Regeneration

in the different parts. In this connection the most important fact is that the growth takes place most rapidly where it will bring about the new form. This problem, which is one of the most fundamental in connection with the phenomena of development and of regeneration, will be more fully discussed in a later chapter. A number of assumptions have been made in the above attempt to give an analysis of the formation of a head at the side of an oblique surface. That these assumptions are not entirely arbitrary, but have a certain amount of evidence in their favor, can, I think, be shown. The new material that first appears is supposed to be totipotent, in the sense that any part of it may produce any part of the structure that develops from this material. That this is probable is shown by the following experiment. If a cross-piece is cut from a worm, and then split lengthwise into halves, each half will produce a new head at the anterior edge of the piece. This result shows, at least, that from the tissue lying to the right or to the left of the middle line new material may be formed from which a whole head may develop. The new head does not stand at first with its middle axis in line with the middle of the old piece, i.e. it does not stand squarely at the anterior end of the half-piece, but more towards the inner side of the piece. It may appear that the old part has sufficient influence on the new part to shift the axis of the latter toward the old middle line, but while some such influence may be present, it is probable that the position of the head is in part the outcome of another factor, viz. the presence at the inner side of the piece of an undeveloped new side, with which the explanation of the less development of the inner side of the head is also connected.

If a cross-piece is cut from a worm and kept until a small amount of new tissue appears over the anterior and posterior cut-surfaces, and if then the piece is split in two lengthwise, there will develop from each piece a new head out of the new material over the anterior surface. The result shows /\ mg tO the Conditions. It ^^_Planariamaculata. A. Cross-piece, allowed to is possible, of COUrse, that regenerate, then cut in two lengthwise, as indicated by

head may have begun at the time of the experiment, but if it had, the development had not gone so far that a new arrangement was impossible. If, however, the piece is not cut lengthwise until just before the formation of a head (Fig. 23, A), then each half-piece produces at first a half-head, that completes itself later at the cut-side. Another experiment shows even more satisfactorily that the material over an anterior cut-edge may produce one or more new heads according to the conditions, and that the result is not connected with the region from which the new material is derived. If the anterior end of a planarian is cut off and then an oblong piece is removed from the middle of the worm, as shown in Fig. 24, A, it will be found, if

FIG. 24. — Planaria lugubrls. A. Showing where a piece, 4, was removed from middle of a worm, a, b. Regeneration of a single head, c , c1. Regeneration of two heads. D, E, F. Re- generation of small piece, 4, that was cut out. the side parts are kept from fusing together in the middle line, that a new head develops at the anterior end of each part, as shown in Fig. 24, c, cl. If, on the other hand, the two sides come together and fuse in the middle line, as shown in Fig. 24, a, b, the new material that appears over their anterior ends becomes continuous and produces a single head. In this case, although the middle part of the old tissue has been removed, a single head develops that is normal in all respects, and the eyes are not nearer together than when the middle part is present, as when regeneration takes place from an anterior cross-cut surface.

The assumption that the lateral position of the head on an oblique surface is connected with the more anterior region of the old material that is found at that side, can be made at least more intelligible by the following experiment : If the head of a planarian is cut off obliquely, as indicated in Fig. 21, B, so that one of the " ears " is left at one side, the new head arises at the side in connection with the part of the old head that lies at that side. The new head does not extend over the entire cut-surface, which is longer of course than a cross-cut would be, but lies at one side, as in the other cases just described. In this case we can see that if the new head cannot, on account of certain conditions, extend over the entire cut-surface, one side of it may be determined by the presence of a part of the old head,

and this influence may be stronger than any other that might tend to locate the new head in the original middle line. If we suppose that similar conditions prevail in all cases when oblique surfaces are present in these worms, we have a formal solution of the problem. The argument cannot be convincing unless we can give a further explanation of the nature of this influence that the old part has upon the new. In other cases, as in the regeneration from an oblique surface in the tail of the tadpole and of a fish, we must assume that the factor that determines the middle of the new part has a stronger influence on the new material than has the most posterior part of the old tissue.

The influence of an oblique cut-surface on the position of the new parts is shown in a different way in the hydroid, tubularia. The conditions are different in this case inasmuch as there is no proliferation from the cut-end, but the old part produces the new hydranth. Driesch found that if the stem of tubularia is cut in two obliquely, the new tentacles, that develop as two rings around the tube near its cut-end, stand obliquely on the stem,1 as shown in Fig. 20, A. In most cases, both the distal and the proximal circles of tentacles lie obliquely to the long axis of the stem, but there is some variability in the result, and occasionally one or the other, especially the proximal circle, may be squarely placed, although, as a rule, the influence of the oblique cut-end can be seen. It can be shown, I think, that the oblique position of the rings of tentacles in tubularia is the outcome of factors different from those that are found in the regeneration of the tail of the tadpole and of the head and tail of the planarian. Driesch suggested that the distance of the tentacle-rings from the cut-end is the result of some sort of " regulation " that determines their position at a given distance from the region at which the surrounding water acts on the exposed end. Hence, if the exposed surface is an oblique one the rings will also be formed in an oblique position. On the other hand, I have suggested that we can imagine the regulation to result from other factors. At the beginning of the development, and before the tentacles appear, there is a withdrawal of tissue from the cut-end that leaves the region from which the proboscis develops quite thin.

If this material withdraws at a uniform rate and to the same distance at all points from the end of the piece, as observation shows to be the case, and if, as appears also to be true, the outer end of the distal ring of tentacles lies at the inner end of the proboscis region, then it too will assume an oblique position if the cut-end is oblique. If we imagine a similar series of regulations taking place throughout 1 The same holds good for the basal hydranth if it arises near an oblique end.

FIG. 25. — Piece of stem of Tubular ia mesembryanthemum split in two lengthwise. Formation of whole hydranth that turned away from contact with old perisarc. the piece, we can account for the results. On this hypothesis the action of the water on the free end need not be a factor in the result, but the oblique end is itself sufficient to determine the series of regulations, or mass-relations, that lead to the laying down of an oblique hydranth.

When the hydranth protrudes from the stem it assumes an oblique position, as shown in Fig. 20, A1. Driesch supposed the oblique position of the hydranth to be due to an oblique zone that develops behind the hydranth, but the result can best be explained, as certain other experiments that I have made seem to show, as due to the negative thigmotropism of the hydranth at the time it protrudes from the old perisarc. It turns away from the projecting side of the oblique end of the perisarc, as it does from any solid body with which it comes in contact. That this is the case is best shown by splitting the stem lengthwise into halves. In this case, although the two circles of tentacles may be laid down squarely (Fig. 25, A), the new hydranth protrudes at right angles to the old perisarc, as shown in Fig.

In a few cases it has been discovered that the presence of certain organs at the exposed surface is necessary in order that regeneration may take place. The following experiment that I have recently carried out shows, for instance, the influence of the nerve-cord on the regenerating part. A fev/ of the anterior segments of the earthworm are cut off, as shown in the left-hand figure in Fig. 26, and then a piece of the mid-ventral body wall of the worm is cut out, a part of the ventral nerve-cord being removed with the piece. The cut-edges meet along the mid-ventral line and fuse, closing the wound. As a result of the operation there is left exposed, at the anterior end of the worm, a cut-surface with all of the internal organs present except the nervous system. The anterior end heals over, but I have not observed the development of a new head at this level, although the exposed end is in a region at which, under ordinary circumstances, a new head readily regenerates. In several cases a new head developed at the point where the cut-end of the nervous system is situated, i.e. at the level B in the figure.

A variation of the same experiment shows still more conclusively the importance of the nervous system for the result. A few anterior segments are cut from the anterior end as before. A cut is made, as shown in the right-hand figure in Fig. 26, to one side of the mid-ventral line (indicated by the black line in the figure at the level A). Then, at the posterior end of this cut a piece is removed from the mid-ventral line as in the former experiment (shown by the stippled area in the figure). A portion of the ventral nerve-cord is removed with the piece. As a result of this operation, two anterior ends of the nervous system are left exposed (shown by the black dots in the figure). At the anterior end of the worm, i.e. at A, there is one exposure, and at the posterior end of the region from which the piece was removed there is another. Two heads develop in successful cases, one at the anterior end of the anterior cut-surface, i.e. at A, and the other at B.

The results show that in the absence of the cut-end of the nervous system at an exposed surface a new head does not develop ; and conversely, the development of a new head takes place when the anterior FIG. 26. — Left-hand figure X shows how, alter cutting oft" the anterior endofA/Mo- bophora fcetida, a piece of the ventral wall (including a part of the nerve-cord) is cut out. Right-hand figure Y illustrates a more complicated operation, in which the piece of the ventral wall that is cut out is a little behind the anterior end.

end of the nervous system is present at a cut-surface, even when such a surface is not at the anterior end of the worm. We may perhaps be able to extend this statement, and state that as many heads will develop as there are exposed anterior ends of the nervous system. In two other cases, at least, a somewhat similar conclusion may be drawn, although it appears that in these cases other organs than the nervous system may be the centres around which the new parts develop. Tornier has shown that when the vertebrae of the tail of the lizard are injured, the new material proliferated by the wounded surfaces serve as centres l for the regeneration of new tails ; and Barfurth has found that the notochord in the tail of the tadpole plays a similar r61e in the formation of a new tail. These experiments will be more fully described in connection with the formation of double structures, but from what has been said it will be seen that the cases are parallel to that of the earthworm.

Until more has been discovered in regard to the internal factors of regeneration, it would be venturesome to make any general statement based on these few cases, but there is opened here a wide field for experimental work. By eliminating one by one the different organs that are present in the old part, it may be possible to discover much more in regard to the internal conditions that are necessary in order that the process of regeneration may take place.

There are certain results connected with the amount of new material which is produced during regeneration, that should be considered in connection with the question of internal factors. It has been pointed out that when one segment only is removed from the anterior end of the earthworm only one new one returns ; when two are cut off two come back, and this holds good up to five segments. Beyond this, no matter how many are removed, only five at most come back. The latter result seems to be connected with the amount of material that is formed over the cut-surface before differentiation begins. When only one or two segments have been cut off, the new material that is formed is soon sufficient in amount for the production of one or two new segments, but when three to five are cut off somewhat more material is formed before differentiation begins. When more than five are cut off the new material is at best only sufficient to produce five new ones, and in some cases even a smaller number is formed. This hypothesis assumes that there is a lower limit of size for the formation of new segments below which a segment

1 Although it is by no means certain that the results may not be due in part, at least, to injuries to the nervous system. cannot develop. The interpretation is fully in accordance with what we know to be the case for small pieces of hydra and of other forms that, below a certain minimal size, do not regenerate. The question as to how many segments are formed out of the new part is determined, not only by the amount of new material, but also by the number of segments to be replaced, at least up to five segments. Beyond this limit we may think of the maximum possible number of segments appearing in the new material. That a relation of some sort obtains between the old and the new parts, that may have an influence on the number of the new segments which are formed, is shown by the fact that, when one, two, three, four, or five are cut off, just this number comes back. A sort of completing principle exists as a factor in the result, but when so much has been cut off that the old part cannot complete itself in the new material that is formed, then other factors must determine how many segments will be produced.

In planarians we find a similar phenomenon. If much of the anterior end is cut off, only a head is formed at the anterior cutsurface of the posterior piece, and the intermediate region is absent. I interpret this in the same way as the similar case in the earthworm. As soon as enough new material has been formed for the anterior end to appear, it begins to develop, and since it cannot develop below a certain minimal size, or rather, since the tendency to produce a head approaching the maximum size is stronger than the tendency to produce as much as possible of the missing anterior end, all the new material goes into the new head. In the planarian the possibility of subsequently replacing the missing region behind the head exists, and the intermediate part is later produced, the head being carried farther forward. The same is true of the new posterior end of the earthworm, in which a growing region is established at a very early stage in front of the tip of the tail, but no such growing region is present at the anterior end in the earthworm. These differences appear to be connected with the general phenomena of growth in these forms. In the planarian interstitial growth can take place in any part of the body, hence the possibility of producing a missing region is present in all parts of the worm ; but in the earthworm we never find new segments intercalated at the anterior end during normal growth, nor does this take place during regeneration. At the posterior end of the earthworm we find a region of growth in which new segments are produced, and we find the same thing is true in the regeneration of the posterior end. In other words, the growing region in front of the last segment is also regenerated.

It has been found in several forms that pieces below a certain size do not regenerate. In those cases in which a small piece dies soon after its removal from the rest of the body we have no direct means of knowing whether or not the piece has potentially the power to regenerate, but in some other cases, in which small pieces may be kept alive for some time, they may not regenerate. Furthermore, the regeneration of small pieces that are just above the minimal size is often delayed and is sometimes imperfect. These small pieces seem to meet with a greater difficulty in regenerating than do larger pieces. Peebles has shown that pieces of hydra that measure less than \ mm. in diameter (= about ^-<nr °f tne volume of hydra) do not regenerate, although if very small pieces are taken from a developing bud they may regenerate, even when only \ mm. in diameter. Very small pieces that are, however, just above the minimal size, while they may assume a hydra-like form, produce only one or two tentacles. The failure of the smallest pieces to regenerate is not due to their dying, since they may live for a much longer time than would suffice for larger pieces to regenerate. Isolated tentacles of hydra do not produce new hydras, although they may remain alive for some time. A single tentacle is larger than the minimal piece, so that its failure to regenerate is probably connected with the differentiation of the tentacle, rather than with its size. The lack of power to regenerate in the smallest pieces of hydra cannot be connected with the absence of any special organ, since these pieces contain both ectoderm and endoderm. In tubularia also, Driesch and I have found that pieces below a certain size do not regenerate (Fig. 27). There is likewise in planarians a lower limit of regeneration, even for pieces that contain all the elements which, being present in larger pieces, make regeneration possible. Lillie has found that nucleated pieces of the protozoon stentor FIG. 27.

— Tubularia mesembryfail to regenerate if they are below the mini- p£ce'"that pro™unceTaStydmal size. He places this minimal size at 80 [i. dranth B, c. Pieces below diameter, which he calculates as 9V of the duced by closing of small volume of the stentor from which the piece has come. I have obtained a slightly smaller piece that regenerated, and since it came from a larger stentor it represents about g1^ of the whole animal. The lack of the power of development of these smallest pieces seems to be due to the absence of sufficient material for the production of the typical form. We can give no other explanation of the phenomenon at present, especially since the pieces contain material that we know from other experiments has the power of producing any part of the organism. The superficial area of small pieces is relatively greater than that of larger pieces,

but there is no evidence that this relation can in any way influence the result. Whether the difference in surface tension could prevent the small piece from assuming the typical form and hold it, as it were, in a spherical form is not known, but there is little probability that this is the explanation of the phenomena. The regeneration of small pieces of animals and of plants may often fail to take place, because, as Vochting has pointed out, the injury caused by the cutting may extend so far into the small piece that its repair may be impossible. In other cases there may be an insufficient reserve supply of food stuff, although, if a proportionate form of any size could be produced, it is difficult to see how this could be the case. There can be no doubt, however, that pieces taken from parts of the body that are dependent on other parts for their food, oxygen, etc., will die for lack of these things, and even if they can live for some time their further development may not take place in the absence of sufficient food to carry on the process. After these possibilities have been given due weight, there remain several cases in which there can be little doubt that the failure of a small piece to regenerate is owing to the lack of sufficient material to produce even the smallest possible form for that sort of material, i.e. for the organization to be formed on so small a scale.

There are some facts in connection with the regeneration of small pieces of tubularia that have an important bearing on this question of organization size. If long pieces of the stem are cut off, the new hydranth, that develops out of the old tissue at the end of the piece, occupies, within certain limits, a region of definite length. If pieces of the stem are cut off that are only twice the length of the hydranthforming region, the length of the latter will be reduced to half the length that it has in longer pieces, and if still smaller pieces are cut off, the hydranth-forming region may be reduced, as Driesch has shown, to seventy per cent of the normal length. The hydranths that develop from the smaller pieces have also a reduced number of tentacles, as I have found. It was first shown by Bickford, and later by Driesch, and by myself, that in many cases very short pieces of the stem of tubularia produce only the distal parts of a hydrantli. This happens most often when the length of the piece is less than the average normal length of the hydranth-forming area, but it may also take place in pieces that are much longer than the minimal size of the least hydranth-forming region. Driesch made the further discovery, which I have confirmed, that pieces from the distal end of the stem are more likely to produce these partial structures than are pieces from the more proximal part. Some of these partial structures are represented in Fig. 28, C—G. Sometimes the inner tube, or coenosarc, which is composed of the two layers of the body, ectoderm and

endoderm, draws away from the chitinous perisarc, as shown in Fig. 28, B. A hydranth with a short stalk is then produced. In other cases, Fig. 28, C, almost all of the coenosarc is used up to form the hydranth, and only a short, dome-shaped knob represents the stalk. In still other cases there may be no stalk at all (Fig. 27, Z>), but only the hydranth. Forms like the last two are more often produced from pieces of the FIG. 28. — Tubularia mesembryanthemum. Products of regeneration of short pieces. A. Piece that regenerated a hydranth in same way as do longer pieces, but with fewer tentacles. B. Pieces whose stem drew away from wall of old perisarc (cylinder in figures). C. Hydranth with almost no stalk. D. Hydranth without stalk. JS. Distal part of hydranth with one long proximal tentacle. El. Similar, but more reduced. E"*. Similar, with two tentacles at side. F. Proboscis with reproductive organs. G. Proboscis without reproductive organs.

distal end of the stalk. From very small pieces, forms like those shown in Figs. 28, E-E2, that represent only proboscides with a reduced number of tentacles, are sometimes formed. Reproductive organs may be present at the base of these pieces. A further reduction is shown in Figs. 28, F, G, that are proboscides with only the distal circle of tentacles ; in one of these, reproductive organs are present around the base. Partial forms more reduced than these have not been found.

If we examine the factors that determine the production of the partial structures, we find, in the first place, that the size of the piece is of the greatest importance. The reduced forms appear most often in pieces that are shorter than the average length of the hydranthforming area. A second factor is connected with the region of the stem from which the piece is taken. Larger pieces from the distal end produce partial structures, especially hydranths with very short stalks (Fig. 28, C), or with none at all (Fig. 28, D). There are certain facts connected with this distal region, which lies just behind the hydranth, that should be mentioned in this connection. It was first discovered by Dalyell that a hydranth-head lives for only a limited time, and that when it dies a new head is regenerated from the region behind the old one. The stalk of the new hydranth continues to elongate for some time after the new hydranth has been formed. Whether this continuous growth in the distal end, or the normal formation of a new hydranth by it from time to time, can in any way be connected with the development of partial structures from this region cannot at present be stated. The distal part of the stem contains more of the red-pigment, that gives color to the stem and to the hydranth, than does any other part. Loeb first advanced the view that the redpigment in the stem acts as a formative substance in Sachs' sense, and determines the production of a new hydranth by accumulating near the cut-end of the piece. Driesch also assumes the red-pigment to be a factor in the result, but supposes that it acts quantitatively, rather than in determining the quality of the result. If this red-pigment acted in the way supposed either by Loeb or by Driesch, it might act as one of the factors in the production of these partial structures.

This red-pigment is contained in the form of reddish granules in the cells of the endoderm. The granules are of various sizes, the largest being easily seen even with low powers of the microscope. When a piece of the stem is cut off, the ends close by the drawing in of the cut-edges over the open-end. A circulation of the fluid contained in the piece then begins. In the fluid, globules appear very soon that contain red-pigment granules like those in the endoderm. The globules appear to be endodermal cells, or parts of cells, that are set free in the central cavity. The circulation continues for about twentyfour hours. At about this time one end of the stem becomes reddish, owing to the presence in it of a larger number of red-pigment granules than before. The ridges that are the rudiments of the tentacles appear (Fig. 30, A), and a new hydranth very rapidly develops. At the time when the hydranth begins to appear the globules in the circulating fluid disappear. They disappear at the time when the red-pigment of the forming hydranth is rapidly increasing in quantity, and not unnaturally one might suppose that the pigment of the circulating

fluid had been added to the wall where the hydranth is produced. The globules disappear in the region of the new hydranth, but, I think, it can be shown that they do not form any essential part of the hydranth. They may be found stuck together in a ball that lies in the digestive tract of the new hydranth, and when the hydranth is fully formed the pigment is ejected, as Stevens has shown, through the mouth. The development of the new hydranth begins several hours before the red-pigment globules have disappeared from the circulation. The walls in the region of the future hydranth begin to thicken, and, later, pigment develops in the endoderm of this region. The new pigment is formed in the new cells of the endoderm, and does not come from the circulating globules, as shown by the development of very short pieces of the stem. In these the amount of new pigment that develops in the new hydranth may be far greater than that in the whole original piece (Fig. 30, D\ and in this case there can be no question but that new pigment is made in the endodermal cells of the hydranth. The formation of a hydranth, that usually takes place after another twenty-four hours, from the basal end of a long piece, shows that a hydranth may develop when there are no granules in the circulating fluid. These basal hydranths may contain as much pigment as do the distal ones.

Driesch suggested that the red-pigment in the circulating fluid determines quantitatively by its presence how much of a hydranth is formed, or the size of the hydranth in relation to the rest of the piece. There seems to be no evidence in favor of this view and much against it. Loeb has not stated specifically whether he means that it is the pigment in the circulating fluid or that in the walls which acts as a formative stuff; the presumption is that he meant the latter. An examination of the piece during regeneration gives no evidence in favor of the view that the pigment moves into the region of the new hydranth. On the contrary, it remains constant in amount at all points except where the new hydranth is developing, and there is in this region unquestionably a large development of new pigment.

The evidence for and against the idea that the red-pigment of tubularia is a formative stuff, or even building material, has been considered at some length, because it is the only case in which the hypothetical formative stuff has been definitely located in a specific, recognizable substance that can be followed during the process of regeneration. It is well, I think, to give the question full consideration, especially as the hypothesis often appears to give an easy solution of some of the problems of regeneration. In a later chapter the subject will be more fully treated.

Since the red-pigment hypothesis does not explain the phenomenon of the formation of the partial structures in tubularia, we must look for another explanation. As the matter stands at present we can only assume that there is a predisposition of a very small piece to form a larger partial structure than a smaller whole one. This problem of the method of development of small pieces of the stem of tubularia is further complicated by the development in many cases of double hydranths, or double parts of hydranths, as shown in Fig. 29, A-E.