REGENERATION IN EGG AND EMBRYO
Not only do adult organisms have the power of regeneration, but embryos
and larval forms possess the same power, and even portions of the
segmenting, and also the unsegmented, egg may be able not only to
continue their development, but in many cases to produce whole
organisms. Haeckel observed in 1869-1870 that pieces of the ciliated
larvæ of certain medusæ, and even pieces of the segmented egg, could
produce whole organisms. The more recent experiments of Pflüger (’83)
and of Roux (’83) on the frog’s egg mark, however, the beginning of a
new epoch in embryological study. The explanation of this is to be
found, I think, not only in the introduction of experimental methods,
but also in the fact that Pflüger and Roux realized the important
theoretical questions involved in their results.
Pflüger’s experiments were made by changing the conditions under which
the egg develops in order to determine what factors control the
development. Since these experiments were made with whole eggs, the
problems of regeneration were not directly involved in his results,
although his conclusions are of great importance in connection with
questions concerning the regeneration of the egg. A part of Roux’s work
dealt directly with the development of a new organism from a piece of
the egg or of the embryo. Roux’s principal discovery[107] (’88) was that
a half-embryo develops from either of the first two blastomeres of the
frog’s egg, if the other blastomere has been injured or destroyed, but
that subsequently the missing half of the embryo is “post-generated.”
Roux was led to this experiment by his discovery that the plane of the
first cleavage of the egg corresponds very often to the median plane of
the body of the embryo.[108] This relation suggested that there might be
some causal connection between the two phenomena in the sense that the
first cleavage plane divides the material for the right side of the body
from that of the left side. In a descriptive sense this would be, of
course, true if the two planes do really correspond, and if there was no
later shifting of material across the middle line, but whether the two
phenomena are causally connected, or are merely due to a coincidence,
could only be determined by further experiment. The observations
themselves are not beyond question, for the two planes do not always
coincide, and may be even ninety degrees apart. These cases of
divergence were thought by Roux to be due to an unobserved shifting of
the developing embryo, but it is improbable that all cases can be
accounted for in this way.
[Illustration: FIG. 61.--After Roux. _A._ Section of semi-blastula of
frog’s egg. _B._ Half-embryo. _C._ Cross-section of last (reversed right
and left in _B_ and _C_). _D._ Anterior half-embryo.]
Roux carried out his experiment by plunging a hot needle into one of the
first two blastomeres, so that it is injured to such an extent that its
development is prevented. The same needle, without heating again, was
used for one or two other eggs, for, if the needle had been so hot in
the first instance that both blastomeres had been injured by the heat,
this might not happen in the second or the third egg. It was found that
amongst the eggs that had been operated upon in this way, some had been
so much injured that neither blastomere developed, others had been so
little injured that both blastomeres developed, but in the successful
operations the uninjured blastomere developed, while the injured one did
not. In the last case the uninjured blastomere divided, and produced a
large number of cells. A segmentation cavity was present in the upper
part of the hemisphere (Fig. 61, _A_). The injured half remained in
contact with the other, completing the sphere, but it did not segment. A
half-embryo developed from the uninjured half, as shown in Fig. 61, _B_,
_C_. This embryo has a half-medullary fold along the side in contact
with the injured half. At the anterior end somewhat more than half a
head is present, and at the posterior end there is a half-blastopore.
The cross-sections[109] (Fig. 61, _C_), through the embryo, show that
beneath the half-medullary fold a rod-like notochord is present, which
is made up apparently of fewer cells than the normal notochord, but it
has, in cross-section, a round and not a half form. At the side, the
mesoderm is present, as in the normal embryo, and it has produced the
characteristic mesoblastic somites. An archenteron is formed in the
half-embryo, and, since it is smaller than the normal, it may, perhaps,
be called a half-archenteron. The embryo is, therefore, in most respects
a half-structure. The head is, however, nearly a whole head, but whether
this is due to a whole head developing out of material derived entirely
from one of the two blastomeres, or whether, as Roux supposes, a portion
of the material of the injured blastomere has been worked over, _i.e._
“post-generated,” remains, I think, an open question.
The results of this experiment seem to confirm Roux’s conjecture that
the material of each of the first two blastomeres is of such a sort that
it gives rise to half the embryo, and, if so, there would be some
probability that there is a causal connection between the first cleavage
and the separating out of the parts of the embryo. In fact, Roux drew
this conclusion, and even attempted to show how such a qualitative
division is brought about. It should not be overlooked, however, that
this conclusion goes beyond the legitimate bounds of deduction from the
results, since the half-development takes place while the injured half
retains its connection with the developing half, the former still
remaining alive. On the other hand, the presence of the injured half
makes the experiment more suitable to demonstrate that each of the first
blastomeres gives rise, under normal circumstances, to half of the
embryo. If one half had been removed, we can foresee that its absence
might lead to other complications that would affect the result.
The most important outcome of this experiment is, I think, to show that
a half-structure may develop by itself, _i.e._ that there is a certain
amount of independent power of development in the parts of the egg.
Roux also tried to show that if, after the second cleavage has been
completed, the two blastomeres that lie on opposite sides of the first
cleavage plane are killed by a hot needle, the remaining two produce
either an anterior or a posterior half of an embryo. An embryo derived
from the two “anterior” blastomeres is represented in Fig. 61, _D_. The
anterior half of the body is present. Posteriorly the half-embryo abuts
against the injured half. It is possible, I think, that this embryo may
represent the anterior half of a whole embryo of half size that has been
prevented from closing in posteriorly by the mass of injured material of
the undeveloped blastomere. Roux did not determine positively whether
the two “posterior” blastomeres could give rise to posterior
half-embryos; one embryo in his opinion appeared to bear out this
interpretation. This part of Roux’s work is, it seems to me, not so
satisfactory as the part dealing with the first two blastomeres, and we
may leave it, for the present, out of the discussion, and consider only
the result of the first experiment, in which one of the first two
blastomeres was injured. Since the problems involved in the two cases
are essentially the same, nothing will be lost by dealing with the first
case alone.
The uninjured blastomere first gives rise to a half-embryo. After this
has been accomplished, other changes take place that “reorganize,”
according to Roux, the material of the injured half in such a way that
the missing half of the embryo is formed by a process that Roux calls
“post-generation.” This process can be studied only by means of
sectioning the embryos, and since the eggs may be injured to a varying
extent, there must be some uncertainty in making out the sequence of
events. It is found that the yolk of the injured blastomere is
vacuolated in places, and that the protoplasm in the path of the needle
has been killed (Fig. 61, _A_). Irregular pieces of chromatin are found
in the protoplasm, which seem to come from an irregular breaking up of
the nucleus.
The changes that lead to the reorganization of the injured half may take
place at different times in different eggs. Roux describes three kinds
of reorganization phenomena. The first includes the formation of new
cells in the injured half. Nuclei, surrounded by finely granular
protoplasm, appear in the protoplasm of the injured blastomere. These
nuclei arise from two sources: in part from the scattered chromatin of
the injured blastomere itself, and in part from nuclei, or from cells
without walls that have emigrated from the developing half. Around these
nuclei, as centres, the protoplasm (with its contained yolk) of the
injured half breaks up into cells. This cellulation of the yolk may take
place in different eggs at different times. In some cases it may not
have appeared as late as the gastrula stage of the uninjured half; in
others, it may take place at the time when the uninjured half is
segmenting.[110] The formation of the cells in the injured half begins
always near the developing half, and extends thence into the injured
parts. The new cells are of different sizes, but are larger than those
of the uninjured half.
The cellulation of the yolk takes place only in the least injured parts
of the protoplasm. Where the protoplasm and yolk have been much injured,
they are changed over by the second method of reorganization. This part
of the blastomere is either actually devoured by wandering cells, or is
slowly changed under the influence of the neighboring cells, so that it
becomes a part of these cells.
The surface of the injured half is covered over by ectoderm that grows
directly from the developing half (third method of reorganization),--at
least this happens where the protoplasm has been much injured. In other
parts of the injured half the new cells that have appeared in this part,
and that lie at the surface, become new ectoderm.
Post-generation now begins in the reorganized and cellulated half; the
cells become changed over into the different layers and organs that make
the new half-embryo. A few hours or a night is sometimes sufficient to
change a hemi-embryo into a whole embryo. The new half-medullary fold
develops from the new ectoderm to supplement the half already present.
The mesoblast appears over the side. Its upper part seems to come from
the uninjured mesoderm that has grown over to the other side, but this
is added to at the free edge by cells that belong to the newly
cellulated part. The new differentiation is, in general, in a
dorso-ventral direction. The lacking half of the archenteron arises in
connection with the half of the archenteron already present in the
hemi-embryo. The yolk cells arrange themselves radially, and a split
appears in the post-generated part, extending from the archenteron of
the hemi-embryo. The split opens, and the new half-archenteron appears.
In general, Roux states, the post-generation of the organs of the
injured half proceeds from the already differentiated germ-layers of the
hemi-embryo. The post-generation begins where the exposed surfaces of
the germ-layers of the hemi-embryo touch the newly cellulated regions of
the injured half.
It is most difficult to account for these post-generative changes, since
the new part has, according to Roux, a double and even a three-fold
origin. The pieces of the old nucleus, he admits, may take a part in the
formation of the new cells; wandering cells migrate from the yolk mass
of the old half into the new, and the cells of the formed germ-layers
may be pushed over to the other side. Since a certain share, and perhaps
a large share, of the new cells comes from the hemi-embryo, it is clear
that, in addition to the power of self-differentiation shown by the
uninjured blastomere, we must also ascribe to it certain _regenerative_
powers, at least to the extent that each kind of cell that comes from it
can give rise in the injured half to cells like itself, and produce
similar structures in the other half.
If then, as Roux supposes, the development of the egg consists in an
orderly, qualitative series of changes that lead to the subsequent
differentiation, we must also suppose that the new parts are gifted with
latent powers by virtue of which they can re-create all parts of the
other half. Roux supposes, in fact, that each cell carries with it a
sort of reserve-plasm, that is dormant in ordinary development but is
awakened when any disturbance of the normal development takes place.
Objections have been made to this subsidiary hypothesis, since the
addition of this to the original assumption of a series of qualitative
changes involves such complications that the view can hardly be
considered a probable one. This objection is, I think, not as strong as
certain critics believe, since the facts of development show beyond a
doubt that although the egg has the power of progressive change it has
also, as certain experiments show, the power of reorganization, if the
ordinary course of events is interrupted. This admission by no means
throws us back upon Roux’s hypothesis, for, as will be shown later, a
different conception of the development may better account for both
phenomena.
Inasmuch as a good deal of discussion has taken place in regard to the
process of post-generation described by Roux, it should be stated that
Endres and Walter reëxamined the process, and found, as had Roux, that
the reorganizing cells migrate from the uninjured to the injured side,
and around them the protoplasm of that side makes new cells. They found
that the injured half is directly overgrown by the ectoderm from the
developing half. When the material of the injured blastomere is only
incompletely reorganized, there is formed, after post-generation, an
embryo that has a protrusion of yolk in the dorsal part of the body.
When the injured material is completely worked over, a perfectly formed
embryo may result. The typical half-embryos that Roux obtained were also
obtained by Endres and Walter. They deny that whole embryos develop from
one of the first two blastomeres, as Hertwig affirms.
[Illustration: FIG. 62.--After O. Hertwig. _A._ Section through a frog’s
egg (blastula stage) in which one blastomere had been killed. _B._ Same.
Gastrula stage. _C._ Later gastrula stage. _D, E._ Surface view of
embryos from one of first two blastomeres. _F._ Same as last (_E_).
Dorsal view. _G._ Ventral view of last. _H._ Dorsal view of another
embryo, lying in a very eccentric position. _I._ Later stage of embryo
from one blastomere. Other injured blastomere nearly covered over. _J._
Section through gastrula stage of embryo from one of first two
blastomeres. _K._ Cross-section of the embryo shown in _F_ and _G_.]
Hertwig repeated Roux’s experiment and obtained results entirely
different from those of Roux. He injured one of the first two
blastomeres of the frog’s egg with a hot needle, or by means of a
galvanic current. Hertwig states that after the operation the egg turns
so that the uninjured part lies uppermost. This is owing, he thinks, to
the appearance of a blastula or of a gastrula cavity in the developing
part. The segmentation cavity is found in many cases surrounded by the
cells of the segmenting half (Fig. 62, _A_), but at other times at the
border between the new and the old parts. In still other cases the
cavity may lie eccentrically, and in some cases the floor of the cavity
may be bounded by the yolk substance of the injured half. An embryo
appears on the upper, uninjured part, though it is not, according to
Hertwig, a half-embryo, but a whole embryo, or at least one approaching
that condition (Fig. 62, _D_, _E_, _F_, _G_, _H_). It is shorter than
the normal embryo, and its posterior end is incomplete. When these
embryos are cut into sections, it is found that the part that has
developed corresponds to the dorsal part of a normal embryo, but the
ventral part is continuous with the yolk substance of the injured half
(Fig. 62, _B_, _C_, _J_, _K_). Hertwig interprets these embryos as forms
in which the yolk portion of the developing half, together with the
whole of the injured blastomere, represents a yolk mass that has not yet
been enclosed by the margin of the developing part.
In nearly all the embryos that Hertwig has described, the medullary
folds appear eccentrically on the developing half (Fig. 62, _D_, _F_,
_K_), and in some cases they may lie so far to one side that they are
situated almost at the edge; and the less development of one of the
folds makes the embryo appear almost like the hemi-embryos obtained by
Roux. In fact, one embryo seems to have been a true hemi-embryo.
Hertwig attributes the eccentric position of the embryo to the eccentric
position of the blastopore of an earlier stage, but he does not attempt
to account for the eccentricity of the latter.
It is significant in this connection to find that Hertwig obtained other
embryos that show a condition of “spina bifida.” In these there is an
exposure of yolk in the mid-dorsal line between the halves of the
medullary folds. Still other embryos in the same series of experiments
were only slightly injured, and developed nearly normally. In these
cases, Hertwig thinks, the blastomere that was stuck had been only
slightly injured, and had partly developed. I have also often observed
in this experiment that the injured blastomere may segment and add cells
to the developing half, but in such cases the development of the injured
half may be less regular than is that of the uninjured half. It seems to
me not improbable that in several of the embryos described by Hertwig
both blastomeres have taken part in the development. The main points of
difference between the results of Roux and of Hertwig cannot, however,
be explained in this way, and the explanation is to be found in another
direction.
Hertwig emphasizes the view that the injured blastomere is not dead, but
exerts an influence upon the other half--an influence of the same kind
as that which the yolk of a meroblastic egg has on the protoplasmic
portion of the egg from which the embryo arises. He ventured to prophesy
that if the injured yolk mass could be entirely removed, the uninjured
blastomere would produce a normal embryo without defect, and one like
the normal embryo in every respect except in size.[111]
[Illustration: FIG. 63.--_A._ After Wetzel. Section through an egg
(blastula stage) reversed at two-celled stage. _B._ After Schultze.
Double embryo, from reversed two-celled stage, united ventrally. _C,
C¹._ Two views of another double embryo (united dorsally). _C²._
Cross-section through last. _D._ After Wetzel. Double embryo united
laterally. _D¹._ Section through same.]
Roux interprets Hertwig’s results as due to the sudden partial
post-generation of a part of the injured half of the egg. He thinks that
a half-embryo had first developed, and then to this there has been
quickly added a part of the missing side. This reply fails, however, to
meet Hertwig’s description of the method of development of the embryos.
Later work, however, has put us in a position to give a more
satisfactory account of the differences between the results of Roux and
Hertwig. It seemed to me that the two kinds of embryos might be due to
the different positions of the eggs after the operation. It had been
shown by Schultze (’94) that if a normal egg in the two-celled stage is
turned upside down and held in that position two embryos develop from
the egg (Fig. 63, _B_, _C_, _D_). These embryos are united in various
ways, and arise presumably one from each of the first two blastomeres.
These results have been confirmed by Wetzel, who examined more fully
into the early development of the twin embryos. He showed with much
probability that the protoplasm rotates in each blastomere, so that in
many cases the lighter part flows, or starts to flow, toward the upper
hemisphere of the egg. In this way similar protoplasmic regions of the
two blastomeres may become separated, and under these circumstances each
blastomere gives rise to a whole embryo. A cross-section through one of
the segmentation stages of one of these eggs is shown in Fig. 63, _A_.
The smallest cells are found at the outer side of each half, and the two
segmentation cavities lie one in the upper region of each hemisphere.
Some of the different kinds of embryos that develop from inverted eggs
are shown in Fig. 63, _B_, _C_, _D_. They are united in Fig. 63, _B_, by
their ventral surfaces, and in Fig. 63, _C_, _C¹_, _C²_, by their
dorsal surfaces, and in Fig. 63, _D_, _D¹_, at the sides. These
differences are probably accounted for by the different ways in which
the protoplasm of the first two blastomeres rotated before the egg
divided.
A consideration of these results led me to carry out the following
experiment on eggs operated upon by Roux’s method. After sticking one of
the first two blastomeres, some of the eggs were placed so that the
uninjured blastomere kept its normal position, _i.e._ with the black
hemisphere upward. Other eggs were turned, so that more or less of the
white hemisphere was upward. From the two kinds of eggs two kinds of
embryos were obtained. From those with the black hemisphere upward the
embryo was a half-embryo like that described by Roux, while from the
eggs with the white hemisphere upward embryos developed that were in
many respects whole embryos of half size.[112] The explanation of this
difference will be obvious from what has been said. When the _black_
hemisphere is uppermost the contents of the uninjured blastomere remain
as in the normal egg, and a half-embryo results. When the _white_
hemisphere is uppermost the contents of the uninjured blastomere rotate,
so that it generally shifts its relation to the protoplasm in the other
injured half, and a whole embryo develops, as in Schultze’s experiment.
In one case I obtained a half-embryo from an inverted egg. The result
did not appear to be due to a lack of rotation of the protoplasm,
because the medullary folds were white, showing that the protoplasm must
have changed its position. The result can possibly be explained as due
to the protoplasm rotating in each blastomere along the line between the
halves, so that it still retains the same relation as that of the normal
two-celled stage.
The whole embryos of half size are generally imperfect in certain
respects on account of their union with the other half. They resemble in
all important points the embryos described by Hertwig, and I see no
grounds for interpreting them as embryos of a meroblastic type, but
rather as whole embryos of half size, whose development posteriorly and
ventrally has been delayed or interfered with by the presence of the
other blastomere.
It has not been possible to separate the first two blastomeres of the
frog’s egg, for if one is removed the other collapses. In the
salamander, that has a mode of development similar to that of the
frog,[113] it has been possible to separate the first two blastomeres.
Herlitzka, who carried out this experiment, found that each blastomere
gives rise to a perfect, whole embryo of half size. We cannot doubt, I
think, that the same power of producing a whole embryo is also present
in each of the first two blastomeres of the frog’s egg. When the two
remain in contact in their normal relation to each other, each produces
only a half; when like regions of the two blastomeres are separated,
each produces a whole embryo. Thus we see that whatever the factors may
be that determine the development of a single embryo from the egg, still
each half, and perhaps each fourth also, has the power of producing a
whole embryo.
In later papers Roux has stated that he had also, even in his earlier
experiments, found other kinds of embryos than the half-embryos that he
described. Some of these were whole embryos that had developed from the
uninjured blastomere without the injured one taking any part or only a
very small share in their formation. He found, he states, all stages
between those embryos that had used up all the yolk material of the
injured side (though post-generated) and those that had not used any
part of it. The latter kind of embryo he does not recognize as a whole
embryo of half size in the sense that a single blastomere has developed
directly into a smaller whole embryo, but he believes that there must
have been formed at first a half-blastula, half-gastrula, half-embryo,
and that the last stage completed itself laterally without using any
material from the injured half. That the uninjured blastomere may at
first segment as a half is not improbable, but that whole embryos are
formed only by the formation of new material at the side of a
half-embryo is, I think, hardly possible, since the results of Schultze,
Wetzel, Hertwig, and myself show that a whole embryo may develop
_directly_ out of the material of a single blastomere.
Spemann (1900) has carried out some novel experiments on the eggs of
triton, and has shown how in another way double structures may be
produced. If a ligature is tied loosely around the egg at the first
cleavage exactly along the division plane between the first two
blastomeres, it will be found later that the long axis of the single
embryo lies, in the great majority of cases, across the ligature, and
only in a small percentage of cases does the median plane correspond
with that of the ligature, and, therefore, with the first cleavage
plane.
If one of the latter eggs is allowed to develop to the blastula stage,
and the ligature is then drawn tighter, so that the blastula is
completely constricted, an embryo develops from each half.
If one of the former eggs is allowed to develop to a stage when the
medullary plate is laid down, but is not yet sharply marked off, and the
ligature is then tightened, there will be formed (the plane of
constriction being across the medullary plate) from the anterior part a
normal head with eyes, nasal pits, ears, and a piece of the notochord,
and from the posterior part there will be formed, at its anterior end,
another new head just behind the ligature. Ear-vesicles develop in this
part at the typical distance from the anterior end. The brain that
develops has a typical cervical curvature, and eye evaginations appear
at the anterior end. The chorda, that extended at first to the anterior
end of this region, is partially absorbed.
If the ligature is drawn tighter at a later stage, when, for instance,
the medullary plate is plainly visible but is still wide open, a
different result is obtained. The posterior part no longer forms a new
head at its anterior end, but develops into those structures that it
would form normally. In some cases it was found that the region from
which the ear develops had been pinched in two, and in consequence a
small vesicle appears in front of the constriction and another behind
it.
In those cases in which the ligature lies in the median plane of the
embryo, it is found that a double anterior end is produced. As the
embryo develops it tends to elongate, and in consequence the material is
pushed forward on each side of the ligature. A double head is the
result. The extent of the doubling depends on the depth of the
constriction between the halves. In the most extreme cases two complete
heads are formed with an inner nasal pit, eye, and ear on each head, as
well as the normal outer ones. The results show that even such
complicated structures as the eyes and ears, etc., may arise from parts
of the body where they never appear under normal conditions.
[Illustration: FIG. 64.--Sea-urchin egg and embryo. _A._ Two-cell stage.
_B._ Same, with blastomeres separated. _G._ Two half-sixteen-cell
stages. _C._ Open half-blastula stages. _D._ One of last, later stage,
closed blastula of half size. _E._ Gastrula of half size. _F._ Whole
pluteus of half size. _H._ A half-sixteen cell dividing in same way as a
whole egg (eight cell). _I._ Whole egg at sixteen-cell stage.]
A series of experiments that have been made on the eggs of sea-urchins
has led to equally important results. The earliest experiments are those
of O. and R. Hertwig, who, in addition to studying the effect of
different drugs on the developing egg, found that fragments of the eggs
of sea-urchins, obtained by violently shaking the eggs in a small vial,
could give rise, if they contained a nucleus, to small whole embryos.
Boveri made the important discovery in 1889 that if a non-nucleated
piece of the egg of the sea-urchin is entered by a single spermatozoon,
the piece develops into a whole embryo of a size corresponding to that
of the piece. Fiedler, in 1891, separated the first two blastomeres by
means of a knife, and found that the isolated blastomere divides as a
half, but he did not succeed in obtaining embryos from the halves.
Driesch has made many experiments, beginning in 1891, with the eggs and
embryos of the sea-urchin. He separated the first two blastomeres (’91)
by means of Hertwig’s method of shaking the eggs, and studied the
development of the isolated blastomeres. He found that the cleavage was
strictly that of a half, and not like that of a whole egg. The normal
egg divides into two, four, and eight equal parts. At the next division,
four of the cells divide very unequally, producing four very small
cells, the micromeres, at one pole. The four cells of the other
hemisphere divide equally (Fig. 64, _I_). The isolated blastomere
divides at first into two equal parts, then again into equal parts. At
the next division two of the cells produce micromeres and two divide
equally (Fig. 64, _G_). This is exactly what happens at this division in
each half, if the blastomeres are not separated. In later stages a
half-sphere is formed that is equivalent to half of the normal sphere
(Fig. 64, _C_). The open side corresponds to the side at which the half
would have been united to the other half. Thus up to this point a
half-cleavage and a half-blastula have appeared.[114]
In later stages the open half-blastulæ close in, producing a whole
sphere that becomes perfectly symmetrical (Fig. 64, _D_). A symmetrical
gastrula develops (Fig. 64, _E_) by the invagination of a tube at one
pole, and a symmetrical embryo is formed (Fig. 64, _F_) that resembles
the normal embryo except in point of size.
Driesch has also found that a number of twin embryos arise from the
shaken eggs. They arise from eggs whose blastomeres have been disturbed
or shifted, so that each produces a small whole embryo, the two embryos
being united to each other in various ways.
In a second paper, published in the following year, Driesch extended his
experiments, and attempted to discover how far the “independence” of the
blastomeres extends; _i.e._ he tried to find out if all the blastomeres
resulting from the cleavage are alike. When one of the first four cells
is separated from its fellows by shaking, it continues to divide, in
most cases as a quarter, and produces later a small spherical blastula.
Many of these blastulæ, although apparently healthy, never develop
further, although they may remain alive for several days. In one
experiment only eight out of twenty-six reached the pluteus stage, with
a typical digestive tract and skeleton.
From these experiments Driesch drew the important conclusion that the
cleavage cells or blastomeres of the sea-urchin’s egg are equivalent, in
the sense that if they were interchanged a normal embryo would still
result. A somewhat similar view is expressed in the dictum that the
position of a blastomere in its relation to the others determines what
part it will produce, if its position is changed it gives rise to
another part, etc.,--or, expressed more concisely, the prospective
value of a blastomere is a function of its position.[115] Driesch
extended these experiments further in 1893. His aim was to separate
different groups of cells at the sixteen-cell stage in order to see
whether the cells around the micromere pole (or “animal pole”) if
separated from those of the opposite (or “vegetative pole”) could
produce a whole embryo, etc. Eggs whose membranes had been removed by
shaking immediately after fertilization were allowed to develop normally
to the sixteen-cell stage and were then shaken into pieces. Amongst the
groups of cells that were present those that contained the micromeres
were picked out. It was found that they give rise to whole embryos. In
order to obtain cells that belong to the vegetative hemisphere, the
blastomeres were shaken apart at the eight-cell stage, and those groups
of cells that in later divisions did not produce micromeres were
isolated. From these also whole embryos develop. The results show that
the cells of both hemispheres are able to produce whole embryos, and
that at the sixteen-cell stage the different parts of the egg are still
capable of producing all parts of the embryo. It is important to observe
that the results of the experiment do not show that if the normal
development goes on undisturbed any part of the egg may become any part
of the embryo, for it is highly probable that a definite region of the
egg may always produce a definite part of the embryo. The results do
show, however, that, even if this is true, any cell has the power of
producing any or all parts of the embryo if the normal conditions are
changed.
In connection with these experiments Driesch discussed the factors that
determine the axial relations of the embryo. If all the cells have the
power of producing all parts, what determines in the normal development,
and also in the development of a part of the whole, the axial relations
of the embryo? Driesch assumed that the egg has a polar structure, and
that the same polarity is found in all parts of the protoplasm. Around
this primary axis all the parts are alike or isotropous.[116] The origin
of the mesenchyme and the position of the archenteron, that develop at
one pole, are determined by the polarity of the protoplasm. The plane of
bilateral symmetry may appear in any one of all the possible radial
planes around the primary axis. The selection of a particular one is due
to some accidental difference in the structure of the protoplasm, or to
some external factor. In later papers Driesch modified this view, and
assumed that along with the primary polarity a bilateral structure also
exists in the protoplasm.
Wilson (’93) studied the development of isolated blastomeres of
amphioxus, and found that it agreed in all essential respects with the
mode of development of the blastomeres of the sea-urchin. The isolated
blastomeres of the two-cell and four-cell stages produce whole embryos,
but the blastomeres of the eight-cell stage develop only as far as the
blastula. The blastomeres segment, after separation, in most cases not
as a part, but as a whole egg would divide, although the cleavage of the
one-eighth blastomere only approaches that of the entire egg, but is
never identical with it. Incompletely separated blastomeres give rise to
twins, triplets, etc. Wilson agreed with the Hertwig-Driesch conception
of the value of the early blastomeres, and accepted the view that the
fate of each is a function of its position, and that at first they are
qualitatively alike. During the early cleavage he supposed that a change
takes place that is slight at the two-cell stage, greater at the
four-cell stage, and in the eight-cell stage the differentiation has
gone so far that the blastomere can no longer return to the condition of
the ovum. “The ontogeny assumes more and more the character of a mosaic
work as it goes forward.”
Loeb (’94) showed that if the eggs of the sea-urchin are placed in sea
water, diluted by distilled water, the egg swells and bursts its
membrane, so that a part of its protoplasm protrudes. Into this
protrusion some of the first-formed nuclei pass, and from both the part
remaining in the egg membrane, as well as from the protruding part, an
embryo is produced, the two embryos often sticking together. In several
cases two to eight separate groups of blastomeres are formed from one
egg and develop into whole embryos.[117]
The question of the number of cells which are produced by the one-half
and one-fourth embryos had not up to this time been determined. Until
this was known it could not be stated whether the smaller embryos were
miniature copies of the normal embryos in all respects, or whether they
assumed the typical form with fewer cells. I found (’95) that the
blastula from one of the first two blastomeres contains half the number
of cells produced by the whole embryo, and that in the later stages also
it contains only about half the normal number. The one-fourth blastomere
produces only a fourth of the whole number of cells, and yet can develop
with this number, in many cases, into a whole embryo. The one-eighth
blastomere produces one-eighth the normal number of cells. In most cases
I found that these one-eighth blastomeres do not produce embryos, but
occasionally they produce a gastrula, and probably a young pluteus
stage.
The development of nucleated fragments of the egg was also studied in
order to find out if they too produce a smaller number of cells than
does the whole egg, and a number in proportion to their size. The
problem is different in this case, because the nucleus has not divided
before the piece is separated, and the results ought to show whether
there is a prescribed number of divisions for the egg nucleus, or
whether the number of times it divides is regulated by the amount of the
protoplasm. It was found that the number of cells produced by each
fragment is in proportion to the size of the piece. This shows that the
division of the nucleus is brought to an end when the protoplasm has
become subdivided to a certain point.
A further examination of the number of cells that are invaginated in
these smaller “partial” larvæ to produce the archenteron seemed to show
that they often use relatively more than their proportionate number. The
normal blastula of _Sphærechinus granularis_ contains about five hundred
cells and turns in fifty cells, or one-tenth the total number. The
one-half and one-fourth embryos, and some of the small embryos from the
egg fragments, seemed to invaginate more than one-tenth of their total
number of cells.
Driesch (1900) reëxamined this point, and found that the embryos from
isolated blastomeres may use the proportionate number of cells. I have
made a new study of the problem on a larger scale and have found that my
earlier statement, as well as that of Driesch, is substantially correct,
and that the difference that we found is due to the time at which the
embryos gastrulate. Thus the one-half embryos and even the one-fourth
embryos, that gastrulate as soon as (or only a little later than) the
normal, whole embryos, turn into the archenteron about one-half and
one-fourth the number of cells invaginated in the whole embryo; but
those partial embryos that gastrulate later (as most of them do) turn
into the archenteron more than a half or a fourth of the number of cells
turned in _at first_ by the whole embryo. This difference between the
early and the retarded partial embryos is in large part due to a slow
increase of cells that takes place during the delay in development.
Driesch (’95) found that pieces of the blastula wall of the sea-urchin,
_if large enough_, can also produce a gastrula and embryo. I found that
the number of cells in these pieces does not increase appreciably after
they are cut off (if the operation has been carried out at the end of
the cleavage period), and that the new embryo is organized out of the
cells present at the time of removal of the piece from the wall. There
is, therefore, in this case no chance for “post-generation” by means of
new cells produced at the side, which Roux has supposed to take place in
the frog embryo.
The development of pieces of the blastula wall, if they are not too
small, also shows that the lack of power to develop, found in some of
the one-fourth and in many of the one-eighth blastulæ, is not the result
of any special differentiation that they have undergone during the
cleavage period, but is due to their size.
A recent series of experiments by Driesch (1900) on the development of
isolated blastomeres of the sea-urchin’s egg has given more exact data
in regard to their limit of power to produce embryos, and has shown the
possibilities in these respects of different parts of the egg. By means
of a method discovered by Herbst (1900) it is possible to obtain
isolated blastomeres more readily than by the somewhat crude shaking
process. If the eggs, after fertilization and after the removal of the
membrane by shaking, are placed in an artificial sea water, from which
all calcium salts have been left out, the eggs divide normally, but the
blastomeres are not held firmly together, and readily fall apart if the
egg is disturbed. By means of a fine pipette any desired blastomere or
group of blastomeres can be picked out. If these are returned to sea
water they continue to develop.
Driesch found that the one-half and one-fourth blastomeres develop into
proportionate gastrulæ and larvæ; that the one-eighth blastomeres, both
of the animal and the vegetative hemispheres, sometimes produce
gastrulæ, and even the beginning of the larval stage with the rudiments
of a skeleton. There are certain differences between the one-eighth
larvæ that come from the animal hemisphere and those from the vegetative
half. More of the one-eighth blastomeres from the animal part of the egg
die than from the opposite part, but of those that remain alive a larger
percentage reach the gastrula stage than in the case of those from the
vegetative pole; their protoplasm moreover is not so clear as is that of
the larvæ from the other hemisphere. These “animal pole” blastomeres
develop faster than those of the other sort. The gastrulæ from the
one-eighth blastomeres of the vegetative hemisphere do not die so often
after separation, the protoplasm of the larvæ is clearer, and they often
produce long-lived blastulæ with long cilia. The blastulæ often develop
into gastrulæ without mesenchyme. These results show that although whole
larvæ may be produced from the one-eighth blastomeres of both
hemispheres, yet there are certain characteristics that may be referred
with great probability to differences that are present in the protoplasm
of the two hemispheres of the egg. The differences are not in all cases
sufficient to interfere with the production of all the characteristic
structures of the embryo, yet traces of the origin of the larvæ can be
found in their structure. It is probable that the so-called animal (or
micromere) pole corresponds to that part of the egg from which the
archenteron is produced. Hence the one-eighth blastulæ from this
hemisphere gastrulate sooner and in proportionately larger numbers than
do those from the opposite hemisphere. The vegetative hemisphere would
correspond to that part of the egg from which the wall of the normal
gastrula is derived, and this may account for the clearer protoplasm of
these embryos, their inability in many cases to gastrulate, their larger
cilia, and the absence of mesenchyme in some of them. Driesch finds that
the number of cells that go into the mesenchyme of the partial larvæ is
in proportion to the total number, and that the number of cells in the
archenteron is probably also proportionate.[118]
The smallest blastomeres that produce gastrula are the one-sixteenth
products. Out of a total of 139 cases only 31 produced true gastrulæ, 5
produced gastrulæ with evaginated archenteron, and 103 remained blastulæ
with long cilia. The one-thirty-second blastomeres were not observed to
gastrulate.
Driesch (’95) has also made a study of the potentialities of the
blastula and gastrula stages of sphærechinus, echinus, and asterias. If
a blastula is cut in half before the mesenchyme cells are produced, both
pieces produce gastrulæ and larvæ. Since some of the pieces probably
come from the animal hemisphere, and others from the vegetative
hemisphere, it follows that all parts of the blastula possess the power
of producing whole embryos, and in this respect the potentialities are
the same as for the blastomeres. If the experiment is made at a stage
just before the archenteron has begun to develop (Fig. 65, _A_), the
results may be different. A half that contains the region from which the
archenteron is about to develop will produce a gastrula and a larva
(Fig. 65, _A_, lower row to right of _A_). A half that contains only the
opposite regions of the egg (Fig. 65, _A_, upper row) may in some cases
gastrulate,[119] often abnormally, but as many as half of the pieces do
not gastrulate. They may remain alive for a week or more, and even
produce a typical ciliated ring with a mouth in the centre, but do not
form a new archenteron. These important results show that after the
formation of the mesenchyme and archenteron at one pole, the other cells
of the blastula wall are no longer able to carry out a process that the
same cells were able to carry out at a slightly younger stage, but
whether this loss of power is connected with the previous formation of
the archenteron, or due to some other change which has by this time
taken place in the cells, cannot be determined from the experiment. It
is also important to note that these small ectodermal blastulæ can still
develop whole, typical, ectodermal organs, the ciliated ring and the
mouth, and that the former especially has the characteristic structure
of the whole normal ring.
[Illustration: FIG. 65.--_A._ Blastula of sea-urchin beginning to
gastrulate. Cut in half as indicated by line. Two rows of figures to
right show development of upper and lower halves. _B._ Later gastrula
cut in half. Two rows of figures to right show later development. _C._
End of gastrulation process. Embryo cut in half. Two rows of figures to
right show later stages of each half. _D._ Formation of endodermal
pouches from inner end of archenteron. Embryo cut in two. Two rows of
figures to right show later stages.]
Similar phenomena have been made out by Driesch in the development of
the archenteron of the same forms. At the end of the normal gastrula
period of the starfish embryo, there is produced from the inner part of
the archenteron two outgrowths, or pouches, that later constrict off to
give rise to the cœlom sac and water-vascular system. If the gastrula
is cut in two in such a way that the inner end of the archenteron,
_i.e._ the part from which the pouches develop, is cut off (Fig. 65,
_C_), it is found that the piece containing the posterior part of the
archenteron closes in, forms a new sphere, and from the present inner
end of the archenteron (that has also healed over) a pair of pouches is
produced (Fig. 65, _C_, lower row to right of _C_). These pouches have
arisen, therefore, from a more posterior part of the archenteron than
that from which the pouches normally arise.
If the same experiment is made at a later stage, when the pouches have
been given off from the archenteron (Fig. 65, _D_, lower row to right of
_D_), no new pouches are formed. This means that after the archenteron
has once produced its pouches it loses throughout all its parts the
power to repeat the process, although these parts possessed this power
at an earlier stage. It is a very plausible view that the result is
directly connected with the formation of the normal pouches, although it
is of course possible that some other change has taken place in the
archenteron that prevents the formation of the pouches.
In order to give as nearly as possible a consecutive account of the
experiments on the eggs of the frog and of the sea-urchin, a number of
other discoveries have been passed over. Let us now examine some of the
results on other forms.
Chabry, as early as 1887, experimented with the eggs of an ascidian. By
means of an ingenious instrument he was able to prick and kill
individual blastomeres. The results of his experiments were not
described very clearly, and later writers have interpreted his results
in different ways.[120] Chabry stated that he obtained half-embryos from
one of the first two blastomeres, but his figures show, especially in
the light of later work, that the embryos were whole embryos of half
size, although certain organs, as the papillæ and the otolith, may be
lacking.
Driesch (’95) reëxamined the development of isolated blastomeres in one
of the ascidians, _Phallusia mammalata_, and found that the cleavage of
blastomeres, isolated by shaking, is neither like that of the whole egg,
nor is it like that of half the normal cleavage, although it shows some
characteristics of the latter. A symmetrical gastrula is produced, and
from this a typical whole larva of half size. These larvæ lack, however,
one or more papillæ, and the otolith rarely develops. The absence of
these organs Driesch ascribes to the rough treatment that the egg has
received, since embryos from whole eggs may sometimes lack these organs
if the development has taken place under unfavorable conditions. The
isolated one-fourth blastomere may also produce a whole larva.
Crampton (’97) has also studied the development of the isolated
blastomeres of another ascidian, _Molgula manhattensis_. He has more
fully worked out the cleavage, and finds that the isolated blastomere
segments as a part, _i.e._ as it would have segmented had it remained in
connection with the rest of the egg. In general appearance the
half-cleavage seems to differ from the half of the complete cleavage,
because rearrangements of the blastomeres occur, but despite these
shiftings the form of the division is always like that of a part. A
whole embryo develops, although there may be defects in certain organs,
which are due, he suggests, to the smaller amount of material available
for the development of the larva.
Zoja showed in 1894-1895 in a number of jellyfish that the isolated
blastomeres produce whole larvæ of smaller size.[121] In one form,
liriope, the endoderm that forms the digestive tract is normally
delaminated at the sixteen-cell stage, each cell of the blastula wall
dividing into an inner and an outer part. In the blastula from the
one-half blastomere this delamination also takes place when sixteen
cells are present, and not at the preceding cleavage when only eight
cells are present. In this form, therefore, the whole number of cells
develops before the delamination takes place, and the one-half larva is
composed of the same number of cells as is the normal embryo at this
stage, but the cells are only half as large. In other species the
endoderm appears to begin to develop in the half-larvæ when only half
the total number of cells is present.
The conditions in the egg of the bony fishes are very different from
those in the preceding forms. The protoplasm, from which the embryo is
produced, accumulates at one pole to make the blastodisc. After the
cleavage of this blastodisc, the blastoderm that has resulted grows over
the yolk sphere at the same time that the embryo is forming along one
meridian. I carried out some experiments, in 1895, on the eggs of
_Fundulus heteroclitus_. If one of the first two blastomeres of the egg
of fundulus is destroyed, the remaining one produces a whole embryo. If
three of the first four blastomeres are removed, the remaining one may
produce a whole embryo of small size. The problem of development is, in
the case of the fish, different from the other cases described, inasmuch
as the whole yolk sphere is left attached to the remaining blastomere
and is covered over by cells derived from this blastomere. The smaller
embryo that is formed lies on a yolk of full size.[122]
Wilson’s work on amphioxus has been already described in connection
with the experiments on the sea-urchin’s eggs. Later I (’96) also
obtained whole larvæ from one-half and one-fourth blastomeres, and I
also found that the one-eighth blastomeres do not develop beyond the
blastula stage. The number of cells of which the one-half larva is
composed is half that of the normal larva, and the one-fourth larva is
made up of one-fourth of the total number of cells.
[Illustration: FIG. 66.--Ctenophore-egg and embryo. _A._ Normal
sixteen-cell stage. _B._ Half-sixteen-cell stage. _C._ Later
half-segmentation stage. _D_. Later half-embryo. _E._ Corresponding
whole embryo. _F._ Half-embryo seen from side. _G._ Same seen from
apical end. In _F_ and _G_, four rows of paddles present, three
endodermal sacs and ectodermal stomach.]
In all the preceding cases in which the blastomeres have been
_separated_, a whole embryo has developed, although the cleavage was
often like that of a part. In one form, however, it has been found that
a whole embryo does not develop. Chun (’92) first showed that the
isolated one-half blastomere of the ctenophore egg produced a
half-larva. He also inferred from certain incomplete embryos caught in
the sea, that these incomplete larvæ could subsequently regenerate the
missing parts. Driesch and Morgan (’95) studied the development of the
isolated blastomeres of another ctenophore, _Beroë ovata_. They found
that the isolated one-half blastomere divides exactly as a half of the
whole egg (Fig. 66, _A_, _B_, _C_). It remains more or less a
half-structure, even after the ectoderm has grown over the whole surface
(Fig. 66, _D_). The invagination of ectoderm, to form the so-called
stomach, that takes place at the lower pole of the whole embryo, is
formed at one side of the lower pole in the half-embryo (Fig. 66, _F_,
_G_). It pushes into the endodermal yolk mass, and lies not in the
middle, but somewhat to one side. In the normal embryo there are formed
four endodermal sacs or pouches in the central yolk mass that become
connected with the inner end of the ectodermal stomach, around which
they lie symmetrically. In the half-embryo two sacs are formed, and in
addition a smaller third sac, which always lies on the side of the
stomach that is nearest the outer wall (Fig. 66, _F_, _G_). The embryo
is, therefore, somewhat more than half the normal embryo in regard to
the number of its endodermal sacs.
There are present eight meridional rows of paddles in the normal embryos
of the ctenophore. They lie symmetrically on the sides, converging
towards an apical sense organ. In the one-half larva there are always
only four of these rows of paddles that are not equally distributed over
the surface, since on one side there is a wider gap between two of the
rows than elsewhere (Fig. 66, _G_). The sense plate also lies somewhat
eccentrically, _i.e._ more towards the side corresponding to that at
which the other blastomere lay.
If the one-fourth blastomeres are separated, each continues to segment
as though still a part of the whole. A one-fourth embryo develops that
has an unsymmetrical stomach, with _two_ endodermal sacs. There are only
two rows of paddles. The embryos are, therefore, in several respects
one-fourth embryos, but the presence of two endodermal sacs, instead of
only one, shows that in this particular, at least, the embryo is more
than a fourth of the whole.
The part of the work of Driesch and Morgan, that has a special bearing
on the _interpretation_ of the one-half and one-fourth development of
the isolated blastomeres, is that in which some experiments are
described which consisted in cutting off portions of the unsegmented
egg. If a fertilized but unsegmented egg is cut in two by means of a
small pair of scissors, the part that contains the nucleus may segment,
and give rise to an embryo. The division is generally like that of a
part, and in such cases an incomplete embryo develops. The embryo may
have fewer rows of swim-plates than has the normal embryo, and fewer
endodermal sacs, and the stomach may be in an eccentric position. The
embryos resemble in every respect the incomplete embryos from isolated
blastomeres. It is important to note that although the embryos from
isolated blastomeres resemble those from pieces of the segmented egg, in
the former case the nucleus has divided once, and each blastomere
contains half of the original nucleus, while in the latter case the
entire segmentation nucleus is present in the piece. These facts seem to
show that in this egg the incomplete development is directly connected
with the protoplasm, and not with the nucleus,--a view that is
maintained by Driesch and Morgan in connection with these experiments.
It was found in one or two instances that the nucleated pieces divided
in the same way that the whole egg did, except that the blastomeres are
proportionately smaller. From pieces of this kind whole embryos of small
size developed. In this case we must suppose that the protoplasm has
succeeded in rearranging itself into a new whole of smaller
proportions.[123]
Crampton (’96) has shown in a mollusk, _Ilyanassa obsoleta_, that when a
blastomere is separated from the rest, the cleavage proceeds as though
the blastomere or its products were still present, and the larva is
defective in those organs that are normally derived from that
blastomere. These results are in line with those on the ctenophore egg.
Fischel (1900) has also made some experiments on the segmented egg of
the ctenophore, and has confirmed several of the results obtained by
Driesch and Morgan. In addition he has tried the effect of disturbing
the first-formed cells by pushing them over each other, so that their
relative positions are changed. He finds as a result that the paddles,
sense organ, etc., appear in unusual positions, and the latter may be
doubled. This shows that we must regard the material or structural basis
of the organs as present very early in the different parts of the egg,
and that the organs develop without much regard to their relation to
other organs.
Ziegler (’98) has also made some observations on the egg of this same
ctenophore, that bear directly on some of the questions here raised. His
study of the cleavage shows that the micromeres arise from the part of
the egg that is opposite the pole at which the first cleavage furrow
appears--the animal pole. Fischel’s results have shown that the paddles
and the sense organs arise from these micromeres, for, if the latter are
displaced the former are also.
Ziegler performed the experiment of cutting off that part of an egg
(which has just begun to divide) lying opposite the region in which the
first furrow has appeared. In this way there was removed from the
unsegmented egg the part from which the micromeres develop. Ziegler
found that the micromeres still arise, and that from such pieces larvæ
develop that have _eight_ rows of paddles and _four_ endodermal sacs. In
one case two of the sacs were smaller than the others; in another case
one of the four was very much smaller than the rest. In another
operation a large piece was cut from the egg, leaving a small nucleated
piece that divided into two blastomeres of unequal size. An embryo
developed from this small piece with four endodermal sacs, and only
_four_ well-developed rows of paddles. The four rows of paddles that
were lacking were represented by two groups of a few plates each.
Ziegler gives a different interpretation of these results from that
which Driesch and Morgan have offered. He interprets the last
experiment, in which after the operation the piece divided into two
unequal parts, and only four rows of paddles appeared, as meaning that
the development of these organs on the smaller part is suppressed on
account of the small size of the part. If the part had been still
smaller all trace of the missing paddles might disappear, as he thinks
was the case in certain experiments of Driesch and Morgan. There can be,
I think, little doubt that if a piece is small enough, the result would
follow as Ziegler supposes. It does not seem probable, however, that the
pieces were really below the lower limit in the experiments of Driesch
and Morgan, since the smaller blastomere was in one case as large as the
whole piece (_i.e._ as both blastomeres taken together) in one of
Ziegler’s experiments.
Ziegler’s results show very clearly that we are not obliged to think of
the substance of the micromeres as laid down in the protoplasm of the
egg, and hence there is no ground for supposing the substance of the
paddles is _necessarily_ present in the vegetative hemisphere of the
egg. His results show that if the vegetative part is cut off, micromeres
and paddles are still formed, although that part of the egg substance
from which they normally arise has been removed. It should be pointed
out, in this connection, that Driesch and Morgan did not suppose that
the bases of the micromeres, or of the paddles, are actually laid down
in a definite part of the protoplasm of the egg. They had also observed
that in some cases whole embryos arose after a part of the egg had been
removed, and this they attributed to the symmetrical position of the cut
in relation to the organization of the egg. Ziegler’s operations were
made more or less in this symmetrical plane, excepting the one that gave
rise to an incomplete embryo. Driesch and Morgan held that the formative
factors become localized in the protoplasm, rather than arise from the
nucleus, but pointed out that these observations do not lead to His’s
conclusion of localized germ areas in the egg.