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THREE LECTURES

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EXPERIMENTAL EMBRYOLOGY

OXFORD UNIVERSITY PRESS
LONDON EDINBURGH GLASGOW NEW YORK
TORONTO MELBOURNE CAPE TOWN BOMBAY
HUMPHREY MILFORD

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THREE LECTURES

ON

BAPERIMENTAL
EMBRYOLOGY

BY

J. W. JENKINSON, M.A., D.Sc.

SOMETIME UNIVERSITY LECTURER IN EMBRYOLOGY
AND FELLOW OF EXETER COLLEGE, OXFORD

WITH A BIOGRAPHICAL NOTE
BY

R. R. MARETT, M.A., DSc.

FELLOW OF EXETER COLLEGE, OXFORD

OXFORD
AT THE CLARENDON PRESS
1917

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IN MEMORY

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PREFACE

THE manuscript of these lectures was found amongst
my husband’s papers. He had, I know, intended to
publish them, but had laid them aside to await final
revision before going to press. In these circumstances
the best course seemed to be to publish the text
exactly as he had left it, while adding a few illustra-
tions to take the place of the lantern slides used at
the lectures when these were delivered at University
College, London.

I am glad to have the opportunity of acknowledging
here my deep indebtedness to all who have so kindly
helped me in seeing this book through the Press. My
thanks are especially due to Miss Kirkaldy, to Mrs.
Cuthbert Baines, to Dr. Marett, to Professor Ramsay
Wright, and to the Delegates of the University Press,
who one and all by their valuable help and kindly
sympathy have made it possible for me to carry out
one of my husband’s last wishes.

CONSTANCE JENKINSON.

OXFORD.

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CONTENTS

BIOGRAPHICAL Notre. By R. R. MARETT

CHAPTER I

PAGE

Xl

INTRODUCTORY: GROWTH. STRUCTURE OF THEGERM-CELLS Il

CHAPTER II
CLEAVAGE , i : : : ; : ‘
CHAPTER III
DIFFERENTIATION
LITERATURE

APPENDIX. CHARTS DEALING WITH TROUT LARVAE

INDEX

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~ 123

JOHN WILFRED JENKINSON

To remember is, during this devastating war-time, almost
the sole part left to Oxford. ‘How doth the city sit solitary
that was full of people! how is she become as a widow!’ Of
the fallen, most will be remembered as gallant youths who
laid down life while it was yet all promise, a garden of
blossoming hopes. But some were senior men, with their
faculties already mature, and their life-work in active process
of achievement. Among such elder sons of the University
who fought and are gone was John Wilfred Jenkinson. His
name will live on in his written works; for at least some-
thing of the rich fruitage of his mind has been harvested,
though how much more has been lost can never be known.
Yet a man is more than his books. So let these few lines
be dedicated to the memory of the man himself—by one
who, as his tutor first and his colleague afterwards, learnt
to know him for the noble spirit that he was, and to admire
and esteem him accordingly.

A contrast is sometimes drawn between the man of thought
and the man of action; but it is possible for these diverse
characters to unite harmoniously in the same individual.
Jenkinson’s outstanding quality was strength of purpose.
Nothing in the form of work could daunt him. His was the
ascetic, the morally no less than physically athletic, tempera-
ment that rejoices in hard, clean striving for its own sake.
Such a mind must be up and doing. Its activities will,
therefore, naturally issue in research, in bringing truth to
light. But Jenkinson was likewise a thinker in whom the
purely contemplative mood craved for satisfaction. The pre-
condition of his research must be some vision of the whole.

xl BIOGRAPHICAL NOTE

As it was, the manner of his education was such as to
minister conjointly to both sides of his nature, to foster in
him the man of science and the philosopher at once and
together.

The Oxford School of Jiterae Humaniores is sometimes
accused of trying to be all things to all men; and this
impeachment is perhaps best met by the plea that such
a policy would seem to answer. Here was a man who in
his first term of ‘Greats’ attended biological courses at the
Museum, and that with his tutor’s blessing. As all roads ©
lead to Rome, so every intellectual avenue might be held to
lead to philosophy. And thus it turned out with Jenkinson.
He might devote time that he could ill spare to those special
problems of biology which were to mean so much for him in
after life. He might even be so deeply impressed by the need
in that context of empirical methods, of a reasoning resting
solidly on observation and experiment, as to be unduly
suspicious of the dialectical flights to which philosophy is
prone. But a mind so broad and so thorough could not
ignore the further issues which the biological theory of
development involves. How Becoming in general is related
to Being was the question that soon dominated his attention,
in those early years when he was still finding himself, still
seeking to evolve a sense of cosmic direction.

There was a College society, then in its heyday, which met
for purposes of philosophical discussion. Jenkinson was one
of the leading spirits in it. He had not yet, it is true,
acquired that power of lucid utterance which was afterwards
to make him one of the most impressive of University
lecturers. But his very earnestness brought him to the front
in these youthful debates. In the myriad processes of life
which it was his passion to study he divined the workings of
law. The organic world was for him no welter of aimlessly
competing forms, chance-begun and chance-ended. Somehow
there must be determination towards an end, a movement

BIOGRAPHICAL NOTE xiii

and growth responsive to a central principle of order; or
why the human instinct for truth, why science at all? It
was typical of Jenkinson that he never strayed from this
path of philosophy, once his feet were firmly planted on it.
With him thought and character were so much at one that
he had no need to cast about for a leading. Singly-deter-
mined as he was in himself towards a life of duty, he saw his
world as a counterpart in which every natural process tended
towards the ultimate fulfilment of a purpose.

A paper of his exists which well deserves publication if
only because it is so much more than a clever arrangement of
words, being the expression of a faith by which a man has
lived, and died, nobly. It may draw much of its inspiration
from Aristotle and Hegel; but is nevertheless original in the
fullest sense as proceeding from one who thought for himself
—who owned whatever he affirmed. The paper in question first
examines the postulates to which the inductive sciences owe
their power of advance. In biology no less than in physics
strict causation must be assumed. Jenkinson, indeed, is at
the level of science so thoroughgoing a determinist that he
deprecates the attempt to resort in a biological context to
teleological explanations of any kind. Material and efficient
causes are the only necessary conditions which science as such
has any need to recognize. On the other hand, materialism
cannot suffice as a philosophy. ‘There is one fact which the
materialist forgets to analyse, and which materialism abso-
lutely fails to explain, which is indeed the hardest to under-
stand of all, and that is knowledge; and, with knowledge,
those other facts of self-consciousness, feeling, and will.’
He concludes, with the idealist, that ‘the phenomena which
constitute the object of knowledge ’—in a word, nature and
its laws—‘are the creation of the mind itself’. He goes on to
urge that ‘this mind must be regarded not so much as some-
thing outside individual minds, differing from them in capacity,
but related to them as they are to each other; but rather as

xiv BIOGRAPHICAL NOTE

an inner harmonizing activity between them, of which they
are so to speak the expressions, and in which they are
included’. In such a faith he finds a common justification
for the Jaborious investigations of the man of science and for
the speculative efforts of the philosopher. Herein, too, for
both of them, lies the secret of happiness—if they be content
to ‘strive and hold cheap the strain; learn, nor account the
pang; dare, never grudge the throe’. So Jenkinson himself
strove and iearnt and dared, and no one who knew him can
doubt that he lived and died happy.

The time at length came when, his course of the humanities
accomplished, he must qualify for that scientific career on
which he had set his heart. The omens were by no means
propitious. As a schoolboy at Bradfield he had botanized
with zeal, some of his finds being recorded in Druce’s Flora
of Berkshire. At Oxford, again, though as Scholar of Exeter
he had taken the usual classical schools, he was allowed, as
has been mentioned, to attend a few biological classes by way
of an intellectual luxury. Such being his entire record as
a student of science, he must evidently start from the very
bottom of the ladder as regards the special instruction in
biology which he now required. Nor can it be said that Oxford
makes adequate provision in the way of endowments for
those who, having obtained an education in general culture,
seek after graduation a technical training in some particular
branch of learning or research.

Jenkinson, however, was the last man to he turned aside
from an ideal aim by material considerations. Working hard
and spending little, he studied zoology for some time at
University College, London, under the late Professor Weldon.
In the latter he found a kindred spirit—a man whose whole
nature found its satisfaction in the pursuit of knowledge.
And so he continued, immersed in research, and, as his letters
to his friends at Oxford showed, perfectly happy in his
chosen vocation, until at length he was appointed to the

BIOGRAPHICAL NOTE XV

teaching staff of the Department of Comparative Anatomy
at Oxford. Such junior posts carry with them a mere pit-
tance in the way of remuneration; but, fortunately for the
University, there is no lack of young men of first-rate talent
ready to serve in the ranks of science on a slender ration.
It may even be that hard conditions help to make the
pioneer. Certainly, for a man of Jenkinson’s strenuous and
disinterested temper, the way was clear ahead, once a definite
function was assigned him,

Six years after taking his Bachelor’s degree he published,
as the firstfruits of his researches, a study entitled ‘ Early
Stages of the Development of the Mouse’;! this being partly
the result of work done at Utrecht in the laboratory of the
illustrious Dutch zoologist, the late Professor Hubrecht.
Five years later he became Doctor of Science, a distinction
awarded at Oxford for writings held to display originality in
some high degree. Almost from the first his chief interest
had lain in embryological questions, and it was appropriate
that, in the year after he had received his doctorate, the
post should be created for him of University Lecturer in
Comparative and Experimental Embryology. A few years
later, in 1909, his College, Exeter, on the staff of which he
had already been serving as lecturer, was proud to elect him
to its sole Research Fellowship, which, tenable as it is for but
a limited term of years, can boast a long list of distinguished
holders. This year was marked by the publication of his
Experimental Embryology, the first comprehensive English
text-book on the subject, and one that is not likely soon to be
superseded. In 1913 another text-book, of no less excellent
quality, appeared under the title Vertebrate Embryology. So
much must suffice as a bare record of the steps by which he
was steadily mounting upwards in the scale of usefulness and
honour.

Then the war broke out. Jenkinson was now past forty years

1 Quart. Journ. Micr. Sci., 1900.

XV1 BIOGRAPHICAL NOTE

of age. He was married, and most happily married, his wife
taking a keen and active interest in his scientific pursuits.
He was recognized not only in his own University but in the
world at large as a foremost authority in regard to his special
subject. Yet, though on so many grounds he might have
rightfully disregarded the call to arms, he did not hang back
fora moment. Soon after the declaration of war he enrolled
himself in the Oxfordshire Volunteer Training Corps, and in
the following January accepted a commission as Lieutenant
in the 12th Battalion of the Worcestershire Regiment, being
gazetted Captain three months later. He was one of six
officers specially selected for service in the Dardanelles, and
left for Gallipoli early in May. Upon landing there he was
attached to the 2nd Battalion of the Royal Fusiliers; and,
after a little more than a week’s experience of the trenches,
he took part in the general advance of June 4, and fell in
action on that day.

Of such an end one can only say that it was worthy of his life.
He had always given the best of himself to some ideal cause,
whether it was that of science or of his country. This, indeed,
was the very secret of his charm for those who were his
personal friends—that he was utterly single-natured, simple
and strong, at one with the reason and law which he instine-
tively discerned in the seeming chaos of nature and human
life. Oxford will remember him sadly, yet proudly, as a true
son of hers, who, thanks to her ancient kindly discipline of
mingled work and play, learnt ‘to philosophize without soft-
ness ’—at once to love wisdom and to play the man.

R. R. MARETT.

CHAPTER I

INTRODUCTORY: GROWTH. STRUCTURE OF
THE GERM-CELLS

EMBRYOLOGY is the study of development, and when used
in its widest sense signifies the inquiring into the reproduction
of specific form in the individual organism, by whatever means
that reproduction may be brought about, whether by the
development of a single cell, of a bud, or by the regeneration
of a lost part or parts.

But in a narrower and perhaps commoner sense Embryology
is the investigation of the first of these processes, of that
series of changes by which there is produced from a single
cell a new organism which is like the parents that gave it
birth. This cell is usually, though not always, a fertilized
egg-cell produced by the union of the two germ-cells, the
ovum and the spermatozoon ; while these cells therefore are
the material basis, the development of the product of their
union is the mechanism, of inheritance.

To the inquirer into the phenomena of development two
methods are open. Either he may observe and describe the
sequence of changes in as many forms of animals as possible—
and in that way Comparative Embryology has been built
up—or he may, looking upon development as one of the func-
tions of the organism, add to observation experiment with the
deliberate intention of discovering the causes of each step and
so of the whole process, of establishing general laws expressing
the relation between the various antecedents—given in the
initial structure of the germ-cells and in the external environ-
ment—and their consequents, under which general laws fresh
particulars may be subsumed, by which they may be explained,
and from which predictions may be made. This is indeed the

19638 B

2 INTRODUCTORY J

aim and scope of Experimental Embryology, and our duty in
these lectures will be to discover what progress recent research
has made towards the achievement of that ideal.

In development three processes may be discerned, quite
distinct from one another but taking place concurrently.
These are growth, or increase of size, nuclear and cell-division,
and differentiation or, as Herbert Spencer defined it long ago,
increase of structure.

A few well-known examples will serve to illustrate this.

The unripe but full-grown ovum of the Sea-urchin (Strongy-
locentrotus lividus) is a minute spherical body, of a faint orange
colour due to the presence of a pigment at the surface (Fig. 1).
The large nucleus is excentrically placed on that side on which
there is a passage—the micropyle—in the external jelly mem-
brane. After maturation, the female pronucleus, much smaller
than the original nucleus, lies at first excentrically under the
micropyle, into which the polar bodies have been extruded,
but later wanders from this position. At the same time the
pigment is withdrawn from the larger part of the surface, and
concentrated in a superficial zone which is placed—usually
though not always—subequatorially, that is parallel to the
equator and nearer the vegetative pole. The equator is of
course the plane passing through the centre of the egg and at
right angles to the axis, while the axis is the line passing
through the micropyle and the centre of the egg, the micro-
pyle end of the axis being known as the animal, the opposite
as the vegetative pole. We shall have occasion to see later on
that this polarity of the ovum depends also upon the intimate
structure of the cytoplasm.

The egg is now ready for fertilization. Shortly after
fertilization it begins to segment, and the planes of division
pass through the egg-substance in a perfectly definite and
regular way. ‘The first division is meridional (including the
egg-axis), and therefore separates the egg into two equal
blastomeres, each of which has a similar share of the large
unpigmented region of the animal hemisphere, the pigment-
ring, and the smaller unpigmented region around the vege-
tative pole (provided, of course, the ring has its usual

Fig. 1—Normal development of the sea-urchin Strongylocentrotus
lividus. (After Boveri, 1901.)

The animal pole is uppermost in all cases, and in the first two figures
the jelly with the canal (micropyle) is shown.

a, primary oocyte, the pigment is uniformly peripheral.

b, ovum after extrusion of polar bodies. The pigment now forms
a subequatorial band. The nucleus is ex-axial.

c, d, first division (meridional).

e, 8 cells, the pigment almost wholly in the vegetative blastomeres.

J, formation of mesomeres (animal cells) by meridional division: the
vegetative cells have divided into macromeres and micromeres,

g, blastula. h, mesenchyme blastula.

i, j, k, invagination of the pigmented cells to form the archenteron of
the gastrula. In j the primary mesenchyme is separated into two
groups, in each of which, in &, a spicule has been secreted. In k the
secondary, pigmented mesenchyme is being budded off from the inner
end of the archenteron.

B2

4 INTRODUCTORY I

subequatorial position). The second divisions, or more properly
the two divisions of the second phase, are again meridional,
and there are now four blastomeres, each of which has a
similar share of the three regions of the egg. But at the
next cleavage, which is equatorial, four unpigmented animal
cells are separated from four vegetative cells, each of which
has one quarter of the pigmented ring and of the smaller
unpigmented area.

The direction of the divisions of the fourth phase is different
in the animal and in the vegetative hemispheres. In the
former it is meridional, producing a ring of eight cells, while
in the latter it is latitudinal (parallel to the equator) and
unequal, resulting in four large blastomeres (macromeres),
which take the pigment, and four small ones, the micromeres,
round the vegetative pole.

Division proceeds with regularity and synchrony, at least
in some regions of the ovum, for some little time yet, but we
need not follow the details. Eventually the divisions become
irregular. The final result of segmentation is the blastula, or
hollow sphere of cells, disposed in a single layer round the
central blastocoel or segmentation cavity. The cells are
ciliated, and the blastula escapes from the egg-membrane.
In the blastula the same three regions are present as in the
unsegmented egg, the large unpigmented, the pigment ring,
and the small unpigmented. Segmentation has therefore
merely cut up the unlike material of the egg into small
pieces, and beyond the segmentation cavity and the cilia
there has been formed no new structure, there has been no
differentiation.

The next step is the development of the primary mesen-
chyme. One by one the cells of the small vegetative un-
pigmented area become amoeboid and migrate into the
blastocoel. There they quickly arrange themselves in two
groups, of which one lies upon the right, the other upon the
left, of the future median plane. Each group secretes a tri-
radiate calcareous spicule, which is the rudiment of the
skeleton of the Pluteus larva. The two groups are connected
by two curved lines of cells, one of which, on the future

I GROWTH OF THE GERM-CELLS 5

ventral side, consists of a single cell-row, the other, dorsal, of
two or more rows. The embryo is now visibly bilateral.

By the immigration of the primary mesenchyme the pig-
mented cells are naturally brought down to the vegetative
pole, and they are now invaginated into the interior. The
inner tube so formed is the archenteron, the aperture of
invagination the blastopore. The archenteron contains the
material for the body cavity and water-vascular system and
the alimentary canal, the blastopore persists as the anus.
Cells are budded off from the inner end of the archenteron to
form the secondary mesenchyme, and then this inner end
enlarges, divides into two, and these are nipped off as the
right and left coelom-sacs. Each of these later divides into
three—pre-oral coelom, hydrocoel, and posterior coelom ; but
that is a process which we cannot follow here.

The rest of the archenteron is now the gut. It quickly
becomes divided into the fore-, mid-, and hind-guts. It is
inclined towards one, the ventral, side, where the fore-gut
soon unites with an ectodermal depression, the stomodaeum.
By perforation at the point of union the mouth is formed.

Meanwhile the ventral or oral side has become flattened
and square, the opposite dorsal side convex, and the ‘ prism ’
form is attained. A sense-organ—a tuft of long cilia borne
by a patch of thickened ectoderm—is developed at the
anterior end.

The edges of the square oral side now become thickened,
while their cells put out long cilia, so producing the cireum-
oral ciliated ring, by means of which the larva swims. The
four corners of the ring are then pushed out into the four
primary arms of the Pluteus, two anterior or antero-lateral,
two posterior or postero-lateral, and each is supported by
a rod of the skeleton. Of the three radii of the primary
triradiate spicule, one grows into the postero-lateral arm on
each side, one forms a horizontal bar reaching to the middle
line below the gut, and the bar supporting the antero-lateral
arm is a branch of this. The third radius passes dorsally to
the convex side, and becomes the apical or body rod. Its
extremity is swollen and club-shaped. The arms and their

6 INTRODUCTORY I

supporting skeletal rods quickly lengthen, and the young
Pluteus assumes its ‘easel’ shape (Fig. 2). Into the later deve-
lopment of additional arms, ciliated epaulettes and so on, and
into the formation of the body of the urchin on the left side
of the Pluteus, we need not stay now to inquire.

As a second example we shall take another type whose egg
has been the subject of frequent and indeed classical experi-
ments, the Frog.

The full-grown but unfertilized egg of the Frog is a spherical

Fig. 2.—Pluteus of Echinus microtuberculatus from in front and from
the side. (After Boveri, 1896.)

body, with a polarity which depends on the disposition of
yolk and cytoplasm, on the arrangement of the pigment, and
on the position of the nucleus. The egg is telolecithal; that
is, the yolk is so disposed that the yolk-granules on one side
are larger and more numerous, on the other side smaller and
less numerous, and consequently the cytoplasm more abundant
on the latter, less so on the former side; the distinction between
the two being, however, not sharply marked but gradual. There
is in fact a gradual increase of cytoplasm in passing from one

1 GROWTH OF THE GERM-CELLS 7

side to the other, a gradual increase of yolk in passing in the
opposite direction. Since the egg is spherical one line, which
is different from all others, may be drawn through the centre
of the protoplasmic portion at the surface, the centre of the
ege itself, and the centre of the yolk portion ‘at the surface.
This line is the egg-axis, and its ends or poles are obviously
unlike, the protoplasmic pole being known as the animal, the
yolk-pole as the vegetative. The equator of the egg is the
plane passing through the centre at right angles to the axis.
It will be evident that since the cytoplasm and the yolk are
symmetrically distributed round the axis, the egg may be
divided into precisely similar halves by any plane that
includes the axis, but by none other. In other words, the egg
is radially symmetrical about its axis.

This radial symmetry is further emphasized by the arrange-
ment of the pigment, which is deposited in the form of minute
granules in a superficial layer in the animal portion and
extends some little way, to a variable extent in different eggs,
below the equator into the vegetative hemisphere. The
boundary between pigmented and unpigmented portions is
thus a circle parallel to the equator. Lastly, the nucleus—
the germinal vesicle in the immature egg, the female pro-
nucleus in the mature egg, and the two pro-nuclei in the
fertilized egg—lies in the axis, but excentrically, in the animal
hemisphere.

As a result of fertilization, however, this primitive radial
symmetry is lost, and replaced by a bilateral symmetry, for,
opposite the point of entrance of the spermatozoon (which is
somewhere in the animal hemisphere), there appears on the
border of the pigmented area a grey crescent (Fig. 3), due to
the disappearance of the superficial pigment into the interior.
The egg is now divisible into similar halves only by a plane
including the axis and passing through the middle point of
the grey crescent, and about this plane it is now bilaterally
symmetrical.

We shall see that this plane of symmetry of the fertilized
but unsegmented egg is approximately coincident with the
median plane of the embryo, since the side of the grey

8 INTRODUCTORY I

crescent becomes dorsal, the opposite side (on which the
spermatozoon had entered) ventral, while the anterior end is
just above the animal pole, the posterior end therefore just
below the vegetative pole.

Segmentation now occurs, the divisions of the first few
phases passing through the egg-substance in a_ perfectly
regular and definite way, the first being meridional, the

Fig. 3.—Formation of the grey crescent in the frog’s egg (R. tem-
poraria). A,B from the side; c,D from the vegetative pole. In A,
there is no crescent, in B, D a part of the border of the pigmented area
has become grey.

second meridional and at right angles to the first, the third
latitudinal, the fourth meridional, the fifth latitudinal. This
is the typical sequence, but variations from this radial type
are frequent. Subsequent divisions are irregular, tangential
divisions occur, and a segmentation cavity is formed. It is
noticeable from the very beginning that the animal region of
the egg divides more rapidly than the vegetative region, owing
to the retarding influence of the yolk. Hence at the end of
cleavage the animal hemisphere consists of small cells with

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BS z

¥ eonse! a

R
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ttal sections through the fro

Sag
and closure of the blasto

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10 INTRODUCTORY I

small yolk-granules disposed in about four layers in the roof
of the segmentation cavity, the vegetative hemisphere of large
cells with large and abundant yolk-granules in the floor of the
segmentation cavity. The distinction between the two regions
is not however abrupt, but gradual: about the equator are
cells of intermediate size and structure. The superficial animal
cells receive the pigment. The segmentation cavity lies in the
animal hemisphere. Apart from the formation of this cavity
there has been no differentiation during cleavage: the unlike
material of the ovum has merely been cut up into unlike ©
pieces, the characters of which in each region depend directly
on the structure of that region in the unsegmented egg. But
after cleavage comes differentiation, and the first step in this
is the formation and closure of the blastopore, during which the
material for the germinal layers is laid down, and a new cavity,
the archenteron, developed (Fig. 4). The blastopore is formed
and closed bilaterally, since the dorsal lip appears first (on the
side of the grey crescent, just below the equator), the right
and left lateral lips next, the ventral lip last, and since the
overgrowth of the fold of pigmented cells is most rapid at the
dorsal, least rapid at the ventral, and at an intermediate rate
at the lateral lips in between. The archenteron, the cavity
developed between the blastoporic fold and the yolk which it
covers up, is therefore also most extensive below the dorsal
lip, that is, anteriorly. As the yolk-cells are pushed into the
segmentation cavity the latter becomes obliterated, while the
archenteron is enlarged. From its thin roof are formed
the notochord, the dorsal mesoderm, and the roof of the gut ;
from its thick floor (the yolk-cells), the ventral mesoderm and
the floor of the gut.

The blastopore, which has meanwhile been reduced to a
small circle, now closes by approximation of its lateral lips,
the yolk-plug being withdrawn. The nervous system arises
in the form of the pear-shaped medullary plate of thickened
ectoderm upon the dorsal side. The medullary folds rise up
along the edges of this plate, bounding the medullary groove,
which by the coalescence of the folds becomes converted into
the closed canal of the central nervous system. Gill-plates

I GROWTH OF THE GERM-CELLS 11

and sense-plates are developed at the sides of the head,
a stomodaeum is pushed in in front, a proctodaeum behind,
and the head becomes separated by a slight constriction from
the neck. The tail grows out above the proctodaeum, the
external gills appear, the olfactory pit, eyes, auditory vesicles,
heart, gill-slits, coelom, pronephros are developed, and the
embryo is ready to hatch out as the tadpole. We need follow
the development no farther, but enough has been said to
illustrate the occurrence of the three processes of growth,
cell- and nuclear division, and differentiation.

The third example will be one of those cases in which it is
possible to trace back each organ or system of organs in the
body to a particular cell or cell-group in the segmenting
ovum, to state in fact its cell-lineage. This is the round
worm, Ascaris megalocephala. The egg of Ascaris is very
small and spherical. In it may be distinguished, in addition
to the cytoplasm, some yolk globules (specifically lighter than
the cytoplasm) massed together on one side: the arrangement
is therefore telolecithal and the symmetry similar to that of
the frog’s egg. There are also some spherules of a clear
substance, placed in the animal region.

The first division (Fig. 5) is equatorial, separating an animal
cell AB or S, from a vegetative cell P,. When the spindles
appear for the second division, it is seen that in the cell 8,
the spindle axis is placed at right angles to the egg-axis, while
in P, the spindle lies in the egg-axis. S, is therefore about
to divide meridionally, P, latitudinally, and when the division
has been accomplished, the cells A and B resulting from the
division of S, and S, or # M St and P, resulting from the divi-
sion of P,, are arranged in a characteristic T shape, the cell P,
being nearest the vegetative pole at the bottom of the stem
of the T.

The very remarkable phenomenon of the diminution of the
chromosomes can at this stage (metaphase of the mitosis
prior to the division) be seen in the cell S, or AB. As the
four Y-shaped chromosomes lie in the equator of the spindle
the middle portion of each becomes transversely divided into
a row of small granules, while the ends are swollen. The

12 INTRODUCTORY i!

whole row then undergoes longitudinal fission, and the
daughter rows pass to the opposite centrosomes in the ana-
phase of the mitosis, to form the daughter nuclei. The
swollen ends are however cast out into the cytoplasm, quite
irregularly, passing sometimes into both, sometimes into one
only of the blastomeres A and B, and disintegrate and dis-
appear. The cell in which the chromosomes are thus
diminished is purely somatic; it gives rise to the ectoderm
of the embryo. In the other cell, P,, the chromosomes
remain intact. This cell is the parent not merely of certain
somatic cells, but also of the future germ-cells, and we shall
see that in the course of segmentation each cell which con-
tains in itself the material for the future germ-cells, or lies
in the germ-track, retains intact chromosomes, while in every
cell that is destined to give rise only to some part of the soma,
the chromosomes undergo diminution. The cell # M St is such
a cell; it contains the material for the endoderm, some of the
mesoderm, and the stomodaeum, while from its sister cell,
P,, the gonad, as well as some mesoderm, is to be derived.

The P, cell now shifts round to one side (the future posterior
end), so that the four cells lie in a rhombus. They prepare
for the next division. A and B are divided lengthways into
right and left moieties, the chromosomes appearing in the
‘diminished’ form as granules.

EM St (S,) divides transversely into an anterior cell, M St
(mesoderm and stomodaeum), and a posterior cell, # (endo-
derm). In the mitosis, diminution of the chromosomes occurs.
P, divides with intact chromosomes into a lower anterior
cell, P,, and an upper posterior cell, C (or 8,). In this cell
diminution will occur prior to its division. It gives rise to
mesoderm.

By further divisions the number of ectoderm cells is
increased (derivatives of AB), and of endoderm cells likewise,
the mesoderm (J/) is separated from the stomodaeum (St?)
material, and P, divides into P, and D (or S,). The latter is
somatic (mesoderm), and diminution of the chromosomes occurs
init. P,is now purely germinal, and the chromosomes remain
intact in all its descendants.

I GROWTH OF THE GERM-CELLS 13

Fie. 5.—The process of chromatin diminution as seen in the somatic
cells of Ascaris megalocephala. (After Boveri, 1899.)

1. Mitosis in the 2-celled stage. In the first somatic cell (S, or AB),
the primary ectoderm, the chromatin undergoes diminution, not in the
germ-cell (P,). 1a, chromosomes being ‘diminished ’.

2. 4-cell stage, T-shape. In A and B the discarded masses of chroma-
tin are seen. S, (HM St), second somatoblast or endomesostomodaeal
rudiment.

3. 4-cell stage, lozenge-shape. In A and B the next mitosis is begin-
ning, in P,and HE M St the nuclei are in the resting stage. A is anterior,
A and B are dorsal, all four cells lie in one plane, the sagittal plane of
the embryo.

4. In FE M St the chromatin is being diminished. Division of P, into
P, and S,(C), the secondary ectoderm. a, b, primary ectoderm of right,
a, B, primary ectoderm of left side.

5. The endoderm (E,, F,) has now been separated from mesoderm and
stomodaeum. P, has just divided into P, and S,(d’, tertiary ectoderm.

6. Diminution of chromatin in S,(D). The four endoderm cells (£)
beginning to be invaginated: on each side two mesoderm cells (J) in
which granular chromosomes may be seen, and two stomodaeal cells (St).
Ventral view.

14 INTRODUCTORY I

When segmentation is concluded the blastula stage is
reached, with a segmentation cavity. Into this pass the
mesoderm cells, while the endoderm is invaginated to form
the gut. The blastopore is shifted to the anterior end, where
it is encircled by the stomodaeal cells.

The cell-lineage which we have just described may be
displayed in diagrammatic form thus: the cells in which the
chromosomes remain intact being indicated by a cross.

ovum
Ss oe
P. Ss. TAB)
a eee:
P, 8,(E MSt,)

O
FP S,(C)) Mt.
ea
‘®) O
P, S, (D) M St M St.
COG O
M St. M St.

The egg of Ascaris has been made the subject of important
experiments by Boveri.

And now let us turn to our main thesis, the inquiry into the
causes of growth, cell-division, and differentiation.

We shall begin with growth.

Growth is increase in size, and may be measured by increase
in weight, sometimes by increase in other dimensions, such as
stature, girth, and so on. Growth is partly the increase of
living material, in part the increase of substances secreted by
that living material. These secretions may be intra-cellular,
for instance the contractile substance of a muscle fibre, or
extra-cellular, as the chitinous cuticle of an Arthropod; they
may be organic, as in the examples quoted, or inorganic, such
as sponge spicules or the calcareous matrix of bone. Growth
obviously depends upon the assimilation of food-material,
which in early stages is usually provided by the yolk. It

I GROWTH OF THE GERM-CELLS 15

also depends upon the absorption of water, as Davenport’s
investigations upon tadpoles have shown. The percentage of
water in a tadpole’s body, for example, increases during the
first fortnight after hatching from 50% to over 80%: after
that the water percentage decreases slightly.

The same high proportion of water in the tissues in early
stages, followed later by a decline, has been demonstrated for
other embryos, for example the human embryo, as may be
gathered from the sixth column in the accompanying table
(Table I). At the present moment, however, the main interest
in growth undoubtedly centres in the way in which the rate
of growth alters during the progress of development.

There is some difference of opinion as to the method by
which this rate should be measured.

The table gives the actual weight (x) at certain times, the
increments from time to time (4x), the average increments

per unit of time for each of these intervals (Ep and the

“percentage increments, or these increments expressed as a
percentage of the weight at the beginning of the interval

= % <. It is this last magnitude which Minot has
proposed to employ as the measure of growth-rate, and if the
figures in the fifth column of the table be examined it will be
seen at once that the rate is high initially, diminishes abruptly
at first, thereafter more slowly to a final minimal value.

The same abrupt descent from an initial high value, followed
by a more gradual decline, can be seen in many other cases ; for
example, in the figures for the change in the post-natal growth-
rate of man (Table II). There is a slight but temporary rise
about the time of puberty. The graph constructed from the
figures resembles therefore a logarithmic graph or curve.

The parts of the body obey the same law as the whole,
although of course all the parts are not growing at the same
rate. The latter may be illustrated by the changes undergone
by the index-value of the parts (the dimension of the part
expressed as a percentage of that of the whole) during the
course of development.

16 INTRODUCTORY ]

TABLE I. HumMAN EmBryo, (FEHLING.)

Weekly Weekly %

Age in Weight in | tacrement increment | increment | % of

weeks. | ‘e7abimes (Ax). Ax Oa 100,| water.
(x). ( —— je ( —_ x —
At at x

6 0-975 97:5

17 36-5 39:525 3-23 331-2 91-8

22 100-0 63-5 12-7 34-8 92-0

24 242-0 142-0 71-0 71-0 89-9

26 569-0 327-0 163-5 67-6 86-4

30 924.0 355-0 88-75 15-6 83-7

39 1640-0 716-0 79-56 8-6 74-2

TABLE II. WEIGHT OF MALE BELGIANS IN KILOGRAMS.
(QUETELET.)

| Time in Weight Increment Increment /% increment

: Ax Ax 100
years. (x). (Az). pertime (=~). Ge xi
Birth ol
1 9-0 5:9 5:9 190-3
2 11-0 2-0 2-0 22-2
3 12-5 1-5 1-5 13-6
4 14-0 1-5 1:5 12-0
5 15-9 1.9 1-9 13-6
6 17:8 1-9 1-9 11-9
7 19-7 1-9 1-9 10-7
8 21-6 1-9 1-9 9-6
9 23-5 1:9 1-9 8:3
10 25-2 1-7 1-7 7:2
11 27-0 1-8 1:8 71
12 29-0 2:0 2-0 7:4
13 33:1 4-] 4-1 14:1
14 37-1 4-0 4-0 12-1
15 41-2 4-1 4-] 11-1
16 45.4 4-2 4:2 10-2
17 49-7 4-3 4:3 9°5
18 53-9 4:2 4-2 8-5
19 57:6 3-7 3°7 6-9
20 59-5 1-9 1-9 3°3
21 61-2 1-7 1:7 2-9
22 62-9 1-7 1-7 2:8
23 64:5 1-6 1:6 2-5
25 66-2 1-7 0:85 1-2
27 65-9 —0-3 —0:15 —0-2
30 66-1 0-2 —0-07 0-1

I GROWTH OF THE GERM-CELLS 17

It has however been proposed by Robertson that the average
increment for a short interval of time, namely, the ratio =
would be the more proper measure of the rate, the velocity of
a chemical or physical reaction being indeed expressed in
this way.

Now if the graph of the rate as so measured be constructed
it will evidently be of a totally different character, rising from
a minimum to a maximum, and then descending to a minimum
again. In the human being, as a matter of fact, there are
four periods in each of which a maximum rate is thus
attained: the first before birth, the second at the time of
birth, the third about the sixth year, and the fourth about
the sixteenth.

Now such a graph resembles that constructed for the rate of
an autocatalytic unimolecular chemical reaction, as Robertson
has pointed out. Robertson, indeed, following Loeb, suggests
that the growth of the body depends on the synthesis of
nucleins, and that this involves reactions of this kind. But
however that may be, it is not difficult to put to the test the
supposed resemblance of the change in the animal growth-
rate to the change in the rate of the kind of chemical reaction
alluded to. The velocity of such a reaction at any moment is
given by the equation

dx

oT ia ka (A—«),

where « is the amount of change that has been accomplished
at that moment (¢), A the final amount to be accomplished,
and A— consequently the amount that still remains to be
accomplished at the moment in question.

It follows from this that where «= = that is, when the

reaction is half over, the velocity attains a maximum, since

ele A
which is equal to zero when «# = 3° and since

1963 Cc

18 INTRODUCTORY I

dk (A—2a) _
dx if

Hence, if we suppose the growth-rate of the animal organism
to depend in the same way upon a, the amount of growth
already accomplished, and upon A—«w, the amount still to be
accomplished at any particular moment, then the theory may
readily be checked by ascertaining whether the rate is indeed
at a maximum when the growth is half done.

Robertson, calculating from the data given for human and
other growth, has found that this is very approximately true.
One instance will suffice here, the growth of the human being
during the fourth period (second post-natal) from the ninth
to the thirtieth year.

If a graph be constructed whose ordinates are the succes-

— 2k, that is, is negative.

: Ax ! :
Sive rates Ga) and whose abscissae are the successive weights

(a), it will be found that the maximum rate occurs when half
the increase of weight to be achieved during this period has
been attained. From the formula the theoretical rates for
the successive weights may be calculated, and their graph
compared with that of the observed rates.

There are means by which the value of the hypothesis may
be tried. The value of the constant & for instance may be
found, the theoretical weights (a) then calculated and compared
with the actual ones. Robertson has found the agreement to
be very fairly good. |

A botanical instance may be quoted here.. The measure-
ments made, many years ago, by Errera for the growth of the
sporangium-bearing hypha of the fungus Phycomyces show
very clearly that the rate ascends to a maximum when the
growth is half completed. i

The body of a Metazoon is often compared to the sum-total
of all the individuals produced by the repeated division of
a single Protozoon, and it might perhaps be thought that the
rate of growth of this total would alter, in the same way as
the growth-rate of a Metazoon. But this is apparently not
the case. We know indeed from the researches of Calkins,
Woodruff, and others that the rate of division of Protozoa

I GROWTH OF THE GERM-CELLS 19

(Infusoria) changes, periods of rapid division alternating with
periods of depression, or slow division, and it would seem from
the figures given by Woodruff for the mean rate of division
for a succession of five-day periods, that at any moment the
rate is roughly proportional to the number of divisions that
have taken place since the last period of depression, and to the
number that will occur before the next period of depression is
reached : also that the rate is at a maximum when half the
divisions have occurred. In other words, the rate of division
of the Protozoon changes in the same way as the rate of
growth of the Metazoon. It follows, of course, that the rate
of growth of the former, as expressed by the average incre-
ment of the total weight (or other dimension) of all the indi-
viduals produced by division of one, cannot obey the same
law. At the same time, some figures published by Popoff for
the growth of a single individual of an Infusorian (Frontonia)
between two divisions, indicate that the growth-rate rises to
a maximum in the middle of the process, and may therefore
possibly depend at any moment on the amount grown already,
and the amount still to be grown. If this should prove after
further investigation to be really the case, then the growth of
the individual Protozoon would be comparable with the growth
of the individual Metazoon ; in both cases the rate at any
moment would depend on the two factors named. A further
problem remains. While we can understand that the rate
should depend upon the amount of living and growing sub-
stance, x, it is not so easy to see what meaning is to be
attached to the other factor, A—z. In the case of the chemical
reaction this is the amount of substance left unchanged.
Robertson has suggested that in the organism this is cytoplasm,
or some ingredient in the cytoplasm from which nuclein can
be synthesized, the synthesis of nuclein being the supposed
autocatalytic unimolecular reaction upon which growth
depends. The idea is based upon Loeb’s assumption that there
is such a synthesis during segmentation in the (Sea-urchin)
ovum. Thisis, however, erroneous, since Masing has shown that
there is as much nucleic acid in the unsegmented as in the fully
segmented egg. In the former it lies in the cytoplasm, into
02

20 INTRODUCTORY I

which it had passed from the nucleus, as ‘ yolk-nucleus’, during
the growth of the oocyte; in the latter it lies in the nuclei,
which apparently have taken it up. Still, there may be
synthesis of nucleins in later stages, and the idea may be
substantially correct.

Again, the internal factor A, which gives the definite limit
to which the growth can proceed, might possibly be looked for
in the capacity of the fertilized ovum to divide a given number
of times and no more. If this were so, then the rate of cell-
division in the Metazoa would not be governed by the same law
as in the Protozoa, if in the latter the rate of division is given by
km (n—m), where m is the number of divisions that have taken
place at any moment, n the total number that can take place,
while in the former the rate of growth is given by y2™ (2”—2"),
where y is the initial size of the plasma of the ovum.

More probably it will be necessary to look for a wider for-
mula, which will include both these, as for instance that the
processes of cell-division, of synthesis of nucleins, and of syn-
thesis of all those cytoplasmic substances and secretions on
which growth depends, are all conditioned by the presence of
certain extra-cellular ferments of a certain intensity, whose
activities become progressively diminished by the combination
of the ferments with the products of the reaction, the reversi-
bility of the reaction, and other causes.

Before bringing this part of the subject to a conclusion it
may be pointed out that Minot’s expression for the growth-
rate (the percentage increment) states the facts in another way,
though it fails in not calling attention to both the factors in-

aa

volved. The equation a = ka (A —«x)

may be approximately written
At hw (A tie ee = BAe)
Bin ee ane , Ato) @

The expression on the left is proportional to Minot’s percent-
age increment, that on the right the velocity of a unimolecular
reaction, the graph for which (logarithmic) resembles the
graph of rates constructed by Minot’s method.

The next feature of interest presented by growth is the

I GROWTH OF THE GERM-CELLS 21

relation between the growth-rate on the one hand, and the
variability of the organism and the degree of correlation
between its various parts on the other. With regard to the
first, it has been found by various observers that as the growth-
rate rises and falls so does the magnitude of the variability
(which we may measure by the standard deviation (c), or better

by the coefficient of variability G x 100, M being the mean).

A few instances will suffice.

The way in which the variability goes up and down with the
growth-rate will be seen at once ENGLISH ARTISANS.
from the accompanying graph of | , WEIGHTS (ROBERTS)
the alteration of these quantities
during the last growth-cycle of
the human being. The data have
been taken from the measurements
of Roberts for the weights of
English artisans.
_ Again, the same general agree-
ment between these two magni- pr ha eee rar ane
tudes is found in the Trout during AGE IN YEARS
the short period, the first ten weeks after hatching, for which
data are available, and this is true not only for the growth
and variability of the whole body (total length) but for those
of the parts as well, eye diameter for instance, length of head
and length of caudal fin, The graphs do not indeed run parallel
throughout their course, but the fall or rise in variability is
accompanied by a fall or rise in the growth-rate. The agree-
ment is perhaps as close as could be expected when it is
remembered that a very small portion of the total life of the
animal has been under observation, and that this small portion
possibly includes one of the points of transition from one
growth-cycle to another (see the graphs of the growth-rate
of the total length in appendix).!

1 The agreement between growth-rate and variability is much improved
by taking the mean increment and not the percentage increment as the
measure of the former. I mention this as I was much puzzled by finding
that a rise of variability at the end of the period was not accompanied
by a rise in the growth-rate when the latter was expressed, as I now
believe erroneously, by the percentage increment.

22 INTRODUCTORY I

Secondly, it seems that there is a similar relation between
growth-rate and correlation, as Boas has pointed out in the
case of the human being.

The accompanying table (Table III), which gives the value
of the correlation coefficient between several pairs of organs
in the young Trout, shows that in many cases—total length and
breadth of caudal fin, total length and length of anterior dorsal
fin, total length and length of head, head length and eye
diameter—there is a significant diminution in the value during
the time that shows a decrease in growth-rate. The increases
seen in certain cases in the table are within the limits of error.

TABLE III, Trout LARVAE. CORRELATION COEFFICIENTS (p).

Weeks | Total length | Total length | Total length | Total length
after and Breadth | and Length | and Length | and Length

hatching.| caud. fin. | ant. dors. fin. | post. dors. fin. head.
2 0-89+0-01 0.76 +0-02 0-50 + 0-05 0-95 + 0-00
10 0:73 + 0-08 0:60 + 0-03 0°52 + 0-04 0-85+0-01

Weeks | Head length | Length ant. | Length ven- | position of

after and eye oaedeluoiin Leela pectoral and
hatching.| diameter. ae : nt ; ae by of pelvic fin.
2 0:94+0-01 0-43 + 0-06 0-52 + 0-05 0-87+0-01
10 0-65 + 0-03 0-39 + 0-05 0:58 + 0-04 0-83 + 0-02

Boas has brought forward mathematical proof that the
magnitudes of the variability and of the correlation coefficient
are necessary consequences of those of the growth-rate, rising
and falling with it.

But apart from this, considerable interest attaches to this
question of the change in the degree of correlation during

I GROWTH OF THE GERM-CELLS 23

development, for high correlation probably points to depen-
dence in development of one part upon another, a factor we
shall see to be of the greatest importance, and what throws
light upon one may help to explain the other.

The same may be said of the change in variability, for
a knowledge of what brings about this change is a step
towards the knowledge of the causes of the phenomenon.

We turn next to the second process in development, the
division of the ovum, preceded by the karyokinetic division of
the nucleus. A discussion of the problems presented by these
phenomena requires, however, a brief preliminary account of
the structure of the germ-cells, and of their union in the act
of fertilization.

The germ-cells—the ovum and the spermatozoon—though
very dissimilar in structure, except in one respect, are the
products of a history which is almost identical in the two
sexes. In this history there are three periods, of multiplica-
.tion, of rest and growth, and of maturation. In the first
period the young germ-cells derived from the primordial germ-
cells divide many times, showing at each division of the nucleus
the same number of chromosomes as is seen in the tissue-cells.
This number, since it is usually even, we shall speak of as 27.
These cells are spoken of as oogonia and spermogonia respec-
tively.

After a while the divisions come to an end and each cell
grows into a primary oocyte or a primary spermocyte as the
case may be. It is at this point that the difference between
the two sexes becomes manifest.

In the male the growth is not great, the nucleus passes
through the prophases of the first maturation division—lep-
totene, synaptene, pachytene, and formation of heterotypic
chromosomes—and then immediately proceeds to the two actual
divisions of the third or maturation period. Asis well known,
the number of chromosomes is reduced from the somatic
number (27) to the germ number (7), and very possibly this
occurs, as many hold, by a separation of the dissimilar halves
of the n bivalent heterotypic chromosomes in the first division,

24 INTRODUCTORY I

while the single chromosomes, thus reduced in number, are
longitudinally split in the ordinary manner in the second
(homoeotypic) division. But however that may be, the number
is reduced and each secondary spermocyte (produced in the
first division), and again each spermatid (formed from the
division of these), contains in its nucleus only one-half of
the somatic number of chromosomes. Each spermatid is then
metamorphosed into a spermatozoon. This process shows
a remarkable similarity in the many forms in which it has
been studied: we may take a flagellate vertebrate spermatozoon
—that of a Salamander—as fairly typical (Fig. 6). The centro-
some of the spermatid divides into two, and these leave the
sphere of attraction. The two centrosomes place themselves
radially, while the sphere moves round to the opposite (or
anterior as 1t will be) end of the cell. Here it becomes altered
to form the acrosome or perforatorium. Of the two centro-
somes the inner or anterior places itself close to the nucleus,
in the hinder wall of which it becomes embedded. Here it
enlarges and elongates to form the so-called middle-piece or
neck. The posterior centrosome, from which the axial fila-
ment of the tail has in the meantime grown out, becomes trans-
formed into a ring. The ring is eventually broken in two,
one half remaining attached to the anterior centrosome, the
other travelling a little way down one (the ventral) side of the
tail. On the other (dorsal) side of the tail, the fin grows out
along the axial filament. The nucleus elongates, and becomes
finally dense and very chromatic.

In the female, on the other hand, the growth in the second
period is very much greater, since it is at this time that the
yolk granules are deposited in the cytoplasm. The nucleus,
which has previously passed through the prophases of the first
maturation division, makes certain contributions to the cyto-
plasm ; that is to say, substances, which may be solid or
liquid, chromatic or achromatic, and are generally spoken of
as ‘yolk-nucleus’, pass from the nucleus into the cytoplasm
and are there concerned in the metabolism of yolk-secretion.
When growth has come to an end, the enlarged nucleus or
germinal vesicle breaks down and its contents are cast into the

I GROWTH OF THE GERM-CELLS 25

cytoplasm, of which they form henceforward a definite and

integral part, of great importance, as we shall segin the early

differentiation of theembryo. The spindle of the first matura-
®

8 9

Fig. 6.—Metamorphosis of the spermatid into the spermatozoon in the
Salamander (after Meves). 1-6, the whole cell; 7-9, the anterior end;
10-138, the posterior end of the head.

tion division and the heterotypic chromosomes, which are
derived from only a very small part of the contents of the
nucleus, go to the surface and the two maturation divisions
occur. Whereas in the male these divisions are equal, in the
female they are very unequal. By the first the first polar body,
by the second (which is homoeotypic) the second polar body,

26 INTRODUCTORY I

is extruded from the ovum (Fig. 7). The polar bodies are of
course minute compared to the egg-cell. The first often divides
intotwo. ‘Thus four cells are produced as in the male, but one

eee ERATE FG SET rere Seeieereceammennsbaremcere: : :
bravest TSS Spe es ee NPT een he ee

Fie. 7.—The maturation divisions in the female (Axolotl). 1, First
polar spindle with heterotypic chromosomes ; 2, Extrusion of first polar
body ; 3, Appearance of second polar spindle; longitudinal division of
chromosomes in egg and in first polar body ; 4, Second polar spindle radial ;
homoeotypic chromosomes on equator (metaphase) ; 5, Polar view of the
same ; 6, Anaphase; 7, Extrusion of second polar body; 8, Second polar
body with resting nucleus; 9, Female pronucleus in resting condition,
closely surrounded by yolk-granules.

is large, the actual ovum, the others small (potential ova).
Each of these four contains, as does each spermatid, only one-
half the ordinary number of chromosomes.

The ripe egg-cell possesses a definite structure,and frequently
this structure is polar, as in the case we have been describing,

I GROWTH OF THE GERM-CELLS 27

where the axis with unlike poles (animal and vegetative) is
‘determined by the elongation of the ovarian egg, the forma-
tion of the micropyle, and liberation of the contents of the
germinal vesicle into the cytoplasm. All telolecithal eggs
(e.g. Vertebrate eggs) have a similar polar structure similarly
determined by the disposition of yolk, and sometimes by the
presence (Frog) of pigment as well, and by the position of the
nucleus. Such an egg is divisible into similar halves by any
plane which includes the axis. But the egg may be centro-
lecithal (most Coelenterates), and here the axis is only deter-
minable by the excentricity of the nucleus, though in one
interesting case (Carmarina) there is an excentric jelly-plasm
near the vegetative pole. In some cases (Cephalopods, Insects)
the egg is bilateral.

When the germ-cells meet and unite in the act of fertiliza-
tion the full number of chromosomes (2 7) is of course restored,
but that is not the only nor even the chief event involved in
the process. In fertilization four distinct events occur,and some-
~ times a fifth. The first of these is the extrusion by the ovum
of a fluid, the perivitelline fluid; the second is the entrance
of the spermatozoon ; the third is the appearance of the defini-
tive centrosome and its division into two to form the cleavage
apparatus of asters and spindle; the fourth is the union of the
male and female pronuclei. To these must be added, in some
cases at least, a fifth, the alteration of the structure and
symmetry of the egg.

1, The moment a spermatozoon comes in contact (by its
acrosome) with the surface of the egg the latter extrudes a
fluid, termed perivitelline. This process is often accompanied
by the formation of a membrane, the fertilization membrane,
as in the Sea-urchin, which prevents the entrance of more
spermatozoa. This membrane, in the Sea-urchin, is not pre-
formed, but is to be regarded as the surface-layer of the egg
itself, gradually lifted up and separated from the egg by the
collection beneath it of a fluid—the perivitelline fluid—which
is hypertonic to sea-water. The membrane so formed quickly
becomes spherical.

In the Polychaet worm (Nereis) the mechanism is rather

28 INTRODUCTORY I

different. Here the membrane is preformed—the vitelline
membrane. Immediately below it there is a peripheral layer
of radial alveoli filled with a gelatinous material. Internal
to this is the cytoplasm with yolk-spherules and oil globules,
and of course the nucleus. Immediately upon contact of a
spermatozoon with the surface of the vitelline membrane the
gelatinous material of the peripheral alveoli is extruded as
a broad zone of jelly. Sea-water enters and occupies the
alveoli, whose walls remain as strands or lamellae crossing
the perivitelline space between the membrane and the ovum.
The perivitelline space is therefore, as in the Sea-urchin, in
a sense a part of the ovum.

In the Frog, a fluid is excreted, and this pushes away from
the ovum the vitelline membrane, which remains adherent to
the jelly. The ovum is now free to turn in the fluid, and
does so until the axis assumes a vertical position with the
heavy white pole below. This appears about half an hour
after insemination.

2. As soon as the acrosome of the spermatozoon has
perforated the surface-layer of the egg-cytoplasm there |
begins to collect around it a substance, hyaline in appearance
(in the Axolotl) and apparently of a more watery consistence
than the rest of the cytoplasm. It increases quickly in amount
and assumes the form of a cone, the apex of which is directed
towards the interior of the egg. It is known as the entrance
funnel (Fig. 8). Its base usually projects slightly from the
surface: this is the entrance cone (erroneously termed cone of
attraction). The entrance-funnel, still increasing, moves on
into the interior of the ovum, and the spermatozoon is carried
impassively with it. In the Axolotl (and others) the tail is
also carried in, but in some cases (Mus, Nereis, for instance) it
remains outside. Even when taken into the egg it degenerates:
it has no part to play in the later stages of fertilization. The
acrosome now seems to get caught in the side of the entrance-
funnel and, the inward streaming movement of the latter
continuing, the head of the spermatozoon is driven on and
rotated through 180°, so that the neck (containing the centro-
some) now lies at the inner end of the entrance-funnel, while

I GROWTH OF THE GERM-CELLS 29

the head and tail are both directed to the outside. The
entrance is now completed. It is effected by the formation,
under the influence of the acrosome, of a substance which, by
virtue apparently of its capillary properties, moves into the
egg and carries the sperm bodily with it. The sperm is in all
cases rotated through 180° in the process, so that the middle-
piece is inwardly directed.

The sides of the entrance-funnel are lined, in the Axolotl
and Frog egg, by pigment dragged in from the surface. This
pigment remains as a streak marking the position of the funnel
when the latter has disappeared. The entrance-funnel is
known in these eggs as the first part of the sperm-path.

A clear, yolk-free area is now developed round the middle-
piece or centrosome: this is the sperm-sphere, and from its
periphery radiations soon arise and pass out between the
yolk-granules, the sperm-aster. The formation of the sperm-
sphere is probably also due to the withdrawal of water from
the cytoplasm by the centrosome. The centrosome is in any
- case used up during the process, and entirely disappears. The
sperm-head, now detached from the tail, quickly shortens and
thickens to become the sperm-nucleus, or male pronucleus.

3. The definitive centrosome is now developed. It arises
in the Axolotl from the sperm-nucleus, The nuclear mem-
brane opens on the inside, and there emerges a dense rounded
body, or more probably there emerges something (? nucleic acid)
which produces this body by precipitation of the proteins of
the egg. However formed, this body is the definitive centro-
some. The sperm-nucleus is now moving along what is known
as the second part of its path to meet the female pronucleus.
The latter has moved from its position at the animal pole
towards the centre of the egg, usually but not necessarily
along the egg-axis. Thus the second part of the path may or
may not lie in the same meridional plane as the first part
(entrance-funnel), and in the former case it usually makes an
angle with the first part, since it is directed to a point approxi-
mately in the axis and at a fixed distance from the animal
pole, while the point of entrance of the spermatozoon may be
anywhere in the animal hemisphere (see below).

Fig. 8.—Fertilization in the Axolotl.

A and B, Meridional sections of the whole egg. a, Formation of entrance-
funnel (first part of sperm-path). B, Formation of sperm-sphere and aster ;
o7 male pronucleus; 9 female pronucleus; p.b., the two polar bodies.

c, Formation of the sperm-sphere round the middle piece (anterior
centrosome); parts only of the head (black) and tail are shown.

D, Formation of the sperm-aster. The centrosome has disappeared ; the
head, beginning to be vacuolated, is separated from the tail.

E, Further shortening and vacuolation of the sperm-nucleus. There is
still no centrosome.

F, Appearance of the definite centrosome. G, H, Division of the
centrosome.

(In c-H the arrow marks the direction of entrance of the spermatozoon.)

1, Approach of the two pronuclei. Formation of spindle-fibres.

J, Formation of asters, elongation of spindle, further enlargement of
pronuclei, and appearance of chromosomes,

K, Further elongation of spindle, and formation of a centrosphere
round each centrosome. The pronuclear membranes are breaking down
and the spindle-fibres passing in.

L, The fully-formed fertilization spindle. In the equator are the chromo-
somes, now longitudinally split, and attached to large spindle-fibres, In
each centrosome the centriole has divided.

32 INTRODUCTORY I

The definitive centrosome now divides at right angles to
this second part of the path, and in a plane parallel to the
equator of the egg. Preceded by its two centrosomes and
the sperm-aster, the sperm-nucleus continues to move towards
the female pronucleus. When they meet, the line joining
them, that is the second part of the sperm-path, is naturally at
right angles to the line uniting the two centrosomes (Fig. 8).

The ovum itself has no centrosome, and it is an invariable
rule for the female centrosome, even though present in the
polar divisions, to disappear. There is also little doubt that
in all cases the definitive centrosome is of male origin, though
whether derived from the centrosome present in the middle
piece, or developed de novo, as in the Axolotl, is not certain.
It may be said, however, that while there is very little
evidence for the continued persistence of the original centro-
some in any case, a conclusive demonstration has been given
recently by Lillie of the origin of the cleavage centrosome
from the sperm-nucleus, in Nereis. For in Nereis, first the
middle piece is normally left outside with the tail, but a
centrosome nevertheless is seen later in the sperm-aster; and
secondly Lillie has shown by a very beautiful experiment that
any fragment of sperm-head introduced into the egg rotates,
and develops at its inner end an aster and a centrosome. The
experiment consists in centrifuging the egg during the entrance
of the sperm. The jelly, being lighter than the egg itself, is
dragged off towards the centripetal end of the tube, and
removes with itself any part of the spermatozoon that may
still be outside the egg. It is possible in this way to remove
the hind end of the head but allow the front end to go in.
The part that enters behaves as described. Sometimes it is
by the violence of the operations divided into two: each piece
then rotates, and forms aster and centrosome.

With the evidence before us it would hardly be too rash to
suppose that it is a general rule for the definitive centrosome
to be not only a sperm-centrosome, but one deyeloped de novo
from the sperm-nucleus.

4. The pronuclei now lie side by side between the two
centrosomes. From the latter spindle-fibres grow out and

I GROWTH OF THE GERM-CELLS 33

impinge upon the nuclear membranes, while in other direc-
tions similar outgrowths constitute the asters. The spindle
_ quickly elongates while its fibres break through the nuclear
membranes and pass continuously from pole to pole. The
chromosomes are formed independently in each nucleus,
and the two sets, paternal and maternal, each to the
number of vn, lie side by side in the equator, There they
are divided lengthways in the usual fashion and their
halves pass to the spindle-poles. The nucleus of each of
the first two blastomeres and eventually of each cell in
the body thus receives a full set of chromosomes from each
parent.

It is not unnatural that the union of the two pronuclei
should have led the discoverers of the fact to regard it as
being the essence of fertilization. The opinion was further
supported by the phenomena of conjugation in certain
Infusoria, where apparently, there is merely an exchange of
micronuclear material between the gametes, and by the
similarity of the two germ-cells in respect of their nuclei,
the latter being conceived as the sole vehicles for the trans-
mission of the inheritable characters of the species, a task
believed to be performed equally by the two sexes.

Now whatever views we may come to hold as to the réle of
the nucleus in inheritance, it is assuredly not true that both
nuclei are necessary for the production of a normal individual.
For in the first place in parthenogenesis, natural and artificial,
only the female nucleus is present, and yet a normal embryo
or larva is developed and reared.

In the second place in what is called merogony, the enucleate
fragment of an ovum may be fertilized and give rise to a
normal larva.

We are obliged therefore to look for the meaning of fertiliza-
tion elsewhere, and we find it in the restoration of the lost
power of nuclear and cell-division, The ripe germ-cells are
cells which have come to the end of their power of reproduc-
tion by division: in the act of fertilization that power is
restored. It is mutually restored, for in ordinary fertiliza-
tion we see the egg-cell with both sets of chromosomes divide

1963 D

34 INTRODUCTORY I

as a result of this stimulus, while in merogony we see the
paternal chromosomes alone so stimulated. Nor is the
mechanism of the process far to seek. The male cell intro-
duces or manufactures a centrosome, the female cell provides
the cytoplasm wherein the centrosome can make the necessary
division-apparatus. The two cells are thus mutually comple-
mentary. The experiments we shall have to describe in the
next lecture suggest that the stimulus so conveyed to the
egg by the spermatozoon may be of a physical or chemical
nature.

5. Finally in some cases the spermatozoon produces a
change in the cytoplasmic structure of the egg, the original
radial being replaced by a bilateral symmetry.

A very good instance of this is the formation of the grey
crescent in the Frog’s egg, already alluded to (p. 7).

Another is provided by the Ascidian Cynthia, in the im-
mature egg of which there is a peripheral yellow substance
surrounding a central grey yolk; the large germinal vesicle
is near the animal pole (Fig. 9). Upon the entrance of the sper-
matozoon—near the vegetative pole—the germinal vesicle
breaks down and maturation occurs. The contents of the
germinal vesicle are discharged as a clearsubstance into the cyto-
plasm. Meanwhile the yellow layer has moved to the vegetative
extremity of the egg, followed by most of the clear substance,
only a small portion of which remains near the animal pole
surrounding the female pronucleus. The animal hemisphere
is thus occupied by the grey yolk. The egg has still a radial
symmetry about its axis, but this is now replaced by a
bilaterality, for the yellow and clear substances move to one
side below the equator: this will be the posterior end, while
the grey yolk, thus displaced, moves into the opposite side
of the vegetative hemisphere. The pronuclei meet on the
posterior side in the clear area, but finally move, taking this
substance with them, into the animal hemisphere. We shall
see that this bilateral symmetry of the egg is identical not
merely with the symmetry of the bilateral cleavage but also
with that of the embryo.

Other instances are known of the spermatozoon producing

ia GROWTH OF THE GERM-CELLS 35

Fra. 9.—Normal development of the egg of Cynthia partita. Maturation
and fertilization. (After Conklin.)

A, Unfertilized egg before the fading of the germinal vesicle (clear),
showing central mass of grey yolk (lightly dotted), peripheral layer of
yellow protoplasm (thickly dotted), test-cells and chorion.

B, After the entrance of the spermatozoon the yellow protoplasm has
streamed to the vegetative pole (v); in it, excentrically, is the sperm
nucleus (sp.n). The clear protoplasm derived from the germinal vesicle
partly forms a layer over the yellow protoplasm, in part remains at the
animal pole (a). The grey yolk occupies the animal part of the egg.

c, The yellow and clear protoplasmic substances have both streamed
to what will be the posterior side (P). A, anterior, Vv, ventral (animal
pole), D, dorsal (vegetative pole).

D, View of the same egg from behind. The two pronuclei are seen
side by side in the clear area. R, right; L, left.

pe

36 INTRODUCTORY I

a similar alteration in egg structure; future investigations
must show how far it is of general occurrence.

In a later lecture we shall see that the cy toplasmic sub-
stances so rearranged are causally related to the development
of the various parts of the embryonic body, are in fact
determinants of inheritance.

‘® , 1 J ; “< >
no ett ie 2 ner
CHAPTER II oN er
ie vs ' et D fi De" o"% ¥
f f x ra
CLEAVAGE ne

THE fertilized ovum is ready to develop, and the first step
in development is segmentation or cleavage.

The segmentation of the ovum may be said to present to
us, on the whole, four problems. The first of these is to
discover the reason why the egg divides at all. The second
is to find out why, when it does divide, it exhibits its own
particular pattern of cleavage. In the third place we have
to inquire into the causes which bring segmentation as such
to an end. The fourth question asks whether cleavage is or
is not in itself a process of differentiation.

These problems we shall discuss in order.

1. It is hardly necessary to say that any knowledge we
may have of the nature of the causes which provoke the
division of the egg is due to the genius of the American
physiologist Jacques Loeb, for Loeb has shown that it is
possible to replace the stimulus which is normally given to
the egg by the spermatozoon, by the action of a solution, that
is, by a physical or chemical agency. It is true that others
had previously attempted in this way to cause the egg to
divide, notably Hertwig and Morgan, but these met with
little suecess. To Loeb belongs the honour of the achieve-
ment.

Since the methods employed have undergone some modi-
fication we may briefly recapitulate the history of these
experiments.

The subject was in the first instance the egg of a Sea-
urchin (Arbacia, and in later experiments, Strongylocen-
trotus), and it was found possible to incite it to segment and
develop by temporarily immersing it in sea-water to which

38 CLEAVAGE II

some magnesium chloride had been added. The superiority
of the results obtained by the use of this salt led Loeb
at first to attribute the effect to some specific activity of the
magnesium ion.

Since however the solution used was hypertonic to sea-
water, the question arose whether the determining cause
might not be the increased osmotic pressure. Experiments
were therefore instituted in which the eggs were subjected
temporarily to the action of solutions of a number of sub-
stances in sea-water, the solutions being all hypertonic to
sea-water but all isotonic with one another. The substances
employed were not only salts like magnesium chloride, sodium
chloride, and potassium chloride, but also non-electrolytes
such as cane-sugar and urea. After temporary immersion in
any of these solutions the eggs segmented and developed.
The hypothesis was then adopted that the increased osmotic
pressure, resulting in a withdrawal of water from the eggs,
was responsible for the result. The hypertonic solutions must
be alkaline and contain oxygen.

It is to be noticed that development was not perfect in any
of these cases, for the fertilization membrane was not formed,
the cleavage was very irregular—simultaneous division into
several cells, and multipolar mitoses occurred—and the larvae
sank to the bottom instead of swimming at the surface.

Further investigation showed however that solutions which
were physically isotonic did not produce precisely the same
effect, were not in fact physiologically equivalent, and con-
versely that solutions which had the same physiological effect
had not the same osmotic pressure, as may be seen from the
accompanying table (Table IV).

TABLE IV.
Optimal concentration Osmotic pressure
Solution. in gram-molecules. Dissociation. in atmospheres.
Cane Sugar 0-96 m. — 21-53
Grape Sugar 1-04 m. aa 23-33
CaCl, 0-50 m. 64 % 25-57
MgCl, 0-49 m. OF 26-47
LiCl 0-74 m. 66 % 27-59
NaCl 0-79 m. (Gy A 30-28

KCl 0-78 m. fii s - 80-95

II CLEAVAGE 39

The reason for this is that the eggs are not surrounded by
a semi-permeable membrane or coating, but that their surface
is permeable to these different substances in varying degree.
Since then the eggs admit the entrance of the substances to
a greater or less extent, the effect may be due in part at least
to some other cause than the osmotic pressure of the solution.
And indeed the role attributed to these solutions in the later,
improved method, in which they are also employed, is
different.

In the improved method the egg is first submitted to the
action of an agent which incites the formation of the fertiliza-
tion membrane, a feature characteristic of the normal process,
but absent, as we have seen, in the earlier experiments.

The brothers Hertwig had, many years before, caused the
formation of this membrane by the use of chloroform. Loeb
was thus led to try esters and fatty acids, and of the latter
butyric acid was found to be the most reliable.

The procedure adopted is briefly as follows: the eggs are
placed for a short time (from 14 to 24 minutes) in sea-water
1D
10
butyric). They are then removed to sea-water (which must
be alkaline), washed, and kept in sea-water (alkaline) for
from 15 to 20 minutes. The membrane is now formed in
precisely like manner to the normal membrane. Should the
eggs remain longer in the sea-water, however, cytolysis occurs;
that is, droplets of clear cytoplasm begin to protrude from
the surface, are separated off, and this continues until the
whole egg is broken up into fragments. To prevent this
some corrective must be employed. The eggs are accordingly
placed either in a solution hypertonic to sea-water (made
usually by the addition of NaCl, for example, 50c.c. sea-
water+8c.c. 24n NaCl), which must also both be alkaline
and contain oxygen, or in a dilute solution of potassium
cyanide (for instance, 50 cc. sea-water+2c.c. sg per cent.
KCN).

The eggs, after remaining some time (half an hour or more)
in the corrective solution, are finally retransferred to sea-water,

containing a little butyric acid (55¢.c. sea-water+3 cc. es

40 CLEAVAGE II

where they segment normally’ and develop into swimming
larvae. |

The larvae produced by this improved method of artificial
parthenogenesis may be reared through the metamorphosis
and reach the adult condition. It should not be forgotten
that the credit of first accomplishing this belongs to Yves
Delage (who used, however, a different method), and that at
a time when the means now employed at Plymouth for
rearing larvae had not come into use. The individual first
successfully raised by Delage was a male, with ripe gene-
rative products. .

The success of Loeb’s experiments naturally gave an impetus
to the inquiry into this problem, and artificial partheno-
genesis has been induced in the eggs of many animals, by
many methods in the hands of various investigators.

Thus, Delage used for Sea-urchins (Strongylocentrotus) acids
and alkalis alternately, for Asterias, carbon dioxide: Lefevre
has employed acids for Thalassema, Loeb, saponin for Polynoe.
Osmotic pressure (hypertonic solutions) has given good results
with Mactra (Kostanecki), Chaetopterus (Loeb, Mead), Amphi-
trite (Scott), Nereis (Fischer), Ophelia (Bullot). Greeley has
found cold sufficient, Lillie heat for Asterias, while Mathews
used merely mechanical agitation for the same form. Lastly,
in Vertebrates, Bataillon found that while hypertonic solu-
tions would cause the eggs of Petromyzon and Rana, to pass
through only a few of the cleavage divisions, the eggs of the
latter might be stimulated not only to segment but to de-
velop by being punctured with a fine needle. From the
punctured egg a perivitelline fluid was exuded, in it a grey
crescent appeared at the normal time, the eggs segmented
and a few of the larvae produced lived almost to the meta-
morphosis.

Though the very diversity of the means—mechanical,
physical, chemical—by which artificial parthenogenesis may
1 That is, the first three furrows are meridional, meridional, and

equatorial. We are not told whether in the next division the charac-
teristic mesomeres, macromeres, and micromeres are produced.

II CLEAVAGE 4]

be brought about suggests that the mechanism of the process
is not to be understood by reference to the factors involved
in one method alone, the theories which his own experi-
ments have induced Loeb to adopt, are well worth a brief
discussion.

As Loeb points out, the whole action falls into two phases.
In the first the egg is subjected to the influence of a sub-
stance which not only determines the formation of the mem-
brane but also sets in motion certain changes which ultimately
lead to the destruction of the egg by cytolysis. In the second
phase it is rescued from the disastrous effects of the first
solution by being immersed in a second.

It appears that the chain of events set in motion by the
first solution (the butyric or other fatty acid) consists in part
at least of the oxidation of substances in the egg. The
membrane is also formed, but the mechanism of that is
another process, to which we shall return in a moment.

It is known (Warburg) that the unfertilized egg undergoes
.a@ very slow oxidation, which is increased many times not
only by normal fertilization, but also by the use of the
membrane-producing reagent. It is these oxidations that
lead to cytolysis, since by them are formed certain decomposi-
tion products which are toxic to the egg.

The function of the second reagent is either to stop this
harmful oxidation or else to counteract it. The first is ac-
complished by the use of potassium cyanide, which inhibits
oxidation generally, and the part played by this substance
in the rescue of the egg from death is not hard to understand.
But it is more difficult to form a conception of the réle of
the alternative agent, namely a hypertonic solution, for, as
pointed out already, this must be alkaline but must also contain
oxygen. If potassium cyanide be added to the hypertonic
sea-water, or if the oxygen be removed and replaced by
hydrogen, then the solution becomes ineffective or at least
requires a much longer sojourn of the eggs in it before they
can be made to develop on being replaced in sea-water.

Hence the evil effects that follow on the oxidations incited
by the first solution are counteracted by another oxidizing

42 | CLEAVAGE II

agent, which is supposed to render the egg immune, or to
carry off the toxic substances.

It must be admitted that theory here leaves us in the lurch.

It is interesting to notice that the same two phases, de-
structive, involving the formation of the membrane, and
counteractive, permitting of segmentation and development,
can be distinguished in fertilization by a spermatozoon.
When the eggs of a Sea-urchin are inseminated with the
sperm of a Starfish, they all form membranes. This is due
to the contact of the sperm with the egg surface. But while
all the sperms touch the egg, they are not all able to enter -
and complete the process. If so able, then the egg develops
normally ; but if the sperm remain outside, the egg under-
goes cytolysis, from which, however, it may be saved by
timely treatment with hypertonic sea-water. On removal
to ordinary sea-water, it develops. The spermatozoon there-
fore normally conveys to the egg first the membrane-forming
substance and then the counteractor.

While we still await a more satisfactory explanation of
the workings of these stimulants to parthenogenesis, we have
been able to gain rather more insight into the mechanism by
which the membrane is thrown off.

The hypothesis adopted by Loeb is based on the fact that
the agent employed (a fatty acid) is lipoid-soluble, and upon
a certain conception of the structure of the egg-cytoplasm.
This structure is supposed to be alveolar, and the contents
of the alveoli are supposed to be prevented from coalescing
with one another by a coating of a lipoid (lecithin perhaps).
The fatty acid destroys this coating, the superficial alveoli
coalesce (the fatty acid having only penetrated a _ short
distance below the egg surface), absorb water, and the accu-
mulating perivitelline fluid, being hypertonic to sea-water,
throws off the surface-layer of the egg—inter-alveolar sub-
stance—as the membrane.

When the membrane is fully expanded the perivitelline
fluid is practically pure sea-water. If there be added to the
sea-water a substance which increases the osmotic pressure,

II CLEAVAGE 43

but to which the membrane is impermeable (Mammalian
blood-serum, for instance), the membrane at once collapses.
Should the lipoid-soluble reagent be allowed to penetrate still
further into the egg, the deeper parts of the latter become
liquefied and cytolysis occurs.

It is, however, doubtful whether this hypothesis is entirely
satisfactory. Solution is a physical process, but the magni-
tude of the temperature coefficient or quotient points to a
chemical reaction, as Harvey and indeed Loeb himself have
shown.

The temperature quotient for an interval of 10°C. for
a chemical reaction is at least 2: for a physical action it is
much lower.

The following table shows that its magnitude for this
reaction is about 2 (taken from Harvey, for various Sea-
urchins).

TABLE V.
Optimum concentration
Hi feante ii in ¢.c. of S acetic to
minutes. Temperature. 50 c.c. sea-water.

13 23° 6

BS 33° 2-3
3 23° 3-4
3 33° 11-2
6 23° 3

6 33° 1i

The solution of the lipoids by the fatty acid must then be
rejected. A change in the surface-tension of the egg might
be suggested, but surface-tension is not altered much by
temperature, nor is the rate of diffusion, nor the dissociation
of the fatty acid.

A chemical explanation is therefore put forward, namely,
that the acid combines with the protein at the egg-surface,
and that the compound so formed is more permeable than the
cytoplasm of the unfertilized egg. It is supposed then that
substances (protein) pass out through this surface-layer and
are immediately coagulated by the water to form the mem-
brane. Or else it may be imagined that the altered surface-
layer itself becomes the membrane. Through this membrane

A4 CLEAVAGE II

—presumed to be permeable to water and salts, but imperme-
able to the proteins and sugars in the egg—sea-water is then
absorbed and the membrane raised from the surface.

There are facts which support this view. In the first
place it is known that all haemolytic agents—all agents that
cause the diffusion of haemoglobin from a blood corpuscle—
are also capable of inciting the formation of the membrane in
these ova. Such are electricity, heat, hydroxy] ions, hydrogen
ions, distilled water, fat solvents, bile-salts, soaps, glucosides,
such as saponin, digitalin, solanin, and blood-sera. Haemolysis
must depend on an alteration of the permeability of the
surface-layer. |

In the second place Lillie has shown that the order of
effectiveness of neutral salts of potassium and sodium in
causing membrane formation is the same as the order of their
effectiveness in causing the liberation of the red pigment
from the egg of the Sea-urchin Arbacia, and the latter must
depend on an increased permeability of the peripheral layer
of the egg.

The following table gives the salts in the order of effective-
ness :

COOCH, is least effective
Cl

Br

ClO,

NO,

if

CNS is most effective.

The hypothesis seems peculiarly applicable to Nereis, for
here, as we have already seen, there is a preformed membrane
which becomes separated from the egg by a perivitelline fluid
as the result of insemination. This fluid is simply sea-water
which passes through the membrane and fills the superficial
alveoli from which their gelatinous contents have escaped
and diffused out. It will be noted that the membrane
must have become more permeable, and that the walls of the
alveoli remain, whereas on the lipoid-solution theory they
should disappear.

II CLEAVAGE 45

The membrane formed by these agents is apparently exactly
like that produced normally by the spermatozoon. We have
now to inquire what resemblance the division apparatus of
these parthenogenetic eggs bears to that which is made by the
sperm-centrosomes in a fertilized egg.

The details have been revealed to us by the investigations
of Wilson into the cytology of magnesium chloride eggs, of
Hindle into that of those stimulated by the later butyric acid
method.

The two accounts are in essential agreement as to the
origin of the division centres, but we shall follow Hindle in
the main, since the segmentation of these eggs at any rate
approaches the normal.

The species used was Strongylocentrotus purpuratus.

The following changes occur in the short interval (fifteen to
twenty minutes) between the removal from the butyric and
the immersion in the hypertonic solution.

The membrane having been thrown off, the nucleolus
of the female nucleus, previously chromatic, loses its affinity
for basic dyes, and its definite shape; it may fragment.
Meanwhile there appears a clear perinuclear zone of cytoplasm
from which radiations pass out in all directions. In the
hypertonic solution the nucleus enlarges, while the perinuclear
zone almost disappears, but, on removal to sea-water, reappears,
while the nucleus enlarges still more.

Two cleavage asters are now developed with a spindle
between them ; each contains a centrosome. They are formed
by division of one aster and centrosome; Wilson has shown
that the centrosome originates from, or at least at the
surface of, the nucleus. There are also in the cytoplasm
independent cytasters, each with its centrosome, which in
this case is not of nuclear origin.

If exposure to the hypertonic solution is not too long, these
cytasters disappear and division takes place across the equator
of the ‘fertilization’ spindle, the female nucleus having first
broken up into chromosomes, which are present in the
reduced number (7), a number which persists at least as far
as the blastula stage.

45 CLEAVAGE Il

If, however, exposure to the hypertonic solution is too
prolonged, the cytasters become very numerous, and united
by spindles to one another as well as to the cleavage amphi-
aster. The chromosomes become scattered not only on the
cleavage spindle, but on the other spindles too, and division
occurring in all the equators of the multipolar figure, the egg
is irregularly divided into several cells at once, as was
always the case in the earlier experiments with magnesium
chloride. Since division is not restricted to those spindles on
which chromosomes are cast, but may occur in their absence,
it happens that some of the cells produced are enucleate.

The centrosomes seen in these ova—not only those in the
cytasters, but also those in the cleavage asters—are new
formations. The foci for the formation of the cytasters may
possibly be the chromatic particles in the cytoplasm (remains
of the ‘ yolk-nucleus’), while the development of centrosome
from the nucleus is paralleled by the mode of origin of the
definitive centrosome in ordinary fertilization. There seems
to be no ground for believing that the centrosome of the
maturation divisions persists and is revivified.

In other cases also which have been examined [the
Kchiuroid Worm Thalassema (Lefevre), the Polychaet Amphi-
trite (Scott) | normal cleavage seems to depend on the presence
of one amphiaster or cleavage spindle. In Thalassema the
two centres with their asters appear simultaneously upon the
nuclear membrane.

It appears therefore that just as the development of a division
apparatus in ordinary fertilization is a necessary condition
of cleavage, so also in artificial parthenogenesis a bi-polar
spindle is a pre-requisite of normal, that is, binary segmenta-
tion. The centrosomes about and between which this appa-
ratus is produced in the cytoplasm are in the first case of male,
in the second of female nuclear origin.

In conclusion, a word may be said of the part played by
this spindle in the division of the nucleus and the cell. In
mitosis the chromatic material of the nucleus (which alone is
divided) is thrown into the form of chromosomes, These bodies

Ty CLEAVAGE 47

divide independently, they are not divided by the spindle.
The function of the spindle-fibres is merely to pull apart
their halves. But the spindle does take an active part in
cell-division. In plants the cell-plate, the future cell-wall,
arises by equatorial thickening of the spindle-fibres, and
there is a parallel phenomenon in the animal cell, for after
the separation of the daughter chromosomes there arises a
plate of material in the spindle equator which has a less
surface-tension than the remainder of the cytoplasm. It is
the reduction of the surface-tension, or rather the greater
surface-tension of the remainder, which pulls the two blasto-
meres apart. This not only appears to be a theoretical
necessity but is experimentally demonstrable. For if a drop
of rancid olive oil be floated on water, or better on a mixture
of alcohol and water, and a thread soaked in weak potash
be laid across a diameter of the drop, the soap now formed
along this diameter having a less surface-tension than the
remainder, the drop divides. The experiment is due to
_Robertson : it may be readily verified.

We have finally to inquire whether the last event of ordinary
fertilization, the alteration of the egg symmetry, finds a
parallel in artificial parthenogenesis. In only one sense, so
far, has it at present been found possible to give an answer
to this question, in the frog’s egg, where the grey crescent
appears as a result of fertilization on that side of the egg
opposite to the point of entrance of the spermatozoon.
Brachet has shown that in the eggs stimulated by puncture
a grey crescent appears, and at the same time as in the
controls, but that it bears no definite relation to the point of
puncture, being variably on the same side, on the opposite
side, or at right angles to the latter. There is on the other
hand an invariable coincidence between the plane of symmetry
of the egg, as thus defined, and the median plane of the
embryo, for the dorsal lip of the blastopore always appears
in the region of the grey crescent. Brachet is therefore
driven to the somewhat remarkable conclusion that the
unfertilized egg is only apparently radially symmetrical, and

48 CLEAVAGE II

really, though invisibly, bilateral. In ordinary fertilization
this weak primary bilaterality is superseded by the far
stronger bilaterality imposed by the sperm, a bilaterality
which persists as that of the embryo. In artificial par-
thenogenesis the primary bilaterality remains, becoming
manifest as the result of the stimulation.

It must be pointed out that the number of cases on which
Brachet relies are really too few to support any safe conclu-
sion, and further that the coincidence between the plane of
symmetry of the fertilized egg and the median plane of the
embryo is not absolute but only approximate, as the accom-_
panying table of the frequencies of the angle between the two
planes will readily show (Table VI). The eggs used in this
experiment were placed on the slides with their axes vertical
and the white pole below, to avoid any disturbing influence
of gravity, and spaced to avoid the influence of pressure.

TABLE VI. FREQUENCY.

Angle. Positive. Negative. Total frequency.
Q°-~15° 68 59 127
15°=30" 36 a2 68
30°-45° 10 13 Ze
45°-60° 4 5 9
60°—75° 1 8 9
75°-90° 3 1 4
90°-105° 1 — 1
105°-120° = = =
120°-135° 1 — 1
135°-150° = ze —
150°-165° 2 — 2
165°-180° 1 — 1

It is clear that while there is a very strong tendency for
the two planes to coincide, it may happen that they diverge
a good deal, even to the extent of 180°.

Some caution should therefore be exercised in drawing con-
clusions as to the coincidence of the planes of fertilization,
egg-symmetry, and embryonic symmetry from a small number
of observations.

2. The second problem presented by cell-division is con-
cerned with the causes which determine the particular pattern
of cleavage in each case.

II CLEAVAGE 49

First, however, a word on the conditions of there being
a system of cleavage at all.

It is an established physical principle that drops of fluid
will only cohere to form a system of drops without fusing
with one another if they are coated with a layer or film
which is insoluble both in the surrounding medium and in the
fluid of the drops themselves. If the former condition be not
observed then the drops separate, if the latter condition be
absent then the drops fuse. In neither case is there a system.

A beautiful experiment of Herbst’s has shown that the
possibility of the blastomeres into which an egg divides
forming a coherent system is governed by a like condition.
Around and between the blastomeres there is visible in many
cases (the Sea-urchin egg for instance) a definite coating film.
This is insoluble in ordinary sea-water, but if the egg be
placed in an artificial sea-water from which the calcium has
been omitted, then the film is seen to become dissolved, and
the blastomeres separate. So far, therefore, the blastomeres
behave like drops of other fluids. We shall see now that the
pattern assumed by the cells in cleavage may also be explained,
in some cases at least, by reference to the principles of surface-
tension.

There are three principal patterns of cleavage, the radial,
the bilateral (including the iso-bilateral), and the spiral. The
chief features of these types have been often described, but
may be briefly recapitulated here.

The radial type of cleavage is distinguished by the fact that
more than three, usually four or eight, surfaces of contact
between adjacent blastomeres may meet in one line; for in-
stance, in the four-celled stage of a Frog’s egg the four surfaces
in question intersect in the egg-axis,and so on. The planes
of division in this type also either include, are parallel to, or
at right angles to the egg-axis. The same must be said of
cleavages of the second type, with the addition that they are
symmetrically disposed on each side of one plane, often that of
the first furrow, as in the Cephalopod egg (Fig.10), but it may be
another plane, for instance in Ascaris the plane including the
first four blastomeres. In the iso-bilateral form there are, of

1963 EK

50 CLEAVAGE II

course, two such planes, for example in the Ctenophore egg, and
the Teleostean egg. In the spiral type, firstly, the successive
cleavages are oblique to the egg-axis; secondly, never more
than three surfaces of contact intersect in one line, so that
cross- or polar-furrows are developed between opposite blasto-
meres in for instance the four-celled stage; while in the third
place, successive quartettes of micromeres are thrown off at

ie aan 2

Fria. 10.—-Three segmentation stages in the blastoderm of Sepia off-
cinalis ; the segmentation is of the bilateral type. J, left; 7, right; I-V,
first to fifth cleavages. The top sides of the figures are anterior. (After
Vialleton, from Korscheit and Heider.)

the third and following phases of cleavage towards the animal
pole, alternately in a right-handed and a left-handed direction
(Fig. 11). In later stages spirally segmenting eggs assume the
characters proper to the first and second types or may do so.
The law of alternation of direction of cleavage at successive
divisions, just alluded to, holds good for a considerable time in

II CLEAVAGE a

cleavage, and for the divisions of the micromeres of the various
quartettes. It is, as a matter of fact, only a special case of
a rule which is seen in segmentations of the other types also,
namely, that successive divisions are at right angles to one
another. It is known as Sachs’ rule, since this botanist first
formulated it for plant cell-divisions, and depends, in part at
least, on the factthat the centrosomes, in preparation for the next
division, divide at right angles to the previous spindle-axis.
The pattern of cleavage is conditioned obviously by the

Fie. 11.—Diagram of a spirally segmenting egg in the 16-cell stage.
2a-2D macromeres; 2 a—-2d micromeres of second quartette; lal, la2-
1d1, 1d2 micromeres of first quartette.

shapes, sizes, and arrangement of the cells, and these in turn
on the rate and direction of division, and the movements of the
cells on one another; and these again on the relation between
the dividing nucleus with its centrosomes and the cytoplasm,
and on the properties of that plasma and its inclusions.

These relations are expressed in certain rules, one of which,
that of Sach’s, has just been referred to. Another is Balfour’s
rule that yolk impedes division, hence the more rapid division
and smaller size of animal cells in telolecithal eggs. Hertwig’s
rules state that the nucleus lies in the centre of the cytoplasm,
and that the dividing nucleus or spindle elongates in the direc-
tion of greatest protoplasmic mass or, as Pfliiger worded it, the
direction of least resistance, resistance being offered by the yolk.
These ‘rules explain a good many of the features of division in
the various types. The properties of the cytoplasm which play

E 2

52 CLEAVAGE II

a part in the ordering of the pattern appear to be, sometimes,
the physical properties which they possess as liquids; for in
spirally segmenting eggs, where never more than three surfaces
of contact between adjacent blastomeres intersect in one line,
making angles of 120° with one another, the cells are merely
obeying the laws of surface-tension as enunciated by Plateau
for systems of drops, for example soap-bubbles. In the other
types of cleavage, it is true, these capillary laws are not obeyed,
but even here as Roux has shown it is possible to cause drops
of oil to imitate the arrangement of a radial system by enclos-
ing them in a boundary; in the radial eggs the membrane
may represent such a boundary.

More than this need not be said at the moment since these
matters may be found discussed in the text-books.

3. Nuclear and cell-division continue of course throughout
the life of the organism ; and during all but the earliest stages
of development they are accompanied by the processes of
growth and differentiation. There is, however, an earliest
stage of all in which the material of the ovum is simply cut
up into small pieces, the cells, without the concurrence of any
growth or of any differentiation other than the formation of
the segmentation cavity, and such physical and chemical altera-
tions as may be taking place in the blastomeres under perhaps
the influence of their nuclei. This early stage is the stage of
segmentation, and we have now to discuss the nature of those
causes which bring segmentation as such to an end and deter-
mine the beginnings of differentiation.

Following out an idea which originated with Richard
Hertwig, Boveri has suggested that initially the cytoplasm is
too large for the nucleus, but that by the process of nuclear
and cell-division the ratio of plasma to nucleus is reduced
until it reaches a certain value, the attainment of which
marks the end of cleavage, and the commencement of other
processes.

The evidence adduced by Boveri in support of this hypo-
thesis consists in the experimental demonstration that at a given
stage of development the ratio in question has always a given

IT CLEAVAGE 53

value, whatever be the initial size of the nucleus, which may
be arbitrarily altered, while the cell-size is kept constant.

The number of chromosomes can be altered in the following
ways:

1. In artificial parthenogenesis the number is n (maternal)
(Thelykaryotic).

2. In merogony thenumber is n(paternal) (Arrhenokaryotic).

3. In monaster eggs the number is raised to 4n (Diplo-
karyotic). These are fertilized eggs in which the division of
the centrosome has been delayed by shaking. The 2 chromo-
somes nevertheless divide, but return into the condition of
a resting nucleus. From this, 41 chromosomes emerge when
the centrosome does divide, and this number persists.

4, If the egg is kept for twenty-four hours in sea-water
while the sperm is treated with dilute alkali, then upon fertili-
zation the sperm-nucleus lags behind its centrosome, which
divides to form a spindle in which only the female nucleus is
included. The latter breaks up into chromosomes which are
divided in the ordinary way, but the male nucleus passes un-
divided to one pole (Partial Thelykaryosis), Hence, after
cell-division, one blastomere has 27 chromosomes (is Amphi-
karyotic) while the other has only , which are maternal (is
Thelykaryotic).

5. In dispermy it may happen that the spindles formed by
the division of the two sperm-centrosomes remain apart, with-
out uniting in the usual tetraster. The female nucleus lies in
the equator of one spindle, together with one male nucleus, the
other male nucleus lies in the other spindle. The twospindles
are parallel, or may be, and division takes place in the plane
including their equators, and also between them (if not at
once, then eventually). Hence on one side of the egg there
are Amphikaryotic nuclei with 2, on the other Arrhenokary-
otic with n chromosomes. This is Partial Arrhenokaryosis.

6. The normal egg is Amphikaryotic, with 2 7 chromosomes.

The following examples will suffice to show how the rela-
tion between the number of chromosomes and the number
of the nuclei is determined, cell (cytoplasm) size remaining
constant.

54 CLEAVAGE II

A. Nucleate and enucleate ego-fragments are taken of the
same size; both are fertilized, and develop. The number of
cells in equal areas of the same germ-layer, at the same stage,
is then counted.

For example:

Nucleate fragment §Enucleate fragment

2n (2+ 07). n (07)
Ectoderm of anal area . 167 317
Ectoderm of ciliated ring 86 163

B. The number of cells in larvae from monaster eggs is
compared with that in normal larvae.
For example:
Monaster (47). Normal (2 n).
43

Blastula . 5 : : 28
Pluteus (animal ectoderm) 37 (gs

It is clear that at the same stage in larvae derived from the
same original quantity of cytoplasm, the number of the cells in
equal areas of the same tissue is inversely proportional to the
number of chromosomes in the nuclei.

C. In partial Arrhenokaryosis and partial Thelykaryosis the
larva is obviously composed of two regions (separated usually
by the median plane) in which the size of the nuclei is differ-
ent. The number of small (n chromosomes) nuclei is to the
number of large (2 7) as 2:1.

We turn to the relation between the number of chromo-
somes and the size of the nuclei as measured not by their
diameters but by their surface-areas. The initial amount of
cytoplasm in each case is constant as before.

A. Merogonic and nucleate egg-fragments.

Nucleate fragment Enucleate fragment
2n (2+ 07). n (07)
In gastrula stage 42 21
In pluteus stage. 46 25
B. Partial Arrhenokaryosis. 2n (9+ 07). n (97).
The two sides of the pluteus are compared 27 14
C. Partial Thelykaryosis. 2n (94+ 07). n (9).
The two sides of the pluteus are compared 30 14

D. Diplokaryosis ; the monaster egg is com-
pared with the normal. Monaster (4). Normal (2 7).

66 33

II CLEAVAGE 55

It is clear that the surface-area of the nucleus is directly
proportional to the number of chromosomes contained in it.

It follows therefore at once that the size of the nucleus (as
measured by its surface-area) varies inversely with the number
of cells. But in equal areas of like tissues (which are of the
same thickness) the number of cells must be inversely propor-
tional to the size (volume) of the cells. Hence, the cell-volume
is directly proportional to the surface-area of the nucleus, as
well as to the number of chromosomes contained in it, or the
ratio of plasma to nucleus has in the blastula stage, and again
in each tissue at later stages, some constant value.

It is suggested that the attainment of this constant value as
a result of the multiplication of the nuclei during segmenta-
tion brings this process as such to an end.

The converse of this experiment is seen if the number of
cells is compared in larvae reared from fertilized nucleate egg-
fragments of different sizes.

Two cases may be quoted.

Ratios of surfaces Ratio of numbers of nuclei
of gastrulae. in gastrulae.
| 1:6:5 1:1-48
2. 1:11-5:28 1: 1.42 : 2.82

The number of nuclei being proportional to layers of equal
thickness the cell-volume has, at a given stage, a constant
value. This is, of course, a re-statement of Driesch’s rule that
in larvae developed from isolated blastomeres the number of
cells and the surface-area are both directly proportional to the
germinal value.

It will be observed that the value of the constant plasma-
nucleus ratio has not been given by Boveri, and further that
the actual volume of the cells has not been determined. This
lacuna in our knowledge has been filled by the researches of
Fraulein Erdmann, who has investigated in the same form
as that employed by Boveri (Strongylocentrotus lividus) the
changes in dimensions undergone by nucleus, cell, and chromo-
somes, during early development.

In the accompanying table (Table VII) the results for one
temperature—10° C.—are given, for a series of stages.

56 CLEAVAGE II

TABLE VII.
Volumes in cubic p of
Stage. Nucleus. Cell. Chromosomes.
2 cells 10037-0 106250-0 19-17
4 cells 1605-8 51063-0 10-83
8 cells 1081-0 26290-0 8-32
16 cells 837-8 9973-0 7.24
82 cells 803-6 6023-0 5-46
64-132 cells 529.7 2685-5 4-51
Blastula 1 460-5 1343-0 3-59
Blastula 2 332-4 549-7 2-77
Gastrula 1 117-4 292.5 1-92
Gastrula 2 62-9 180-7 1-00
Pluteus 28-7 118-0 0-41

It seems therefore that not only the cell-volume but also the
nuclear volume decreases during segmentation, gastrulation,
and the development of the larva, and it is obvious that the
nucleus does not grow to the original size after each division.
As we should expect from Boveri’s work, diminution in the
volume of the chromosomes is accompanied by decrease in the
size of the nucleus; since, according to Boveri, the size of the
nucleus depends on the number of the chromosomes, originally
given to it, and the mean size of the paternal and maternal
chromosomes is at least nearly the same.

At higher temperatures (15°-16° and 20°) the same diminu-
tion of the nucleus and its chromosomes is seen: the absolute
values for any stage diminish as the temperature rises. At one
and the same stage the number of cells is greater, the higher
the temperature. The total amount of chromatin present in
the embryo at any one stage (blastula or gastrula) is, however,
constant at all temperatures,

From the data the ratios are at once obtained (Table VIII).

TABLE VIII,
Ratios of volumes.

ae Cell. Cell. Nucleus.
ico Chromosome. Nucleus. Chromosome.
2 cells 348-0 10-5 32-8
4 cells 294-0 31-8 9.3
8 cells 187-0 24.3 8-1
16 cells 86-0 11-9 7-2
32 cells 68-0 7-4 9.2
64-182 cells 37-0 5-0 7:3
Blastula 1 23-0 2-9 8-0
Blastula 2 12-0 1-6 7-5
Gastrula 1 9.4 2-5 3-9
Gastrula 2 11-2 2-9 3-9
Pluteus 19-0 4.2 4.3

II CLEAVAGE 57

The ratios given are for a temperature of 10°, but the same
diminution with a slight rise at the end occurs at the higher
temperatures. The final values reached are practically the
same at all temperatures, and this suggests some causal rela-
tion between the amount of chromatin and the size of the
nucleus and cell. The ratio of plasma to nucleus diminishes,
as originally suggested by Boveri, but not at anything like
the rate which a growth of the nucleus to its original size
after each division would involve. At a low temperature this
ratio reaches a lower value, that is the nucleus is relatively
larger than at a high temperature. This is in accordance
with a rule which the studies of R. Hertwig and his pupils
on Protozoa seem to have established.

Lastly, the ratio of the surface-area of the nucleus to the
chromosome volume does not remain constant, but diminishes
and increases again.

TABLE IX.
Ratio of nuclear surface
chromosome volume.
Stage. 10° 15° 20°
2 cells 7-8 6-4 7-4
8 cells 3-6 3-6 3-8
82 cells 4.6 4.4 6-4
Blastula 1 5-0 2-6 2-0
Gastrula 1 3-6 2.2 2-0
Pluteus 6-8 6-0 5-2

As we have already seen, the nuclear surface diminishes as
development proceeds, though of course not as fast as the
nuclear volume. The nuclear surface, therefore, is determined
neither by the number nor by the volume of the chromosomes
taken alone.

This, however, does not necessarily invalidate Boveri's state-
ment, which was based on a comparison of nuclear surfaces at
corresponding stages with a varying number of chromosomes,
not on a comparison of successive stages with the same number
of chromosomes.

In the studies just considered the plasma whose volume is
determined includes the yolk-granules as well as the actual
living cytoplasm. It is the great merit of Conklin to have
attempted to measure the volume of the cytoplasm as distinct
from the yolk, at successive stages, and in the different cells,

08 CLEAVAGE II

The egg used was that of Crepidula, a Molluse, which segments
spirally. As segmentation progresses the plasma increases at
the expense of the yolk.
The table gives the value of the nucleo-plasma ratio
(|= == volume
plasma volume
when the nuclei first become spherical after division.

i as calculated from measurements taken

TABLE X.

Cell. Ratio.
Before cleavage 1:27-5
AB or CD 1:13-5
A, B, Cor D 1:14.5
1A4-1D | ae Ey da
la-ld 1:145
2A-2D 1:12-7
2a-2d 1: 25-6
la'—-l1d' 1:35-7
la’?-1d 1:14-6
3A-—3D i We ea
3a—-3d 1:14.5
2a'-2d) 1:10-3
2a°-20? 1:10-8
1la—1q'" 1:14-6
1 qi: — 1 qd}? 1 : 7-0

There are great differences, 1t will be seen, in the value of
the ratio in different parts of the egg. The nuclear size is
apparently determined partly by the length of the resting
period—the value of the ratio increases where this is pro-
longed, as in 3 A—3 D—partly by the amount of plasma
present, and partly by the number of chromosomes. The
third factor is of course constant in all the instances quoted
in the table, but Conklin has shown that the chromosomes
may be scattered by the use of hypertonic solutions, and that
the nuclei so formed from less are smaller than those formed
from more chromosomes. The influence of the second factor
can be demonstrated by experiment, for if the egg be centri-
fuged the yolk may be unequally distributed between the
first two blastomeres. The blastomere with the larger share
of eytoplasm has also a larger nucleus and a larger
centrosome.

The mean value for the nucleo-plasma ratio of all the
blastomeres is 1:15. Measurements made on adult tissue

II CLEAVAGE 59

cells—intestinal epithelium, gastric epithelium, liver epi-
thelium, ectoderm, epithelium of gills, ganglion cells—give
a mean ratio of 1:10-5. There is therefore apparently an
increase.

The mean volume of all the nuclei does not, however,
increase to its original size after each division, but diminishes
at first to reach its original value once more by the 70-cell
stage. The total amount of nuclear material has by this time
therefore increased. In the Ascidian Cynthia there is similarly
an eventual increase in the total volume of nuclei in spite of
the diminution of the mean volume. These results are in
exact agreement with those obtained by Fraulein Erdmann
for the Sea-urchin.

4. The fourth question is whether the nuclear and cell-
division of cleavage are themselves processes of differen-
tiation. |

According to a well-known theory, which is or rather was
associated with the names of Roux and Weismann, while the
cytoplasm of the ovum was regarded as isotropic or equivalent
in all its parts, the internal causes of differentiation were placed
in the nucleus, that is, the determinants or different materials
on which the appearance in the offspring of the inheritable
characters ultimately depend, were imagined to reside in the
chromosomes of the nucleus, but to be gradually separated
from one another in successive divisions. Nuclear division
in other words was qualitative, and through its agency the
qualitatively different determinants were distributed to the
different cells, there to call forth in the cytoplasm the histo-
logical characters to which each was appropriated, the charac-
ters which are to the observer the actual sign of differentiation,
which is therefore actually produced by cell and nuclear
division, and would not occur without it. Further, each cell
having received in this way certain determinants and those
only, can alone give rise to certain structures, and the causes
for the production by it of those structures lie wholly within
itself, that is, in its nucleus. Development is therefore a
process of self-differentiation of each part of a mosaic-work.

60 CLEAVAGE dh)

This theory was founded upon an observation and an experi-
ment made by Roux upon the egg of the Frog. Roux believed
that observation told him that the first furrow of the egg
invariably coincided with the median longitudinal plane of
the embryo, the second with the transverse plane (putting
those cases aside in which the first furrow by an ‘anachronism ’
occupied the position of the second). Hence of the two blasto-
meres one contained the determinants for the right half, the

Fig. 12.—A and B. Normal Frog embryos with medullary folds (m),
open (A) and closed (B). C. Hemiembryo dexter with almost complete
post-generation of the ectoderm; u, yolk-plug. D. The same, older, but
with less post-generation. H. Hemiembryo anterior (?) with beginning
post-generation. (From Korschelt and Heider, after Roux.)

other those for the left half of the body. This was confirmed
by experiment, for when one of the blastomeres was killed by
means of a hot needle the survivor was found to give rise toa
half-embryo, right or left as the case might be (Fig. 12). Later
investigation has, however, been able wholly to confirm neither
the observation nor the experiment, while the institution of
experiments on the ova not only of the Frog but of other
animals has shown that a cell-division is not the cause of

II CLEAVAGE 61

differentiation in the cytoplasm, for the simple reason that
that differentiation exists before cleavage takes place.

We may begin this discussion by a re-examination of the
facts on which Roux’s own theory was based.

The first statement is that the first furrow in the Frog’s egg
always coincides with the sagittal plane. This is certainly
not the case, for if a sufficiently large number of eggs be
examined it will be found that while in the majority the
angle between the two planes is small, it is nevertheless
possible for that angle to have any value.

One example will be enough. The following table gives
the frequencies for different values of this angle under the
most favourable circumstances, that is when the disturbing
influences of mutual pressure and gravity have been removed.

TABLE XI.
Angle between
First Furrow and Sagittal Plane. Frequency.
— 90° —75° 6
75° — 60° 10
60° — 45° 20
45° — 30° 31
30°— 15° 70
15°— 0° 82
+ 0°-15° 102
15° — 30° 50
30° — 45° 31
45° — 60° 9
60° —75° 7
75° — 90° 10

Total 428

The tendency of the two planes to coincide is measured by
the standard deviation (o), which in this case is 31-45° + 0-73.

If now under the same conditions the standard deviation of
the angle between the plane of symmetry (plane of the grey
crescent) and the sagittal plane be determined it is found to
have a value of 26-80°+0-82. In other words there is a greater
tendency of the sagittal plane to coincide with the plane of
symmetry than with that of the first furrow. The standard
deviation of the angle between the plane of symmetry and
the first furrow is highest of all, namely 34-46° + 1-07.

The relation between these planes may also be stated by

62 CLEAVAGE i

finding the value of the correlation coefficient (p). This
is, between the plane of symmetry and the sagittal plane
0-451 + 0-035, between the first furrow and the sagittal plane
0-364 + 0-033, and between the plane of symmetry and the
first furrow 0-186 +0-043. This confirms the first result. We
shall return later on to the relation between the plane of
symmetry and the sagittal plane, but for the present it is
sufficient to say that a statistical inquiry does not bear out
Roux’s assertion.

In the second place Roux claimed to have produced from
one of the first two blastomeres a half-embryo. It must be
conceded that this sometimes occurs, but not always. Oscar
Hertwig, repeating the experiment, found that frequently the
living blastomere developed into much more than a half-
embryo, being apparently only impeded in its differentiation
by the presence of the inert mass of the other. Morgan
suggested that the capacity of the survivor to develop into
a whole depended on the position it occupied with regard to
the dead cell; when the egg turned over so that the dead
blastomere lay underneath the living, the latter became a
whole, and it was indeed found by the same observer that if
the egg were turned upside down, the living blastomere gave
rise to a whole embryo. Precisely the same result is seen in
the experiment, due to Schulze, in which each of the two
blastomeres is made to develop totally and the egg to give
rise to a double monster, by merely turning the egg upside
down in the two-celled stage. The further analysis of this
experiment, by Wetzel, shows that under the influence of
gravity the contents of the blastomeres are redistributed, the
heavy yolk-granules sinking, the lighter cytoplasm rising,
until each blastomere has acquired a new polarity of its own;
and in such a way that now the two polarities are opposed,
the two blastomeres like two eggs united by their vegetative
poles, and the two components of the double monster united
by the persistent yolk-plugs of their dorsal sides. There can
be little doubt that if it were possible to separate the two
blastomeres completely, each would give rise to an independent
larva, as happens in the Newt, where the separation can be

II CLEAVAGE 63

effected by means of a noose of hair tied round the egg in the
first furrow (Fig. 13).

It is highly probable, however, that even then due attention
would have to be paid to the position of the first furrow with
regard to the grey crescent. For Brachet has shown that if
one blastomere be killed, the fate of the other depends upon
the angle made by the first furrow with the symmetry plane.
When the planes coincide, the survivor becomes a right or left
half-embryo; when the angle is 90° the survivor (when it

ff

Fie. 13.—Three stages in the production of a double monster by
strong median constriction of the Newt’s egg. (After Spemann, 1903.)
a. Beginning of gastrulation; there is a separate lip in each half.
b. l. and r, Med., Medullary folds of left and right embryos; *, point
where the medullary grooves separate; Bl, blastopore. c. The double-
headed larva.

includes the grey crescent) becomes a postero-dorsal half-
embryo; while when the angle is between 0° and 90° the
survivor develops into an embryo which is defective on the
right or left, anteriorly cr posteriorly as the case may be.

This experiment demonstrates in the most convincing way
the closer dependence of the embryonic symmetry upon the
plane of egg-symmetry than on the first furrow.

In the Frog’s egg, therefore, the factors that determine the
symmetry of the embryo must be distinct from those that

64 CLEAVAGE Mi

determine the plane of the first and therefore of subsequent
furrows, or as we may put it, the symmetry of segmentation,
and in fact we know, with some degree of precision, the nature
of these factors and the causes of their divergence.

The unfertilized egg has a radial symmetry about its axis:
as a result of fertilization it becomes bilateral, since the grey
crescent appears on the side of the egg opposite the point of
entrance of the spermatozoon. It appears that the position
of the grey crescent is determined not by the actual point
of entrance of the spermatozoon, but by the position of the
whole of the first part of the sperm-path, the entrance funnel. |
We also know that there is a considerable tendency for the
median plane of the embryo to coincide with the plane of

D

Fria. 14.—Roux’s diagrams to show the relation of the sperm-path
(Pig.) to the first furrow in the Frog’s egg. In the furrow includes the
sperm-path, in B it is parallel to it, in c it is parallel to the inner portion

of the path (copulation path), in pD it includes only the very last portion
of the copulation path. (From Korschelt and Heider, after Roux.)

embryonic symmetry, for the dorsal lip of the blastopore to
be formed on the side of the grey crescent. We may assume
then that the entrance of the spermatozoon imposes a bilateral
structure upon the cytoplasm, a structure which persists as
the bilaterality of the embryo.

In the second part of its path the sperm-nucleus moves to
meet the female-nucleus. This second part may but need
not lie in the same meridional plane as the first part (Fig. 14).
When the first part is directed towards the egg-axis, and when
the female pronucleus lies in the egg-axis, then both parts do
lie in the same meridional plane, but when, as may happen, the
female pronucleus is not in the egg-axis, or the sperm entrance
path is not directed towards the axis, or possibly on account
of other disturbances (gravity, &c.), then the meridional planes

II CLEAVAGE 65

of the two parts of the whole path diverge to a greater or
less degree.

Now it is upon the second part of the path that the posi-
tion of the first furrow depends, since it falls in the equator
of the fertilization spindle, and the spindle is developed
between the two centrosomes which are produced by the
division of the sperm centrosome at right angles to that
meridional plane which includes the line of union of the
pronuclei. Hence, while the first furrow may and does in-
deed tend to coincide with the plane of symmetry and the
sagittal plane, it need not do so.

One more point must be briefly alluded to. As has been
mentioned the correlation between the sagittal plane and the
plane of symmetry is not complete, even when disturbing
agencies are removed. At the same time there is some cor-
relation between the first furrow and the sagittal plane. It
is worth while suggesting that the elongation of the spindle
in a certain direction with the concomitant radiation of its
asters, may itself impose some bilateral structure upon the
egg material, and that the plane of embryonic symmetry may
be a resultant of the separate influences exerted by the mitotie
figure and the sperm-entrance.

With regard to the production of the grey crescent, which
is due to the retreat of superficial pigment into the interior,
it seems probable that this in turn depends on streaming
movements in the cytoplasm set up by the aggregation of
a watery material to form the entrance funnel.

We turn next to the theory of the qualitative division of
the nucleus. Experiment compels us to reject this also. For
if the egg of the Frog be subjected to pressure the sequence
of the divisions is altered. When, for instance, the pressure
is in the direction of the axis, while the first two furrows
preserve their normal meridional directions at right angles
to one another, the third furrows, instead of being latitudinal
are again meridional or parallel to the first, while the fourth
are latitudinal or parallel to the second. It follows that the
arrangement of the nuclei produced by these successive
divisions is also abnormal, and if the nuclei were really

1968 F

66 CLEAVAGE Il

qualitatively different,then the distribution of the determinants
contained in them would be abnormal, and should cause an
abnormal differentiation of the cytoplasm. As a matter of
fact the egg develops into a normal tadpole.

The theory of the qualitative division of the nucleus is
therefore proved untenable from more than one side. It is
only fair to Roux to state that he has some time since
- abandoned an impossible position.

It only remains for us to review briefly the evidence of
a like nature drawn from experiments on the eggs of othe
forms.

Experiments similar to that just quoted have shown that
the pattern of cleavage may be altered by pressure without
interfering with the normality of development in, for in-
stance, the eggs of the Sea-urchin (Driesch) and of the worm
Nereis (Wilson). In Nereis the egg segments into a flat
plate of eight cells. On releasing the pressure, an octette
of micromeres is formed instead of the usual quartette, and
a normal trochophore is developed. The pattern of cleavage
may be altered by other means. Thus in Cerebratulus (a
Nemertine) in calcium-free sea-water the third division is
meridional, then a first, and later a second, octette of micro-
meres are formed. Again, in certain cases the egg divides
simultaneously into three, a meridional division produces
six cells in a ring, sextettes of micromeres are then given
off. In both cases a normal Pilidium is produced (Yatsu).
In Sea-urchin eggs the blastomeres may be deranged by heat,
shaking, and diluting the sea-water to any extent or almost
so, without prejudice to the eventual normality of develop-
ment.

In artificial parthenogenesis again, an irregular segmenta-
tion may be followed by a regular development.

Secondly, isolated blastomeres frequently segment as parts,
that is, as though the remaining blastomeres were present.
Thus a half-blastomere of Echinus gives rise to four meso-
meres (animal cells), two macromeres, and two micromeres,
and a quarter-blastomere to two mesomeres, one macromere,
and one micromere. The isolated blastomeres of spirally

II CLEAVAGE 67

segmenting eggs (Nemertines, Molluscs) behave in the same
way. The quarter-blastomere of Patella, for instance, pro-
duces one micromere of the first quartette, one of the second,
and so on. But the fate of these isolated and partially
segmenting cells differs, and depends indeed on the kind of
material they contain. The half- or quarter-blastomere of
a Sea-urchin egg, being produced by meridional divisions,
possesses a proper share of all the materials present in the

Fig. 15.—Dentalium. Cleavage after removal of the first polar lobe ;
a, first division: the isolated polar lobe is seen below; 8, 4-cell stage,
from animal pole; c, 8-cell stage, from animal pole; d, formation of
second quartette, from vegetative pole ; e, same stage as last, open type;
J, same stage, from animal pole. (After Wilson.)

egg, since these are distributed symmetrically about the axis,
and is able to develop into a whole pluteus. The half- or
quarter-blastomere of the Nemertine egg can do the same.
In the Mollusca (I/yanassa, Patella) the developmental capa-
cities of the cells are usually strictly limited (Crampton,
Wilson), but in Dentaliwm while the AB half-blastomeres and
the A, B, and C quarter-blastomeres not only segment but
develop partially, the CD cell or the D cell, in which is
F2

68 CLEAVAGE II

included the polar lobe, which other experiments have shown
to contain the material necessary for the formation of the
sense-organ and post-trochal region, develops into a complete
larva.

In these cases a partial segmentation is followed by a total
development.

The converse is seen in the total cleavage, at low tem-
peratures, of blastomeres of Jlyanassa which are still unable
to form whole larvae, and again in the complete segmentation
of the Ctenophor egg from which a piece of the vegetative
hemisphere has been removed, though the larva that ‘is
derived from such a fragment is defective (Driesch and
Morgan).

Thirdly, it is well known that in the spirally segmenting
egos of Nemertines, Turbellarians, Molluscs, and Annelids,
it is possible to trace the several organs and tissues of the
embryonic body each back to an origin in some particular
cell or group of cells in the cleavage system, and that as
a general rule homologous organs, say the prototroch or the
mesoderm, arise from cells which occupy identical positions
and come into being by the same sequence of divisions in
all eases. Thus the mesoderm usually is derived from the
cell 4d; 2d, the first somatoblast, gives rise to the ectoderm
of the ventral plate; the first quartette of micromeres is
ectodermal and so forth. But there is no necessity that
identical cells or cell-groups should have the same destiny,
and there are many exceptions known to the general rule.
One instance will suffice.

In the earthworm the nephridia, being derived from the
ectoderm of the ventral plate can be traced back to 2 d, while
in another Annelid, the leech, the same organs are mesodermal
in origin and spring from 4d. These need cause no per-
plexity, for, as we shall see more fully in a moment, the
organs depend for their development upon the presence of
some factor in the cytoplasm, but this factor (a material
factor) need not be present in that position in which the
organ to which it is appropriate will arise. Thus the factor
which conditions the differentiation of nephridia, being in

II CLEAVAGE 69

both cases in the D quadrant, may readily in one case pass
into the cell 2d, in another into 4d, and is then dubbed
ectodermal in the one case, mesodermal in the other. It is
none the less the same organ, homologous in the two forms,
since its homology depends eventually on the conditioning
factor, the cytoplasmic substance which is specific for it, and
not, as has been sometimes supposed, on the cell into which
that substance happens to pass.

And fourthly, it is possible to suppress cleavage without
preventing differentiation. At least Lillie has been able, by
the use of potassium chloride, to show that the unsegmented
egg of Chaetopterus will put out cilia, while certain cyto-
plasmic substances undergo a change of position. In particular
certain granules assume the position normally taken by them
in the cells of the prototroch. It is interesting to note that in
these ova the nucleus enlarges, so that the plasma-nucleus
ratio is reduced.

‘But though the factors on which the pattern and symmetry
of segmentation depend are distinct from those which condi-
tion the symmetry of the embryo, it must not be forgotten
that they may coincide. Thus we have seen that in the
Frog’s egg, the plane of symmetry, the first furrow, and the
sagittal plane may sometimes all be coincident, and there are
other cases. In Amphioxus (Cerfontaine) and in Ascidians
(Conklin) this occurs and again in the Cephalopod Mollusca,
though it remains for a statistical examination to show that
the coincidence is invariable. In spiral eggs also, the median
plane of the embryo usually has a definite relation to the
cleavage system, the D quadrant being as a rule posterior.

Experimental analysis, however, shows the difference. The
causes which determine a particular pattern of cleavage, as we
know, consist in the physical properties of the cytoplasm,
and in the relation between the cytoplasm with its con-
stituents and the dividing nuclei with their centrosomes.
The causes of differentiation on the other hand, as we are
now to see more fully, reside in the first instance in specitic
organ-forming cytoplasmic materials.

CHAPTER III
DIFFERENTIATION

WE shall be obliged to limit ourselves to a discussion of the
internal factors. These are to be sought, firstly, in the initial
structure of the germ, that is, of the fertilized ovum, and,
secondly, in the interactions of the developing parts.

I. The ovum comprises the cytoplasm and the nucleus, and
each of these constituents has its part to play in the determi-
nation of inheritable characters.

A. THE CYTOPLASM.

The evidence which we have just been reviewing has shown
us not only that the division of the nucleus is not qualitative,
but also that the cytoplasm is not the homogeneous isotropic
body imagined by the ‘ Mosaik-theorie ’, at least as originally
propounded. We know now that the different parts of the
cytoplasm are related causally to the formation of the various
organs of the embryo, are therefore factors in differentiation
and determinants of inheritance.

The testimony of experiment is conclusive on this point,
since it is known that the abstraction of a certain part of the
cytoplasm involves the absence of certain embryonic organs.
As the facts are fairly well known they need only be briefly
recapitulated here.

The development of experimentally isolated parts of the
ovum—they may be pieces of the unsegmented ovum, or blasto-
meres removed during cleavage—has been observed in a
number of forms, and it has been shown that while it is
possible in certain cases to obtain a whole embryo or larva
from such a portion, the capacity of a part to develop into
a whole is conditioned by the structure of the egg-cytoplasm.
Thus in the Hydromedusae among the Coelenterates this eyto-

Il DIFFERENTIATION 71

plasm consists usually of a finely granular ectoplasm, and
a coarsely granular endoplasm. The first four divisions—
meridional, meridional, equatorial, and meridional—are all
perpendicular to the surface, and each blastomere has a share
of each of these two substances in the same proportions as
they exist in the whole egg. Experiment shows that when
separated from one another half-, quarter-, one-eighth-, and
even one-sixteenth-blastomeres will develop into whole
hydroids or medusae as the case may be. In Carmarina, how-
ever, there is in addition a jelly-plasm, a spherical hyaline
mass placed excentrically near the vegetative pole. This is
really a precociously secreted mesogloea and passes normally
into the exumbrella of the medusa. Isolated half- and quarter-
blastomeres, being produced by meridional divisions, naturally
each include a portion of this as well as of the other two
materials, and they can develop into whole medusae. But
the one-eighth-blastomeres, which must either contain none or
relatively too much of the jelly-plasm, can only develop par-
tially. In Aegineta, another medusa, there is also a jelly-
plasm in the egg, but centrally placed. The one-eighth-blasto-
meres are here totipotent.

Amongst Vertebrates it is possible to obtain a whole larva
from each of the first two blastomeres in the Newt, at any
rate when the first furrow lies in the (invisible) plane of egg-
symmetry, and in the Frog we know that a double monster
can be produced by turning the egg upside down in the two-
celled stage, and so causing each blastomere, by rearranging
its contents, to acquire a new polarity of itsown. In both
these cases, since the first furrow is meridional, each of the
blastomeres has a share of the various substances of the cyto-
plasm which are placed symmetrically around the axis of the
telolecithal egg. We should expect perhaps that after the
third, latitudinal, division the potentialities of animal and
vegetative blastomeres would differ. The evidence—slender
though it is—shows that this is not altogether so, for a
blastopore and archenteron may be developed not only from
the four vegetative cells when the others have been destroyed
(Morgan), but also from the animal cells alone (Samassa).

72 DIFFERENTIATION III

It is, of course, from the former that the structure in question
is derived in the development of the whole egg.

We shall discuss the significance of this later on. It has
been pointed out above that the egg-cytoplasm acquires its
definitive bilateral structure as a result of fertilization. It is
interesting that the removal of a part of the cytoplasm before
the appearance of the grey crescent has far less serious conse-
quences than after the bilateral symmetry has been estab-
lished. In the first case the embryo is normal, in the second
while it may be normal, it is frequently defective in certain
respects or altogether unable to develop (Brachet). )

In Amphioxus, Echinoids, and Nemertines the egg has
a telolecithal structure and a polar radial symmetry. In
Amphioxus the radial is replaced by a bilateral symmetry as
a result of fertilization (Cerfontaine) exactly as in the Frog
and in Ascidians (Conklin). At what moment the bilaterality
is determined in the other two cases is not known.

In all three groups the first two and again the first four
blastomeres have similar shares of the parts of the polar struc-
ture, since the first two divisions are meridional, and in all three
isolated half- and quarter-blastomeres give rise to whole
embryos or larvae of reduced size. But by compressing the
egg of Cerebratulus at right angles to its axis the second
division may be made equatorial; the quarter-blastomeres are
then either animal or vegetative, and behave when isolated
like the one-eighth-blastomeres of the normal egg (Yatsu).
The next division, however, separates animal from vegetative
one-eighth-blastomeres, and these are no longer totipotent, at
least not invariably so. In Amphioxus neither the animal nor
the vegetative cells can gastrulate; in the Nemertines the
vegetative cells gastrulate but form no sense-organ, the animal
cells form a sense-organ but no gut; and in the Sea-urchin
the animal cells usually give rise to long ciliated blastulae,
though they may gastrulate, while the vegetative cells de-
velop more frequently into gastrulae with a pair of skeletal
spicules.

_ There is therefore a difference between the cells derived

III DIFFERENTIATION 73

from the animal and from the vegetative hemisphere in respect
of their developmental capacities.

In the Nemertines there is a similar difference between the
capacities of animal and vegetative egg-fragments, removed
prior to fertilization (Yatsu) ; the vegetative quarter of the egg
gives rise to a larva without sense-organ, the animal fragment
of about the same size, to a larva with defective gut and also
without the apical organ; so that apparently the factor on
which the development of this organ depends is somewhere in
the centre of the egg, that is, not in its definitive position. Into
that position it moves after the first cleavage, since removal of
the animal regions of both blastomeres does not interfere with
its development. At an earlier stage still (prior to the break-
ing down of the germinal vesicle) any nucleated piece of an
ovum can give rise to a normal larva, whatever be the direc-
tion of the cut by which it is removed; that is, since the
nucleus is in the animal hemisphere, a meridional, an oblique
or an animal fragment.

The development of egg-fragments of Sea-urchins is said to
be always complete, provided the fragment be not too small
(Driesch).; but this is a matter which requires re-investigation.
In the Ctenophora the development of isolated blastomeres is
always partial, at least in respect to the costae, 4, 3, 3,3 blasto-
meres giving rise to larvae with respectively 2, 1,3,5 costae, and
soon. The stomodaeum, however, of 4 and j larvae is complete;
and a #larva has four and not merely three endodermal canals.
In the Mollusca as a rule a cell when isolated gives rise to no
more than it would have done had it remained in connexion
with its fellows, except that the cell-mass produced from it
may form a closed vesicle (for instance, isolated 1 a—1ld cells
of Patella (E. B. Wilson)),butin those peculiar cases (I/yanassa,
Dentaliwm), where there is a polar lobe the cell in the two-
celled stage or four-celled stage which possesses it, i.e. either
CD or D, can produce a whole larva. Removal of the polar
lobe from the whole egg involves absence of mesoderm in
Ilyanassa, absence of the post-trochal region, and of the apical
sense-organ in Dentaliwm. Removal of the lobe from the CD
cells involves in the latter genus the same consequences, but

74 DIFFERENTIATION Bi

the larva reared from the lobeless D cell has an apical organ
though still devoid of a trunk. The polar lobe of the egg,
therefore, contains some factor on which the development
of the apical organ, as well as that of the trunk, depends,
and the factor for the sense-organ moves from its original
position in the vegetative hemisphere to its definitive posi-
tion at the animal pole between the first and the second
divisions.

In Ascaris again where the cells from which certain organs
come can be distinguished from the beginning the isolated
blastomeres have only a partial development (Stevens), Thus
S, (Fig. 5, p. 13) gives rise to an ectoblastic vesicle, P, to P,
and # M St and the derivatives of these, and so on. Removal
of one ectodermal cell (A) in the four-celled stage does not,
however, very seriously interfere with the development of the
remaining three into a normal embryo. Strictly speaking,
the blastomeres are not isolated, but one or more is killed by
ultra-violet light.

If, however, the egg be centrifuged it divides meridionally,
and each cell then segments like a whole egg, with diminu-
tion of the chromosomes in the somatic cells. Lastly, in
Ascidians the cytoplasm has a very definite structure (easily
visible in Cynthia, only with the help of reagents in Phallusia),
the parts of which assume their definitive bilateral arrange-
ment as a result of fertilization. This bilaterality persists as
that of the embryo, and since the first furrow (meridional)
coincides with the plane of symmetry and the sagittal plane,
and the subsequent furrows (meridional, latitudinal, and so on)
have very definite positions with regard to the first, the
various regions of the cytoplasm necessarily pass into particu-
lar cells. It is not therefore surprising that a cell should never
be able, when the others have been killed, to give rise to any
more than it would have done in the whole egg. As in
Ascaris and in the Mollusca the potentialities of isolated cells
are restricted ab initio.

While the cytology of the ovum shows us that its cytoplasm
has always a certain structure, though different in different
cases, a structure which assumes its definitive form during

Il DIFFERENTIATION 75

maturation and, in some instances, fertilization, the experi-
ments just reviewed demonstrate the necessity of the parts of
this structure for the development of organs of the embryonic
body ; removal of this or that part entails the absence of this
or that organ. The materials therefore on which the forma-
tion of these organs depends may be said to be preformed in

Fie. 16.—Cynthia partita. Half and three-quarter embryos. (After
Conklin.) a, right half gastrula, dorsal view. The neural plate, chorda,
and mesoderm cells are present only on the right side and in their
normal position and numbers. 8, left half of young tadpole, dorsal
view. The notochord is normal except for size and number of cells;
the muscle and mesenchyme cells are present only on one side; the
neural plate (n.p.) is abnormal in form but not in position. c, right
half of young tadpole, dorsal view; slightly younger than 6. m’ch.,
mesenchyme, ms., muscle-cells. d, left anterior three-quarter embryo,
dorsal view. The anterior half is entirely normal with anterior mesen-
chyme (m’ch.) on both sides. Posterior mesenchyme and muscle-cells
upon the left side only.

76 DIFFERENTIATION Ill

the cytoplasm, though the organs themselves of course are not.
But though preformed they are not prelocalized since—the
apical sense-organ in Cerebratulus and Dentaliwm—they need
not be initially present in their ultimate position.

On the other hand it is possible to divide the ovum into
totipotent parts, which may be either fragments of unseg-
mented eggs or isolated blastomeres. This possibility obviously
depends on the arrangement in the cytoplasm of the various
necessary materials, which must be such that a fragment or
blastomere can receive a portion of each. It decreases with
time in both cases, though for different reasons. The totipo-
tence of egg-fragments diminishes apparently because there
is a redistribution of the materials during maturation and
fertilization, when for instance a bilateral replaces a previously
radial symmetry, but isolated blastomeres cease to be able to
give rise to whole embryos or larvae so soon as the cell-
divisions fall in such a way as to divide the cytoplasm into
unlike portions. Where the cell-plasma is highly differentiated
this occurs at once, as in the bilateral eggs of Cynthia and the
Ctenophores and usually in Molluscs: where the structure is
polar, but not markedly bilateral, cells separated by meridional
divisions are totipotent, those produced by equatorial or lati-
tudinal divisions less so or not at all (Sea-urchins, Nemertines,
Amphioxus). The case of Ascaris is very instructive: nor-
mally the first division is equatorial, but in the centrifuged
egg meridional: in the former the half-blastomeres develop
partially, in the latter totally. The egg of Cerebratulus pro-
vides a similar example ; for the second furrow, which is nor-
mally meridional, may be made equatorial by pressure at right
angles to the egg-axis (Yatsu). The quarter-blastomeres
which in the first case are totipotent, are now no longer so,
since two are animal and two vegetative. Lastly, where the
egg-structure is radially symmetrical about the centre (as in
Hydromedusae, with the exception of Carmarina), the cells
remain totipotent as long as the divisions are perpendicular to
the surface, but when tangential divisions occur by which the
endoderm is delaminated from the ectoderm, this capacity is
lost; at least it is known that these two germ-layers cannot

Ill DIFFERENTIATION 77

replace one another in regeneration. The power of giving rise
to whole structures therefore only disappears. because the
cleavage takes a certain course, and, were the divisions to con-
tinue in one kind of direction, might conceivably be indefinitely
retained, just as the compressed ege of Nereis, or the egg
of Cerebratulus in calcium-free sea-water produces octettes
instead of quartettes of micromeres.

The limitation of potentialities seen in later stages is due
again to the original regional differences in the egg-material.
Thus endoderm and ectoderm can as little replace one another
in a Sea-urchin gastrula as in a regenerating Hydra, or in
a Hydra that has been turned inside out (the experiment of
the Abbé Trembley is well known). It is stated indeed
(Driesch) that any fragment of a blastula of a Sea-urchin can
give rise to as normal a larva as any other, but this needs
re-examination.

On the other hand, organs normally formed by one part of
the embryo can be developed by another. The posterior half-
gastrula of a Nemertine develops an apical organ, that of
a Sea-urchin an apical organ, a stomodaeum, and a tripartite
gut; and here we are in the presence of the phenomenon
which is characteristic of all regeneration, the develop-
ment of a whole structure from a part of a differentiated
structure.

When a (meridional) half- or quarter-blastomere of a polar
egg is differentiated into a complete larva, that appears to be
due to its possessing a share of each necessary substance; but
when a worm regenerates a head, or a newt the lens of the
eye, or a crayfish a limb, this simple explanation will not
avail. Regeneration is not within our present scope, but the
behaviour of certain blastomeres when isolated, raises precisely
the same question.

We have seen that in the Frog not only from the four
vegetative but also from the four animal cells a blastopore
and archenteron can be developed. Similarly in the Sea-
urchin, not only a vegetative, but also an animal, blastomere
can gastrulate, though the latter does so neither as well nor
as frequently as the former. In other words, a structure can

78 DIFFERENTIATION Il

be formed by a part of the germ which is not ordinarily
devoted to that end. We must therefore assume provisionally
some such disposition of the organ-forming materials in the
germ as that suggested by Boveri in his ‘stratification’
hypothesis. According to this conception the substances in
question are distributed through the cytoplasm, but with con-
centrations decreasing in opposite directions, which directions
are, in polar eggs, along the axis. Thus a substance on which
the formation of the ectoderm depends is assumed to be most
concentrated at the animal pole, less in the equator, and least
at the vegetative pole, while there is a similar decrease of
endoderm-forming substance in the reverse direction along
the axis. Both animal and vegetative cells therefore possess
the requisite materials for developing these structures, though
not in the same proportions as in the entire ovum, nor as one
another. Hence the differences in their capacities.

Such a stratification can of course in some cases be actually
observed, for instance in the Amphibian egg, where yolk and
cytoplasm are graded in opposite directions in this way, and
even where not easily visible may be made apparent by the
use of the centrifuge, since the several constituents of the
cytoplasm are frequently of different specific gravities.

The effect of separating these constituents in this way
has now been investigated on several eggs and much light
thereby thrown on the significance of each one in develop-
ment.

Kver since Born’s cytological examination of the Frog’s egg
forcibly inverted in Pfliiger’s original experiment, it has been
known that the heavy yolk-granules sank in the viscid cyto-
plasm while the lighter plasma rose to replace them, so con-
ferring a new polarity upon the egg ; and ever since Hertwig’s
experiment it has been known that by this use of the centrifuge
the yolk could be driven much more rapidly to one side of the
egg than under the influence of gravity alone. Such eggs
frequently develop abnormally, with meroblastic segmenta-
tion, spina bifida of the embryo, and so on. The development
of such centrifuged eggs has been more recently studied by
Konopacka. The eggs, placed on slides in water, are centri-

Il DIFFERENTIATION 79

fuged either slowly for a long time (f= 10g to 12g) or
rapidly for a short time (f= 228 ), and various stages are
used, namely, unfertilized eggs, fertilized but unsegmented
eggs, two-celled stages and eight-celled stages.

1, The eggs are centrifuged while still in the oviduct
(presumably during the second maturation division), then
removed and fertilized.

A. Slowly for five hours. The direction of the centrifugal
force makes any angle with the egg-axis, and some eggs have
the white pole uppermost. The first furrow appears at the
normal time, but is often not meridional, the blastomeres
being unequal. While normal embryos may be developed,
the blastopore sometimes fails to close, and in a few cases
only half the egg is segmented and develops into a half-
embryo.

B. Rapidly for thirty minutes. At the centripetal pole—
which may or may not be the original animal pole—there is
an extrusion of hyaloplasm. The development of these eggs
is as in the previous experiment.

It is to be noted that in these eggs the axis of centrifuging
may make any angle with the original axis, that by the
redistribution of yolk and cytoplasm a new polarity may
therefore be conferred on the egg, and that the symmetry of
the embryo is related to this new and not to the original
polarity, the anterior end being developed at the centripetal
pole, in precisely the same way as in Pfliiger’s forcibly
inverted eggs.

2. Since the perivitelline fluid has been exuded the egg is
free to turn inside the jelly membrane, and places itself upon
the machine with its yolk-pole outwards. The egg is centri-
fuged, from fifteen minutes to one and a half hours after
insemination.

A. Slowly for five hours. The results are very much the
same as before; open blastopores and half-embryos occur.

B. Rapidly—i. Fifteen minutes after insemination for from
ten minutes to thirty minutes. Three layers or strata appear,
at right angles to the egg-axis (which coincides with the axis
of centrifuging); at the animal (centripetal) pole is a layer

80 DIFFERENTIATION III

of yellow hyaloplasm, next this a layer of pigment, and
finally the unchanged yolk may be seen to come to the
surface at the vegetative (centrifugal) pole.

The embryos have depigmented heads, or are headless
altogether, the anterior end being occupied by a swollen
vesicle. The degree of deformity depends on the length of
exposure to the centrifugal force.

ii. When the eggs are centrifuged one and a half to three
hours after insemination for fifteen minutes or longer a fourth
white stratum appears, between the yellow hyaloplasm and
the pigment. The first division is usually unequal, and the
small blastomere segments more rapidly than the large one.
Many eggs die before gastrulation. Amongst those that do
develop half-embryos and headless monsters are frequent.

3. The egg is centrifuged in the two-celled stage.

A. Slowly for five hours. The first division is unequal,
and there is a tendency to meroblastic segmentation.

B. Rapidly for from five to twenty-five minutes. The same
strata appear in each blastomere as in the second experiment,
and, with the longer exposures, there are abnormalities of
development, such as meroblastic segmentation.

4, The eggs and anterior half-embryos are centrifuged
rapidly. After the completion of the third furrow the strata
appear in each of the eight cells. There is a tendency to
meroblastic segmentation, open blastopores, and anterior half-
embryos. These experiments make it perfectly clear that
derangement of the substances of the cytoplasm involves
abnormality in development. In the first place the yolk is
driven to the vegetative pole, and the distinction between the
protoplasmic and deutoplasmic regions of the egg thereby
increased, The yolk fails to segment, but the cytoplasm of
the animal hemisphere divides, and gives rise to a blastoderm,
The displacement of the yolk entails later on malformation of
the posterior end, normally developed near the vegetative pole,
with consequent persistence of the blastopore and restriction
of differentiation to the anterior half of the embryo. The
heads of these embryos are depigmented and the pigment is
therefore presumably inessential, but when the derangement

= ER DIFFERENTIATION 81

is more serious, possibly because some other material is driven
from the animal pole into the interior of the egg, there is no
head at all,

Hertwig and Wetzel have studied the effect of centrifugal
force upon the unfertilized egg.

Similar experiments have been performed on other ova.
We turn first to those, carried out by Lyon, Morgan, and
Spooner upon the eggs of the Sea-urchin Arbacia, in which
there is a diffuse red pigment.

If the ripe but unfertilized ovum be strongly centrifuged
(f = 6400 g) four strata appear. The pigment passes to the
centrifugal pole, next to this is a grey granular layer,
blackened by osmic acid, then a fluid hyaline layer in which
lies the nucleus, while the centripetal pole is occupied by
a cap of opaque white material. The new axis of stratifica-
tion which is thus produced by the operation may make
any angle with the original axis as determined by the
micropyle.

When removed from the centrifuge the strata begin to
remingle, but the first and fourth return to their original
positions very slowly if at all. The second and third layers
on the other hand intermingle with one another rapidly, and
it is apparently necessary that they should do so before
segméntation and development can occur; for if the egg be
broken into two portions between them, then neither portion
can be fertilized.

In segmentation it is the axis of stratification which deter-
mines the direction of the furrows, since the first three, which
are at right angles to one another as in the normal egg, either
include or are at right angles to this axis, or, the axis of
stratification coincides with one line of intersection between
some two of these three divisions. At the next division
the micromeres are formed at that intersection of two
furrows which is at the anti-micropylar pole or nearest to
it (when the axis of stratification is oblique to the original
egg-axis).

It appears therefore that some invisible polarity of the egg
has remained unaffected by the centrifugal force, and this

1963 G

82 DIFFERENTIATION III

determines the symmetry of the embryo, since the micromere
pole becomes the blastopore pole, and the original egg axis the
gastrula axis, or as nearly so as possible. The pigment is found
in any part of the larva, right or left, dorsal or ventral, anterior
or posterior. It is not therefore essential to development. It
may be added that the yellow pigment band of Strongy-
locentrotus is equally unnecessary. Normally it is subequa-
torial and passes into the archenteron, but it may be meridional
or oblique to the egg-axis, and so become incorporated wholly
or partly in the ectoderm (Garbowsk)).

Experiments of a like kind on other eggs have yielded like
results; for while the existence of an invisible structure has
been revealed, a structure which is not disturbed by the centri-
fuge and is definitely related to the subsequent differentiation,
that differentiation has been shown to be independent of the
distribution of some at least of the visible constituents of
the cytoplasm.

Thus Lillie, by centrifuging the egg of Chaetopterus during
the first maturation division, produced in it three layers:
a small grey cap at the centripetal pole, a clear layer, and
a yellow granular hemisphere (on the centrifugal side). These
strata, it was found, might occupy any position with regard
to the egg-axis (as defined by the polar bodies), yet in
fertilization the sperm always entered at the vegetative pole,
and cleavage was always normally related to that axis. The
grey cap is derived from the contents of the germinal vesicle,
the clear band fromthe mié¢rosomes of the endoplasm, and the
yellow granules from the coarser endoplasmic constituents.
It would be interesting to know the further history of these
centrifuged eggs.

This we do know in other cases.

The ovum of the Lamellibranch Cumingia contains a red
pigment and an oily green material both scattered through
the cytoplasm. When the egg is centrifuged during the
first polar division (Morgan) these go to opposite poles, the
red pigment to the centrifugal, the green oil to the centri-
petal. Between the two is a broad hyaline layer. Maturation
proceeds and the polar bodies are extruded.

III DIFFERENTIATION 83

With the egg-axis, as so determined, the axis of stratifica-
tion may make any angle. Fertilization occurs, and in the
subsequent cleavage the planes of division bear the normal
relation to the axis of the egg. The strata persist, so that
the red pigment may be in AB or CD and so on, the green
oil on the opposite side. Development follows and these two
coloured materials are found opposite to one another in any
position in the trochophore larva, the structure of which has
the normal relation to the cleavage system. There does,
however, appear to be some tendency for the green oil to
redistribute itself.

So again in Pulmonate eggs (Physa, Planorbis, Limnaea)
Conklin has, by the same means, produced three strata: a
grey finely granular zone at the centripetal pole, a narrower
clear zone, and a yellow granular centrifugal hemisphere.
When segmentation and development take place the strata
make any angle with regard to the first and subsequent
furrows, and any angle with the principal planes of the
embryo. Conklin has, however, added the important observa-
tion that the possibility of obtaining a normal development
is largely dependent on the redistribution of some of the
disturbed cytoplasmic materials, for it is only when the
operation is performed prior to maturation or during its
earlier stages—only, that is, when some time elapses between
the operation and cleavage—that development is afterwards
normal. Eggs centrifuged during the extrusion of the first
polar body, or later, either die or give rise to monstrous
embryos. It appears further that during the interval, the
clear substance disappears into the grey or the yellow layer
or both, a readjustment which cannot occur unless sufficient
time be allowed. In another Mollusc, Crepidula, on the
other hand, and in the Ascidian Cynthia, Conklin has found
it possible, by prolonged centrifuging, to shift the original
polar axis (which in the experiments just quoted is left
unaltered) without prejudice to normal development. The
symmetry of cleavage and of differentiation are, it seems,
determined by the new polarity as in the Frog’s egg.

Soa Cyclops (Spooner) the centrifuge separates the cytoplasm
G2

84. : DIFFERENTIATION III

into three similar zones: a greenish-white layer at the centri-
petal pole, a middle clear stratum, and the blue yolk-granules.
These eggs develop normally even when continuously centri-
fuged.

In the Rotifer Hydatina (Whitney) the polar zones are
pink and grey, the middle clear as in the foregoing instances.
The stratification may have any relation to the original axis ;
the first cleavage is, as in the normal egg, transverse to this
axis, and normal young are precyee become mature, and
reproduce in their turn.

Lastly, in the centrifuged egg of Ascaris similar strata
appear. The normal egg, as pointed out already, is telo-
lecithal. There are in the cytoplasm also some clear spherules
and pigment granules. The layers that appear after centri-
fuging are a layer of yolk at the centripetal end (the yolk
is here lighter than the cytoplasm), a layer of clear spherules,
protoplasm, and finally, at the centrifugal pole, a cap of brown
pigment. When strongly centrifuged, the egg becomes flat-
tened against the slide on which it is placed (the centrifugal
force is perpendicular to the slide), and, if still subjected to
the action of the force, the fertilization spindle places itself
at right angles to the direction of the force, that is, parallel
to the stratification (and in the clear zone) and the division
is meridional. If, on the other hand, the egg is removed from
the machine it resumes its spherical shape and the spindle
returns, more or less completely, to its proper axial position
and the first division is equatorial (or oblique). It is sug-
gested by Boveri and Miss Hogue (to whom the experiments
are due) that there is an invisible polarized structure in the
cytoplasm which is not affected by the operation, and with
the axis of which the stratification of the movable substances
_ can make any angle. Into the axis of this invisible polarity
the spindle is supposed to return, if and when the egg is
allowed to resume its spherical shape. The facts do not
appear to necessitate this view, for when placed on the
machine the whole egg rotates inside its shell until the
heavier animal pole is centrifugal and then the stratification
of the cytoplasmic materials begins. As long as the force is

II DIFFERENTIATION 85

operating, the spindle is compelled to place itself parallel to
the stratification, but when released from the force, returns
or attempts to return to its normal position, namely, in the
egg (i.e. in the stratification) axis. The obliquity of the
spindle, in those cases where the return to the normal
position is not complete, would then be the result of two
tendencies at right angles to one another, the one urging the
spindle to place itself perpendicular, the other parallel, to the
stratification.

When the spindle returns more or less completely to its
normal situation, the division is equatorial or oblique and
a normal embryo is developed in spite of the stratification.
When, however, the spindle remains in the stratification plane,
the first division is meridional and each cell behaves as the
P, (vegetative) cell of an entire ovum. The greater part of
the pigment zone is usually extruded from these eggs at
the centrifugal pole, as a ‘ball’. Each half-blastomere divides
into two, which can be recognized as HM St and P, by the
chromosomes being diminished in the one and intact in the
other, and by their subsequent behaviour, and so gives rise
to.what is essentially a blastula without ectoderm (see Fig. 5,
p. 13). It might be imagined that the ectodermal material
had been extruded with the ‘ball’, but apparently this is not
so since the development is the same when (as may happen)
no ball is extruded.

It must be admitted that it is at the moment very difficult
to build any very definite hypothesis upon the results of
these various experiments with the centrifuge. It is obvious
that by the rearrangement of some at least of the constituents
of the cytoplasm a stratified polar structure may be easily
imposed upon the egg, and that it is certainly not necessary
that all of these materials should return to their original
positions in order that development may be normal. The
evidence does, however, indicate here and there (the Frog, the
Sea-urchin, the Pulmonate) that time must be allowed for
a recovery of some kind before development can be normal.
The axis of the new polarity may entirely replace the original
axis in the determination of embryonic symmetry (as in the

86 DIFFERENTIATION Ill

Frog) but certainly does not in other cases, where it appears
that the old axis persists unaffected by the operation, not
marked by any visible differentiation of materials, but causally
related nevertheless to the symmetry of cleavage and develop-
ment. That this axis has not been affected by the centrifuge
hitherto does not, however, justify the assumption that it
cannot be, and indeed Conklin has succeeded in shifting it
in Crepidula and Cynthia. The polar structure to which it
belongs may, therefore, eventually prove to be dependent on
‘the heteropolar arrangement of certain odplasmic substances’,
though these are indistinguishable to the eye, and need not
of course be of sufficiently different specific gravities to allow
the force applied to overcome their viscosities.

Taking all the results together, it seems that since it
has been demonstrated that the different regions of the
cytoplasm do play definite but different parts in development,
are in fact determinants of characters which form an integral
portion of the total inheritance of the species, the time is
ripe for the physical and chemical investigation of the pro-
perties of the egg-plasma.

There remains for discussion Boveri's description of the
development of dispermic eggs in Ascaris, the history of
which shows clearly the influence upon the nucleus of the
different regions of the cytoplasm.

These doubly fertilized eggs divide simultaneously into
four cells, arranged in a tetrahedron.

The subsequent cleavage is of either one of three types
according as there are one, two, or three P, cells, each at the
end of a T-piece (Fig. 5, p. 138).

I. One of the four cells divides so that of its two products
the outer touches its sister cell but no other cell in the germ.
This cell is P, and divides into EMSt and P,. The three
remaining cells are together equivalent to the AB of normal
development and give rise to ectoderm. The somatic cells
are recognized of course by the diminution of their chro-
matin, while the germ-cells have whole chromosomes.

II. Of the four cells two behave as P,, two as S, and there

III DIFFERENTIATION 87

are therefore, after the next division, two figures of 7, each
of which continues to segment in every respect like a
whole egg.

III. This type occurs frequently in centrifuged eggs. Of
the four cells one is large and yolkless (animal), and behaves
as S,; while the remaining three are small and full of yolk,
and each develops as a P,.

The disperm eggs continue to segment, but give rise to
irregular cell masses incapable of development beyond the
gastrular stage, and that only in type I. Two primordial
germ-cells were found in these gastrulae.

The number of chromosomes in these eggs is of course 3 7.
There are four centres between which spindles are developed in
various ways to form a quadripolar figure, the chromosomes
being irregularly distributed on the equators of these. There
they divide, and their halves are pulled in the ordinary way
to the spindle-poles. The four cells may and do receive,
therefore, different shares of the available 6 , i. e. twelve chro-
mosomes. Now in the normal egg there are always 2n
chromosomes that remain intact, in each cell in the germ-
track. If, therefore, the diminution were an intrinsic pro-
perty of certain chromosomes (namely, the somatic) while
conversely the intact chromosomes remained so by virtue of
some internal cause, then there should be in a dispermic
egg exactly six intact, and six diminished, chromosomes,
and these would be irregularly distributed over the four cells
and their descendants. The reverse of this is the case. In
certain cells, the number of which differs in the three types,
the chromosomes remain entire, whatever their number may
be (it may be any number from two to twelve), while in the
other cells the chromosomes are diminished, whatever their
number. We know further that the diminution occurs in
the dispermic, as in the normal egg, in the cells of the animal
region, while in the vegetative cell or cells it is absent. It
is necessary therefore to suppose that it is some difference
in the cytoplasm of the animal hemisphere which causes the
diminution,

We shall see in the next section that while there is evidence

88 DIFFERENTIATION III

that the chromosomes of the germ-cells are unlike one
another, yet the entire set contributed by each germ-cell is
handed on to every cell in the body, and indeed must be if
development is to be normal. The experiment which has
just been described suggests that the cytoplasm, different
in the various cells of the body, might incite to activity
different elements of the chromatin in each case.

B. THr NUCLEUS.

The experiments we have so far considered have shown us
that while the nucleus is not qualitatively divided during
segmentation and subsequent development, the cytoplasm, far
from being isotropic, is heterogeneous, and its several parts
causally related to the differentiation of certain elementary
organs.

It does not, however, follow for a moment that the nucleus
has no role to play in the process of differentiation, and there
are indeed very sound reasons for believing that it is a vehicle
of inheritable characters.

1. For in the first place experiments on Protozoa have
proved that the nucleus, though not essential for irritability
and locomotion, nor even for the ingestion of food, is yet
necessary for the functions of metabolism and reproduction.

An enucleate piece of an Amoeba can ingest and even
partially digest food, but the products of digestion cannot
be assimilated. Any portion of an Infusorian (that is not
too small), provided it contains a piece of the nucleus, can
replace those parts of the whole structure which it lacks, can
reproduce the original form.

2. Secondly, the study of the maturation of the germ-cells
has shown that these elements, while unlike in every other
respect, are yet identical in the number and size of the
chromosomes of their nuclei (with the exception only of the
heterochromosomes or sex chromosomes in Insects and others).
Similar characters being inheritable from either parent, the
determinants of such characters have naturally been imagined
to reside in the nuclei, that is, in the chromosomes of the

Ill DIFFERENTIATION 89

germ-cells. Moreover, in fertilization the acrosome, sperm-
head or nucleus, and centrosome, alone are essential, for the
tail may be left outside. The functions of the acrosome—
ensuring the entrance of the spermatozoon—and of the
centrosome—making the sperm-sphere—are known: hence
the nucleus is the seat of the determinants of those characters
that can be transmitted by the male, and these are similar
to those transmissible by the nucleus of the female. At the
same time we know from the phenomena of merogony and
artificial parthenogenesis that both nuclei are not necessary,
but that one set of n chromosomes will suffice.

We have now to consider the evidence for believing that
these n chromosomes are really different from one another.

3. Roux long ago pointed out that karyokinesis looked like
an apparatus for simultaneously dividing and distributing
to two cells a number of qualitatively unlike bodies, but the
experimental proof of the dissimilarity has only recently
been brought forward by Boveri.

This proof is based on the behaviour of dispermic eggs of
the Sea-urchin Strongylocentrotus lividus.

The dispermy is caused, not by treatment with a poison
(as in the experiments of the brothers Hertwig), but merely
by adding a large quantity of sperm to the ova.

Of these dispermic eggs the following types may be dis-
tinguished :

I. The tetraster (each sperm produces a centrosome which
divides), followed by simultaneous quadripartition. There
are spindles between all four centres.

a. Plane tetraster. The four cells lie in one plane, parallel
to the equator of the egg.

Another meridional division gives eight cells ina ring. An
equatorial cleavage separates animal from vegetative blasto-
meres, and there follows the formation of sixteen mesomeres,
eight macromeres, and eight micromeres.

b. 'Tetrahedral tetraster.

There are never eight micromeres and macromeres, but
either six or four of each.

II. The double spindle.

90 DIFFERENTIATION III

One sperm nucleus with its two centres and the spindle
between them remains apart from the other. The latter
conjugates with the female pronucleus. |

a. Both spindles lie in the same plane.

b. They are at right angles to one another, that is tetra-
hedrally placed.

These eggs usually divide into two, occasionally (Teichmann)
into four. On one side of the egg are nuclei with 2, on the
other nuclei with 2 chromosomes. (‘These double-spindle eggs

Fig. 17.—Diagram of one case of irregular chromosome distribution
in a doubly fertilized egg ; ay, b,, cy, and dy; dg, bg, co, and d,, and dg, bs,
cz, and d, are the three complete, specific sets of unlike chromosomes.
(After Boveri, 1904.)

have been referred to already in another connexion. To our
present purpose they are irrelevant.)

III. The triaster.

By shaking the eggs the division of one centre is prevented.

a. Triaster proper, with spindles between all three centres.

6. Amphiaster-monaster, in which one centre remains apart.

The triaster divides simultaneously into three by meridional
divisions, and then into six by another meridional cleavage.
An equatorial division is followed by the production of twelve
mesomeres, SIX macromeres, and six micromeres.

IV. Both centres may fail to divide.

a. An amphiaster is formed between them.

III DIFFERENTIATION 91

b. They remain apart.

The first and third types are those which immediately
interest us. In these tetracentric and tricentric, simul-
taneously quadripartite and tripartite ova, the 8n chromo-
somes divide and the 6” chromosomes are then thrown at
random on the equators of the spindles connecting the various
centres. In the anaphase of the mitosis the daughter chromo-
somes pass to the four or three centres, and the four or three
cells consequently receive a random number of the elements.
Assuming for the moment that the chromosomes are qualita-
tively unlike, each cell also receives a perfectly random assort-
ment of them, and the chance of every cell receiving at least
one of each kind of chromosome is very small indeed, but
greater of course for the tripartite than for the quadripartite
ova. The chance, however, that one cell will receive a full
complement is much larger.

A study of the development shows that the quadripartite
practically never develop normally, the tripartite sometimes
but not often, while, if the four cells of the former or the
three cells of the latter be isolated and allowed to develop
independently, a very fair percentage of normal larvae is
obtained. All four, or three, develop differently. In the first
case one or even two may give normal larvae, but never all
four, while of the three cells of the triasters one or two, rarely
all three, reach the pluteus stage.

The same differences between the blastomeres are seen when
they develop in connexion with one another.

The tripartite ova gave a mean of 8% normal larvae, out of
828 reared in all.

The normal larva consists of three regions, marked by the
different size of the nuclei, dependent on the different number
of chromosomes in the original three cells. The egg-axis,
segmentation-axis, and gastrula-axis being all coincident, the
boundaries between the three regions meet in the blastopore
at one end, the animal pole (anterior end) at the other. One
boundary is generally in the median longitudinal plane.

Special cases are (1) where the nuclei are not of different
sizes and the regions therefore indistinguishable; (2) where

92 DIFFERENTIATION Ii

the dimensions (surfaces) of the nuclei are as 1: 2:3; this is
accounted for by supposing only two of the centres to have
been united by spindles (this was actually observed) one of
which was occupied by one sperm-nucleus, the other by the
second male and the female nucleus: on division the centres
would receive only 2” and 3n chromosomes. (8) By supposing
the numbers of chromosomes on the three spindles to have

been 55 and 2n, the centres, on the division of these,

would receive n, 24n, and 2in. This would account for the
ratios of the dimensions of the nuclei observed in the three
regions.

Lastly, amongst these larvae are asymmetrical forms. It
is suggested that this is due to some slight difference between
the sperms, such differences having been in fact observed
amongst larvae of the same monosperm culture.

In the remainder of the embryos or larvae reared from
tripartite ova, more or less serious defects were found. Thus
the skeleton is incomplete or wanting in one part, the pigment-
cells may be entirely absent from some one region, but never
from the same part. The defective region is always charac-
terized also by the size of its nuclei, that is, is derived from
one of the original three cells. In other cases one-third, two-
thirds, or all three are pathological, that is, break up into cells
which pass into the blastocoel and there degenerate. The
remainder (if two-thirds) may develop into a normal larva
if the pathological one-third has been got rid of at an early
stage, but if not till later, then the two-thirds larva shows
irregularities in skeleton. These irregularities are supposed
to depend on (1) the relation of the triaster to the (assumed)
plane of bilateral symmetry in the ovum, and (2) the position
of the degenerate cell, whether dorsal, ventral, right, left,
anterolateral, or posterolateral. The nuclei in the two regions
of the sound part may be of different sizes.

When two-thirds are degenerate only stereoblastulae or
stereogastrulae are produced.

Of 1,600 quadripartite ova kept under observation, only
thirteen reached the pluteus stage, and only three of these

II DIFFERENTIATION 93

really deserved the name of pluteus. Of these three one had
to be removed to the category of double-spindled eggs, another
was probably a # egg, while the third had an abnormal
skeleton. In this last there were three sizes of nuclei, small
in one quarter, medium in one quarter, and large in one half
of the larva, and a supposed distribution of chromosomes in
the tetraster is suggested to account for this.

In the remaining ten, which did not deserve the name and
style of pluteus, there were disintegrated cells in the blastocoel,
imagined to be derived from one of the four blastomeres, while
three areas characterized by the different size of their nuclei,
and each one-third of the whole, were found in the larva.

Usually, however, only one half or one quarter of the egg
develops.

The abnormality of development is considered by Boveri to
be directly due to the irregular distribution of chromosomes,
which is such that the chance of each cell in a quadripartite
tetraster egg receiving a full set at least of the n chromosomes
is practically negligible. The chance of each cell of a tripartite
triaster egg receiving a complete set is however appreciable,
and a certain percentage of these develop normally. The
chance of one cell in either case receiving the total comple-
ment is greater still, and these when isolated give rise to
normal plutei in a fair proportion of the cases.

It is concluded, therefore, that the chromosomes are different,
and that at least one complete set of the n chromosomes of the
species is necessary, not merely in the ovum, but in every cell
into which that ovum divides in order that its development
may be normal.

Such alternative hypotheses, as that the abnormality
observed is due merely to irregularities in the number of
chromosomes, are negatived by the known normal develop-
ment of plutei with nuclei of different sizes in the different
regions of the body, the sizes being determined (see above) by
the number of the chromosomes, and being such that the
number may have been less than n, or greater than n, and in
the latter case either a multiple of n or not.

The chromosomes are therefore different, and probably them-

94 DIFFERENTIATION III

selves made up of unlike elements, the elements which conju-
gate in the synapsis prior to the maturation divisions. For
normal development of the whole and of each part a complete
set must be present in every cell. In sexual reproduction two
such sets are present, but one suffices.

To the evidence thus brought forward by Boveri must be
added certain cytological observations on the different size
and form of one or more of the chromosomes. Thus Sutton
found that in the Insect Brachystola the chromosomes of the
spermogonia could be arranged in 7 pairs according to their
sizes, and that in the maturation divisions the members of the
several pairs conjugated and then separated from one another,
Baltzer again and Tennent have found straight, hook-shaped,
and horse-shoe shaped chromosomes in Echinids, while Wilson
and others have demonstrated the heterochromosomes in many
Insects and possibly some other animals.

In what way these various chromatic elements call forth
the development of those characters which they transmit is
unknown, but it seems certain that the differential activity of
nuclei which are alike must depend on differences in the
environment in which they are placed, that is, on differences
in the cytoplasm. Such we know to exist, and we know also
that they can provoke dissimilar behaviour in similar nuclei.
We are therefore brought back to the structure of the cyto-
plasm as a necessary condition of the transmission of characters
by the nuclei.

It remains for us to consider exactly what kind of characters
are handed on by the plasma directly and by the nuclei.
Experiments on heterogeneous hybridization enable us to give
at least a provisional answer to this question.

The possibility of fertilizing an ovum with the spermatozoon
of an animal of quite a different kind was the discovery of
Loeb, who found that by the addition of a small quantity
of calcium chloride and sodium hydrate to sea-water, the
egos of the Sea-urchin Strongylocentrotus purpuratus would
permit the entrance of spermatozoa of the Starfish Asterzas
ochracea. From these eggs pluteus larvae with the charac-

Ill DIFFERENTIATION 95

teristic skeleton are developed. The starfish larva is of course
the Bipinnaria, without skeleton. The problem was taken up
next by Godlewski, who used the Crinoid Antedon as the male
parent, the Sea-urchins Sphaerechinus, Echinus, and Strongy-
locentrotus as the female. The sexual elements must first be
treated with hyperalkaline sea-water.

_ Fertilization is perfectly normal with production of a
vitelline membrane, rotation of the sperm-head, union of the
male and female pronuclei, and so forth. Cleavage is of the
Echinoid type with formation of micromeres at the fourth
division; no such micromeres occur in Antedon, where the
fourth cleavage is meridional in both hemispheres. Primary
mesenchyme (absent in Antedon) is formed, gastrulation
follows, and then a typical pluteus with the characteristic
skeleton is developed. The larva of Antedon has no skeleton.
The Antedon chromosomes persist, and the nuclei of the
hybrids are intermediate in size between those of the parent
forms. Baltzer, who has repeated the experiment, confirms
this as well as all other details.

Enucleate egg-fragments of Echinus were also fertilized by
Godlewski with Antedon sperm. The details of fertilization
were normal, but segmentation was irregular, and after gastru-
lation development ceased. Primary mesenchyme was differen-
tiated and the archenteron inclined to the oral side, both
Kchinoid characters.

In spite, therefore, of the persistence of the male chromo-
somes the larva is of the pure maternal type.

The Sea-urchin egg may also be fertilized by the spermato-
zoa of Molluscs and Worms.

Loeb, employing the Mollusc Chlorostoma, found that a
membrane was extruded, that cleavage was normal in form
and rate, and that normal plutei were developed. The cytology
was not investigated.

A more extensive series of experiments is due to Kupelwieser.
If the egg of Strongylocentrotus or Echinus be fertilized with
the sperm of the Mussel Mytzdus, no membrane is formed and
polyspermy is frequent. The sperm-nucleus having rotated,
preceded by its sphere, moves towards the egg-nucleus.

96 DIFFERENTIATION III

Segmentation is irregular, but the swimming blastulae be-
come spherical and give rise to normal plutei, at the normal rate.

Though the male nucleus unites without fusing with the
female nucleus, in the spindle developed between the two
sperm-centrosomes it does not break up into chromosomes,
and eventually passes undivided to one pole and into one
cell. The female chromosomes divide in the usual way. The
sperm-nucleus degenerates ultimately. It may not even have
approached the female nucleus in the earlier stages, but have
remained apart from the beginning.

The same author has found that membrane formation can
be incited in the Sea-urchin (HZchinus) egg by spermatozoa
of a number of Molluses (Lithodomus, Mactra, Modiolaria,
Pecten, Venus, Patella, Gibbula, Murex, Nassa, Trochus,
Fusus) and by those of the Polychaets Aricoa and Audow-
nia. Only when Mytilus, Mactra, Patella, Aricia, and
Audouinia are used does development follow. It has been
shown elsewhere that membrane formation and the incite-
ment to develop are two independent phases of fertilization.

For ensuring fertilization with Audouinia sperm treatment
with alkali is unnecessary. The sperm enters, the head swells
up, and a centrosome and aster are developed. The male
nucleus may then unite more or less completely with the
female nucleus, or remain apart. In the latter case it passes
into one blastomere and degenerates, as does the Mytilus
sperm in the Strongylocentrotus egg. In the former case
when the spindle is developed and the female chromosomes
are placed upon it, the sperm chromatin is seen in the form
not of chromosomes but of one or more irregular lumps lying
outside, on, or inside, the spindle.

When in the anaphase the female chromosomes become
vesicular, the male lumps of chromatin do the same; they
are distributed sometimes to both blastomeres, sometimes to
one only. In the blastomeres the sperm chromatin may unite
with the (female) nuclei but need not do so, and there is
always a tendency for it to become gradually eliminated, for
it is found in fewer and fewer cells as development proceeds,
and the nuclei are found to be of half the size (surface) of the

III DIFFERENTIATION 97

amphikaryotic nuclei of normally fertilized ova. The irregu-
larity in the behaviour of the male chromatin sometimes
affects the female, and one or more of the cells may fail to
obtain a complete set, and this is put forward as the cause of
the irregular development of some of the larvae.

The segmentation of these ova is of the usual Echinoid
type (in a certain percentage of cases) and swimming blastulae,
and later, plutei, are produced from them. The rate is
slow.

Godlewski has found that when Echinoid eggs are fertilized
with the sperm of Chaetopterus or Dentalium the elimination
of what is presumably the male chromatin takes place after
the male and female pronuclei have fused, but before the
chromosomes and the spindle appear.

In all these eases the larva is of the purely maternal type
in every respect; it is a pluteus, and shows no trace of any of
the characters of the larva of the male parental form. The
earlier processes of cleavage and gastrulation are also those
characteristic of the ovum employed. The male chromosomes
may persist or be sooner or later eliminated. In no case do
they exert the slightest influence.

Identical results have been obtained by Moenkhaus and
Loeb with the heterogeneous hybridization of fishes. The
egg of Fundulus heteroclitus is employed, while the distinct
genera, Menidia, Ctenolabrus, and Stenotomus have served as
the male parent. In fertilization the male (Menidia) chromo-
somes have been shown by Moenkhaus to be properly formed,
and to persist in later stages. From the egg arises an embryo,
which is however defective with cyclops eye, small head,
pigment not distributed over the blood-vessels, and circula-
tion not established though the heart is formed. The pig-
ment-cells, red and black, are those characteristic of the
female parent; red pigment not being present in any of the
forms used as males. It appears, however, that the red pig-
ment may be transmitted by the sperm, since in the reverse
cross of Menidia ? x Fundulus o7 it appeared in two cases.

It does not seem possible at this moment to say whether
the total failure of the male characters to appear in these

1963 H

98 DIFFERENTIATION If

heterogeneous hybrids is due to the inability of the male
chromosomes to exert their activity in a cytoplasm to which
they are not adapted, or to the fact that such characters as
appear are not carried by the chromosomes at all. If the
latter were true then it might be supposed—since the germ-
cells of the two sexes each carry a complete set of the
necessary specific chromosomes—that some at least of the
characters of the larvae were not carried by the female
chromosomes either, but simply by the cytoplasm, a supposi-
tion which is certainly strengthened by the appearance of
certain Echinoid characters—the primary mesenchyme, and
the inclination of the archenteron to the oral side—in the
gastrulae reared from enucleate Sea-urchin egg-fragments ferti-
lized with Antedon sperm. In addition to this there is the
evidence from the defective development of ova from which
certain parts of the cytoplasm have been removed.

While therefore we know that the determinants for some
characters reside in the cytoplasm, the results of heterogeneous
hybridization certainly suggest that these characters are the
large ones, those that put the organism in its phylum, class,
order, and family, the characters that make it an Echinoderm
and not a Worm or Mollusc, a Sea-urchin and not a Starfish
or a Feather-star, while the smaller characters, generic, specific,
individual are transmitted by the nucleus. The former are
perpetuated therefore by the female parent alone, the latter
equally by both parents.

In the last resort, as we know, the cytoplasm is indebted to
the nucleus for several contributions to its structure—the ‘ yolk-
nucleus’ and the contents of the germinal vesicle at matura-
tion—but these are processes which find no counterpart
during the formation of the male cells, except perhaps in the
seemingly insignificant chromatoid accessory bodies.

It has been suggested (by Driesch) that the characters
handed on through the cytoplasm are those that appear early
in development, while those that arise later are carried by
the nuclei. This is probably in the main true, as we have
known, since von Baer taught us, that the general appears
before the particular—‘aus dem allgemeineren Typus bildet

III DIFFERENTIATION 99

sich also der speziellere hervor’. It is also in accord with
the hypothesis put forward above that the various determinants
comprised in the chromosomes will only be able to become
active under the influence of different parts of the cytoplasm—
only, that is, after the nuclei have been distributed during
segmentation.

Further, early characters, such as the rate of cleavage, are
known to be sometimes transmitted by the male cell as in
hybrids of Fundulus (Newman); the rate may very well
depend on the male centrosome, but that is of nuclear origin
though not derived from the chromosomes.

It seems preferable therefore to base the distinction between
the parts played by the two germ-cells in inheritance on the
magnitude of the characters concerned.

The cytoplasm may of course carry some specific characters,
such as the pigment of the egg and of the embryo, for instance
in Amphibia; the cuticle, which may persist as that of the
larva in Polychaets and the size of the egg, which may be
correlated with that of the embryo, though this last is not
necessarily so, since Conklin has found that the larger species
of Crepidula usually have the smaller eggs.

It is to be observed that the characters which common
experience and the experiments of breeders and those interested
in inheritance suggest are transmitted by the male as well as
by the female, and therefore through the intermediation
of the nucleus, are just these smaller individual, varietal,
specific and generic characters. The larger characters have
always been tacitly omitted from consideration, for the simple
reason that the investigation of them could only become
possible by the discovery of some means of bringing about
heterogeneous fertilization.

Into the evidence, based on common experience and scientific
experiment, for the transmission of characters of some sort
by the male parent, and therefore by the nucleus, it is quite
unnecessary to go at length; but the hybrids between various
genera of Sea-urchins have been such classical objects for
investigations of this kind, ever since the days of Boveri’s
supposed production of a dwarf larva with the characters of

H 2

100 DIFFERENTIATION III

the male parent (Zchinus) from a fertilized enucleate frag-
ment of a Sphaerechinus egg, that brief mention may be made
of the more recent researches.

It will be recalled that the plutei of the two genera Echinus
and Sphaerechinus differ typically from one another, according
to Boveri. The latter (Fig. 18) is short and squat, the oral
lappet not divided into lobes, and the skeleton provided with
a fenestrated anal arm—produced by three long parallel bars
united by numerous cross-bars—an apical branch to the oral
arm and, at the apex, a square ‘frame’ formed by the union of
twigs from the last-mentioned and from the apical arms. The
former (Fig. 19) is long and lank, the oral lappet is deeply

2) -€

Fra. 18.—Pluteus of Sphaerechinus granularis from in front and from
the side. (After Boveri, 1896.)

cleft, and in the skeleton the anal arm is not fenestrated ;
there is no apical branch to the oral arm, and the extremity
of the apical arm is thickened and club-shaped.

The hybrid larvae (Fig. 20) were described by Boveri as
being intermediate in form, shorter and broader than those
of Echinus, longer and narrower than those of Sphaerechinus,
and having the oral lappet slightly divided; while in the
skeleton, the extremity of the apical arm was swollen and
branched, the anal arms double but not fenestrated, and the
oral arms sometimes provided with an apical branch.

Seeliger, however, pointed out that there is much variability

Ill DIFFERENTIATION 101

in the parental types—the extremity of the apical arm of
Echinus, for instance, is not always club-shaped—and that
in the hybrid cultures there are to be found, together with
variable larvae of more or less intermediate type, individuals
with purely paternal characters, though the pure Sphaerechi-
mus type never occurs. This contention has been upheld
by Morgan and by Steinbriick, the latter of whom has used
Strongylocentrotus, the pluteus of which is hardly distinguish-
able from that of Hchinus, as the male parent. Steinbriick
i

Fie. 19.—Pluteus of Beahnus microtuberculatus from in front and from
the side. (After Boveri, 1896.)

finds that the pluteus of Strongylocentrotus is variable (1) in
the termination of the apical arm, (2) in the anal arm, which
may fork, or be double or even treble throughout, though
cross-bars are not seen, and (3) in the length of the oral and
of the transverse arms. In the pluteus of Sphaerechinus
(1) the apical ‘frame ’ may be lacking, (2) the apical arms may
fork and meet one another, but not be met by the oro-apical
branches, and (3) the cross-bars of the anal arm may be few
or absent. Most of the hybrids are intermediate, there is

102 DIFFERENTIATION III

usually an emargination between the oral arms, and the apex
of the body is prismatic. In the skeleton the extremities of the
apical arms are simple or branched and antler-like, but never
club-shaped ; they may be wide apart, touching, or even fused ;
the anal arm may be single, double, or treble, cross-pieces are
rare; the oro-apical branch is usually lacking, and seldom
reaches the apical, and the transverse arms generally cross.
While, therefore, it is possible to say of a whole culture
whether it is of the Strongylocentrotus, of the Sphaerechinus,
or of the hybrid type, no such assertion can be made of an
individual larva.

Fria. 20.—Pluteus of the cross Sphaerechinus granularis ? x Echinus
microtuberculatus 97, from in front and from the side. (After Boveri,
1896.)

Under these circumstances the difficulty of determining
whether a given character is transmissible from the male
parent is obvious. Vernon, however, using Strongylocentrotus
for the male and Sphaerechinus for the female parent, found
it possible to obtain a culture of purely paternal type in
respect of the skeleton, in the winter, when the sexual
maturity of the male parent is ata maximum. This is stated
by Doncaster to be due merely to the lower temperature, and
the same conclusion is reached by Herbst, who has examined
the effect of temperature changes on the larvae of the pure
parental forms and the hybrid larvae simultaneously.

The temperatures used were from 11°-19° and from 24°-27°.
Thus in the hybrids the cross-bars of the anal arms are more

III DIFFERENTIATION 103

abundant at the higher temperature. This is an inheritance
from the female parent (Sphaerechinus), in the pure type of
which a rise of temperature has the same effect. Of the
number of roots in the anal arms it is impossible to make
a definite statement since heat increases these in both parents.
The ratio of the length of the apical to the length of the
anal arm is 1 in Strongylocentrotus, and greater than 1 at
the higher temperature, 0-5 in Sphaerechinus and decreased
at the higher temperature, while in the hybrids it approaches
0-5 at higher, 1 at lower temperatures; this appears therefore
to be determined from the female side.

Other means have been used by Herbst to displace the
inheritance to the female side, namely, the combination of
artificial parthenogenesis with cross-fertilization. The egg is
thus given an initial impetus towards the development of the
maternal characters: the paternal are then superadded.

To this end the ova (of Sphaerechinus) are treated with
butyric or other fatty acid, and subsequently with sea-water,
in such a way that while the fertilization membrane is not
thrown off, the nucleus yet enlarges and becomes indistinct,
suggesting the passage of substances from it into the cyto-
plasm. After a suitable interval they are fertilized (by
Strongylocentrotus sperm). Irregular segmentation follows
and larvae are produced which have a greater similarity to
larvae of the pure Sphaerechinus type than have the ordinary
hybrids, though they are not completely of that type. The
increase of maternal characters is evinced by the greater
number of cross-bars in the anal arms, the greater number of
roots to the anal arms, the greater length of the oro-apical
branch, the branched extremity of the apical arm and by
the decrease in the ratio of apical to anal arm (less than 0-5).

Cytological examination shows that the sperm always
enters, rotates, and develops its sphere and centrosome. The
female nucleus is in some cases only just resolving itself into
chromosomes, in others this has occurred and the monaster
has appeared.

In the first case the male and female nuclei unite and
a fertilization spindle appears. On it are thrown both sets of

104 DIFFERENTIATION III

chromosomes, but the male are retarded and smaller, so that
while the daughter female chromosomes are passing to the
spindle-poles and becoming vesicular, the male are not so far
advanced and still le about on the spindle. They may divide,
and their halves pass to the poles, but they often fail in this,
and then either pass undivided to one pole or the other, or
else get left behind to disintegrate in the cytoplasm. One
cell may therefore easily fail to obtain a full set of male
chromosomes, and the preponderance of female over male
chromatin in one cell accounts for the smaller nuclei and
preponderance of female over male characters on one side of
the larva. This is incompletely partial thelykaryosis. Com-
pletely partial thelykaryosis also occurs—that is, all the male
chromosomes go into one cell—and the larva has Sphaerechinus
characters on one side, hybrid on the other, with small nuclei
in the first and large in the second half.

In the second case the female nucleus has advanced as far
as the monaster condition by the time that the spermatozoon
enters—that is, the female chromosomes have divided and
there are radiations about the female nucleus.

The female nucleus is now reconstituted from the chromo-
somes; the male nucleus approaches but does not as a rule
unite with it.

The female monaster now degenerates, the 2” female
chromosomes reappear and are thrown on the equator of the
spindle formed in the meantime between the two sperm-centres.
The male nucleus may (a) break up into chromosomes which
lie either with or apart from the female chromosomes on the
spindle, or (8) (and this is more usual) remain intact and lie on
the spindle-equator, near one spindle-pole, or remotely in the
cytoplasm. 7

Should male chromosomes have been formed they are
dragged out as irregular strings and so divided (transversely) ;
if not, then the nucleus is either divided amitotically, or
else passes undivided into one blastomere. Except in the
last contingency, the paternal chromatin must be irregu-
larly distributed to the two blastomeres, and even when the
nucleus does pass entire to one cell and fuse with the female

III DIFFERENTIATION 105

nucleus there it becomes irregularly divided in the next or
some following cleavage. Partial thelykaryosis therefore
never occurs. Hence also ultimately no cell in the body can
have the full set of paternal chromosomes. Occasionally com-
posite spindles are found in these eggs—that is, between the
female monaster and an undivided male centre.

There seems little room for doubt, when all these results are
considered, that the characters of the skeleton and body-shape
at least, by which one genus of pluteus is distinguishable from
another, are transmissible from the male as well as from the
female parent, and therefore by the nucleus.

The experiments of others, while they lead to the like
conclusion, illustrate also the difficulty of discovering the
causes which determine whether the characters transmitted
by the nuclei shall or shall not become manifest in the
hybrid.

Thus MacBride, crossing Hchinocardiwm cordatum 3} with
Echinus esculentus o7, finds that the hybrid larva is smaller
than that of either pure type, that the aboral spike charac-
teristic of the skeleton of the pluteus of the female parent is
absent, but that a male parental character, the bending in
of the ends of the apical rods, is present, while the skeleton of
the post-oral (anal) arms may be of the maternal, of the
paternal or of an intermediate type.

Shearer, De Morgan, and Fuchs employ not the highly
variable skeletal characters of the early plutei, but the stable
features of the later larvae, such as the posterior pedicellaria
found in Echinus esculentus and acutus but not in Echinus
miliaris, the posterior epaulettes found in the two former but
not in the latter, and the green pigment found in miliaris but
not in the other two. When esculentus or acutus is crossed
with miliaris the posterior epaulette appears, whether the
parental form which possesses it is used as a male or female,
while the green pigment of miliaris is not developed by the
hybrid, whether this form has been used as male or female.
This result, confirmed by Debaisieux, is not however invari-
able, for on previous occasions the authors referred to had
found that the characters in question were always transmitted

106 DIFFERENTIATION III

through the ovum, never through the spermatozoon. The
later results obtained by these authors and by Debaisieux
suggest that, under certain conditions at least, the posterior
epaulettes are dominant, the green pigment recessive, and
a similar alternate inheritance of either a paternal or
a maternal character, but in any case in its entirety, was
reported by Loeb, King, and Moore in the hybrid Strongylo-
centrotus franciscanus o% x Strongylocentrotus purpwra-
tus 9. So Tennent has found that in the cross Toxopneustes
variegatus x Hipponoe esculenta the characters of the latter
are always dominant in the hybrid, whether it is used as-
the o7 or ? parent. The dominance is not, however, absolute,
since only about 70% of the plutei have the Hipponoe struc-
ture (Sphaerechinus type, while Toxopneustes has a larva like
that of Hchinus), and even then it is not pure.

Tennent makes the interesting observation that the domi-
nance can be altered by decreasing the alkalinity of the sea-
water. Under these conditions the percentage of hybrids of
the Hipponoe type sinks to about 20%. Cytological investi-
gation has shown that while in one cross (Hipponoe 2 x
Toxopneustes o7) there is some elimination of chromosomes,
though whether paternal or maternal is not known, there is
no such elimination in the reciprocal cross.

Baltzer, who has studied the various crosses of Arbacia
pustulosa, Strongylocentrotus lividus, Echinus esculentus, and
Sphaerechinus granularis, finds that elimination of chromo-
somes occurs in certain cases only, and has further suc-
ceeded in identifying the eliminated chromosomes as paternal,
and in showing that in these cases the skeleton of the hybrid
pluteus is of the maternal type, whereas otherwise it is inter-
mediate.

Thus the cross Sphaerechinus 9 x Strongylocentrotus o7
gives plutei with intermediate skeleton, as we know, but the
plutei reared from the reciprocal cross are of the pure Stron-
gylocentrotus type. The cytological examination shows that
fertilization is normal up to the metaphase, but that then
certain chromosomes begin to lag behind, and so are cast out
instead of entering into the daughter nuclei. This elimina-

Ill DIFFERENTIATION 107

tion continues in later divisions until ultimately about sixteen
or seventeen chromosomes in all are got rid of. The germ-
number in Strongylocentrotus is eighteen, and among these is
a long hook-shaped chromosome, which is always found in the
hybrids; the germ-number in Sphaerechinus is twenty, and
amongst these are two very long chromosomes; these are
never found in the hybrids.

In the larva reared from the disperm egg there should
therefore be 18 + 20 + 20 chromosomes, but the same elimina-
tion occurring, there are only twenty-six, while the monosperm
larva has twenty-two. In other words, the disperm larva has
lost thirty-two while the monosperm has lost sixteen, the
disperm has retained 18 + 4 + 4, while the monosperm has
retained 18 + 4, Hence it is the paternal chromosomes which
have been eliminated, all but four. In the cross Xchinus o7 x
Sphaerechinus 2 there is no elimination of chromosomes, and
the larva is intermediate. In the reciprocal cross the elimi-
nation occurs, but the paternity of the lost chromosomes has
not been demonstrated, though it is known that the charac-
teristic long hook and horse-shoe of Echanus remain.

In the cross Strongylocentrotus 2 (or Echinus 2?) x
Arbacia o7, the larva is of the maternal type, and there is
elimination of chromosomes. This, however, does not occur
until the blastula stage is reached. It is not possible to be
certain that the eliminated elements are paternal. In the
reciprocal cross there is also elimination but the pluteus stage
is never. reached.

The discussions of the foregoing section may now be very
shortly summarized.

1. Experiment shows that the removal of certain parts of
the cytoplasm of the ovum entails the absence or at least the
defective development of certain organs of the embryo or
larva. Hence there are in the cytoplasm certain material
factors on which the formation of certain characters depends.
These characters are part of the total inheritance.

2. Every visible substance in the cytoplasm is not, however,
necessarily such an organ-forming substance as experiments
with the centrifuge demonstrate.

108 DIFFERENTIATION III

3. Experiments on heterogeneous hybridization indicate
that it is the large characters—those of the phylum, class,
order, family to which the animal belongs—that are carried
by the cytoplasm, and, this means, transmitted through the
female germ-cell alone.

4, At the same time the cytoplasm is, during pre-matura-
tion stages, indebted to the nucleus for certain elements in its
structure. In the female, therefore, these nuclear elements of
the cytoplasm are concerned in the transmission of inheritable
characters, as well as the chromosomes.

5. In the chromosomes, the germ-cells of the two sexes are ©
alike, and these chromosomes are certainly concerned in the
transmission of some characters.

6. It is known (a) by observation, (8) by experiment that
there are qualitative differences between individual chromo-
somes, and that a complete set of these different chromosomes
must be possessed by every cell in the body if development is
to be normal. It is further probable that the chromosomes
themselves are heterogeneous.

7. The different activities of the different chromatic elements
are probably only called forth by differences in their environ-
ment, that is, in the cytoplasm to which they are distributed.
It is known that differential behaviour of nuclei can be incited
by cytoplasmic dissimilarity.

8. Hybridization experiments on nearlyrelated forms make
it certain that the smaller characters—generic, specific, varietal,
and individual—can be transmitted as easily from the father
as from the mother, and therefore through the nuclei.

II. THE INTERACTION OF THE PARTS UPON
ONE ANOTHER.

The evidence discussed so far has taught us that the un-
developed egg possesses a structure, both in its cytoplasm
and in its nucleus, the parts of which are definitely related
to the formation of certain organs of the embryo. It does not,
however, follow that every separately inheritable character

Ill DIFFERENTIATION 109

of the animal is represented by a separate unit or deter-
minant in the germ, whether in the cytoplasm or in the
nucleus, and there is indeed evidence to show that certain
characters are caused not by the independent development
of any such determinant, but by the action upon one another
of parts already in existence. The importance of this concep-
tion is obvious. The initial structure of the germ need not
be unnecessarily complicated by imagining it to comprise an
enormously large number of such separate material factors or
determinants. Given a certain number to start with, the rest
will follow by the actions and interactions of these upon one
another. Such interactions may probably be of the nature, not
of grossly mechanical influences, but of responses to stimuli.

Thus in the fish Fundulus the yolk-sac is deeply pig-
mented, the chromatophores being especially closely aggre-
gated around the blood-vessels. When the creature is de-
prived of oxygen the pigment disappears, and it is supposed
that under normal conditions the pigment-cells are chemo-
tactically attracted by the oxygen in the blood (Loeb).

Again, Herbst has found that when the larvae of Sea-
urchins are placed in solutions of lithium salts or in sea-water
deprived of the sulph-ion, or devoid of the carbon-ion, the
skeleton of the pluteus is either distorted in the first two
cases, or absent in the last. The distortion takes the form of
a multiplication of the tri-radiate spicules and the arms are
correspondingly multiplied. In water devoid of CO, there
are no spicules and no arms. Herbst has therefore urged that
normally the outgrowth of the ciliated ring into the arms is
due to a stimulus—thigmotropic perhaps—exerted by the
tip of the spicule. The converse of this is seen later on when
the arms diminish in length as the calcium carbonate of the
pluteus skeleton is made use of by the developing urchin.

A third case is the lens of the vertebrate eye, which it is
said depends for its formation upon a stimulus given by the
optic vesicle or optic cup to the overlying ectoderm. This
statement rests upon evidence brought forward by Spemann
and more particularly by Lewis and Le Cron, that when (in
the tadpole) the optic vesicle is experimentally injured or

110 DIFFERENTIATION EEE

destroyed, the lens is not developed, and that the lens may
be developed from ectoderm taken from another part of the
body or even from another tadpole, and grafted over the
optic vesicle in place of the normal lens-forming cells, or from
the ectoderm lying over an optic vesicle transplanted to some
other region of the body.

Again, in tadpoles grown in certain solutions (sodium
chloride, bromide, and nitrate) the optic vesicle may be some
distance from the ectoderm, and the lens is then absent, and
Stockard has found that in solutions of magnesium chloride
a single median eye may be developed in embryos of the |
fish Fundulus instead of the two normal lateral eyes. Such
median eyes have a lens which is obviously not developed
from the ordinary lens-forming ectoderm.

It seems therefore quite clear that under certain conditions
a lens may be formed over the optic vesicle but from other
cells than those usually devoted to its formation. On the
other hand it is urged that in certain cases lenses are un-
doubtedly developed from the usual cells and in the usual
positions, in spite of the absence of the optic vesicle (King,
Mencl, Stockard).

Spemann has found that the lens, if formed under these
conditions, is small, but that by inserting an optic vesicle to
replace that destroyed, a lens of full size is produced. An
optic vesicle of Bombinator may even be used to replace one
of Rana.

A fourth case is the formation of the cornea in Amphibia.
Spemann and Lewis have both found that in the absence of
the optic cup the corneal ectoderm does not ‘clear’ and that
Descemet’s membrane is not formed.

The development of the ear provides another instance.
The auditory vesicle or membranous labyrinth is formed first
and subsequently surrounded by the cartilage of the periotic
or auditory capsule. The latter, of course, conforms to the
shape of the former. The vesicle may be removed in an
early stage and transplanted to another individual. Here
it continues its development, and is surrounded by a capsule
developed from connective tissue which would of course

III DIFFERENTIATION TEL

otherwise have had a very different fate. The normal vesicle
and capsule are developed alongside the transplant. It is
even possible to graft a vesicle of Rana on to a larva of
Amblystoma. The foreign vesicle then becomes enveloped
in a periotic derived from Amblystoma cells. The normal
Amblystoma labyrinth is present as well (Spemann, Lewis,
Streeter),

Our last example is furnished by the experimental situs
inversus viscerum et cordis produced by Spemann and
Pressler in Amphibian larvae (Rana and Bombinator). A
square piece is cut outof the back of the embryoin the medullary
fold stage, turned right round so that its anterior end faces
posteriorly and its right side to the left, and grafted in again.
The part removed involves nervous system, notochord, some
mesoderm, and the roof of the gut, but not the floor of the
latter nor of course the heart or any subjacent structures.
The embryo continues to develop, and shows inverted viscera
and inverted heart. The part of the gut removed and inverted
is in the region of the duodenum, but betore the dorsal
pancreas was developed; nevertheless, not only is the dorsal
part of the intestine inverted, but the whole of the alimentary
canal, that is, parts posterior and ventral to that which was
turned round. In other words, the liver is pushed over to the
left instead of to the right, the dorsal and ventral pancreas
unite on the left instead of on the right, the stomach is on
the right and the duodenum on the left, while the intestine
passes into the rectum on the right. Moreover, the heart is
inverted. The right is larger than the left vitelline vein, and
the ventricle is bent to the left, the left auricle is larger than
the right. The spiral valve starts at the right instead of at
the left side of the truncus, so that the cavum aorticum is on
the left, the cavum pulmonale on the right. The heart and
viscera of the tadpoles operated on are the mirror images
of the normal organs, the situs inversus viscerum et cordis is
complete, and it is clear that an experimental alteration of
the normal position of some parts has incited a corresponding
change in other parts which were not immediately touched
in the operation.

112 DIFFERENTIATION

These examples are all taken from ontogeny. The study
of Regeneration will give a few more. Thus in earthworms,
new structures—heads and tails—are developed over the cut
ends of the nerve-cord, and by exposing more than one cut
surface of this part, the number of regenerated heads or tails
may be correspondingly increased. So in the regenerating
tail of vertebrata (Urodela). Injury to the nerve-cord starts
a new formation, the cut end of this structure exerting appa-
rently some stimulus upon surrounding tissues.

When aneurogenic limb buds of Amphibian embryos—i.e.
limb buds removed before the ventral root (motor) nerves —
have grown into them—are transplanted on to strange posi-
tions in normal individuals, the posterior limb bud for instance
on to the back of the head of another tadpole, they continue
their development and acquire a normal nerve-supply to their
normally differentiated muscles; the nerve-supply is derived
from the nervous system of the host, the fibres growing into
the graft and being guided in their course by the developing
muscles of the latter (Harrison). The directive stimulus so
exerted by one part upon another is seen again in the rege-
nerating appendages of Insect larvae (Agrionidae), where the
joints of the exoskeleton only appear after the insertion of
the tendons of the newly differentiated muscle fibres.

Though the instances which at present can be cited of this
interaction of parts to develop new structures in ontogeny
and regeneration are lamentably few, they at least indicate
the existence of a factor of the utmost importance ; for it is
clear that with this factor the whole process of development
must be immensely simplified, though of course as truly
determinate as if there were in the germ a unit representative
of each separately inheritable character.

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LITERATURE 115

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LITERATURE 117

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Mech. x, 1900.

Mor@an, T. H. The relation between the normal and abnormal
development of the Frog as determined by the removal of the upper
blastomeres of the Frog’s egg. Arch. Ent.-Mech. xix, 1905.

Mora@ay, T. H., and E. P. Lyon. Relation of substances of the egg,
separated by a strong centrifugal force, to the location of the embryo.
Arch. Ent.-Mech. xxiv, 1907.

Mora@an,T.H. Cytological studies of centrifuged eggs. 1. Cumingia.
Journ. Exper. Zool. ix, 1910.

Minot, ©. S. Senescence and rejuvenation. Journ, Phys. xii, 1891.

Minot, C. S. The problem of age, growth, and death. Pop. Sci.
Monthly, 1907.

Newman, H.H. The process of heredity as exhibited by the develop-
ment of Fundulus hybrids. Journ. Exp. Zool. v, 1907, and viii, 1910.

Priijiger, E. Ueber die Einwirkung der Schwerkraft und andere
Bedingungen auf die Richtung der Zelltheilung. Pfliiger’s Arch, xxxiv,
1884.

PLATEAU, J. Statique des liquides. Paris, 1873.

Poporr, M. Experimentelle Zellstudien, II. Archiv fir Zellforschung
iii, 1909.

PRESSLER, K. Beobachtungen und Versuche iiber den normalen und
inversen Situs viscerum et cordis bei Anurenlarven. Arch. Ent.-Mech.
xxxii, 1911.

QUETELET, A. Anthropométrie. Bruxelles, 1870.

13

118 LITERATURE

RoBErtTs, C. Manual of anthropometry. London, 1878.

RoBertTsoN, T. B. On the normal rate of growth of an individual and
its biochemical significance. Arch. Ent.-Mech. xxv, xxvi, 1908.

RoBeRtTson, T. B. Note on the chemical mechanics of cell-division.
Arch. Ent.-Mech. xxvii, 1909.

ROBERTSON, T. B. Further remarks on the chemical mechanics of
cell-division. Arch. Ent.-Mech. xxxii, 1911.

Roux, W. Ueber Mosaikarbeit und neuere Entwicklungshypothesen.
Anat. Hefte, 1. Abt., ii, 1892-3.

Roux, W. Ueber die erste Theilung des Froscheies und ihre Be-
ziehungen zu der Organbildung des Embryo. Anat. Anz. viii, 1893.

Roux, W. Ueber die Abhingigkeit der Kerngrésse und Zellenzahl
der Seeigellarven von der Chromosomenzahl der Ausgangszellen. Jena,
1905.

ScHuuzE, O. Die kiinstliche Erzeugung von Doppelbildung bei
Froschlarven mit Hilfe abnormer Gravitationswirkung. Arch. Ent.-
Mech. 1, 1895.

Scort, J. W. Morphology of the parthenogenetic development of
Amphitrite. Journ. Exp. Zool. iii, 1906.

SEELIGER, 0. Giebt es geschlechtlich erzeugte Organismen ohne
miitterliche Higenschaften? Arch. Ent.-Mech. i, 1895.

SEELIGER, 0. Bemerkungen iiber Bastardlarven der Seeigel. Arch.
Ent.-Mech. iii, 1896.

SHEARER, C., W. DE MoraaAn, and H. M. Fucus. Preliminary
notice on the experimental hybridization of Echinoids. Journ. Mar.
Biol. Ass. ix, 1911.

SPEMANN, H. Ueber Linsenbildung bei defekter Augenblase. Anat. |
Anz. xxiii, 1903.

SPpeMANN, H. Ueber eine neue Methode der embryonalen Transplan-
tation. Ver. Deut. Zool. Ges., 1906.

SPooneR,G. B. Embryological studies with the centrifuge. Journ.
Exper. Zool. x, 1911.

Stevens, N. M. The effect of ultra-violet light upon the developing
eggs of Ascaris megalocephala. Arch. Ent.-Mech. xxvii, 1909.

STEINBRUCK, H. Ueber die Bastardbildung bei Strongylocentrotus
lividus o7 und Sphaerechinus granularis9. Arch. Ent.-Mech. xiv, 1902.

StocKaRD, C. Theartificial production of a single median cyclopean
eye in the fish embryo by means of sea-water solutions of magnesium
chloride. Arch. Ent.-Mech. xxiii, 1907.

STREETER, G. L. Some factors in the development of the Amphibian
ear vesicle and further experiments on equilibration. Journ. Exper.
Zool. Baltimore, iv, 1907.

STREETER, G. L. Experimental observations on the development of
the Amphibian ear vesicle. Anat. Ree. Philad. iii, 1909.

LITERATURE 119

Sutton, W. S. On the morphology of the chromosome group in
Brachystola magna. Biol. Bull. iv, 1902.

TEICHMANN, E. Ueber Furchung befruchteter Seeigeleier ohne
Beteiligung des Spermakerns. Jen. Zeitschr. xxxvii, 1903.

TENNENT, D.H. The dominance of maternal or of paternal characters
in Echinoderm hybrids. Arch. Ent.-Mech. xxix, 1910.

Vernon, H. M. The relations between the hybrid and parent forms
of Echinoid larvae. Phil. Trans. Roy. Soc. Exc. B, 1898.

Vernon, H. M. Cross-fertilization among Echinoids. Arch. Ent.-
Mech. ix, 1900.

WarBurG, O. Beobachtungen tiber die Oxydationsprozesse im See-
igelei. Zeitschr. Physiol. Chem. \vii, 1908.

WETZzEL, G. Zentrifugierversuche an unbefruchteten Hiern von Rana
fusca. Arch. Mikr. Anat. 1|xiii, 1904.

Wuitney, D. D. The effect of a centrifugal force upon the develop-
ment and sex of parthenogenetic eggs of Hydatina Senta. Journ. Exp.
Zool. vi, 1909.

Witson, E. B. Cleavage and mosaic work. Arch. Ent.-Mech. iil,
1896.

Witson, E. B. A cytological study of artificial parthenogenesis in
Sea-urchin eggs. Arch. Ent.-Mech. xii, 1901.

Wison, E. B. Experiments on the cleavage mosaic in Patella and
Dentalium (11). Journ. Exp. Zool. i, 1904.

Witson, E. B. Studies on chromosomes. Journ. Exp. Zool. 11, 1905,
and iii, 1906.

Wooprurr, L. An experimental study of the life-history of Hypo-
trichus Infusoria. Journ. Exper. Zool. ii, 1905.

Yatsu, N. Experiments on the development of egg-fragments in
Cerebratulus. Biol. Bull. vi, 1904. '

Yatsu, N. An experiment on the localization of developmental
factors in the Nemertine egg. Science (2), xxv, 1907.

120 APPENDIX

TROUT LARVAE. |TOTAL| LENG
Ax and |&x 100 +—<-={

WEEKS AFTER HATCHING

APPENDIX 121

LENGTH OF HEAD
re and

OF CAUDAL FIN

WEEKS AFTER HATCHING

122 APPENDIX

TROUT LAIRVAE.
LENGTH OF CAUDAL

oa

At

WEEKS AFTER HATCHING

6 .
Wwe EKS AFTER HATCHING

INDEX a

Abnormality, in centrifuged eggs, 73,
80 ; in dispermic eggs, 93 ; in eggs
under pressure, 65; in hybrid
larvae, 97.

Absorption of water, see Water.

Acids in artificial parthenogenesis,
40,

Acrosome, 24, 27; function of, 89.

Aegineta, 71.

Agronidae, 112.

Alkalinity of sea-water, affecting
hybrid larvae, 106.

Alkalis in artificial parthenogenesis,
40,

Alternation in direction of division,
59 ; in spiral cleavage, 51.

Alveolar structure of egg cytoplasm,
42.

Amblystoma, 111.

Amoeba, 88.

Amphiaster, 46,

Amphiaster-monaster, 90.

Amphibia, formation of cornea in,
110; inversion experiments in,
111; stratification in eggs of, 78.

Amphikaryotic, 53,

Amphioxus, 69; symmetry of, 72,

Amphitrite, 40, 46.

Animal pole, 2, 7.

Annelids, 68.

Antedon, hybridization, 95.

Apical organ, 5; absence of, in larvae
from egg-fragments, 73 ; formed in
posterior half-gastrulae, 77; pre-
formation of, 76.

Arbacia, artificial parthenogenesis,
37,106 ; centrifuged eggs of, 81;
cross-fertilization, 106, 107; effect
of salts on egg of, 44.

Archenteron, 3, 5, 10, 71, 95.

Arrhenokaryosis, 53, 54.

Artificial parthenogenesis, see Par-
thenogenesis,

Ascaris megalocephala, cell-lineage
in, 14; centrifuged eggs of, 84;
cleavage of, 11, 13 (fig.), 49 ; dimi-
nution of chromosomesin, 11; di-
spermic eggs of, 86 ; isolated blas-
tomeres of, 74; totipotence of egg-
fragments of, 76.

d ga \ yf “hig
Le >’ Bre . -
a 4 £, id oe ~~
f 4
4 ao \ 4 4

Ascidian, 59, 69, 72, 74.

Asterias, 40; hybridization, 94.

Auditory vesicle, 11; transplanta-
tion of, 110,

Audouinia, 96.

Axes of embryo, see Embryonic axes.

Axis of egg, see Egg-axis ; of polarity,
change in, 85, 86.

Axolotl, definitive centrosome in,
32 ; fertilization, 28, 30, 31 (figs.) ;
maturation division, 26 (fig.).

Baltzer, F., 94, 95, 106.

Bataillon, E., 40.

Bilateral cleavage, see Segmentation.

Bilateral-symmetry, see Symmetry.

Bipinnaria, 95.

Blastomeres, behaviour of isolated,
63, 66, 67, 71; coating film of, 49;
effect of gravity on, 63; killed by
hot needle, 61; killed by ultra-
violet light, 74; separation in cal-
cium-free sea-water, 49; totipo-
tence of isolated, 72, 76.

Blastopore, 5, 14; closure of, 9 (fig.),
10; formed from animal cells, 71 ;
open in centrifuged eggs, 79 ; per-
sistence of, 80.

Blastula : Echinoderm, 4,

Blood-serum, 43,

Boas, F., 22.

Bombinator, optic vesicle of, 110.

Born, G., 78.

Boveri, T., 52.

Brachet, A., 47, 72, 63.

Brachystola, 94.

Bullot, G., 40.

Butyric acid, 39, 103; method of
causing artificial parthenogenesis,
39, 45.

Calcium chloride, 94; effects of ab-
sence of, 49, 66.

Calkins, G., 18.

Cane-sugar, artificial parthenogene-
sis, 38.

Carbon dioxide to induce artificial
parthenogenesis, 40, 109.

Carmarina, 27,

124

Cell-division, 52 sqq.; Ascaris, 11,
86; in diagrammatic form, 14; at
low temperatures, 68 ; see Segmen-
tation.

Cell lineage, 12, 68.

Cell volume, 55.

Cells, enucleate, 46 ; potentialities of
isolated, 74; ratios of volumes of,
56.

Centrifuged eggs, 74, 76, 78, 80, 82, 84.

Centrolecithal, see Egg-structure.

Centrosome, 29, 31; division of, 24,
27; function of, 89; origin in par-
thenogenetic eggs from 9 nucleus,
45, 46.

Cephalopod, 27, 49, 69.

Cerebratulus, apical sense-organ of,
76; compression of egg, 72; effect
of calcium-free sea-water on eggs
of, 66, 77; effect of pressure on
egg-axis, 76.

Cerfontaine, P., 69, 72.

Chaetopterus, artificial partheno-
genesis, 40; centrifuged eggs of,
82; cross-fertilization, 97; differ-
entiation in eggs without cleavage,

Chloride of sodium, magnesium,
potassium, 38; of calcium, 49, 66,
94; of magnesium, 110.

Chloroform and membrane forma-
tion, 39.

Chlorostoma, 95.

Chromatin, diminution in Ascaris, 11,
13; elimination of, 96, 97.

Chromosomes, alteration of number,
11, 23, 27, 53; behaviour in arti-
ficial parthenogenesis and cross-
fertilization, 103; differences in,
89, 93 ; diminution of, 11, 18, 74 ;
division of, 33, 47 ; distribution of,
in dispermic eggs, 90 (fig.), 93;
elimination of, 106, 107; hetero-
typic, 23, 25; in inheritance, 108 ;
numbers in dispermic eggs, 53, 87 ;
number in monaster eggs, 53;
ratios of volumes, 56, 57 ; various
size and form of heterochromo-
somes, 94.

Cleavage, see Segmentation.

Coefficient of correlation, 22, 62; of
variability, 21.

Coelenterates, 27.

Cold, artificial parthenogenesis, 40.

Conjugation, Infusoria, 33.

Conklin, E. G., 56, 69, 72, 86, 99.

Cornea, formation in Amphibia, 110.

Correlation, coefficients, trout larvae,
22; and development, 23; between
first furrow and sagittal plane, 62,

INDEX

65; between growth and varia-
bility, 21; between symmetry and
sagittal planes, 62, 65.

Crampton, H. E., 67.

Crepidula, 58, 99; centrifuged eggs
of, 83, 86.

Crinoid, Antedon in cross-fertiliza-
tion, 95.

Cross-fertilization, see Hybridization.

Ctenolabrus, 97.

Ctenophora, 50, 68, 73;
blastomeres, 73.

Cumingia, centrifuged eggs of, 82.

Cyclops, centrifuged eggs of, 83.

Cynthia partita, centrifuged eggs
of, 88, 86; development of, 35
(fig.) ; half and three-quarter em-
bryos of, 75 (fig.) ; maturation in,
84; pigmentation of eggs of, 34;
volume of nuclei in, 59.

Cytasters in parthenogenetic eggs, 45.

Cytolysis, 39, 41.

Cytoplasm, change in structure, 34 ;
characters transmitted by, 98, 108 ;
deposit of yolk granules in, 24; in
egg-fragments and isolated blasto-
meres, 76; heterogeneity of, 88;
measurement of volume of, 57;
organ-forming substances in, 70,
107; part played by different
regions, 86; physical properties
of, 69; removal of part of, 72;
streaming movements in, 65;
structure of, 71, 74.

isolated

Davenport, C. B., 15.

Debaisieux, G., 106.

Delage, Yves, 40.

De Morgan, W., 105.

Dentalium, apical organ of, 78, 76 ;
cross-fertilization, 97; isolated
cells of embryo, 67 (fig.) ; removal
of polar lobe, 67.

Determinants, 59, 66, 86, 88, 98.

Development, abnormality of, due
to irregular distribution of chromo-
somes, 93; of Ascaris megalo-
cephala, 11; of centrifuged eggs,
78 sqq.; of definitive centrosomes,
29, 82; of dispermic eggs, 86, 91 ;
effect of derangement of cytoplasm
upon, 80; of egg-fragments, 73;
of isolated blastomeres, 76; of par-
thenogenetic eggs, 40 sqq.; of
Rana temporaria, 8 sqq.; of
Strongylocentrotus lividus, 2, 3
(fig.) ; three distinct processes in, 2.

Developmental capacities of cells,
67, 73.

Differentiation, 2, 10, 11; causes_of,

INDEX

52 sqq., 69; internal factors of,
70 sqq.; of nephridia, 68 ; with-
out cleavage, 69.

Diminution of chromosomes in
Ascaris, 11, 12, 13 (fig.), 74; in
dispermy, 87.

Diplokaryotic, 53.

Dispermy, 53; in Ascaris, 86 sqq. ;
development of embryos, 91; in
Strongylocentrotus, 89sqq.

Division, cell-, 2, 11, 52; of centro-
some, 32; of chromosomes, 33;
equatorial, 4, 8, 11; karyokinetic,
23; latitudinal, 4, 8, 11; meri-
dional, 4, 8, 11; maturation, 23;
nuclear, 2, 11, 52; power of, re-
stored by fertilization, 33; simul-
taneous in parthenogenetic eggs,
38 ; see Segmentation.

Dominant characters, 106.

Doncaster, L., 102.

Driesch, H., 66, 68, 73, 77.

Drops of fluid, coherence of, to form
system, 49; of oil, radial system
of, 52.

Earthworm, nephridia of, 68; re-
generation in, 112.

Echinocardium cordatum, 105.

Echinoderms, archenteron, 5; arms
of pluteus, 5, 100, 101, 109; arti-
ficial parthenogenesis in,37,40; arti-
ficial parthenogenesis with cross-
fertilization, 103 ; blastula, 4, 72;
centrifuged eggs of, 81, 85 sqq. ;
cleavage altered by pressure, &c.,
66 ; cross-fertilization, 94; develop-
ment of egg-fragments, 73, 77;
development in salt solutions,
109 ; dispermy in, 89; fertilization
membranes of, 27, 42; hybridiza-
tion, 95 sqq. ; isolated blastomeres
of, 67; membrane formation in,
45, 96; normal development of,
3 (fig.); number of chromosomes
in eggs, 54; plasma, nuclear ratio
in, 55; pluteus larva, 6 (fig.), 100
(fig.), 102 (fig.) ; separation of blas-
tomeres of, 49; symmetry of, 72;
various shaped chromosomes in,
94

Echinoids, see Echinoderms.

Echinus acutus, 105.

— esculentus, 105, 106.

— microtuberculatus, 6 (fig.), 101.

— miliaris, 105.

Ectoderm, Ascaris, 12 ; corneal, 110;
in dispermic eggs, 85, 86; in Echi-
noderms, 77; in Hydromedusae

125

76; lens-forming, 110; of ventral
plate, 68.

Egg, fertilization of, 30, 31 (fig.), 32 ;
maturation of, 24, 26 (fig.) ; strati-
fication of, 78.

Egg-axis, 49, 91; in Ascaris, 11; in
centrifuged eggs, 80, 83; in di-
spermic eggs, 86; in Echinoderms,
2; in Frog, 7, 9, 64.

Egg-fragments, enucleate, see Mero-
gony ; isolated, 70; segmentation
of, see Segmentation ; totipotence
of, 72, 76.

Egg-structure, centrolecithal, 27; of
Cynthia, 34, 35 (fig.) ; of Frog, 6 ;
of Hydromedusae, 70; of Strongy-
locentrotus, 2; telolecithal, 6, 11,
27, 51, 71 ; of vertebrates, 27.

Embryology, comparative, 1 ; experi-
mental, 2.

Embryonic symmetry, see Symmetry.

Endoderm, in_ Ascaris, 12; in
Echinoderms, 77; -forming sub-
stance, 78 ; in Hydromedusae, 76.

Entrance funnel, 28, 30; position of
grey crescent determined by, 64, 65.

Epaulettes, in pluteus, 6; in hybrid
larvae, 105.

Equator of egg, 2, 7.

Equatorial cleavage, see Segmenta-
tion.

Erdmann, R., 55.

Errera, L., 18.

Ferments, extra-cellular, 20.

Fertilization, 27, 33, 46; of Axolotl,
28, 30 (fig.), 31 (fig.), 32 ; of centri-
fuged eggs, 84; cross-, 42, 94 sqq.;
spindle, 31(fig.); and symmetry, 64.

Fertilization membrane, 27; arti-
ficial formation of, 39.

Fischer, M. A., 40.

Fishes, heterogeneous hybridization,
97.

Frequencies, of angle between first
furrow and sagittal plane, 61
(tab.) ; of angle between symmetry
plane of egg and embryo, 48 (tab.).

Frog, artificial parthenogenesis, 40 ;
blastopore formation in, 9 (fig.) ;
centrifuged eggs of, 79, 85; cleav-
age, 8, 49; development of, 6;
double monster, 71; first furrow
and sagittal plane, 61, 65; grey
crescent in egg of, 7, 8 (fig.), 64,
65; half-embryos, 60 (fig.) ; inver-
sion of eggs, 71, 78; inversion
of embryonic organs, 111; optic
vesicle of, 110; position of axis in
eggs of, 28; effect of pressure on

126

eggs of, 65; sperm-path in, 64
(fig.) ; symmetry, 47, 63.
Frontonia, 19.
Fuchs, R. F., 105.
Fundulus, 97, 109 ; median eye, 110.
Furrow, first, and sagittal plane, 61 ;
first, and sperm-path, 64.
Furrows, cross- or polar, 50.
Fusus, 96.

Garbowski, M. T., 82.

Germ-cells, 1, 23, 88 ; of Ascaris, 12;
development of, 25, 26 (fig.); in
dispermic eggs, 87 ; in fertilization,
27, 338, 89; in inheritance, 99;
maturation of, 23 sqq., 88.

Gibbula, 96.

Godlewski, E., 95, 97.

Gravity, influence of, 62.

Greeley, A. W., 40.

Grey crescent, 7, 8 (fig.), 64, 65 ; and
embryonic symmetry, 47, 63; in
punctured eggs, 40, 47.

Growth, 11, 14, 16 (fig.), 24, 25; ab-
sorption of water during, 15.

Growth-rate, 15,18, 22; measurement
of, 17; and variability, 21 (fig.).

Gut, formation from animal frag-
ments, 73 ; in Echinoderms, 5; in
Frog, 10 ; in half-gastrula, 77 ; in-
version of, 111.

Haemolysis, 44.

Half-blastomeres, 71, 72, 77.

Half-embryos from centrifuged eggs,
79, 80; of Frog, 60 (fig.), 62.

Harrison, R. G., 112.

Harvey, E. N., 43.

Heat, in artificial parthenogenesis,
40; effect on Sea-urchin eggs, 66.

Herbst, C., 49, 103, 109.

Hertwig, O., 62, 78, 81.

Hertwig, R., 37, 39, 52, 57.

Heterochromosomes, 94.

Heterogeneous hybridization, 94sqq.

Hindle, E., 45.

Hipponoe esculenta, cross-fertiliza-
tion, 106.

Hogue, M. J., 84.

Hyaloplasm layer
eggs, 79, 80.

Hybrid larvae, 100, 101; effect of
temperature on, 102.

Hydatina, centrifuged, 84.

Hydra, regeneration in, 77.

Hydrogen in hypertonic solutions,
Al

in centrifuged

Hydromedusae, 70; totipotence of
eggs of, 76.
Hypertonic solutions, 38, 40.

INDEX

Ilyanassa, egg-fragments of, 73; iso-
lated blastomeres, 67, 68.

Increments, growth-rate, 15, 21.

Infusoria, 19, 33.

Inheritance, 107; determinants of,
59, 66, 70, 86; and germ-cells, 99 ;
mechanism of, 1; part played by
nucleus in, 33, 88, 94.

Iso-bilateral cleavage, see Segmenta-
tion.

Interaction of parts, 108 sqq.

Inversion of Frog’s egg, 62, 78; of
organs in Frog, 111.

Jelly-plasm, 27; in Carmarina, 71.

Karyokinesis, 89.

King, H. D., 110.
Konopacka, B., 78.
Kostanecki, K., 40.
Kupelwieser, H., 95, 96.

Lamellibranchs, pigment in egg of
Cumingia, 82, 83.

Le Cron, 109.

Leech, 68.

Lefevre, G., 40, 46.

Lens formation in vertebrate eye,
109.

Lewis, W. H., 109, 111.

Lillie, F. R., 32, 69, 82.

Lillie, R. S., 44.

Linnaea, centrifuged eggs, 83.

Lipoid, soluble, 42, 43.

Lithium salts, 109.

Lithodomus, 96.

Loeb, J., 17, 37, 40, 41, 94, 115.

Lyon, E. P., 81.

MacBride, E. W., 105.

Macromeres, in dispermic eggs, 89;
in Echinoderms, 4; in spiral cleav-
age, 51 (fig.).

Mactra, 40, 96.

Magnesium chloride, in artificial par-
thenogenesis, 38, 45; effect on
Fundulus embryos, 110.

Masing, E., 19.

Mathews, A. P., 40.

Maturation of germ-cells, 23, 88; in
the Axolotl, 26 (fig.) ; in Cynthia,
34; in the Salamander, 25 (fig.).

Mead, A. D., 40.

Measurements of nucleus, cell, and
chromosomes, 56 (tab.).

Mechanical agitation, to induce arti-
ficial parthenogenesis, 40.

INDEX

Mechanism of membrane formation,
42, 43; inheritance, 1.

Medusa, 71.

Membrane, Descemet’s, 110; of egg,
28 ; formation in artificial partheno-
genesis, 41-4; by chloroform, 39;
by haemolytic agents, 44; by neutral
salts of K and Na, 44 (tab.); by
various spermatozoa, 96.

Mencl, E., 110.

Menidia, 97.

Meridional division, 65, 76. See Seg-
mentation.

Merogony, 33, 53, 89.

Mesenchyme,in Echinoderms, 3(fig.),
4,5, 95; in half-embryos of Cynthia,

75.

Mesoderm, 111; absence of, in Ilya-
nassa, 73; in " Ascaris, 12; in Frog,
10, 111; in spirally-segmenting
eggs, 68.

Metazoa, growth-rate, 18-20.

Micromeres, in compressed egg of
Nereis, 77; in dispermic eggs, 89;
in Echinoderms, 4; in spiral cleav-
age, 50, 51 (fig.).

‘Micropyle, 2, 27, 81.

Minot, C. S., 15, 20.

Mitosis, 10, 12, 13, 91; multipolar,
38.

Modiolaria, 96.

Moenkhaus, W. J., 97.

Mollusca, cell-lineage in, 68; hetero-
geneous hybridization, 96; isolated
blastomeres of, 67, 73.

Monaster eggs, 53, 54.

Monsters, double, 63 (fig.), 71 ; head-
less, from centrifuged eggs, 80.

Morgan, T. H., 37, 62, 71.

Mosaic-theory, 59, 60, 70.

Murex, 96.

Mus, fertilization in, 28.

Mytilus, cross-fertilization, 95.

Nassa, 96.

Nemertines, cell-lineage, 68 ; develop-
mental capacities of cells of, 73;
effect of calcium-free sea-water on
eggs, 66; isolated blastomeres, 67,
72, 77.

Nereis, artificial parthenogenesis, 40;
cleavage centrosome, 32 ; fertiliza-
tion, 27, 28; segmentation under
pressure, 66, 77; vitelline mem-
brane, 28, 44.

Newman, H. H., 99.

Newt, double monster, 63 (fig.) ; iso-
lated blastomeres, 71; regeneration
of lens of, 77.

127

Notochord formation, in Frog, 10;
in half-embryo of Cynthia, 75;
inversions of, 111.

Nuclear division, see Nucleus.

Nucleins, synthesis of, 17, 19.

Nucleo-plasma ratio, 52, 55, 69; in
adult tissues, 59; in Crepidula egg,
58 (tab.).

Nucleus, 88; in artificial partheno-
genesis, 45; characters transmitted
by, 89, 98 : ; in dispermy, 53, 86;
division of, 33, 52, 59; in Echino-
derms, 2; ; in fertilization, 29, 30,
31 (fig.) ; in inheritance, 33, 88, 94,
108 ; karyokinetic division of, 23,
89; position in Frog’s egg, 7; in
Protozoa, 88; qualitative division
of, 59, 65, 70, 88; size and number
of chromosomes, 54, 91, 93; surface
area of, 54, 55; volume, decrease
during development, 56.

Nervous system in Frog, 10; ine
verted, 111.

Oil-drops, 47; radial system of, 52.

Ontogeny, 112.

Ophelia, 40.

Optic cup and lens, 109.

Organ-forming cytoplasmic
rials, 69, 78, 107.

Osmotic pressure cause of artificial
parthenogenesis, 38, 40.

Oxidation of substances in the egg,
41,

Oxygen, chemotactic effect on pig-
ment-cells, 109; necessity for, in
hypertonic solutions, 41.

mate-

Parthenogenesis, artificial, 33, 37, 89;
alteration of symmetry in, 47; with
cross-fertilization, 103; cytolysis,
41; irregular segmentation, 66 (fig.) ;
Loeb’s theories of, 41; membrane
formation in, 41 sqq.; number of
chromosomes in, 53; various
methods of inducing, 38, 40.

Patella, isolated blastomeres, 67, 73.

Pecten, 96.

Percentage increments, 15-20.

Perivitelline fluid, 27, 42, 79; space,
28.

Petromyzon,
genesis, 40.

Pfliiger, E., 51, 79.

Phallusia, 74.

Phycomyces, growth-rate of hyphae,
18.

Physa, centrifuged eggs of, 83.

Pigment, in Arbacia, 81; in Ascaris,
84; in centrifuged eggs, 80, 81, 82,

artificial partheno-

128

84; in Cumingia, 82; in Cynthia,
34, 35 (fig.); in Echinus miliaris,
105; in entrance-funnel, 29; in
Frog, 6, 80 ; in Strongylocentrotus,
2, 82; transmission of, 97.

Pilidium, 66.

Planorbis, centrifuged eggs of, 83.

Plateau, J., 52.

Pluteus larva, abnormal skeletons of,
100,109; arms of, 5, 100 sqq.; from
4- and 1-blastomeres, 67; of Echi-
nus microtuberculatus, 6 (fig.) ; of
hybrids, 101, 102, 105, 107; of
Sphaerechinus granularis, 100
(fig. ).

Polar eae 2, 25, 26 (fig.); eggs,
78; furrows, 50; lobe, 67 (fig.),
73, 74.

Polarity, alteration of, 71, 78; of
centrifuged eggs, 81, 85; of ovum,
in Sea-urchin, 2.

Polychaeta, Amphitrite, 46; in
hybridization, 96; Nereis, 27.
Polynoe, 40.

Popoff, M., 19.

Potassium chloride, 38, 69; cyanide,
39, 41.

Potentiality of animal and vegetative
blastomeres, 71, 72, 73; of isolated
cells, 74; limitation of, 77.

Pressler, K., 111.

Pressure, effect of, on segmentation,
65, 66; osmotic, 38, 40.

Protoplasmic or animal pole, 7.

Protozoa, rate of cell-division, 18, 20 ;
role of nucleus in, 88.

Pulmonates, eggs centrifuged, 83, 85.

Punctured eggs, artificial partheno-
genesis, 40; grey crescent formed
in, 47.

Quadripartite ova, 91; development
of, 92.

Qualitative division of nucleus, 59,
88.

Quarter blastomeres, 66, 67, 71, 76,
77

Quetelet, A., 16.

Radial cleavage, see Segmentation.
Radial symmetry, see Symmetry.
Rana, 110. See Frog.

Rate of growth, 15, 16; maximum in
man, 16 (tab.), 17; of cleavage,
99.

Ratio, cell to chromosome number,
54, 56,57; cell, nuclear and chro-
mosome volumes, 56 (tab.); cell-
volume to surface area of nucleus,
55; nuclear size to chromosome

INDEX

number, 54; nuclear surface to
chromosome volume, 57; nucleo-
plasma, 52, 55, 58, 59, 69.

Recessive characters, 106,

Redistribution of materials in cyto-
plasm, 76, 83.

Regeneration, 77, 112.

Roberts, C., 21.

Robertson, T. B., 17, 47.

Roux, W., 52, 59, 60, 89.

Sagittal plane of Frog’s egg, 61(tab.) ;
and first furrow, 65.

Salamander, development of sper-
matozoon, 24, 25 (fig.).

Salts, artificial parthenogenesis, 38
sqq.; effect on segmentation, 66, —
69; in heterogeneous hybridiza-
tion, 94; in membrane formation,
41, 44; skeleton of pluteus, 109.

Samassa, P., 71.

Saponin, 24.

Schulze, O., 62.

Scott, J. W., 40, 46.

Sea-urchin, see Echinoderms.

Sea-water, absorption by egg, 44;
artificial, 49; effects of diluting,
66; hyper-alkaline, in hybridiza-
tion, 95.

Secretion of substances in growth,
14,

Seeliger, O., 100.

Segmentation, 37 sqq., 52; alterna-
tion of directions of, 50; in artifi-
cial parthenogenesis, 38, 40, 45,
46; in Ascaris, 11, 13 (fig.}, 76,
84, 86; bilateral type of, 49, 50;
causes of end of, 52, 55 ; cavity, 4,
8, 10, 14, 52; of centrifuged eggs,
74, 79, 80, 83; in Dentalium, 67
(fig.) ; of dispermic eggs, 86, 87,
91; in Echinoderms, 2, 3 (fig.) ;
of egg-fragments, 76; in Frog, 8,
49; of hybrids, 95; irregular, 66,
88; iso-bilateral type of, 40; of
isolated blastomeres, 66, 67, 738,
76; at low temperatures, 68; nu-
cleo-plasma ratio during, 58; par-
tial, 68 ; patterns of, 37, 48, 49, 66,
69; under pressure, 66, 72, 76;
radial type of, 23, 49; rate of, 99;
of Sepia, 50 (fig.) ; sequence of, 8;
spiral type of, 49, 50, 51 (fig.), 68;
suppression of, 69.

Sense organ, see Apical organ.

Sepia officinalis, 50 (fig.).

Sequence of divisions in Frog’s egg, 8.

Shaking, effects of, 53, 66, 90.

Shearer, C., 105.

Skeleton of pluteus, 5, 6 (fig.); of

INDEX

hybrids, 105 sqq. ; from tripartite
ova, 92.

Soap-bubbles, 52.

Solutions, hypertonic and isotonic,
38 ; of varying osmotic pressures,
38 (tab.).

Somatic cells of Ascaris, 12, 13 (fig.),
86

Specific characters, 98, 99.

Spemann, H., 109, 110, 111.

Spermatozoon, 23, 89 ; acrosome, 24,
27, 89; axial filament of, 24; deve-
lopment of, 24, 25 (fig.) ; entrance
of, 28, 29; relation to first fur-
row, 64 (fig.) ; sperm-aster, 29, 32 ;
sperm-path, 29. See Centrosome.

Sphaerechinus granularis, 100; cross-
fertilization with, 95; hybrid plu-
teus, 102 (fig.); normal pluteus,
100 (fig.).

Spicules, 4, 5, 109.

Spina bifida, centrifuged eggs, 78.

Spindle of Axolotl, 30, 31 (figs.), 32 ;
bi-polar, necessary for normal seg-
mentation, 46; in centrifuged eggs,
84, 85; double and quadruple in di-
spermic eggs, 89, 90 (fig.).

Spindle-fibres, function of, 47.

Spiral type of cleavage, 51 (fig.). See
Segmentation.

Spooner, G. B., 83.

Standard deviation, 21.

Starfish, 42, 94.

Steinbriick, H., 101.

Stenotomus, 97.

Stevens, N. M., 74.

Stimuli, in artificial parthenogenesis,
37; exerted by organs in develop-
ment, 109 ; in regeneration, 112.

Stockard, C., 110.

Stomodaeum, in Ascaris, 12; in
Echinoderms, 5; in Frog, 11; de-
veloped from isolated blastomeres,
wy ie

Stratification, 78 ; axis of, 81, 83.

Streeter, G. L., 111.

Strongylocentrotus, abnormal deve-
lopment of, 96, 98; artificial par-
thenogenesis, 37, 40, 45; cross-
fertilization, 94, 95,103; dispermic
eggs of, 90 (fig.); Franciscanus,
106; lividus, normal development
of, 2, 3 (fig.); purpuratus, 45, 106.
See Echinoderms.

Surface-tension, 47,49; in membrane
formation, 43.

Sutton, W. S., 94.

Symmetry, alteration of, by centri-
fuging, 34, 47, 79, 83, 85; altera-
tion of, at fertilization, 34, 47, 72,

128

76; in artificial parthenogenesis,
47, 48; of Ascaris egg, 11; bilateral,
7, 48, 64, 72; of egg and embryo,
7, 48, 63; of embryo, 48, 63, 64,
69; of Frog’s egg, 7, 69; plane and
first furrow, 61, 62; plane and sa-
Ag plane, 61, 62; radial, 7, 64,

Tadpole, 11 ; optic vesicle, 109 ; pres-
sure experiments, 66.

Teichmann, E., 90.

Teleostean egg, 50.

Telolecithal, see Egg-structure.

Temperature, 56, 68; affecting hybrid
larvae, 102; coefficient, 43; quo-
tient, 43 (tab.).

Tennent, D. H., 94, 106.

Tetrahedron of cells, 86.

Tetrasters, 89.

Thalassema, 40, 46,

Thelykaryosis, 53, 54, 104.

Theory, of Loeb, artificial partheno-
genesis, 41; of Robertson, syn-
thesis of nucleus, 17,19; of Roux,
mosaic-theory, 59, 60, 61; of Weis-
mann, 59.

Totipotence, see Egg-fragments.

Toxopneustes, 106.

Transmission of characters, 94, 96,
98, 99; by nuclei, 33.

Transplantation, of auditory vesicle,
110; of limb-bud, 112; of optic
vesicle, 110.

Trembley, Abbé, 77.

Tripartite embryos, 92; ova, 91.

Trochophore, 66, 83.

Trochus, 96.

Trout larvae, correlation coefficients,
- (tab.) ; growth and variability,

Turbellarians, 68.

Urea, artificial parthenogenesis, 38.
Urodela, regeneration in, 112.

Variability, coefficient of, 21; and
growth-rate, 21.
Vegetative pole, 2, 7.

Venus, 96.
Vernon, H. M., 102.
Vertebrates, artificial partheno-

genesis, 40 ; egg-structure, 27; in-
teraction of parts in, 109sqq.
Viscera, inverted, 111.

Warburg, O., 41.

130 INDEX

Water, absorption of, in growth, 15; | Woodruff, L., 18, 19.
withdrawn in membrane forma-
tion, 42; by osmotic pressure, 38.
Weights of English artisans, 21 | Yatsu, N., 66, 72, 73, 76.
(fig.) ; increase during growth, 14, | Yolk, in centrifuged eggs, 80; glo-

15; of male Belgians, 16 (tab.). bules, 11; granules, redistribution
Weismann, A., 59. of inverted eggs, 62; influence on
Wetzel, G., 62, 81. segmentation, 8, 58; nucleus, 20,
Whitney, D. D., 84. 24, 46, 98; plug, 9, 62; pole or

Wilson, E. B., 45, 66, 67, 738, 94. vegetative pole, 7.

PRINTED IN ENGLAND

AT THE OXFORD UNIVERSITY PRESS

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