, 71, 74 et seq., 191
— , lethal 74, 75, 82
— ■ hormones 5, 87 et seq., 157

— substances 88, 89
Genetic constitution 139
Germ bands 51, 53, 54, 191

— cells 7, 8, 69

Germinal vesicle 7, 10, 16, 65,

66, 191
Gibbs phenomenon 103
Gill 140, 141, 152, 159

field 140

slits 140

Glycogen 106, 107, 118
Gonad 8, 69

Gradient 42, 104, 169, 170, 191
— , axial 42, 47, 48, 49, 50, 62
— , cortical 49, 50, 62, 104, 105,

125
field 5, 42 et seq., 63, 125,

147, 170

, in Limnaea 47

, in sea urchins 44 et seq.

— , spatial 43

— , surface 43

— , yolk 43, 48, 49, 104, 105, 125

Gravity, influence of 48

Growth 65, 80, 154 et seq., 156,

157, 158, 170, 171, 174, 175

— rate 155

Gut 94, 99, 111, 140, 153
Gynogamones 11, 191

Habrobracon 87, 89
Hand 180

plate 141

Haploid 8, 25, 67, 72, 173, 191
Head, regeneration of 160 et

seq., 170
Heart 153, 154
Hind-limb 175, 177, 179, 181
Histogenesis 2, 92, 150, 192
Hormones 157, 192
Humerus 175, 176
Hyaluronic acid 11
Hyaluronidase 11
Hybridisation 11, 15, 75 et seq.
Hybrids 75 et seq., 192
— , false 79, 191
— , lethal 79
Hydra 167

Hydranth 163, 164, 168, 169
Hydroids 163, 164, 167, 168,

169, 171, 172
Hyperblastula 101
Hypertonicity 19, 20, 23, 25, 26
Hypertrophy 158, 192

Id 34, 35

Illumination 20, 168
Immunological reactions 11
Inclusions of cytoplasm 33, 39
Incompatibility 78, 138
Indicator dyes 43
Induction 5, 6, 111 et seq., 128

et seq., 182, 192
— , assimilative 120, 125, 189
— , contact 114 et seq., 125, 135,

136, 144, 145, 147, 149
— , in regeneration 164, 166,

167, 168, 169
— , progressive 114, 120 et seq.
— shock 20, 41
Inductor 114, 117, 139, 144,

147, 192
— , abnormal 146
Inhibitory influence 164, 170,

171
Insects, cleavage 70, 92
— , egg 39, 40
— , gene hormones 87 et seq.

SUBJECT INDEX

211

Insemination 13
Insertion method 114, 115, 116
Interfacial tension 63, 103
Invagination 93 et seq., 101,

102, 104, 106, 124, 128, 129,

131, 132, 136, 138
Invasion of nerve fibres 174
Iris 129

Irradiation, mesothorium 84
— , radium 23
— , ultraviolet 53, 71
— , X-rays 161, 173, 176
Isolation of cleavage cells 28
et seq., 35, 44, 56, 59, 63, 72
— , physiological 171

Janus green 43
Jaw 139, 158
Jelly, egg 13, 14

Kidney 108, 146, 158
Kynurenin 89

Labyrinth 138

Lateral line sense organs 157
Laws of nature 185, 186
Leg 149

Lens 129, 132 et seq., 136, 145,
146, 155, 178

— epithelium 134

— fibres 134

— groove 135

— placode 129, 134, 135

— proteins 135

— vesicle 129, 132, 134, 135
Limbs 80, 117, 141 et seq., 156,

157
Limb-bud 141 et seq.

— primordia 116, 142, 143, 148,
152, 153, 155

— regeneration 172 et seq.
Limnaea 18, 99

— , gradient-field 47
Lipids 33, 34
Lytechinus 78

Machine theory 183, 184
Magnesium 34

Mammals 11, 152, 174

Man 174

Marginal zone 95 et seq., 98 et

seq., 101, 106 et seq., Ill et seq.
Matrocline hybrids 78, 192
Maturation 8 et seq., 16, 18, 58
Maturity 2
Mechanics, developmental 1,

28, 183
Mechanism 184
Median plane 40, 41, 192
Melanin 86
Melanophores 81, 192
Meridional plane 39, 40, 41, 92,

192
Merogony 11, 66, 82 et seq., 192
— , heterospermic 82 et seq., 191
— , hybrid 82 et seq.
— , parthenogenetic 66
Mesenchyme 46, 59, 83, 84, 93,

109, 129, 140, 141, 192
Mesoderm 98 et seq., 113 et

seq., 120 et seq., 142, 143, 192
Mesonephros 153
Mesoplasm 58, 60, 192
Mesothorium 84
Metabolism 1, 5, 34, 47, 48,
60, 61, 63, 65, 66, 79, 90,
118, 120, 126, 144, 147, 164,
171
— and determination 47, 60
— , axial gradient 43, 48, 171
— , carbohydrate 47, 79, 118
— , protein 47, 89, 118
Metamorphosis 19, 85, 174
Metanephros 153
Methylene blue 43, 171
Microdissection 15, 192
Micromeres 51, 52, 192
Microsomes 25, 33, 34, 65, 86,

119, 192
Migration, cell 81, 92, 105, 106.

128, 138, 161, 162, 173, 182
Mitochondria 33, 65, 192
Mitosis 21, 22, 23, 24, 72, 192
Modes of operation 185 et seq.
Modulation 154, 192
Molluscs 10, 15, 31, 92

212

SUBJECT INDEX

Molluscs, cleavage 51
— , polar lobe 55 et seq., 66
Monaster 19 et seq.
Monospermy 14, 193
Morphailaxis 165 et seq., 175,

193
Morphogenesis 174, 193
Mosaic 6, 28, 60, 62, 91, 137,
149, 151

— eggs 32, 63

— stage 151 et seq.
Moth 87
Motility 99

Motor nerves 157, 174

— neurones 156

Mouth 94, 98, 139, 146, 147, 166

— plate 139

Multiplicity, extensive 3, 4, 31,
38, 39, 63, 182, 183, 191

— , intensive 3, 4, 32, 34, 39, 63,
183, 188, 192

— , spatial 2 et seq., 5, 27, 28,
33, 34, 35, 50, 63, 91, 106,
126, 128, 149, 182, 183, 184

Muscle 108, 157, 158, 173, 176,
181

Mussel 23, 24

Mutants 87 et seq., 193

Mutation 75, 87, 89, 90, 193

Mytilus 23, 24

Narcotics 164, 168
Negativity, wave of 14
Nematodes 33, 68
Neoblasts 161, 162, 193
Neo-epigenesi'S 28, 193
Neo-evolutio 3, 193
Neo-preformation 28
Nereis 10, 14
Nerve 156, 157, 173, 174, 175

— cord 164

— fibres 156, 174

Nervous system 156 et seq., 174
Nervus ischiadicus 175
Neural crest 138, 140, 147

— folds 80, 81, 98, 138

— plate 98 et seq., 109 et seq.,
128, 130, 131, 138, 145

Neural plate, determination

111 et seq.
, secondary 112

— tube 98, 111 et seq., 128, 136
Neurospora 90

Neurula 109, 112 et seq., 132,

138, 193
Neurulation 98 et seq., 102, 106

et seq., 128, 146, 193
Neutral red 43
Newts 22, 139, 155, 159, 172,

175
— , exogastrulation 102
— , fusion of eggs 30
— , heterospermic merogony

84 et seq.
— , hybridisation 79 et seq.
— , isolation of blastomeres 29,

35
— , pronuclei 17 et seq.
— , retarded nucleation 35

et seq.
Nile blue hydrochloride 43
Notochord 58, 59, 98, 100, 103,

108, 111 et seq., 120 et seq.,

126, 193
Nuclear factors 64 et seq.
Nucleoplasm 10, 11, 22 et seq.,

65, 193
Nucleotides 118

Nucleus 3, 5, 7, 8, 23, 25, 26, 47

— of oocyte 8, 10, 19 et seq., 58
— , role in development 34 et

seq., 64 et seq.

Oil 33

Olfactory organs 135 et seq.

— pits 117, 134, 136, 144, 145,
146, 156

— placodes 136
Oligonucleotides 119
Omentum 152, 193
Omnipotent 109, 162, 193
„Onde de g^lification" 26
Oocyte 7, 8, 9, 10, 40, 58, 65,

66, 119, 193
Oogonium 7, 193
Optic nerve 129

SUBJECT INDEX

213

Orderliness 186 et seq.
Organ-field 148, 149, 179

— primordia 2, 5, 6, 79, 95,
99. 100, 122, 128 et seq., 151

et seq.
Organisation 128 et seq., 164,
166, 167, 168, 169, 171, 193

— centre 113 et seq., 131, 193

— field 121 et seq., 137, 142,
145, 147, 148, 149, 151, 167
et seq., 179, 180, 181

Organiser 6, 112 et seq., 167,

168, 170, 181, 193
Organisine 117, 125
Organogenesis 128 et seq., 193
Orienting rotation 41
Osmosis 13, 186, 187
Osmotic pressure 20
Ovary 7, 9, 11, 40, 193
Oxidases 51, 60, 85, 193
Oxidation 65, 118
Oxygen 159, 164, 171

Paracentrotus 30, 76, 77, 78, 83
Parthenogenesis, artificial 18

et seq., 66
Parthenogenetic merogony 66
Patrocline hybrids 77, 193
Penetration of sperm 12, 14 et

seq., 22, 23, 41
Peristome 167, 193
Perivitelline cavity 12, 13, 193

— fluid 19, 41

Permeability 5, 10, 21, 44, 118
Peroxidases 60, 61, 193

pH of cytoplasm 43, 44, 63
Pharynx 164 et seq., 194

— sheath 166
Phosphatase, alkaline 107
Phosphate bonds 65
Phosphatids 33, 34
Phosphorylation 65
Pigment 87, 89, 91

— cells 80, 81, 85, 86. 87

— epithelium 129, 131
Pigmentation 89, 140, 173, 176
~, Amphibia 80, 84, 85, 86

— , eggs 40, 41

Planaria 161, 163, 164, 166,

167, 170
Planarians, regeneration 160

et seq., 170 et seq., 173
Plasmagenes 86, 194
Platycnemis 69, 70
Pleurodeles 116
Pluteus 29, 30, 44, 45, 76 et

seq., 83 et seq., 95, 194
Polar bodies 8 et seq., 39, 48,

55, 99, 194

— lobe 55 et seq.
Polarisation microscope 33
Polarity 4, 39 et seq., 63 91,

92, 134, 137, 140, 143, 'l60,
161, 182, 194
Pole, animal 10, 39 et seq.,
50 et seq., 189

— -plasms 50 et seq., 60
— , vegetative 39 et seq., 50

et seq., 69, 195
Polycelis 160, 165
Polychaetes 10, 14, 31, 162
Polypeptides 33, 34
Polyploids 67, 194
Polyps 163, 167, 168, 169, 171,

172
Polysaccharides 11, 14
Polyspermy 14 et seq., 17, 194
Posttrochal region 57
Potassium 10, 44
Potency 6, 44, 45, 54, 59, 60,

62, 63, 92, 107, 131, 132, 137,

140, 142, 144, 145, 147, 148,

149, 151, 194
Potencies, differentiation 107

et seq.
— , histogenetic 107 et seq.
— , topogenetic 101 et seq., Ill
Potential, electric 169
Predetermination 136, 139,

147, 194
Preformation 3, 27, 34, 194
Pretrochal region 57
Proliferation 139, 151, 170,

173, 176
Pronucleus 9, 15 et seq., 22 et

seq., 194

214

SUBJECT INDEX

Proteins 47, 119

— in cytoplasm 33

— in egg cortex 21, 34

— in fertilisation membrane 14

— in yolk 7, 44, 48

— , metabolism 47, 89, 118
— , structure 47
— , synthesis 34, 47, 65
Protoplasm, structure 33
Psammechinus 76, 77, 78, 83, 94

Rabbits 152, 159

Rana 40, 79, 116, 132, 133

Rats 152

Reaction field 132

— system 114, 120, 135, 144,
145, 147, 194

Reactivity 145

Realisation of nuclear factors

64 et seq., 92
Redox dyes 171

— potential 43
Reduction division 8, 194
Regeneration 2, 6, 126, 151, 152,

158, 160 et seq., 182, 194

— blastema 162, 165, 166, 172,
173, 174

— bud 160, 162, 169, 172, 174,
177, 178, 179

— cells 161, 162

— cone 179

— , total 172, 174, 175
Regional differentiation 115,

119
Regulation 32, 63, 67, 121 et
seq., 148, 151, 152, 184, 194

— eggs 32, 63

— , in fertilisation 20 et seq.
Removal of organ primordia

151 et seq., 175
Reorganisation 163, 165, 166,

194
Reproduction, sexual 7
— , vegetative 1, 171, 195
Respiration 79, 159
Respiratory quotient 118
Restitution 160, 162, 182
Retarded nucleation 36, 37

Retina 129, 131, 132, 147, 149,

178
Reversal of polarity 161, 166
— of symmetry 143
Revitalisation 79
rH 43, 63
Ribonucleic acid 25, 33, 34, 65,

79, 107, 118, 119
Ribonucleoproteins 119

Sabella 162, 165, 166, 168

Salts 33

Sea urchin 10, 183

artificial parthenogene-
sis 18, 19, 21, 25, 26
cell size and nuclear size
66

centrifugation 49
cleavage 44
— under compression

35, 36
combinations of blasto-
meres 45
double fertilisation
71 et seq.

fertilisation 11, 12, 13,
14, 15, 16

fusion of eggs 30
gastrula 44, 45
gastrulation 93 et seq,
gradient fields 44 et seq.
heterogeneous fertili-
sation 23, 24
heterospermic merogony
82 et seq.

hybridisation 76 et seq.
influence of chemicals
46 et seq.

isolation of blastomeres
28, 29, 44

skeleton of plutei 77,
83, 84, 87, 95
metabolism 47
parthenogenetic mero-
gony 66

pluteus 29, 30, 44, 45,
77, 83, 95

SUBJECT INDEX

215

Sea urchin, potencies of

blastomeres 45
, structure of cytoplasm

33, 34
Segmentation nuclei 5
Segments 161, 162, 165, 166,

169
Segregation 44
Self-differentiation 108, 109
Senility 2
Sense organs 156
Sensory nerves 174

— neurones 156
Serological methods 135
Skeleton 172, 173, 175, 176,

181
Skin 174, 176, 181
Snail 17, 62, 99
Sodium 15, 19, 46, 47
Somatoblasts 51, 52, 53, 54,

56, 194
Somites 98, 100, 103, 111 et

seq., 120 et seq., 126
Specificity in induction

145 et seq.
Sperm 1, 4, 5, 7 et seq., 41, 195
Spermatid 8
Spermatocyte 8
Spermatozoon 7 et seq.
Sphaerechinus V6, 78
Spinal cord 115, 116, 146, 156

— ganglion 156

Spindle 8, 10, 16, 22, 25, 52, 72

Sponges 99

Spongioplasm 33, 195

Starfish 9, 10

— , activation 19, 21

— , fertilisation 12

— , maturation divisions 9

Sterols 118

Stimulation, local 144, 164, 175

bcomodaeum 94, 195

Stratification 31, 48

Strongylocentrotus 13

Sub-fields 149

Substances, determining 5, 6,

50 et seq., 91, 182
— , fertilisation 11

— , gene 88, 89

— , inducing 117, 145

— , organ-forming 50, 60

Suckers 139

Sulphydryl compounds 34, 65,

107, 118, 119
Superficial cleavage 70
Symmetry, bilateral 4, 39 et

seq., 63, 91, 92, 94, 95, 134,

182, 189
— , determination of 41

— of auditory organs 137

— of limbs 142 et seq.
Syncytium 83, 195
Synkarion 64, 195
Synthesis, ribonucleic acid 79
— , protein 34, 47, 65

Tail-bud 113, 151, 152

— , regeneration 172 et seq.

Taste-buds 157

Teeth 139

Teloblasts 53, 195

Temperature, influence of 10,

20, 53, 77, 84, 146, 155, 163,

164, 168, 171
Tendencies 101, 105, 144, 147
— , differentiation 108
Tendons 158
Tension 158
Tentacles 167
,,Territoire de regeneration"

175
Testis 8, 195
Tetraster 72, 73, 195
Thoracic segments 165, 166,

168
Threshold 145
Tissue culture 154, 158

— differentiation 150, 154, 182
Toad 22, 116

Topc^enesis 2, 5, 6, 91 et seq.,
115, 144, 182, 195

Trabeculae 158

Transplantation 80, 81, 85, 86,
87, 88, 107, 108, 109, 128 et
seq., 152, 155, 166, 167, 169,
170, 173, 175 et seq., 195

216

SUBJECT INDEX

Transplantation, heteroplastic
110, 111, 116, 120, 133, 134,
140, 142, 155, 167, 191

— , xenoplastic 116, 134, 136,
138, 139, 195

Triploid 71, 72, 195

Triton 18, 22, 37, 79, 80, 84
85, 101, 110, 111, 112, 116,
122, 123, 133, 139, 140, 141,
142, 143, 144, 146, 152, 176

Triturus 80, 81, 85

Trochophore 56, 195

Trophic influence of nervous
system 157

Trypaflavin 22, 23, 25

Tryptophane 89

Tubifex 50 et seq., 57, 60

Tubularia 163

Ultracentrifugation 34, 195
Ultraviolet light 53, 71
Urodeles 100, 134, 136, 138,
139, 142, 143, 144, 174

Vascular system 158 et seq.
Vermilion 87 et seq^
Veins 159

Viscosity 10, 21, 33, 83, 84
Vital force 184

— stains 99
Vitalism 184, 185
Vitamins 61, 195
Vitelline membrane 12, 14
Vitreous body 130
Vitro, in 153, 195

Wasp 87, 89

Worms 15, 33, 50, 51. 92, 162,

169
— , polar lobe 55 et seq.

X-chromosome 74, 195
X-rays 161, 173, 176

Yolk 7, 33, 39, 44, 48, 154
factor 105

— -gradient 43, 48, 89, 105
plug 96

sac 152

— , shifting 44

Zygote 5, 7, 195

— nucleus 17, 37, 39, 64, 70,
71, 91, 195