Regeneration

the water, regeneration takes place at the cut-ends. If a piece is suspended with its apical end upwards (Fig. 14, B), a new stem develops at the upper cut-end, and new roots from the lower cut-end. If a piece is suspended with its basal end upwards (Fig. 14, C), there is formed at its upper (basal) end a new stem with its branches also slanting upwards as shown in the figure. Roots appear at the lower (apical) end. Since gravity is the only force that acts in a vertical direction under the conditions of the experiment, Loeb concluded that it plays an important rôle in determining the kind of regeneration that takes place. Its action is of such a nature that a new stem develops from the upper cut-end, and roots from the lower end, regardless of whether the upper end is the basal or the apical end of the piece. Similar results are also obtained, according to Loeb, if the pieces are suspended obliquely. In a piece of this sort, it is found that new stems arise along the upper surface of the old stem, and roots from the lower surface as well as from the lower cut-end (Fig. 14, D, E). If a piece of the stem is placed horizontally on the bottom of an aquarium, the branches that come off from the under surface of the stem begin to grow downwards at their ends, and where they come in contact with a solid body they fasten themselves to it, thus showing that they are true roots (Fig. 14, F). One or more stems may arise from the upper side of the main stem. These stems grow vertically upwards, and produce lateral branches. Only in one case did a new stem, or stem-like structure, arise from one of the vertical branches, as shown to the left in Fig. 14, F.

Loeb found it also possible to change the character of the growth of the apex of the normal stem and to transform it into a root. A long piece of the hydroid was cut off and suspended vertically with the basal end upwards. From the upper end a new stem began to grow, and then the entire piece was reversed, so that the new stem pointed downwards. Under these circumstances the young stem did not bend around and begin to grow upwards, as a young plant might have done, but it ceased to grow as a stem, and at its apex one or more roots developed. Loeb concludes: “I cannot imagine by what means the place of the formation of organs in antennularia is determined in connection with the orientation of the animal except by means of gravity.”

The response of antennularia to the action of gravity is, I think, conclusively demonstrated by Loeb’s results, but that the phenomenon may be complicated by other factors is shown, I think, by the following experiments. Driesch found that if pieces of antennularia are cut off and placed between horizontal plates, so that both ends are free, roots are produced by the basal end.[23] If the basal end with its new roots is cut off, new roots may appear, but sometimes a thin stem also. If the end is again cut off, a larger stem, and also one or two roots, may appear, and if the operation is repeated again only a stem is formed. The factor that brings about this change is not shown by the experiment. The piece had been kept in a horizontal position throughout the whole time. The apical end died in most cases without producing roots, but it is not stated whether or not roots appear on the stem between the plates of glass. If they develop they may affect the result, as certain experiments that I have made seem to show.

In my experiments, made at a different time of year from that at which Loeb’s experiments were made, pieces of the stem were suspended vertically,—some with the apical end upwards, others with the basal end upwards. In nearly all cases roots were formed by both the upper and lower ends. In a few cases, in which the apical end was upwards, a new stem developed at that end. Pieces suspended in a horizontal position also produced roots at both ends. After removing the ends with their new roots from the pieces suspended vertically, I found that roots again appeared at both ends in nearly every case. The difference between these results and those of Loeb may be due to the time of the year at which the experiments were made, or possibly to some other difference, but the results show that the response to gravity is not always so constant as Loeb’s results indicate.

In a few cases in my experiments the basal end of the hydroid was left attached to the stem on which it had grown, and the piece was put into the same aquarium used for the preceding experiments. In those pieces that lay on the bottom of the aquarium, with the stem standing vertically, a new shoot, and not new roots, appeared on the upper end. Other pieces were hung at the top of the water of the aquarium with the stem turned downwards, and the basal, attached end of the piece upwards. These pieces produced neither a stem nor roots from the apical end. The results show that the presence of roots at one end has an influence on the regeneration at the other end. The same thing was shown in one case in which a short piece sank to the bottom of the dish and, developing roots at its basal end, became fixed: a stem grew out of the apical end.

A number of other experiments that I made, in which pieces of antennularia were fixed to a rotating wheel, gave negative results, since neither roots nor stems appeared on the pieces. The rubbing of the ends of the piece against the water as the wheel turned round, or else the agitation of the water, prevented, most probably, the regeneration from taking place.

How gravity acts on antennularia has not as yet been determined. The only suggestion that we can offer at present is that it brings about a rearrangement of the lighter and heavier parts of the tissues. A rearrangement of this sort has been demonstrated when the egg of the frog is inverted, and in consequence certain changes are brought about in the development that will be described in another chapter.

EFFECT OF CONTACT

The contact of a newly forming part with a solid body has been shown by Loeb in a few cases, at least, to be a factor in regeneration. If a piece is cut from the stem of the tubularian hydroid Tubularia mesembryanthemum, and the piece held so that its basal end comes in contact with a solid body, a root develops at that end. If a piece is held in a similar position, but with its apical end in contact with a solid body, a root does not develop from this end. Evidently the development of a root in this form is also connected with an internal factor; but that there is in reality a reaction in this case, and not simply the development of a root at the basal end, is shown by the following experiment: If a piece is cut from the stem and suspended so that both ends are surrounded by water—it makes no difference whether the piece is vertical or horizontal—a hydranth develops first on the apical end, and then another on the basal end (Fig. 15, B). When the apical end of a piece is stuck in the sand, leaving the basal end free, a hydranth develops on the latter, but not on the end in the sand.

In another hydroid, Margelis carolinensis, studied by Loeb, the effect of contact is more easily demonstrated. If a branch of margelis is put into a dish of water and is kept from all motion, the parts that come in contact with the dish produce roots that attach themselves. Even the apical end of the stem may grow out as a root, as shown in Fig. 15, A. Those parts of the branch that are not in contact with any solid object give rise to new hydranths. Another hydroid, Pennaria tiarella, also shows, according to Loeb, the same response to contact. In this connection it is interesting to find that a growing hydranth of pennaria, if brought in contact with a solid body, turns away from the region of contact and bends at right angles to the body which it touches. We find, once more, that a factor having an influence on the growth of the animal has also a similar influence on the regeneration.

Loeb has found that if pieces of the hydroid Campanularia are cut off and placed in a dish filled with sea water, all the hydranths that touch the bottom of the dish are absorbed and transformed into the substance of the stem. The cœnosarc may creep out of the stem wherever it comes in contact with the glass, and produce stolons that give rise to new polyps on their upper surfaces. Loeb shows that growth takes place at the end of the stolon that pushes out of the perisarc, and this growing region draws the rest of the cœnosarc after it. If a new hydranth appears along the old piece, the cœnosarc is drawn towards the hydranth.

EFFECT OF CHEMICAL CHANGES IN THE ENVIRONMENT

Temperature, light, gravity, and contact are the most familiar kinds of external physical agencies that have a direct influence upon the growth of organisms. Food, though coming from the outside, yet acts only after it has entered the body. Organisms that live in water may be affected by the quantity and the kinds of the salts contained in the water, and also by the dissolved gases. The only experiments that have been made to show the influence of this last class of agents on animals are those made by Loeb. He placed pieces of the stem of tubularia in sea water of different degrees of concentration. After eight days the pieces, that had meanwhile produced hydranths, were measured. It was found that the maximum growth in length takes place, not in normal sea water, but in a much diluted solution. Loeb interprets this result to mean that the cells of tubularia must have a certain amount of turgidity in order to grow, and this is possible so long as the concentration does not pass a certain limit. This limit is reached by the addition of 1.6 grams of sodium chloride to each 100 c.c. of sea water. With a decrease in the concentration, the cells become more turgid, the maximum point corresponding to the maximum amount of growth. Below this point the solution is supposed 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.[24]

GENERAL CONSIDERATIONS

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 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.

CHAPTER III

THE INTERNAL FACTORS OF REGENERATION IN ANIMALS

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

the anterior surface of a short posterior piece, correspond to a different part of the worm from that which would be expected to develop, if the polarity of the piece is taken into account. Another reversed head develops on the posterior cut-surface of the anterior piece, and another tail on the anterior end of the posterior piece. The polarity of the new part is in this case reversed, as compared with that of the piece from which it arises. In the earthworm there is a marked delay in the regeneration of these heteromorphic parts. Even in tubularia in which heteromorphosis takes place, there is usually a delay of twenty-four hours in the formation of the reversed head. In Planaria lugubris, in which a reversed head develops, if a piece is cut from the anterior end just behind the eyes, the delay in the formation of the reversed head is very slight, if indeed there is any delay at all.

In the earthworm and in the planarian the production of reversed structures appears to be connected with the part of the body through which the cut is made, and to be due to internal factors. The question arises whether the presence of certain organs at the exposed surface can account for the result. It is conceivable that if such organs are present, and produce new cells that go into the new part, the presence of such cells may be the factor that determines what the new part will become; and in consequence the polarity of the part may be reversed. For example, the presence of the cut-end of the œsophagus or of the pharynx at the posterior surface of the anterior piece of the earthworm may determine that a new pharynx develops at the cut-end, and this may in turn act on the rest of the new tissues in such a way that a head rather than a tail is formed. When a posterior piece is cut off, the presence of the stomach-intestine at the cut-end may influence the new part, so that a tail is produced. It can be shown, however, that a new head may arise at the anterior end of a piece that contains only the stomach-intestine, as sometimes occurs when the worm is cut in two anterior to the middle; and it is not improbable that a tail can be produced from the posterior end of a piece that contains the old œsophagus, and perhaps even the old pharynx. In the planarian I have especially examined this point, but I have not yet found that the result can be referred to the cut-surface passing through any particular organ, or to the absence of any organs at the cut-end.

If, instead of referring the result to any one organ, we assume that the tissues near the cut-ends are specialized in such a way that they can only produce their like, and that the sum total of tissues of this sort making up the new part determines the result, we can only suggest that this may be so, but we cannot show at present that it is so, or that the result could be brought about in this way.

We might make an appeal to the hypothesis of formative stuffs, and assume that there are certain substances present in the head, and others in the tail, of such a sort that they determine the kind of differentiation of the new part; but this view meets also with serious objections. In the first place, it gives only the appearance of an explanation because it assumes both that such stuffs are present, and that they can produce the kind of result that is to be explained. Until such substances have been found and until it can be shown that this kind of action is possible, the stuff-hypothesis adds nothing to the facts themselves, and may withdraw attention from the real solution of the problem.

Bonnet, who first proposed the hypothesis of specific stuffs, went further and assumed also that they move in definite directions in the body, the head-stuff flowing forward and the tail-stuff flowing backward. It was necessary to assume definite movements of the stuffs in order to account for the development of the head at the anterior end of a piece and of a tail at the posterior end. In cases of heteromorphosis of the sort described above, these stuffs, if they brought about the results, would have to move in opposite directions from those assumed in the hypothesis; or else that part of the hypothesis that postulates the movement of the substances must be dropped, and in its place there must be substituted the idea of the excessive amount of such substances in the ends accounting for the heteromorphosis. An hypothesis that must be changed in this fundamental way to explain both classes of facts cannot be given very serious consideration. Of these possible ways in which it has been attempted to account for the phenomenon of heteromorphosis, the first one suggested seems to me simpler and more probable, but which organs are to be made responsible for the result cannot at present be stated. The fact that both Bardeen and I have obtained heteromorphosis in planarians in other regions than in the head indicates at least that other factors than the presence of head tissues or of head substances may bring about the development, and if it can be discovered what produces the result in regions remote from the head we may be in a position to explain the result in the head region in the same way, although it may be, of course, that the same result may be brought about by different factors, when the internal conditions are somewhat different.

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.

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.

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

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.

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.

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.

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.