Table Of ContentMORPHOGENESIS:
THE ANALYSIS OF
MOLLUSCAN DEVELOPMENT
by
CHR. P. RAVEN
Professor of Zoology in the
University of Utrecht
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Chr. P. Raven
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Preface
THIS book has arisen from a series of lectures that the author gave in the
years 1954-1956 for advanced biology students of the University of
Utrecht. It soon became clear that the not inconsiderable amount of
time and labour invested in collecting and working-through the relevant
literature, might be made profitable to a wider circle.
The ever-rising flood of publications makes it a hard task for the
expect to keep abreast of his own specialism ; for the outsider in a certain
field it has become impossible altogether. Science threatens to fall apart
into innumerable super-specialisms. Under these circumstances, it
appears that it is up to the specialist from time to time to draw up the
balance-sheet of his own field of activity, in order to make clear to his
fellow-scientists where we are standing, what has been accomplished up
to the present, and which are the problems to be tackled in the im-
mediate future.
A survey of the present state of affairs in the field of experimental
embryology can be established in different ways. It may be centred
around certain general problems, which are illustrated by drawing one's
examples freely from all groups of the animal kingdom. In this way it is
possible to draw in broad outline the general trends and laws of animal
development, as far as present knowledge enables one to see them
clearly.
This is the usual procedure, and it is a very valuable one, too. But it
has some drawbacks, which make it desirable to approach the matter
also in another way. There is the danger of unfounded generalization
which is always imminent in this method of approach. Moreover, it is
hardly possible to be exhaustive in this kind of survey, so that certain
new data, the importance of which has not yet been generally recog-
nized, may be easily overlooked. Finally, it is practically impossible to
include an extensive survey of normal development which, however,
must of necessity form the foundation on which all causal analysis must
be based.
Therefore, it is important that such reviews of a comprehensive
nature are supplemented with monographs restricting themselves to one
branch of the animal kingdom only. Here the causal analysis of the
experimental data can be preceded by a detailed description of the
xi
Xll PREFACE
phenomena of normal development, so that the problems can be for-
mulated with greater accuracy, and the peculiarities of the group can be
taken into account. Examples of this kind are v. UBISCH'S review papers
on the development of the sea urchins and the Monascidia, respectively
(Verhandl. Kon. Ned. Akad. v. Wetensch. Amsterdam, Afd. Natuurk. (II),
47, 1950; (II) 49, 1952). The present book also belongs to this category.
In writing it, I have tried to reflect as accurately as possible the
present state of our knowledge with respect to molluscan development,
whereby the literature up to about the middle of 1956 could be taken
into account. I have not restricted myself to the mere facts, however,
but wherever possible I have mentioned existing hypotheses or tried to
frame new ones, in order, on the one hand, to explain the facts, and, on
the other hand, to permit the formulation of new questions and the
design of new experiments. I have often marshalled the facts in such a
way that they might serve to illustrate the leading ideas. I realize that
this lends a certain touch of subjectivity to the exposition. But in my
opinion an author has a right to be subjective, as long as he does not
strain the objectivity of the factual data.
Since my main interest is centred on the causal analysis of morpho-
genesis, the description of normal development has been subordinated
to this purpose. Comparative embryological points of view have hardly
been taken into consideration, but the cytological and cytochemical
aspects of development have been especially emphasized. On the other
hand, purely biochemical investigations, unless they had a direct bearing
on morphogenesis, have also been left out.
It has not been my aim to give a complete bibliography of molluscan
development. Papers of a descriptive nature have only been explicitly
mentioned when necessary. But I have tried to include the literature on
cytochemical and experimental embryological investigations of the last
half century as completely as possible.
As regards taxonomy, as a rule only generic names have been men-
tioned, unless specific differences might be important in the context.
The taxonomic names used by the authors have as a rule been taken
over as such ; no attempts have been made to bring them into harmony
with the present state of nomenclature. Only in very obvious cases
(Lymnaeus - Lymnaea - Limnaea; Paludina - Vivipara - Viparus;
Bythinia - Bithynia) I have sometimes departed from this rule.
I am greatly indebted to many colleagues for their kind permission
to make use of figures copied from their papers. To the Scientific
Committee of the Zoological Society of London my thanks are due for
the same reason. CHR. P. RAVEN
Utrecht December 1956
f
Addition to Preface
In this paperback edition the literature on molluscan descriptive and
experimental embryology up to about the end of 1964 has been taken
into account. The new material has been added as a separate chapter at
the end of the book. Besides keeping the price of the book as low as
possible, this had the additional advantage that it permits one to evalu-
ate the progress in our understanding of molluscan development during
the latter eight and half years. It is linked up with the original text by
references.
September 1965 CHR. P. R.
xiii
CHAPTER I
Oogenesis
1. The ripe egg
THE egg cells of most molluscs are spherical. The animal and vegetative
sides are often clearly indicated. As a rule, the uncleaved egg is radially
symmetrical around the main axis running through the animal and the
vegetative pole.
This description holds also for some cephalopods (Sepia). In most
cephalopods, however, the eggs are oblong, the long axis corresponding
to the main axis of the egg cell. Moreover, e.g. in Loligo, the eggs are
bilaterally symmetrical, one of the sides being flattened. Later, this
flattened side becomes the hind-part of the embryo, the animal pole
corresponding to its dorsal side.
The size of the ripe egg cells differs very much. To mention some
examples : the egg diameter in Mytilus ediilis is about 60/x ; in Paludina
18/x, Limnaea stagnate 120/x, Littorina littorea 130ft, Litt, obtusata and
rudis 200jLt, Sycotypus canalliculatus 1 mm, Fulgur carica 1-7 mm; Argo-
nauta 1-3 mm, Eledone 8-15 mm, Nautilus 40-50 mm.
There is also a great diversity in yolk content. In general, the eggs of
Lamellibranchiata, Polyplacophora and Solenoconcha are poor in yolk.
In Gastropoda, yolk content differs much: little yolk e.g. in Paludina,
much in Nassa, Fulgur, Purpura, Buccinum, Murex. The same holds for
Cephalopoda, where yolk content increases according to the series
Argonauta<Octopus, Loligo<Sepia<Eledone<Nautilus. In general,
increase in egg size and in yolk content go hand in hand.
The superficial part of the egg cell is formed by a thin yolk-free ecto-
plasmic layer. Local thickenings of this layer may occur near the animal
and vegetative poles, forming the so-called pole plasms. Animal pole
plasms have been described, e.g. in Cumingia, Dentalium, Crepidula,
Fulgur, Physa, Planorbis, Limnaea and Limax; vegetative pole plasms
in Cumingia, Dentalium and Limnaea. It must be emphasized, however,
that these pole plasms of different species differ greatly as to their size,
composition and yolk content. It is not permissible, therefore, to con-
sider them as homologous cytoplasmic differentiations without further
proof; the term 'pole plasm' has only a descriptive meaning. In this
sense, the 'germinal disc' of cytoplasm at the animal pole of the ripe
cephalopod egg may also be called an animal pole plasm.
1
2 DEVELOPMENT OF THE MOLLUSC
The egg cells may be surrounded by three kinds of membranes:
(1) Primary egg membrane or vitelline membrane. This is secreted in the
ovary by the egg cell itself. In many cases (e.g. most Lamelli-
branchiata) it is the only egg membrane. Often it is very thin and
hardly visible. There are many reports in literature of molluscan eggs
being laid without a vitelline membrane (e.g. cephalopods) ; it is
probable, however, that in these cases the extremely thin membrane
has been overlooked. Moreover, in some cases the vitelline mem-
brane is thrown off very soon after laying, e.g. Mytilus, Dreissensia,
Ostrea, Dentalium. Often there is a small opening in the membrane ;
in Lamellibranchiata and Gastropoda this is always situated at the
vegetative pole. It is called a micropyle. The use of this term does
not imply, however, that it is used by the fertilizing sperm as a port
of entrance; this is not the case, as a rule. Rather, this so-called
micropyle represents a scar of the connecting stalk by which the egg
cell has been attached to the ovary.
(2) Secondary egg membrane or chorion, formed in the ovary by the
follicle cells surrounding the egg. It may be a thick and firm mem-
brane, as in the Polyplacophora, where its outer side is covered with
numerous warts or spines. In the cephalopods, the chorion is
thickened at the animal pole, and pierced by a funnel-shaped hole,
representing a true micropyle.
(3) Tertiary egg membranes, formed by the wall of the oviduct. They are
highly variable in shape and composition. Horny egg-shells are
found, e.g. in Sepia; calcareous shells in many land pulmonates
(Helix); various kinds of egg masses, egg ribbons and cocoons, in
which a certain number of egg cells, varying from a few to many
thousands, are embedded in a common jelly-mass, are found in
other gastropods.
2. Oogenesis
In molluscs various types of egg formation in the ovary occur.
In the Lamellibranchiata and Solenoconcha, egg formation is of the
'solitary' type, according to the classification by KORSCHELT and HEIDER
(1902). We may describe as an example the process in Cyclas, as ob-
served by STAUFFACHER (1894).
In Cyclas, the wall of the ovary consists of a regular columnar epi-
thelium. Single cells of this germinal epithelium round off and change
into oogonia with a large spherical nucleus with large nucleolus, and a
thin layer of cytoplasm. Then they begin to grow, and protrude into the
central cavity of the ovary; their connection with the basal membrane
OOGENESIS 3
of the ovarian wall is narrowed to a slender stalk. On the surface of that
part of the cell that projects freely into the cavity a vitelline membrane
is formed, presumably as a reaction of the cell surface to the contact
with the medium. In the stalk an accumulation of dark granules (mito-
chondria, according to WOODS, 1932) appears, reaching towards the
nucleus which lies at the base of the stalk. This may indicate the food
stream entering the growing oocyte from the ovarian wall. The nucleolus
shows signs of considerable activity, with formation of buds constricted
off from the main nucleolus. The adjacent cells of the germinal epi-
thelium lengthen and partly cover the stalk of the oocyte ; presumably,
they assist in transferring food substances to the latter. (According to
WOODS (1932) some of these cells are in Sphaerium wholly or partly
incorporated into the substance of the growing oocyte.) Later, these
cells withdraw from the stalk. The oocyte nucleus now moves towards
the free end of the cell. Finally, the oocyte is set free into the ovarian
cavity; a hole in the vitelline membrane (the so-called micropyle, cf.
above p. 2) indicates the place of its last attachment.
The tgg stalks, by which the growing oocytes are attached in the
ovary, may become very long and slender in certain Lamellibranchiata
(e.g. Scrobicularia).
In Polyplacophora and Gastropoda, less clearly also in Aplacophora,
egg formation is of the 'follicular' type. FAHMY (1949) has described the
process in the pulmonate Eremina. The undifferentiated germinal epi-
thelium consists here of a continuous layer of flat cells with flat oval
nuclei. Differentiation begins with the formation of male germ cells.
Then some of the cells of the germinal epithelium differentiate into
nurse cells. They extend on the inner side of the germinal epithelium,
forming a second layer towards the lumen of the ovotestis. Finally the
remaining cells of the germinal epithelium form oogonia, which begin
their development, therefore, beneath a layer of nurse cells, and remain
separated from the lumen by them during the whole period of oogenesis.
When the oocyte begins to grow and protrudes into the cavity, the
nurse cells form a follicle around it. This becomes thinner during growth
of the oocyte, and finally disappears just before ovulation.
According to ANCEL (1903), the follicle in Helix is not formed by the
nurse cells, but by indifferent cells of the peripheral layer secondarily
surrounding the oocyte. This author first advanced the idea that the
differentiation of the germinal epithelium cells into either male or
female germ cells is dependent on the absence or presence, respectively,
of differentiated nurse cells at that time.
This idea was somewhat modified by BURESCH (1911). Male and
female germ cells develop in Helix side by side in the same acinus of the
B
4 DEVELOPMENT OF THE MOLLUSC
ovotestis, but the developing oocytes lie always near one or more nurse
cells. Development in the male or female direction depends on the
conditions of nutrition of the indifferent germ cell. The follicle is at
first formed by the cytoplasm of the adjacent nurse cells; other cells
from the germinal epithelium, also differentiating into nurse cells, may
later be added.
In Cavolinia, different relationships were found by VITAGLIANO (1950).
Here the germ cells develop as far as the pachytene stage in the region
close to the protogonial zone of the gonad. From this stage onwards they
may either remain adjacent to those nurse cells, highly charged with
ribonucleic acid, lying in the male region of the gonad, in which case
they develop into spermatocytes ; or they migrate towards the centre of
the gonad, where the nurse cells contain little RNA; in this case they
become oocytes. Only during the last stage of their development (ad-
vanced vitellogenesis), do the oocytes pass into regions where the nurse
cells are rich in RNA. Then meiosis continues. It is suggested that the
sexual differentiation of the germ cells is determined by their spatial
relationships to the two kinds of nurse cells, and that the process of
meiosis is dependent on the intense production of RNA by the nurse
cells.
In Limnaea (BRETSCHNEIDER and RAVEN, 1951) the egg cells develop
likewise between the connective tissue capsule of the gonad and a layer
of epithelium cells. They pass first through a period of amoeboid
motility, during which they disperse over the acini. Then they become
sedentary, begin to grow and a follicle forms around their protruding
part in the same way as in Eremina, while the basal part of the oocyte
remains in contact with the connective tissue layer (Fig. 6). The nucleus
now takes an eccentric position in the apical half of the oocyte. After the
growth period of the oocyte has come to an end, there is a rest phase of
variable duration ; then the follicle is autolysed and ovulation takes place.
The cytological changes taking place during oogenesis in the egg cells
of the Polyplacophora have been described by GABE and PRENANT
(1949). The primordial oocyte in the germinal epithelium has a big
nucleus and a very thin layer of cytoplasm, which shows but little baso-
phily. Later, a cap of basophil substance is formed against one side of
the nucleus. It extends rapidly through the cell, and soon the whole
cytoplasm is strongly basophil. With further growth the cytoplasm be-
comes heterogeneous; clear areolae appear, first in the neighbourhood
of the stalk, and rapidly increase in number until the whole cytoplasm is
filled with them. Then yolk formation begins in contact with the areolae;
at the same time, the basophily of the cytoplasm decreases strongly.
Finally the full-grown oocyte is completely filled with yolk.
OOGENESIS 5
Egg formation in cephalopods is also of the follicular type. In con-
nection with the large size of the ripe egg, however, it exhibits the
peculiarity that the follicle shows a temporary folding (Ussow, 1881;
VIALLETON, 1888). The early oocytes are surrounded by one layer of
flat follicle cells; later, these cells become columnar and form the
membrana granulosa. This is surrounded by a second layer of connective
tissue cells, the theca. In the meantime, the egg has protruded from the
surface of the ovary, the theca forming a connecting stalk, in which
blood vessels develop. Then the granulosa begins to fold inwards ; both
transverse and longitudinal folds are formed, together forming a reti-
culate pattern. Blood vessels lie in the folds. Yolk is secreted by the
granulosa. It accumulates especially on the side of the stalk, pushing
the cytoplasm with the germinal vesicle towards the free end of the cell,
where the germinal disc is formed in this way. However, a very thin
ectoplasmic lamella remains around the whole periphery of the yolk.
When the oocyte has reached its final size, a chorion is formed beneath
the granulosa. It is thickened at the free pole, where a lens-shaped
thickening of the theca protruding into the granulosa prepares the for-
mation of the micropyle. Then the folds of the granulosa disappear, and
ovulation takes place through rupture of the follicle.
Presumably in all molluscs the side where the food stream reaches
the growing oocyte becomes the vegetative side of the egg. Thus the
polarity of the tgg appears to be determined by its position in the ovary.
3. The behaviour of various cell components during oogenesis
a. THE NUCLEUS
In the nucleus of the developing egg cell one functional phase is
followed by another. During the earlier part of oogenesis, the generative
function of the nucleus predominates; during the later part, it is the
vegetative function which especially attracts attention.
The first phase of premeiotic phenomena coincides with the very first
stages of egg formation : oogonium and early oocyte. It is described by
FAHMY (1949) for Eremina. The cells of the germinal epithelium have a
nucleus containing irregular chromatin granules. At the beginning of
female differentiation, a haploid number of double prochromosomes is
formed by condensation of the chromatin. At the same time a nucleolus
appears. The prochromosomes stretch to leptotene threads, each pro-
chromosome leaving a globular remnant. The threads unite in pairs to
zygotene bivalents, and then wind around each other into double strep-
sitene spirals, which are arranged radially around the central nucleolus
(Fig. 2a). They shorten to diplotene bivalents, arranged peripherally be-
