The Organism as a Whole, from a Physicochemical Viewpoint

1 Loeb, J., Artificial Parthenogenesis and Fertilization, Chicago, 1913. I. The writer in a former book (Dynamics of Living Matter, 1906, p. i), 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. Uexkull 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 book1 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.

Boveri1 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 concen-

tration 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^ FlG I0 are determined. 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 FIG. ii is a diminutive whole egg since each contains the three layers in the normal arrangement (Fig. n). 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,

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. 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 mi- -pic. I3 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 nsxt* sfep is the formation of a skeleton by the formation of crystals consisting of the CaCO3by 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. 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. Lyon1 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 larvae 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.y 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.

3. Driesch1 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 gastrulae 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 purpuratus1 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 f ollowing three

constituents o f t h e 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 blastulae 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. x 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 paper1 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 view-point. It is, therefore, only fair to point out that Boveri2 has furnished the facts for a simpler explanation, which seems to have escaped the notice of antimechanists. 3

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 3v. Uexkull makes in his last book (Bausteine zu einer biologischen Weltanschauung, Munchen, 1913, p. 24) the following statement: " Driesch suceeded in showing that the germ cell has no trace of a machine-like structure but consists entirely of equivalent parts." This is not correct.

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 waterimmiscible 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 waterimmiscible phase, like oleic acid or lecithin.1 In his earlier writings 2 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 2 Loeb, J., The Dynamics of Living Matter. New York, 1906. Introductory Remarks. 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. I 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.1 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 LymncBUS turn to the right, those of Planorbis to the left. It was observed by Crampton1, 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. 2

E. B. Wilson3 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.4 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 setae, 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 ample 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. VThat 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, T in the egg just before fertilization when the surface layer of the egg protoplasm 1 Conklin, E. G., Heredity and Environment in the Development of Man. Princeton University Press, 1915. The reader is referred to this book for the literature and main facts on the structure of the egg; it should also be stated that Conklin's book is one of the best introductions to modern biology in the English literature.

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 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.