When the yolk is very much increased in amount, the nuclei produced by the division of the zygote nucleus are unable to bring about a surface tension sufficient to divide the cytoplasm, and so we get a multiplication of nuclei without the formation of blastomeres. When this happens segmentation is confined to the animal pole of the egg and results in the formation of a thin disc of blastomeres termed the "blastoderm," resting on an unsegmented “yolk." Such eggs (for instance the hen's egg) are termed " meroblastic (gr. epos, a part) in contradistinction to eggs, like those of the frog, which are completely divided and are termed "holoblastic."

In centrolecithal eggs, like those of the crayfish, the egg appears to be completely divided into cells, but although division may at first be complete, the lowered surface tension of the inner yolky ends of the blastomeres is unable to keep them apart and they flow together so as to form a common inner yolky mass. Such eggs are said to exhibit superficial segmentation. Later, the outer protoplasmic ends of these incomplete blastomeres become completely cut off, so as to form a skin of cells of blastoderm surrounding a central “yolk." A still further modification of this type is found in the eggs of insects in which the yolk is so abundant as to prevent all segmentation. The zygote nucleus alone divides and gives rise to daughter nuclei each surrounded by an island of protoplasm; these are at first dispersed throughout the "yolk" but they gradually migrate to the surface and here form a blastoderm.

In primitive alecithal eggs segmentation results in the formation of a hollow ball of cells one layer thick. This ball is termed the "blastula" and its cavity the blastocoele," "segmentation-cavity" or primary body-cavity." The formation of the blastula marks the accomplishment of an important step in development. Although typically formed only in alecithal eggs, it appears in a modified form in telolecithal eggs, even in those in which there is so much yolk that they have meroblastic segmentation. Thus in the case of the frog the blastula is a hollow ball of which the roof is two cells thick and the floor is many cells thick, whilst in the case of the pigeon the blastula is represented by a stage in which the blastoderm is one layer thick and forms the roof and is separated by a slit-like cavity from the immense mass of the unsegmented yolk forming the floor in the uppermost layer of which are a few nuclei. These nuclei are representatives of the cells which should constitute the vegetative pole of the blastula but they are utterly unable to cut the yolk up into cells.

Formation of Germ Layers.-As soon as the blastula stage has been attained, the formation of layers" begins. The cells at the vegetative pole become turned inwards, forming a tube-like structure which projects into the blastocoele and partially obliterates it. This tube is the primitive gut or "archenteron" and the cells forming it are termed "endoderm," whereas the cells forming the outer wall of the blastula give rise to the primitive skin and are termed "ectoderm." Driesch has shown that until the archenteron begins to be formed all the cells of the blastula of Echinus are alike in their potencies; any sufficiently large piece of it, if cut off, will round itself off and form a blastula and ultimately a perfect larva of diminished size; after a region has been delimited as the centre of the formation of the endoderm the rest of the blastula wall, if cut off, can no longer form an archenteron and so it follows that when the endoderm is differentiated at one place, the rest of the blastular wall becomes changed into definitive ectoderm.

When the archenteron has been formed the developing egg has assumed the shape of a double-walled cup, the opening into which is termed the " blastopore." This stage is clearly and sharply marked in the development of almost all eggs in which the yolk is small in amount, and it can be recognized in an obscured and altered form in the development of large yolky eggs. It is of equal importance to the blastula stage, and it is termed the "gastrula.'

The primary body-cavity has now become reduced to the slit intervening between the wall of the archenteron and the outer wall of the gastrula and this slit becomes largely filled up by the development of the third germ layer, the "mesoderm." We have defined this layer as the primitive peritoneum or lining of the bodycavity, but the body-cavity now indicated is termed the "coelom or "secondary body-cavity" in order to distinguish it from the primary body-cavity. In the eggs of primitive animals, where the yolk is small in amount, the coelom is always formed as a series of pouch-like outgrowths of the archenteron which become cut off from this tube. It follows that the mesoderm is differentiated from the primary endoderm. Driesch has shown that if the front half of the gastrula of the starfish which includes the apex of the archenteron be cut off, the hinder half will heal up and will form a perfect larva, forming, of course, the coelom in the normal way. If, however, this operation be performed after a swelling of the tip of the archenteron -the first rudiment of the coelom-has appeared, then, although the hinder half will heal up and form a larva, it never forms a coelom. Driesch concludes from this experiment that at first all parts of the archenteric wall have the power of giving rise to a coelom, that is of forming mesoderm, but that later a definite portion of this wall becomes set aside as the rudiment of the coelom and that then

1 H. Driesch, "Zur Analysis der Potenzen embryonaler Organzellen," Archiv für Entwicklungsmechanik, vol. ii., 1896.

the rest of it becomes the definitive endoderm devoid of this coelomforming power. In Echinodermata the coelom arises as a single pouch from the apex of the archenteron; in primitive Vertebrata it originates as five pouches of which one is apical and four are paired and lateral; in Chaetognatha and Brachiopoda as a lateral pair of pouches. The remnant of the primary body-cavity becomes almost filled up with cells budded from the wall of the coelom which are termed "mesenchyme." These cells may become joined to one another by their processes and thus constitute a network which becomes converted into connective tissue by the secretion of fibres; or they may remain separate from one another, and then they become developed into blood and lymph cells, the remnants of the primary body-cavity constituting the blood-spaces. In the Coelenterata, in which no coelom is formed, similar cells are budded from both ectoderm and endoderm; in Annelida and Mollusca, in addition to the mesenchyme given off from the coelomic wall, some is likewise budded from the ectoderm, and to this the name mesectoderm has been given. In Vertebrata the most recent research indicates that no mesenchyme is given off from the ectoderm.

Organogeny. Turning now to the third stage of development, viz. the formation of special organs, we find that from the ectoderm are derived the central nervous system and the sense organs, and also the lining of the mouth-cavity and of the terminal portion of the alimentary canal near the anus. The endoderm gives rise to the middle portion of the gut and to the glands which are developed from it, and in Vertebrata to the primitive elastic axis of the back-bone or "notochord." From the mesoderm arise the majority of the muscles, the connective tissue, and, in Vertebrata and Echinodermata, the internal calcareous skeleton which is derived from the connective tissue. The mesoderm also gives rise to the genital organs and their ducts in all Metazoa above the rank of Coelenterata and in Mollusca and Vertebrata to the kidney tubules.

Now we have pointed out that, in telolecithal eggs, segmentation proceeds most rapidly at the animal pole; here the second stage of development rapidly supervenes, and the archenteron is begun before segmentation is even initiated at the vegetative pole. In meroblastic eggs the upper pole of the egg may become converted into an embryo in which all the important organs of the adult are mapped out before the lower pole is even invested with cells. Finally in Amniota (reptiles, birds, and mammals) the lower pole of the egg, after all the yolk has been absorbed from it, is torn from the rest of the embryo at birth and cast off as a useless embryonic membrane. In the earlier article a strong attempt was made to show that the primitive germ-layers do not correspond to one another in different eggs; in a word, that the same name has been given to different things. Some of the arguments adduced are the diverse origins of the mesoderm in various animals, and the alleged origin of the epithelium of the alimentary canal of insects and some other Arthropoda from the ectoderm. The result of the labours of embryologists during the last 15 years has been to establish the universal homology of the germ-layers on an ever firmer basis, and to show that the difficulties alluded to were based on faulty observations.

If, for instance, we define the mesoderm as the wall of the coelom then it is found that this organ originates in one of two ways, viz.: either as a pouch or a mass of cells. The pouch (recognizable in Chaetognatha, Brachiopoda, Echinodermata, Enteropneusta and the lowest Vertebrata) quite clearly originates as an outgrowth from the endoderm; the mass of cells can be traced back to its source in one large cell, the mother mesoderm-cell. This cell, as was first shown by Shearer 3 in the annelid Hydroides and by Conklin in the mollusc Crepidula, originally forms part of the wall of the archenteron and its ejection from this wall is evidently a modification of the more primitive method of coelom-formation by the outgrowth of a gut-pouch. Attempts which have been made by Meissenheimer and Harms to show that in Mollusca the coelom originates from cells budded from the ectoderm are based on obvious blunders in missing out stages in reconstructing the life-history—that most fertile source of error in embryology. Later workers have exposed this error and have shown that in the Mollusca, with which Meissenheimer and Harms dealt, the mother mesoderm cell gives rise to the pericardium which is representative of the coelom in these animals. We have already alluded to the presence of mesectoderm in Annelida and Mollusca; this gives rise to some superficial muscles, but to confound this with the coelomic wall and its derivatives by calling both mesoderm and then to complain that the mesoderm is not an homologous structure in various groups of animals is to introduce a perfectly gratuitous confusion.

We may now turn to the alleged origin of the gut epithelium of certain Arthropoda from the ectoderm. In the earlier article the statement was made that in the embryo of that most primitive of all land Arthropoda Peripatus there is a large slit-like blastopore which later

3 C. Shearer, "On the development and structure of the Trochophore of Hydroides," Quart. Journ. Micr. Sci.,vol. xiii. (N.S.), 1911. Conklin, The Embryology of Crepidula," Journal of Morphology, vol. xiii., 1897.

4

Meissenheimer, Entwicklungsgeschichte von Dreissensia polymorpha," Zeitschrift f. wissenschaftliche Zoologie, vol. Ixix., 1901. 6 Harms, "Postembryonale Entwicklungsgeschichte der Unioni

becomes divided by a constriction into mouth and anus, and that a portion of the gut epithelium, viz. that forming the midventral portion, is formed from ectoderm turned in round the edges of the slit. It is practically certain that this last statement also rests on an error of observation. In the primitive annelid Polygordius, Woltereck1 has described a similar slit-like blastopore and he has followed the process of its closure in great detail describing the division of every cell involved. In this case the midventral epithelium of the gut is formed by the union of endoderm cells lying at the sides of the blastopore whilst the ectoderm cells lying in the blastopore lips by their union reconstitute the midventral skin. No reasonable doubt can be entertained that a renewed investigation with a more modern technique would show that this is also true of Peripatus.

It must never be forgotten that embryological research is based on a comparison of embryos of different ages with one another-not, as would be the ideal method, on a continuous observation of the progress of one and the same embryo. It follows that too large an age-difference between the embryos examined may give rise to a totally wrong conception of the process which is taking place. So are to be explained the statements which crop up from time to time, such as those of Heymons that the mid-gut of the higher insects is entirely formed from ectoderm, and of Watase who made a similar assertion about the mid-gut of the cephalopod Loligo. Hirschler has shown how the error of Heymons originated, and Watase has been corrected by Faussek; and should further statements of this kind occur in the literature the strong presumption is that they also are founded on mistakes.

3

Organ-forming Substances.-We have arrived at the conclusion that the establishment of the validity of the germ-layer theory is one of the great achievements of embryological research, and we now turn to the question of how the differences which distinguish the layers from one another are brought about. We have learnt that in primitive alecithal eggs like those of Echinodermata all portions of the blastula wall are alike in their potencies and that the differentiation of ectoderm from endoderm only begins when the first traces of gut-formation are visible. We have likewise learnt that all parts of the primitive gut or archenteron are alike in their powers, and that the separation of endoderm from mesoderm only becomes apparent when the first indication of the coelom appears. But this progressive differentiation of the embryo might be due to a differentiation of the nuclei of various regions or of the cytoplasm or of both. We have, however, learnt from the development of the polyspermic frog's egg that there is a strong presumption that the nuclei of the embryo are alike in their nature and that the differentiation of the layers must be due to the separation of organ-forming cytoplasmic substances from one another. This conclusion is confirmed by a large number of observations on many different kinds of eggs; a few of the more striking may be given here.

Hertwig allowed frogs' eggs to develop under pressure between glass plates and in capillary tubes. Under these circumstances the divisions took place by planes normal to the pressure and flat plates and rows of cells were produced. When the pressure was removed, however, these deformed embryos recovered, multiplication of cells took place and the normal form was regained and normal development proceeded. It was easy to show that nuclei which under undisturbed conditions would have occupied certain definite regions of the embryo had been forced into quite other regions, and yet perfectly normal embryos resulted. Hertwig concludes that the nuclei could be juggled about like a handful of balls without affecting the formation of the embryo.

7

In many eggs the differentiation of the layers is indicated at a far earlier period than that at which it occurs in the eggs of the Echinodermata or even of the lower Vertebrata like the frog. The egg of the ascidian Cynthia partita which has been studied in great detail by Conklin may be adduced as an example. This egg when it develops becomes converted into an elongated blastula consisting 1 Woltereck, Beitrag zur praktischen Analyse der Polygordiusentwicklung," Archiv f. Entwicklungsmechanik, vol. xviii., 1903. 2 Heymons, Über die Bildung der Keimblätter bei den Insecten." Sitzungsb. der Preussischen Akad. der Wiss., vol. i., 1894. * Watase, Observations on the Development of Cephalopods," Studies from the Biol. Lab. Johns Hopkins Univ., Baltimore., vol. vi.,

1888.

• Hirschler, "Die Embryonalentwicklung v. Donacia crassipes," Zeitschrift für wissenschaftliche Zool., vol. xcii., 1909.

44

* Faussek, Untersuchungen über die Entwicklung der Cephalopoden," Mitteilungen a. d. Zool. Station zu Neapel, vol. xiv., 1920. Hertwig," Ueber den Werth der ersten Furchungszellen für die Organbildung des Embryos," Archiv f. mikroscopische Anatomie, vol. xlii., 1893.

7 Conklin, The Orientation and Cell-lineage of the Ascidian Egg." Journ. Acad. Sciences, Philadelphia. Series 2., vol. xiii., 1905.

of few cells; this blastula changes into a gastrula in the typical way, and though no distinct coelomic pouches are formed large portions of the archenteric wall are directly converted into muscles which lie at the sides of the tail of the tadpole-like larva. In this species the nucleus of the unripe egg is as usual a vesicle filled with fluid (the so-called germinal vesicle). The cytoplasm contains numerous yolk globules of a slaty-blue colour and also larger yellowish globules which are concentrated in its superficial layer. When the maturation divisions of the nucleus occur the nuclear wall is dissolved and the fluid contents escape and form a cap of clear material at the animal pole of the egg. When fertilization takes place profound rearrangements of the substances in the cytoplasm are effected. The yellow globules stream downwards to meet the spermatozoon which enters at the vegetative pole, and they finally form a crescentic layer of yellow material round the lower pole of the egg. As the egg develops first into a blastula and then into a gastrula, and finally into the characteristic ascidian tadpole, it becomes evident that the clear substance forms the ectoderm, the slaty-blue material the endoderm, whilst the yellow material forms the masses of mesoderm which give rise to the tail muscles. When the egg is in the fourcell stage the yellow material is confined to the two posterior cells; if one of these be killed the remainder of the egg will give rise to a larva with muscles only on one side of the tail. That the nuclei have nothing to do with this separation of substances is shown by what occurs at the lip of the blastopore. Here we find an arc of what Conklin calls neurochordal " cells. Each of these has of course a single nucleus, but the cytoplasm of each consists of two zones, one clear and one slaty-blue. At the next division two daughter cells are produced from each neurochordal cell; one of these contains the clear substance and is added to the nerve plate which is a part of the ectoderm; the other is composed of the blue substance and forms part of the notochord which in Cynthia as in other Vertebrata is a derivative of the endoderm,

From this development we conclude that the germinal layers owe their origin to the segregation of cytoplasmic substances in the growing egg; that these substances assume their final arrangement under the influence of the spermatozoön, which thus on its path to meet the female pronucleus determines the symmetry of the embryo. Brachet has shown that this is also true of the frog's egg. It was for long a puzzle why competent observers like Roux and Hertwig" should differ so profoundly on the results of killing one of the first two blastomeres of the frog's egg. Roux asserted that the surviving blastomere gave rise to a half blastula which developed into a half tadpole, whilst Hertwig maintained that it tended to form a normal tadpole. being only impeded in its development by the mass of dead material constituted by the other blastomere. Brachet has shown that both are right, for the plane separating the first two blastomeres need not by any means coincide with the future median plane of the embryo, but may make any angle up to a right angle with it. If it coincides with this plane by killing one blastomere Roux's result is obtained; if it is oblique the result accords with Hertwig's researches. Thus the potency of each of the first two blastomeres of the frog's egg depends entirely on the cytoplasm it happens to include and in no way on the nucleus. Brachet has shown that the fixing of the median plane of symmetry in the frog's tadpole, as in the ascidian tadpole, is effected by the spermatozoon. As the spermatozoon penetrates the egg in its path towards the female pronucleus, it leaves behind a trail of pigment which persists for a considerable time and can be detected at a much later period in the development of the egg. It is found that on the opposite surface of the egg to that at which the spermatozoon enters it, there is formed the so-called grey crescent." This is in reality the upper lip of the blastopore: it is here that the differentiation of ectoderm from endoderm begins. Therefore we conclude that the arrangement of the organ-forming substances in the frog's egg is caused by the spermatozoon.

44

44

In the mollusc Dentalium when the egg has reached the four-cell stage one of the blastomeres emits a protuberance termed the 'yolk-lobe" or polar lobe." This lobe is devoid of a nucleus and before the attainment of the eight-cell stage is reabsorbed into the blastomere. Nevertheless, if this lobe be cut off, the remainder of the egg develops into a larva which is fatally devoid of mesoderm.

That the materials which form the basis of the different substances embodied in the germinal layers are formed in the growing egg under the influence of emissions from the nucleus is rendered certain first by the close relationship of the nucleus to assimilation and secondly by the fact (see CYTOLOGY) that the nucleolus of the unripe egg breaks up into fragments and is extruded into the cytoplasm. It is, however, a surprising fact that the nuclei of the segmenting egg are alike and apparently without influence on the differentiation of the primary organs. In fertilization a second nucleus of alien origin is introduced and portions of this nucleus, as we have already seen, are 8 Brachet, loc. cit.

44

Roux, Über das entwicklungsmechanische Vermögen jeder der beiden ersten Furchungszellen des Eies," Verhandlungen der anatomischen Gesellschaft, 1892.

10 Hertwig. "Über den Werth der ersten Furchungszellen fur Organbildung des Embryos," Archiv für mikroscopische Anatomie. vol. xlii., 1893.

11 Brachet, loc. cit.

incorporated in all these "segmentation" nuclei. Now it is common knowledge that the influence of the father is as potent as that of the mother in heredity and therefore there must arrive a period of development at which the nuclei again influence the cytoplasm.

An attempt to determine this period was made by the writer1 by fertilizing the eggs of Echinocardium with the sperm of Echinus. As we have seen, the result of this cross is in most cases to produce cytolysis of the egg, but in a minority of cases a hybrid develops. The egg of Echinocardium is oval whereas that of Echinus is spherical and the shape of the blastula of each species follows that of the egg. The blastula of the hybrid is oval, like the maternal blastula, and the gastrula is also like that of Echinocardium. But the typical larva (the four-armed echinopluteus) resembles in several points the larva of Echinus; in the vast majority of cases it is totally devoid of a large aboral club supported by a special skeleton which is characteristic of the larva of Echinocardium. It is clear therefore that at this stage the paternal nucleus is influencing the structure of the organism. When the eggs of Echinus are fertilized with sperm of a still more divergent character, such as that of the crinoid Antedon, a hybrid occasionally develops as far as the gastrula stage, but it always resembles the larva developed from the normally fertilized egg in every detail and shows no trace of paternal influence. Nuclei and Cytoplasm.-We are thus led to the conception of an intermittent action of the nuclei on the cytoplasm, and in this it seems as if we had reached the deepest point to which analysis of development will lead us. Perhaps it would be more accurate to speak of an intermittent reaction between cytoplasm and nucleus, for in some embryos there is evidence that the nuclei undergo alteration as development proceeds. It is on cases like these that Weismann's theory of development was founded. According to this theory, as growth proceeds, differential division of the nuclei takes place, some becoming specialized as ectodermal nuclei, others as endodermal nuclei and so on, whilst some retain the constitution of the original zygote nucleus; these last give rise by division to others like themselves which eventually engender the nuclei of the germ cells. The lineage or line of descent leading from these germcell nuclei back to their ancestors amongst the nuclei of the first blastomeres is termed the "germ-track." Now in the Nematode worm Ascaris megalocephala the zygote nucleus contains only four chromosomes, but as the egg divides into blastomeres, the nucleus of one blastomere after another undergoes the change termed diminution of the chromatin. This change involves the nipping-off of the ends of the chromosomes, and these portions are ejected into the cytoplasm and are absorbed; the remainder of each chromosome becomes fragmented into a large number of minute granules. These granules act as chromosomes in the next nuclear division. The nucleus of one blastomere remains exempt from this change and this blastomere eventually gives rise to the genital organs.

Boveri has shown that the fact that one nucleus undergoes diminution of the chromatin whilst another does not is not the consequence of a differential division of the mother nucleus of them both, but is due to the fact that one nucleus takes up its position in a region occupied by a particular cytoplasmic substance. This he proves in two ways, viz. (1) by considering the case of eggs fertilized by two spermatozoa, and (2) by the results obtained by subjecting eggs about to segment to the action of strong centrifugal force.

In doubly fertilized eggs the extra spermatozoon forms an independent nucleus whilst the other fuses with the female pronucleus to form the zygote nucleus. The first division of the egg results in the formation of four nuclei and four blastomeres. In the development of the normally fertilized egg one of the two first nuclei undergoes diminution, and the cell containing it gives rise to a large part of the dorsal ectoderm; the other nucleus remains undiminished and amongst the progeny of the cell containing it are found the genital cells. Now amongst the four cells produced by the division of the doubly fertilized egg, three may contain nuclei which undergo diminution, and one may remain undiminished-in such cases the egg develops into a single embryo with an unusually abundant ectoderm. In other cases only two of the nuclei undergo diminution -such eggs form twin embryos of normal aspect; whereas in still other cases one nucleus alone may undergo diminution and in these cases a monstrous triple embryo is formed. These differences are accounted for on the assumption that one region of the egg contains a substance which induces diminution and one, two or three nuclei of the doubly fertilized egg may be in it.

When eggs about to segment are exposed to the action of longcontinued and intense centrifugal force the plane separating the first two blastomeres will in some cases be found to lie along a radius of the circle of rotation, and in these cases a small mass of material will be found to be ejected from the egg which then becomes divided

1E. W. MacBride, "Studies on the Development of Echinoidea," (II) "The early larva of Echinocardium cordatum and the result of crossing this species with Echinus esenlentus," Quarterly Journ. Micr. Science, vol. lviii., 1912.

2 A. Weismann, The Germ-Plasm. A Theory of Heredity (1893). 3 Th. Boveri, "Die Potenzen der Ascaris-Blastomeren bei abgeänderter Furchung," Festschrift zum 60 ten Geburtstag Richard

into two appreciably equal and similar blastomeres, the nucleus of neither of which undergoes reduction. This suppression of reduction must be attributed to the even distribution of the cytoplasmic materials under the stress of the centrifugal force, so that no region of the egg contains more of the peculiar substance than any other. Diminution of the chromatin apparently results from the action of an excess of this substance on any nucleus contained in it.

Regeneration. In the phenomena of regeneration and of budding we meet with evidence of the renewed influence of the nuclei in causing the formation of cytoplasmic substances.

We have already learnt that when one of the first two blastomeres into which a frog's egg divides is killed the survivor frequently develops into a half gastrula which may even grow into a half tadpole. Roux, however, has shown that if this half tadpole survives it becomes a whole tadpole by what he calls the "post-generation of the missing half. This is effected by the multiplication of the cells lying at the edges of the half embryo. The nuclei increase in number and confer on the cytoplasm in their neighbourhood new powers. In this case it might be objected that each kind of tissue in the old half gives origin only to the same kind of tissue in the new half. But Morgan has shown that if the head (including the pharynx) of the annelid Nereis be cut off, a new head with pharynx will be regenerated from the stump; whereas, however, the original pharynx was formed by an intucking of ectoderm, the new pharynx is formed by an outgrowth from the endodermal tube in the stump. The new powers thus conferred on the cytoplasm of the endodermal gut can only be explained as the result of the calling-forth of new potentialities in the nuclei lying in the cut edge. More remarkable evidence still has cropped up in connexion with the regeneration of the lens of the eye of the newt. In the embryo the lens is formed as a thickening of the ectoderm on the side of the head. But if the original lens be torn out, a new lens is developed either from the edge of the iris or of the retina-tissues that have no connexion with the skin of the head. Some try to meet this difficulty by the phrase that in these cases the organism acts as a whole, independently of the germlayers into which we analyse it. But what meaning can be attached to this phrase, except that the organism under different circumstances uses different means in order to effect a restoration of its integrity, it would be difficult to say. In fact we approach very closely to the celebrated "entelechy " of Driesch; that is an indwelling thing" in an organism which strives to realize a purpose. Vitalism, and the Theory of an Entelechy.-It may be argued that such an idea is unscientific, because it introduces "vital force and similar mystical ideas amongst our biological conceptions. It may be answered that in the last resort all explanation is comparison, and that those who reject vitalism seek to compare all the activities of living beings to phenomena which go on outside the body in testtubes. But this is equivalent to referring all the phenomena of life to structure, in other words the juxtaposition of definite chemical substances in a definite spatial arrangement; in regeneration, however, we encounter phenomena where structure appears to be irrelevant. If we are to do justice to such phenomena we must have some working hypothesis similar to that of Driesch. Whether the assumption of an entelechy" is better or worse than the statement that all the nuclei in the body are totipotent and that varying potentialities are called forth seems to be a matter of taste.

some

Budding. Regeneration is in many respects akin to budding, since buds in many cases may be regarded as portions of the mother organism restored after natural amputations.

In the growth of buds we often meet with a wide divergence between the materials used to build up certain organs, and those used to construct similar organs in the embryo. To give an example the bud of the ascidian Botryllus begins its existence as a little two-layered vesicle very similar to the gastrula of the same species. But in the embryo the central nervous system is developed from the outer layer as it is in all other Vertebrata. In the bud, on the contrary, it is formed as an outgrowth from the inner layer. Hjort,' who described this phenomenon, suggested as the explanation for it the fact that the outer layer of the bud is an outgrowth of the adult maternal ectoderm, which is specialized for the secretion of the cellulose "mantle and not sufficiently plastic to be turned into nervous tissue. This is only another way of saying that the formative nuclei act differently in different cases and distribute the organforming cytoplasmic substances in a different manner in the bud from their arrangement in the egg.

One or Two Embryos.-The primary organs, i.e. the germlayers, are the material out of which the higher organs are built up, and one of the most remarkable of recent discoveries in embryology is the fact that the question of whether this material Roux, loc. cit.

Morgan, Regeneration (1901).

H. Driesch, Zwei Vorträge zur Naturphilosophie (1910); see also Gifford lectures for 1907 and 1908.

7

Hjort, "Germ-layer Studies based on the Development of

shall be used to build up one embryo or two depends on the special relations which these primary cytoplasmic substances sustain to one another.

If the eggs of a frog be placed dry on the surface of a slide with their animal poles uppermost and fertilized in that position by the addition of small quantities of the fluid extracted from the seminal vesicles of a male; if then another slide be placed on top of them and the two slides clamped together by rubber bands; if when the eggs have divided into two blastomeres the whole preparation be inverted and left in water in a shallow dish for five or six days tadpoles with two heads or two tails will be developed. The materials in the unsegmented egg are of different specific gravities; the first furrow often (see above) divides them into two symmetrical halves; when the two-cell stage is inverted they tend to rearrange themselves in each cell in the same manner as they would have in the whole egg had it been inverted. Nothing has been added or taken away, yet the altered position of the materials in each cell has led to the formation of two organs where normally only one would have been formed. In the case of the newt's egg a similar procedure leads to the formation of two complete embryos, whilst if the blastula of the newt be constricted longitudinally by a hair a two-headed monster is formed. When a lizard's tail is broken off, if the little regenerating bud which forms at the wounded surface be indented the animal will regenerate two tails instead of one.

Internal Environment. When the higher organs begin to develop we can in many cases prove that the whole course of their growth is governed by what may be called their internal environment, i.e. by influences emitted by other organs.

This may be clearly seen in the development of the common sea-urchin Echinus miliaris. The echinopluteus" larva of this species is a transparent bilaterally symmetrical free-swimming creature. It is provided with a complete alimentary canal consisting of oesophagus, stomach and rectum, and at the sides of the oesophagus are situated two flattened coelomic sacs. As development proceeds each sac becomes divided into anterior and posterior portions, and the latter move backwards so as to be pressed against the stomach. Still later from the posterior end of the left anterior sac a little bud termed the "hydrococle" grows out. This is the rudiment of the water vascular system of tubes in the adult. The ectoderm lying over this bud becomes depressed so as to form a sac (the "amniotic cavity") from the floor of which grow up the spines which will cover the test of the future sea-urchin.

External Environment.-We now approach the subject of the possible influence of the external environment on the course of development. In the earlier article the attention of the reader was called to the fact that development presents itself under two principal aspects, viz. the embryonic and the larval. In the embryonic phase the young organism is sheltered from the external world, either within an egg-shell or in the mother's womb, whereas in the larval phase it leads a free life, using its larval organs to seek its own food and escape its enemies.

It was further pointed out that if we compare two nearly allied animals such as Salamandra atra and Salamandra maculosa, in the first of which development is mainly embryonic whereas in the second it is largely larval, we arrive at the conclusion that the embryonic phase is secondarily derived from the larval phase, since the organs such as gills which are functionless in the embryo are functional in the larva. It was also pointed out that larval organs frequently resemble the adult organs of other animals of simpler and more primitive structure.

On these facts was founded the celebrated biogenetic law first enunciated by Haeckel3 which affirms that "the embryo in its development recapitulates the ancestral history of the race." It is the law which provides a large part of the fascination of embryological research, but it was vigorously attacked in the earlier article and an effort was then made to show that it is not

valid, since it was maintained that whilst it is true that larvae retain ancestral characters, the same is true of adults, and that larvae in their structure are not more reminiscent of the former history of the race than are adults.

Now the outcome of recent investigation has in large measure tended to reinstate the doctrine of recapitulation in its former position of preeminence, to show in fact that recapitulation forms the central thread in every life history, although it has been believers in the biogenetic law have from the first admitted. blurred and deflected by secondary influences, as indeed all

The first point to which we wish to direct the reader's attention is that larval and embryonic phases occur in all life histories. Every animal begins its existence as an egg which is quite incapable of feeding or of defending itself and this egg is always protected by an egg-shell although this shell may be very thin, and no animal upon leaving its early shelter and beginning to seek its own food attains at once the structure of the sexually ripe adult. Hence every animal in the course of its development may be said to pass first through an embryonic and then through a larval phase, although the latter phase may be very short and the difference in structure between the larva and the adult inconsiderable. Now, the larval phase being the later is the most recent addition to the life history and therefore the least likely to be modified by secondary factors; if therefore the biogenetic law be valid, it is the larval phase which will possess most ancestral significance. But in the earlier article attention is called to the fact that the identification of a larva as the representative of an ancestor must always be hypothetical because we have no direct knowledge of what the ancestor of any living animal was like. It behooves us therefore to look a little more closely at the rea sons which actually do induce us to regard a given stage as ancestral.

First, it has been claimed quite recently that direct experimental proof of the validity of the biogenetic law has been obtained. Kammerer placed young specimens of Salamandra maculosa which had just completed their metamorphosis in cages the floors and walls of which were coloured differently in different cases. The larva of this species has a skin of a uniform dark-greyish tint, but the skin of the adult is gaily coloured with bright yellow patches on a black background. The salamanders which were confined in cages having a floor of moist yellow loam and walls coloured yellow became yellower as they grew to maturity-a process which occupies between three and four years. The yellow patches, in a word, increased in number and size and tended to become joined together in bands. Those confined in cages with blackened walls and a floor of black size. When the salamanders had attained sexual maturity and were garden earth became darker since the yellow patches dwindled in allowed to pair, it was found that the offspring of two which had been reared in yellow surroundings, if they continued to live in the Haeckel, Allgemeine Morphologie (1866). 4 P. Kammerer,

3

Vererbung erzwungener Farbveränderungen. IV. Das Farbkleid des Feuersalamanders (Salamandra maculosa) in seiner Abhängigkeit von der Umwelt," Archiv für Entwicklungsmechanik, vol. xxxvi., 1913.

same environment, became still yellower than their parents until the black pigment had been almost entirely displaced; whilst the offspring of two which had become darker, if reared in cages with black walls and floor, became practically completely black by the time they reached maturity so that they came to resemble the mountain species Salamandra atra. If, however, the offspring of two salamanders reared in yellow surroundings were allowed to grow up under black surroundings, they nevertheless for the first six months of their lives became progressively yellower; then and only then did the influence of the black environment begin to tell-the yellow patches became invaded by numerous small black spots and grew smaller. In short, the young recapitulated the process of "yellowing" that their parents had undergone.

If these results are confirmed the doctrine of recapitulation will change its status from that of an hypothesis to that of a proved fact; and further proof will be furnished that changes acquired by the individual in response to the demands of the environment are to a certain extent at least inherited.

adult stage is attained, but in the majority of life histories when a new phase is added there is a tendency for some of the older phases to be pushed back into the embryonic period, so that as an animal passes from stage to stage in evolution it leaves behind a trail of stages at first larval and then becoming embryonic.

Secondary Modifying Factors.-We may now glance at the principal factors which modify and tend to obscure the recapitulatory factor. It is only possible to define these factors by a truly comparative embryology based on a wide survey.

One of these factors is "tachygenesis " or precocious development; that is to say, we find that organs originally developed as a response to the stimulus of a new environment come in course of time to be

The typical Ctenophora are ovoid organisms of a glassy transparence which swim in a vertical position in the sea. Their locomotor organs are eight vertical rows of vibratile combs, each comb consisting of a short horizontal row of powerful cilia fused together at their bases. A certain creeping organism resembling a flat worm, named Coelo plana, had been believed by some zoologists to exhibit ctenophore affinities but its relationships were very obscure. Quite recently a Japanese zoologist1 has described its development. Its larva is a small typical ctenophore with eight rows of perfectly formed combs; these it discards after swimming for a few hours— it sinks to the bottom and flattens out and gradually assumes the adult structure. Another extraordinary organism, named by its discoverer Tjalfjellia,2 was discovered amongst dredgings collected in the Arctic Ocean. This creature superficially resembled a sponge or an ascidian. It was gelatinous and sessile and seemed to consist of a pair of upright tubes like towers whence proceeded smaller tubes which ramified in its substance. In pockets connected with these smaller tubes were discovered groups of the larvae. These were small ovoid creatures of typical ctenophore structure with the eight vertical rows of combs.

If recapitulation of ancestral history forms an unquestionable element in the life history of some animals, is it not probable that it constitutes a factor in all life histories? To this question it seems to us only an affirmative answer is conceivable.

Change of Habits.—If we then regard the reality of recapitulation as proven we may now reflect on its meaning. We have seen that the recapitulatory element is most obvious in the latest larval stage of development, the most recently added page of the life history. Now the organs of the larva are adapted to its environment; therefore this environment in its broad outlines at least must represent the ancestral environment of the race. The present condition of the race both as regards structure and habits has been produced as a consequence of migration from the original haunts of the race. Change of habits therefore reveals itself as the great driving-force in evolution, and change in habits usually means the choice of a different type of food.

We may conclude that the period of life at which this change most frequently occurred was when the adult organs had developed but before sexual maturity had been attained-in a word, at the stage of what we may call the young adult. As one change of habits succeeds to another in the course of evolution, the life history is not lengthened in the same proportion, since the new phase takes the place of the sexual phase in the previous condition of the race. In some Crustacea, e.g. in the shrimp Penaeus, at least four larval stages are passed through before the

1 Taku Komai," Notes on Coeloplana bocki and its development," Annotationes Zoologicale Japonenses, vol. ix., 1920.

44

2 Mortensen, Čtenophora," Danish Ingolf Expedition, vol. v.,

A second powerful modifying factor is the change from the larval to the embryonic phase, so far as the development of a particular organ is concerned. This change of phase is sometimes caused by an unfavourable alteration in the environment of the larva. It was actually effected artificially in the development of Salamandra maculosa by Kammerer. This species is viviparous and normally gives birth to between 30 and 40 young which are provided with gillslits and long gills and which live in the water for six weeks before they metamorphose into land animals. If the parents are exposed to successively colder and drier conditions, the number of young produced at a birth diminishes with each breeding-period, and these young are born at a progressively more advanced stage of development. If these young are reared to maturity under similar conditions of coolness and dryness, they will in turn give birth to young which will be still fewer in number than those produced by their parents and which are born at a still more advanced stage of development. The process goes on till only three or four are born at one time and these are provided with the merest stumps of gills; such young never enter the water at all but at once take up the adult mode of life. This is the normal mode of development of Salamandra atra.

The change of phase from the larval to the embryonic type entails many other changes. The embryo must be fed and it obtains its food from one of three sources, (a) devouring its sisters; (b) secretions from the mother's womb; (c) inclusions of yolk in its own cytoplasm. When the embryo devours its own sisters, this, as in the case of Salamandra atra, may entail little change of structure because the habit is one recently acquired; but where, as in the case of the playhelminth worms, the habit is of old standing then the embryo may be distorted out of all recognition. In these worms one viable egg is shut up in a capsule along with thousands of small sterile ones; and it is difficult to find in the embryo any vestige of resemblance to the larva of these Playhelminthes which lay their eggs singly.

When the embryo derives its nourishment from the mother's womb then it frequently develops organs of adhesion to the wall of this. To this category belongs the placenta which profoundly distorts the ventral surface of the human embryo, so that this surface gives rise to a treelike outgrowth whilst the dorsal surface is moulded into a ludicrously exact copy of the early tadpole of the amphibian. When the embryo is fed by yolk, this, as we have already pointed out, modifies all the processes of development; cell division becomes slow and the cells produced few and large, and folding which plays a large part in the development of small alecithal eggs becomes impossible and is replaced by solid outgrowths of cells.

Still a third factor which tends to hide the recapitulatory element is the development of special larval adaptations. This occurs when changed. These special adaptations have been developed in thouthe larva retains its free life but when its circumstances become sands of insect larvae. So generally is this the case that Balfour denied to these larvae any ancestral significance at all; but modern research has succeeded in revealing the original ancestral larval type beneath the secondary modifications.

ancestors of insects were creeping myriapod forms-scavengers All the evidence at our disposal points to the conclusion that the

3 Kammerer, "Vererbung erzwungener Fortpflanzungsanpassungen I & II. Die Nachkommen der spätgeborenen Salamandra maculosa und der frühgeborenen S. atra," Archiv für Entwicklungsmechanik, vol. xxv., 1908.