Regeneration

posed to act as a poison. The most important result of this experiment is to show that the maximum growth does not take place in sea water in which the animal is accustomed to live, but in a much more dilute solution. Normal sea water contains about 3.8 per cent of salts ; the maximum growth takes place in a solution containing only 2.2 per cent. Not only is the length of the stem greater in the latter solution, but the thickness of the stem is also greater. The stem is smaller in a solution containing more salt than that contained in ordinary sea water.

There is another variant in these solutions which Loeb takes into account. With the increase in concentration of the solution its power of absorbing oxygen decreases, but the difference is too slight to affect the main result. Not only does the amount of salts in solution affect the osmotic condition of the cells, but the salts also play a part in the metabolism of the animal. As the result of a series of experiments, the details of which may be here omitted, Loeb has shown that the regeneration of tubularia takes place only when the salts of potassium and of magnesium are present. A very little of the potassium salt is necessary, too much retards, and still more prevents regeneration.

There must be also a certain amount of oxygen dissolved in sea water in order that regeneration may take place. If a piece of the stem of tubularia is cut off and one end pushed into a small tube that fits the stem closely, and if the tube is then stuck into the sand at the bottom of an aquarium, a hydranth develops only at the free end of the piece, and none at the end in the tube. The result appears to be due to the lack of oxygen. If the piece is then taken from the tube, a hydranth may appear at the end that has been in the tube.

Another experiment shows the same result even more clearly. If a piece of the stem is suspended freely in the water, so that its lower end is almost in contact with the surface of the sand, but does not quite touch it, no regeneration takes place at the lower end. This result is interpreted by Loeb as due to the lack of oxygen in the water near the surface of the sand.1 In connection with the action of external factors on regeneration it is evident that in some cases they may not be in themselves necessary for the growth of a new part, yet when growth takes place they may determine what sort of a part is produced. For instance, if gravity

1Jacobson has shown that the layer of water just above the sedimentary layer at the bottom is poor in oxygen. determines the kind of regeneration in antennularia, it is possible that if the regenerating piece were placed on a rotating wheel, the piece might still produce a new stem at the apical end, and roots at the lower end. In an experiment of this sort that I made, the pieces did not, it is true, regenerate at all, but this was probably due not to the change of position in regard to gravity, but to agitation of the water, or to the rubbing of the cut-end against the water. It is also possible that in this form the attachment of the piece at one end may be a factor that may counterbalance the action of gravity. Other factors, such as food, or temperature, or oxygen, appear not to determine the kind of product that results, but only the rapidity with which the change takes place. The salts in solution seem also to act on the rate and extent of the new growth, but possibly other cases may be found in which the kind of regeneration may also be affected by the salts.

It is important to find that those animals whose growth and regeneration are influenced by such external factors as light, gravity, and contact are attached animals that stand in a constant relation to these physical agents. They form only a very small part of the entire number of animals in which regeneration takes place. Animals that constantly move about are not, as a rule, influenced during their growth and regeneration by gravity and contact, and under natural circumstances they are always changing their position in regard to these agents. Temperature, and food, and substances in solution act alike on fixed and free forms, and they are, it appears, both influenced in the same way by these agents. The most significant fact that has been discovered in connection with the influence of external factors on regeneration is that the same factors that influence the normal growth of the organism also affect in the same way the regeneration.

As yet an analysis of the external factors that influence growth has not been made out as completely for animals as for plants, especially in those cases in which the result is determined by several factors at the same time. An examination of the factors that influence regeneration in plants will be made in a later chapter. First, however, the internal factors of regeneration in animals will be considered. 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.

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.1 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

1 "There is thus manifested in the formative force of the tubularia-stem a well-marked polarity, which is rendered very apparent if a segment be cut out from the centre of the stem." Allman ('64). 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

FlG. 16. — A. Head of Planar ia 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 faetida regenerating a new tail at its anterior end. C1. Enlarged anterior end of last with new tail. C*. Tip of new tail. D. Anterior end of one individual of A. fastida, 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. fxtida united by anterior ends. One end was subsequently cut off and a new tail regenerated. G. End of a developing piece of Tubular ia mesembryanthemum that had been cut off; it has regenerated, at its proximal end, another proboscis.

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 Ingnbris, 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 stomachintestine, 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.

the polarity of a piece is shown by Ceri- FIG. 17. - After vTigt. pianarian anthns mcmbmnaccons. 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. 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.

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. It has been shown by Loeb that if the incurrent siphon of the ascidian dona intestinalis be partially cut off, new eye-specks develop around the margin of the cut, as shown in Fig. 18, A. I have repeated 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.

this experiment and obtained the same result, and found, as had Loeb also, that the same holds true for the excurrent siphon (Fig. 1 8, 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. 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. 13^, 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 outgrowthsfrom 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. 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 sur- FIG. 1*9. -indicating how a piece is cut off face at all (Fig. 19, A)j yet a head from side of Planar ia maculata. a, 6. develops at the anterior end of the c. Regeneration of last. a. Regenera- .11 i

tion of single head at side. *. Regeneranew material that appears at the 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).

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

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 struc- FlG. 20. — A, A*. After Driesch. A. Piece of stem of tubularia cut off obliquely, showing oblique position of tentacles. A1. Same, later stage. D. 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. £. Piece of planaria, cut off by two oblique cuts, regenerating new head and tail, f, fl, /•'2. Three stages in the development of a new head (of a piece of bipalium) at anterior end of oblique surface.

ture 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.

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 FlG. 21. — Planaria lugubris. Upper row. A. Part of head cut off obliquely; a-a*. Regeneration of new head. Lower row. B. More of head cut off obliquely ; b-b*. Regeneration of same.

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.

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 cutsurface. 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 FlG. 22. — Two upper rows Planar ia lugubris. Lower row Planar ia 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.

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, /?). 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