Regeneration

The development of pieces of the blastula wall, if they are not too small, also shows that the lack of power to develop, found in some of the one-fourth and in many of the one-eighth blastulae, is not the result of any special differentiation that they have undergone during the cleavage period, but is due to their size. A recent series of experiments by Driesch (1900) on the development of isolated blastomeres of the sea-urchin's egg has given more exact data in regard to their limit of power to produce embryos, and has shown the possibilities in these respects of different parts of the egg. By means of a method discovered by Herbst (1900) it is possible to obtain isolated blastomeres more readily than by the somewhat crude shaking process. If the eggs, after fertilization and after the removal of the membrane by shaking, are placed in an artificial sea water, from which all calcium salts have been left out, the eggs divide normally, but the blastomeres are not held firmly together, and readily fall apart if the egg is disturbed. By means of a fine pipette any desired blastomere or group of blastomeres can be picked out. If these are returned to sea water they continue to develop.

Driesch found that the one-half and one-fourth blastomeres develop into proportionate gastrulae and larvae ; that the one-eighth blastomeres, both of the animal and the vegetative hemispheres, sometimes produce gastrulae, and even the beginning of the larval stage with the rudiments of a skeleton. There are certain differences between the one-eighth larvae that come from the animal hemisphere and those from the vegetative half. More of the one-eighth blastomeres from the animal part of the egg die than from the opposite part, but of those that remain alive a larger percentage reach the gastrula stage than in the case of those from the vegetative pole ; their protoplasm moreover is not so clear as is that of the larvae from the other hemisphere. These "animal pole" blastomeres develop faster than those of the other sort. The gastrulae from the one-eighth blastomeres of the vegetative hemisphere do not die so often after separation, the protoplasm of the larvae is clearer, and they often produce long-lived blastulae with long cilia. The blastulae often develop into gastrulae without mesenchyme. These results show that although whole larvae may be produced from the one-eighth blastomeres of both hemispheres, yet there are certain characteristics that may be referred with great probability to differences that are present in the protoplasm of the two hemispheres of the egg. The differences are not in all cases sufficient to interfere with the production of all the characteristic structures of the embryo, yet traces of the origin of the larvae can be found in their structure. It is probable that the so-called animal (or micromere) pole corresponds to that part of the egg from which the archenteron is produced. Hence the one-eighth blastulae from this hemisphere gastrulate

sooner and in proportionately larger numbers than do those from the opposite hemisphere. The vegetative hemisphere would correspond to that part of the egg from which the wall of the normal gastrula is derived, and this may account for the clearer protoplasm of these embryos, their inability in many cases to gastrulate, their larger cilia, and the absence of mesenchyme in some of them. Driesch finds that the number of cells that go into the mesenchyme of the partial larvae is in proportion to the total number, and that the number of cells in the archenteron is probably also proportionate.1

The smallest blastomeres that produce gastrulae are the onesixteenth products. Out of a total of 139 cases only 31 produced true gastrulae, 5 produced gastrulae with evaginated archenteron, and 103 remained blastulae with long cilia. The one-thirty-second blastomeres were not observed to gastrulate. Driesch ('95) has also made a study of the potentialities of the blastula and gastrula stages of sphaerechinus, echinus, and asterias. If a blastula is cut in half before the mesenchyme cells are produced, both pieces produce gastrulae and larvae. Since some of the pieces probably come from the animal hemisphere, and others from the vegetative hemisphere, it follows that all parts of the blastula possess the power of producing whole embryos, and in this respect the potentialities are the same as for the blastomeres. If the experiment is made at a stage just before the archenteron has begun to develop (Fig. 65, A), the results may be different. A half that contains the region from which the archenteron is about to develop will produce a gastrula and a larva (Fig. 65, A, lower row to right of A). A half that contains only the opposite regions of the egg (Fig. 65, A, upper row) may in some cases gastrulate,2 often abnormally, but as many as half of the pieces do not gastrulate. They may remain alive for a week or more, and even produce a typical ciliated ring with a mouth in the centre, but do not form a new archenteron. These important results show that after the formation of the mesenchyme and archenteron at one pole, the other cells of the blastula wall are no longer able to carry out a process that the same cells were able to carry out at a slightly younger stage, but whether this loss of power is connected with the previous formation of the archenteron, or due to some other change which has by this time taken place in the cells, cannot be determined from the experiment. It is also important to note that these small ectodermal blastulae can still develop whole, typical, ectodermal organs, the ciliated ring and the mouth, and that the former especially has the characteristic structure of the whole normal ring.

1 Driesch's figures seem to show, nevertheless, that the archenterons are proportionately too large. 2 These may be pieces that were cut obliquely, as Driesch suggests, so that they contain a part of the archenteric region. Similar phenomena have been made out by Driesch in the development of the archenteron of the same forms. At the end of the nor- FlG. 65. — A. Blastula of sea-urchin beginning to gastrulaie. Cut in half as indicated by line. Two rows of figures to right show development of upper and lower halves. B. Later gastrula cut in half. Two rows of figures to right show later development. C. End of gastrulation process. Embryo cut in half. Two rows of figures to right show later stages of each half. D. Formation of endodermal pouches from inner end of archenteron. Embryo cut in two. Two rows of figures to right show later stages.

mal gastrula period of the starfish embryo, there is produced from the inner part of the archenteron two outgrowths, or pouches, that later constrict off to give rise to the coelom sac and water-vascular system. If the gastrula is cut in two in such a way that the inner end of the archenteron, i.e. the part from which the pouches develop, is cut off (Fig. 65, C\ it is found that the piece containing the posterior part of the archenteron closes in, forms a new sphere, and from the present inner end of the archenteron (that has also healed over) a pair of pouches is produced (Fig. 65, C, lower row to right of C). These pouches have arisen, therefore, from a more posterior part of the archenteron than that from which the pouches normally arise.

If the same experiment is made at a later stage, when the pouches have been given off from the archenteron (Fig. 65, D, lower row to right of D}, no new pouches are formed. This means that after the archenteron has once produced its pouches it loses throughout all its parts the power to repeat the process, although these parts possessed this power at an earlier stage. It is a very plausible view that the result is directly connected with the formation of the normal pouches, although it is of course possible that some other change has taken place in the archenteron that prevents the formation of the pouches.

In order to give as nearly as possible a consecutive account of the experiments on the eggs of the frog and of the sea-urchin, a number of other discoveries have been passed over. Let us now examine some of the results on other forms. Chabry, as early as 1887, experimented with the eggs of an ascidian. By means of an ingenious instrument he was able to prick and kill individual blastomeres. The results of his experiments were not described very clearly, and later writers have interpreted his results in different ways.1 Chabry stated that he obtained half-embryos from one of the first two blastomeres, but his figures show, especially in the light of later work, that the embryos were whole embryos of half size, although certain organs, as the papillae and the otolith, may be lacking.

Driesch ('95) reexamined the development of isolated blastomeres in one of the ascidians, Phallusia mammalata, and found that the cleavage of blastomeres, isolated by shaking, is neither like that of the whole egg, nor is it like that of half the normal cleavage, although it shows some characteristics of the latter. A symmetrical gastrula is produced, and from this a typical whole larva of half size. These larvae lack, however, one or more papillae, and the otolith rarely develops. The absence of these organs Driesch ascribes to the rough treatment that the egg has received, since embryos from whole eggs may sometimes lack these organs if the development has taken place under unfavorable conditions. The isolated one-fourth blastomere may also produce a whole larva.

Crampton ('97) has also studied the development of the isolated 1 Driesch, Hertwig, Roux, Weismann, Barfurth. For review see Driesch ('95). blastomeres of another ascidian, Molgnla manhattcnsis . He has more fully worked out the cleavage, and finds that the isolated blastomere segments as a part, i.e. as it would have segmented had it remained in connection with the rest of the egg. In general appearance the half-cleavage seems to differ from the half of the complete cleavage, because rearrangements of the blastomeres occur, but despite these shiftings the form of the division is always like that of a part. A whole embryo develops, although there may be defects in certain organs, which are due, he suggests, to the smaller amount of material available for the development of the larva.

Zoja showed in 1894-1895 in a number of jellyfish that the isolated blastomeres produce whole larvae of smaller size.1 In one form, liriope, the endoderm that forms the digestive tract is normally delaminated at the sixteen-cell stage, each cell of the blastula wall dividing into an inner and an outer part. In the blastula from the one-half blastomere this delamination also takes place when sixteen cells are present, and not at the preceding cleavage when only eight cells are present. In this form, therefore, the whole number of cells develops before the delamination takes place, and the one-half larva is composed of the same number of cells as is the normal embryo at this stage, but the cells are only half as large. In other species the endoderm appears to begin to develop in the half-larvae when only half the total number of cells is present.

The conditions in the egg of the bony fishes are very different from those in the preceding forms. The protoplasm, from which the embryo is produced, accumulates at one pole to make the blastodisc. After the cleavage of this blastodisc, the blastoderm that has resulted grows over the yolk sphere at the same time that the embryo is forming along one meridian. I carried out some experiments, in 1895, on the eggs of Funduliis hetcroclitus. If one of the first two blastomeres of the egg of fundulus is destroyed, the remaining one produces a whole embryo. If three of the first four blastomeres are removed, the remaining one may produce a whole embryo of small size. The problem of development is, in the case of the fis-h, different from the other cases described, inasmuch as the whole yolk sphere is left attached to the remaining blastomere and is covered over by cells derived from this blastomere. The smaller embryo that is formed lies on a yolk of full size.2

Wilson's work on amphioxus has been already described in con- 1 Bunting ('94) also found that isolated blastomeres of hydractinia make whole embryos. 2 If the yolk of the dividing egg is partially withdrawn without disturbing the blastomeres, the form of the cleavage may be altered, but a normal whole embryo develops over the smaller yolk sphere. nection with the experiments on the sea-urchin's eggs. Later I ('96) also obtained whole larvae from one-half and one-fourth blastomeres, and I also found that the one-eighth blastomeres do not develop beyond the blastula stage. The number of cells of which the onehalf larva is composed is half that of the normal larva, and the one-

FlG. 66. — Ctenophore-egg and embryo. A. Normal sixteen-cell stage. B. Half-sixteen-cell stage. C. Later half-segmentation stage. D. Later half-embryo. E. Corresponding whole embryo. F. Half-embryo seen from side. G. Same seen from apical end. In F and (/, four rows of paddles present, three endodermal sacs and ectodermal stomach. fourth larva is made up of one-fourth of the total number of cells. In all the preceding cases in which the blastomeres have been separated, a whole embryo has developed, although the cleavage was often like that of a part. In one form, however, it has been found that a whole embryo does not develop. Chun ('92) first showed that the isolated one-half blastomere of the ctenophore egg produced a

half-larva. He also inferred from certain incomplete embryos caught in the sea, that these incomplete larvae could subsequently regenerate the missing parts. Driesch and Morgan ('95) studied the development of the isolated blastomeres of another ctenophore, Bcroe ovata. They found that the isolated one-half blastomere divides exactly as a half of the whole egg (Fig. 66, A, B, C). It remains more or less a half-structure, even after the ectoderm has grown over the whole surface (Fig. 66, D). The invagination of ectoderm, to form the so-called stomach, that takes place at the lower pole of the whole embryo, is formed at one side of the lower pole in the half-embryo (Fig. 66, F, G). It pushes into the endodermal yolk mass, and lies not in the middle, but somewhat to one side. In the normal embryo there are formed four endodermal sacs or pouches in the central yolk mass that become connected with the inner end of the ectodermal stomach, around which they lie symmetrically. In the half-embryo two sacs are formed, and in addition a smaller third sac, which always lies on the side of the stomach that is nearest the outer wall (Fig. 66, F, G). The embryo is, therefore, somewhat more than half the normal embryo in regard to the number of its endodermal sacs.

There are present eight meridional rows of paddles in the normal embryos of the ctenophore. They lie symmetrically on the sides, converging towards an apical sense organ. In the one-half larva there are always only four of these rows of paddles that are not equally distributed over the surface, since on one side there is a wider gap between two of the rows than elsewhere (Fig. 66, G}. The sense plate also lies somewhat eccentrically, i.e. more towards the side corresponding to that at which the other blastomere lay.

If the one-fourth blastomeres are separated, each continues to segment as though still a part of the whole. A one-fourth embryo develops that has an un symmetrical stomach, withtwo endodermal sacs. There are only two rows of paddles. The embryos are, therefore, in several respects one-fourth embryos, but the presence of two endodermal sacs, instead of only one, shows that in this particular, at least, the embryo is more than a fourth of the whole.

The part of the work of Driesch and Morgan, that has a special bearing on the interpretation of the one-half and one-fourth development of the isolated blastomeres, is that in which some experiments are described which consisted in cutting off portions of the unsegmented egg. If a fertilized but unsegmented egg is cut in two by means of a small pair of scissors, the part that contains the nucleus may segment, and give rise to an embryo. The division is generally like that of a part, and in such cases an incomplete embryo develops. The embryo may have fewer rows of swim-plates than has the normal embryo, and fewer endodermal sacs, and the stomach may be in an

eccentric position. The embryos resemble in every respect the incomplete embryos from isolated blastomeres. It is important to note that although the embryos from isolated blastomeres resemble those from pieces of the segmented egg, in the former case the nucleus has divided once, and each blastomere contains half of the original nucleus, while in the latter case the entire segmentation nucleus is present in the piece. These facts seem to show that in this egg the incomplete development is directly connected with the protoplasm, and not with the nucleus, — a view that is maintained by Driesch and Morgan in connection with these experiments.

It was found in one or two instances that the nucleated pieces divided in the same way that the whole egg did, except that the blastomeres are proportionately smaller. From pieces of this kind whole embryos of small size developed. In this case we must suppose that the protoplasm has succeeded in rearranging itself into a new whole of smaller proportions.1 Crampton ('96) has shown in a mollusk, Ilyanassa obsoleta, that when a blastomere is separated from the rest, the cleavage proceeds as though the blastomere or its products were still present, and the larva is defective in those organs that are normally derived from that blastomere. These results are in line with those on the ctenophore egg. Fischel (1900) has also made some experiments on the segmented egg of the ctenophore, and has confirmed several of the results obtained by Driesch and Morgan. In addition he has tried the effect of disturbing the first-formed cells by pushing them over each other, so that their relative positions are changed. He finds as a result that the paddles, sense organ, etc., appear in unusual positions, and the latter may be doubled. This shows that we must regard the material or structural basis of the organs as present very early in the different parts of the egg, and that the organs develop without much regard to their relation to other organs.

Ziegler ('98) has also made some observations on the egg of this same ctenophore, that bear directly on some of the questions here raised. His study of the cleavage shows that the micromeres arise from the part of the egg that is opposite the pole at which the first cleavage furrow appears — the animal pole. Fischel's results have shown that the paddles and the sense organs arise from these micromeres, for, if the latter are displaced the former are also.

Ziegler performed the experiment of cutting off that part of an egg (which has just begun to divide) lying opposite the region in which the first furrow has appeared. In this way there was removed from the unsegmented egg the part from which the micromeres 1 We offered as a possible explanation in this case that the egg had been cut in two symmetrically with reference to the eccentric nucleus. develop. Ziegler found that the micromeres still arise, and that from such pieces larvae develop that have eight rows of paddles and four endodermal sacs. In one case two of the sacs were smaller than the others ; in another case one of the four was very much smaller than the rest. In another operation a large piece was cut from the egg, leaving a small nucleated piece that divided into two blastomeres of unequal size. An embryo developed from this small piece with four endodermal sacs, and only four well-developed rows of paddles. The four rows of paddles that were lacking were represented by two groups of a few plates each.

Ziegler gives a different interpretation of these results from that which Driesch and Morgan have offered. He interprets the last experiment, in which after the operation the piece divided into two unequal parts, and only four rows of paddles appeared, as meaning that the development of these organs on the smaller part is suppressed on account of the small size of the part. If the part had been still smaller all trace of the missing paddles might disappear, as he thinks was the case in certain experiments of Driesch and Morgan. There can be, I think, little doubt that if a piece is small enough, the result would follow as Ziegler supposes. It does not seem probable, however, that the pieces were really below the lower limit in the experiments of Driesch and Morgan, since the smaller blastomere was in one case as large as the whole piece (i.e. as both blastomeres taken together) in one of Ziegler's experiments.

Ziegler's results show very clearly that we are not obliged to think of the substance of the micromeres as laid down in the protoplasm of the egg, and hence there is no ground for supposing the substance of the paddles is necessarily present in the vegetative hemisphere of the egg. His results show that if the vegetative part is cut off, micromeres and paddles are still formed, although that part of the egg substance from which they normally arise has been removed. It should be pointed out, in this connection, that Driesch and Morgan did not suppose that the bases of the micromeres, or of the paddles, are actually laid down in a definite part of the protoplasm of the egg. They had also observed that in some cases whole embryos arose after a part of the egg had been removed, and this they attributed to the symmetrical position of the cut in relation to the organization of the egg. Ziegler's operations were made more or less in this symmetrical plane, excepting the one that gave rise to an incomplete embryo. Driesch and Morgan held that the formative factors become localized in the protoplasm, rather than arise from the nucleus, but pointed out that these observations do not lead to His's conclusion of localized germ areas in the egg.

THE experimental work that Pfluger carried out in 1883 on the effect of gravity on the cleavage of the frog's egg, and the conclusions that he drew from his experiments, mark the startingpoint for the modern study of experimental embryology.1 We can trace the influence of Pfluger's results through most of the more recent work, and one of the conclusions reached by Pfluger, namely, that the material of the egg may be divided by the cleavage planes in any way whatsoever without thereby altering the position of the embryo on the egg, is, I think, one of the most important results that has yet been reached in connection with the experimental work on the egg. Pfluger's analysis of the factors that direct the development has also an important bearing on the interpretation of the development of a whole embryo from a part of an egg.

Pfluger found that in whatever position the frog's egg is turned before it begins to divide, the first two planes come in vertically, and the third horizontally, and that later the smallest cells are always formed in the upper hemisphere. He concluded, therefore, that gravity has some sort of influence in determining the position of the planes of cleavage. Pfluger observed that the position of the median plane of the body of embryos that have developed from eggs turned into unusual positions does not, as a rule, correspond to the plane of the first cleavage, but that the embryo generally lies on that meridian of the egg that passes through the primary egg axis and the highest point of the egg in its new position. Since any meridian may happen to be placed uppermost, the embryo may, therefore, develop upon any one of the primary meridians, and hence the material must be isotropous around the primary axis. Furthermore, since the embryo appears always below the middle of the egg, in whatever position the egg may lie, we must conclude that in each meridian the material is also isotropic.

It may be pointed out that while more recent work has substantiated, on the whole, the latter conclusions2 of Pfluger, just stated, still 1 These experiments have been quite fully described in my book on The Development of the Frog's Egg. the results of studies of regenerative phenomena of organisms show that the conclusions are not necessarily the only ones deducible from the experiments; for, although it may be true that any possible primary meridian of the egg may become the median plane of the body of the embryo, it does not follow that there is no one organized plane always present in the normal egg, i.e. the egg may not be entirely isotropic. That this may be the case is shown in the regeneration of pieces of adult animals in which a piece cut to one side of the old median plane may develop a new plane of symmetry of its own. This possibility must be also admitted for the egg. If we substitute the term " totipotence," meaning that any meridian of the egg has the possibility of becoming the median plane of the embryo, in place of Pfliiger's term " isotropy," we remove this element of possible error from his statement.

Roux and Born have shown that the only action that gravity has on the frog's egg is to bring about a rearrangement of the contents of the egg, a phenomenon that Pfliiger had not observed. The lighter part flows to the highest region of the egg, and the heaviest to the bottom of the egg, hence the change in the position of the cleavage planes observed by Pfliiger that begin in the upper, more protoplasmic part of the egg. Another series of experiments, that we also owe, in the first place, to Pfliiger ('84), consist in compressing the egg before and during its cleavage. The position of several of the cleavage planes may be altered, yet a normal embryo develops from the egg. The same experiment has been repeated by Hertwig ('93), and by Born ('93), on the frog's egg, and by Driesch ('92), Ziegler ('94), myself ('93), and others, on the egg of the sea-urchin, with substantially the same results. The value of the experiment lies not so much in showing that the coincidence between the first cleavage planes and the orienting planes of the body may be lost, as in showing that under these circumstances the nuclei have a different distribution in the protoplasm from that which they hold in the normal egg. Any theory of development that depends on the qualitative distribution of nuclear products during the cleavage period meets with great difficulties in the light of these results, and in order to overcome them will be obliged to add qualifications of such a kind as materially to alter its simplicity. Roux's theory, for instance, comes into this category. Roux ('83) suggested that since the complicated karyokinetic division of the nucleus is carried out in such a way as to insure a precise division of the chromatin, and since the qualities of the male are transmitted to the egg through the chromatin of the spermatozoon, it is probable that the division of the chromatin is a qualitative process, by means of which the elements are distributed to different parts of the egg.

According to Roux, the first division of the frog's egg divides the material of the right half of the embryo from that of the left ; the second division separates the material of the anterior half from that of the posterior half. Roux limited, to a certain extent, his hypothesis to these two divisions of the frog's egg, and stated further that it is not improbable that during the later stages of development there may take place an interaction of the parts on each other, and this interaction would be another factor in the development. Weismann has adopted Roux's hypothesis, and has extended it to all organisms, and to most of the divisions of the developing egg, at least to all those divisions in which the qualities of the layers, tissues, organs, etc., are separated. On this slight basis he has constructed his theory of development and of regeneration It is important, therefore, to examine critically the evidence furnished by experimental embryology for or against this hypothesis of a qualitative division of the egg during the cleavage period.

The development of a half embryo from one of the first two blastomeres of the frog's egg, in Roux's experiment, seemed to support Roux's hypothesis, but it was not long before it was seen that the presence of the other blastomere vitiated the evidence to such an extent as to render it worthless, so far as this hypothesis is concerned. Then followed the experiments with the isolated blastomeres of the sea-urchin, amphioxus, jelly-fish, teleost, ascidian, triton, etc., in which each blastomere, when completely separated, gives rise to a whole embryo. From these experiments Driesch and Hertwig drew the opposite conclusion, namely, that during the cleavage there is a quantitative division of the egg into blastomeres that are equivalent, or at least totipotent. Roux attempted to meet the results of these experiments in two ways. He pointed out that in several of these cases the isolated blastomere divides as a half or as a fourth of the egg, and that in the sea-urchin this leads to the formation of an open half-blastula. In the second place, Roux brought more to the front his subsidiary hypothesis of the reserve germ plasm. He supposed that along with the early qualitative division of the nucleus, by means of which each part receives its particular chromatic substance, there is also a quantitative division of a sort of reserve germ plasm contained in the nucleus. Each cell may receive also a part of this material, and hence each cell may contain the potentialities of the whole egg. This reserve plasm may be awakened by any change that alters the normal development, as, for instance, when the blastomeres are separated. It may take some time for this reserve stuff to wake up, as shown by the half-development of the sea-urchin's egg that goes on for some time after the separation of the blastomeres. This hypothesis cannot be objected to on purely