Regeneration

But even granting that there is an historical, embryonic l connection, its small importance for the scientific problems connected with embryonic development, and budding and regeneration has been shown by a number of recent discoveries, and nowhere more clearly than in the cases of the formation of new individuals by budding. As an example may be cited the method of development of the ascidian from the egg, and by means of buds. The work of Kowalevsky, Delia Valle, Seeliger, and Van Beneden on the budding process of ascidians showed that there are some discrepancies between the bud development and the embryonic development. The more recent papers of Hjort, Oka, Pizon, Salensky, Lefevre, and others have shown very clearly that the germ-layer theory is inapplicable to the bud development in this group. The bud arises as a doublewalled tube, or rather a tube within a tube, with a space between. The outer tube comes in all cases from the ectoderm of the animal ; the inner tube has a different origin in different species. In perophora, didemnum, and clavellina, the inner tube comes from endoderm ; in botryllus it arises from the ectoderm of the larval peribranchial or

1 That is, one not depending on inheritance through adult forms. atrial cavity. In all these forms the inner tube gives rise to the new pharyngeal cavity of the bud, while this same cavity comes from the endoderm of the archenteron of the embryo. In the bud embryo the peribranchial space is also derived from the inner tube ; hence it is endodermal in the first series, and ectodermal in botryllus. In the egg embryo it is ectodermal. In regard to the development of the nervous system there is some difference of opinion. A number of investigators have found that the new brain arises from the outer part of the inner or branchial tube, which has in most cases an endodermal origin. Seeliger and Lefevre believe the nervous system to arise from mesodermal cells that lie between the two tubes. It appears, nevertheless, that in several forms the brain really comes from the inner tube, which also gives rise to the branchial sac. Therefore, in those cases in which the inner tube is endodermal the brain has the same origin, and in the case in which the inner tube is ectodermal, the brain is ectodermal, but the pharyngeal sac has also an ectodermal origin. There is obviously no definite relation between the origin of these structures in the bud and in the egg embryo.

A similar difficulty is met with in the Bryozoa in regard to the development of the egg embryo and the bud embryo. Braem, who has made a critical examination of the germ-layer theory,1 has found it impossible to give a morphological definition of a germ-layer, and has adopted a physiological criterion. He thinks that in whatever way a germ-layer arises, whether by folding, or by delamination, etc., it exists independently of its method or place of origin. A layer is not endodermal because it forms the inner wall of a gastrula, but it is endodermal because it develops into the digestive tract. The germ-layers of different forms are only similarly placed, but whether they are homologous will depend on other things. On this view the inner tube of the ascidian bud that gives rise to both digestive tract and to the nervous system is simply an indifferent layer until it gives rise to these structures. Its cells may be looked upon as indifferent, as are those of the blastula. Thus the difficulty of the morphologist is not solved, but the knot is cut. For Braem the germ-layers are convenient terms, since he rejects any historical significance that they may have, and it is just this side of the question that the morphologist has attempted to work out. While the evidence shows that the germ-layers cannot have any such final attributes as embryologists have attempted to assign to them, and that Braem has called attention to the real and important problems connected with the study of development, yet it may still be admitted without endangering the newer point of view, that there may be also an historical question in connection with the germ-

layers, if not in the sense of a repetition of an ancestral adult gastraea, yet in the sense that similarity in embryonic development may in some cases find its historical explanation in a common descent. If in the light of this discussion we turn to the phenomena of regeneration, we again find evidence showing that the germ-layer theory fails to apply in all cases. It has been pointed out that in lumbriculus, and in the naids, the new mesoderm is derived from the ectoderm, and does not come from the old mesodermal tissues. The mesoderm of the embryo in annelids is derived from one, and later from two, superficial cells of the blastula,1 that push in about the time of gastrulation. They cannot, at this time, be referred to one layer rather than to the other. It cannot be affirmed, therefore, that in regeneration, the mesoderm arises from a different layer from that in the embryo, but neither can this be denied. The most important point in this connection is that the new mesoderm comes from the ectoderm that is already differentiated, and not from the mesodermal tissues. It is clear, however, that while the lining of the pharynx in the embryo is ectoJermal, it is endodermal in the regenerated part.

It is true that these cases are very exceptional, and that generally the new organs come from similar organs in the old part, but one established exception is sufficient to show that the traditional conception of the germ-layers may be of little value, and since the hypothesis itself, out of which the idea in regard to regeneration from definite germ-layers has been formed, has been proven to be insufficient in other directions, the time is ripe to look for a more secure footing. It need hardly be added that the idea of a supposed necessity for an organ to arise from a definite germ-layer is so empty of all significance that we may well rejoice to be able to set it aside as a naive view that has had its day. Furthermore, a new series of problems has arisen in connection with the experimental work to be described in a later chapter. If, as seems probable, the question of the germlayers will be merged into the much broader question of the origin of the specification of the tissues, we can in the future more profitably direct our attention to the experimental evidence that bears on the latter question.

It has been claimed that at times ontogenetic, and even phylogenetic, processes are repeated during regeneration. Fraisse, for instance, who advocates this point of view, thinks that it has been 1 A small amount of embryonic mesenchyme may come from some of the ectodermal quartettes of the embryo and produce the branching muscles of the head, but not the characteristic muscles of the trunk. too much neglected, and calls attention to several instances of what he believes to be cases in point. He thinks that Biilow is correct in his comparison between the method of development of the new tissue at the end of the tail in certain naids, and the method of gastrulation and formation of the mesoderm in the embryo. Later results have shown, however, that in several points Billow's observations are incorrect. The in-turning of ectoderm that Biilow compares with the process of gastrulation is connected with the formation of the ectodermal proctodaeum, and is not comparable with the development of the endoderm in the embryo.

Gotte also, as we have seen, cites a case of resemblance between the regeneration of the limbs of the salamander and their mode of embryonic development. He finds the resemblances less marked as the animal becomes older. The resemblance is, however, not very close and of a rather general sort, and since the same structures develop in both cases out of the same kind of substance, it is not surprising that there should be some resemblances in the processes. This evidence is counterbalanced by the mode of regeneration of the tail in the adult of certain forms, and in the regeneration of the lens of the eye from the iris.

Carriere finds that the eye of snails regenerates from the ectoderm in much the same way as the young eye develops. Granted that the eye is to come from the ectoderm in both cases, and that the same structure develops, it is not to be wondered at that the two processes have much in common. The mistake, I think, is not in stating that the two processes are sometimes similar, or even identical, but in stating the matter as though the regenerative process repeats the embryonic method of development. If the same conditions prevail, then the same factors that bring about the embryonic development may be active in bringing about the regenerative processes. In fact, we should expect them to coincide oftener than appears to be the case, but this may be due to the conditions being different in the young and in the adult.

It has been claimed also that in some cases there is regenerated a structure like that possessed by the ancestors of the animal. The stock example of this process is Fritz Miiller's result on the regeneration of the claw of a shrimp, Atypoida protiminim^ Fraisse and Weismann and others have brought forward this case as demonstrative. The animal is said to regenerate a claw different from any of those in the typical form, and one that resembles the claw of another related genus, Carodina. The value of evidence of this sort is not above question. Przibram has shown in other Crustacea that when

a maxilliped is cut off a structure different in kind often regenerates, but that after several months the typical structure returns. Do we find here an ancestral organ that first appears, and then gives way to its more modern representative ? If it resembled the maxilliped of any other crustacean, the evidence would, no doubt, be accepted by those who accept the evidence furnished by Miiller. What then shall we say to the case, first discovered by Herbst, in which the eye of certain prawns being cut off, an antenna-like organ regenerates? Since these antennae are similar to those possessed by the same animal, shall we assume that it once had antennae in place of eyes ?

Another comparison, that Fraisse has made, is worth quoting as showing how far credulity may be carried. In the regeneration of the tail of certain lizards pigment first appears in the ectoderm of the new part and then sinks deeper into the layers. Fraisse found a lizard on Capri in which the tail is pigmented throughout life, and although he did not know whether or not the pigment is in the skin he suggests that this lizard represents an ancestral condition, that is repeated by the regenerating tails of other forms.

Boulenger ('88) pointed out that the scales over the regenerated tail of several lizards have a different arrangement from that of the normal tail, and furthermore, the new arrangement is sometimes like that found in other species. He claims that this shows that such forms are related, even where no evidence of their relation is forthcoming. That the conditions in the new tail may be different from those in the normal tail is shown by the absence of a vertebral column, etc. ; therefore that the scales also should have a new arrangement is not surprising, but the facts fail, I think, to show that there need be any genetic relation between the forms in question. That the conditions in the new tail might be like those in an ancestral form may be admitted, but this is very different from assuming that the results show a genetic relation actually to exist. The main point is that, even if the results should be nearly identical, it may be entirely misleading to infer that ancestral characters have reappeared.

In some cases an extra digit or toe may regenerate on the leg of a salamander, and this too has been interpreted as a return to an ancestral condition. But Tornier has shown, as has been stated, that several additional digits, or even a whole extra hand, may be produced by wounding the leg in certain ways, and these too would have to be interpreted as ancestral, if the hypothesis is carried out logically. It has been shown by King that one or more additional arms may be produced in a starfish by splitting between the arms already present, and if we accepted evidence of this sort as having any value in interpreting lines of descent we should conclude * that

the ancestors of the starfish had six, seven, or more arms according to the number that can be produced artificially, etc. Therefore, until further evidence of a more convincing kind is forthcoming, we can safely, I think, decline to accept the results, so far known, as having any value in interpreting the relationships or the descent of the animals. NOT only do adult organisms have the power of regeneration, but embryos and larval forms possess the same power, and even portions of the segmenting, and also the unsegmented, egg may be able not only to continue their development, but in many cases to produce whole organisms. Haeckel observed in 1869-1870 that pieces of the ciliated larvae of certain medusas, and even pieces of the segmented egg, could produce whole organisms. The more recent experiments of Pfliiger ('83) and of Roux ('83) on the frog's egg mark, however, the beginning of a new epoch in embryological study. The explanation of this is to be found, I think, not only in the introduction of experimental methods, but also in the fact that Pfliiger and Roux realized the important theoretical questions involved in their results.

Pfliiger's experiments were made by changing the conditions under which the egg develops in order to determine what factors control the development. Since these experiments were made with whole eggs, the problems of regeneration were not directly involved in his results, although his conclusions are of great importance in connection with questions concerning the regeneration of the egg. A part of Roux's work dealt directly with the development of a new organism from a piece of the egg or of the embryo. Roux's principal discovery l ('88) was that a half-embryo develops from either of the first two blastomeres of the frog's egg, if the other blastomere has been injured or destroyed, but that subsequently the missing half of the embryo is " post-generated." Roux was led to this experiment by his discovery that the plane of the first cleavage of the egg corresponds very often to the median plane of the body of the embryo.2 This relation suggested that there might be some causal connection between the two phenomena in the sense that the first cleavage plane divides the material for the right side of the body from that of the left side. In a descriptive sense this would be, of course, true if the two planes do really correspond, and if there was no later shifting of material

1 Roux's earlier experiments in 1885, in which the unsegmented or segmented egg was stuck and a part of its contents removed, the remaining part making a whole embryo, will be considered in another connection. across the middle line, but whether the two phenomena are causally connected, or are merely due to a coincidence, could only be determined by further experiment. The observations themselves are not beyond question, for the two planes do not always coincide, and may be even

FlG. 61. — After Roux. A. Section of semi-blastula of frog's egg. B. Half-embryo. C, Crosssection of last (reversed right and left in B and C). D. Anterior half-embryo. ninety degrees apart. These cases of divergence were thought by Roux to be due to an unobserved shifting of the developing embryo, but it is improbable that all cases can be accounted for in this way. Roux carried out his experiment by plunging a hot needle into one of the first two blastomeres, so that it is injured to such an extent

that its development is prevented. The same needle, without heating again, was used for one or two other eggs, for, if the needle had been so hot in the first instance that both blastomeres had been injured by the heat, this might not happen in the second or the third egg. It was found that amongst the eggs that had been operated upon in this way, some had been so much injured that neither blastomere developed, others had been so little injured that both blastomeres developed, but in the successful operations the uninjured blastomere developed, while the injured one did not. In the last case the uninjured blastomere divided, and produced a large number of cells. A segmentation cavity was present in the upper part of the hemisphere (Fig. 61, A). The injured half remained in contact with the other, completing the sphere, but it did not segment. A half-embryo developed from the uninjured half, as shown in Fig. 61, B, C. This embryo has a halfmedullary fold along the side in contact with the injured half. At the anterior end somewhat more than half a head is present, and at the posterior end there is a half-blastopore. The cross-sections1 (Fig. 61, C), through the embryo, show that beneath the half-medullary fold a rod-like notochord is present, which is made up apparently of fewer cells than the normal notochord, but it has, in cross-section, a round and not a half form. At the side, the mesoderm is present, as in the normal embryo, and it has produced the characteristic mesoblastic somites. An archenteron is formed in the half-embryo, and, since it is smaller than the normal, it may, perhaps, be called a half-archenteron. The embryo is, therefore, in most respects a half-structure.

The head is, however, nearly a whole head, but whether this is due to a whole head developing out of material derived entirely from one of the two blastomeres, or whether, as Roux supposes, a portion of the material of the injured blastomere has been worked over, i.e. "post-generated," remains, I think, an open question. The results of this experiment seem to confirm Roux's conjecture that the material of each of the first two blastomeres is of such a sort that it gives rise to half the embryo, and, if so, there would be some probability that there is a causal connection between the first cleavage and the separating out of the parts of the embryo. In fact, Roux drew this conclusion, and even attempted to show how such a qualitative division is brought about. It should not be overlooked, however, that this conclusion goes beyond the legitimate bounds of deduction from the results, since the half-development takes place while the injured half retains its connection with the developing half, the former still remaining alive. On the other hand, the presence of the injured half makes the experiment more suitable to demonstrate that each of the first blastomeres gives rise, under normal circumstances, to half of the

1 The cross-section C is reversed as compared with the half-embryo B. embryo. If one half had been removed, we can foresee that its absence might lead to other complications that would affect the result. The most important outcome of this experiment is, I think, to show that a half-structure may develop by itself, i.e. that there is a certain amount of independent power of development in the parts of the egg. Roux also tried to show that if, after the second cleavage has been completed, the two blastomeres that lie on opposite sides of the first cleavage plane are killed by a hot needle, the remaining two produce either an anterior or a posterior half of an embryo. An embryo derived from the two "anterior" blastomeres is represented in Fig. 61, D. The anterior half of the body is present. Posteriorly the half-embryo abuts against the injured half. It is possible, I think, that this embryo may represent the anterior half of a whole embryo of half size that has been prevented from closing in posteriorly by the mass of injured material of the undeveloped blastomere. Roux did not determine positively whether the two "posterior" blastomeres could give rise to posterior half-embryos ; one embryo in his opinion appeared to bear out this interpretation. This part of Roux's work is, it seems to me, not so satisfactory as the part dealing with the first two blastomeres, and we may leave it, for the present, out of the discussion, and consider only the result of the first experiment, in which one of the first two blastomeres was injured. Since the problems involved in the two cases are essentially the same, nothing will be lost by dealing with the first case alone.

The uninjured blastomere first gives rise to a half-embryo. After this has been accomplished, other changes take place that " reorganize," according to Roux, the material of the injured half in such a way that the missing half of the embryo is formed by a process that Roux calls "post-generation." This process can be studied only by means of sectioning the embryos, and since the eggs may be injured to a varying extent, there must be some uncertainty in making out the sequence of events. It is found that the yolk of the injured blastomere is vacuolated in places, and that the protoplasm in the path of the needle has been killed (Fig. 61, A). Irregular pieces of chromatin are found in the protoplasm, which seem to come from an irregular breaking up of the nucleus.

The changes that lead to the reorganization of the injured half may take place at different times in different eggs. Roux describes three kinds of reorganization phenomena. The first includes the formation of new cells in the injured half. Nuclei, surrounded by finely granular protoplasm, appear in the protoplasm of the injured blastomere. These nuclei arise from two sources : in part from the scattered chromatin of the injured blastomere itself, and in part from

nuclei, or from cells without walls that have emigrated from the developing half. Around these nuclei, as centres, the protoplasm (with its contained yolk) of the injured half breaks up into cells. This cellulation of the yolk may take place in different eggs at different times. In some cases it may not have appeared as late as the gastrula stage of the uninjured half; in others, it may take place at the time when the uninjured half is segmenting.1 The formation of the cells in the injured half begins always near the developing half, and extends thence into the injured parts. The new cells are of different sizes, but are larger than those of the uninjured half.

The cellulation of the yolk takes place only in the least injured parts of the protoplasm. Where the protoplasm and yolk have been much injured, they are changed over by the second method of reorganization. This part of the blastomere is either actually devoured by wandering cells, or is slowly changed under the influence of the neighboring cells, so that it becomes a part of these cells. The surface of the injured half is covered over by ectoderm that grows directly from the developing half (third method of reorganization), — at least this happens where the protoplasm has been much injured. In other parts of the injured half the new cells that have appeared in this part, and that lie at the surface, become new ectoderm.

Post-generation now begins in the reorganized and cellulated half ; the cells become changed over into the different layers and organs that make the new half-embryo. A few hours or a night is sometimes sufficient to change a hemi-embryo into a whole embryo. The new half-medullary fold develops from the new ectoderm to supplement the half already present. The mesoblast appears over the side. Its upper part seems to come from the uninjured mesoderm that has grown over to the other side, but this is added to at the free edge by cells that belong to the newly cellulated part. The new differentiation is, in general, in a dorso-ventral direction. The lacking half of the archenteron arises in connection with the half of the archenteron already present in the hemi-embryo. The yolk cells arrange themselves radially, and a split appears in the post-generated part, extending from the archenteron of the hemi-embryo. The split opens, and the new half-archenteron appears. In general, Roux states, the post-generation of the organs of the injured half proceeds from the already differentiated germ-layers of the hemiembryo. The post-generation begins where the exposed surfaces of the germ-layers of the hemi-embryo touch the newly cellulated regions of the injured half.

1This difference is due, I suppose, to the amount of injury that the nucleus of the injured half may have suffered. It is most difficult to account for these post-generative changes, since the new part has, according to Roux, a double and even a threefold origin. The pieces of the old nucleus, he admits, may take a part in the formation of the new cells ; wandering cells migrate from the yolk mass of the old half into the new, and the cells of the formed germ-layers may be pushed over to the other side. Since a certain share, and perhaps a large share, of the new cells comes from the hemi-embryo, it is clear that, in addition to the power of self-differentiation shown by the uninjured blastomere, we must also ascribe to it certain regenerative powers, at least to the extent that each kind of cell that comes from it can give rise in the injured half to cells like itself, and produce similar structures in the other half.

If then, as Roux supposes, the development of the egg consists in an orderly, qualitative series of changes that lead to the subsequent differentiation, we must also suppose that the new parts are gifted with latent powers by virtue of which they can re-create all parts of the other half. Roux supposes, in fact, that each cell carries with it a sort of reserve-plasm, that is dormant in ordinary development but is awakened when any disturbance of the normal development takes place. Objections have been made to this subsidiary hypothesis, since the addition of this to the original assumption of a series of qualitative changes involves such complications that the view can hardly be considered a probable one. This objection is, I think, not as strong as certain critics believe, since the facts of development show beyond a doubt that although the egg has the power of progressive change it has also, as certain experiments show, the power of reorganization, if the ordinary course of events is interrupted. This admission by no means throws us back upon Roux's hypothesis, for, as will be shown later, a different conception of the development may better account for both phenomena.

Inasmuch as a good deal of discussion has taken place in regard to the process of post-generation described by Roux, it should be stated that Endres and Walter reexamined the process, and found, as had Roux, that the reorganizing cells migrate from the uninjured to the injured side, and around them the protoplasm of that side makes new cells. They found that the injured half is directly overgrown by the ectoderm from the developing half. When the material of the injured blastomere is only incompletely reorganized, there is formed, after post-generation, an embryo that has a protrusion of yolk in the dorsal part of the body. When the injured material is completely worked over, a perfectly formed embryo may result. The typical half-embryos that Roux obtained were also obtained by Endres and Walter. They deny that whole embryos develop from one of the first two blastomeres, as Hertwig affirms.

Hertwig repeated Roux's experiment and obtained results entirely different from those of Roux. He injured one of the first two blastomeres of the frog's egg with a hot needle, or by means of a galvanic current. Hertwig states that after the operation the egg turns so that the uninjured part lies uppermost. This is owing, he thinks, to the appearance of a blastula or of a gastrula cavity in the developing FIG. 62. — After O. Hertwig. A. Section through a frog's egg (btastula stage) in which one hlastomere had been killed. B, Same. Gastrula stage. C. Later gastrula stage. D, E. Surface view of embryos from one of first two blastomeres. F. Same as last (£). Dorsal view. G. Ventral view of last. H. Dorsal view of another embryo, lying in a very eccentric position. /. Later stage of embryo from one blastomere. Other injured blastomere nearly covered over. J. Section through gastrula stage of embryo from one of first two blastomeres. K. Cross-section of the embryo shown in F and G.

part. The segmentation cavity is found in many cases surrounded by the cells of the segmenting half (Fig. 62, A), but at other times at the border between the new and the old parts. In still other cases the cavity may lie eccentrically, and in some cases the floor of the cavity may be bounded by the yolk substance of the injured half. An embryo appears on the upper, uninjured part, though it is not, according to Hertwig, a half-embryo, but a whole embryo, or at least one approach-