Regeneration

Not only does regeneration take place in an antero-posterior direction, but in many animals also at the side. The regeneration of the limb of the salamander is, of course, a case of lateral regeneration in relation to the animal as a whole, but in a longitudinal direction in regard to the limb itself. Lateral regeneration of the limb would take place if the limb was split lengthwise into two parts and one of the parts removed. If the entire salamander were cut in two lengthwise, each half would most certainly die without regeneration, if for no other reason than that the integrity of the median organs is necessary for the life of the different parts. If, however, a planarian is cut lengthwise into a right and left half, each piece will complete itself laterally and make a new worm (Fig. 13^, A—D). Even a narrow piece cut from the side will produce a new worm by regenerating laterally, as shown in Fig. 19, a, b, c. In hydra, also, a half-longitudinal piece produces a new animal, but in this case not by the addition of new material at the side, but by the cut-edges meeting to make a tube of smaller diameter. Subsequently the piece changes its form into that characteristic of hydra.

In most of the preceding examples the behavior of the larger piece of the two that result from the operation has been described ; but there are some important facts in connection with the regeneration of the smaller end-pieces. The leg, or the tail, that has been cut from the salamander soon dies without regenerating. The life of the leg can be maintained only when the part is supplied with certain substances from the body of the animal. It does not follow, of course, that, could the leg or the tail be kept alive, they would regenerate a salamander. In fact, there is evidence to show, in the tail at least, that, although it may regenerate a structure at its anterior end, the structure is not a salamander, but something else. This has been definitely shown in certain experiments with the tail of the tadpole. It is possible to graft the tail of one tadpole in a reversed position, i.e. with its anterior end free, on the tail of another tadpole (Fig. 54, A-D\ or even on other parts of the body. Regeneration takes place from the free end, i.e. from the proximal end of the grafted tail. The new structure resembles a tail, and not a tadpole. If it be objected that the experiment is not conclusive because of the presence of the old tail, or of the use of the newly developing part, the objection can be met by another experiment. If, as shown in Fig. 56, A, a triangular piece is cut out of the base of the tail of a young tadpole, the cut being made so deep that the nerve-cord and notochord are cut in two, there develops from the proximal end of the tail a new tail-like structure that is turned forward, or sometimes laterally. In this case the objections to the former experiment do not apply, and the same sort of a structure, namely, a tail, is produced.

In the earthworm also we find some interesting facts connected with the regeneration of the terminal pieces. If one, two, three, four, or five segments are cut from the anterior end, they will die without regenerating. Pieces that contain more segments, six to ten, for example, may remain alive for a month or longer, but do not regenerate (Fig. 3, A, B^). That this lack of power to regenerate at the posterior end is not due to the smallness of the piece can be shown by removing from a piece of five segments one or two of its anterior segments. These will be promptly regenerated. Another experiment

FIG. 5. — Hydra viridis. A. Normal hydra. Lines indicate where piece was cut out. B, 1-4. Changes in a piece of A, as seen from the side. C, 1-4. Same as seen from the end. D, E, F. Later stages of same piece, drawn to same scale. has shown, however, that if these small pieces can be kept alive for a long time, and also supplied with nourishment, regeneration will take place at the posterior end. If, for instance, a small piece of eight or ten segments has its anterior three or four segments cut off, and is grafted by its anterior end to the anterior end of another worm, as shown in Fig. 3, F, the piece will begin, after several months, to regenerate at its exposed posterior end, but in the one instance in which this experiment has been successfully carried out, a new head, and not a tail, appeared on the exposed free end. The result ic not due to the grafting, or to the anterior position of the posterior end, but to

some peculiarity in the piece itself. We find the converse of this result in an experiment with the tail region of the earthworm, where the outcome is more clearly seen to be connected with the nature of the piece itself. If a piece less than half the length of the worm is cut off from the posterior end, there is generally formed from its anterior cut-surface, not a head, but another tail (Fig. 2, /). The result is similar to that described by Bonnet for one of the fresh-water annelids. A parallel case to that of the head of the earthworm is found in one of the planarians. If the head of Planaria lugubris is cut off just behind the eyes (Fig. 4, F), there is produced, at the posterior cut-edge of the head, a new head turned in the opposite direction, as shown in Fig. 4, F1.

In the regeneration of some of the lower animals, the transformation of a piece into a new animal of smaller size is brought about by a change in form of the piece itself, rather than through the production of new material at the cut-ends. If a ring is cut from the body of hydra, as shown in Fig. 5, A, the open ends of the ring are soon closed by the contraction of the sides of the piece, and in the course of a few hours the ring has become a hollow sphere ; or, if the piece is longer, a closed cylinder. After a day or two, the piece begins to elongate, and four tentacles appear near one end (Fig. 5, B, C, D}. The piece continues to elongate until it forms a small polyp, having the typical proportions of length to breadth (Fig. 5, E, F). It has cjianged into a new cylinder that is longer than the piece cut off, but correspondingly narrower. In this case there cannot be said to be a replacement of the missing parts, but rather, through the transformation of the old piece, the formation of a new whole. In planarians also the formation of a new worm from a piece involves a change in the form of the old part, as well as the addition of new material at the cut-end. If a crosspiece is cut out, as shown in Fig. 4, D, new material appears at the ends, but the old piece also becomes narrower and longer (Fig. 4, Dl-D*\ If the old hi it produces new material at its posterior end (Fig. 4, E, E1), and also becomes smaller

stripe injured at two points (see circles in A). .#. Regeneration of same piece. as the new part grows larger (Fig. 4, E2, Es). In a land planarian, Bipalium kewense, a piece is transformed into a new worm, as shown in Fig. 6, A, B. In this case the old pigment stripes of the piece are carried directly over into the new worm, the piece elongating during the transformation. A similar change takes place in pieces of unicellular animals, as best shown by cutting off pieces of stentor. If Stentor cceruleus is

FlG. 7. — Stentor caeruleus. A, Normal, fully expanded individual. A1. Same contracted. Line a-a indicates where it was cut in two. B, C. Pieces after division. B1, B1, B%. Re- generation of three distal pieces (B) containing old peristome. C1, C2. Regeneration of two proximal or foot pieces (Q. cut in two pieces, as indicated in Fig. 7, each piece makes a new individual of half size, but of proportionate form. The old peristome remains on the anterior piece, but becomes reduced in size as the piece changes its shape, and although it may be at first too large for the length of the new piece, it ultimately reaches a size about proportionate to the rest of the animal. The posterior piece is at first too long

for the size of the new peristome that is formed, but the latter becomes larger, until the characteristic form has been reached. The change in form of the stentor may take place in a few hours, and the result FIG. 8. — After Gruber. Stentor cceruleus. A. Cut into three pieces. /?. This row shows regeneration of anterior piece. C. This row shows regeneration of middle piece. D. This row shows regeneration of posterior piece. is brought about, not by the development of new protoplasm over the cut-end, but by a change of the old protoplasm into the new form. A similar experiment is shown in Fig. 8, in which a stentor was cut into three pieces, each piece containing a part of the old nucleus.

In the higher plants the production of a new plant from a piece takes place in a different way from that by which in animals a new individual is formed. The piece does not complete itself at the cutends, nor does it change its form into that of a new plant, but the leaf-buds that are present on the piece begin to develop, especially those near the distal end of the piece, as shown in Fig. 32, A, and roots appear near the basal end of the piece. The changes that take place in the piece are different from those taking place in animals, but as the principal difference is the development of the new part near the end, rather than over the end, and as in some cases the

new part may even appear in new tissue that covers the end, and, further, since the process seems to include many factors that appear also in animals, we are justified, I think, in including this process in plants under the general term regeneration. FIG. 9. — After Vochting. A, A1, A2. Pieces of thallus of Lvnularia communis regenerating at the apical end. D. Piece of thallus cut in two in the middle line. Z?i. Same split at side of middle. C. An oblique piece extending to middle line. C1, C2. Oblique pieces not extending to middle line. D. Fruiting stalk stuck into sand, producing new thallus above sand. Z)l. Same laid horizontally regenerating near base. E. Same with fruiting head cut off. Regenerating at base. £i. Twisted piece regenerating at two points. F. Piece of ray of head regenerating near base. />'*. Same with distal end of ray cut off. Also regenerating at base.

In the lower plants, such as the mosses, the liverworts, the moulds, and the unicellular forms, regeneration also takes place. Vochting has shown that pieces from any part of the thallus of a liverwort 1 produce new plants. If a cross-piece is cut off, there appears a small outgrowth from the middle of the anterior cut-edge, as shown in Fig. 9, A, A2, that gradually enlarges to form a new thallus. It will be seen from the figures that the whole anterior edge does not grow forward, but a new thallus arises from a group of cells at, or near, the anterior edge. These cells are the least-differentiated cells in the piece, and have softer cell walls than have the other cells.

FIG. io. — After Pringsheim. A. A piece of seta of sporophore of Hypnum cupressiforme, sending out protonema-threads. B. Longitudinal section of a piece of the seta of sporophore of Bryum catspitosum. C. Piece of same of Hypnum cupressiforme. Moss-plant arising from new protonema. D. Piece of same of Hypnum serpens with protonema and moss-plant arising from it. Pringsheim has shown that if a piece of the stalk of the sporangium of certain mosses is cut off, it produces at its ends thread-like outgrowths which are like the protonema-stage of the moss, and from this protonema new moss-plants may arise (Fig. io, A, B, C, D).

Braefeld has obtained a somewhat similar result in one of the moulds, in which a piece of the sporangium stalk gives rise to a mycelium from which new sporangia may be produced. Regeneration takes place not only in adult organisms, but also in embryos, and larvae of many animals. It is often stated that the power of regeneration is more highly developed in embryos than in adults, but the facts that can be advanced in support of this view are not numerous. One of the few cases of this sort known to us is that of the leg of the frog, that does not regenerate, while the leg of the tadpole is capable of regenerating.

FIG. ii. — A. Blastula of Sea-urchin. Dotted lines indicate where pieces of wall were cut off. To the right are shown stages in the development of these pieces. B. Two-cell stage of egg of sea-urchin. One blastomere isolated. Its development shown in figures to right of B. C. Fertilized but unsegmented egg. Dotted line indicates where it was cut in two. Upper row of figures to right shows development of nucleated piece ; lower row shows the fertilization and development of non-nucleated piece.

The early stages in the development of the sea-urchin, or of the starfish, may be taken to illustrate the power of regeneration in embryos. If the hollow blastula of the sea-urchin is cut into pieces (Fig. n, A), each piece, if not too small, may produce a new blastula. The edges of the piece come together, and fuse in the same way in which a piece of hydra closes. A new hollow sphere of small size is formed, which then passes through the later stages of development as does the whole normal blastula.

Still earlier stages of the sea-urchin, or of the starfish, have the power of producing embryos if they are cut into pieces. If the segmenting egg is separated into a few parts, each part will continue to develop. Even the first two blastomeres or cells will, if separated, produce each a whole embryo (Fig. 11, B). The power of development of a part does not even end here, for, if the undivided, fertilized egg is cut into pieces, the part that contains the nucleus will segment and produce a whole embryo (Fig. 11, C, upper row). If the egg is cut in two or more pieces before fertilization, and then each part is fertilized, it has been found that not only the nucleated, but even the non-nucleated fragments (if they are entered by a single spermatozoon) may produce embryos (Fig. 11, C, lower row).

It may be questioned whether the development of parts of the embryo, or of the egg, into a whole organism can be included in the category of regenerative processes. There are, it is true, certain differences between these cases and those of adult forms, but as there are many similarities in the two cases, and as the same factors appear in both, we cannot refuse, I think, to consider all the results from a common point of view. Finally, there are certain normal changes that occur in animals and plants that are not the result of injury to the organism, and these have many points in common with the processes of regeneration. They are generally spoken of as processes of physiological regeneration. The annual moulting of the feathers of birds, the periodic loss and growth of the horns of stags, the breaking down of cells in different parts of the body after they have been active for a time, and their replacement by new cells, the loss of the peristome in the protozoon, stentor, and its renewal by a new peristome, are examples of physiological regeneration. This group of phenomena must also be included under the term " regeneration*" since it is not sharply separated from that including those cases of regeneration after injury, or loss of a part, and both processes appear to involve the same factors.

The older writers used such terms as " replacement of lost parts," " renewal of organs," and " regeneration " to designate processes similar to those described in the preceding pages. The term regeneration has been for a long time in general use to include all such phenomena as those referred to, but amongst recent writers there is some diversity of opinion as to how much is to be included in the term, and the question has arisen as to the advantage of applying new names to the different kinds of regeneration. There can be little doubt of the advantage, for the sake of greater clearness, of the use of different terms to designate different phenomena, but I think that there is at the same time the need of some general term to cover the whole field, and the word regeneration, that is already in general use, seems to fulfil this purpose better than any other.

Roux l points out that Trembley, and later Nussbaum, showed that a piece of hydra regenerates without the formation of new material. Roux adds that since during development the piece takes no nourishment, the regeneration must be brought about by the rearrangement of the cells present in the piece.2 The change may, or may not, involve an increase in the number of the cells through a process of division. In consequence of this method of development a re-differentiation of the cells that have been already differentiated takes place. This process of regeneration, Roux points out, is very similar to the " post-generation " of the piece of the blastula of the sea-urchin embryo, and he concludes that " regeneration may be brought about entirely, or very largely, through the rearrangement and re-differentiation of cells without any, or with very little, proliferation taking place." In the adults of higher animals regeneration by proliferation preponderates, but rearrangement and re-differentiation of cells occur in all processes of regeneration, even in higher vertebrates. The two kinds of regeneration that Roux distinguishes are, he says, essentially quantitative.3

2 The fact that the piece does, or does not, take in food has no bearing on the question, since many animals that do not feed while the regeneration is going on produce new cells to form the new part. 3 These two kinds of regeneration are post-generation and regeneration proper. The distinction that Roux attempts to make between these two processes is to a certain extent artificial and rests at present on a very unsafe basis, at least in so far as the post-generation of the frog's embryo is taken as a representative case of this process. Roux states that in the process of regeneration the injured tissues produce each their like in the new part, while in the process of post-generation of the frog's egg the new cell-material arises in part from the nuclei and yolk-material of the injured half and in part through the accidental position of the nuclear material of the uninjured half. In order more fully to understand this distinction the original description of the process of post-generation given by Roux in his account of the development of half embryos of the frog's egg must be referred to. In later papers Roux pointed out that the missing half of the frog embryo, as well as of other forms, may be post-generated without any new material appearing at the open side of the embryo. It is unfortunate, I think, that the original term should have been extended to include these other processes that do not partake of the nature of post-generation as at first denned, but are more like the true process of regeneration as described by Roux.

Barf urth1 has defined regeneration as " the replacement of an organized whole from a part of the same." If the part is given by nature, there is a process of physiological regeneration ; if the part is the result of an artificial injury, the process is one of pathological regeneration. Barfurth includes in the latter category the production of a new, entire individual from a piece, as in hydra ; regeneration by proliferation, as in the earthworm; and also the development of pieces of an egg or of an embryo.

Barfurth's definition of regeneration is unsatisfactory, since an egg is itself a portion of an organism that makes a new whole, and this sort of development is not, of course, as he himself points out, to be included in the term regeneration. Nor does the use of the word " replacement " save the definition, since in many cases the kind of part that is lost is not replaced. The use of the word "pathological" to distinguish ordinary regeneration from physiological regeneration is, I think, also unfortunate, since it implies too much. There is nothing necessarily pathological in the process, especially in such cases as hydra, or as in the development of a piece of an egg where the piece is transformed directly into a new organism. Furthermore, in those cases in which (as in some annelids and planarians) a new head is formed after or during the process of natural division, there is little that suggests a pathological process ; and in this instance the regeneration takes place in the same way as after artificial section.

Driesch, in his AnalytiscJic Theorie, states that Fraisse and Barfurth have established that during regeneration each organ produces only its like. Driesch defines regeneration, therefore, as the re-awakening of those factors that once more bring into play, by means of division and growth, the elementary processes that had ceased to act when the embryonic development was finished. This is regeneration in the restricted sense, but Driesch also points out that this definition must be enlarged, since, when a triton, for example, regenerates its leg, not only does each tissue produce its like, but later a reconstruction and differentiation takes place, so that a leg and foot are formed, and not simply a stump containing all of the typical tissues. Driesch holds that regeneration should include only those cases in which a proliferation of new tissue precedes the development of the new part, and suggests that other terms be used for such cases as those of pieces of hydra, pieces of the egg, etc., in which the change takes place in the old part without proliferation of new tissue. It seems to me unwise to narrow the scope of the word regeneration as Driesch proposes, for it has neither historical usage in its favor, nor can we make any fundamental distinction between cases in which proliferation takes place and those in which it does not. As will be shown later,

the factors that are present in the two cases appear to be in large part the same, and while it may be convenient to put into one class those cases in which proliferation precedes the formation of the new organs, and into another class those cases in which the change takes place without proliferation, yet, since the distinction is one of subordinate value, it is necessary to have one word to include both groups of cases ; and no better word than regeneration has, I think, been as yet suggested.

Driesch has made use of two other descriptive terms. The word " reparation " is used to describe the development of the hydranth of tubularia. The new hydranth is formed in this case out of the old tissue at the end of the piece (Fig. 20, A). The change appears to be the same as that which takes place in a piece of hydra, etc. The word "reparation" does not seem to me to express very satisfactorily this sort of change, or sharply separate it from those cases in which the animal is repaired by adding what has been taken away ; but in this latter sense Driesch does not use the term. I have not made use of the word, in general, except as applied to Driesch's work.

Another term, "regulation," used by Roux^and also by Driesch and others, is used in a sort of physiological sense to express the readjustments that take place, by means of which the typical form is realized or maintained. By inference we may extend the use of the word to include the changes that take place in the new material, that is proliferated in forms that regenerate by this method. Driesch uses this term, regulation, to include a much more general class of phenomena than those included in the term regeneration, as for instance, the regulation of metabolism and of adaptation, etc. One of the subdivisions of the term regulation is called "restitution." This word also is used where I should prefer to use the word regeneration as a general term, and the word reorganization when reference is made to the internal changes that lead to the production of a typical form.

Both Roux and Driesch also speak of " self-regulation," by which is meant, I suppose, that the changes taking place are due to readjustments in the part itself, and are not induced by outside factors. The expression " self-regulation " is not, I think, a very happy one, since all change is ultimately dependent upon a relation between inside and outside conditions. Hertwig2 defines regeneration as the power of replacement of a part of the organism. He states that in all cases the beginning of the process is the same, viz. the appearance of a small protuberance composed of cells, that is the rudiment of the new part. It is evident

1 As used in connection with other terms, see his Ges. Abhandl., Vol. II, page 41. that Hertwig has taken into account only one side of the process. Those cases in which a rearrangement or reorganization takes place in the old part are not even considered.1 Goebel2 points out that in plants the fully formed cells are, as a rule, incapable of further growth after they have once served as a basis of an organ of the body, but often some of the cells may remain in a latent condition, and grow again, when the intercellular interactions are disturbed. This is the case, he thinks, in regeneration. Goebel speaks of regeneration by means of adventitious buds in those cases in which the buds had not previously existed before the removal of the part. In those cases in which the buds are in existence before the piece is removed, as in the leaves of Asplenium, Begonia, etc., the development is not the result of regeneration, Goebel thinks, but the buds represent a stage in the development of the species. It may be pointed out, however, that it is certainly a remarkable fact that often the conditions that lead to the unfolding of an existing bud are the same as those that lead to the development of a new bud.

The preceding account will suffice to illustrate some of the principal ideas that are held in regard to the process of regeneration. Since many new facts have come to light in the last few years, it may not be amiss to point out what terms will be used in the following pages to include each kind of process. The word " regeneration " has come to mean, in general usage, not only the replacement of a lost part, but also the development of a new, whole organism, or even a part of an organism, from a piece of an adult, or of an embryo, or of an egg. We must include also those cases in which the part replaced is less than the part removed, or even different in kind.