DETERMINISM IN THE FORMATION OF AN ORGANISM FROM AN EGG

1. The writer in a former book (Dynamics of Living Matter, 1906, p. 1), defined living organisms as chemical machines consisting chiefly of colloidal material and possessing the peculiarity of preserving and reproducing themselves. Some authors like Driesch, and v. Uexküll seem to find it impossible to account for the development of such machines from an undifferentiated egg on a purely physico­chemical basis. A study of Driesch’s very interesting and important book[116] shows that he assumes the eggs of certain animals, e. g., the sea urchin, to consist of homogeneous material; and he concludes that nature has solved, in the forma­tion of highly differentiated organisms from such undifferentiated material, a problem which does not seem capable of a solu­tion by physico­chemical agencies alone. But the supposi­tion of a structureless egg is wrong, since Boveri has demonstrated the existence of a very simple but definite structure in the unfertilized egg of the sea urchin; and a similar simple structure has been demonstrated by other authors, especially Conklin, in the eggs of other forms.

In this chapter we shall attempt the task among others of showing how, on the basis of the simple physico­chemical structure of the unfertilized egg, the main organ of self-preserva­tion of the organism, the intestine, is formed through the mere process of cell division and growth. Cell division is the most general of the specific func­tions of living matter and it is the basis underlying the differentia­tion of the comparatively simple structure of the egg into a more complex organism. If cell division and growth were equal in all parts of the egg no differentia­tion would be possible, but the different regions of the unfertilized egg contain different constituents and these, probably on account of their chemical difference, do not all begin to grow or divide simultaneously and equally.

Fig. 9

Boveri[117] found that in the unfertilized egg of the sea urchin Strongylo­centrotus lividus at Naples a definite structure is indicated by the fact that the yellowish-red pigment is not equally distributed over the whole surface of the egg but is arranged in a wide ring from the equator almost to one of the poles. Thus three zones can be recognized in the egg (Fig. 9), a small clear cap A at one pole, a pigmented ring B, and the rest again unpigmented C. Observa­tion has shown that each one of these regions gives rise to a definite constituent of the egg: A furnishes the mesenchyme from which the skeleton and the connective tissue originate; B is the material for the forma­tion of the intestine, and C gives rise to the ectoderm.

The pigment is only at the surface of the egg, and its collec­tion at B indicates only that the material in B differs physicochemically from A and C. The real determiners of the three different groups of organs are three different groups of substances whose distribu­tion is approximately but probably not wholly identical with the regions indicated by distribu­tion of pigment. The intestine-forming material is probably not entirely lacking in C but is contained here in a lower concentra­tion and probably the more so the greater the distance from B; and the same may probably be said for the substances determining mesenchyme and ectoderm forma­tion. Hence the unfertilized egg contains already a rough preforma­tion of the embryo inasmuch as the main axis of the embryo and the arrangement of its first organs are determined.

Fig. 10   Fig. 11

After the egg is fertilized the cell divisions begin. The first division is as a rule at right angles to the stratifica­tion of the egg, each of the two cells contains one-half of the pigment ring (and of each of A and C) (Fig. 10), and after the next division each contains one-fourth of the pigmented part. Each of the four cells is a diminutive whole egg since each contains the three layers in the normal arrangement (Fig. 11). The next divisions bring about an unequal division of the material. Four cells will be formed of ectoderm material C and only little intestine material B, the other four cells containing B and A. These latter form at the next division four very small colourless cells, the so-called micromeres, A (Fig. 12), from which the mesenchyme, skeleton, and connective tissue are formed, four larger cells, B, from which the intestine is formed, and eight cells, C, from which the ectoderm will arise. The separa­tion of the three groups of substances is probably not as complete as our purely diagrammatic drawing (Fig. 12) indicates.

Fig. 12

The cell division proceeds and the cells become smaller and smaller and all gather at the surface of the egg, thus forming a hollow sphere. It is not known what brings about this gathering of the cells at the surface, whether it is protoplasmic creeping or streaming or whether the cells are held by a jelly-like layer which covers the surface of the egg (hyaline membrane) (Fig. 13). Then the cilia are formed at the external surface of these cells and the egg begins to swim; we say it has reached the first larval, the so-called blastula stage. This happens according to Driesch after the tenth series of cell divisions, when the number of cells is theoretically 1024, in reality not quite so many (between 800 and 900). The next step consists in the cells derived from the material A (mesenchyme and micromeres) gliding into the hollow sphere, where they form a ring, the physico­chemical process responsible for this gliding being yet unknown. At the opening of this ring an active growing of the cells of the entoderm into the hollow sphere takes place and the hollow cylinder formed by this growth is the intestine (Fig. 14). Why the cells grow into the hollow sphere and not into the opposite direc­tion is unknown. The next step is the forma­tion of a skeleton by the forma­tion of crystals consisting of the CaCO3 by the mesenchyme cells surrounding the intestine. For the establishment of the principle in which we are interested the descrip­tion of morphogenesis need not be carried farther.

Fig. 13    Fig. 14

This principle which is under discussion here is the development of a purposeful arrangement of organs out of the egg. If we assume that the egg consists of homogeneous material we are indeed confronted with a riddle. Since the facts contradict such an assump­tion but show, as Boveri has pointed out, a prearrangement which allows us to indicate in the unfertilized egg already the exact spot where the intestine will grow into the blastula cavity, we are on solid physico­chemical ground, although many ques­tions of detail cannot yet be answered. Such a preforma­tion as Boveri has demonstrated is only conceivable if the material of the egg has not too high a degree of fluidity; we may consider it as consisting essentially of a semi-solid gel which is not homogeneous throughout the egg but divided into three strata.

2. Lyon[118] tried to ascertain whether by centrifuging the sea-urchin egg it was possible to modify its structure and thereby affect the later embryo. He and subsequent experi­menters found that it only is possible to change the posi­tion of the nucleus and the distribu­tion of the pigment in the egg. It follows from this that the nucleus and the pigment are suspended in rather fluid material, the former in the centre, the pigment at or near the surface. The posi­tion of the nucleus determines the first plane of segmenta­tion, since the nuclear division precedes the division of the cytoplasm of the egg and the plane of nuclear division becomes also the plane of the division of the whole egg—a point which need not be discussed here. It was found, however, by Lyon and the subsequent investigators that the place where the micromeres are formed and where the intestine of the embryo later originates is little influenced by the centrifuging of the egg. The localiza­tion of this spot must therefore be determined by a structure sufficiently solid not to be shifted by the centrifugal force. The intestinal stratum in the egg contains the forerunners of the tissues which secrete hydrolyzing enzymes, e. g., trypsin into the digestive tract.

When the surrounding solu­tion is altered in constitu­tion or when the temperature is too high, the intestine instead of growing into the hollow sphere grows outside, we get an evagina­tion instead of an invagina­tion of the intestine. Such larvæ may live for a few days but they cannot grow into a living organism. The forces which make the intestine grow into the hollow sphere are unknown; it may possibly be only the difference between the tension on the external and internal surfaces of the hollow sphere; under normal condi­tions, the resistance on the inner surface being smaller, the intestine grows into the hollow sphere.

The intestine is one of the organs required for the self-preserva­tion of a more complicated organism, in fact a higher organism without a digestive tract is not capable of living for any length of time. In the gastrula—i. e., the blastula with an intestine—we have an organism which is durable, but the processes leading up to the forma­tion of the intestine are so simple that it is difficult to understand why the assump­tion of a “supergene” should be required in this case.

Fig. 15

3. Driesch[119] was the first to show that if we isolate one of the first two cells of a dividing egg each develops into a whole embryo of half size. This is perfectly intelligible, since each of the two cells contains all the three layers in the normal arrangement (Fig. [10]). The cells divide and the cells having the tendency to creep to the surface of the mass arrange themselves in a hollow sphere, the blastula. Since micromeres and intestine material are present and in their normal posi­tion an intestine will grow into the blastula and a whole organism will result. All of this is as necessary as is the forma­tion of one embryo from the whole egg material. Yet the two half-embryos betray their origin from two cleavage cells of the same egg, in that the two gastrulæ formed are often if not always symmetrical to each other (Fig. 15), as the writer had a chance to observe in the egg of Strongylo­centrotus purpuratus[120] in the following experi­ment. The eggs of the sea urchin Strongylo­centrotus purpuratus are put soon after fertiliza­tion into solu­tions which differ from sea water in two points; namely that they are neutral or very faintly acid (through the CO2 absorbed from the air) instead of being faintly alkaline, and second, that one of the following three constituents of the sea water is lacking; namely: K, Na, or Ca. When the eggs are allowed to segment in such a solu­tion the first two cleavage cells are as a rule in a large percentage of cases—often as many as ninety per cent.—separated from each other, and when the eggs are put into normal sea water (about twenty minutes after the cell division) each cell develops into a normal embryo. In a number of cases the embryos remained inside the egg membrane and did not move until after the invagina­tion of the intestine was far advanced; in such cases it was found quite often that the invagina­tion began at the plane of cleavage at symmetrical points of the two embryos, and the growth of the intestine was symmetrical in both embryos.

This symmetry is probably due to the following fact: the first cleavage plane goes through that spot where the intestine grows into the blastula cavity. If the micromere material does not change its posi­tion after the two cleavage cells are separated and the new blastulæ do not become completely spherical the symmetry which we observed is bound to occur. The occurrence is a confirma­tion of Boveri’s observa­tion. It is natural that Driesch also found that each cell in the four-cell stage should give rise to a full embryo, since each of these cells is in reality a diminutive egg containing the three strata in the right arrangement. When, however, the cells of the eight- or sixteen-cell stage were isolated Driesch’s results were different. In this case the isolated cells from the ectoderm material did no longer all form a gastrula; when such a cell still formed a gastrula it was probably due to the fact that it contained some entoderm material; while the cells taken from the entoderm region all formed embryos and therefore contained ectoderm material.[121] The isolated ectoderm cells of a blastula could no longer form an intestine; they were lacking the entoderm material. It looks as if a gradual migra­tion of all the entoderm material from the ectoderm into the entoderm took place during the blastula forma­tion.

When the contents of the egg are displaced by pressure the result will be determined by the loca­tion of the main mass of the intestine-forming material; where the main mass of this body is located the invagina­tion of the intestine will take place. In his earlier work Driesch assumed from pressure experi­ments that the egg had a great power of “regula­tion.” In a later paper[122] he expressed to a large extent his agreement with Boveri who denied this power of “regula­tion” and showed that the existence of the structure of the egg—i. e., a division into three strata, one forming the ectoderm, the second the entoderm, and the third the mesoderm—was sufficient to explain the various phenomena of apparent “regula­tion.” Driesch’s idea of a regula­tion in this case has often been used to insist upon the non-explicability of the phenomena of development from a purely physico­chemical viewpoint. It is, therefore, only fair to point out that Boveri[123] has furnished the facts for a simpler explana­tion, which seems to have escaped the notice of antimechanists.[124]

The objection may be raised that in accepting Boveri’s facts and interpreta­tion we pushed the miracle only one step farther and that we now have to explain the origin of the structure in the unfertilized egg. This Boveri has done by showing that the egg grows from the wall of the ovary and that that part of the egg which is connected with the wall of the ovary gives rise to the ectoderm layer, while the opposite part gives rise to the mesenchyme and the intestine. This shows a connec­tion between the orienta­tion of the egg in the wall of the ovary and its stratifica­tion. While this does not solve the problem of stratifica­tion in the egg it gives the clue to its solu­tion.

The ultimate origin of stratifica­tion probably goes back to the fact of the presence of watery and water-immiscible substances, such as fats. The experi­ments by Beutner and the writer have shown that the electromotive forces which are observed in living tissues originate at the boundaries between a watery and a water-immiscible phase, like oleic acid or lecithin.[125] In his earlier writings[126] the writer had thought that the colloids had special significance and this idea seems to prevail today; but the actual observa­tions have shown that the phase boundary fat-water is of greater importance. Needless to say the fats if not present in the cell from the beginning can be formed in the metabolism.

4. All the “regulation” in the egg is of a purely physico­chemical character; it consists essentially of a flow of material. If this idea is correct, the apparent power of “regula­tion” of the blasto­meres should differ according to the degree of fluidity and the possibility of different layers separating, and this assump­tion is apparently supported by facts. The first plane of segmenta­tion of the egg is usually the plane of symmetry of the later organism and where the degree of fluidity is less than in the sea-urchin egg, a separa­tion of the two first blasto­meres should easily result in the forma­tion of two half-embryos instead of two whole embryos.

This is the case for the frog’s egg as Roux showed in a classical experi­ment. Roux destroyed one of the two first cleavage cells of a frog’s egg with a hot needle and found that as a rule the surviving cell developed into only a half-embryo.[127] The frog’s egg consists of two substances, a lighter one which is on top and a heavier one below. Although viscous, the two substances are not too viscous to prevent a flow if the egg is turned upside down. O. Schultze found that if a normal egg is turned upside down in the two-cell stage and held in that posi­tion, two full embryos arise, one from each of the two blasto­meres. Through the flow of the lighter liquid in the egg upwards the two halves of the protoplasm on top become separated and develop independently into two whole embryos instead of into two half-embryos. In Roux’s experi­ment this flow of protoplasm was avoided. Morgan showed that if Roux’s experi­ment is repeated with the modifica­tion that the egg is put upside down after the destruc­tion of the one cell, the intact cell will give rise not to a half but to a whole embryo.[128] These experi­ments prove that each of the first two cleavage cells of the frog’s egg represents one-half of the embryo and that a whole embryo can develop from each half only when a redistribu­tion of material takes place, which in the egg of the frog can be brought about by gravita­tion since the egg consists of a lighter and a heavier mass.

When, therefore, in the egg of the sea urchin each of the first two blasto­meres naturally gives rise to a whole embryo it is due to a greater degree of fluidity of the protoplasm and not to a lack of preforma­tion of the embryo in the cytoplasm. This idea is confirmed by the observa­tions on the egg of Ctenophores whose cytoplasm seems to be more solid than that of most other eggs. Chun found that the isolated blastomere of the first cell division produced a half-larva, possessing only four instead of the eight locomotor plates of the normal animal.

It seems that in the egg of molluscs, also, the simple symmetry rela­tions of the body are already preformed. It is well known that there are shells of snails which turn to the right while others turn in the opposite direc­tion. The shells of Lymnæus turn to the right, those of Planorbis to the left. It was observed by Crampton[129], Kofoid, and Conklin that the eggs of right-wound snails do not segment in a symmetrical, but in a spiral, order, and that in left-handed snails the direc­tion of the spiral segmenta­tion is the reverse of that of the segmenta­tion in the right-handed snails. Conklin was able to show that the asymmetrical spiral structure is already preformed in the egg before cleavage. The asymmetry of the body in snails is therefore already preformed in the egg.[130]

E. B. Wilson[131] has found a marked differentia­tion in the eggs of some annelids and molluscs. He isolated the first two blasto­meres of the egg of Lanice, an Annelid. These two blasto­meres are somewhat different in size; from the larger one of the first two blasto­meres, the segmented trunk of the worm originates. Wilson found that

when either cell of the two-cell stage is destroyed, the remaining cell segments as if it still formed a part of an entire embryo.[132] The later development of the two cells differs in an essential respect, and in accordance with what we should expect from a study of the normal development. The posterior cell develops into a segmented larva with a prototroch, an asymmetrical pre-trochal or head region, and a nearly typical metameric seta-bearing trunk region, the active movements of which show that the muscles are normally developed. The pre-trochal or head region bears an apical organ, but is more or less asymmetrical, and, in every case observed, but a single eye was present, whereas the normal larva has two symmetrically placed eyes. The development of the anterior cell contrasts sharply with that of the posterior. This embryo likewise produces a prototroch and a pre-trochal region, with an apical organ, but produces no post-trochal region, develops no trunk or setæ, and does not become metameric. Except for the presence of an apical organ, these anterior embryos are similar in their general features to the corresponding ones obtained in Dentalium. None of the individuals observed developed a definite eye, though one of them bore a somewhat vague pigment spot.

This result shows that from the beginning of development the material for the trunk region is mainly localized in the posterior cell; and, furthermore, that this material is essential for the development of the metameric structure. The development of this animal is, therefore, to this extent, at least, a mosaic work from the first cleavage onward—a result that is exactly parallel to that which I earlier reached in Dentalium, where I was able to show that the posterior cell contains the material for the mesoblast, the foot, and the shell; while the anterior cell lacks this material. I did not succeed in determining whether, as in Dentalium, this early localiza­tion in Lanice pre-exists in the unsegmented egg. The fact that the larva from the posterior cell develops but a single eye, suggests the possibility that each of the first two cells may be already specified for the forma­tion of one eye; but this interpreta­tion remains doubtful from the fact that the larva from the anterior cell did not, in the five or six cases observed, produce any eye.

Conklin has established the existence of a definite structure in the unfertilized eggs of Ascidians, Amphioxus, and many molluscs. In all cases the results of the isola­tion of the first blasto­meres seem to agree with the demonstrable structure of the unfertilized egg.

5. These examples may suffice to show that the egg has from the beginning a simple structure, and we will now point out by which means further differentia­tion may come about. Sachs suggested that all differentia­tion and the forma­tion of every organ presupposes the previous existence of specific substances responsible for the forma­tion. These substances which are now called internal secre­tions or hormones develop gradually during embryonic development. What exists first is a jelly-like block of protoplasmic material with a varying degree of viscosity and with just enough differentia­tion to indicate head and tail end, a right and left, and a dorsal and ventral side of the future embryo.

Aside from such simple differences phenomena of protoplasmic streaming contribute to the further differentia­tion. Such streaming begins, according to Conklin,[133] in the egg just before fertiliza­tion when the surface layer of the egg protoplasm

streams to the point of entrance of the sperm, and these movements may lead to the segrega­tion of different kinds of plasma in different parts of the egg and to the unequal distribu­tion of these substances in different regions of the egg.

One of the most striking cases of this is found in the Ascidian Styela in which there are four or five different kinds of substances in the egg which differ in colour, so that their distribu­tion to different regions of the egg and to different cleavage cells may be easily followed and even photographed while in the living condi­tion. The peripheral layer of protoplasm is yellow and when it gathers at the lower pole of the egg where the sperm enters it forms a yellow cap. This yellow substance then moves following the sperm nucleus, up to the equator of the egg on the posterior side and there forms a yellow crescent extending around the posterior side of the egg just below the equator. On the anterior side of the egg a grey crescent is formed in a somewhat similar manner and at the lower pole between these two crescents is a slate-blue substance, while at the upper pole is an area of colourless protoplasm. The yellow crescent goes into cleavage cells which become muscle and mesoderm, the grey crescent into cells which become nervous system and notochord, the slate-blue substance into endoderm cells, and the colourless substance into ectoderm cells.

Thus within a few minutes after the fertiliza­tion of the egg and before or immediately after the first cleavage, the anterior and posterior, dorsal and ventral, right and left poles are clearly distinguishable, and the substances which will give rise to ectoderm, endoderm, mesoderm, muscles, notochord, and nervous system are plainly visible in their characteristic posi­tions.[134]

We may finally allude briefly to the fact that when once a number of tissues are differentiated each one may influence the other by calling forth tropistic reac­tions. Thus the writer showed that in the yolk sac of the fish Fundulus the pigment cells lie at first without any definite order but that they gradually are compelled to creep entirely on the blood-vessels and form a sheath around them with the result that the yolk sac assumes a tiger-like marking.[135] Driesch[136] has pointed out that the mesenchyme cells are directed in their migra­tion; and it seems that the direc­tion of the growth of the axis cylinder is determined by the tissues into which it grows. The idea of tropistic reac­tions in the forma­tion of organs has been discussed by Herbst.[137]

6. As a consequence of further changes definite anlagen or buds originate later in the embryo which are destined to give rise to definite organs. Thus in the tadpole early mesenchyme cells are formed which are the anlagen for the four legs, which will grow out under the proper condi­tions. These anlagen are specific inasmuch as from the anlage of a foreleg only a foreleg, and from the anlage for a hindleg only a hindleg, will develop. Braus[138] has proved this by transplanting the anlage of a foreleg to different parts of the body. No matter into which part of the body they are transplanted the mesenchyme cells for the foreleg will give rise to a foreleg only; even if they are transplanted into the spot from which the hindlegs grow out under natural condi­tions. There is therefore nothing to indicate “regula­tion.”

The same is true for the forma­tion of the eye and probably in general. We have to consider the forma­tion of the various organs of the body as being due to the development of specific cells in definite loca­tions in the organisms which will grow out into definite organs no matter into which part of the organism they are transplanted. It is at present unknown what determines the forma­tion of these specific anlagen. They may lie dormant for a long time and then begin to grow at definite periods of development. We shall see later that we know more about the condi­tions which cause them to grow.

7. The fact that the egg, and probably every cell, has a definite structure should determine the limits of the divisibility of living matter. In most cases the complete destruc­tion of a cell means the cessa­tion of life phenomena. A brain or kidney which has been ground to a pulp is no longer able to perform its func­tions; yet we know that such pulps can still perform some of the characteristic chemical processes of the organ; e. g., the alcoholic fermenta­tion characteristic of yeast can be caused by the press juice from yeast; or characteristic oxida­tions can be induced by the ground pulp of organs. The ques­tion arises as to how far the divisibility of living matter can be carried without interfering with the total of its func­tions. Are the smallest particles of living matter which still exhibit all its func­tions of the order of magnitude of molecules and atoms, or are they of a different order? The first step toward obtaining an answer to this ques­tion was taken by Moritz Nussbaum,[139] who found that if an infusorian be divided into two pieces, one with and one without a nucleus, only the piece with a nucleus will continue to live and perform all the func­tions of self-preserva­tion and development which are characteristic of living organisms. This shows that at least two different structural elements, nucleus and cytoplasm, are needed for life. We can understand to a certain extent from this why an organ after being reduced to a pulp, in which the differentia­tion into nucleus and protoplasm is definitely and permanently lost, is unable to accomplish all its func­tions.[140]

The observa­tions of Nussbaum and those who repeated his experi­ments showed that although two different structures are required, not the whole mass of an infusorian is needed to maintain its life. The ques­tion then arose: How small a fraction of the original cell is required to permit the full maintenance of life? The writer tried to decide this ques­tion in the egg of the sea urchin. He had found a simple method by which the eggs of the sea urchin (Arbacia) can easily be divided into smaller fragments immediately after fertiliza­tion. When the egg is brought from five to ten minutes after fertiliza­tion (long before the first segmenta­tion occurs) into sea water which has been diluted by the addi­tion of equal parts of distilled water, the egg takes up water, swells, and causes the membrane to burst. Part of the protoplasm then flows out, in one egg more, in another less. If these eggs are afterward brought back into normal sea water those fragments which contain a nucleus begin to divide and develop.[141] It was found that the degree of development which such a fragment reaches is a func­tion of its mass; the smaller the piece, the sooner as a rule its development ceases. The smallest fragment which is capable of reaching the pluteus stage possesses the mass of about one-eighth of the whole egg. Boveri has since stated that it was about one twenty-seventh of the whole mass. Inasmuch as only the linear dimensions are directly measurable, a slight difference in measurement will cause a great discrepancy in the calcula­tion of the mass. Driesch’s results disagree with the statement of Boveri and support the observa­tion of the writer.

If we raise the ques­tion why such a limit exists in regard to the divisibility of living matter, it seems probable that only those fragments of an egg are capable of development into a pluteus which contain a sufficient amount of material of each of the three layers. If this be correct, it would certainly not suffice to mix the chemical constituents of the egg in order to produce a normal embryo; this would require besides the proper chemical substances a definite arrangement or structure of this material. The limits of divisibility of a cell seem therefore to depend upon its physical structure and must for this reason vary for different organisms and cells. The smallest piece of a sea-urchin egg that can reach the pluteus stage is still visible with the naked eye, and is therefore considerably larger than bacteria or many algæ, which also may be capable of further division.

8. The most important fact which we gather from these data is that the cytoplasm of the unfertilized egg may be considered as the embryo in the rough and that the nucleus has apparently nothing to do with this predetermina­tion. This must raise the ques­tion suggested already in the third chapter whether it might not be possible that the cytoplasm of the eggs is the carrier of the genus or even species heredity, while the Mendelian heredity which is determined by the nucleus adds only the finer details to the rough block. Such a possibility exists, and if it should turn out to be true we should come to the conclusion that the unity of the organism is not due to a putting together of a number of independent Mendelian characters according to a “pre-established plan,” but to the fact that the organism in the rough existed already in the cytoplasm of the egg before the egg was fertilized. The influence of the hereditary Mendelian factors or genes consisted only in impressing the numerous details upon the rough block and in thus determining its variety and individuality; and this could be accomplished by substances circulating in the liquids of the body as we shall see in later chapters.