Zoology
Totipotency of Blastomeres
Hans Adolf Edward DRIESCH. From Der werth der beiden ersten furchungszellen in der echinodermentwicklung. Experimentelle erzeugung von theil- und doppelbildungen, in Zeitschrift für Wissenschaftliche Zoologie, vol. 53, p. 160, Leipzig, 1892; tr. as The potency of the first two cleavage cells in echinoderm development. Experimental production of partial and double formations by L. Mezger, M. and V. Hamburger, and T. S. Hall for this volume.
"Granting that the primordium of a part originates during a certain period, one must, for greater accuracy, describe this by stating that the material for the primordium is already present in the blastoderm while the latter is still flat but the primordium is not as yet morphologically segregated and hence not recognizable as such. By tracing it back we shall be able for every primordium to determine its exact location even in the period of incomplete or deficient morphological organization; indeed, to be consistent, we should extend this determination back to the newly fertilized, even the unfertilized, egg. The principle according to which the blastoderm contains organ primordia preformed in a flat pattern and, vice versa, every point in the blastoderm can be rediscovered in a later organ, I call the principle of organ-forming germareas."
In these words, he formulated the principle so designated by him. Continuing this train of thought, Roux1 discussed in a perceptive manner the difference between evolution, or the metamorphosis of manifoldness, and epigenesis, or the new formation of manifoldness; in his well-known experiments on "half-embryos" (of which only the first part concerns us here) he decided the question under consideration, for the frog egg, in favor of evolution.
A not very generally known work by Chabry is the only further investigation of this kind known to me. His specific explanations and figures make it clear that his results are fundamentally contrary to those of Roux. I wish to mention here that I came to know of Chabry’s work only after the completion of my own experiments.
As to these, I was interested in repeating Roux’s experiments on material which would be resistant, easily obtainable, and readily observable; all three of these conditions are most satisfactorily fulfilled by the Echinoids, which had already served as a basis for so many investigations. My own experiments were carried out upon Echinus microtuberculatus.
The investigations were made in March and April of 1891. They have led me to many other problems closely connected with the present one, problems whose eventual solution will deepen materially our understanding of the part already solved. Nevertheless, I present my results at this time because they have decided with certainty, for my material, the cardinal point, that is, the potency of the two first blastomeres.
MATERIALS AND METHODS
The first week of my stay in Trieste was lost, inasmuch as I obtained almost exclusively useless material. Whereas the following work follows the above mentioned experiments of Roux in content, the method was taken from the excellent cellular researches of the Hertwig brothers. These investigators, by shaking unfertilized eggs, split off pieces and raised them successfully. It is well known that Boveri used the same method for the production of his "organisms produced sexually without maternal characters," although other factors prevented him from carrying out the procedure exactly.
I therefore went to Trieste with the intention of obtaining one of the first half-blastomeres of Echinus by shaking at the two-cell stage, in order to see, provided it lived, what would become of it.
At an average temperature of about 15º C., cleavage of Echinus eggs occurred 1½ to 2 hours after artificial fertilization. Good material, and only such was used, displayed in only a very few instances immediate division into four cells, an inevitable result, according to Fol and Hertwig, of bispermy.
Shaking was done in small glass containers 4 cm long and about 0.6 cm in diameter. Fifty to one hundred eggs were placed in a small quantity of water. In order to obtain results, one must shake as vigorously as possible for five minutes or more; even then one obtains at best only about ten isolated blastomeres and about as many eggs whose membranes are still intact but whose cells are more or less separated within these membranes.
If shaking is done at the moment of completion of first cleavage, events are, so to speak, reversed; the furrow disappears and one obtains a sausage-shaped body whose two nuclei again show connections. In these recombined eggs the furrow reappears in a short time and normal development follows. On the other hand if one shakes too late, the second cleavage occurs prematurely during the shaking. It is therefore necessary to watch carefully for the right moment.
About one half of the blastomeres are, in addition to being isolated, dead; nevertheless I obtained about fifty capable of development. This appears not unfavorable considering the strength of the mechanical treatment, and considering the fact that the isolated blastomeres are in direct contact with the water on at least one side,—a completely abnormal situation. Isolation is obviously possible only where the membrane bursts.
During cleavage the preparations were observed microscopically as often as possible, and during later development usually once every morning and evening.
One more thing about the treatment of the isolated cells. The contents of the glass used for shaking must be poured into fresh sea water as soon as possible since the water has naturally warmed and evaporated.
It was to be expected that the small quantity of water would not be exactly beneficial, nor the bacteria which were especially numerous toward the end of my experiment and were encouraged by disintegrating pieces which had died.
At any rate my method guarantees that one is observing the same pieces on successive days. Unfortunately, Boveri, in his very important experiments, did not succeed in this respect.
But here I anticipate my results. I turn now to a systematic presentation of findings starting with
CLEAVAGE
First a few words about the normal course of events as revealed in Selenka’s excellent investigations.
Following two meridional cleavages there is an equatorial one and the germ now consists of eight cells of equal size. Four of these now give off, toward one pole, four smaller cells, and at the same time the others divide approximately meridionally.
The germ now consists of 16 cells and shows a marked polarity with the four small cells, easily recognized, occupying one pole. Further divisions lead to stages with 28, 32, 60, and 108 ceils (Selenka). The four small cells which originated at the 16-cell stage clearly indicate the animal pole for a long time. I was unable to establish certainly any differences between the cells of the blastula. At a later stage of development, but before the epithelial flattening due to close union of cells has led to the blastula proper, the Echinus germ, especially in the half containing the smaller-celled pole, consists of cellular rings.
How, then, do the blastomeres of the first division stages after isolation by shaking accomplish cleavage, assuming they survive?
I shall first describe the behavior observed in a majority of cases. Not once did I observe a completely spherical rounding up of the isolated cell. It is true that the normally flat surface tends toward sphericalness but its radius of curvature always remains greater than that of the original free surface of the hemisphere. The cell now divides into two and then, perpendicularly to this, into four parts. Normal controls fertilized at the same time now have eight similar cells the same size as our four. Simultaneously fertilized normal controls have at this time eight similar cells.
In the Echinoids no "gliding" of cells normally occurs either in the four-cell stage nor the ½ eight-cell stage (i.e., my four-cell stage). This is significant because it facilitates considerably the interpretation of the following fact.
About 5½ hours after fertilization occurs, untreated germs have divided into 16 parts, as described above, and isolated blastomeres into 8 parts.
At this point begins the really interesting part of my experiment in that the last-mentioned division brings into existence a typical single half of the 16-cell stage as described; that is, it behaves in the way expected of it according to absolute self-differentiation; it is actually a half of what Selenka’s figure shows.
I will now go on to a description of the normal division of my blastomeres, later speaking about the abnormal cases (about 25 %).
I carefully followed the formation of a half-germ of 16 cells, i.e., a typical ½ 32-cell stage. Each of the normal concentric cell rings is present, but each consists of half its normal number of cells. The entire structure now presents the appearance of an open hemisphere with a polarly differentiated opening.
In the majority of cases here referred to as normal, the half-germ presented, on the evening of the day of fertilization, the appearance of a typical, many-celled, open hemisphere, although the opening often seemed somewhat narrowed. As especially characteristic, I will mention here a case upon which I chanced in doing the Roux-Chabry experiment. Instead of one of the blastomeres being isolated, it was killed by the shaking. The living one, which had developed in the above manner into a typical half-formation, was in the afternoon attached to the dead one in the shape of a hemisphere; but by evening its edges were already clearly curled inward.
The cleavage of isolated blastomeres of the two-cell stage of Echinus microtuberculatus is accordingly a half-formation as described by Roux for operated frog’s eggs.
As already mentioned, this is by far the most frequent behavior. One will not be surprised to find modifications of it in view of damage caused by the strong mechanical insult due to shaking. A few words about these exceptions:
In some cases, germs consisting of about 32 cells (½ 64-cell stage) presented by late afternoon a spherical appearance; development was here more compact, so to speak, though following the typical scheme. This occurs because of a closer union of the cells and is a phenomenon possibly similar to Chabry’s "gliding." Normally, the blastomeres of Echinus make contact in only small areas, until shortly before blastula formation.
In other cases—nine were observed in all there was from the outset (i.e. from the 8 or half 16-cell stage) little to be seen of the usual scheme except as to cell number; specifically, the half germ was spherical from the very beginning, and "gliding" was even more pronounced. I wish to mention especially a case in which the eight cells (half 16) were of almost equal size. Had the role of first cleavage here been different and had I here, to put it briefly, perhaps separated the animal from the vegetal pole instead of the left from the right? By analogy with the experiments of Rauber, Hallez, etc., this seems not unlikely.
The first time I was fortunate enough to make the observations described above, I awaited in excitement the picture which was to present itself in my dishes the next day. I must con less that the idea of a free-swimming hemisphere or a half gastrula with its archenteron open lengthwise seemed rather extraordinary. I thought the formations would probably die. Instead, the next morning I found in their respective dishes typical, actively swimming blastulae of half size.
I have already described how toward the evening of the day of fertilization the, as yet not epithelial, hemisphere had a rather narrowed opening and I have emphasized that tracing of individual cells and hence of the side of the opening corresponding to the animal pole proved impossible. True, I occasionally saw two smaller cells somewhere along the edge but attached no meaning to them. The question as to the actual mode of closing of the blastula must for the time being, therefore, remain unsolved. I may perhaps be briefly permitted to indicate the significance of this.
Now another general question the solution of which I intend soon to undertake: how far does the totipotency of the blastomeres go? That is, up to what stage are blastomeres still able to produce a complete, small organism? In the future I shall call these "part-formations" in contrast to Roux’s "half-formations." The polar course of the cleavage, as well as the above hypothesis concerning the closure of the blastula, suggested that perhaps elements of all concentric rings must be present; that would mean, however, that the four-cell stage would be the last from which isolated cells could produce part-formations, since the equatorial cleavage (namely, the third) divides the material into north and south polar rings, so to speak. This is, as stated, for the time being still merely a question; the totipotency of the cells of the four-cell stage seems to me probable in view of the three-quarter + one-quarter blastulae which will be briefly mentioned later. If, on the other hand, the above-mentioned assumption concerning differences in the effect of the first cleavage should prove true, the latter hypothesis, that material from all three rings is necessary for part-formation, would no longer hold.
But let us leave these conjectures and return to the facts. Thirty times I have succeeded in seeing small free-swimming blastulae arise from cleavage as described above of isolated blastomeres; the rest, about 20 cases, died during cleavage or were sacrificed so I could inspect them under higher magnification. Almost all of them at this stage were still transparent and entirely normal structurally though half-sized. I was not, by a method of estimation, able to discover any difference in size between these cells and those of the normal blastula; therefore, the number of cells is probably half the normal number, which is also to be expected from their cleavage behavior.
At the end of the second day, the fate of the experimental cases seemed to be sealed; they showed the effects of strong mechanical insult and of the small amount of water. For germs still transparent at this time, one could count on raising them further; unfortunately, this was the case with 15 specimens only, that is half the total.
The Gastrula and Pluteus
In healthy specimens invagination at the vegetal pole usually begins at the end of the second day; on the morning of the third day little gastrulae swam about actively in the dishes. As stated, I succeeded in observing 15 such specimens.
Three of the formations finally became actual plutei, differing from the normal only in size.
Therefore, these experiments show that, under certain circumstances, each of the first two blastomeres of Echinus microtuberculatus is able to produce a normally developed larva, whole in form and hence a part-, not half-, formation.
This fact is in fundamental contradiction to the theory of organ-forming germ areas, as the following simple consideration specifically demonstrates.
Imagine a normal blastula split along the median plane of the future pluteus; let us now examine one of the hemispheres preserved this way, for instance the left (see Fig. 12). The material at MoMu would normally supply material for the median region, that at L material for the left side. But suppose that we imagine the hemisphere closing, as explained above, to form a sphere but still maintaining polarity along BC. Then Mo will come to lie upon Mu , and hence possibly upon the right side of the future part-formation. Or, if in closure the original median areas supplied materials for the median region of the part-formation, then this could be thought of only as the upper or lower median region. If it is thought of as the upper, then the lower would come from a part which would otherwise have formed the left side. However one regards it, one cannot escape the fundamental difference in the role which identical material is called upon to play depending upon whether one whole- or two part-formations arise from it,—something which can be brought about artificially. "I’l n’est pas des lors permis de croire que chaque sphere de segmentation dolt occuper une place et jouer un role, qui sont assignés a l’avance" (Hallez); not, at any rate, in Echinus.
FIG. 12.
That this is a particularly pleasing result one could scarcely contend; it seems almost a step backward along a path considered already established.
When compared with Roux’s, my results reveal a difference in behavior in the sea urchin and frog. Yet perhaps this difference is not so fundamental after all. If the frog blastomeres were really isolated and the other half (which was probably not dead in Roux’s case) really removed, would they not perhaps behave like my Echinus cells? The cohesion of the blastomeres, conforming to the law of minimal surface formation, is much greater in the frog than in my object.
I have tried in vain to isolate amphibian blastomeres; let those who are more skillful than I try their luck.
It will not have escaped the reader that the results described might throw light on at least one aspect of the theory of
Double Formation
On this subject, I am in a position to supplement what has already been said. If, from one isolated cell of the two-cell stage, a perfect embryo of half-size is formed (namely a part-formation, in contrast to mere division which yields half-formations as in the case of Roux’s frog embryos), it follows unequivocally that both cells of this stage if they are isolated and kept intact will form separate embryos, or twins.
It is highly probable that the separation of blastomeres by shaking was the direct cause of double formation and that without shaking whole-formation would have resulted.
This is certain since part-formations show that an isolated blastomere, pro?? vided it lives at all, always develops into a structure which differs from th?? normal only in size. With other twins, the situation is different, since they are too numerous to be considered accidental formations of this kind, such having never been seen in thousands of larvae observed by the Hertwig brothers and me.
Roux’s theory of double formation, must, together with the principle of organ-forming germ-areas previously discussed, be discarded, at least in its general form. I have already remarked that, for our theoretical conceptions, this might be considered a backward rather than a forward step if establishment of facts did not always constitute progress.
Whether or not mechanical isolation or separation of the first two cleavage cells is the only way to obtain twin formation will be left open at this time.
It is an old controversy whether double-formation takes place by fusion or fission; birds and fish would, as mentioned, elsewhere, provide rather unfavorable material for a solution of this problem which in its usual formulation is rather a descriptive than a fundamental one. The observations communicated by other workers as well as my own experiments establish splitting as a cause, to which I may add on the basis of my results, splitting without postgeneration.
Obviously fission- and fusion-double-formation would be two quite different things, hence twinning could be of a dual nature. It is certain that the above mentioned double-fertilization modifies cleavage in such a way that immediate four-cell formation occurs; this is in support of our position, as shown before.
What forces come into play when the blastula closes? Can perhaps part of this process be understood in physical terms? The cell mass takes the form of a sphere, the form, that is, which possesses minimal surface. Further, why is it, after all, that a strong pulling apart of the half blastomeres without destroying them results in two individuals? These and other questions present themselves, but it were futile to indulge in idle speculation without actual facts.
Summary
If one isolates one of the first two blastomeres of Echinus microtuberculatus it cleaves as if for half-formation but forms a whole individual of half-size which is a part-formation.
Therefore the principle of organ-forming germ-areas is refuted for the observed species while the possibility of artificial production of twins is demonstrated
Addendum: In proof-reading, I will briefly add that I have just succeeded in killing one cell of the two-cell stage of Spherechinus by shaking and in raising from the other half a small pluteus after half-cleavage.
Naples, October, 1891
1Beitrage zur Entwicklungsmechanik des Embryo. I. Zeitschr. f. Biol. Bd. XXI. III. Breslauer ärztl. Zeitschr. 1885. V. Virchow’s Arch. Bd. CXIV.