THE SEGMENTATION.
I have not been fortunate enough to obtain an absolutely complete series of
eggs during segmentation.
In the cases of Pristiurus and Scyllium only have I had any considerable
number of eggs in this condition, though one or two eggs of Raja in which
the process was not completed have come into my hands.
In the youngest impregnated Pristiurus eggs, which I have obtained, the
germinal disc was already divided into four segments.
The external appearance of the blastoderm, which remains nearly constant
during segmentation, has been already well described by Leydig[79].
Footnote 79: _Rochen und Haie._
The yolk has a pale greenish tinge which, on exposure to the air, acquires
a yellower hue. The true germinal disc appears as a circular spot of a
bright orange colour, and is, according to Leydig's measurements, 1-1/2m.
in diameter. Its colour renders it very conspicuous, a feature which is
further increased by its being surrounded by a narrow dark line (Pl. 6,
fig. 2), the indication of a shallow groove. Surrounding this line is a
concentric space which is lighter in colour than the remainder of the yolk,
but whose outer border passes by insensible gradations into the yolk. As
was mentioned in my preliminary paper (_loc. cit._), and as Leydig (_loc.
cit._) had before noticed, the germinal disc is always situated at the pole
of the yolk which is near the rounded end of the Pristiurus egg. It
occupies a corresponding position in the eggs of both species of Scyllium
(stellare and canicula) near the narrower end of the egg to which the
shorter pair of strings is attached. The germinal disc in the youngest egg
examined, exhibited two furrows which crossed each other at right angles in
the centre of the disc, but neither of which reached its edge. These
furrows accordingly divided the disc into four segments, completely
separated from each other at the centre of the disc, but united near its
circumference.
I made sections, though not very satisfactorily, of this germinal disc. The
sections shewed that the disc was composed of a protoplasmic basis, in
which were imbedded innumerable minute spherical yolk-globules so closely
packed as to constitute nearly the whole mass of the germinal disc.
In passing from the coarsest yolk-spheres to the fine spherules of the
germinal disc, three bands of different-sized yolk-particles have to be
traversed. These bands graduate into one another and are without sharp
lines of demarcation. The outer of the three is composed of the
largest-sized yolk-spherules which constitute the greater part of the ovum.
The middle band forms a concentric layer around the germinal disc, and is
composed of yolk-spheres considerably smaller than those outside it. Where
it cuts the surface it forms the zone of lighter colour immediately
surrounding the germinal disc. The innermost band is formed by the germinal
disc itself and is composed of spherules of the smallest size. These
features are shewn in Pl. 6, fig. 6, which is the section of a germinal
disc with twenty-one segments; in it however the outermost band of
spherules is not present.
From this description it is clear, as has already been mentioned in the
description of the ripe unimpregnated ovum, that the germinal disc is not
to be looked upon as a body entirely distinct from the remainder of the
ovum, but merely as a part of the ovum in which the protoplasm is more
concentrated and the yolk-spherules smaller than elsewhere. Sections shew
that the furrows visible on the surface end below, as indeed they do on the
surface, before they reach the external limit of the finely granular matter
of the germinal disc. There are therefore at this stage no distinct
segments: the otherwise intact germinal disc is merely grooved by two
furrows.
I failed to observe any nuclei in the germinal disc just described, but it
by no means follows that they were not present.
In the next youngest of the eggs[80] examined the germinal disc was already
divided into twenty-one segments. When viewed from the surface (Pl. 6,
fig. 3), the segments appeared divided into two distinct groups--an inner
group of eleven smaller segments, and an outer group of segments
surrounding the former. The segments of both the inner and the outer group
were very irregular in shape and varied considerably in size. The amount of
irregularity is far from constant and many germinal discs are more regular
than the one figured.
Footnote 80: The germinal disc figured was from the egg of a
Scyllium stellare and not Pristiurus, but I have also sections
of a Pristiurus egg of the same age, which do not differ
materially from the Scyllium sections.
In this case the situation of the germinal disc and its relations to the
yolk were precisely the same as in the earlier stage.
In sections of this germinal disc (Pl. 6, fig. 6), the groove which
separates it from the yolk is well marked on one side, but hardly visible
at the other extremity of the section.
Passing from the external features of this stage to those which are
displayed by sections, the striking point to be noticed is the persisting
continuity of the segments, marked out on the surface, with the floor of
the germinal disc.
The furrows which are visible on the surface merely form a pattern, but do
not isolate a series of distinct segments. They do not even extend to the
limit of the finely granular matter of the germinal disc.
The section represented, Pl. 6, fig. 6, bears out the statements about the
segments as seen on the surface. There are three smaller segments in the
middle of the section, and two larger at the two ends. These latter are
continuous with the coarser yolk-spheres surrounding the germinal disc and
are not separated from them by a segmentation furrow.
In a slightly older embryo than the one figured I met with a few completely
isolated segments at the surface. These segments were formed by the
apparent bifurcation of furrows as they neared the surface of the germinal
disc. The segments thus produced are triangular in form. They probably owe
their origin to the meeting of two oblique furrows. The last-formed of
these furrows apparently ceases to be prolonged after meeting the
first-formed furrow. I have not in any case observed an example of two
furrows crossing one another at this stage.
The furrows themselves for the most part are by no means simple slits with
parallel sides. They exhibit a beaded structure, shewn imperfectly in
Pl. 6, fig. 6, but better in Pl. 6, fig. 6_a_, which is executed on a
larger scale. They present intervals of dilatations where the protoplasms
of the segments on the two sides of the furrow are widely separated,
alternating with intervals where the protoplasms of the two segments are
almost in contact and are only separated from one another by a very narrow
space.
A closer study of the germinal disc at this period shews that the cavities
which cause the beaded structure of the furrows are not only present along
the lines of the furrows but are also found scattered generally through the
germinal disc, though far more thickly in the neighbourhood of the furrows.
Their appearance is that of vacuoles, and with these they are probably to
be compared. There can be little question that in the living germinal disc
they are filled with fluid. In some cases, they are collected in very large
numbers in the region of a furrow. Such a case as this is shewn in Pl. 6,
fig. 6_b_. In numerous other cases they occur, roughly speaking,
alternately on each side of a furrow. Some furrows, though not many, are
entirely destitute of these structures. The character of their distribution
renders it impossible to overlook the fact that these vacuole-like bodies
have important relations with the formation of the segmentation furrows.
Lining the two sides of the segmentation furrows there is present in
sections a layer which stains deeply with colouring reagents; and the
surface of the blastoderm is stained in the same manner. In neither case is
it permissible to suppose that any membrane-like structure is present. In
many cases a similar very delicate, but deeply-stained line, invests the
vacuolar cavities, but the fluid filling these remains quite unstained.
When distinct segments are formed, each of these is surrounded by a
similarly stained line.
The yolk-spherules are so numerous, and render even the thinnest section so
opaque, that I have failed to make satisfactory observations on the
behaviour of the nucleus. I find nuclei in many of the segments, though it
is very difficult even to see them, and only in very favourable specimens
can their structure be studied. In some cases, two of them lie one on each
side of a furrow; and in one case at the extreme end of a furrow I could
see two peculiar aggregations of yolk-spherules united by a band through
which the furrow, had it been continued, would have passed. The connection
(if any exists) between this appearance and the formation of the fresh
nuclei in the segments, I have been unable to elucidate.
The peculiar appearances attending the formation of fresh nuclei in
connection with cell-division, which have recently been described by so
many observers, have hitherto escaped my observation at this stage of the
segmentation, though I shall describe them in a later stage. A nucleus of
this stage is shewn on Pl. 6, fig. 6_c_. It is lobate in form and is
divided by lines into areas in each of which a deeply-stained granule is
situated.
The succeeding stages of segmentation present from the surface no fresh
features of great interest. The somewhat irregular (Pl. 6, figs. 4 and 5)
circular line, which divides the peripheral larger from the central smaller
segments, remains for a long time conspicuous. It appears to be the
representative of the horizontal furrow which, in the Batrachian ovum,
separates the smaller pigmented spheres from the larger spheres of the
lower pole of the egg.
As the segments become smaller and smaller, the distinction between the
peripheral and the central segments becomes less and less marked; but it
has not disappeared by the time that the segments become too small to be
seen with the simple lens. When the spheres become smaller than in the
germinal disc represented on Pl. 6, fig. 5, the features of segmentation
can be more easily and more satisfactorily studied by means of sections.
To the features presented in sections, both of the latter and of the
earlier blastoderms, I now return. A section of one of the earlier germinal
discs, of about the age of the one represented on Pl. 6, fig. 4, is shewn
in Pl. 6, fig. 7.
It is clear at a glance that we are now dealing with true segments
completely circumscribed on all sides. The peripheral segments are, as a
rule, larger than the more central ones, though in this respect there is
considerable irregularity. The segments are becoming smaller by repeated
division; but, in addition to this mode of increase, there is now going on
outside the germinal disc a segmentation of the yolk, by which fresh
segments are being formed from the yolk and added to those which already
exist in the germinal disc. One or two such segments are seen in the act of
being formed (Pl. 6, fig. 7, _f_); and it is to be noticed that the furrows
which will eventually mark out the segments, do so at first in a partial
manner only, and do not circumscribe the whole circumference of the segment
in the act of being formed. These fresh furrows are thus repetitions on a
small scale of the earliest segmentation furrows.
It deserves to be noticed that the portion of the germinal disc which has
already undergone segmentation, is still surrounded by a broad band of
small-sized yolk-spherules. It appears to me probable that owing to changes
taking place in the spherules of the yolk, which result in the formation of
fresh spherules of a small size, this band undergoes a continuous
renovation.
The uppermost row of segmentation spheres is now commencing to be
distinguished from the remainder as a separate layer which becomes
progressively more distinct as segmentation proceeds.
The largest segments in this section measure about the 1/100th of an inch
in diameter, and the smallest about 1/300th of an inch.
The nuclei at this stage present points of rather a special interest. In
the first place, though visible in many, and certainly present in all the
segments[81], they are not confined to these: they are also to be seen, in
small numbers, in the band of fine spherules which surrounds the already
segmented part of the germinal disc. Those found outside the germinal disc
are not confined to the spots where fresh segments are appearing, but are
also to be seen in places where there are no traces of fresh segments.
Footnote 81: In the figure of this stage, I have inserted
nuclei in all the segments. In the section from which the
figure was taken, nuclei were not to be seen in many of the
segments, but I have not a question that they were present in
all of them. The difficulty of seeing them is, in part, due to
the yolk-spherules and in part to the thinness of the section
as compared with the diameter of a segmentation sphere.
This fact, especially when taken in connection with the formation of fresh
segments outside the germinal disc and with other facts which I shall
mention hereafter, is of great morphological interest as bearing upon the
nature and homologies of the food-yolk. It also throws light upon the
behaviour and mode of increase of the nuclei. All the nuclei, both those of
the segments and those of the yolk, have the peculiar structure I described
in the last stage.
In specimens of this stage I have been able to observe certain points which
have an important bearing upon the behaviour of the nucleus during
cell-division.
Three figures, illustrating the behaviour of the nucleus, as I have seen it
in sections of blastoderms hardened in chromic acid, are shewn in Pl. 6,
figs. 7_a_, 7_b_ and 7_c_.
In the place of the nucleus is to be seen a sharply defined figure
(Fig. 7_a_) stained in the same way as the nucleus or more deeply. It has
the shape of two cones placed base to base. From the apex of each cone
there diverge towards the base a series of excessively fine striæ. At the
junction between the two cones is an irregular linear series of small
deeply stained granules which form an apparent break between the two. The
line of this break is continued very indistinctly beyond the edge of the
figure on each side.
From the apex of each cone there diverge outwards into the protoplasm of
the cell a series of indistinct markings. They are rendered obscure by the
presence of yolk-spherules, which completely surround the body just
described, but which are not arranged with any reference to these markings.
These latter striæ, diverging from the apex of the cone, are more
distinctly seen when the apex points to the observer (Fig. 7_b_), than when
a side of the cone is in view.
The striæ diverging outwards from the apices of the cones must be carefully
distinguished from the striæ of the cones themselves. The cones are bodies
quite as distinctly differentiated from the protoplasm of the cell as
nuclei, while the striæ which diverge from their apices are merely
structures in the general protoplasm of the cell.
In some cells, which contain these bodies, no trace of a commencing line of
division is visible. In other cases (Fig. 7_c_), such a line of division
does appear and passes through the junction of the two cones. In one case
of this kind I fancied I could see (and have represented) a coloured
circular body in each cone. I do not feel any confidence that these two
bodies are constantly present; and even where visible they are very
indistinct.
Instead of an ordinary nucleus a very indistinctly marked vesicular body
sometimes appears in a segment; but whether it is to be looked on as a
nucleus not satisfactorily stained, or as a nucleus in the act of being
formed, I cannot decide.
With reference to the situation of the cone-like bodies I have described I
have made an observation which appears to me to be of some interest. I find
that bodies of this kind are found in the yolk _completely outside_ the
germinal disc. I have made this observation, in at least two cases which
admitted of no doubt (vide Fig. 7, _nx´_).
We have therefore the remarkable fact, that whatever connection these
bodies may have with cell-division, they can occur in cases where this is
altogether out of the question and where an increase in the number of
nuclei can be their only product.
These are the main facts which I have been able to determine with reference
to the nuclei of this stage; but it will conduce to clearness if I now
finish what I have to say upon this subject.
At a still later stage of segmentation the same peculiar bodies are to be
seen as during the stage just described, but they are rarer; and, in
addition to them, other bodies are to be seen of a character intermediate
between ordinary nuclei and the former bodies.
Three such are represented in Pl. 6, figs. 8_a_, 8_b_, 8_c_. In all of
these there can be traced out the two cones, which are however very
irregular. The striation of the cones is still present, but is not nearly
so clear as it was in the earlier stage.
In addition to this, there are numerous deeply stained granules scattered
about the two figures which resemble exactly the granules of typical
nuclei.
All these bodies occupy the place of an ordinary nucleus, they stain like
an ordinary nucleus and are as sharply defined as an ordinary nucleus.
There is present around some of these, especially those situated in the
yolk, the network of lines of the yolk described by me in a preliminary
paper[82], and I feel satisfied that there is in some cases an actual
connection between the network and the nuclei. This network I shall
describe more fully hereafter.
Footnote 82: _Loc. cit._
Further points about these figures and the nuclei of this stage I should
like to have been able to observe more completely than I have done, but
they are so small that with the highest powers I possess (Zeiss, Immersion
No. 2 = 1/15 in.) their complete and satisfactory investigation is not
possible.
Most of the true nuclei of the cells of the germinal disc are regularly
rounded; those however of the yolk are frequently irregular in shape and
often provided with knob-like processes. The gradations are so complete
between typical nuclei and bodies like that shewn (Pl. 6, fig. 8_c_) that
it is impossible to refuse the name of nucleus to the latter.
In many cases _two nuclei_ are present in one cell.
In later stages knob-like nuclei of various sizes are scattered in very
great numbers in the yolk around the blastoderm (vide Pl. 7). In some cases
it appears to me that several of these are in close juxtaposition, as if
they had been produced by the division of one primitive nucleus. I do not
feel absolutely confident that this is the case, owing to the fact that in
the investigation of a knobbed body there is great difficulty in
ascertaining that the knobs, which appear separate in one plane, are not in
reality united in another.
I have, in spite of careful search, hitherto failed to find amongst these
later nuclei cone-like figures, similar to those I found in the yolk during
segmentation. This is the more remarkable since in the early stages of
segmentation, when very few nuclei are present in the yolk, the cone-like
figures are not uncommon; whereas, in the latter stages of development when
the nuclei of the yolk are very common and obviously increasing rapidly,
such figures are not to be met with.
In no case have I been able to see a distinct membrane round any of the
nuclei.
I have hitherto attempted to describe the appearances bearing on the
behaviour of the nuclei in as objective a manner as possible.
My observations are not as complete as could be desired; but, taken in
conjunction with those of other investigators, they appear to me to point
towards certain definite conclusions with reference to the behaviour of the
nucleus in cell-division.
The most important of these conclusions may be stated as follows. In the
act of cell-division the nuclei of the resulting cells are formed from the
nucleus of the primitive cell.
This may occur:--
(1) By the complete solution of the old nucleus within the protoplasm of
the mother cell and the subsequent reaggregation of its matter to form the
nuclei of the freshly formed daughter cells,
(2) By the simple division of the nucleus,
(3) Or by a process intermediate between these two where part of the old
nucleus passes into the general protoplasm and part remains always
distinguishable and divides; the fresh nucleus being in this case formed
from the divided parts as well as from the dissolved parts of the old
nucleus.
Included in this third process it is permissible to suppose that we may
have a series of all possible gradations between the extreme processes 1
and 2. If it be admitted, and the evidence we have is certainly in favour
of it, that in some cases, both in animal and vegetable cells, the nucleus
itself divides during cell division, and in others the nucleus completely
vanishes during the cell-division, it is more reasonable to suspect the
existence of some connection between the two processes, than to suppose
that they are entirely different in kind. Such a connection is given by the
hypothesis I have just proposed.
The evidence for this view, derived both from my own observations and those
of other investigators, may be put as follows.
The absolute division of the nucleus has been stated to occur in animal
cells, but the number of instances where the evidence is quite conclusive
are not very numerous. Recently F. E. Schultze[83] appears to have observed
it in the case of an Amoeba in an altogether satisfactory manner. The
instance is quoted by Flemming[84]. Schultze saw the nucleus assume a
dumb-bell shape, divide, and the two halves collect themselves together.
The whole process occupied a minute and a half and was shortly followed by
the division of the Amoeba, which occupied eight minutes. Amongst vegetable
cells the division of the nucleus seems to be still rarer than with animal
cells. Sachs[85] admits the division of the nucleus in the case of the
parenchyma cells of certain Dicotyledons (Sambucus, Helianthus, Lysimachia,
Polygonum, Silene) on the authority of Hanstein.
Footnote 83: _Archiv f. Micr. Anat._ XI. p. 592.
Footnote 84: "Entwicklungsgeschicte der Najaden," LXXI. Bd.
_der Sitz. der k. Acad. Wien_, 1875.
Footnote 85: _Text-Book of Botany_, English trans. p. 19.
The division of the nucleus during cell-division, though seemingly not very
common, must therefore be considered as a thoroughly well authenticated
occurrence.
The frequent disappearance of the nucleus during cell-division is now so
thoroughly recognised, both for animal and vegetable cells, as to require
no further mention.
In many cases the partial or complete disappearance of the nucleus is
accompanied by the formation of two peculiar star-like figures. Appearances
of the kind have been described by Fol[86], Flemming[87], Auerbach[88] and
possibly also Oellacher[89] as well as other observers.
Footnote 86: "Entw. d. Geryonideneies." _Jenaische
Zeitschrift_, Bd. VII.
Footnote 87: _Loc. cit._
Footnote 88: _Organologische Studien_, Zweites Heft.
Footnote 89: "Beiträge z. Entwicklungsgeschichte der
Knochenfischen." _Zeit. für Wiss. Zoologie_. Bd. XXII. 1872.
These figures[90] are possibly due to the streaming out of the protoplasm
of the nucleus into that of the cell[91]. The appearance of striation may
on this hypothesis be explained as due to the presence of granules in the
protoplasm. When the streaming out of the protoplasm of a nucleus into that
of a cell takes place, any large granule which cannot be moved by the
stream will leave behind it a slack area where there is no movement of the
fluid. Any granules which are carried into this area will remain there, and
by the continuation of a process of this kind a row of granules may be
formed, and a series of such rows would produce an appearance of striation.
In many cases, _e.g._ Anodon, vide Flemming[92], even the larger
yolk-spherules are arranged in this fashion.
Footnote 90: The memoirs of Auerbach and Strasburger
(_Zellbildung u. Zelltheilung_) have unfortunately come into my
hands too late for me to take advantage of them. Especially in
the magnificent monograph of Strasburger I find drawings
precisely resembling those from my specimens already in the
hands of the engraver. Strasburger comes to the conclusion from
his investigations that the modified nucleus always divides and
never vanishes as is usually stated. If his views on this point
are correct part of the hypothesis I have suggested above is
rendered unnecessary. The striæ of the protoplasm, which in
accordance with Auerbach's view I have considered as being due
to a streaming out of the matter of the nucleus, he regards as
resulting from a polarity of the particles in the cell and the
attraction of the nucleus. My own investigations though, as far
as they go, quite in accordance with those of Strasburger, do
not supply any grounds for deciding on the meaning of these
striæ; and in some respects they support Strasburger's views
against those of other observers, since they demonstrate that
in Elasmobranchii the modified nucleus does actually divide.
Footnote 91: This is the view which has been taken by Auerbach
(_Organologische Studien_).
Footnote 92: _Loc. cit._
On the supposition that the striation of these figures is due to the
outflow from the nucleus, the appearances presented in Elasmobranchii admit
of the following explanation.
The central body consisting of two cones (figs. 7_a_, 7_c_) is almost
without question the remnant of the primitive nucleus. This is shewn by its
occupying the same position as the primitive nucleus, staining in the same
way, and by there being a series of insensible gradations between it and a
typical nucleus. The contents must be supposed to be streaming out from the
two apices of the cones, as appears from the striæ in the body converging
on each side towards the apex, and then diverging again from it. In my
specimens the yolk-spherules are not arranged with any reference to the
radiating striation.
It is very likely that in the cases of the disappearance of the nucleus,
its protoplasm streams out in two directions, towards the two parts of the
cell which will eventually become separated from each other; and probably,
after the division, the matter of the old nucleus is again collected to
form two fresh nuclei.
In some cases of cell-division a remnant of the old nucleus is stated to be
visible after the fresh nuclei have appeared. These cases, of which I have
not seen full accounts, are perhaps analogous to what occasionally happens
with the germinal vesicle of an ovum. The whole of the contents of the
germinal vesicle become at its disappearance mingled with the protoplasm of
the ovum, but the resistant membrane remains and is eventually ejected from
the egg, vide p. 215 _et seq_. If the remnant of the old nucleus in the
cases described is nothing more than its membrane, no difficulty is offered
to the view that the constituents of the old nucleus may help to form the
new ones.
In many cases the total bulk of the new nuclei is greater than that of the
old one; in such instances part of the protoplasm of the cell necessarily
has a share in forming the new nuclei.
Although, in instances where the nucleus vanishes, an absolute
demonstration of the formation of the fresh nuclei from the matter of the
old one is not possible; yet, if cases of the division of the old nucleus
to form the new ones be admitted to exist, the derivation in the first
process of the fresh nuclei from the old ones must be postulated in order
to maintain a continuity between the two processes of formation; and, as I
have attempted to shew, all the circumstantial evidence is in favour of it.
Admitting the existence of the two extreme processes of nuclear formation,
I wish to shew that my results in Elasmobranchii tend to demonstrate the
existence of intermediate steps between them. The first figures I described
of two opposed cones, appear to me almost certainly to represent nuclei in
the act of dissolution; but though a portion of the nucleus may stream out
into the yolk, I think it impossible that the whole of it does[93].
Footnote 93: After Strasburger's observation it must be
considered very doubtful whether the streaming out of the
contents of the nucleus, in the manner implied in the text,
really takes place.
I described these bodies in two states. An earlier one, in which the two
cones were separated by an irregular row of deeply stained granules; and a
later one in which a furrow had already appeared dividing the cones as well
as the cell. In neither of these conditions could I see any signs of the
body vanishing completely. It was as clearly defined and as deeply stained
as an ordinary nucleus, and in its later condition the signs of the
streaming out of material from its pointed extremities were less marked
than in the earlier stage.
All these facts, to my mind, point to the view that these cone-like bodies
do not disappear, but form the basis for the new nuclei. Possibly the body
visible in each cone in the later stage, was the commencement of this new
nucleus. Götte[94] has figured structures somewhat similar to these bodies,
but I hardly understand either his figure or his account sufficiently
clearly to be able to pronounce upon the identity of the two. In case they
are identical, Götte gives a very different explanation of them from my
own[95].
Footnote 94: _Entwicklungsgeschite der Unke_, Pl. 1, fig. 18.
Footnote 95: As I before mentioned, Strasburger (_Zellbildung
u. Zelltheilung_) has represented bodies precisely similar to
those I have described, which appear during the segmentation in
the egg of _Phallusia mammillata_ as well as similar figures
observed by Butschli in eggs of _Cucullanus elegans_ and
_Blatta Germanica_. The figures in this monograph are the only
ones I have seen, which are identical with my own.
A second of my results, which points to a series of intermediate steps
between division and solution of the nucleus, is the distribution in time
of the peculiar cone-like bodies. These are present in fair abundance at an
early period of segmentation, when there are but few nuclei either in the
blastoderm or the yolk. But at later periods, when there are both more
nuclei, especially in the yolk, and they are also increasing in numbers
more rapidly than before, no bodies of this kind are to be seen. This fact
becomes the more striking from the lobate appearance of the later nuclei of
the yolk, an appearance which exactly suits the hypothesis of the rapid
budding off of fresh nuclei.
The observations of R. Hertwig[96] on the gemmation of _Podophrya
gemmipara_, support my interpretation of the knobbed condition of the
nuclei. Hertwig finds (p. 47) that
The horse-shoe shaped nucleus grows out into numerous
anastomosing projections. Over the free ends of the
projections little knobs appear on the surface of the body,
into which the lengthening ends of the processes of the
nucleus grow up. Here they bend themselves into a horse-shoe
form. The newly-formed nucleus then separates from the
original nucleus, and afterwards the bud containing it from
the body.
Footnote 96: _Morphologisches Jahrbuch_, Bd. 1. pp. 46, 47.
From the peculiar arrangement of the net-work of lines of the yolk around
these knobbed nuclei, it is reasonable to conclude that interchange of
material between the protoplasm of the yolk and the nuclei is still taking
place, even during the later periods.
These facts about the distribution in time of the cone-like bodies afford a
strong presumptive evidence of a change in the manner of nuclear increase.
The last argument I propose urging on this head is derived from the bodies
(Pl. 6, fig. 8_a_, _b_, _c_) which I have described as intermediate between
the true cone-like bodies and typical nuclei. They appear to afford
evidence of less and less of the matter of the nucleus streaming out into
the yolk and of a large proportion of it becoming divided.
The conclusion to be derived from all these facts is that for
Elasmobranchii in the earlier stages of segmentation, and during the
formation of fresh segments, a partial solution of the old nucleus takes
place, but all its constituents serve for the reconstruction of the fresh
nuclei.
In later periods of development a still smaller part of the nucleus becomes
dissolved, and the rest divides; but the two fresh nuclei are still derived
from the two sources. After the close of segmentation the fresh nuclei are
formed by a simple division of the older ones.
The appearance of the cone-like bodies in the yolk outside the germinal
disc is a point of some interest. It demonstrates in a conclusive manner
that whatever influence (if any) the nucleus may have in ordinary cases of
cell division, yet it may undergo changes of a precisely similar character
to those which it experiences during cell division, without exerting any
influence on the surrounding protoplasm[97]. If the lobate nuclei are also
nuclei undergoing division, we have in the egg of an Elasmobranch examples
of all the known forms of nuclear increase unaccompanied by cell division.
Footnote 97: Strasburger's (_loc. cit._) arguments about the
influence of the nucleus in cell division are not to my mind
conclusive; though not without importance. It is difficult to
reconcile his views with the facts of cell division observable
during the Elasmobranch segmentation; but even if their truth
be admitted they do not bring us much nearer to a satisfactory
understanding of cell division, unless accompanied (and at
present they are not so) by a rational explanation of the
forces which produce the division of the nucleus.
The next stage in the segmentation does not present so many features of
interest as the last one. The segments are now so small, as to be barely
visible from the surface with a simple lens. A section of an embryo of this
stage is represented in Pl. 6, fig. 8. The section, which is drawn on the
same scale as the section belonging to the last stage, serves to shew the
relative size of the segments in the two cases.
The epiblast is now more distinct than it was. The segments composing it
are markedly smaller than the remainder of the cells of the germinal disc,
but possess nuclei of an absolutely larger size than do the other cells.
They are irregular in shape, with a slight tendency to be columnar. An
average segment of this layer measures about 1/700 inch.
The cells of the lower layer are more polygonal than those of the epiblast,
and are decidedly larger. An average specimen of the larger cells of the
lower layer measures about 1/400 in. in diameter, and is therefore
considerably smaller than one of the smallest cells of the last stage. The
formation of fresh segments from the yolk still continues with fair
rapidity, but nearly comes to an end shortly after this.
Of the nuclei of the lower layer cells, there is not much to add to what
has already been said. Not infrequently two nuclei may be observed in a
single cell.
The nuclei in the yolk which surrounds the germinal disc are more numerous
than in the earlier periods, and are now to be met with in fair numbers in
every section (fig. 8, _n´_).
These are the main features which characterise the present stage, they are
in all essential points similar to those of the last stage, and the two
germinal discs hardly differ except in the size of the segments of which
they are composed.
In the last stage which I consider as belonging to the segmentation, the
cells of the whole blastoderm have become smaller (Pl. 6, fig. 9).
The epiblast (_ep_) now consists of a very marked layer of columnar cells.
It is, as far as I have been able to observe, never more than one cell
deep. The cells of the lower layer are of an approximately uniform size,
though a few of those at the circumference of the blastoderm considerably
exceed the remainder in the bulk.
There are two fresh features of importance in germinal discs of this age.
Instead of being but indistinctly separated from the surrounding yolk, the
blastoderm has now very clearly defined limits.
This is an especially marked feature of preparations made with osmic acid.
In these there may frequently be seen a deeply stained doubly contoured
line, which forms the limit of the yolk, where it surrounds the germinal
disc. Lines of this kind are often to be seen on the surface of the yolk,
or even of the blastoderm, but are probably to be regarded as products of
reagents, rather than as organised structures. The outline of the germinal
disc is well rounded, though it is occasionally broken, from the presence
of a larger cell in the act of being formed from the yolk.
It is not probable that any great importance is to be attached to the
comparative distinctness of the outline of the germinal disc at this stage,
which is in a great measure due to a cessation in the formation of fresh
cells in the surrounding yolk, and in part to the small and comparatively
uniform size of the cells of the germinal disc.
The formation of fresh cells from the yolk nearly comes to an end during
this period, but it still continues on a small scale.
The number of the nuclei around the germinal disc has increased.
Another feature of interest which first becomes apparent during this stage
is the asymmetry of the germinal disc. If a section were made through the
germinal disc, as it lay _in situ_ in the egg capsule, parallel or nearly
so to the long axis of the capsule, one end of the section would be found
to be much thicker than the other. There would in fact be a far larger
collection of cells at one extremity of the germinal disc than at the
other. The end at which this collection of cells is formed points towards
the end of the egg capsule opposite to that near which the yolk is
situated. This collection of cells is the first trace of the embryo; and
with its appearance the segmentation may be supposed to terminate.
The section I have represented, though not quite parallel to the long axis
of the egg, is sufficiently nearly so to shew the greater mass of cells at
the embryonic end of the germinal disc.
This very early appearance of a distinction in the germinal disc between
the extremity at which the embryo appears and the non-embryonic part of the
disc, besides its inherent interest, has a further importance from the fact
that in Osseous Fishes a similar occurrence takes place. Oellacher[98] and
Götte[99] both agree as to the very early period at which a thickening of
one extremity of the blastoderm in Osseous Fishes is formed, which serves
to indicate the position at which the embryo will appear. There are many
details of development in which Osseous Fish and Elasmobranchii agree,
which, although if taken individually are without any great importance, yet
serve to shew how long even insignificant features in development may be
retained.
Footnote 98: _Zeitschrift für Wiss. Zoologie_, Bd. XXIII.
1873.
Footnote 99: _Archiv für Micr. Anat._ Bd. IX. 1873.
* * * * *
The segmentation of the Elasmobranch egg presents in most of its features
great regularity, and exhibits in its mode of occurrence the closest
resemblance to that in other meroblastic vertebrate ova.
There is, nevertheless, one point with reference to which a slight
irregularity may be observed. In almost all eggs segmentation commences by,
what for convenience may be called, a vertical furrow which is followed by
a second vertical furrow at right angles to the first. The third furrow
however is a horizontal one, and cuts the other two at right angles. This
method of segmentation must be looked on as the normal one, in almost all
the important groups of the animal kingdom, both for the so-called
holoblastic and meroblastic eggs, and the gradations intermediate between
the two. The Frog amongst vertebrates exhibits a most typical instance of
this form of segmentation.
In Elasmobranchii the first two furrows are formed in a perfectly normal
manner, but though I have not observed the actual formation of the next
furrow, yet from the later stages, which I have observed, I conclude that
it is parallel to one of the first formed furrows; and it is fairly certain
that, not till a considerably later period, is a furrow homologous with the
horizontal furrow of the Batrachian egg formed. This furrow appears to be
represented in the Elasmobranch segmentation by the irregular
circumscription of a body of central smaller spheres from a ring of
peripheral larger ones (vide Pl. 6, figs. 3, 4 and 5).
In the Bird the representative of the horizontal furrow appears relatively
much earlier. It is formed when there are eight segments marked out on the
surface of the germinal disc[100]. From Oellacher's[101] account of the
segmentation in the fowl[102] it seems certain, as might be anticipated,
that this furrow is nearly parallel to the surface of the disc, so that it
cuts the earlier formed vertical furrows and causes the segments of the
germinal disc to be completely circumscribed below as well as at the
surface. In the Elasmobranch egg this is not the case; so that, even after
the smaller central segments have become separated from the outer ring of
larger ones, none of the segments of the disc are completely circumscribed,
and only appear to be so in surface views (vide Pl. 6, fig. 6).
Segmentation in the Elasmobranch egg differs in the following particulars
from that in the Bird's egg:
(1) The equivalent of the horizontal furrow of the Batrachian egg appears
much later than in the Bird.
(2) When it has appeared it travels inwards much more slowly.
Footnote 100: Vide _Elements of Embryology_, p. 23.
Footnote 101: _Stricker's Studien_, 1869, Pt. I, Pl. II. fig. 4.
Footnote 102: Unfortunately Professor Oellacher gives no
account of the surface appearance of the germinal discs of
which he describes the sections. It is therefore uncertain to
what period his sections belong.
As a result of these differences, the segments of the germinal disc of the
Birds' eggs are much earlier circumscribed on all sides than those of the
Elasmobranch egg.
As might be expected, the segmentation of the Elasmobranch egg resembles in
many points that of Osseous Fishes (vide Oellacher[103] and Klein[104]). It
may be noticed, that with Osseous as with Elasmobranch Fishes, the furrow
corresponding with the horizontal furrow of the Amphibian's egg does not
appear at as early a period as is normal. The third furrow of an Osseous
Fish egg is parallel to one of the first formed pair.
Footnote 103: _Zeitschrift für Wiss. Zool._ Bd. XXII. 1872.
Footnote 104: _Monthly Microscopical Journal_, March, 1872.
In Oellacher's[105] figures, Pl. 23, figs. 19-21, peculiar beadings of the
sides of the earlier formed furrows are distinctly shewn. No mention of
these is made in the text, but they are unquestionably similar to those I
have described in the Elasmobranch furrows. In the case of Elasmobranchii I
pointed out that not only were the sides of the furrow beaded, but that
there appeared in the protoplasm, close to the furrows, peculiar
vacuole-like cavities, precisely similar to the cavities which were the
cause of the beadings of the furrows.
Footnote 105: _Loc. cit._
The presence of these seems to shew that the molecular cohesion of the
protoplasm becomes, as compared with other parts, much diminished in the
region where a furrow is about to appear, so that before the protoplasm
finally gives way along a particular line to form a furrow, its cohesion is
broken at numerous points in this region, and thus a series of vacuole-like
spaces is formed.
If this is the true explanation of the formation of these spaces, their
presence gives considerable support to the views of Dr Kleinenberg upon the
causes of segmentation, so clearly and precisely stated in his monograph
upon Hydra; and is opposed to any view which regards the forces which come
into play during segmentation as resident in the nucleus.
I have not observed the peculiar threads of protoplasm which Oellacher[106]
describes as crossing the commencing segmentation furrows. I have also
failed to discover any signs of a concentration of the yolk-spherules,
round one or two centres, in the segmentation spheres, similar to that
observed by Oellacher in the segmenting eggs of Osseous Fish. The
appearances observed by him are probably connected with the behaviour of
the nucleus during segmentation, and are related to the curious bodies I
have already described.
Footnote 106: _Loc. cit._
With reference to the nuclei which Oellacher[107] has described as
occurring in the eggs of Osseous Fish during segmentation, there can, I
think, be little doubt that they are identical with the peculiar nuclei in
the Elasmobranch eggs.
Footnote 107: _Loc. cit._
He[108] says:
In an unsegmented germ there occurred at a certain point in
the section ... a small aggregation of round bodies. I do
not feel satisfied whether these aggregations represent one
or more nuclei.
Fig. 29 shews such aggregation; by focusing at its optical
section eleven unequally large rounded bodies measuring from
0.004 - 0.009 mm. may be distinguished. They lay as if in a
multilocular gap in the germ mass, which however they did
not quite fill. In each of these bodies there appeared
another but far smaller body. These aggregations were
distinguished from the germ by an especially beautiful
intense violet gold chloride colouration of their elements.
The smaller elements contained in the larger were still more
intensely coloured than the larger.
Footnote 108: _Loc. cit._ pp. 410, 411, &c.
He further states that these aggregations equal the segments in number, and
that the small bodies within the elements are not always to be seen with
the same distinctness.
Oellacher's description as well as his figures of these bodies leaves no
doubt in my mind that they are exactly similar bodies to those which I have
already spoken of as nuclei, and the characteristic features of which I
have shortly mentioned, and shall describe more fully at a later stage. A
moderately full description of them is to be found in my preliminary
paper[109].
Footnote 109: _Loc. cit._ p. 415. [This Edition, p. 64.]
Their division into a series of separate areas each with a deeply-stained
body, as well as the staining of the whole of them, exactly corresponds to
what I have found. That each is a single nucleus is quite certain, though
their knobbed form might occasionally lead to the view of their being
divided. This knobbed condition, observed by Oellacher as well as myself,
certainly supports the view, that they are in the act of budding off fresh
nuclei. Oellacher conceives, that the areas into which these nuclei are
divided represent a series of separate bodies--this according to my
observations is not the case. Nuclei of the same form have already been
described in Nephelis, and are probably not very rare. They pass by
insensible gradations into ordinary nuclei with numerous granules.
One marked feature of the segmentation of the Elasmobranch egg is the
continuous advance of the process of segmentation into the yolk and the
assimilation of this into the germ by the direct formation of fresh
segments out of it. Into the significance of this feature I intend to enter
fully hereafter; but it is interesting to notice that Oellacher's
descriptions point to a similar feature in the segmentation of Osseous
Fish. This however consists chiefly in the formation of fresh segments from
the lower parts of the germinal disc which in Osseous Fish is more
distinctly marked off from the food-yolk than in Elasmobranchii.
I conclude my description of the segmentation by a short account of what
other investigators have written about its features in these fishes. One of
the earliest descriptions of this process was given by Leydig[110]. To his
description of the germinal disc, I have already done full justice.
Footnote 110: _Rochen u. Haie._ It is here mentioned that
Coste observed the segmentation in these fishes.
In the first stage of segmentation which he observed 20-30 segments were
already visible on the surface. In each of these he recognized a nucleus
but no nucleolus.
He rightly states that the segments have no membrane, and describes the
yolk-spherules which fill them.
The next investigator is Gerbe[111]. I have unfortunately been unable to
refer to this elaborate paper, but I gather from an abstract that M. Gerbe
has given a careful description of the external features of segmentation.
Footnote 111: "Recherches sur la segmentation des products
adventifs de l'oeuf des Plagiostomes et particulièrement des
Raies." Robin, _Journal de l'Anatomie et de la Physiologie_, p.
609, 1872.
Schenk[112] has also made important investigations on the subject. He
considers that the ovum is invested with a very delicate membrane. This
membrane I have failed to find a trace of, and agree with Leydig[113] in
denying its existence. Schenk further found that after impregnation, but
before segmentation, the germinal disc divided itself into two layers, an
upper and a lower. Between the two a cavity made its appearance which
Schenk looks upon as the segmentation cavity. Segmentation commences in the
upper of the two layers, but Schenk does not give a precise account of the
fate of the lower. I have had no opportunity of investigating the
impregnated ovum before the commencement of segmentation, but my
observations upon the early stages of this process render it clear that no
division of the germinal disc exists subsequently to the commencement of
segmentation, and that the cavity discovered by Schenk can have no
connection whatever with the segmentation cavity. I am indeed inclined to
look upon this cavity as an artificial product. I have myself met with
somewhat similar appearances, after the completion of segmentation, which
were caused by the non-penetration of my hardening reagent beyond a certain
point.
Footnote 112: "Die Eier von Raja quadrimaculata innerhalb der
Eileiter." _Sitz. der k. Akad. Wien._ Vol. LXXIII. 1873.
Footnote 113: _Loc. cit._ My denial of the existence of this
membrane naturally applies only to the egg after impregnation,
and to the genera Scyllium and Pristiurus.
Without attempting absolutely to explain the appearances described by
Professor Schenk, I think that his observations ought to be repeated,
either by himself or some other competent observer.
Several further facts are recorded by Professor Schenk in his interesting
paper. He states that immediately after impregnation, the germinal disc
presents towards the yolk a strongly convex surface, and that at a later
period, but still before the commencement of segmentation, this becomes
flattened out. He has further detected amoeboid movements in the disc at
the same period. As to the changes of the germinal disc during
segmentation, his paper contains no facts of importance.
Next in point of time to the paper of Schenk, is my own preliminary account
of the development of the Elasmobranch Fishes[114]. In this a large number
of the facts here described in full are briefly alluded to.
Footnote 114: _Loc. cit._
The last author who has investigated the segmentation in Elasmobranchii, is
Dr Alexander Schultz[115]. He merely states that he has observed the
segmentation, and confirms Professor Schenk's statements about the amoeboid
movements of the germinal disc.
Footnote 115: "Die Embryonal Anlage der Selachier. Vorläufige
Mittheilung," _Centralblatt f. Med. Wiss._ No. 33, 1875.
EXPLANATION OF PLATE 6.
Fig. 1. Section through the germinal disc of a ripe ovarian ovum of the
Skate. _gv._ germinal vesicle.
Fig. 2. Surface-view of a germinal disc with two furrows.
Figs. 3, 4, 5. Surface-views of three germinal discs in different stages of
segmentation.
Fig. 6. Section through the germinal disc represented in fig 3. _n._
nucleus; _x._ edge of germinal disc. The engraver has not accurately copied
my original drawings in respect to the structure of the segmentation
furrows.
Figs. 6_a_ and 6_b_. Two furrows of the same germinal disc more highly
magnified.
Fig. 6_c_. A nucleus from the same germinal disc highly magnified.
Fig. 7. Section through a germinal disc of the same age as that represented
in fig. 4. _n._ nucleus; _nx._ modified nucleus; _nx´._ modified nucleus of
the yolk; _f._ furrow appearing in the yolk around the germinal disc.
Figs. 7_a_, 7_b_, 7_c_. Three segments with modified nuclei from the same
germinal disc.
Fig. 8. Section through a somewhat older germinal disc. _ep._ epiblast;
_n´._ nuclei of yolk.
Figs. 8_a_, 8_b_, 8_c_. Modified nuclei from the yolk from the same
germinal disc.
Fig. 8_d_. Segment in the act of division from the same germinal disc.
Fig. 9. Section through a germinal disc in which the segmentation is
completed. It shews the larger collection of cells at the embryonic end of
the germinal disc than at the non-embryonic. _ep._ epiblast.