Regeneration

Barfurth has studied in detail the regeneration of the tail in some amphibia ; and his results, while not covering as much ground as do those of Fraisse, yet give a more detailed account of the origin of the new tissues. Barfurth's results on triton and siredon are not essentially different from those of Fraisse. In the tadpole of the frog, Barfurth finds that the notochord regenerates from the sheath of the old notochord. In the larval urodele, he finds that the new notochord arises as in the tadpole, and not from the skeletal sheath, as Fraisse maintains. In very young larvae of siredon the chordal cells themselves seem to give rise to the cells of the new notochord. In older larvae, in which the skeletal tissue is developed around the notochord, regeneration takes place both from this tissue and also from the sheath of the notochord. He concludes that in the regeneration of the new notochord, and also of the skeleton, the origin of the cells depends upon the developmental stage of the supporting tissues.

In regard to the regeneration of the muscles, Barfurth comes to the following conclusions : In very young larvae of siredon, the degenerative changes in the muscle cells are often very slight. Regeneration takes place by growth from and the displacement of the old muscles. During this time bud-like terminal and lateral formations occur in the muscle fibres. These outgrowths contain nuclei and form sarcoblasts ; and these pass into the new part, where they make the new muscle fibres in the same way as do the cells of the embryo. In older larvae of the frog, and in mature animals in general, the changes are more complicated. Two processes can be distinguished: (a) degenerative and (b} regenerative, (a) Broken-down muscle fibres that have been cut, and torn-off pieces of muscle fibres, are found present. There follows an accumulation of leucocytes and of giant cells. The nuclei in the degenerating muscle fibres atrophy, and the substance of the fibres breaks down, (b) The muscle fibres split lengthwise to form spindle fibres, and there is an increase in the number of nuclei at the same time. Sarcoblast-like outgrowths of the old muscle fibres are formed, which produce the sarcoblasts that become new muscle fibres.

Barfurth agrees with Fraisse in two main points, viz. that all the tissues of the tail have the power of regeneration, and that each tissue produces only tissue like itself. The law which Kolliker attempted to establish, viz. that the elements of the formed tissues have lost the power of producing other kinds of tissue, — the law of the specification of the tissue, — is supported by these results of Fraisse and of Barfurth, but is contradicted, as has been shown above, by the results on the earthworm, and also as we shall see even in the amphibia, as for instance in the regeneration of the lens of the eye.

Spallanzani1 was the first to study the regeneration of the limb in salamanders, and found that the skeleton in the new part is like that in the normal limb. Bonnet, Philipeaux,2 as well as other naturalists,3 also examined the regeneration of the limbs of salamanders. Gotte ('79) has studied the embryonic development and the regeneration of the limb of triton, especially in regard to the origin of the new bones. He found that the skeleton develops in much the same way in the embryonic limb and in the regenerated limb, and the process in the latter may be said to repeat that in the former. This is especially true for the regeneration of the limb of a very young larva, but the older the larva the more it departs from the embryonic type of development. If the limb is cut off through the upper arm, or through the thigh, new tissue develops over the cut-end. If the larva is quite young, so that formation of the cartilages in the leg has not gone very far, the new tissue differs very little from the old ; but if the leg of an older larva is amputated, the difference between the old and the new parts is more striking. If the bones of the leg have

2 Philipeaux, Comptes rendus de I'Acad. des sciences de I'Institut de France, Annee 8 Todd (Quarterly Journal of Science, Literature, and Arts, Vol. XVI), Blumenbach, Treviranus, Von Siebold. become ossified, the transition from the old to the new part is at first very sharp. The new tissue, that will make the new cartilages of the new limb, develops as a cap over the cut-end of the old bone. Gotte does not give an explicit statement in regard to the origin of the new cartilage, but his account leads one to suppose that it develops from the old cartilage or from some part of the bone. This is, in fact, the case, as I have observed in preparations of the regenerating leg of Plethodon cincrcns, in which the new cartilaginous tissue comes from the periosteum of the old bone. Gotte shows that two long rods of tissue are formed, that are separate for the greater part of their length. They give rise to the two bones of the lower leg, or forearm, as the case may be. The broken end of the femur or humerus also completes itself by a short cartilaginous cap, which is at first continuous with the two rods just described. The ends of these two rods break up into a series of pieces that form the tarsalia, or the carpalia, and the digits. Two digits are first formed, and the others are added as outgrowths from the side of one of the two rods. It is important to note that the new cartilages are formed, in large part, out of a continuous substratum (or rather of two) which separates into proportionate parts to produce the elements of the new limb.

The regeneration of the muscles of the limb of an adult animal, plethodon, has been recently worked out by Towle. The leg was cut off in the middle of the forearm. Extensive changes take place in all the muscles that extend across the level of the cut. The old fibres in the lower end of the muscle, i.e. those near the cut-end, disintegrate, and the number of nuclei greatly increases. The division of the nuclei seems to be direct, each retaining some of the old muscle substance about itself. From some of these cells the new muscle tissue is formed in the new part. Higher up in the forearm the muscle fibres break down to a smaller extent, and still higher up some of the old fibres may remain intact. New muscle fibres are also formed in the old muscle, especially in the region near the cut-end.

The process of regeneration has not been so fully worked out in any other vertebrates as in those described in the preceding pages, although the regeneration of single tissues or organs in the vertebrates has been extensively investigated. In all such cases it is found that like tissues give rise to like. In the planarians it has been found that during regeneration the ectoderm covers the exposed surface, and from it arises the new ectoderm ; the digestive tract appears to come in part from the old tract and in part from the middle-layer cells ; the nervous system appears also to develop out of the middle-layer cells that are found scattered through the body. These cells seem to form a sort of reserve supply

that gives rise to the digestive tract, nervous system, and middle-layer cells in the new parts. From them also arise the new pharynx, and the lining of the pharynx chambers, as well as some other structures. It is impossible to say at present whether one and the same kind of cell may give rise to all these structures, or whether different kinds of cells are present in the middle layer, that cannot be distinguished from each other by the methods at present at our command.

The changes taking place in the tissues of those animals that regenerate by morphallaxis have been only quite recently carefully investigated. Bickford stated that in tubularia the old differentiated tissue changes over directly into the tissue of the new part, and Driesch confirmed this statement. Stevens has studied by means of serial sections the different changes that take place. Division of both ectodermal and endodermal cells is found to occur, but especially the ectodermal. Whether all the ectodermal cells divide, or only some of them, is difficult or impossible to state, but whether this happens or not, all the old region goes over into the new hydranth.

The changes that take place in hydra have been recently worked out in my laboratory by Rowley, who finds that a certain amount of division takes place in the old cells, especially in the ectoderm. The division of the cells is not a very active process, and it seems not improbable that many of the old cells go over without dividing into the new part. One of Trembley's most celebrated experiments was that in which hydras were turned inside out (Fig. I, A, B\ so that the ectoderm came to line the inner cavity and the endoderm to cover the outer wall. The tentacles were not everted but remained sticking out of the mouth of the everted animal. Their openings, or arm-holes, therefore, appear on the outer surface of the body. In order to prevent the everted hydra from turning itself back again, as it tends to do, Trembley pushed a small bristle crosswise through the wall of the body. Finding the hydras still sticking on the bristles the next day, he concluded that they had not returned to their former condition, but that the outer layer (the endoderm) had changed its character so that it became ectoderm, and the inner layer (the ectoderm) became endoderm.1 The experiment seemed to show that the two layers could change their specific character and be transformed into 3ach other according to their position in the animal. These remarkable results were not challenged until 1887, when Nussbaum repeated the experiment and showed that Trembley had overlooked an important fact. It was found that even the bristle pushed through the body does not prevent the hydra from regaining its original condition, although it may delay the turning back. If the turning back can be

1 How the tentacles could have gotten into their normal position is not explained. prevented, the animal dies. Nussbaum showed how the turning back takes place in an animal while it remains on the bristle. The everted foot-end begins first to turn back, pushing into the central cavity. When it comes to the bristle it passes to one side of it, and continuing to turn back the foot passes out of the mouth, drawing the rest of the body after it.1 The last act of the turning can take place only by tearing away through one or both sides, and this is often done. The bristle may still remain sticking to the body through one side, or even remain through both sides if the body has, after tearing through, healed up around the bristle. The process of turning back may take place quite quickly, and had been overlooked by Trembley, who trusted too confidently to the presence of the bristle sticking through the animal.

The method by which the turning back of the layers takes place was not, it appears, clearly described by Nussbaum in his first paper, for his account seems to imply, in certain passages, that the ectoderm may slide over the endoderm during the process, rather than that both layers always turn together. Ischikawa, who studied the problem later, gave a clearer account of the method of turning back. Nussbaum has stated^ in a later paper that he had described essentially the same process.

In conclusion, it can be definitely stated that a transformation of ectoderm into endoderm cannot take place in hydra. Ischikawa also tried removing the endoderm from a piece by spreading it out and then killing the inner layer by weak acid applied with a brush, but pieces of this sort failed to regenerate a new endoderm. Tower has recently stated that if a living hydra is put into a strong light from an arc lamp of 52 volt 12 ampere capacity, that is focussed on the animal (after passing through an alum cell), the ectoderm cells fly off, but if the animal is kept, it subsequently produces a new ectoderm. Whether all the ectoderm is lost, or only the larger neuro-muscular cells, was not made out.

One of the most unexpected discoveries of recent times in connection with the problem of regeneration is the renewal of the extirpated eye of triton and salamandra. Colucci first discovered in 1891 that if the eye is partially removed a new eye develops from the piece that remains and that the neiv lens develops from tJie margin of the bulb. Wolff, a few years later, not knowing of Colucci's results, also found that after extirpation of the lens of triton, by making an incision in the cornea, a new lens develops from the edge of the old iris. Wolff pointed out the great theoretical importance of this result. The experiment has been repeated and confirmed by

1 The foot sometimes pushes out through one of the slits made by the bristle instead of out of the mouth. a number of more recent workers, so that there remains no question as to its accuracy. After the removal of the old lens the wound in the cornea quickly heals, and in the course of two or three weeks a thickening appears at one point at the edge of the iris (Fig. 60, A). The cells that produce this thickening are the ordinary deeply pigmented cells of the iris, where the outer layer of cells of the iris becomes continu-

FlG. 60. — After Wolff. Regeneration of lens of eye of Triton. A. Edge of iris with beginning lens. B, C, D. Later stages of same. E. After Fischel. Whole eye with regenerating lens. ous with the inner layer. The cells increase in number and produce a spheroidal ball that hangs down into the space formerly occupied by the lens (Fig. 60, E\ The cells become clearer by absorbing their pigment and arrange themselves concentrically as in the normal lens. When fully formed the new lens separates from the iris and occupies the normal position.

The most surprising fact in connection with the development of the new lens is that it arises from a part of the body from which the lens of the eye never develops in the embryo of this form or of any other vertebrate. In the embryo the lens develops from the ectoderm at the side of the head and only secondarily unites with the optic cup, that has come from an evagination of the anterior wall of the fore brain. In the regeneration of the adult lens, however, the ectoderm covering the eye takes no part in the formation of the new lens, — in fact, it is separated from the eye by the thick inner, mesodermal layer of the cornea. The lens develops, as has been stated, from the already differentiated layers of the iris. It is a point of further interest to notice that the cells that form the transparent lens come from the iris cells that are in part at least filled with black pigment. If this pigment remained in the cells the new lens, while it might be structurally perfect, would be physiologically useless. The pigment disappears, however, as the lens develops. In this case we find a highly specialized organ, the lens, developing out of tissue also specialized in another direction. It does not simplify the problem to point out that the lens and the iris are both parts of the eye, since they have arisen from different parts of the body and have only secondarily come into apposition with each other. Colucci was contented to point out that both the embryonic lens and the regenerated one come from ectoderm and that the result can be brought into harmony with the " germ layer " hypothesis.

Wolff has called attention to the fact that the new lens arises from the upper edge of the iris, and that this is obviously the most advantageous position in which it could develop from the iris, since by its own weight it falls into place as it develops. If the lens had developed from any other point of the margin, its position would be less advantageous, as it might not be brought into its proper position. Fischel, who has more recently studied the regeneration of the lens in the larvae of Salamandra maculata, finds that after the removal of the lens the iris is thrown into wrinkles or folds and may stick at first to the cut-edge of the cornea. After the cornea has healed, the iris returns to its normal position. He finds that the first changes are more or less alike around the entire rim of the iris and involve a partial absorption of the pigment, a separation of the inner and outer layers at the edge, and a swelling of the margin. These changes go only a little way in those parts that do not produce a lens, but at the upper edge of the iris they go farther and lead to the formation of a lens in that region. He finds also that a new lens develops in animals kept in the dark as well as in those kept in the light, and in the same way.

Fischel also tried the effect of removing a part of the upper edge of the iris at the time when the lens was extirpated, in order to see if, in the absence of this part, the lens would develop from other parts of the uninjured margin of the iris. He found that the new lens still 'comes from the upper edge of the iris from the part left after the operation and not from the intact edge in other parts. This seemed to show that an injury to the iris is in itself a stimulus that starts the formation of a lens. This conclusion is made probable by the results of other experiments in which the iris was stuck at several points, when new lenses began to develop at several of these regions of injury. In some cases Fischel found that two or more lenses began to develop when the iris had not been intentionally injured; but it is not improbable that some sort of injury may have been effected when the lens was removed. Fischel, as has been said, removed extensive portions of the upper part of the iris and found that a new lens could be formed at the cut-edge, even in the region of the pars ciliaris ; and, even after the removal of the entire upper part of the iris, lens-like structures may appear in the inner or retinal layer of the remaining region.

If instead of removing the lens it is displaced by pressing on the cornea until the lens leaves its normal position and comes to lie in the vitreous humor, a new lens develops from the edge of the iris, as though the old lens had been entirely removed from the eye, but in the experiments' in which this was done the new lens was not well developed. The result shows that it is not necessary that the old lens be removed from the eye in order to induce the regeneration of a new one, but only that the lens lose its normal position in the eye.

In regard to the stimulus that determines the development of the lens, Fischel agrees with Wolff that gravity has a share in producing the result. The absence of the old lens from its normal «position, as well as the wrinkling of the cornea, may also enter in as factors. Fischel takes issue with Wolff as to the interpretation of the result as an adaptation, and states that "the organism always responds to a change of relation in only one way, whose direction is already determined by internal structural relations, without regard to whether the result is adaptive or not. The response follows each stimulus in a way determined by the limited possibilities of the cells. With such a uniformity in the reaction, the idea of a fundamental adaptability cannot be connected, since the reaction that appears to us to be adaptive in a series of complicated changes may be non-adaptive in another series."

Whether Fischel has here really met Wolff's argument is, I think, open to question. It does not alter the result to show that factors already existing enter into the process, so long as the organism is so constructed that just those factors are present that bring about a useful response. That the response may be sometimes imperfect does not affect seriously the argument — in fact, it makes the case all the more remarkable if these imperfect attempts are in the direction of useful responses. Fischel sums up his conclusions as follows : " It is not necessary, and it is irreconcilable with the facts, to describe the formation of the lens in a teleological sense, and to bring this case forward as a proof of the universal application of a teleological principle. As has been already stated, the facts in regard to this case show much more clearly that the organism reacts to each change always in a manner that corresponds to its limited possibilities without regard to a teleological principle. A planarian, for instance, responds to a stimulus and makes a new head, even when it possesses one or more already ; a tubularian produces a hydranth at its basal end, if this end is freely surrounded by water ; an actinian forms a new mouth on the side of its body, etc. ; so also do the cells of the pars ciliaris, and the pars iridica retince differentiate into lens fibres. Working blindly, without respect to the consequences as far as they concern the whole, the one thing only is produced for which the conditions are present that bring about its formation in the cells."

Our examination of the origin of the tissues and organs in the new parts has shown that in most cases the old tissues give rise to the same kind of tissue in the new part; or in some other cases, as in the nervous system, the regenerating organs arise from the same " layer " as that from which they develop in the embryo. These facts have led many writers to state that the tissues and organs in the regenerated part arise from the same germ-layers as do the same parts in the embryo. It is supposed that ectoderm gives rise to ectoderm, and to those structures that arise from the ectoderm in the embryo, as, for instance, the nervous system, stomodaeum, etc. The endoderm is supposed to give rise to endoderm, and to endodermal structures, and the mesoderm to mesoderm and its derivates. So fixed has this opinion become that it is not uncommon to find investigators proclaiming the triumphant success of their results, because they have been able to trace the organs in the regenerated part to the same germ-layers that give rise to these organs in the embryo. Before deciding as to the value of this point of view, let us examine briefly the foundations of the so-called germ-layer hypothesis.

The origin of this hypothesis goes back at least to 1/59, when £. F. Wolff maintained his thesis that the digestive tract of the chick exists as a flat, leaf-like structure that subsequently rolls up into a tube. He thought it probable that other embryonic organs might arise in the same way. His views made at the time no impression on his contemporaries, and lay buried until 1812, when Meckel republished Wolff's work in a German translation. Pander, in 1817, distinguished two layers in the early embryo, a serous and a mucous, and stated that later a third, vascular layer appears between the other two. Von Baer published in 1829 his celebrated memoir on the development of the chick, in which he made out two primary layers in the germ, the animal and the vegetative layer, and held that each of these separates into two to produce the four embryonic layers. Remak, in 1851-1855, gave a more precise description of the germ-layers, and stated that from the innermost layer, the epithelium and glandular cells of the digestive tract arise (including the lining of the glands that open into the digestive tract). From the outermost layer he showed that the integument and sense organs and the nervous system develop, and from the two middle layers develop the muscles, blood, excretory, and reproductive organs. By the term " germ-layers " was meant at this time only that the embryo is formed out of sheets.

Huxley in 1849 pointed out that a medusa is made up of two layers, an outer and an inner, and called attention to their possible equivalency to von Baer's serous and mucous layers. This idea of a resemblance between the layers of an embryo and of an adult of a lower form furnished the starting-point for the more modern formulation of the germ-layer hypothesis. Kowalevsky's work on the development of a number of the lower animals showed that there is present in many forms a two-layered stage, or gastrula, formed by an in-turning of the wall of the hollow blastula. In this way two germ-layers are established, an outer and an inner, that correspond to the ectoderm and to the lining of the digestive tract, or endoderm. While Kowalevsky's work did much toward laying the foundation of the modern study of embryology, he himself indulged in very little of the sort of speculation that came into vogue a few years later. Kowalevsky's discovery of the gastrula stage in the embryos of many different groups has been fully confirmed and extended, but the elaborate speculations that have been built up on this as a basis have gone far beyond the evidence, and, for a time, drew the attention of embryologists away from more important problems. Haeckel took a more extreme position than most of his contemporaries, and assumed that the gastrula stage that occurs in so many of the groups of metazoa corresponds to an ancestral, two-layered adult animal, the gastraea, from which all the higher forms have descended. The presence of the gastrula in the development was interpreted as a " repetition " of this ancestral adult stage. Thus the two primary layers are sup-

posed to have an historical meaning.1 Embryologists soon began a search for a similar mode of interpreting the middle germ-layer, or layers, which led, amongst other views, to the formulation of the "gut-pouch hypothesis." From this point of view the body cavities, or ccelomes, are supposed to have been originally sac-like outgrowths from the digestive tract of an ancestral adult animal. Later, these coelome sacs are supposed to have been shut off from connection with the digestive tract — their cavities becoming the body cavities, and their walls giving rise to the mesodermal organs. The formation of pouches from the walls of the archenteron of the embryo in several groups of animals has been interpreted as a repetition of the ancestral adult animal.

A comparison of the germ-layers in different forms very soon led to an attempt to " homologize " the layers in different animals. If the layers have had historically the same origin, or appear in the same way in the embryos, or give rise to the same organs, they are said to be homologous. In the absence of a knowledge of the first two of these conditions it is generally considered sufficient, if it can be shown that similar organs arise from a layer, to " homologize " that layer in the two forms. The study of embryology soon became a search for homologies. The results led to inextricable difficulties and innumerable contradictions until, a reaction setting in, many embryologists became sceptical in regard to the value of this entire method of study.

The results of a detailed study of the process of cleavage in a number of groups have helped, perhaps, to clear the way for a sounder conception. It has been found that the cleavage of the egg in members of the groups of annelids, mollusks, and turbellarians is extremely similar — so similar, in fact, that it seems hardly possible that they could be due to chance, especially as the series of cleavages is quite complicated. The discovery of these similarities led at once to comparison, and comparison to the establishment once more of homologies, and the homologies led again to contradictions, until at present scarcely any two workers agree as to a criterion of homology.2 Leaving this question aside, however, and fixing our attention only on the similarity of the process of cleavage, we are justified, I think, in looking for an explanation of the similarity in some sort of an historical connection. We can eliminate, I think, without discussion the possibility of this type of cleavage representing an ancestral

1 I have given elsewhere (The International Monthly, March, 1901) a fuller treatment of the gastnea theory from the historical point of view. 2 It may be pointed out that there may he really several kinds of homology, such as homology due to similar origin of the blastomeres, or to their position, or to their fate, etc. The confusion that has arisen may in part result from the attempt to make homologous parts agree in all points. adult animal. So far as the question of descent enters the problem, we can infer with some degree of probability that the groups in question may have come from a common group in which the egg divided in much the same way as we find it dividing at the present time. As a formal hypothesis this view meets with no serious difficulty, since a chain of forms, or a continuous living substance, connects the present animals with those living in the past ; and we may assume that the same factors peculiar to the egg of the ancestors are still present in the eggs of their descendants. This sort of explanation gives us no causal knowledge of the way in which the egg divides, nor does it preclude the possibility of new changes coming in that may entirely alter the form of the cleavage. Moreover, since we are dealing with a question of historical probability only, we cannot be certain that the same type of cleavage may not have arisen quite independently in each group.

The argument in favor of the gastrula stage also representing an ancestral larval stage may be admitted as a remote possibility, but on evidence even far less satisfactory than that for the similarities of cleavage being accounted for by a common descent. That this gastrula was ever an adult form we have no means of deciding, even as a matter of probability, and even if this could be made plausible it by no means follows that such an adult stage would become an embryonic stage of later forms. Consequently that part of the germ-layer theory that rests on such a supposed connection cannot be looked upon as much more than a fiction.