Thomas Hunt Morgan

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

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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 foetida 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. foetida, 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. foetida 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 oesophagus 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 oesophagus, 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.

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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.

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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.

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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

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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.

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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.

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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.

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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.

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.

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.

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

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 ? 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 coenosarc, which is composed of the two layers of the body, ectoderm and

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 coenosarc 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.

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

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.

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 coeruleus. 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 amoeba 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 amoeba 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 coenosarc, 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 coenosarc, 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.