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 eight-cell 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.[117]
The question of the number of cells which are produced by the one-half 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 one-eighth 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.
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” larvæ to produce the archenteron seemed to show that they often use relatively more than their proportionate number. The normal blastula of Sphærechinus granularis 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) reëxamined 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 sea-urchin, 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 “post-generation” by means of new cells produced at the side, which Roux has supposed to take place in the frog embryo.
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 blastulæ, 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 gastrulæ and larvæ; that the one-eighth blastomeres, both of the animal and the vegetative hemispheres, sometimes produce gastrulæ, and even the beginning of the larval stage with the rudiments of a skeleton. There are certain differences between the one-eighth larvæ 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 larvæ from the other hemisphere. These “animal pole” blastomeres develop faster than those of the other sort. The gastrulæ from the one-eighth blastomeres of the vegetative hemisphere do not die so often after separation, the protoplasm of the larvæ is clearer, and they often produce long-lived blastulæ with long cilia. The blastulæ often develop into gastrulæ without mesenchyme. These results show that although whole larvæ 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 larvæ 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 blastulæ 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 larvæ is in proportion to the total number, and that the number of cells in the archenteron is probably also proportionate.[118]