Regeneration

ing that condition (Fig. 62, D, E, F, G, H\ It is shorter than the normal embryo, and its posterior end is incomplete. When these embryos are cut into sections, it is found that the part that has developed corresponds to the dorsal part of a normal embryo, but the ventral part is continuous with the yolk substance of the injured half (Fig. 62, B, C, J, K), Hertwig interprets these embryos as forms in which the yolk portion of the developing half, together with the whole of the injured blastomere, represents a yolk mass that has not yet been enclosed by the margin of the developing part.

In nearly all the embryos that Hertwig has described, the medullary folds appear eccentrically on the developing half (Fig. 62, D, F, K\ and in some cases they may lie so far to one side that they are situated almost at the edge ; and the less development of one of the folds makes the embryo appear almost like the hemi-embryos obtained by Roux. In fact, one embryo seems to have been a true hemiembryo. Hertwig attributes the eccentric position of the embryo to the eccentric position of the blastopore of an earlier stage, but he does not attempt to account for the eccentricity of the latter.

It is significant in this connection to find that Hertwig obtained other embryos that show a condition of " spina bifida." In these there is an exposure of yolk in the mid-dorsal line between the halves of the medullary folds. Still other embryos in the same series of experiments were only slightly injured, and developed nearly normally. In these cases, Hertwig thinks, the blastomere that was stuck had been only slightly injured, and had partly developed. I have also often observed in this experiment that the injured blastomere may segment and add cells to the developing half, but in such cases the development of the injured half may be less regular than is that of the uninjured half. It seems to me not improbable that in several of the embryos described by Hertwig both blastomeres have taken part in the development. The main points of difference between the results of Roux and of Hertwig cannot, however, be explained in this way, and the explanation is to be found in another direction.

Hertwig emphasizes the view that the injured blastomere is not dead, but exerts an influence upon the other half — an influence of the same kind as that which the yolk of a meroblastic egg has on the protoplasmic portion of the egg from which the embryo arises. He ventured to prophesy that if the injured yolk mass could be entirely removed, the uninjured blastomere would produce a normal embryo without defect, and one like the normal embryo in every respect except in size.1

1 The development of isolated blastomeres of the ctenophore egg shows that this need not be the case. Roux interprets Hertwig's results as due, to the sudden partial post-generation of a part of the injured half of the egg. He thinks that a half-embryo had first developed, and then to this there has been FIG. 63. — A. After Wetzel. Section through an egg (blastula stage) reversed at two-celled stage. B. After Schultze. Double embryo, from reversed two-celled stage, united ventrally.

C, C1. Two views of another double embryo (united dorsally). C2. Cross-section through last. D. After Wetzel. Double embryo united laterally. Z?1. Section through same. quickly added a part of the missing side. This reply fails, however, to meet Hertwig's description of the method of development of the embryos. Later work, however, has put us in a position to give a more satisfactory account of the differences between the results of Roux and Hertwig. It seemed to me that the two kinds of embryos might be due to the different positions of the eggs after the operation. It had been shown by Schultze ('94) that if a normal egg in the twocelled stage is turned upside down and held in that position two embryos develop from the egg (Fig. 63, B, C, D). These embryos are united in various ways, and arise presumably one from each of the first two blastomeres. These results have been confirmed by Wetzel, who examined more fully into the early development of the twin embryos. He showed with much probability that the protoplasm rotates in each blastomere, so that in many cases the lighter part flows, or starts to flow, toward the upper hemisphere of the egg. In this way similar protoplasmic regions of the two blastomeres may become separated, and under these circumstances each blastomere gives rise to a whole embryo. A cross-section through one of the segmentation stages of one of these eggs is shown in Fig. 63, A. The smallest cells are found at the outer side of each half, and the two segmentation cavities lie one in the upper region of each hemisphere. Some of the different kinds of embryos that develop from inverted eggs are shown in Fig. 63, B, C, D. They are united in Fig. 63, B, by their ventral surfaces, and in Fig. 63, C, C1, C2, by their dorsal surfaces, and in Fig. 63, D, D 1, at the sides. These differences are probably accounted for by the different ways in which the protoplasm of the first two blastomeres rotated before the egg divided.

A consideration of these results led me to carry out the following experiment on eggs operated upon by Roux's method. After sticking one of the first two blastomeres, some of the eggs were placed so that the uninjured blastomere kept its normal position, i.e. with the black hemisphere upward. Other eggs were turned, so that more or less of the white hemisphere was upward. From the two kinds of eggs two kinds of embryos were obtained. From those with the black hemisphere upward the embryo was a half-embryo like that described by Roux, while from the eggs with the white hemisphere upward embryos developed that were in many respects whole embryos of half size.1 The explanation of this difference will be obvious from what has been said. When the black hemisphere is uppermost the contents of the uninjured blastomere remain as in the normal egg, and a half-embryo results. When the white hemisphere is uppermost the contents of the uninjured blastomere rotate, so that it generally shifts its relation to the protoplasm in the other injured half, and a

whole embryo develops, as in Schultze's experiment. In one case I obtained a half-embryo from an inverted egg. The result did not appear to be due to a lack of rotation of the protoplasm, because the medullary folds were white, showing that the protoplasm must have changed its position. The result can possibly be explained as due to the protoplasm rotating in each blastomere along the line between the halves, so that it still retains the same relation as that of the normal two-celled stage.

The whole embryos of half size are generally imperfect in certain respects on account of their union with the other half. They resemble in all important points the embryos described by Hertwig, and I see no grounds for interpreting them as embryos of a meroblastic type, but rather as whole embryos of half size, whose development posteriorly and ventrally has been delayed or interfered with by the presence of the other blastomere. It has not been possible to separate the first two blastomeres of the frog's egg, for if one is removed the other collapses. In the salamander, that has a mode of development similar to that of the frog,1 it has been possible to separate the first two blastomeres. Herlitzka, who carried out this experiment, found that each blastomere gives rise to a perfect, whole embryo of half size. We cannot doubt, I think, that the same power of producing a whole embryo is also present in each of the first two blastomeres of the frog's egg. When the two remain in contact in their normal relation to each other, each produces only a half ; when like regions of the two blastomeres are separated, each produces a whole embryo. Thus we see that whatever the factors may be that determine the development of a single embryo from the egg, still each half, and perhaps each fourth also, has the power of producing a whole embryo.

In later papers Roux has stated that he had also, even in his earlier experiments, found other kinds of embryos than the half-embryos that he described. Some of these were whole embryos that had developed from the uninjured blastomere without the injured one taking any part or only a very small share in their formation. He found, he states, all stages between those embryos that had used up all the yolk material of the injured side (though post-generated) and those that had not used any part of it. The latter kind of embryo he does not recognize as a whole embryo of half size in the sense that a single blastomere has developed directly into a smaller whole embryo, but he believes that there must have been formed at first a half-blastula, half-gastrula, half-embryo, and that the last stage completed itself laterally without using any material from the injured half.

1 The plane of the first cleavage has been shown in two urodeles to correspond, not to the median longitudinal plane, but to a cross-plane of the embryo. That the uninjured blastomere may at first segment as a half is not improbable, but that whole embryos are formed only by the formation of new material at the side of a half-embryo is, I think, hardly possible, since the results of Schultze, Wetzel, Hertwig, and myself show that a whole embryo may develop directly out of the material of a single blastomere.

Spemann (1900) has carried out some novel experiments on the eggs of triton, and has shown how in another way double structures may be produced. If a ligature is tied loosely around the egg at the first cleavage exactly along the division plane between the first two blastomeres, it will be found later that the long axis of the single embryo lies, in the great majority of cases, across the ligature, and only in a small percentage of cases does the median plane correspond with that of the ligature, and, therefore, with the first cleavage plane.

If one of the latter eggs is allowed to develop to the blastula stage, and the ligature is then drawn tighter, so that the blastula is completely constricted, an embryo develops from each half. If one of the former eggs is allowed to develop to a stage when the medullary plate is laid down, but is not yet sharply marked off, and the ligature is then tightened, there will be formed (the plane of constriction being across the medullary plate) from the anterior part a normal head with eyes, nasal pits, ears, and a piece of the notochord, and from the posterior part there will be formed, at its anterior end, another new head just behind the ligature. Ear-vesicles develop in this part at the typical distance from the anterior end. The brain that develops has a typical cervical curvature, and eye evaginations appear at the anterior end. The chorda, that extended at first to the anterior end of this region, is partially absorbed.

If the ligature is drawn tighter at a later stage, when, for instance, the medullary plate is plainly visible but is still wide open, a different result is obtained. The posterior part no longer forms a new head at its anterior end, but develops into those structures that it would form normally. In some cases it was found that the region from which the ear develops had been pinched in two, and in consequence a small vesicle appears in front of the constriction and another behind it.

In those cases in which the ligature lies in the median plane of the embryo, it is found that a double anterior end is produced. As the embryo develops it tends to elongate, and in consequence the material is pushed forward on each side of the ligature. A double head is the result. The extent of the doubling depends on the depth of the constriction between the halves. In the most extreme cases two complete heads are formed with an inner nasal pit, eye, and ear on each head, as well as the normal outer ones. The results show that even such complicated structures as the eyes and ears, etc., may arise

from parts of the body where they never appear under normal conditions. A series of experiments that have been made on the eggs of seaurchins has led to equally important results. The earliest experiments are those of O. and R. Hertwig, who, in addition to studying the effect of different drugs on the developing egg, found that fragments of the eggs of sea-urchins, obtained by violently shaking the eggs in a small vial, could give rise, if they contained a

FIG. 64. — Sea-urchin egg and embryo. A. Two-cell stage. B. Same, with blastomeres separated. G. Two half-sixteen-cell stages. C. Open half-blastula stages. D. One of last, later stage, closed blastula of half size. £. Gastrula of half size. F. Whole pluteus of half size. H. A hall-sixteen cell dividing in same way as a whole egg (eight cell) . /. Whole egg at sixteencell stage. nucleus, to small whole embryos. Boveri made the important discovery in 1889 that if a non-nucleated piece of the egg of the sea-urchin is entered by a single spermatozoon, the piece develops into a whole embryo of a size corresponding to that of the piece. Fiedler, in 1891, separated the first two blastomeres by means of a knife, and found that the isolated blastomere divides as a half, but he did not succeed in obtaining embryos from the halves. Driesch has made many experiments, beginning in 1891, with the eggs and embryos of the seaurchin. He separated the first two blastomeres ('91) by means of

Hertwig's method of shaking the eggs, and studied the development of the isolated blastomeres. He found that the cleavage was strictly that of a half, and not like that of a whole egg. The normal egg divides into two, four, and eight equal parts. At the next division, four of the cells divide very unequally, producing four very small cells, the micromeres, at one pole. The four cells of the other hemisphere divide equally (Fig. 64, /). The isolated blastomere divides at first into two equal parts, then again into equal parts. At the next division two of the cells produce micromeres and two divide equally (Fig. 64, G). This is exactly what happens at this division in each half, if the blastomeres are not separated. In later stages a half-sphere is formed that is equivalent to half of the normal sphere (Fig. 64, C). The open side corresponds to the side at which the half would have been united to the other half. Thus up to this point a half-cleavage and a half-blastula have appeared.1

In later stages the open half-blastulae close in, producing a whole sphere that becomes perfectly symmetrical (Fig. 64, D}. A symmetrical gastrula develops (Fig. 64, E) by the invagination of a tube at one pole, and a symmetrical embryo is formed (Fig. 64, F) that resembles the normal embryo except in point of size. Driesch has also found that a number of twin embryos arise from the shaken eggs. They arise from eggs whose blastomeres have been disturbed or shifted, so that each produces a small whole embryo, the two embryos being united to each other in various ways.

In a second paper, published in the following year, Driesch extended his experiments, and attempted to discover how far the " independence " of the blastomeres extends ; i.e. he tried to find out if all the blastomeres resulting from the cleavage are alike. When one of the first four cells is separated from its fellows by shaking, it continues to divide, in most cases as a quarter, and produces later a small spherical blastula. Many of these blastulae, although apparently healthy, never develop further, although they may remain alive for several days. In one experiment only eight out of twentysix reached the pluteus stage, with a typical digestive tract and skeleton.

From these experiments Driesch drew the important conclusion that the cleavage cells or blastomeres of the sea-urchin's egg are equivalent, in the sense that if they were interchanged a normal embryo would still result. A somewhat similar view is expressed in the dictum that the position of a blastomere in its relation to the others determines what part it will produce, if its position is changed it gives rise to another part, etc., — or, expressed more concisely, the

1 In some cases, especially in sphserechinus, even at the eight-celled stage, the blastomeres seem to shift their position, so that a whole sphere of half size is formed. prospective value of a blastomere is a function of its position.1 Driesch extended these experiments further in 1893. His aim was to separate different groups of cells at the sixteen-cell stage in order to see whether the cells around the micromere pole (or "animal-pole ") if separated from those of the opposite (or "vegetative pole") could produce a whole embryo, etc. Eggs whose membranes had been removed by shaking immediately after fertilization were allowed to develop normally to the sixteen-cell stage and were then shaken into pieces. Amongst the groups of cells that were present those that contained the micromeres were picked out. It was found that they give rise to whole embryos. In order to obtain cells that belong to the vegetative hemisphere, the blastomeres were shaken apart at the eight-cell stage, and those groups of cells that in later divisions did not produce micromeres were isolated. From these also whole embryos develop. The results show that the cells of both hemispheres are able to produce whole embryos, and that at the sixteen-cell stage the different parts of the egg are still capable of producing all parts of the embryo. It is important to observe that the results of the experiment do not show that if the normal development goes on undisturbed any part of the egg may become any part of the embryo, for it is highly probable that a definite region of the egg may always produce a definite part of the embryo. The results do show, however, that, even if this is true, any cell has the power of producing any or all parts of the embryo if the normal conditions are changed.

In connection with these experiments Driesch discussed the factors that determine the axial relations of the embryo. If all the cells have the power of producing all parts, what determines in the normal development, and also in the development of a part of the whole, the axial relations of the embryo ? Driesch assumed that the egg has a polar structure, and that the same polarity is found in all parts of the protoplasm. Around this primary axis all the parts are alike or isotropous.2 The origin of the mesenchyme and the position of the archenteron, that develop at one pole, are determined by the polarity of the protoplasm. The plane of bilateral symmetry may appear in any one of all the possible radial planes around the primary axis. The selection of a particular one is due to some accidental difference in the structure of the protoplasm, or to some external factor. In later papers Driesch modified this view, and assumed that along with the primary polarity a bilateral structure also exists in the protoplasm.

1 Hertwig had a year before expressed a similar view in regard to the equivalency of the blastomeres. Wilson ('93) studied the development of isolated blastomeres of amphioxus, and found that it agreed in all essential respects with the mode of development of the blastomeres of the sea-urchin. The isolated blastomeres of the two-cell and four-cell stages produce whole embryos, but the blastomeres of the eight-cell stage develop only as far as the blastula. The blastomeres segment, after separation, in most cases not as a part, but as a whole egg would divide, although the cleavage of the one-eighth blastomere only approaches that of the entire egg, but is never identical with it. Incompletely separated blastomeres give rise to twins, triplets, etc. Wilson agreed with the Hertwig-Driesch conception of the value of the early blastomeres, and accepted the view that the fate of each is a function of its position, and that at first they are qualitatively alike. During the early cleavage he supposed that a change takes place that is slight at the two-cell stage, greater at the four-cell stage, and in the eightcell stage the differentiation has gone so far that the blastomere can no longer return to the condition of the ovum. "The ontogeny assumes more and more the character of a mosaic work as it goes forward."

Loeb ('94) showed that if the eggs of the sea-urchin are placed in sea water, diluted by distilled water, the egg swells and bursts its membrane, so that a part of its protoplasm protrudes. Into this protrusion some of the first-formed nuclei pass, and from both the part remaining in the egg membrane, as well as from the protruding part, an embryo is produced, the two embryos often sticking together. In several cases two to eight separate groups of blastomeres are formed from one egg and develop into whole embryos.1

The question of the number of cells which are produced by the onehalf and one-fourth embryos had not up to this time been determined. Until this was known it could not be stated whether the smaller embryos were miniature copies of the normal embryos in all respects, or whether they assumed the typical form with fewer cells. I found ('95) that the blastula from one of the first two blastomeres contains half the number of cells produced by the whole embryo, and that in the later stages also it contains only about half the normal number. The one-fourth blastomere produces only a fourth of the whole number of cells, and yet can develop with this number, in many cases, into a whole embryo. The one-eighth blastomere produces oneeighth the normal number of cells. In most cases I found that these one-eighth blastomeres do not produce embryos, but occasionally they produce a gastrula, and probably a young pluteus stage.

1 The evidence to show that more than four and certainly more than eight such groups that come from a single egg can produce a pluteus is, I think, insufficient, and the result improbable. The development of nucleated fragments of the egg was also studied in order to find out if they too produce a smaller number of cells than does the whole egg, and a number in proportion to their size. The problem is different in this case, because the nucleus has not divided before the piece is separated, and the results ought to show whether there is a prescribed number of divisions for the egg nucleus, or whether the number of times it divides is regulated by the amount of the protoplasm. It was found that the number of cells produced by each fragment is in proportion to the size of the piece. This shows that the division of the nucleus is brought to an end when the protoplasm has become subdivided to a certain point.

A further examination of the number of cells that are invaginated in these smaller " partial " larvae to produce the archenteron seemed to show that they often use relatively more than their proportionate number. The normal blastula of Sphcerechinus grannlaris contains about five hundred cells and turns in fifty cells, or one-tenth the total number. The one-half and one-fourth embryos, and some of the small embryos from the egg fragments, seemed to invaginate more than one-tenth of their total number of cells.

Driesch (1900) reexamined this point, and found that the embryos from isolated blastomeres may use the proportionate number of cells. I have made a new study of the problem on a larger scale and have found that my earlier statement, as well as that of Driesch, is substantially correct, and that the difference that we found is due to the time at which the embryos gastrulate. Thus the one-half embryos and even the one-fourth embryos, that gastrulate as soon as (or only a little later than) the normal, whole embryos, turn into the archenteron about one-half and one-fourth the number of cells invaginated in the whole embryo ; but those partial embryos that gastrulate later (as most of them do) turn into the archenteron more than a half or a fourth of the number of cells turned in at first by the whole embryo. This difference between the early and the retarded partial embryos is in large part due to a slow increase of cells that takes place during the delay in development.

Driesch ('95) found that pieces of the blastula wall of the seaurchin, if large enough, can also produce a gastrula and embryo. I found that the number of cells in these pieces does not increase appreciably after they are cut off (if the operation has been carried out at the end of the cleavage period), and that the new embryo is organized out of the cells present at the time of removal of the piece from the wall. There is, therefore, in this case no chance for " postgeneration " by means of new cells produced at the side, which Roux has supposed to take place in the frog embryo.