Regeneration

that regenerates is composed in part of material derived from one species and in part from that of the other, but each tissue remains true to its kind, and there is found no evidence of an influence of one on the other (Fig. 55, E). These experiments show that even when the two kinds of tissue regenerate side by side, and unite to form a single morphological organ, there is no influence of a specific kind of one tissue on the other. FlG. 55. — A. Rana sylvatica with grafted tail of Rana palustris. Line a-a indicates where tail was cut off. B. Rana palustris with grafted tail of Rana sylvatica. Line a-a indicates where tail was cut off. C. Older stage of a graft like B, Lines indicating two possible operations. D. Another individual with two tails, one composed of both components. E. Later stage of last, when tail was cut off at level a-a.

Another series of experiments in grafting, similar to one of those made by Joest and myself on the earthworm, has been made by Harrison on the tadpole. I have also later made similar experiments. Two tadpoles are united by their posterior ends, as shown in Fig. 54, A, and a day or two after union one of the tails is cut off near the line of union. There is thus left attached to the end of the tail of one tadpole a part of the tail of the other united in a reverse direction, so that the exposed cut-end is the anterior end of the small piece. There grows out from this cut-end a structure that resembles a tail (Fig. 54, B, C, D). It contains a continuation of the notochord and nerve-cord, that taper in a characteristic way to the end of the new structure. The tail is flat and has a central band of muscle tissue, and

a dorsal and ventral fin. The muscles of the normal tail have a characteristic V-shaped arrangement with the apex of the V's turned forward, but unfortunately in the new tail the muscles are so irregular that it is impossible to make out their arrangement (Fig. 54, D}. If the new part is in reality a tail, the V's ought to stand in the same way as do those in the major component, and opposed to the V's on the part from which the new material arises. If the new structure is not a tail at all, but a new growth, or even a suppressed trunk, then the V's should stand as in the small part itself. It has not been possible as yet to obtain a decisive case. Harrison obtained one case in which the arrangement of the muscles in the new part seemed to be more as it should appear if the new part is a heteromorphic tail (Fig. 54, D\ Even if this could be shown to be the case, it may be that under the conditions of the experiment the arrangement of the muscles is determined by the use of the tail, although this does not seem very probable. Harrison, after a careful analysis of the question, left it undecided, but seemed more inclined to the view that the result is due to the development of something new rather than a heteromorphic growth. On the contrary I am strongly inclined to believe that the latter is the true explanation. In another way I have been able to bring about the development of the same structure. A small triangular piece is cut from the upper part of the tail, as indicated in Fig. 56, A, one point of the triangle passing through the notochord, or even through the aorta. If the cut-surfaces are kept apart for a few hours, until the exposed end has been covered over by ectoderm, they may not unite afterward, and two exposed surfaces are left, — one at the distal end of the base of the tail, and the other at the proximal end of the outer part of the tail.

The latter surface corresponds to that in the grafting-experiment. Regeneration may take place from the two surfaces ; both new parts seem to be exactly alike, and both resemble a regenerated tail. The one from the proximal surface of the outer part of the tail contains a notochord, nerve-cord, connective tissue, pigment cells, and muscle tissue (Fig. 56, B\ The arrangement of the muscle fibres is generally very irregular, and the characteristic V-shaped arrangement cannot be detected.

In only a few cases have attempts been made to unite two eggs or two very early embryos, although there are a few casual observations * in which such a fusion has been observed. The problems that arise in connection with the union of two eggs are full of interest. Each egg has the power of producing an embryo of normal size. If two eggs are united into one, will a single giant organism result, or two organisms ? If the former, we must suppose that a new organization is formed of double size. Whether an upper limit of organiza- i Metschnikoff ('86), Herbst ('92).

tion exists can only be determined by such an experiment. If two fused organisms result from the fusion of two eggs, it would show the structure of the egg is of such a kind that two organizations cannot readjust themselves into a single one of double size. Moreover, it is important to discover whether any difference exists as to the stage of development at which the union is brought about, for it is conceivable that while a rearrangement is possible at one stage, it might not be at another.

FIG. 56. — A. Tadpole to show where the V-shaped piece is cut from the tail. B. Later stage of same with a new tail-like outgrowth from the anterior end of tail. It has been shown that two blastulae of the sea-urchin can be united to form a single embryo. I found ('95) that occasionally two blastulae stick together and fuse, so that a single sphere of double size is formed. As a rule two gastrulae and two more or less complete embryos develop from each double blastula, but in a few cases I found that a single embryo may be formed, that shows, however, traces of its double origin. Driesch has more recently (1900) succeeded l in bringing about more readily a union of two segmenting

1 Eggs without membranes were placed in sea water without calcium, to which a few drops of sodium hydroxide have been added. eggs or blastulae, and obtained perfect single individuals from two fused blastulae. He finds that if the fusion takes place at an early stage the resulting embryo is less likely to show its double origin than when older blastula stages are united. Zur Strassen has also observed giant embryos of ascaris that arise by a fusion of two eggs. Loeb has found that the eggs of chaetopterus, which can be made to develop parthenogenetically in certain salt solutions, often stick together and produce giant embryos.

THERE are many difficulties in the way of determining the origin of the cells that make up the new part. The only means at present at our command for studying their source is by serial sections of a number of different stages taken at intervals from different animals. Since there may be differences between the processes in different individuals, and since we can only piece together the information gained from successive stages, much uncertainty exists in regard to the changes that take place during regeneration, even in some of those forms that have been examined over and over again. Were it possible actually to follow out the movements of the living cells in one and the same animal, the problem would offer fewer difficulties, but this cannot be done. It will be more profitable to consider first the better-known and simpler processes, and afterward those that are less well-known.

The regeneration of the head and tail of lumbriculus and of certain naids is a comparatively simple process, and has been studied by several investigators, whose results agree, at least in regard to the most essential features. Semper ('76) described the origin of the new organs in the formation of new individuals by budding in nais. He found that the new brain and nerve-cord develop from the ectoderm, the newmesoderm also from ectoderm, and the new digestive tract from the old one, except the pharynx, which arises by the fusion of two mesodermal " gill-slits." Bulow ('83) studied the regeneration of the tail of lumbriculus. He found the ventral cord in the new part arising from a paired ectodermal thickening, the mesoderm arising from a proliferation of cells. These cells are invaginated in the region between ectoderm and endoderm — the in-turning of the proctodaeum being looked upon as an endodermal invagination.1 The more recent work of Randolph, Rievel, Michel, Hasse, Hepke, and von Wagner on the same or related forms has served to point out certain errors in the earlier work of Semper and Billow, and has added some new and important facts, especially in connection with the origin of the mesoderm in the new part. Without attempting to give a detailed account of these results,

1 The usual interpretation at present is to regard the proctodseal ingrowth as ectodermal. I shall describe the principal changes that have been found to take place. When the anterior end of lumbriculus or of tubifex is cut off, the cut-surface very quickly closes, as a result of the contraction of the body wall. According to some investigators, the circular muscles are chiefly concerned in the closing, but according to others the longitudinal muscles bring about the result. The cut-end of the diges-

FlG. 57. — After Hasse. Regeneration of head of Tubifex rivulorvm. A. Sagittal section of anterior end. Six days after cutting in two. B. Eleven days after cutting in two. C. Crosssection through new part. Five days after operation. D. Fourteen days after operation. E. Sixteen days after operation. tive tract is pulled a little inward, and its end also closes (Fig. 57, A). For a day or two no important changes can be observed to take place, but new ectoderm soon appears over the cut-surface. This ectoderm arises in all cases from the old ectoderm, and as it increases in amount the old ectoderm is pushed back from over the cut-end, leaving a layer composed of a single row of cells over the end. Since nuclei in process of division are rarely present before these initial

processes begin, it is probable that the changes are due, in large part, to an out-wandering of ectodermal cells, or, what amounts to the same thing, to the leaving behind of cells as the old ectoderm withdraws from the cut-end. In the new ectoderm over the end, an active process of proliferation takes place (Fig. 57, B), that leads to the production of a large number of cells lying within the new part. The ectoderm has at this time begun to bulge outward, so that the proliferated cells come to lie within the dome-shaped beginning of the new head. There appears to be some difference in the number and in the location of the proliferations in different species. In general, the new cells arise from the ventral and ventro-anterior region of the dome-shaped ectodermal covering of the new part. Most of this new material gives rise to the brain, commissures, and ventral nerve-cord (Fig. 57, Q. The cells giving rise to these structures in tubifex come from two ventral regions of proliferation that extend along the sides and dorsally to the anterior end in front of the digestive tract. Where the two masses meet above and in front, the brain is formed.1 The cells that do not take part in the formation of the nervous system give rise to the muscles and connective tissue of the new head. These cells lie especially at the outer sides of the proliferated mass. The origin of the new muscles from ectoderm stands in sharp contrast to the current ideas in regard to the origin of new tissues, and yet it is a point on which the more recent investigators are entirely in accord. Michel, Hepke, and von Wagner have arrived at the same conclusion after a careful examination, and there seems to be no reason for refusing to accept their results. The theoretical importance of this discovery will be discussed later.

Soon after the proliferation from the ectoderm has begun, the blind end of the digestive tract starts to push forward (Fig. 57, Z>). The cells in the most anterior part of its wall begin to divide, and the end grows in an anterior direction as a more or less solid rod. This rod extends, in some species, as far forward as the ectoderm, meeting the latter on the inner side of its antero-ventral surface. At this point an in-turning of ectodermal cells, in the form of a blind pit, develops, and later this pit, deepening to become a tube, forms the mouth cavity. Its inner end is from the beginning in contact with the anterior end of the digestive tract, or else it connects with the latter soon after its formation. The two flatten against each other, the cells draw away in the middle of the region of contact, and the cavity of the new mouth becomes continuous with the cavity of the old digestive tract. The mouth lies at first nearly terminal in position (Fig. 57, E), but by the forward growth of the body wall over

1 In some species the two proliferating regions seem to be in contact above from the beginning (Hepke, in Nais~). and in front of the mouth to form the prostomium, the mouth comes later to lie more on the ventral surface. The short tube produced by the in-turned ectoderm forms only a short part of the digestive tract. It leads from the mouth opening to the new pharynx, and forms, therefore, only the buccal cavity. A similar ectodermal tube, the stomodaeum, which develops in the egg-embryo, becomes not only the buccal chamber, but also the lining of the pharynx. The latter is, therefore, considered an ectodermal structure in the embryo. On the other hand, in the regenerated head the lining of the new pharynx arises from the anterior part of the endodermal digestive tract. We find, therefore, that the same organ, the pharynx, may arise in the same animal from distinct "germ-layers." This result also has an important bearing on our ideas concerning the value and meaning of the so-called "germ-layers," and has helped to bring about a revolution of current opinion as to the importance of these layers.

The preceding account of the development of the head has shown that while certain of the new organs and layers arise from the same organs of the old part, yet this is not true for all of them. Thus while the ectoderm gives rise to ectoderm, the new muscles do not appear to come from the old ones, or even from other mesodermal tissues, but from the ectoderm. The old digestive tract gives rise to the greater part of the new one, but the new pharynx comes from the old endoderm, and not from the in-turned ectoderm. The nervous system does not arise from the old ventral cord, but from a proliferation of ectoderm. It has, thus, the same origin as the nervous system of the embryo. The origin of the new blood vessels has not been satisfactorily made out. The seta sacs arise from ectodermal pits as in the embryo.

In regard to the origin of the new mesoderm, the evidence is still insufficient, I think, to show that cells derived from the old muscles or peritoneum take no part in the formation of the new muscles and peritoneum ; but that the greater part of the new muscles, etc., comes from the proliferated cells can scarcely be doubted. This latter discovery loses none of its significance, however, even if it should prove true that the old muscles, etc., contribute something to the new part. It is also not entirely disproven that the ventral nerve-cord does not take a small share in the development of the new cord.

The regeneration of a new tail-end in these same forms appears to take place in much the same way as the head. The cut-end quickly closes ; later a layer of ectoderm appears over the posterior surface, and the new part bulges out and becomes dome-shaped. A paired, or in some species a single, region of proliferation develops irom the ectoderm, that gives rise to the new ventral nerve-cord. Lateral proliferations of ectoderm produce, according to some writers, the material out of which the mesoderm of the new tail is formed. Randolph, on the other hand, has described the new mesoderm as arising from the old, especially from certain large peritoneal cells that are found throughout the body. The cut-end of the digestive tract closes, and later new cells develop at its posterior end. An in-turning of ectoderm, in the form of a pit, fuses with the posterior .end of the digestive tract and establishes communication with the outside.

FIG. 58. — After Hescheler. Regeneration of anterior end of earthworm. A. After four days. B. After eleven days. C. After twenty-five days. D. After twenty-one days (younger individual). The regeneration of the anterior end of the earthworm has been carefully worked out by Hescheler, and although on account of the greater complexity of the process the results are not so decisive as those just described, yet in many respects they are in agreement. In Hescheler's experiments only four or five anterior segments were cut off. The closing of the cut-end is somewhat different from that in lumbriculus. A plug of cells soon forms over the end (Fig.

58, A). The new cells appear to be lymph cells. Although this mass of cells may be quite large, the cells do not seem to form later any of the organs in the new head. The presence of these cells makes it very difficult to work out the origin of the other cells that appear later. Owing to the absence of this lymph plug in lumbriculus and nais it is easier to follow in them the regenerative processes. In the midst of these lymph cells spindle-liker cells soon appear whose origin is obscure, but Hescheler thinks it improbable that they are transformed lymph cells, although they are completely intermixed with the latter. The spindle-cells arrange themselves later in regular bands, that appear to be extensions of the longitudinal muscles. A few days after the operation, the lymph plug is covered over, beginning at the edge, by the ectoderm. The new ectodermal cells arise from the old ectoderm, and seem to extend over the lymph plug by a sort of migration process. Division of the cells does not occur at this time. These covering cells are at first all alike, the characteristic gland cells of the ectoderm being absent. The digestive tract withdraws somewhat from the outer cut-surface, and its end closes. The closed end abuts against the inner surface of the lymph plug. The next changes are initiated by the appearance of karyokinetic divisions in all the tissues of the new part, which lead to a rapid growth and elongation. Dividing cells are found in the new, as well as at the border of the old, ectoderm, where the new and the old parts are continuous. At this stage there appears in the lymph plug another kind of cell, that seems to arise, in part at least, from the ectoderm by an in-wandering of new cells.

Other new cells may come from the edge of the old muscles, but it is not clear whether they come from a transformation of muscle cells, or from undifferentiated cells lying in the old muscles. In addition to these sources of new cells, it appears not improbable that cells may separate from the end of the digestive tract. Nerve fibres push out from the end of the ventral nerve-cord into the new part, and groups of cells, often in process of division, appear in the old ganglia, even in those that lie a long distance from the anterior end. It is not improbable, Hescheler thinks, that new cells, as well as fibres, grow forward from the most anterior end of the nerve-cord into the new part. A mass of nerve cells and fibres appears in front of the old nerve-cord, and extends upwards and around the digestive tract, to meet over the anterior end of the latter in another mass of cells that have arisen from an early in-wandering of ectodermal cells. It is not improbable that the masses around the digestive tract (the commissures) and also the new ventral cord may also include cells that have had the same origin.

A tubular invagination of ectoderm is formed at this time at the anterior end. It meets the anterior end of the digestive tract ; the two fuse, and the communication of the digestive tract with the outside is established. The pharynx develops from the anterior part of the digestive tract, which after Hescheler's operation may contain some of the original ectodermal stomodaeum, since only five of the anterior segments were cut off, and the embryonic stomodaeum extends somewhat behind this region. In another experiment, carried out by Kroeber, somewhat more of the anterior end was removed, but the result was the same (Fig. 59), so that it is clear that the new pharynx may be formed from the old endoderm.

Hescheler leaves several points still unsettled, more especially the origin of the FIG. 59. — After Kroeber. Regeneration of anterior cells that give rise to the new end of Allolobophora fastlda. after removal of six > , !_*.•*.• i segments. The first stomodoeal invagination mUSCUiature, but it IS almost had been destroyed. The new pharynx is develimpossible tO make OUt their oping from the endoderm. ... the presence of the lymph cells. Hescheler's discovery that the cells of the lymph plug do not themselves, in all probability, contribute to the new part, is an important result, and shows that these seemingly undifferentiated cells do not possess the power of giving rise to the different kinds of new tissues. The in-wandering of cells into this solid plug from the ectoderm, and perhaps also from other sources, and their subsequent union to produce the definitive organs, is also a point of capital importance, especially as it puts us on our guard against a too ready acceptation of the view that all cells in a mass that have the same general and undifferentiated appearance have had a similar origin, and in showing that apparently indifferent cells may really carry with them into the new part those characters that determine their fate. Other cells, apparently equally undifferentiated, and lying in the same position, may have quite different possibilities. In the vertebrates, the regeneration of the tail and limbs of amphibia and of the tail of lizards has been studied by a number of investigators. The regeneration of the tail of several urodeles and of the larva of the frog was investigated more fully by Fraisse

('95) and by Barfurth ('91). If we examine first the results of Fraisse's study of the tail of urodeles, which have bony vertebrae, we find the following changes take place. The cut-surface is covered by the skin bending over the exposed part, accompanied by a migration of cells from the edge of the ectoderm. Only the unspecialized cells leave the old ectoderm to wander out over the cut-surface ; gland cells and sense cells are entirely absent from the new ectoderm. These kinds of cells develop later out of the undifferentiated cells over the new part. The development of new vertebrae does not follow the embryonic method of development. In the embryo the endodermal notochord is first laid down, and around this and the nerve-cord mesodermal cells accumulate to form the skeletal tissue. Later the notochord is largely obliterated, as the vertebrae develop, pieces of it being left along the vertebral column. In the regeneration of the tail of the adult animal, the remnants of the old notochord (even if exposed by the cut) do not take any part in the formation of new tissue. In fact, there is no notochord formed at all. From the injured vertebrae, or at least from their covering of skeletal tissue, cells are proliferated, out of which a cartilaginous tube develops, enclosing the new nerve-cord, which is growing out from the cutend of the old cord. In this tube centres of deposition of calcareous material are formed, and the new vertebrae are produced in this way. The new nerve-cord develops from the cut-end of the old cord, and more especially out of the cells of the lining epithelium of the canalis centralis. The new muscles develop from cells that arise from the old muscles.

In the tadpole of the frog the regeneration of the tail takes place essentially in the way just described for the adult urodele, except that, there being only a notochord in the tail, only a notochord is regenerated. According to Fraisse, the new notochord develops from cells that arise from the sheath of the old notochord, and not from the vacuolated cells of the notochord itself. The notochord cells are, he states, derived from the endoderm of the embryo,1 while the sheath arises from the mesoderm ; hence the newly regenerated notochord that arises from the sheath of the old one comes from a different germ-layer. Exception may be taken to this statement, because in the frog's embryo the notochord develops from tissue that is at first perfectly continuous with the mesoderm, and, in fact, may be called mesoderm ; also because it is probable, in the light of more recent research, that both the notochord and its sheath have exactly the same origin.

1 This seems to be true for urodeles, but whether it is true for the anurans is rather a question of definition, as I have pointed out in my book on The Development of the frog's It is known that the tail of lizards breaks off generally at a definite region near the base, and that the break does not occur between the vertebrae, but in the middle of a vertebra — in some species the seventh caudal. The vertebras are thicker at their ends than in the middle, and are firmly held together by intervertebral cartilages. The centres of the caudal vertebras are the weakest links in the chain, or at least the place at which the vertebral column is most easily broken in response to the contraction of the tail-muscles.1 Fraisse and others speak of this arrangement as an adaptation for breaking off the tail.

The new tail that regenerates does not contain a new series of vertebrae, as does the new tail of the salamander, but, instead, a cartilaginous tube that is attached to the half of the broken seventh caudal vertebra. The regeneration of the new tissues of the tail of the lizard takes place as follows : A scab forms over the cut-surface, composed in part of clotted blood, in part of broken-down tissues from the injured cells. In the course of a week the necrotic tissue falls off, and a smooth surface of ectoderm is found covering the end of the tail. The new ectoderm appears to come from the old, but its method of development has not been studied. The deeper layer of the skin of the lizard is composed of mesodermal connective tissue, and in the new part this layer arises from the connective tissue of the old part. The tissue that forms the cartilaginous tube of the new tail develops from the skeletal tissue of the broken vertebra. The remnants of the old notochord, that are present in the vertebra, have nothing to do with the new structure, nor does the new tube represent in any way a notochord, but it appears to be a structure sui generis. In later stages, osseous plates may be formed in the cartilage, but these are too irregular to be compared to vertebrae. A tube grows out from the cut-end of the nerve-cord, which in some forms, as Fraisse shows, is only an extension of the lining epithelium of the nerve-cord. In other forms it is possible that other cells of the old cord may also grow backward, divide, and produce new cells. The fine thread that is formed in this way does not send out any nerve fibres into the surrounding parts. In Anguis fragilis, however, a few ganglion cells are present in the new cord. It is probable, Fraisse states, that while the new tube is morphologically a nerve-cord, yet physiologically it is not functional in any of the reptiles.

The new muscles come from the old ones. Fraisse thinks that the new muscle fibres come from the so-called " spindle fibres " that split off from the primitive muscle bundles. These fibres, Fraisse believes, originate normally during the process of physiological regeneration of 1 The attachments of the muscles may be the cause of the break in the middle of the vertebrae, rather than between two vertebrae. the muscles, and also after injury to the muscles. From these spindle cells the new muscle fibres develop in the same way as the muscle cells of the embryo.

Fraisse sums up the results of his studies of regeneration as follows: (i) Both in amphibians and reptiles, injured tissues can only produce new tissues like themselves. The leucocytes assume only the function of nutrition and of devouring the broken-down parts of tissues. They never become fixed tissues — neither connective tissue nor any other sort. (2) All tissues are capable of regenerating themselves, either directly out of their differentiated elements, or out of a matrix. As a matrix for the epidermis, there is the Malpighian layer of the skin; for the central nervous system, the epithelium of the central canal of the nerve-cord ; and for the musculature, the spindle fibres.

Fraisse also formulates the following general statements : (a) Re- generation is neither a pure recapitulation of the ontogeny nor of the phylogeny. The process is rather a hereditary one, with which complicated adaptations of the tissues are often involved that follow the laws of correlated development, (b) We cannot explain the phenomenon of regeneration, as the result of wounding the tissues, or as the outcome of an increase in the food supply, or as due to the removal of a resistance to growth. Far more important are the principles covered by the former paragraph, (a).