Chapter III: The Internal Factors of Regeneration in Animals (2)

in all these cases the basal hydranth develops about twenty-four hours after the apical one. In the short pieces, however, the two ends develop at the same time, although the development of all the short pieces, whatever structures they may produce, whether single or double, is delayed, and the hydranths may not appear until after the long pieces have produced their basal hydranths. In these double structures both ends develop at the same time (Fig. 30, _D_). If we suppose the influences that start the development of the piece begin first at the distal end, the region affected will lie so near to the proximal end of the piece that the development at this end may be hastened, and under these circumstances the region of new formation will be shared by the two hydranths. The factors that determine that a larger, partial structure is formed in preference to a smaller whole one will no doubt be found to be the same in these double structures and in the single ones.

_THE INFLUENCE OF THE OLD PARTS ON THE NEW_

One of the most striking and general facts connected with the phenomenon of regeneration is that the new part that is built up on the exposed surface is like the part removed. This suggests that an influence of some sort starts from the old part and changes the part immediately in contact with it into a structure that completes the old part in that region. We can imagine that the new part that has been changed in this way may act on the new part just beyond it, and so step by step the new part may be differentiated. It is not difficult to show that the phenomenon is really more complicated than this, and that other factors are also acting on the new part; but, nevertheless, that the old part has some such influence is probable. Under certain conditions, however, this influence may be counteracted by other factors, and something different from the part removed may be formed. One example of this sort has already been discussed, namely, that in which after the removal of much of the anterior end of the earthworm or of a planarian, only the distal end comes back. Another case is that in which something different from the part removed is regenerated. If the tip of the eye of the hermit-crab or of other crustaceans is cut off a new eye is regenerated, but if the eye-stalk is cut off near its base an antenna-like organ develops. Herbst has suggested that the presence of the ganglion at the end of the stalk accounts for the regeneration of a new eye, when only the tip of the stalk is cut off. In the absence of the ganglion at the cut-edge the stalk does not produce an eye, but an antenna, as is shown when the eye-stalk is cut off near the base. The factors that determine the development of an antenna instead of an eye have not been discovered. Przibram has shown that when the third maxilliped of portunas, carcinas, or of other crustaceans is cut off near the base, the new appendage that develops is different from the one removed, and resembles a leg in many ways, but if the animal is kept until it has moulted several times the appendage becomes more and more like the part removed. Another remarkable case has also been described by Przibram for _Alpheus platyrrhynchus_. In this decapod, the claws of the first pair of legs are different from each other, one being much larger than the other and having a different structure.[28] If the larger claw is thrown off at its breaking-joint, and the smaller one left intact, the latter at the next moult (or sometimes after two moults) changes into the characteristic larger claw and the newly regenerated claw is like the smaller one. If the experiment is repeated on this same animal, _i.e._ if the newly acquired large claw is removed, then at the next moult the smaller claw becomes the larger one and the new claw becomes the smaller one--the conditions now being the same once more as at the beginning. If both claws of an animal are thrown off at the same time, two new claws regenerate that are both of the same size, and each is a small copy of the claw that was removed. As yet no experiments have been made that show what factors regulate the development of each kind of claw.

Returning again to the question of the regeneration of parts similar to the ones removed, there are some interesting results that Peebles has obtained in the colonial hydroids, podocoryne and hydractinia. These colonies consist of three principal sorts of individuals: the nutritive, the reproductive, and the protective zooids. Peebles has found that if the stalks of these zooids are cut into pieces, each produces the same kind of zooid as was originally carried by that stalk. Pieces of the stem of the nutritive zooid produce new nutritive zooids at the anterior end of the piece, and sometimes also at the basal end. A similar statement may be made for each of the other kinds. Another method of regeneration sometimes takes place, when, for instance, a piece of the stalk of a nutritive individual is left undisturbed without being supplied with fresh water. It sends out root-like stolons instead of producing a new zooid. The stolons appear first at the ends of the piece, but may later also appear at several points along the piece. They make a delicate network, and the original piece may entirely disappear in the stolons. After several days new feeding zooids grow out at right angles to the stolon network. Pieces of the stalk of protective zooids may also produce stolons, but they spread less slowly, and the formation of new individuals was not observed. In one case a piece of a reproductive zooid made a short stolon, and from it arose a new individual that seemed to be a nutritive zooid. If the latter result proves to be true, we see that a piece may produce a new part that is of a different kind from that of which the piece itself was once a part, but this is brought about by the formation of a stolon that is itself one of the characteristic structures by means of which these colonial forms produce new nutritive zooids. In this case there is a return of the piece to a simpler form, the stolon, and, acting on this, the factors that produce nutritive zooids may bring about new nutritive zooids. The influence of the old structure is lost when the piece assumes a new character.

Another series of experiments gives an insight into an internal factor of regeneration that may prove, I think, to be one of some importance and help in interpreting certain phenomena. If the head-end of a planarian is cut off, the posterior piece split along the middle line, and one side cut off, just above the lower end of the longitudinal cut, as shown in Fig. 31, _A_, it will be found that, if the long and the short sides are kept from uniting along the middle line, each half will produce a new head on its anterior surface (Fig. 31, _C_). If the two halves grow together, and the anterior surface of the shorter piece becomes connected with the anterior surface of the longer piece by means of the new tissue that develops along the inner side of the latter (Fig. 30, _B_), then a head appears only on the anterior half. The development of a head on the shorter half is prevented by the establishment of a connection with the new side. Sometimes an abortive attempt to produce a head is made, but the posterior surface fails to produce anything more than a pointed outgrowth. If we attempt to picture to ourselves how this influence of the new side on the posterior surface is brought about, we can, I think, most easily conceive the influence to be due to some kind of tension or pull of the new material which is of such a sort that it restrains the development of a head at a more posterior level. We can picture to ourselves the same kind of process taking place in the regeneration of the tail of a fish from an oblique surface. The maximum rate of growth is found over that part of the cut-surface that is nearer the base of the tail (Fig. 40). At all other points the growth is retarded, or held in check, and it can be shown that the suppression is connected with the formation of the typical form of the tail in the new part. If we cannot actually demonstrate at present that this is due to some sort of tension between the different parts which regulates the growth, we find, nevertheless, that it is by means of some such idea as this that we can form a clearer conception of how such a relation of the parts to each other is established. In a later chapter this subject will be dealt with more fully.

_THE INFLUENCE OF THE NUCLEUS ON REGENERATION_

The influence of the nucleus on the process of regeneration has been shown in a number of unicellular forms. It was first observed by Brandt in 1877 that pieces of _Actinosphærium eichhornii_ that contain a nucleus assume the characteristic form, but pieces without a nucleus fail to do so. Schmitz (’79) found that when the wall of the many-celled siphonocladus is broken, the protoplasm rounds up into balls, some of which contain one or more nuclei, while others may be without nuclei. The nucleated pieces produce a new membrane, and later become typical organisms, but non-nucleated pieces do not form a new membrane, and soon disintegrate. Nussbaum (’84, ’86) cut into pieces the ciliate infusoria, oxytricha and gastrostyla. Those pieces that contained a nucleus quickly regenerated a new whole organism of smaller size, that had the power of further reproduction, while the pieces that did not contain a part of the nucleus showed no evidence of regeneration; and, although they continued to move about for as much as two days, they subsequently disintegrated. Gruber obtained the same result on another ciliate infusorian, _Stentor cœruleus_. He found that, although the non-nucleated pieces close over the cut-surface, and move about for some time, they eventually die. He further showed that a non-nucleated piece containing a portion of a new peristome in process of formation will continue to develop this new peristome, although a new peristome is never produced by a non-nucleated piece under other circumstances. He believes that if the new peristome has begun to be formed under the influence of the old nucleus, it may continue its development after the piece is severed from its connection with the nucleus. A non-nucleated piece containing a part of the _old_ peristome does not produce a new peristome from the old piece. Gruber observed that a non-nucleated piece of amœba behaves differently from a nucleated piece, and dies after a time.

Klebs found that when certain algæ are put into a solution that does not seriously injure them, but causes the protoplasm to contract into balls, some of these contain nuclei, others not. If, for instance, threads of zygnema, or of spirogyra, are placed in a 16 per cent solution of sugar, the protoplasm of each cell breaks up into one or more clumps, some with nuclei, others without. Both kinds may remain alive for a time; some of the non-nucleated pieces may live for even six weeks. The nucleated pieces surround themselves at once, when returned to water, with a new cellulose wall, but the non-nucleated pieces remain naked. The latter can, nevertheless, produce in the sunlight new starch that is used up in the dark and is made anew on the return to light.[29]

Balbiani (’88) found that non-nucleated pieces of cytrostomum, trachelus, and protodon failed to regenerate, and Verworn (’89 and ’92) obtained similar results on several other protozoa. Similar facts have been made out by Hofer (’89), Haberlandt and Gerassimoff (’90). Palla (’90) found that in certain cases non-nucleated pieces, especially those from cells in growing regions, can produce a new cell wall; while more recently Townsend (’97) has shown in several forms that non-nucleated pieces do not produce a new cell wall unless they are connected by protoplasmic threads with nucleated pieces. The most delicate connection suffices to enable a non-nucleated piece to make a cell wall, even when the nucleated piece lies in one cell and the non-nucleated in another, the two being connected by a thread of protoplasm that passes through the intervening wall.

If we examine somewhat more in detail some of these cases, we find that when a form like stylonychia is cut into three pieces, the two end-pieces without a nucleus fail to regenerate, while the central piece makes a new entire organism of smaller size. If stentor is cut into three pieces, each piece containing one or more nodes of the macronucleus, each produces a new stentor. If, however, a piece is cut off so that it does not contain a part of the macronucleus, it fails to regenerate. Verworn (’95) succeeded in removing the central capsule with its contained nucleus from the large radiolarian, _Thallasicolla nucleata._ The non-nucleated animal remained alive for some time, but eventually died. The nucleated capsule developed a new outer zone with processes like those in the normal animal. If the nucleus is taken from the capsule, the capsule dies, but shows some traces of the formation of an outer zone. If the protoplasm is removed as far as possible from around the nucleus, the latter does not regenerate new protoplasm, but dies after a time. Verworn concludes that the protoplasm cannot carry on all its normal functions without the nucleus, or the nucleus without the protoplasm.

These experiments sufficiently demonstrate that non-nucleated pieces are unable to regenerate. If we attempt to examine further into the meaning of the phenomenon, we find a few things that appear to have a bearing on the result. The behavior of the non-nucleated pieces shows that the metabolism of the cell has been changed after the removal of the nucleus. In some cases the protoplasm is not able to carry out the process of digestion of the included food substances. This process may be due to some interchange that goes on between the nucleus and the protoplasm, which is stopped by the removal of the nucleus, and, in consequence, the metabolism of the cell is changed. The lack of regenerative power may be due to this change in the metabolism. It cannot be claimed, however, that the result is due to a lack of energy in the pieces, for the incessant motion of the cilia in some kinds of pieces, that goes on for several days, shows that a large store of energy is present. Unfortunately, we do not know enough of the relation that subsists between the nucleus and the protoplasm to be able to state to what the lack of regenerative power is due.

Loeb (’99) has suggested that the lack of power of non-nucleated pieces may be due to a lack of oxidation. The nucleus contains substances which, according to Spitzer, are favorable to the process of oxidation. When the nucleus is removed, the oxidation is supposed by Loeb to be too low to allow the process of regeneration to take place. In support of this view, he points out that while non-nucleated pieces of infusoria live for only two or three days, non-nucleated pieces of plants containing chlorophyl may be kept alive for five or six weeks. Non-nucleated pieces containing chlorophyl can obtain a supply of oxygen, owing to the breaking down of carbon dioxide in the chlorophyl-bodies, and the consequent setting free of oxygen. It should be pointed out, on the other hand, as opposed to Loeb’s view, that non-nucleated pieces of amœba have been kept alive for fourteen days; and that despite the better oxidation that may take place in non-nucleated pieces of plants, regeneration does not take place.

It has been found that non-nucleated pieces of the egg of the sea-urchin do not segment or develop, and the result is the same whether the pieces come from fertilized or unfertilized eggs. If, however, a spermatozoon enters one of these pieces, the piece will segment, and, as Boveri and later Wilson have shown, it will produce an embryo.

Boveri also tried fertilizing a non-nucleated piece of the egg of one species of sea-urchin with a spermatozoon of another species. He found that the embryo that develops is of the type of the species from which the spermatozoon has come, and he concluded that the nucleus determines the character of the larva, and that the protoplasm has no influence on the form. The evidence from which Boveri drew his conclusion is not beyond question. It has been shown by Seeliger (’95) and myself (’95) that if whole eggs of the species _Sphærechinus granularis_, used by Boveri, are fertilized by the spermatozoa of the other species, _Echinus microtuberculatus_, there is great variability in the form of the resulting larvæ. Most of them are intermediate in character between the types of larvæ of the two species, but a few of them are like the paternal type. Vernon (’99) has more recently shown that the character of hybrids is dependent upon the ripeness of the sexual products of the two parents. If, for instance, the eggs (sphærechinus) are at the minimum of maturity, the hybrids are more like the male (strongylocentrotus).

It remains, therefore, still to be shown whether or not the protoplasm has any influence on the form of larva that comes from a non-nucleated piece, fertilized by a spermatozoon of another species. That the nucleus of the male does have an influence on the form of the animal is abundantly shown by the inheritance of the peculiarities of the father through the chromatin of the spermatozoon.

_THE CLOSING IN OF CUT-EDGES_

One of the most familiar changes that takes place when a cut-edge is exposed involves the rapid covering over of the exposed tissues. This takes place from the margin of the wound, and a layer of cells, usually the ectoderm at first, covers the surface. The closing in is brought about in many forms by the contraction of the muscles of the outer wall of the body. This seems to be the case in the earthworm and in the planarian, as well as in other animals, such for instance as the starfish, holothurian, etc. But in addition to this purely muscular contraction another process takes place, that is less conspicuous in forms in which the muscles bring about the first closing, but which is evident in forms in which the muscles are absent or little developed. I am able to cite two striking cases that have come under my own observation. When a piece is cut from the stem of tubularia, the ends close in twenty minutes to half an hour. The body wall, the cœnosarc, composed of the two layers of ectoderm and endoderm, withdraws a little from the cut-edge of the outer hard tube, or perisarc, that covers the stem, and then begins to draw across the open end. A perfectly smooth, clean edge is formed that advances from all points to the centre, where the final closing takes place. The closing is not due to an arching over of the cœnosarc, but the thin plate is formed standing nearly at right angles to the outer tube. This plate is composed of two layers of cells, of which there are a number of rows arranged concentrically between the centre and the outer edge. In the absence of muscle-fibres in the stem, the result cannot be due to a muscular contraction, and even if short fibres existed the transportation of cells entirely across the open end would speak against this interpretation.[30] Since the closing over takes place without any support, we cannot suppose the process to be due to any sort of cytotropic effect. The closing takes place equally well in diluted sea water and in stronger solutions. The method of withdrawal of the cells, as best seen when longitudinal pieces are studied, resembles very much the withdrawal or contraction of protoplasmic processes in the protozoa, and so far as one can judge from resemblances of this sort, the two processes appear to be the same.

This closing in of the cut-surface, while a preliminary step in the process of regeneration, cannot, I think, be regarded as a part of the regeneration in a strict sense. That the two processes are not dependent on the same internal factors is shown by the following experiments: If a bunch of tubularia is kept in an aquarium, it will produce new heads two or three times and then cease, and if after the last-formed heads have died, pieces of the stem are cut off, they close as readily as do pieces from fresh hydroids. Moreover, at certain times of year the species _Tubularia (Parypha) crocea_ lose their heads, and only the stalks remain. Pieces of these stalks will not regenerate new heads, at this time, although they close in as quickly as do pieces at other times of the year when the heads are present and when new ones regenerate.

Another equally good illustration of what seems to be the same phenomenon is found in the closing in of wounded surfaces in the young tadpole embryos. If embryos are taken from the jelly membranes, or even after they have been set free, and cut in half, each piece quickly covers over the wounded surface by means of the ectodermal cells. A much more striking illustration of this closing over in the young tadpole is obtained by cutting, with a pair of small scissors, a large piece from the side. The area may be a fourth or more of the entire side, and yet it may be closed over in an amazingly short time. Half an hour or an hour often suffices to cover a large exposed surface. In this case also the wound is covered not by individual cells wandering over the exposed surface, but by a steady advance of the smooth edge of the ectoderm toward a central point. The process is so similar to that which takes place in tubularia that little doubt can remain as to the two being due to the same factors. As there are no muscle fibres present in the part of the frog’s embryo from which the piece is cut off, the result cannot be due to muscular contraction, but appears to be a contractile phenomenon similar to that in tubularia. Even the small piece that is cut from the side of the body shows the same phenomenon. At first it suddenly bends outwards owing to some physical difference between the inner and the outer parts of the piece. Then the edges thicken, bend in, and begin their advance over the inner tissues. The process is seldom completed, since there appears to be a limit to which the ectoderm can be stretched as the edges advance. A most striking phenomenon both in pieces of tubularia and of the frog’s embryo is the entire absence of dead material at the wounded surface. No sooner is the operation performed than the advance begins; and there is not a trace of dying cells or parts of cells to be seen.