The Dynamics of Living Matter

that the solid constituents of the body, e.g. the skeleton, are formed later on, and that its form can, of course, be only indirectly pre- determined in the egg. We are therefore forced to conclude that for the transmission of the hereditary qualities no complicated or morphological structure is required in the sexual cells. This harmonizes with the idea already gathered from the preceding parts of this lecture, that chemical conditions are the bearers of hereditary qualities in the egg, for the instincts as well as the form of the body.

Driesch has shown that when a single cell of the two- or four-cell stage in the development of the egg in the sea urchin is isolated, this cell not only develops into a pluteus, but the mode of development is not essentially different from that of the intact egg.* It would be merely a play on words to speak in such a case of regeneration. The development in the early stages consists in successive divisions of each cell and the creeping of each of these cells to the surface, so that finally the mass of cells thus formed is a sphere with cells at the surface, while a space in the center remains free from cells and is filled with a liquid. The cells at the surface of this hollow sphere then form cilia at their external surface, and in this stage the larva, which now begins to swim through the motion of the cilia, is called a blastula. The next stage in the development is the growing in at one spot of the blastula, of a group of cells, into the hollow space of the sphere; and the cells thus growing in, form finally an inner lining of the cells of the blastula. This process is called the gastrulation, inasmuch as this inner lining is the beginning of the alimentary tract of the larva. At this stage the larva is called the gastrula. Later on large cells are formed, the mesenchyme cells, which creep to certain places in the gastrula, giving rise to the skeleton, probably through the secretion of CaCO3, or of a substance that leads to the formation of CaCO3. According to Driesch, the pro- cess of development of a pluteus from an isolated cell of a two- or four- cell stage of the sea urchin's egg occurs in practically the same way, as in the case of the development of an intact egg; except that the larva developing from a single cell of the two- or four-cell stage is smaller than the normal larva, having only one half or one fourth the mass of the latter.

There may be also slight differences in the development, owing, as I believe, to a kind of hysteresis, inasmuch as the side of the cell which was in contact with the other cells of the egg before the blas- tomeres were separated, acts possibly a little differently from the other sides. These experiments of Driesch are of great importance, inas- * For the extensive literature on this subject, see E. B. Wilson's book, The Cell, New York, 1900 ; or T. H. Morgan's book on Regeneration, New York, 1900.

much as they show how twins, triplets, and quadruplets can originate from one egg. The lack of any complicated structure in the unseg- mented egg is, I believe, evidenced very strikingly in the following observation. I have mentioned in a former lecture that if the egg of a sea urchin (Arbacia) is put into diluted sea water (equal parts of ab (see Figs. 35 and 36) that connects the two spheres.* If this piece is very narrow, as in Fig. 36, twin blastulae will originate from

such an egg; if it is wide, as in Fig. 35, only a single embryo will develop from it. Why this should be so can be readily recognized. We have already stated that the cells have a tendency to creep to the periphery of the egg, thus leaving an empty space in the center which becomes the blastula cavity. When the connecting piece is very narrow, it will be filled with cells, and the two segmentation cavities can and will remain separate, and two blastulae will be formed (Fig.

37). If, however, the piece ab is wide (Fig. 38), an open space will formation of the blastula is concerned there is no preformed structure of any high degree of complication present in the egg ; and this is still more true for the later embryonic formations, which follow the blastula stage. In these deformed eggs the distribution of the nuclear material during cell division is entirely different from that which normally occurs ; yet normal embryos result. Driesch has shown the same in a different way; namely, by submitting the developing eggs to pressure. In eggs thus flattened, the planes of segmentation differ from those of the normal egg, yet normal embryos are formed. These observations exclude the idea that the distribution of the nuclear material through the egg is of importance for the form of the embryo.

Driesch succeeded in causing fertilized sea urchins' eggs to fuse in a number of cases. Such a fusion of the masses of two fertilized eggs into one, resulted in the formation of a sin- gle giant embryo (pluteus).* Such a result would be inconceivable did the egg possess a structure of such a degree of complexity as the adult animal. Zur Strassen \ had already before Driesch's experiments made the observation that the eggs of a parasitic FIG. 38. worm, Ascaris, occasionally give rise to

eggs fuse and that their combined masses now give rise to but one organism. If the egg possessed a complicated structure, the fusion of the masses of two eggs could no more give rise to a single individual of gigantic dimensions than two individual adult animals could be transformed into one by fusing their masses. I have also observed that with the proper chemical treatment the eggs of the starfish and of Ch&topterus can be caused to fuse; that from two or more such eggs a single giant embryo may result.^

Boveri and Driesch assume the existence of a certain simple struc- ture in the unfertilized egg of the sea urchin. According to Boveri, the egg protoplasm consists of three layers occupying different part 5 of the egg (see Fig. 6, p. 31). These three masses can still be recog- nized in the first four cleavage cells, but in the eight-cell stage cells arise which no longer contain all three layers. It is possible that only such isolated cells can give rise to a single embryo, as contain all three layers. This may account for the fact that an isolated blastomere of the four-cell stage can still develop into a normal embryo, while the same is no longer true for the isolated cell of the eight-cell stage. § As far as the possible origin of the differentiation observed by Boveri is concerned, I have noticed in an Ophiurian that the immature eggs

are attached like berries on one side to a tissue which is ramified like the branches of a tree. That side which is attached to the tissue is free from pigment, while the other part is pigmented. I consider it quite possible that the difference in structure observed by Boveri in the egg of the sea urchin, as stated above, is of a purely physical character; namely, that it consists in the fact that different phases are represented in the egg. Two liquids separated by a layer of a more viscous substance might give rise to a differentiation, as noticed by Boveri. If two eggs fuse, the complete union of the corresponding layers in both eggs must be possible, in order to produce single embryos, as would be the case if the contents of two vessels were mixed, each of which contained oil and water.

That the organs which originate later in the larva, e.g. the skeleton, are not preformed in the egg from the beginning is obvious, and re- quires no further discussion. The idea that any structure which may exist in the undivided egg is of the simplest physical kind is also corroborated by the ob- servations made by Chun, Driesch, Morgan,* and Fischel, f on the eggs of Ctenophores. The Ctenophores possess a simple bell-shaped body, the outside of which contains eight ribs or rows of cilia in sym- metrical distribution. The above-named authors found that if the first two cleavage cells of the Ctenophore's egg are isolated, a Cteno- phore originates from each cell; that such a Ctenophore, however, possesses only four ribs. Fischel found that if the egg of a Ctenophore is cut into several pieces, and each piece gives rise to an embryo, the total number of ribs possessed by these larvae never exceeds eight. The body of a Ctenophore consists mainly of a jelly, or gel, and, in all prob- ability, this gel already exists in the egg, and determines its shape and the symmetry relations of the future embryo, inasmuch as the ribs arrange themselves symmetrically on its surface. It is obvious that the degree of preformed structure in this case need not exceed the sym- metry relations due to simple physical conditions. The difference between the Ctenophore's egg and the sea urchin's egg deserves some special mention. An isolated blastomere of the two-cell stage of the sea urchin's egg- gives rise to a whole embryo, while an isolated blas- tomere of the same stage in a Ctenophore's egg gives rise to but a half embryo, as far as the ribs are concerned. This difference is probably due to a difference in the viscosity of the contents of the two eggs, the fluid contents of the sea urchin's egg assuming a spherical shape again after isolation ; while the blastomere of the egg of a Ctenophore, being

* Driesch und Morgan, Arckiv fur Entwickelungsmechanik, Vol. 2, 1895. t Fischel, Archiv fiir Entwickelungsmechanik, Vol. 6, 1897. more viscous and jellylike, keeps the hemispherical shape, even after its isolation. If this idea is correct, it ought to be much more difficult to produce a giant embryo by the fusion of the contents of two eggs, in Ctenophores than in sea urchins, inasmuch as their fusion would be more difficult on account of the jellylike consistency of the main mass of the embryo.

It seems that in the egg of Mollusks, 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 Lymnceus turn to the right, those of Planorbis to the left. It had been observed that the eggs of right- wound snails do not segment in a symmetrical, but in a spiral, order. Crampton and Kofoid discovered independently of each other that in left-handed snails an asymmetrical spiral segmentation occurs also, but the direction of the spiral is the reverse of that in the segmentation of the right-handed snails.* The asymmetry of the body in snails is therefore already preformed in the egg. The conditions which de- termine such an asymmetry may be of a very simple character-!

From the facts we have thus far discussed it is obvious that in eggs whose contents possess a high degree of fluidity not much beyond the simplest symmetry relations can be preformed. A higher degree of preformation is only possible where liquid and solid constituents are contained in different parts of the egg. E. B. Wilson { has recently found a still more marked differentiation in the eggs of some Annelids and Mollusks than the cases thus far discussed. Wilson 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. 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 pos- terior cell develops into a segmented larva with a prototroch, an asym- metrical 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

* Crampton, New York Academy of Sciences, 1894; Kofoid, Proceedings of the Am. Academy of Arts and Sciences, Vol. 29, 1894. has two symmetrically placed eyes. The development of the anterior cell contrasts sharply with that of the posterior. This embryo like- wise 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 Dentaliiim. None of the indi- viduals 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 mate- rial 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 Dental ium, where I was able to show that the posterior cell contains the material for the mesoblast, the foot, and the shell; while the ante- rior cell lacks this material. I did not succeed in determining whether, as in Dentalium, this early localization in Lanice preexists in the un- segmented 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 inter- pretation remains doubtful from the fact that the larva from the ante- rior cell did not, in the five or six cases observed, produce any eye." It should, however, be pointed out that the posterior cell, which in the whole egg only seems to form the segmented trunk of the animal, forms a head if isolated, although the latter in the cases thus far observed was not symmetrical. We do not wish to enter further into this field of experimental embryology, and we refer the reader, in addition to the papers mentioned here, to those published by Chabry, Conklin, Driesch, O. Hertwig, Morgan, Pfliiger, Roux, Schultze, Whitman, Wilson, and many others. It was our intention in this connection only to show that the first structures in the egg do not seem to be beyond the reach of purely physicochemical data.

On the other hand, these data corroborate still further the statement that the early forms of the embryo are determined by the egg, and in no way depend upon the spermatozoon. It has occasionally been suggested that it might be possible to produce an organism from a spermatozoon alone, if the latter were only transplanted into a nutritive medium. This could only be true if the culture medium used possessed also the typical structure of the egg, which is not very likely.

Boveri and others have shown that often very early in the develop- ment, part of the substance of the egg is laid aside as the germ from which the sexual glands develop. While the rest of the egg is transformed into the various organs of the body, this part remains what it is ; namely, embryonic matter. This embryonic matter begins to grow at a certain stage in the development. Miescher* has investigated this phenomenon somewhat in the case of the salmon. The salmon leave the ocean and migrate into rivers to spawn. When they begin to go into the rivers their testicles and ovaries possess little weight, while their muscles are powerfully developed. At this time the testicle is only ToVo to T1j"o °f the weight of the whole animal, while a few months later it is 5 per cent of the body weight. In the female fish the relative weight of the sexual glands is still more considerable. According to Miescher, the salmon do not take up any food while they are in the fresh water. The source of material from which the sexual glands are built up must therefore be in the animal. Possibly through an increase in hydrolytic processes this material gets into the blood and is retained by the sexual glands. Miescher found that the muscles apparently furnished the material from which the glands are built up. The male and female animals behave somewhat differently in regard to the utilization of the material furnished by the muscles. In the sexual gland of the males the protein taken up from the blood is' partly hydrolyzed, and the prod- ucts, according to Miescher, — protamin, guanin, sarkin, — collect in the spermatozoa. In the ovaries this hydrolysis does not occur, and the protein of the blood is utilized for the building up of the eggs whose mass is considerably larger than that of the spermatozoa.

The hydroly- sis of the muscles is due, according to Miescher, to lack of oxygen, caused in his opinion by the diminution in the rapidity of the circula- tion of the blood through the muscles at the time of the growth of the sexual glands. * Miescher, Histochemische und physiologische Arbeiten, Leipzig, 1897. THE investigation of the physicochemical conditions for the for- mation of organs in the egg meets with the difficulty that the germ is too small for a thorough experimental analysis of the processes which occur there. It seems to me that it will be easier to use for such an analysis another series of morphogenetic processes; namely, regen- eration. In many plants and animals when an organ is cut off, a new organ is formed which is identical with the lost organ. The only scien- tific hypothesis of morphogenesis which we thus far possess, — namely, that of Sachs,* — starts with the processes of regeneration.

Sachs takes it for granted that the variety in the form of organs is determined by a corresponding variety in their chemical constitution. As an illustration of the relation between chemical constitution and the formation of organs, he uses his experiences with the influence of light upon the origin of blossoms. If plants, e.g. Tropceolum majus, are put into the dark in spring, their flowering buds which are already formed are not able to develop. In the dark the assimilating power of the green plant is inhibited, and Sachs concluded that the specific substances which are required for the formation of the blossoms cannot be formed by the leaves in the dark. In the light, however, these sub- stances are formed in the leaves, and are carried by the sap from the leaves to the nearest flowering buds. Growth was not restricted in the dark, as was shown by the formation of large (etiolized) shoots in the dark by the same plants. From this fact Sachs concluded that if only the quantity and not the quality of the material circulating in the sap determined the nature of organs, the Tropceolum should have formed flowers; for the mass of the shoots formed in the dark was a multiple of the mass of material required for the production of flowers.

* Sachs, Staff und Form der Pflanzenorgane. Gesammelte Abhandlungen iiber Pflan- zenphysiologie, Vol. 2, p. 1159, Leipzig, 1892. The reader will find a rather complete survey of the literature on Regeneration in Morgan's book on this subject (T. H. Morgan, Regen- eration, New York, 1901). If the tip of the stem alone is put into the dark, while the leaves are exposed to the light, the tip forms blossoms. Therefore Sachs con- cluded that in the light the leaves form substances which are of specific importance for the formation of flowers. He found in support of this view that bulbs of tulips, hyacinths, iris, and crocus, if they are caused to grow in the dark during the spring, produce normal blossoms. In these plants the material and the specific substances necessary for the formation of their flowers were stored up during the preceding year. Sachs generalized this conclusion : Not the quantity of material alone but also the quality is decisive for the formation of organs. There are as many specific substances in a plant as there are different organs. It is obvious that this idea is in full harmony with the experiments of Mendel and De Vries on heredity, inasmuch as this theory ultimately forces us to assume specific substances as the determinants for the hereditary qualities.

To make Sachs's hypothesis as clear as possible, we will quote the following passage: "We may imagine the process (of organization) as being in a way comparable to the successive processes in a chemical factory, where from the original raw material chemical compounds of the greatest variety are formed in succession until the final product is obtained chemically pure, possibly in an extremely small quantity. Although our analyses seem to indicate apparently always the same protoplasm, starch, sugar, fat, we must realize that these substances may themselves differ,* or that traces of other substances may force them to solidify in different organic forms. To give one example, it seems that the formation of flowers and seeds depends upon a storing up of phosphates in these organs, that the plant cannot form flowers and seeds until there is a comparative excess of phosphates com- pared with the other ashes in the saps. . . . An excess of phosphates may force a beet to produce flowers in the first instead of the second year." Sachs applied the same idea to the problem of regeneration. If we cut a piece from the branch of a willow tree, it will under the proper conditions, form roots near the basal and shoots near the apical end. Sachs raises the question as to how it happens that the cutting off of a piece causes the formation of organs in places where it would never occur without this operation or other disturbing conditions. The question is answered by Sachs in harmony with his above-mentioned hypothesis. Duhamel had assumed the existence of two currents of sap in the plant, one ascending, the other descending ; the latter carry- ing root-forming, the former stem-forming, material. Sachs imagines that "as long as a green plant with an upright stem is nourished and

growing, the specific formative substances of the root flow from the assimilating leaves to the root at the lower end of the stem; while the shoot-forming substances flow from the leaves upward to the apices of the twigs. If a piece be cut out from the stem or the root, the cut surfaces form an obstruction where these substances now gather." In such a piece the root-forming substances will therefore gather at the basal end of the twig, and cause here the formation of roots; and the shoot-forming substances will collect at the apical end where they favor the formation of shoots. Sachs's hypothesis finds a beautiful confirma- tion in the phenomena of regeneration in leaves. The leaves are the factories in which the carbohydrates, and perhaps all the specific sub- stances in the sense of Sachs, are formed. The dissolved substances flow from the leaves to the base, and from here to the stem. If pieces be cut from a leaf, e.g. in a Begonia, shoots as well as roots are formed at the basal end of the leaf where the substances flow. Goebel has added a great many beautiful observations on regeneration in plants which support Sachs's conclusion.

The observations of pathologists on compensatory hypertrophia support also in my opinion the idea of Sachs.* If, e.g., one kidney is removed, the other increases in mass. The same seems to hold also for other glandular organs. Pathologists assume that this is due to the greater work now done by the remaining gland. If the hypothesis of Sachs is applicable here, it is possible that certain substances which caused the growth of these glands circulate only in limited quantities in the blood. If a gland be removed on one side of the body, these sub- stances will all flow through the remaining organ which will, therefore, begin to grow.

The only way which seems to lead directly to any information concern- ing the dynamics of regeneration lies in our finding means to substitute at desire one organ for another. The results thus obtained no longer rest upon surmises, but allow us to determine the variables of which the process of regeneration is a function. Sixteen years ago I under- took to bring about such substitutions of one organ for another at desire. I succeeded in a number of hydroids and an ascidian, and called the process heteromorphosis f to discriminate between this phenomenon and the substitution of an organ by an identical one

* Loeb, Untersiichungen zur physiologischen Morphologie der Tiere, Wiirzburg, 1890 and 1891. (Translated in Studies in General Physiology, Chicago, 1905.) t Loeb, loc. cit. regeneration). Since then heteromorphoses have been obtained in a number of animals. Tubularia mesembryanthemum of the Mediterranean and Tubu- laria crocea of the Pacific Ocean are hydroids which consist of an un- divided stem (ss, Fig. 39) from 2 to 6 cm. long, which has a polyp (p, Fig. 39) at the oral, a stolon or foot w at the aboral end. If a piece ab (Fig. 40) is cut from the stem of a Tubularian, according to the observations of Allman, a polyp forms at the oral end a, a stolon at the aboral end b. Allman therefore called this animal "polar- ized," thereby signifying that each element of the stem of the animal possesses a different oral and aboral side. I undertook to bring about a heteromorphosis in this .animal, and succeeded in finding a method by which without fail at j/^ the aboral cut end b of a piece of the stem a polyp can be jf V" produced. This method consisted in putting the piece ab with its oral end a into the sand, while the aboral end b was surrounded by water on all sides. In this case a polyp instead of a stolon was invariably formed at the aboral end b. The oral end which is in the sand does not regenerate, presumably on account of the lack of oxygen. When both ends of a piece ab cut from the stem of a Tubularian are surrounded by sea water, the oral end a invariably forms a polyp. The aboral end b may form a stolon or a polyp. In Tubularia crocea I observed that under such conditions the aboral ends form polyps in about 90 per cent of the cases, and stolons in about 10 per cent. In this case 90 per cent of animals were obtained with a head at either end (see Fig. 41).

There was, however, a marked difference in the velocity of regeneration of the two polyps. The polyp at the oral end forms without exception more quickly than the polyp at the aboral end. The difference in time may vary from two days to two weeks; the difference apparently being less at a high than at a low temperature. Even if the piece of the stem forms a head at either end an intimation of the old polarity still exists, inasmuch as the oral pole is formed earlier than the aboral pole.*

In the following year I was able to show that the formation of the polyp at the aboral end could be accelerated by suppressing the forma- tion of the polyp at the oral end. The mode of procedure of demon- strating this fact was as follows : Pieces ab (Fig. 40) were cut from a number of Tubularian stems ; one half of these pieces were put ver- tically, but reversed, i.e. with the oral end a in the sand, in order to suppress the formation of polyps at this end. The other half of the pieces were also put vertically and reversed into the same aquarium, but in such a way that both cut ends a and b were surrounded by sea water. These latter pieces formed polyps at the oral end a after three or four days, while it took nine days for them to form polyps at the other end. The stems, however, whose oral ends were put into the sand, where consequently the formation of polyps at the oral end was suppressed, formed polyps at the aboral end within three or four days. Hence, the suppres- sion of the forma- tion of the polyp

accelerated con- siderably* the formation of the polyp at the aboral end. The next question was, How can the suppression of the polyp at the oral end accelerate the formation of the polyp at the aboral end? The following experiments were intended to answer this question. r Pieces ab (see Fig. 42) were cut from a number of stems, and each of these pieces was cut in two between c and d. If ~a all four cut ends were surrounded by sea water, polyps were first formed at the oral end of each half; namely, at a and d, where the polyp formation occurred simultaneously. Con- -cd siderably later the polyps were formed at the aboral ends c and b, and here also the polyp formation occurred simul- taneously. It therefore appears as if the polarity in this b case were due to a condition of the nature of a current (e.g. of liquid), by which certain substances were carried through the stem in the direction from the aboral to the oral end. FlG 2 The deposition of certain substances by the current at the anterior end retards the formation of the polyp at the opposite end. The idea that a current is the decisive variable in this case could be tested in another way ; namely, by tying a ligature around the stem in the middle. If this is done, the polarity is done away with and a polyp is formed simultaneously at both ends.f The same experiment

was made from a different point of view independently by Godlewski.* Through the ligature the current which carries away substances from the aboral end is inhibited, and hence the cause for the polarity is removed. When I first made these experiments the hypothesis of Sachs seemed to suggest that in the excised piece of a stem not enough specific polyp- forming material was present to allow the simultaneous forming of two polyps, but the experiments already mentioned exclude this idea. It is further excluded by the observations made by Miss Bickford, who found that even if a stem be cut into a number of small pieces, each oral end of such a piece forms a polyp. Hence it is not a question of lack of material but only a lack of free cut ends, which block the flow of sap, if only one polyp is formed in a piece cut from the stem of a Tubularian. It is therefore apparently the process of streaming itself which may take something away from the aboral end, which is responsi- ble for the fact that a stolon is formed here, or if a polyp be formed that its formation is delayed.