THE INTERNAL FACTORS OF REGENERATION IN ANIMALS
The comparatively few cases in animals in which regeneration has been
shown to be influenced by external factors have been given in the
preceding chapter. In all other cases that are known the factors are
internal. By this is meant that we cannot trace any direct connection
between the result and any of the known external agents that have been
shown in other cases to have an influence on regeneration. Certain
external conditions must, of course, be present, such as a supply of
oxygen, a certain temperature, moisture in some cases, etc., in order
that the process may go on, but they are without influence on the kind
of regeneration, and are necessary for all parts alike.
_POLARITY AND HETEROMORPHOSIS_
Trembley, Spallanzani, and Bonnet knew that, in general, at the end of a
piece of an animal from which a head has been cut off a new head
develops, and from the posterior cut-surface of a piece a new posterior
part is regenerated. Allman was the first to give the name “polarity” to
this phenomenon.[25]
In several animals regeneration takes place more readily from one end
than from the other of the same cut, and this difference seems to be
connected with the kind of new part that is to be regenerated, and not
with the actual power of regeneration of the region itself. For
instance, if a short piece is cut from the anterior end of an earthworm,
a new anterior end is quickly regenerated from the anterior cut-surface
of the posterior piece, but no regeneration takes place, or only after a
long time, from the posterior cut-surface of the anterior piece. These
relations are reversed if the posterior end of a worm is cut off. There
regenerates very quickly a new posterior end from the posterior
cut-surface of the anterior piece, but no regeneration takes place, or
only after a long time, from the anterior cut-surface of the posterior
piece. The new structures that develop after a long time from the
posterior surface of a short anterior piece, and from
[Illustration: FIG. 16.--_A._ Head of _Planaria lugubris_ with line
indicating level at which _A¹_ was cut off. _A¹._ Head of last
regenerating a new head at its posterior end. _B._ Piece of _P.
maculata_ regenerating head at each end. _C._ Posterior end of
_Allolobophora fœtida_ regenerating a new tail at its anterior end.
_C¹._ Enlarged anterior end of last with new tail. _C²._ Tip of new
tail. _D._ Anterior end of one individual of _A. fœtida_, grafted to
anterior end of another worm, leaving posterior end of piece exposed.
This has begun to regenerate. _E._ After Hazen. Similar experiment in
which a _new head_ regenerated at posterior end of grafted piece. _F._
Two longer pieces of _A. fœtida_ united by anterior ends. One end was
subsequently cut off and a new tail regenerated. _G._ End of a
developing piece of _Tubularia mesembryanthemum_ that had been cut off;
it has regenerated, at its proximal end, another proboscis.]
the anterior surface of a short posterior piece, correspond to a
different part of the worm from that which would be expected to develop,
if the polarity of the piece is taken into account. Another reversed
head develops on the posterior cut-surface of the anterior piece, and
another tail on the anterior end of the posterior piece. The polarity of
the new part is in this case reversed, as compared with that of the
piece from which it arises. In the earthworm there is a marked delay in
the regeneration of these heteromorphic parts. Even in tubularia in
which heteromorphosis takes place, there is usually a delay of
twenty-four hours in the formation of the reversed head. In _Planaria
lugubris_, in which a reversed head develops, if a piece is cut from the
anterior end just behind the eyes, the delay in the formation of the
reversed head is very slight, if indeed there is any delay at all.
In the earthworm and in the planarian the production of reversed
structures appears to be connected with the part of the body through
which the cut is made, and to be due to internal factors. The question
arises whether the presence of certain organs at the exposed surface can
account for the result. It is conceivable that if such organs are
present, and produce new cells that go into the new part, the presence
of such cells may be the factor that determines what the new part will
become; and in consequence the polarity of the part may be reversed. For
example, the presence of the cut-end of the œsophagus or of the pharynx
at the posterior surface of the anterior piece of the earthworm may
determine that a new pharynx develops at the cut-end, and this may in
turn act on the rest of the new tissues in such a way that a head rather
than a tail is formed. When a posterior piece is cut off, the presence
of the stomach-intestine at the cut-end may influence the new part, so
that a tail is produced. It can be shown, however, that a new head may
arise at the anterior end of a piece that contains only the
stomach-intestine, as sometimes occurs when the worm is cut in two
anterior to the middle; and it is not improbable that a tail can be
produced from the posterior end of a piece that contains the old
œsophagus, and perhaps even the old pharynx. In the planarian I have
especially examined this point, but I have not yet found that the result
can be referred to the cut-surface passing through any particular organ,
or to the absence of any organs at the cut-end.
If, instead of referring the result to any one organ, we assume that the
tissues near the cut-ends are specialized in such a way that they can
only produce their like, and that the sum total of tissues of this sort
making up the new part determines the result, we can only suggest that
this may be so, but we cannot show at present that it is so, or that the
result could be brought about in this way.
We might make an appeal to the hypothesis of formative stuffs, and
assume that there are certain substances present in the head, and others
in the tail, of such a sort that they determine the kind of
differentiation of the new part; but this view meets also with serious
objections. In the first place, it gives only the appearance of an
explanation because it assumes both that such stuffs are present, and
that they can produce the kind of result that is to be explained. Until
such substances have been found and until it can be shown that this kind
of action is possible, the stuff-hypothesis adds nothing to the facts
themselves, and may withdraw attention from the real solution of the
problem.
Bonnet, who first proposed the hypothesis of specific stuffs, went
further and assumed also that they move in definite directions in the
body, the head-stuff flowing forward and the tail-stuff flowing
backward. It was necessary to assume definite movements of the stuffs in
order to account for the development of the head at the anterior end of
a piece and of a tail at the posterior end. In cases of heteromorphosis
of the sort described above, these stuffs, if they brought about the
results, would have to move in _opposite_ directions from those assumed
in the hypothesis; or else that part of the hypothesis that postulates
the movement of the substances must be dropped, and in its place there
must be substituted the idea of the excessive amount of such substances
in the ends accounting for the heteromorphosis. An hypothesis that must
be changed in this fundamental way to explain both classes of facts
cannot be given very serious consideration. Of these possible ways in
which it has been attempted to account for the phenomenon of
heteromorphosis, the first one suggested seems to me simpler and more
probable, but which organs are to be made responsible for the result
cannot at present be stated. The fact that both Bardeen and I have
obtained heteromorphosis in planarians in other regions than in the head
indicates at least that other factors than the presence of head tissues
or of head substances may bring about the development, and if it can be
discovered what produces the result in regions remote from the head we
may be in a position to explain the result in the head region in the
same way, although it may be, of course, that the same result may be
brought about by different factors, when the internal conditions are
somewhat different.
[Illustration: FIG. 17.--After Voigt. Planarian with three oblique cuts
at side. The most anterior cut (left side), directed forward, produced a
tail. The one on the right side, directed backwards, produced a head.
The most posterior cut (left side) made a head with pharynx, and also a
tail-like outgrowth.]
Another phenomenon connected with the polarity of a piece is shown by
_Cerianthus membranaceous_. When a triangular piece is cut from the side
of the body, a half circle of tentacles appears around the lower edge of
the cut, as shown in Fig. 15, _C_. The presence of a free distal edge on
the lower side of the opening is a sufficient stimulus to call forth the
development of tentacles.
A somewhat similar result is obtained when an incision is made in the
side of the body of a planarian. A lateral head may grow out from the
anterior edge of the cut-surface, as shown in Fig. 17.
[Illustration: FIG. 18.--_A._ After Loeb. Anterior end of _Ciona
intestinalis_ with oral-siphon partially cut off. Eye-specks regenerate,
both on oral and aboral edge. _B._ Same (after T. H. M.), showing
similar result on excurrent siphon.]
It has been shown by Loeb that if the incurrent siphon of the ascidian
_Ciona intestinalis_ be partially cut off, new eye-specks develop around
the margin of the cut, as shown in Fig. 18, _A_. I have repeated this
experiment and obtained the same result, and found, as had Loeb also,
that the same holds true for the excurrent siphon (Fig. 18, _B_). In
these cases the new eyes appear both on the anterior and posterior edges
of the cut. Most probably the result is connected with an external
stimulus, rather than with an internal one. This may be true also for
cerianthus, but probably not for the planarian.
_LATERAL REGENERATION_
Since the most familiar cases of regeneration are those that take place
at the anterior and posterior ends, we not unnaturally come to think of
polarity as a phenomenon connected only with the long axis of the
animal; but there are also many cases of lateral regeneration in which a
similar relation can be shown. In such a case as the regeneration of the
leg of a salamander, or of a crab, we find instances of lateral
regeneration, but since the development takes place in the direction of
the long axis of the leg, the polarity of the leg may be thought of as
substituted for that of the body. In other animals, however, the
regeneration is strictly lateral. I have found that if the anterior end
of an earthworm, or even of lumbriculus, is split lengthwise in halves,
and then one of the half-pieces is removed, the missing half is replaced
by the half left attached to the rest of the worm. Trembley split a
hydra lengthwise into two pieces, and each piece bent inwards to make a
new tubular body. Bickford, Driesch, and I have obtained similar results
with pieces of the stem of tubularia.
In planarians which have a flat, broad body, lateral regeneration takes
place readily. If a worm is split in two along the middle line of the
body (Fig. 13½, _A_), each half regenerates the missing half. This is
brought about by the development of new tissue along the cut-side, and
the extension into the new part of outgrowths from the digestive tract.
Lateral regeneration also takes place if the worm is split lengthwise
into two unequal parts. In this case the larger piece produces new
material along the cut-side, and into this new part the branches of the
old digestive tract extend. The smaller piece also produces new material
along the cut-side, a new pharynx appears along the line between the old
and the new tissue, and a new digestive tract is formed out of the
remains of the old one (Fig. 19, _a_, _b_, _c_). New branches grow out
of the fused part into the new tissues at the side. The new worm that
develops from a piece that is less than half the width of the old worm
is about as wide as the piece that was cut off, for what is gained at
the cut-side is lost in the old part. The piece loses in length also
during regeneration. If the new worm is fed, it increases in size,
gaining in breadth both on the old side, as well as on the new side, and
in time it becomes a full-grown, symmetrical worm.
In the formation of the new part in these cases of lateral regeneration
it is not difficult to understand how some of the old organs, as the
digestive tract, grow out laterally into the new part; but it is more
difficult to see how longitudinal organs, such as the nerve-cord and
genital ducts, are formed anew. Bardeen, who has examined the
development of the new nerve-cord in lateral pieces, thinks that the new
nerve-cord grows backwards in the new part from the brain that develops
at the anterior end, either out of the old brain, if it, or any part of
it, is left, or out of the new brain that develops from the anterior end
of the lateral cord that is present in the piece. What takes place in
pieces cut so far to one side that none of the old cord is present in
the piece he did not make out; but I can state that a new brain develops
even when none of the lateral cord is present.
[Illustration: FIG. 19.--Indicating how a piece is cut off from side of
_Planaria maculata_. _a, b, c._ Regeneration of last. _d._ Regeneration
of single head at side. _e._ Regeneration of two heads at side.]
The development of a new head in pieces cut to one side of the old
median line offers some facts of interest. A piece may be cut from the
side of a planarian of such a shape that it has no anterior surface at
all (Fig. 19, _A_); yet a head develops at the anterior end of the new
material that appears at the side. It stands at first to one side, later
it assumes an anterior position. In this case an axial structure arises
in a lateral position, unless we look upon the new head as arising at
the anterior end of the new part, rather than at the side of the old,
but there is no evidence in favor of such an interpretation, since the
head arises at the same time as does the rest of the new material at the
side. In a small piece all of the new material at the side may be used
to form the new head (Fig. 19, _d_). Sometimes two heads develop (Fig.
19, _e_).
_REGENERATION FROM AN OBLIQUE SURFACE_
There are also certain important facts connected with the regeneration
from an oblique surface. The first case of the sort was described by
Barfurth. He found that if the tail of a tadpole is cut off obliquely,
as shown in Fig. 20, _B_, the new tail that develops stands at first at
right angles to the oblique surface. The angle that the new tail makes
with the axis of the old tail will be in proportion to the obliquity of
the cut-surface. The notochord that occupies the centre of the new tail
begins at the end of the old notochord, and extends to the tip of the
new tail, dividing it in the same proportionate parts as does the
notochord of the normal tail. The other organs occupy corresponding
positions. As the new tail becomes larger it slowly swings around into
line with the old part. This phenomenon of regeneration from an oblique
surface has been found in a number of other forms. It has been described
by Hescheler, and by myself
[Illustration: FIG. 20.--_A, A¹._ After Driesch. _A._ Piece of stem of
tubularia cut off obliquely, showing oblique position of tentacles.
_A¹._ Same, later stage. _B._ After Barfurth. Tail of tadpole
regenerating from oblique surface. _C._ Tail of fundulus regenerating
from oblique surface. _D._ After Hescheler. Anterior end of
allolobophora regenerating from oblique surface. _E._ Piece of planaria,
cut off by two oblique cuts, regenerating new head and tail. _F, F¹,
F²._ Three stages in the development of a new head (of a piece of
bipalium) at anterior end of oblique surface.]
in earthworms (Fig. 20, _D_), both for the anterior and posterior ends.
I have shown that it also takes place in the tail of a teleostian fish,
fundulus (Fig. 20, _C_), and have offered the following explanation of
the phenomenon. The new material that is first laid down is, to a
certain extent, indifferent as regards its axes. A symmetrical structure
is then formed, with the old edge as a basis. The median point of the
cut-edge connected with the median point of the outer surface of the new
edge, gives the axis of symmetry of the new tail. The other regions
assume corresponding positions. In the tail of the tadpole the position
of the new notochord is determined by the cut-end of the old notochord
and the median, outer point of the new material, and since the new
material is at first equally developed along the cut-edge, or at least
symmetrically developed, the new tail must stand at right angles to the
cut-edge. This explanation will cover, I think, all cases of
regeneration from an oblique surface. It assumes a law of symmetry in
the new material that is in accordance with the observed position in
which the new structure appears. The hypothesis makes no pretence to
explain why the new structures _should_ assume a symmetrical position,
but given that they do, the observed result follows.
[Illustration: FIG. 21.--Planaria lugubris. Upper row. _A._ Part of head
cut off obliquely; _a-a⁴._ Regeneration of new head. Lower row. _B._
More of head cut off obliquely; _b-b⁴._ Regeneration of same.]
There are certain peculiarities connected with the regeneration from an
oblique surface in planarians that may be considered in this connection.
If the worm is cut in two by means of an oblique cut, as shown by the
oblique line in Fig. 21, _B_, the new head that appears on the anterior
cut-surface of the posterior piece appears _at one side_ and not in the
middle of the oblique surface (Fig. 21, _B_, _b_). The new head stands
at right angles to the cut-surface. The anterior piece of the worm
produces a new tail at the side of the posterior cut-surface, in the
same way that the tail is formed in Fig. 20, _E_. The tail also stands
at right angles to the cut-surface. The new pharynx that develops in a
piece of this kind appears in the middle of the posterior cut-surface,
between the old and the new parts. It may extend somewhat obliquely in
the new part, and point toward the new tail.
[Illustration: FIG. 22.--Two upper rows _Planaria lugubris_. Lower row
_Planaria maculata_. Upper row. Tail-piece cut off obliquely in front of
genital pore. Figures show mode of regeneration. Middle row. Piece
including old pharynx cut off by two cross-cuts, regenerating head and
tail. Lower row. Piece cut off as last, regenerating head and tail.]
If a piece is cut from the anterior part of a worm by two oblique and
parallel cuts, the new head appears at one side of the anterior
cut-surface, and the new tail at the other side of the posterior
cut-surface. The new pharynx appears in the new material of the
posterior part in the middle line. Thus the middle lines of the new head
and tail and pharynx lie in different positions, yet these parts are
subsequently brought into the same line. This is done by the head
extending more forward and becoming broader, the tail growing backward
and also becoming broader. The old piece becomes narrower at the same
time. These three changes going on simultaneously produce a new
symmetrical worm. In one form, _Planaria lugubris_, the symmetrical form
is reached largely by the forward growth and the enlargement of the
head, and the growth backward and the enlargement of the tail (Fig. 22,
_B_). In _Planaria maculata_ the old part shifts, so that it forms a new
median line connecting the median line of the new head and new tail.
This is best shown when the piece includes the old pharynx (Fig. 22,
_C_). The pharynx is also shifted, so that its anterior end points
towards the side at which the new head lies, and its posterior end
towards the new tail. The result is that a new symmetrical worm is
formed, as shown by the series of figures in Fig. 22, _C_. In _Planaria
maculata_ the changes take place largely in the old part, and the old
material extends throughout the entire length of the new worm. In
_Planaria lugubris_ the change takes place largely in the new parts
(Fig. 22, _B_). The general method in the latter species by which the
symmetry is attained can be best shown by cutting the worm in two by an
oblique cut just in front of the genital pore (Fig. 22, _A_). The
posterior piece produces a new head at the side, and a new pharynx
appears along the border between the new and the old parts, as shown in
these figures. Its posterior end touches the middle line of the old
part, and from this point it extends obliquely across the new tissue
towards the middle of the new head. As regeneration goes on the new head
is carried farther forward, it becomes larger, and the main region of
new growth is found to be, in the figure, to the left side of the new
part. As a result of these changes the new head turns forward, and comes
to lie nearer the middle line of the old part. The pharynx is also
turned more forward, and finally, as the new parts enlarge, the
symmetrical form is produced. The internal factors that are involved in
the development of these oblique pieces are very difficult to analyze.
The position of the new head and tail at one side of the cut-edge is the
most difficult phenomenon of all to explain. We may, I think, safely
regard the first new material that is proliferated along the cut-edge as
totipotent, and our special problem resolves itself into discovering
what factor or factors determine that the new head is to form at the
most anterior end of the new material, and the new tail at the most
posterior end. If we assume that the result is in some way connected
with the influence of the old part on the new, and that this influence
is of such a sort that the more anterior part of the old tissue
determines that one side of the head must be at the most anterior edge,
we have at least a formal explanation of the position of the head at the
side. Given the position of the new head fixed at one side, its breadth
will be determined by the maximum breadth possible for the formation of
a new head. This is also in part an assumption, but it has at least
certain general facts of observation in its favor. The oblique position
of the new head is the result of its symmetrical development in the new
material in the same way that the position of the tail of the fish or of
the tadpole is the result of the symmetrical formation of the new tail
on the oblique surface. The subsequent changes, by means of which a
symmetrical worm is developed, are the result of different rates of
growth in the different parts. In this connection the most important
fact is that the growth takes place most rapidly where it will bring
about the new form. This problem, which is one of the most fundamental
in connection with the phenomena of development and of regeneration,
will be more fully discussed in a later chapter.
A number of assumptions have been made in the above attempt to give an
analysis of the formation of a head at the side of an oblique surface.
That these assumptions are not entirely arbitrary, but have a certain
amount of evidence in their favor, can, I think, be shown. The new
material that first appears is supposed to be totipotent, in the sense
that any part of it may produce any part of the structure that develops
from this material. That this is probable is shown by the following
experiment. If a cross-piece is cut from a worm, and then split
lengthwise into halves, each half will produce a new head at the
anterior edge of the piece. This result shows, at least, that from the
tissue lying to the right or to the left of the middle line new material
may be formed from which a whole head may develop. The new head does not
stand at first with its middle axis in line with the middle of the old
piece, _i.e._ it does not stand squarely at the anterior end of the
half-piece, but more towards the inner side of the piece. It may appear
that the old part has sufficient influence on the new part to shift the
axis of the latter toward the old middle line, but while some such
influence may be present, it is probable that the position of the head
is in part the outcome of another factor, viz. the presence at the inner
side of the piece of an undeveloped new side, with which the explanation
of the less development of the inner side of the head is also connected.
[Illustration: FIG. 23.--_Planaria maculata._ _A._ Cross-piece, allowed
to regenerate, then cut in two lengthwise, as indicated by line.
_a-a⁵._ Regeneration of left half.]
If a cross-piece is cut from a worm and kept until a small amount of new
tissue appears over the anterior and posterior cut-surfaces, and if then
the piece is split in two lengthwise, there will develop from each piece
a new head out of the new material over the anterior surface. The result
shows that the new material is at first totipotent, in the sense that it
may still produce one or more heads according to the conditions. It is
possible, of course, that the formation of the new head may have begun
at the time of the experiment, but if it had, the development had not
gone so far that a new arrangement was impossible. If, however, the
piece is not cut lengthwise until just before the formation of a head
(Fig. 23, _A_), then each half-piece produces at first a half-head, that
completes itself later at the cut-side.
Another experiment shows even more satisfactorily that the material over
an anterior cut-edge may produce one or more new heads according to the
conditions, and that the result is not connected with the region from
which the new material is derived. If the anterior end of a planarian is
cut off and then an oblong piece is removed from the middle of the worm,
as shown in Fig. 24, _A_, it will be found, if the side parts are kept
from fusing together in the middle line, that a new head develops at the
anterior end of each part, as shown in Fig. 24, _c, c¹_. If, on the
other hand, the two sides come together and fuse in the middle line, as
shown in Fig. 24, _a, b_, the new material that appears over their
anterior ends becomes continuous and produces a single head. In this
case, although the middle part of the old tissue has been removed, a
single head develops that is normal in all respects, and the eyes are
not nearer together than when the middle part is present, as when
regeneration takes place from an anterior cross-cut surface.
[Illustration: FIG. 24.--_Planaria lugubris._ _A._ Showing where a
piece, 4. was removed from middle of a worm. _a, b._ Regeneration of a
single head. _c, c¹._ Regeneration of two heads. _D, E, F._
Regeneration of small piece, 4. that was cut out.]
The assumption that the lateral position of the head on an oblique
surface is connected with the more anterior region of the old material
that is found at that side, can be made at least more intelligible by
the following experiment: If the head of a planarian is cut off
obliquely, as indicated in Fig. 21, _B_, so that one of the “ears” is
left at one side, the new head arises at the side in connection with the
part of the old head that lies at that side. The new head does not
extend over the entire cut-surface, which is longer of course than a
cross-cut would be, but lies at one side, as in the other cases just
described. In this case we can see that if the new head cannot, on
account of certain conditions, extend over the entire cut-surface, one
side of it may be determined by the presence of a part of the old head,
and this influence may be stronger than any other that might tend to
locate the new head in the original middle line. If we suppose that
similar conditions prevail in all cases when oblique surfaces are
present in these worms, we have a formal solution of the problem. The
argument cannot be convincing unless we can give a further explanation
of the nature of this influence that the old part has upon the new.
In other cases, as in the regeneration from an oblique surface in the
tail of the tadpole and of a fish, we must assume that the factor that
determines the middle of the new part has a stronger influence on the
new material than has the most posterior part of the old tissue.
The influence of an oblique cut-surface on the position of the new parts
is shown in a different way in the hydroid, tubularia. The conditions
are different in this case inasmuch as there is no proliferation from
the cut-end, but the old part produces the new hydranth. Driesch found
that if the stem of tubularia is cut in two obliquely, the new
tentacles, that develop as two rings around the tube near its cut-end,
stand obliquely on the stem,[26] as shown in Fig. 20, _A_. In most
cases, both the distal and the proximal circles of tentacles lie
obliquely to the long axis of the stem, but there is some variability in
the result, and occasionally one or the other, especially the proximal
circle, may be squarely placed, although, as a rule, the influence of
the oblique cut-end can be seen. It can be shown, I think, that the
oblique position of the rings of tentacles in tubularia is the outcome
of factors different from those that are found in the regeneration of
the tail of the tadpole and of the head and tail of the planarian.
Driesch suggested that the distance of the tentacle-rings from the
cut-end is the result of some sort of “regulation” that determines their
position at a given distance from the region at which the surrounding
water acts on the exposed end. Hence, if the exposed surface is an
oblique one the rings will also be formed in an oblique position. On the
other hand, I have suggested that we can imagine the regulation to
result from other factors. At the beginning of the development, and
before the tentacles appear, there is a withdrawal of tissue from the
cut-end that leaves the region from which the proboscis develops quite
thin. If this material withdraws at a uniform rate and to the same
distance at all points from the end of the piece, as observation shows
to be the case, and if, as appears also to be true, the outer end of the
distal ring of tentacles lies at the inner end of the proboscis region,
then it too will assume an oblique position if the cut-end is oblique.
If we imagine a similar series of regulations taking place throughout
the piece, we can account for the results. On this hypothesis the action
of the water on the free end need not be a factor in the result, but the
oblique end is itself sufficient to determine the series of regulations,
or mass-relations, that lead to the laying down of an oblique hydranth.
When the hydranth protrudes from the stem it assumes an oblique
position, as shown in Fig. 20, _A¹_. Driesch supposed the oblique
position of the hydranth to be due to an oblique zone that develops
behind the hydranth, but the result can best be explained, as certain
other experiments that I have made seem to show, as due to the negative
thigmotropism of the hydranth at the time it protrudes from the old
perisarc. It turns away from the projecting side of the oblique end of
the perisarc, as it does from any solid body with which it comes in
contact. That this is the case is best shown by splitting the stem
lengthwise into halves. In this case, although the two circles of
tentacles may be laid down squarely (Fig. 25, _A_), the new hydranth
protrudes at right angles to the old perisarc, as shown in Fig. 25, _B_.
[Illustration: FIG. 25.--Piece of stem of _Tubularia mesembryanthemum_
split in two lengthwise. Formation of whole hydranth that turned away
from contact with old perisarc.]
_THE INFLUENCE OF INTERNAL ORGANS AT THE CUT-SURFACE ON THE NEW
STRUCTURE_
In a few cases it has been discovered that the presence of certain
organs at the exposed surface is necessary in order that regeneration
may take place. The following experiment that I have recently carried
out shows, for instance, the influence of the nerve-cord on the
regenerating part. A few of the anterior segments of the earthworm are
cut off, as shown in the left-hand figure in Fig. 26, and then a piece
of the mid-ventral body wall of the worm is cut out, a part of the
ventral nerve-cord being removed with the piece. The cut-edges meet
along the mid-ventral line and fuse, closing the wound. As a result of
the operation there is left exposed, at the anterior end of the worm, a
cut-surface with all of the internal organs present except the nervous
system. The anterior end heals over, but I have not observed the
development of a new head at this level, although the exposed end is in
a region at which, under ordinary circumstances, a new head readily
regenerates. In several cases a new head developed at the point where
the cut-end of the nervous system is situated, _i.e._ at the level _B_
in the figure.
[Illustration: FIG. 26.--Left-hand figure X shows how, after cutting off
the anterior end of _Allolobophora fœtida_, a piece of the ventral wall
(including a part of the nerve-cord) is cut out. Right-hand figure _Y_
illustrates a more complicated operation, in which the piece of the
ventral wall that is cut out is a little behind the anterior end.]
A variation of the same experiment shows still more conclusively the
importance of the nervous system for the result. A few anterior segments
are cut from the anterior end as before. A cut is made, as shown in the
right-hand figure in Fig. 26, to one side of the mid-ventral line
(indicated by the black line in the figure at the level _A_). Then, at
the posterior end of this cut a piece is removed from the mid-ventral
line as in the former experiment (shown by the stippled area in the
figure). A portion of the ventral nerve-cord is removed with the piece.
As a result of this operation, two anterior ends of the nervous system
are left exposed (shown by the black dots in the figure). At the
anterior end of the worm, _i.e._ at _A_, there is one exposure, and at
the posterior end of the region from which the piece was removed there
is another. Two heads develop in successful cases, one at the anterior
end of the anterior cut-surface, _i.e._ at _A_, and the other at _B_.
The results show that in the absence of the cut-end of the nervous
system at an exposed surface a new head does not develop; and
conversely, the development of a new head takes place when the anterior
end of the nervous system is present at a cut-surface, even when such a
surface is not at the anterior end of the worm. We may perhaps be able
to extend this statement, and state that as many heads will develop as
there are exposed anterior ends of the nervous system.
In two other cases, at least, a somewhat similar conclusion may be
drawn, although it appears that in these cases other organs than the
nervous system may be the centres around which the new parts develop.
Tornier has shown that when the vertebræ of the tail of the lizard are
injured, the new material proliferated by the wounded surfaces serve as
centres[27] for the regeneration of new tails; and Barfurth has found
that the notochord in the tail of the tadpole plays a similar rôle in
the formation of a new tail. These experiments will be more fully
described in connection with the formation of double structures, but
from what has been said it will be seen that the cases are parallel to
that of the earthworm.
Until more has been discovered in regard to the internal factors of
regeneration, it would be venturesome to make any general statement
based on these few cases, but there is opened here a wide field for
experimental work. By eliminating one by one the different organs that
are present in the old part, it may be possible to discover much more in
regard to the internal conditions that are necessary in order that the
process of regeneration may take place.
_THE INFLUENCE OF THE AMOUNT OF NEW MATERIAL_
There are certain results connected with the amount of new material
which is produced during regeneration, that should be considered in
connection with the question of internal factors. It has been pointed
out that when one segment only is removed from the anterior end of the
earthworm only one new one returns; when two are cut off two come back,
and this holds good up to five segments. Beyond this, no matter how many
are removed, only five at most come back. The latter result seems to be
connected with the amount of material that is formed over the
cut-surface before differentiation begins. When only one or two segments
have been cut off, the new material that is formed is soon sufficient in
amount for the production of one or two new segments, but when three to
five are cut off somewhat more material is formed before differentiation
begins. When more than five are cut off the new material is at best only
sufficient to produce five new ones, and in some cases even a smaller
number is formed. This hypothesis assumes that there is a lower limit of
size for the formation of new segments below which a segment cannot
develop. The interpretation is fully in accordance with what we know to
be the case for small pieces of hydra and of other forms that, below a
certain minimal size, do not regenerate. The question as to how many
segments are formed out of the new part is determined, not only by the
amount of new material, but also by the number of segments to be
replaced, at least up to five segments. Beyond this limit we may think
of the maximum possible number of segments appearing in the new
material. That a relation of some sort obtains between the old and the
new parts, that may have an influence on the number of the new segments
which are formed, is shown by the fact that, when one, two, three, four,
or five are cut off, just this number comes back. A sort of completing
principle exists as a factor in the result, but when so much has been
cut off that the old part cannot complete itself in the new material
that is formed, then other factors must determine how many segments will
be produced.
In planarians we find a similar phenomenon. If much of the anterior end
is cut off, only a head is formed at the anterior cut-surface of the
posterior piece, and the intermediate region is absent. I interpret this
in the same way as the similar case in the earthworm. As soon as enough
new material has been formed for the anterior end to appear, it begins
to develop, and since it cannot develop below a certain minimal size, or
rather, since the tendency to produce a head approaching the maximum
size is stronger than the tendency to produce as much as possible of the
missing anterior end, all the new material goes into the new head. In
the planarian the possibility of subsequently replacing the missing
region behind the head exists, and the intermediate part is later
produced, the head being carried farther forward. The same is true of
the new posterior end of the earthworm, in which a growing region is
established at a very early stage in front of the tip of the tail, but
no such growing region is present at the anterior end in the earthworm.
These differences appear to be connected with the general phenomena of
growth in these forms. In the planarian interstitial growth can take
place in any part of the body, hence the possibility of producing a
missing region is present in all parts of the worm; but in the earthworm
we never find new segments intercalated at the anterior end during
normal growth, nor does this take place during regeneration. At the
posterior end of the earthworm we find a region of growth in which new
segments are produced, and we find the same thing is true in the
regeneration of the posterior end. In other words, the growing region in
front of the last segment is also regenerated.
It has been found in several forms that pieces below a certain size do
not regenerate. In those cases in which a small piece dies soon
[Illustration: FIG. 27.--_Tubularia mesembryanthemum._ _A._
Minimal-sized piece that produced a hydranth. _B, C._ Pieces below
minimal size. _D._ Ring produced by closing of small piece.]
after its removal from the rest of the body we have no direct means of
knowing whether or not the piece has potentially the power to
regenerate, but in some other cases, in which small pieces may be kept
alive for some time, they may not regenerate. Furthermore, the
regeneration of small pieces that are just above the minimal size is
often delayed and is sometimes imperfect. These small pieces seem to
meet with a greater difficulty in regenerating than do larger pieces.
Peebles has shown that pieces of hydra that measure less than 1/6 mm. in
diameter (= about 1/200 of the volume of hydra) do not regenerate,
although if very small pieces are taken from a developing bud they may
regenerate, even when only a 1/9 mm. in diameter. Very small pieces that
are, however, just above the minimal size, while they may assume a
hydra-like form, produce only one or two tentacles. The failure of the
smallest pieces to regenerate is not due to their dying, since they may
live for a much longer time than would suffice for larger pieces to
regenerate. Isolated tentacles of hydra do not produce new hydras,
although they may remain alive for some time. A single tentacle is
larger than the minimal piece, so that its failure to regenerate is
probably connected with the differentiation of the tentacle, rather than
with its size. The lack of power to regenerate in the smallest pieces of
hydra cannot be connected with the absence of any special organ, since
these pieces contain both ectoderm and endoderm. In tubularia also,
Driesch and I have found that pieces below a certain size do not
regenerate (Fig. 27). There is likewise in planarians a lower limit of
regeneration, even for pieces that contain all the elements which, being
present in larger pieces, make regeneration possible. Lillie has found
that nucleated pieces of the protozoon stentor fail to regenerate if
they are below the minimal size. He places this minimal size at 80 µ.
diameter, which he calculates as 1/27 of the volume of the stentor from
which the piece has come. I have obtained a slightly smaller piece that
regenerated, and since it came from a larger stentor it represents about
1/64 of the whole animal. The lack of the power of development of these
smallest pieces seems to be due to the absence of sufficient material
for the production of the typical form. We can give no other explanation
of the phenomenon at present, especially since the pieces contain
material that we know from other experiments has the power of producing
any part of the organism. The superficial area of small pieces is
relatively greater than that of larger pieces, but there is no evidence
that this relation can in any way influence the result. Whether the
difference in surface tension could prevent the small piece from
assuming the typical form and hold it, as it were, in a spherical form
is not known, but there is little probability that this is the
explanation of the phenomena.
The regeneration of small pieces of animals and of plants may often fail
to take place, because, as Vöchting has pointed out, the injury caused
by the cutting may extend so far into the small piece that its repair
may be impossible. In other cases there may be an insufficient reserve
supply of food stuff, although, if a proportionate form of any size
could be produced, it is difficult to see how this could be the case.
There can be no doubt, however, that pieces taken from parts of the body
that are dependent on other parts for their food, oxygen, etc., will die
for lack of these things, and even if they can live for some time their
further development may not take place in the absence of sufficient food
to carry on the process. After these possibilities have been given due
weight, there remain several cases in which there can be little doubt
that the failure of a small piece to regenerate is owing to the lack of
sufficient material to produce even the smallest possible form for that
sort of material, _i.e._ for the organization to be formed on so small a
scale.
There are some facts in connection with the regeneration of small pieces
of tubularia that have an important bearing on this question of
organization size. If long pieces of the stem are cut off, the new
hydranth, that develops out of the old tissue at the end of the piece,
occupies, within certain limits, a region of definite length. If pieces
of the stem are cut off that are only twice the length of the
hydranth-forming region, the length of the latter will be reduced to
half the length that it has in longer pieces, and if still smaller
pieces are cut off, the hydranth-forming region may be reduced, as
Driesch has shown, to seventy per cent of the normal length. The
hydranths that develop from the smaller pieces have also a reduced
number of tentacles, as I have found. It was first shown by Bickford,
and later by Driesch, and by myself, that in many cases very short
pieces of the stem of tubularia produce _only the distal parts of a
hydranth_. This happens most often when the length of the piece is less
than the average normal length of the hydranth-forming area, but it may
also take place in pieces that are much longer than the minimal size of
the least hydranth-forming region. Driesch made the further discovery,
which I have confirmed, that pieces from the distal end of the stem are
more likely to produce these partial structures than are pieces from the
more proximal part. Some of these partial structures are represented in
Fig. 28, _C-G_. Sometimes the inner tube, or cœnosarc, which is composed
of the two layers of the body, ectoderm and
[Illustration: FIG. 28.--_Tubularia mesembryanthemum._ Products of
regeneration of short pieces. _A._ Piece that regenerated a hydranth in
same way as do longer pieces, but with fewer tentacles. _B._ Pieces
whose stem drew away from wall of old perisarc (cylinder in figures).
_C._ Hydranth with almost no stalk. _D._ Hydranth without stalk. _E._
Distal part of hydranth with one long proximal tentacle. _E¹._ Similar,
but more reduced. _E²._ Similar, with two tentacles at side. _F._
Proboscis with reproductive organs. _G._ Proboscis without reproductive
organs.]
endoderm, draws away from the chitinous perisarc, as shown in Fig. 28,
_B_. A hydranth with a short stalk is then produced. In other cases,
Fig. 28, _C_, almost all of the cœnosarc is used up to form the
hydranth, and only a short, dome-shaped knob represents the stalk. In
still other cases there may be no stalk at all (Fig. 27, _D_), but only
the hydranth. Forms like the last two are more often produced from
pieces of the distal end of the stalk. From very small pieces, forms
like those shown in Figs. 28, _E-E²_, that represent only proboscides
with a reduced number of tentacles, are sometimes formed. Reproductive
organs may be present at the base of these pieces. A further reduction
is shown in Figs. 28, _F_, _G_, that are proboscides with only the
distal circle of tentacles; in one of these, reproductive organs are
present around the base. Partial forms more reduced than these have not
been found.
If we examine the factors that determine the production of the partial
structures, we find, in the first place, that the size of the piece is
of the greatest importance. The reduced forms appear most often in
pieces that are shorter than the average length of the hydranth-forming
area. A second factor is connected with the region of the stem from
which the piece is taken. Larger pieces from the distal end produce
partial structures, especially hydranths with very short stalks (Fig.
28, _C_), or with none at all (Fig. 28, _D_). There are certain facts
connected with this distal region, which lies just behind the hydranth,
that should be mentioned in this connection. It was first discovered by
Dalyell that a hydranth-head lives for only a limited time, and that
when it dies a new head is regenerated from the region behind the old
one. The stalk of the new hydranth continues to elongate for some time
after the new hydranth has been formed. Whether this continuous growth
in the distal end, or the normal formation of a new hydranth by it from
time to time, can in any way be connected with the development of
partial structures from this region cannot at present be stated. The
distal part of the stem contains more of the red-pigment, that gives
color to the stem and to the hydranth, than does any other part. Loeb
first advanced the view that the red-pigment in the stem acts as a
formative substance in Sachs’ sense, and determines the production of a
new hydranth by accumulating near the cut-end of the piece. Driesch also
assumes the red-pigment to be a factor in the result, but supposes that
it acts quantitatively, rather than in determining the quality of the
result. If this red-pigment acted in the way supposed either by Loeb or
by Driesch, it might act as one of the factors in the production of
these partial structures. This red-pigment is contained in the form of
reddish granules in the cells of the endoderm. The granules are of
various sizes, the largest being easily seen even with low powers of the
microscope. When a piece of the stem is cut off, the ends close by the
drawing in of the cut-edges over the open-end. A circulation of the
fluid contained in the piece then begins. In the fluid, globules appear
very soon that contain red-pigment granules like those in the endoderm.
The globules appear to be endodermal cells, or parts of cells, that are
set free in the central cavity. The circulation continues for about
twenty-four hours. At about this time one end of the stem becomes
reddish, owing to the presence in it of a larger number of red-pigment
granules than before. The ridges that are the rudiments of the tentacles
appear (Fig. 30, _A_), and a new hydranth very rapidly develops. At the
time when the hydranth begins to appear the globules in the circulating
fluid disappear. They disappear at the time when the red-pigment of the
forming hydranth is rapidly increasing in quantity, and not unnaturally
one might suppose that the pigment of the circulating fluid had been
added to the wall where the hydranth is produced. The globules disappear
in the region of the new hydranth, but, I think, it can be shown that
they do not form any essential part of the hydranth. They may be found
stuck together in a ball that lies in the digestive tract of the new
hydranth, and when the hydranth is fully formed the pigment is ejected,
as Stevens has shown, through the mouth.
The development of the new hydranth begins several hours before the
red-pigment globules have disappeared from the circulation. The walls in
the region of the future hydranth begin to thicken, and, later, pigment
develops in the endoderm of this region. The new pigment is formed in
the new cells of the endoderm, and does not come from the circulating
globules, as shown by the development of very short pieces of the stem.
In these the amount of new pigment that develops in the new hydranth may
be far greater than that in the whole original piece (Fig. 30, _D_), and
in this case there can be no question but that new pigment is made in
the endodermal cells of the hydranth. The formation of a hydranth, that
usually takes place after another twenty-four hours, from the basal end
of a long piece, shows that a hydranth may develop when there are no
granules in the circulating fluid. These basal hydranths may contain as
much pigment as do the distal ones.
Driesch suggested that the red-pigment in the circulating fluid
determines quantitatively by its presence how much of a hydranth is
formed, or the size of the hydranth in relation to the rest of the
piece. There seems to be no evidence in favor of this view and much
against it. Loeb has not stated specifically whether he means that it is
the pigment in the circulating fluid or that in the walls which acts as
a formative stuff; the presumption is that he meant the latter. An
examination of the piece during regeneration gives no evidence in favor
of the view that the pigment moves into the region of the new hydranth.
On the contrary, it remains constant in amount at all points except
where the new hydranth is developing, and there is in this region
unquestionably a large development of new pigment.
The evidence for and against the idea that the red-pigment of tubularia
is a formative stuff, or even building material, has been considered at
some length, because it is the only case in which the hypothetical
formative stuff has been definitely located in a specific, recognizable
substance that can be followed during the process of regeneration. It is
well, I think, to give the question full consideration, especially as
the hypothesis often appears to give an easy solution of some of the
problems of regeneration. In a later chapter the subject will be more
fully treated.
[Illustration: FIG. 29.--_Tubularia mesembryanthemum._ _A._ Short piece
with hydranth at each end. _B._ Double piece with one circle of proximal
tentacles. _C._ Double piece with only two proximal tentacles. _D._
Double proboscis with two sets of reproductive organs. _E-E³._ Double
proboscis.]
Since the red-pigment hypothesis does not explain the phenomenon of the
formation of the partial structures in tubularia, we must look for
another explanation. As the matter stands at present we can only assume
that there is a _predisposition_ of a very small piece to form a larger
partial structure than a smaller whole one. This problem of the method
of development of small pieces of the stem of tubularia is further
complicated by the development in many cases of double hydranths, or
double parts of hydranths, as shown in Fig. 29, _A-E_. The first form
(Fig. 29, _A_) shows two hydranths turned in opposite directions, that
are united at their bases. Another form has only a single circle of
proximal tentacles between the two proboscides (Fig. 29, _B-C_). In
other forms there are only two proboscides, each with its reproductive
organs (Fig. 29, _D_), and often there are simply two proboscides united
at the base (Fig. 29, _E-E³_). It is the rule, even in longer pieces,
that a hydranth appears at each end of the piece, if the piece is
suspended or even lies on the bottom of the water; but
[Illustration: FIG. 30.--_Tubularia mesembryanthemum._ _A._ Short piece
with reduced hydranth-region. _B._ Piece from distal end of stalk
producing a hydranth without a stalk (see Fig. 27, _D_). _C._ Piece
producing hydranth as outgrowth of end. _C¹._ Later stage of last. _D._
Short piece producing double proboscis (see Fig. 28, _E_).]
in all these cases the basal hydranth develops about twenty-four hours
after the apical one. In the short pieces, however, the two ends develop
at the same time, although the development of all the short pieces,
whatever structures they may produce, whether single or double, is
delayed, and the hydranths may not appear until after the long pieces
have produced their basal hydranths. In these double structures both
ends develop at the same time (Fig. 30, _D_). If we suppose the
influences that start the development of the piece begin first at the
distal end, the region affected will lie so near to the proximal end of
the piece that the development at this end may be hastened, and under
these circumstances the region of new formation will be shared by the
two hydranths. The factors that determine that a larger, partial
structure is formed in preference to a smaller whole one will no doubt
be found to be the same in these double structures and in the single
ones.
_THE INFLUENCE OF THE OLD PARTS ON THE NEW_
One of the most striking and general facts connected with the phenomenon
of regeneration is that the new part that is built up on the exposed
surface is like the part removed. This suggests that an influence of
some sort starts from the old part and changes the part immediately in
contact with it into a structure that completes the old part in that
region. We can imagine that the new part that has been changed in this
way may act on the new part just beyond it, and so step by step the new
part may be differentiated. It is not difficult to show that the
phenomenon is really more complicated than this, and that other factors
are also acting on the new part; but, nevertheless, that the old part
has some such influence is probable. Under certain conditions, however,
this influence may be counteracted by other factors, and something
different from the part removed may be formed. One example of this sort
has already been discussed, namely, that in which after the removal of
much of the anterior end of the earthworm or of a planarian, only the
distal end comes back. Another case is that in which something different
from the part removed is regenerated. If the tip of the eye of the
hermit-crab or of other crustaceans is cut off a new eye is regenerated,
but if the eye-stalk is cut off near its base an antenna-like organ
develops. Herbst has suggested that the presence of the ganglion at the
end of the stalk accounts for the regeneration of a new eye, when only
the tip of the stalk is cut off. In the absence of the ganglion at the
cut-edge the stalk does not produce an eye, but an antenna, as is shown
when the eye-stalk is cut off near the base. The factors that determine
the development of an antenna instead of an eye have not been
discovered. Przibram has shown that when the third maxilliped of
portunas, carcinas, or of other crustaceans is cut off near the base,
the new appendage that develops is different from the one removed, and
resembles a leg in many ways, but if the animal is kept until it has
moulted several times the appendage becomes more and more like the part
removed. Another remarkable case has also been described by Przibram for
_Alpheus platyrrhynchus_. In this decapod, the claws of the first pair
of legs are different from each other, one being much larger than the
other and having a different structure.[28] If the larger claw is thrown
off at its breaking-joint, and the smaller one left intact, the latter
at the next moult (or sometimes after two moults) changes into the
characteristic larger claw and the newly regenerated claw is like the
smaller one. If the experiment is repeated on this same animal, _i.e._
if the newly acquired large claw is removed, then at the next moult the
smaller claw becomes the larger one and the new claw becomes the smaller
one--the conditions now being the same once more as at the beginning. If
both claws of an animal are thrown off at the same time, two new claws
regenerate that are both of the same size, and each is a small copy of
the claw that was removed. As yet no experiments have been made that
show what factors regulate the development of each kind of claw.
Returning again to the question of the regeneration of parts similar to
the ones removed, there are some interesting results that Peebles has
obtained in the colonial hydroids, podocoryne and hydractinia. These
colonies consist of three principal sorts of individuals: the nutritive,
the reproductive, and the protective zooids. Peebles has found that if
the stalks of these zooids are cut into pieces, each produces the same
kind of zooid as was originally carried by that stalk. Pieces of the
stem of the nutritive zooid produce new nutritive zooids at the
anterior end of the piece, and sometimes also at the basal end. A
similar statement may be made for each of the other kinds. Another
method of regeneration sometimes takes place, when, for instance, a
piece of the stalk of a nutritive individual is left undisturbed without
being supplied with fresh water. It sends out root-like stolons instead
of producing a new zooid. The stolons appear first at the ends of the
piece, but may later also appear at several points along the piece. They
make a delicate network, and the original piece may entirely disappear
in the stolons. After several days new feeding zooids grow out at right
angles to the stolon network. Pieces of the stalk of protective zooids
may also produce stolons, but they spread less slowly, and the formation
of new individuals was not observed. In one case a piece of a
reproductive zooid made a short stolon, and from it arose a new
individual that seemed to be a nutritive zooid. If the latter result
proves to be true, we see that a piece may produce a new part that is of
a different kind from that of which the piece itself was once a part,
but this is brought about by the formation of a stolon that is itself
one of the characteristic structures by means of which these colonial
forms produce new nutritive zooids. In this case there is a return of
the piece to a simpler form, the stolon, and, acting on this, the
factors that produce nutritive zooids may bring about new nutritive
zooids. The influence of the old structure is lost when the piece
assumes a new character.
Another series of experiments gives an insight into an internal factor
of regeneration that may prove, I think, to be one of some importance
and help in interpreting certain phenomena. If the head-end of a
planarian is cut off, the posterior piece split along the middle line,
and one side cut off, just above the lower end of the longitudinal cut,
as shown in Fig. 31, _A_, it will be found that, if the long and the
short sides are kept from uniting along the middle line, each half will
produce a new head on its anterior surface (Fig. 31, _C_). If the two
halves grow together, and the anterior surface of the shorter piece
becomes connected with the anterior surface of the longer piece by means
of the new tissue that develops along the inner side of the latter (Fig.
30, _B_), then a head appears only on the anterior half. The development
of a head on the shorter half is prevented by the establishment of a
connection with the new side. Sometimes an abortive attempt to produce a
head is made, but the posterior surface fails to produce anything more
than a pointed outgrowth. If we attempt to picture to ourselves how this
influence of the new side on the posterior surface is brought about, we
can, I think, most easily conceive the influence to be due to some kind
of tension or pull of the new material which is of such a sort that it
restrains the development of a head at a more posterior level. We can
picture to ourselves the same kind of process taking place in the
regeneration of the tail of a fish from an oblique surface. The maximum
rate of growth is found over that part of the cut-surface that is nearer
the base of the tail (Fig. 40). At all other points the growth is
retarded, or held in check, and it can be shown that the suppression is
connected with the formation of the typical form of the tail in the new
part. If we cannot actually demonstrate at present that this is due to
some sort of tension between the different parts which regulates the
growth, we find, nevertheless, that it is by means of some such idea as
this that we can form a clearer conception of how such a relation of the
parts to each other is established. In a later chapter this subject will
be dealt with more fully.
[Illustration: FIG. 31.--_Planaria lugubris._ _A._ Showing how worm was
operated upon. _B._ A single head regenerated at anterior cross-cut. It
was united by a line of new tissue along the side of the long half-piece
with the new tissue at the anterior end of the short half-piece. The two
half-pieces reunited along the middle line. _C._ Two heads regenerated,
one from each half cross-cut. The two half-pieces were kept apart along
the middle line.]
_THE INFLUENCE OF THE NUCLEUS ON REGENERATION_
The influence of the nucleus on the process of regeneration has been
shown in a number of unicellular forms. It was first observed by Brandt
in 1877 that pieces of _Actinosphærium eichhornii_ that contain a
nucleus assume the characteristic form, but pieces without a nucleus
fail to do so. Schmitz (’79) found that when the wall of the many-celled
siphonocladus is broken, the protoplasm rounds up into balls, some of
which contain one or more nuclei, while others may be without nuclei.
The nucleated pieces produce a new membrane, and later become typical
organisms, but non-nucleated pieces do not form a new membrane, and soon
disintegrate. Nussbaum (’84, ’86) cut into pieces the ciliate infusoria,
oxytricha and gastrostyla. Those pieces that contained a nucleus quickly
regenerated a new whole organism of smaller size, that had the power of
further reproduction, while the pieces that did not contain a part of
the nucleus showed no evidence of regeneration; and, although they
continued to move about for as much as two days, they subsequently
disintegrated. Gruber obtained the same result on another ciliate
infusorian, _Stentor cœruleus_. He found that, although the
non-nucleated pieces close over the cut-surface, and move about for some
time, they eventually die. He further showed that a non-nucleated piece
containing a portion of a new peristome in process of formation will
continue to develop this new peristome, although a new peristome is
never produced by a non-nucleated piece under other circumstances. He
believes that if the new peristome has begun to be formed under the
influence of the old nucleus, it may continue its development after the
piece is severed from its connection with the nucleus. A non-nucleated
piece containing a part of the _old_ peristome does not produce a new
peristome from the old piece. Gruber observed that a non-nucleated piece
of amœba behaves differently from a nucleated piece, and dies after a
time.
Klebs found that when certain algæ are put into a solution that does not
seriously injure them, but causes the protoplasm to contract into balls,
some of these contain nuclei, others not. If, for instance, threads of
zygnema, or of spirogyra, are placed in a 16 per cent solution of sugar,
the protoplasm of each cell breaks up into one or more clumps, some with
nuclei, others without. Both kinds may remain alive for a time; some of
the non-nucleated pieces may live for even six weeks. The nucleated
pieces surround themselves at once, when returned to water, with a new
cellulose wall, but the non-nucleated pieces remain naked. The latter
can, nevertheless, produce in the sunlight new starch that is used up in
the dark and is made anew on the return to light.[29]
Balbiani (’88) found that non-nucleated pieces of cytrostomum,
trachelus, and protodon failed to regenerate, and Verworn (’89 and ’92)
obtained similar results on several other protozoa. Similar facts have
been made out by Hofer (’89), Haberlandt and Gerassimoff (’90). Palla
(’90) found that in certain cases non-nucleated pieces, especially those
from cells in growing regions, can produce a new cell wall; while more
recently Townsend (’97) has shown in several forms that non-nucleated
pieces do not produce a new cell wall unless they are connected by
protoplasmic threads with nucleated pieces. The most delicate connection
suffices to enable a non-nucleated piece to make a cell wall, even when
the nucleated piece lies in one cell and the non-nucleated in another,
the two being connected by a thread of protoplasm that passes through
the intervening wall.
If we examine somewhat more in detail some of these cases, we find that
when a form like stylonychia is cut into three pieces, the two
end-pieces without a nucleus fail to regenerate, while the central piece
makes a new entire organism of smaller size. If stentor is cut into
three pieces, each piece containing one or more nodes of the
macronucleus, each produces a new stentor. If, however, a piece is cut
off so that it does not contain a part of the macronucleus, it fails to
regenerate. Verworn (’95) succeeded in removing the central capsule with
its contained nucleus from the large radiolarian, _Thallasicolla
nucleata._ The non-nucleated animal remained alive for some time, but
eventually died. The nucleated capsule developed a new outer zone with
processes like those in the normal animal. If the nucleus is taken from
the capsule, the capsule dies, but shows some traces of the formation of
an outer zone. If the protoplasm is removed as far as possible from
around the nucleus, the latter does not regenerate new protoplasm, but
dies after a time. Verworn concludes that the protoplasm cannot carry on
all its normal functions without the nucleus, or the nucleus without the
protoplasm.
These experiments sufficiently demonstrate that non-nucleated pieces are
unable to regenerate. If we attempt to examine further into the meaning
of the phenomenon, we find a few things that appear to have a bearing on
the result. The behavior of the non-nucleated pieces shows that the
metabolism of the cell has been changed after the removal of the
nucleus. In some cases the protoplasm is not able to carry out the
process of digestion of the included food substances. This process may
be due to some interchange that goes on between the nucleus and the
protoplasm, which is stopped by the removal of the nucleus, and, in
consequence, the metabolism of the cell is changed. The lack of
regenerative power may be due to this change in the metabolism. It
cannot be claimed, however, that the result is due to a lack of energy
in the pieces, for the incessant motion of the cilia in some kinds of
pieces, that goes on for several days, shows that a large store of
energy is present. Unfortunately, we do not know enough of the relation
that subsists between the nucleus and the protoplasm to be able to state
to what the lack of regenerative power is due.
Loeb (’99) has suggested that the lack of power of non-nucleated pieces
may be due to a lack of oxidation. The nucleus contains substances
which, according to Spitzer, are favorable to the process of oxidation.
When the nucleus is removed, the oxidation is supposed by Loeb to be too
low to allow the process of regeneration to take place. In support of
this view, he points out that while non-nucleated pieces of infusoria
live for only two or three days, non-nucleated pieces of plants
containing chlorophyl may be kept alive for five or six weeks.
Non-nucleated pieces containing chlorophyl can obtain a supply of
oxygen, owing to the breaking down of carbon dioxide in the
chlorophyl-bodies, and the consequent setting free of oxygen. It should
be pointed out, on the other hand, as opposed to Loeb’s view, that
non-nucleated pieces of amœba have been kept alive for fourteen days;
and that despite the better oxidation that may take place in
non-nucleated pieces of plants, regeneration does not take place.
It has been found that non-nucleated pieces of the egg of the sea-urchin
do not segment or develop, and the result is the same whether the pieces
come from fertilized or unfertilized eggs. If, however, a spermatozoon
enters one of these pieces, the piece will segment, and, as Boveri and
later Wilson have shown, it will produce an embryo.
Boveri also tried fertilizing a non-nucleated piece of the egg of one
species of sea-urchin with a spermatozoon of another species. He found
that the embryo that develops is of the type of the species from which
the spermatozoon has come, and he concluded that the nucleus determines
the character of the larva, and that the protoplasm has no influence on
the form. The evidence from which Boveri drew his conclusion is not
beyond question. It has been shown by Seeliger (’95) and myself (’95)
that if whole eggs of the species _Sphærechinus granularis_, used by
Boveri, are fertilized by the spermatozoa of the other species, _Echinus
microtuberculatus_, there is great variability in the form of the
resulting larvæ. Most of them are intermediate in character between the
types of larvæ of the two species, but a few of them are like the
paternal type. Vernon (’99) has more recently shown that the character
of hybrids is dependent upon the ripeness of the sexual products of the
two parents. If, for instance, the eggs (sphærechinus) are at the
minimum of maturity, the hybrids are more like the male
(strongylocentrotus).
It remains, therefore, still to be shown whether or not the protoplasm
has any influence on the form of larva that comes from a non-nucleated
piece, fertilized by a spermatozoon of another species. That the nucleus
of the male does have an influence on the form of the animal is
abundantly shown by the inheritance of the peculiarities of the father
through the chromatin of the spermatozoon.
_THE CLOSING IN OF CUT-EDGES_
One of the most familiar changes that takes place when a cut-edge is
exposed involves the rapid covering over of the exposed tissues. This
takes place from the margin of the wound, and a layer of cells, usually
the ectoderm at first, covers the surface. The closing in is brought
about in many forms by the contraction of the muscles of the outer wall
of the body. This seems to be the case in the earthworm and in the
planarian, as well as in other animals, such for instance as the
starfish, holothurian, etc. But in addition to this purely muscular
contraction another process takes place, that is less conspicuous in
forms in which the muscles bring about the first closing, but which is
evident in forms in which the muscles are absent or little developed. I
am able to cite two striking cases that have come under my own
observation. When a piece is cut from the stem of tubularia, the ends
close in twenty minutes to half an hour. The body wall, the cœnosarc,
composed of the two layers of ectoderm and endoderm, withdraws a little
from the cut-edge of the outer hard tube, or perisarc, that covers the
stem, and then begins to draw across the open end. A perfectly smooth,
clean edge is formed that advances from all points to the centre, where
the final closing takes place. The closing is not due to an arching over
of the cœnosarc, but the thin plate is formed standing nearly at right
angles to the outer tube. This plate is composed of two layers of cells,
of which there are a number of rows arranged concentrically between the
centre and the outer edge. In the absence of muscle-fibres in the stem,
the result cannot be due to a muscular contraction, and even if short
fibres existed the transportation of cells entirely across the open end
would speak against this interpretation.[30] Since the closing over
takes place without any support, we cannot suppose the process to be due
to any sort of cytotropic effect. The closing takes place equally well
in diluted sea water and in stronger solutions. The method of withdrawal
of the cells, as best seen when longitudinal pieces are studied,
resembles very much the withdrawal or contraction of protoplasmic
processes in the protozoa, and so far as one can judge from resemblances
of this sort, the two processes appear to be the same.
This closing in of the cut-surface, while a preliminary step in the
process of regeneration, cannot, I think, be regarded as a part of the
regeneration in a strict sense. That the two processes are not dependent
on the same internal factors is shown by the following experiments: If a
bunch of tubularia is kept in an aquarium, it will produce new heads
two or three times and then cease, and if after the last-formed heads
have died, pieces of the stem are cut off, they close as readily as do
pieces from fresh hydroids. Moreover, at certain times of year the
species _Tubularia (Parypha) crocea_ lose their heads, and only the
stalks remain. Pieces of these stalks will not regenerate new heads, at
this time, although they close in as quickly as do pieces at other times
of the year when the heads are present and when new ones regenerate.
Another equally good illustration of what seems to be the same
phenomenon is found in the closing in of wounded surfaces in the young
tadpole embryos. If embryos are taken from the jelly membranes, or even
after they have been set free, and cut in half, each piece quickly
covers over the wounded surface by means of the ectodermal cells. A much
more striking illustration of this closing over in the young tadpole is
obtained by cutting, with a pair of small scissors, a large piece from
the side. The area may be a fourth or more of the entire side, and yet
it may be closed over in an amazingly short time. Half an hour or an
hour often suffices to cover a large exposed surface. In this case also
the wound is covered not by individual cells wandering over the exposed
surface, but by a steady advance of the smooth edge of the ectoderm
toward a central point. The process is so similar to that which takes
place in tubularia that little doubt can remain as to the two being due
to the same factors. As there are no muscle fibres present in the part
of the frog’s embryo from which the piece is cut off, the result cannot
be due to muscular contraction, but appears to be a contractile
phenomenon similar to that in tubularia. Even the small piece that is
cut from the side of the body shows the same phenomenon. At first it
suddenly bends outwards owing to some physical difference between the
inner and the outer parts of the piece. Then the edges thicken, bend in,
and begin their advance over the inner tissues. The process is seldom
completed, since there appears to be a limit to which the ectoderm can
be stretched as the edges advance. A most striking phenomenon both in
pieces of tubularia and of the frog’s embryo is the entire absence of
dead material at the wounded surface. No sooner is the operation
performed than the advance begins; and there is not a trace of dying
cells or parts of cells to be seen.