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Jenkinson JW. Experimental Embryology. (1909) Claredon Press, Oxford.

Jenkinson (1909): 1 Introductory | 2 Cell-Division and Growth | 3 External Factors | 4 Internal Factors | 5 Driesch’s Theories - General Conclusions | 6 Appendices
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Chapter IV Internal Factors

1. The Initial Structure Of The Germ As A Cause Of Differentiation


As has been already pointed out, it was in the hands of Roux that the principle of germinal localization first advanced by His assumed the rank and importance of a theory of development. On this ‘ Mosaik-Theorie’ of self-differentiation the precise relation observable between the several parts of the embryo and certain definite regions of the undeveloped germ is not merely customary or normal, but necessary and causal. The germ-cell is endowed with a preformed structure corresponding to the structure of the organism which is to arise from it; each part of this structure is predetermined for the formation of some particular member of the embryo, and out of it no other member can, under ordinary circumstances, be made. The causes of its development, regarded as a specific activity of the organism, as leading to the production of a form which is like that of the parents which gave it birth, lie wholly within this prc—cxisting structure, and of each part within each part. Although the influence of the environment and, in later stages, of the parts on one another is not entirely excluded, the factors on which the differentiation of the whole and of each part depends are essentially internal, and all that happens is that by a continued process of cell-division the parts are separated from one another and the structure thus made palpable and manifest. Cell-division in the embryo is therefore qualitatively unlike.

The supposed proof, by Pfliiger’s experiment with forcibly inverted Frogs’ eggs, of the complete ‘isotropy’ or equivalence of all parts of the cytoplasm, compelled Roux to locate the IV. 1 INITIAL STRUCTURE OF THE GERM 159

selfdifferentiating substance, the Idioplasson, in the nucleus, a. view adopted and elaborated by Weismann, while the facts of regeneration. necessitated the assumption of a reserve Idioplasson endowed with the potentialities of the whole, which is not qualitatively divided during development, but handed on intact to some or all of the tissues of the body.

The hypothesis then assumes the existence in the nucleus of the fertilized ovum of as many separate units as there are separately inheritable characters in the embryo, arranged there according to a plan which conforms to the structural arrangement of the parts which they represent, the separation of these units by continued ‘unlike’ nuclear division, and their distribution to the cytoplasm, where they determine the formation of the structures to which they are beforehand assigned.

For Roux the theory was no mere speculation, but the result of observation and experiment. The natural occurrence of half or partial monsters, the correspondence of defects in the embryo to injuries to the egg, and the coincidence of the first furrow with the sagittal plane in a large percentage (80 Z) of, unfortunately, only a small number of eggs, suggested the selfdiiferentiation of predetermined parts, while the last-mentioned observation led directly to the experiment in which one of the first two blastomeres of the Frog’s egg was killed and a halfembryo produced from the survivor.

By means of a hot needle Roux succeeded in at least partially destroying one of the first two blastomeres. The other continued to segment, and passed through the ordinary stages; first the Hemimorula, with animal and vegetative cells and a segmentation cavity (though the last was sometimes absent), then by continued cell-division the Hemiblastula, followed by the Hemigastrula and Hemiembryo. In all cases the living tissues formed an exact half——either a right half or a left halfof a normal embryo, and ended abruptly at the (sagittal) plane of separation of the living and the dead. The segmentation cavity, however, was observed to extend in some cases across this plane, while in others it was confined wholly within the living cells. ‘

In the Hemigastrula the greatest extent of the archenteron—160 INTERNAL FAC’1‘O1tS IV. 1

produced by the overgrowth of the blastoporic lip—-was parallel to the plane of separation: the yolk-cells were pushed into the segmentation cavity. The Hemiembryo had half a medullary


FIG. 76.—A and B. Meridional sections through Herniblustulae of the Frog: kn, remains of nucleus; In’, large reticular nuclei; 1:, nuclei In the yolk; F,b1astocoe1; 4*, vacuoles. C. Hemigastrulalatemlis. Oblique lon itudinal section. I). Hemiembryo sinister, transverse section; .93, me ian plane ; the right half of the egg is already completely cellulated, post-generation of the germ-layers has begun; ch, notochord, of whole size; mw, medullary fold; u, gut; j, two yolk-cells that have remained embryonic. (From Korschelt and Heider, after Roux.)

tube, mesoderm on one side, half a gut cavity—open towards the dead blastomere——but a whole notochord (attributed by Roux to

premature regeneration). IV. I INITIAL STRUCTURE OF THE GERM 161

Anterior half-embryos—occurring when the second furrow by an ‘ anachronism ’ appears first-—were also observed, but posterior never. Quarter and three-quarter embryos were obtained by killing three or one of the first four cells, and Hemiblastulae superiores by killing the four yolk-cells in the eig-l1t~celled stage.

The self-differentiation of the ldioplasson is thus demonstrated. Subsequently, however, the reserve Idioplasson comes into play, and the missing half is formed by a peculiar process, to which

FIG. 77.——A and B. Normal Frog embryos with medullary folds (m), open (A) and closed (B). C. Hemiembryo dexter with almost complete post-generation of the eotoderm; u,yo1k-plug. D. The same, older, but with less post-generation. E. Hemiemhryo anterior (‘i’) with beginning post-generation. (From Korschelt and Heider, after Roux.)

Roux has given the name of ‘post-generation ’ to distinguish it from regeneration, in which lost parts are formed out of already (liiferentiated tissue.

The injured blastomere first degenerates, is then reorganized, and finally post-generated.

In the first of these phases the cytoplasm and yolk are seen to be vacuolated, while the injured but not completely killed nucleus has given rise to small and large nuclei of normal structure


surrounded by masses of pigment and in addition to irregular chromatic masses —some pale, some deeply staining. Separating it from the uninjured half is athin layer of yolkless protoplasm.

Reorganization is accomplished by any one of three methods. The first is nucleation followed by cellulation, that is to say, masses of cytoplasm arise round normal nuclei, some of which are derivatives of the injured nucleus, while others have migrated across from the living blastomere. The second method involves an abundant immigration of whole cells from the living embryo which resuscitate or feed on the yolk and nuclei of the injured half. In the third method overgrowths take place at various points of the tissues of the living half-embryo, the cells dipping into and feeding on the yolk as they pass across.

In post-generation, which only occurs after the first method of reorganization, each layer of the uninjured reforms the corresponding layer of the injured, the cells of the former exerting a directive stimulus upon the reorganized tissues of the latter, which thus pass through a process of ‘ dependent ’ differentiation. The ectoderm grows over from in front backwards and from below upwards, so that a structure superficially resembling a. yolk-plug is formed at the hinder end; as it does so it becomes difierentiated into the usual two layers. The medullary tube is formed in like manner. The mesoderm is formed from the ventral side and later divided into vertebral and lateral plates. The notochord was a whole from the beginning. The missing half of the gut is formed directly from the half-gut of the living embryo, not by any process of blastoporic invagination.

The conclusions drawn by Roux from this experiment have already been stated. Development is conceived of as a process governed essentially by a factor which is entirely internal, the preformed structure of the nucleus of the germ. Into the validity of this factor we have now to inquire by examining the evidence oifered by the very numerous and similar experiments, performed on the eggs of animals of all kinds, which the ‘ Mosaik-Theorie’ has evoked. We shall take these experiments in order, beginning with the form employed by Roux himself, the common Frog 1.

‘ For literature see following section. IV. I INITIAL STRUCTURE OF THE GERM 163


It will be remembered that the Frog’s egg possesses, when freshly laid, a symmetry which is radial about the axis, the line joining the centres (animal and vegetative poles) of the pigmented and unpigmented portions of the egg; the symmetry is marked internally by the arrangement of yolk and protoplasm, for the latter lies most abundantly in the smaller unpigmentcd, the former mainly in the larger pigmented portion. Since the yolk

FIG. 78.~—Formation of the grey crescent in the Frog's egg (R. temporaria). A, B from the side; 0, D from the vegetative pole. In A, C

there is no crescent, in B, 1) a part of the border of the pigmented area has become grey.

is heavier than the protoplasm the white pole is turned downwards, with the axis vertical, the egg being free to rotate inside its jelly membrane. Shortly after fertilization, however, the radial is replaced by a bilateral symmetry (in Rana flwca and I2. temporm-z'a), a grey crescent being formed on one side of the egg along the border of the pigmented area by the retreat of pigment into the interior (Fig. 78). The grey crescent eventually becomes white and added to the white area. In Rama caculenta M 2 164 INTERNAL FACTORS IV. 1

it is stated that the egg‘-axis becomes inclined to the vertical. This would seem to be an error, due to a confusion between the original white area and the white area enlarged by the addition

FIG. 79.—Diagrams of the closure of the blastopore in the egg of the common Frog (R. tempo:-aria). In A~—D the egg is viewed from the vegetative pole, in E, F from below. The dorsal lip is at the top of the figures. In D the ventral lip has just been formed and the blastopore is circular. In E the rotation of the whole egg has begun, and in F is complete.

of the grey crescent (compare Fig. 44 with Fig. 78). The egg is now bilaterally; symmetrical about the plane which includes the egg-axis and the middle (‘point of the crescent. IV.1 INITIAL STRUCTURE OF THE GERM 165

Since the crescent is formed on the side opposite to the entry of the spermatozoon, the latter is considered by Roux to determine the symmetry-plane of the egg. The first furrow is also stated to generally lie in this plane, and the dorsal lip of the blastopore, which marks, of course, the sagittal plane of the embryo, appears in the region of the grey crescent. The dorsal lip grows through 75°, starting 25° below the equator and passing beyond the vegetative pole ,- with the rotation of the whole egg and therefore also of its axis in the direction opposite to that through which the dorsal lip has travelled, the anterior end of the embryo, whose dorsal side is now uppermost, comes to lie a little above and behind the animal pole, while its posterior end is marked by the now fast-closing blastopore (Fig. 79).

According to Roux the first furrow and the sagittal plane coincide and the first two blastomeres are right and left, unless, by an anaehronism, the second furrow, which separates anterior from posterior (this should be dorsal from ventral), occurs first. The third furrow, therefore, separates dorsal from ventral (correctly speaking, anterior from posterior). In the small number of eggs examined by Roux the percentage of coincidences was found to be high (80 %) and the deviations were attributed to experimental error.

Oscar Hertwig has, however, stated—-on the strength of a small number of observations on eggs compressed between horizontal glass plates—that the angle between the two planes may have any value; while Schulze and Kopsch think it probable that they coincide in the majority of cases. The recent examination by the present author of a large number of cases has shown that the angle in question may indeed have any value from 0° to 90°, but that there is a decided tendency to small values, that is to coincidence, as may be seen from the annexed frequency polygon (Fig. 80), although at the same time there is no correlation, and therefore no causal connexion, between the two planes. Between the plane of symmetry and the sagittal plane, however, the correlation is considerable, and the tendency of the two to coincide much greater (Fig. 81), a result in conformity with the statements of other observers (Roux, Schulze, Morgan). Within certain limits, therefore, the right and left 166 INTERNAL FACTORS IV. 1

halves, the dorsal and ventral sides (since the dorsal lip always

appears in the region of the grey crescent) and the anterior and posterior ends (since the anterior end is always a little above the

-90-oo -10 -so -50 -40 -50 -20 -no

o no '20 '30 Ho 050 +60 910 +00 *9!)

FIG. 80.—-Frequency polygon of the angle between the first furrow of the Frog's egg and the sagittal plane of the embryo. n = 889, M = 2-12°

1-914, 0- = 40-39°:-646.

animal pole) are predetermined in the undivided egg. But the manner in which the material of the egg is cut up by the first two meridional divisions makes no diiference to the result. In IV. 1 INI'1‘IAL STRUCTURE or THE GERM 167

these two divisions the qualitatively distinct parts of the cyto. plasm may be separated from one another in any assignable manner, the nuclear material being distributed to these in any assignable order. The symmetry of segmentation has no relation






50 ‘IO



-90 -80 -70 -no 50 40 '30 '20 -10 o 0-Io v20 950 +40 950 can #70 0-60 9-90

FIG. 81.—F1-equcncy polygon of the angle between the plane of symmetry of the Frog's egg and the sagittal plane of the embryo. n = 509, M = 2-23:-889, rr = 29-75:-629. either to the symmetry of the embryo or to the symmetry of the undivided egg, for the first furrow tends to lie either in or at right angles to the plane of symmetry and is not correlated with

it to any great extent‘. The third furrow, however, being‘ always ‘ See, however, Appendix A. 168 INTERNAL FACTORS IV. I

at right angles to the axis, invariably separates anterior from posterior.

An observation of Morgan’s has some bearing on this question ; even when the first furrow divides the egg unequally a normal embryo results.

Even when the first two blastomeres are equal the cells produced by their subsequent division may migrate across the plane of separation (Kopsch). In Nectmws there appears to be no relation between the first furrow and the sagittal plane (Eycleshymer).

The hypothesis of qualitative nuclear division has been tested

by O. Hertwig in another way.

FIG. 82.—'l‘he first four phases of segmentation of the F1-og’s egg. Normally (the three upper figures) and under pressure between horizontal plates (the three lower figures) I, II, 111, 1 V, the first, second, third, and fourth divisions. 1-16, the cells produced by successive (livisions, numbered as follows : The egg divides into

1”5 3 7 2 6 4J8

1 95'T4:?'T77 52'1“06164?28”'fis

The animal cells in the normal egg and the corresponding cells in the compressed egg are stippled. (After 0. Hertwig’s account.)

The eggs are aflixed to glass plates by their jelly, fertilized, and allowed to rest until the axis has become vertical. Pressure is then applied by a second plate, in the direction of the axis, the plates being horizontal, or at right angles to the axis, the plates being vertical or oblique. In such compressed eggs the direction of elongation of the nuclear spindles is distorted, and IV. 1 INITIAL STRUCTURE OF THE GERM 169

consequently the ‘qualitatively unlike’ nuclei compelled to assume an abnormal arrangement. Between horizontal plates the first two furrows are, as normally, meridional and vertical (Fig. 82) ; the third, however, is parallel to the first (instead of latitudinal), and the fourth latitudinal (instead of meridional).

Between vertical plates the first is meridional and at right angles to the plates, the second latitudinal (horizontal) and near the animal pole, instead of meridional, the third furrows are parallel to the first, and the fourth meridional and at right angles to the first in the four upper blastomeres.

Now if nuclear division is a qualitative process, if, further, the nuclei of the compressed egg are divided in the same way in successive mitoses as are the nuclei of the normal egg (that is to say if the first two nuclei are right and left, each of these then divided into dorsal and ventral, and each of these again into an anterior and a posterior), then as a result of compression their distribution and arrangement must be altered (as the figures show), parts which should be anterior will be lateral or ventral, and vice versa, and a monster will be formed. Such eggs give rise to normal embryos. Nuclear division is therefore not qualitative. From this conclusion there is only one escape; it may be argued that the orientation of the nuclei remains unaltered when the direction of the spindles is changed by the pressure, and that therefore the order of sequence of the cell-divisions may be varied ad libitum, may be as ‘ anachronistic ’ as is pleased, without affecting in the least the mode of distribution of the, qualitatively unlike, parts, a contention, surely, which would only be urged for the sake of supporting a thesis.

0. Hertwig has also repeated Roux’s experiment, killing, or at least injuring, one of the first two blastomeres by a needle or by means of electricity. The uninjured half segments to form a mass of cells lying on the top of the dead blastomere, as a blastoderm lies on the yolk of a meroblastic egg, and separated from it by a segmentation cavity, although the latter may be wholly within the living cells. This is Roux’s Ilemiblastula, but in Hertwig’s case the dead cell lies below, a point, as we shall see, of considerable importance. Later on a blastopore is formed, but always, according to Hertwig, within the bounds of 170 INTERNAL FACTORS IV. 1

the living portion, not at the edge; the blastopore is usually symmetrical to the plane of separation of the two blastomeres. Beneath the lip of the blastopore an archenteron is formed, and notochord and mesoderm are differentiated. The closure of the blastopore is, however, prevented by the resistance ofiered by the dead yolk-mass, which lies ventrally and posteriorly, just as it is retarded by the yolk in a large-yolked Fish egg; in fact, were this dead yolk removed, the living portion, Hertwig maintains, would develop normally. A nearly normal embryo may, in fact, be formed, but more usually there are considerable abnormalities. The mass of dead yolk, though partially enclosed by the growing edge of the ectoderm, is sufficiently great to impede the ultimate closure of the blastopore, and sometimes of the medullary folds as well; the latter frequently diverge round the yolk-plug of dead and living tissue, the chorda being split as well; they may be symmetrical, or one side may be much less developed than the other, owing, it is asserted, to an asymmetry in the resistance offered by the dead cell, and in the more extreme cases this inequality may be so pronounced as to give rise to a condition which does not differ in any way from Roux’s Hemiembryo lateralis ; there is one medullary fold, a notochord, mesoderm on one side, and a gut cavity in the yolk-mass ; to the bare side of the yolk-cells is attached the dead blastomere, only partially covered by extensions of the cctoderm of the living half. Such cases are, however, very rare; in the majority more, sometimes much more, than a half is produced from the living blastomere, for Hertwig denies that the missing parts are post-generated, as Roux maintains, though he admits the overgrowth and immigration of cells, as well as the persistence of living nuclei where the injury has been only partial ,- all these contribute to the formation of the embryo, which would be complete were its development not hindered by the presence of the inert mass of yolk.

The diiferences of interpretation put by Roux and Hertwig on the same phenomena appear to be radical. A reconciliation is, however, possible, for Morgan has observed that the whole or half development of the injured cell depends upon the position it takes up. If the original position—with the black pole IV.J INITIAL STRUCTURE OF THE GERM 171

uppermost-—is retained, then a half-embryo is formed, as is indeed the case, according to Hertwig’s own evidence, when the egg is prevented from turning over by compression.

In such half-embryos Morgan finds no traces of Roux’s postgeneration, although a whole embryo may be eventually formed by regenerative processes, referred by the author to the retarded development of the living parts of the injured half. It should be noticed, however, that Roux’s statements are confirmed by Endres and Walter.

Should, however, the white pole be uppermost, then a whole embryo of half-size results. In Hertwig’s experiments, of course, the original position was usually not retained.

FIG: 83.-.~—Doub1e embryo of Rana fusca, from an egg compressed in the direction of the axis and inverted in the two-cell stage. (After Schultze, from Korschelt and Hcider.)


FIG. 84.—Double monsters of Rana fusca, obtained by the same method. (After Schultzc, from Korschelt and Heidcr.)

This conclusion is still further strengthened by Schultze’s observations on the development of eggs inverted in the twocelled stage and kept so. Each blastomere develops independently and a double monster is produced (Figs. 83, 84). The result is not due to the pressure, for controls similarly compressed developed normally.

The details of development of these eggs have been carefully worked out by Wetzel (Figs. 85, 86). In each inverted blasto172 INTERNAL FACTORS IV. I

mere the yolk sinks next to the plane of separation, while the protoplasm and pigment rises to the outer side. The two cells are now related as two whole eggs united by their vegetative poles, their axes in one and the same (horizontal) straight line; each has, in fact, acquired a totally new polarity of its own. When segmentation has been completed a groove appears, in the plane of separation, and gradually extends round the whole circumference ; the ends of the groove are forked, but the branches of each fork unite as the groove grows round. The groove is, in fact, a blastoporic lip common to the two, the branches the individual lips, the material between them and finally covered over by them a common yolk-plug, the space between the two

FIG. 86.——Section through a. double blastula of the Frog ' (Rana fusca). Ia, blastocoels. FIG. 8-3.—-Double embryo ob- (After Wetzel, from Korschelt

tained by the same method: h, and Heider.) heads; 1», metlullary grooves;

c, line of union of the latter.

(After Wetzel, from Korschelt

and Heider.)

a common archenteron extending into an archenteric space in each individual. Subsequently medullary folds and notochord are developed in each below the groove, that is on the vegetative or postero-dorsal side of each, but anteriorly each grows out free of the other, and in this region medulla, notochord, and gut are single. The result is‘, therefore, two embryos placed back to back, and united by a common yolk-plug.

Experiments in which the four animal or the four vegetative cells of the eight-celled stage are killed are not very conclusive, as the develonment of the survivors does not go very far. IV. I INITIAL STRUCTURE OF THE GERM 173

Samassa has, however, shown that a short archenteron with dorsal lip, together with traces of notochord and mesoderm, may be formed from the four animal cells alone, the dead yolk-cells making a floor to the archenteric cavity and protruding as a large yolk-plug; and Morgan has obtained a similar result with the four vegetative cells‘ alone.

From all these investigations it seems reasonable to infer that each of the first two blastomeres of the Frog’s egg may under certain circumstances acquire the polarity of a whole, and be capable of giving rise to an embryo whose complete development is only prevented by the impediment offered by the presence of the other, whether living or dead. Were it possible to completely separate the two blastomeres, we may surmise that each would become a perfect embryo: a surmise which is raised to a certainty by our knowledge of what happens in the newt. For in this form it is possible to separate the two cells by means of a noose of fine hair tied round a __,,.. the egg in the plane of the first ' i‘ ’ ' _.,furrow, as Herlitzka has shown, },‘1G_ g7__Egg of the newt

and in case gach half seg-- (Triton cristatus), tW0 (E0111pletely normal embryos, obtained

ments as a whole and develops by tying a thread W) round the

into a whole larva of rather more egg the f'li1I‘r0iv§'é ky,ijc1Iy - - - _ mem l'2].:n(3. er erl Z a, .l'Ol'l1 than ha’It'S1ze (F135 87)‘ H01" Korschelt and Heider.)

litzka has further investigated the dimensions of the organs in these embryos ,- those of the me dulla and notochord he finds are the same as in embryos developed from a whole egg, those of myotomes and gut a little less. The size of the nuclei and cells in the medulla and myotomes is the same as in the whole (3%) embryo 1 ; the number of nuclei in the medulla is the same, in the myotomes one-half. He concludes that some organs need a certain minimum number of cells for their differentiation, while the cells must attain to certain dimensions.

1 It will be convenient henceforward to designate the whole egg, or the embryo or larva developed from it, by the symbol }, each of the first two blastomeres and the embryo or larva, whether complete or Incomplete, formed from it by —§, and so on; thus a % embryo means one which arises from three out of the first four blastomeres. 174 INTERNAL morons IV. 1

The discovery of Herlitzka has been followed up by the researches of Spemann, who has employed the same method of constriction, but with variations of degree, direction, and time. These difierences have given the most interesting results.

The first furrow, according to this author, is usually at right angles to the sagittal plane and separates the material for the dorsal and ventral halves of the embryo ; only rarely do sagittal plane and first furrow coincide. Both cases were, however, experimentally investigated.

FIG. 88.—Three stages in the roduction of a double monster by strong median constriction of the §Iewt’s egg. (After Spemann, 1903.) a. Beginning of gastrulation; there is a separate lip in each half. b. I. and 1'. Med., edullary folds of left and right embryos; *, point where the medullary grooves separate; Bl, blastopore. c. The doubleheaded larva.

When the first furrow is in a horizontal plane, a slight constriction in the two-celled stage separates the dorsal lip of the blastopore in one portion from the ventral lip in the other. Medullary folds are developed in the first half only, the second forms a sort of yolk-sac appendage which is later absorbed by the single normal embryo. With tighter, but still incomplete, constriction the dorsal half alone becomes an embryo; it contains either the whole or only the dorsal portion of the blastopore. The ventral half contains either no portion of the blastopore or IV. I INITIAL STRUCTURE OF THE GERM 175

only the ventral lip; it develops mesoderm but undergoes no further dilferentiation, and eventually drops ofi. Should the constriction be delayed until after the dorsal lip has appeared both halves may form an embryo, but the ventral embryo is usually imperfect. These dilferences are attributed by Spemann to differences in the time or place of constriction.

When a. transverse constriction is made after the appearance of the medullary plate the anterior half develops as a whole, with complete nervous system and optic vesicles; the posterior forms a medullary plate, but no folds, and dies. After the appearance of the medullary folds, however, the anterior and posterior halves produced by transverse constriction develop as halves, although a case is described where each had a pair of auditory vesicles.

When the sagittal plane coincides with the first furrow, constriction in the two-celled stage gives rise to double-headed monsters; if the constriction is slight the most anterior organs only are involved (duplicitas anterior), the cerebral hemispheres, epiphysis, hypophysis, and paraphysis being doubled; there may be two complete pairs of eyes, or the median eyes may be more or less fused (Diprosopus triophthalmus). Tighter constriction brings about a reduplication of the chorda, auditory vesicles, and fore limbs (Dicephalus tetrabrachius). Similar effects are obtained by constriction in the blastopore stage, but not later; halves separated in the median plane when the medullary folds have arisen die as halves.

We shall now briefly consider another experiment by which the independence of one another of the parts is demonstrated. Schaper removed the brain, eyes, and, probably, the auditory vesicles from newly hatched tadpoles of Rana esculenta. The wound healed up. The mouth moved to the anterior end and the suckers up the sides of the tadpoles, which lived for nearly a. week, in the course of which they grew 2mm. They then showed signs of weakness and were preserved. It was found that the mouth had opened, that labial cartilages, pterygopalatine bar, jaw-muscles, gill-bars, gills, heart and bloodvessels, trigeminus and vagus ganglia, oesophagus, pronephros and glomus, and dorsal muscles had all been dififerentiated. 176 INTERNAL FACTORS IV. I

There was, however, no operculum. The anterior end was occupied by a mass of mesenchyme. There was no sign of a regeneration of any of the lost organs except the anterior end of the notochord. The nerve ganglia were normal, but the spinal cord underwent degeneration. In spite of this the creatures could execute spontaneous and reflex movements.

What this experiment shows is, of course, that the organs of the trunk are not dependent for their development upon the presence of the brain; fresh researches would be necessary to determine how far in each case the parts are self-difierentiating. In the second place the brain and eyes, when removed at this stage, cannot be remade by the tissues, but remain behind. So far, therefore, the body is at this stage an inequipotential system, as Driesch would call it. We know, however, that at an earlier stage the parts of the body are equipotential. We have, in fact, only another instance of that loss of totipotentiality, of that increase of independence and self-diiferentiation which takes place as development proceeds. Here, however, we are anticipating a conclusion which can only be completely stated after a discussion of the experiments performed on eggs of other types.


H. ENDRES and H. E. WALTER. Anstichversuche an Eiern von Rana fusca, I'°' und lI*°' '1‘ei1, Arch. Eat. Mech. ii, 1896.

A. C. EYCLESHYMER. Bilateral symmetry in the egg of Nectum.9, Anat. Anz. xxv, 1904.

A. HERLITZKA. Contributo allo studio della capacitia. evolutiva dei due primi blastomeri nell’ uovo di tritone (Triton crislatus), Arch. Eur. Mech. ii, 1896.

A. HERLITZKA. Sullo sviluppo di embrioni completi da blastomeri isolati di nova di tritone, Arch. Ent. Mech. iv, 1897.

O. HERTWIG. Ueber den Werth der ersten Furchungszellen fiir die Organbildung des Embryo, Arch. mikr. Anat. xlii, 1893.

F. KOPSCH. Ueber das Verh-liltniss der embryonalen Axen zu den drei ersten Furchungsebenen beim Froseh, Iutemaf. Monatsc-hr. Anal. 14. Phys. xvii, 1900.

T. H. MORGAN. Half-embryos and who1e—embryos from one of the first two blastomeres of the Frog’s egg, Anat. Auz. x, 1895.

T. H. MORGAN and E. TORELLE. The relation between normal and abnormal development (iv) as determined by Roux’s experiment of injuring the first-formed blastomeres of the Frog's egg, Arch. Ent. Mech. xviii, 1904. ' IV. I INITIAL STRUCTURE OF THE GERM 177

T. H. MORGAN. The relation between normal and abnormal development of the embryo of the Frog, (v) as determined by the removal of the upper blastomeres of the Frog's egg, Arch. Ent. Mech. xix, 1905.

W. RoUx. Ueber die Zeit der Bestimmung der Hauptrichtungen des Froechembryo, Leipzig, 1883; also Ges. Abh. 16.

W. RoUx. Ueber die Bestimmung der Hauptriohtungen (lee Froschembryo im Ei und fiber die erste Theilung des Froscheies, Bresl. iirtz. Zeitschn, 1885; also Ges. Abh. 20.

W. ROUX. Uebcr die kiinstliche Hervorbringung halber Embryonen, Virchoufs A1'ch., 1888; also Ges. Abh. 22.

W. Roux. Ueber das entwicklungsmechanische Vermtigen jeder der beiden ersten Furchungszellen des Eies, Verh. Anal. Ges., 1892; also Ges. Abh. 26.

W. RoUx. Ueber Mosaikarbeit und neuere Entwicklungshypothesen, Anat. Hqfte, I" Abt., ii, 1892-3; also Ges. Abh. 27.

W. Roux. Uebcr die Specification der Furchungszellen und iiber die bei der Postgeneration und Regeneration anzunehmenden Vorgiinge, Biol. Centralbl. xiii, 1893; also Ges. Abh. 28.

W. ROUX. Ueber die ersten Theilungen des Froscheies und ihre Beziehungen zu der Organbildung des Embryo, Anat. Anz. viii, 1893; also Gas. Abh. 29.

W. ROUX. Die Methoden zur Hervorbringung halber Frosch-Embryonen, uml zum Nachweis der Beziehung der ersten Furchungeebenen des Froscheies zur Medianebene des Embryo, Anat. Anz. ix, 1894; also Ges. Abh. 31.

W. Rovx. Ueber die Ursachen der Bestimmung der Hauptrichtungen des Embryo i1n Froschei, Anat. Anz. xxiii, 1903.

P. SAMASSA. Studien fiber den Einfluss des Dotters auf die Ga.strula.tion und die Bildung der primiiren Keimbliitter der Wirbelthiere : II. Amphibien, Arch. Ent. Mech. ii, 1896.

A. SCIIAPER. Experimentelle Studien an Amphibienlarven, Arch. Em‘. Mech. vi, 1898.

O. SCHULTZE. Die kiinstliche Erzeugung von Doppelbildungen bei F1'oschla.rven mit Hilfe abnormer Gravitntionswirkung, Arch. Ent. lllech. i, 1895.

0. SCHULTZE. Ueber das erste Auftreten der bilateralen Symmetric im Verlauf der Entwicklung, Arch. mikr. Anat. lv, 1900.

H. SPEMANN. Entwickelungsphysiologische Studien am Triton-Ei, I, II, III, Arch. Ent. Mech. xii, xv, xvi, 1901, 1903.

G. WE'rzEI.. Ueber die Bedeutung der cirkuléiren Furche in der Entwicklung der Schultze'schen Doppelbildungen von Roma fusca, Arch. mikr. Anat. xlvi, 1895. 178 INTERNAL FACTORS IV. 1

§3. P1scEs.

Morgan has shown that in the fishes Ctenola/)ru.9, Serramw, and Fmzdulus there is no definite relation between the first (or second) furrow and the median plane of the embryo.

In Fumiulus when one of the first two blastomeres is removed the other gives rise to a perfect embryo of more than half-size. In segmentation the first furrow is in the plane of what would be the second furrow of the whole egg, the second in that of the third, and the third more or less in that of the fourth ,- but as the successive furrows are at right angles to one another in both cases it is permissible to assert that the segmentation of the half blastomere is total.

Some of the protoplasm of the half that has been removed flows in underneath the other, is nucleated by it and added to its mesoderm. The size of the nuclei is the same in % and -} embryos.

Morgan also found it possible to remove two-thirds of the yolk, but not more, without interfering with the normal development of the whole egg.

Injury to the germ—ring on one side of the dorsal lip resulted in a deficiency in the mesoderm of that side posteriorly ; anteriorly both sides were equally well formed.

So Kastschenko has shown for Elasmobranchs and Kopseh for Teleostei and Elasmobranehs that injury to the lip of the blastopore will only interfere with the normal bilaterality of the embryo when effected quite close to the middle line.

A series of experiments similar to the last has been carried out by Sumner on the eggs of various fish (principally 1*'zmrlulus, also Ewocoetzza, Salvelinus, and Batrac/ms).

By means of needles inserted into various parts of the blastederm the exact mode in which the material of the latter is used in the formation of the embryo is first determined. It is shown that the material for the embryo is brought into position not by a conerescence of the lateral lips of the blastopore (sides of the germ—ring), but rather by an axial concentration of the cells originally situated in that ring at the posterior margin or dorsal lip, the cells so concentrated being continually pushed forwards in the middle line until the anterior end of the embryo comes to lie near the original centre of the blastoderm. IV. 1 INITIAL STRUCTURE OF THE GERM 179

Injuries to various parts of the blastoderm and its margin (the germ-ring) prove that it possesses a certain degree of ‘isotropy’.

Thus the entire embryonic region of the posterior margin was destroyed by electrocautery. The adjacent edges closed up and completed the germ-ring ; at the posterior point of this a new embryonic shield was formed, in which ectoderm, endoderm, and notochord became differentiated. Or, again, a lateral piece of the germ-ring immediately adjacent to the embryo was removed without preventing the formation of a normal, bilaterally symmetrical embryo, although, as Morgan fou11d, the mesoderm might be deficient posteriorly on the injured side.


N. KASTSCHENKO. Zur Entwickelungsgeschichte des Selachierembryos,

Anat. Anz. iii, 1888. F. KoPscH. Experimentelle Untersuchungen iiber die Keimhaut der

Salmoniden, Vcrh. Aunt. Gesell., 1896. F. KOPSCH. Experimentelle Untersuchungen am Primitivstreifen

des Hiihnchens und an Sc;/Ilium-Embryonen, Verh. Auat. GeseIl., 1898.

T. H. MORGAN. Experimental Studies on Teleost eggs, Anat. Anz. viii, 1893.

T. H. MORGAN. The formation of the fish-embryo, Journ. Morph.

x 1895. ’F. B. SUMNER. A study of early fish-development, experimental and

morphological, Arch. Em‘. Mech. xvii, 1904.

§ 4. Amrmoxus.

In this form the blastomeres may be separated by shaking. Their development has been observed by Wilson, whose account

‘ 8 '’GD


FIG. 89. A. Normal } gastrula. of Amphioams. B. Gastrula from blas tomere. C Gastrula from a i‘ blastomere. J). Gastrula of normal size from an egg-fragment. (After Wilson, from Korschelt an Heider.) has since been confirmed by Morgan. The isolated blastomeres soon become rounded; the first division is always transverse to the long axis.


A i‘ blastomere segments like a whole ovum, except that the second division may be unequal, and gives rise to a normal blastula, gastmla, and embryo of half the normal size; % blastemeres behave in the same way (Fig. 89). Incomplete separation of the cells after the first or second division leads to double embryos in the first case, double, triple, or quadruple embryos in the second. The double embryos may make any angle with one another and continue to live for some time (Fig. 90).

A % blastomere segments like a whole egg (the second division is, however, always unequal) and gives rise to a normal blastula and gastrula, more rarely to an embryo of one-quarter size.

FIG. 90. Double nrastrulae of Amphioams, from incompletely separated

blastomeres. 1;‘), u’, separate blastopores; u, common blastopore.

(After Wilson, from Korschelt and Heider.)

A % blastomere will segment normally, or nearly so. A blastula is rarely formed, usually an open curved plate of cells, which are ciliated and swim about but do not gastrulate.

Lastly 115 blastomeres divide but produce only an irregular heap of cells.

It is obvious that the potentiality of a blastomere to form a whole embryo diminishes with its germinal value, the ratio it bears to the whole ovum; but it is not so clear whether this diminished capacity is due to the lack of some necessary, specific, organ-forming substance or merely to the small size and lack of IV. I INITIAL STRUCTURE OF THE GERM 181

undifferentiated material. Morgan has attempted to answer this question by counting the number of cells in the various organs of whole and partial larvae. It appears from his estimate that the number of cells in the archenteron, in the notochord and in the nerve-cord of -1-, 5, and 71,- larvae is constant, and that though the size of the whole body is less, the dimensions of the notochord and nerve-cord are about the same in all three. It would seem, therefore, that there is a minimum size and a minimum number of cells necessary for the formation of these organs. Whether the failure of the % blastomere to develop into a larva is, however, due to more lack of material or the absence of some specific substance needful for the development of some particular organ is still undecided ; for the 1% and :1; cells contain substance from both the animal and vegetative portions of the egg, while the §- are composed of one or the other substance exclusively, the furrows of the third phase being equatorial.


T. H. MORGAN. The number of cells in larvae from isolated blastomeres of Anzphioxzts, Arch. Ent. Mech. iii, 1896.

E. B. WILSON. Amp7n'0.z'us and the Mosaic theory of development, Joum. Morph. viii, 1893.


Many years ago Metschnikoif observed a perfectly normal separation of the blastomeres at a certain period in the development of certain medusae (Oceania). If when the cells reunite in a later stage the order of their rearrangement is not constant, the egg-substance must be in some measure isotropic.‘

More recently the equipotentiality of the blastomeres of these forms has been experimentally demonstrated by Zoja (Fig. 91).

The cells were separated by means of a needle. -3 and :1blastomeres (in the case of Lz'rz'ope, Gem/onia, Mitrocoma, Clytia, and Laozlice) and % and 1-15 blastomeres in the case of the two last mentioned will give rise by normal segmentation to blas ‘ It should not be forgotten that in 1869 Haeckel had cut the blastulae of Crystallodes (a Siphonophor) in pieces and obtained from the larger fra ments normal larvae. The development of small pieces was retar ed and abnormal (Zur En!wickelimysgeschichte der Siplconophovm, Utrecht, 18%). 182 INTERNAL FACTORS IV. 1

tulae with closed blastocoel and these to normal Planula larvae. In Olytia a hydroid was eventually reared and in Liriope a medusa with four primary tentacles from 73 and 3%; cells. It is to be noticed that, since the third division in these eggs is equatorial, the ,1, and 1% blastomeres must be either animal or vegetative.

Zoja also finds that in Laodice and Clytia the number of cells in partial larvae is proportional to the germinal value. In Li;-iope the number of cells in the endoderm is equal to that in the whole larva.


FIG. 91. Partial development

in Coelenterata. (After Zoja, 1896.)

a-cl. Laodice cruciata: a,§ blastomeres: 12, § blastomeres ; c, blastula from blastemere; d, larva during formation of endoderm. e—l. Liriope mucrouara. c— 2'. Development of blastomere : e, division of blaste more into f; -§-; g, -,"5,;; h, end ofendoderm formation in embryo; 1', medusa. reared from blastomere. j—l. Development 01} blastomere: j, 3; k, ,4“; Z, embryo with endodenn.



E. METSCHNIKOFF. Embryologische Studien au Medusen, Wien, 1886. R. ZOJA. Sullo sviluppo dei blastomeri isolati dalle uova di alcune meduse, Arch. Ent. Mach. i, ii, 1895-6. IV. 1 INITIAL STRUCTURE OF THE GERM 183

§ 6. ECHINOD]*Jlh\IA'1‘A.

Easy to obtain in large numbers and eminently suitable for experiment, the eggs of the sea-urchins and starfishes have provided the analytical embryologists with abundance of material; and it is in this group that the possibility of rearing a perfect larva from a single blastomere was first demonstrated by the classical researches of Driesch.

The experiments which have been carried out, mainly by Driesch, but also subsequently by others, are very numerous and fall into two principal classes.

In the first the development of parts—wheth_er isolated blastemeres or groups of blastomeres, fragments of unsegmented eggs fertilized or unfertilized, or pieces of blastulae and ga.strulae—has been observed, the rate of their development recorded, and the relation of the number of cells and dimensions of the partial larvae to their germinal value determined. By the second series of investigations the effect of alteration of the type of nuclear or cell-division and consequent displacement of the blastomeres upon the subsequent development has been discovered.

Before discussing these experiments, however, a word must be said as to the normal behaviour of the whole egg.

The structure, segmentation and formation of the primary germinal layers have been very completely studied by Boveri in the sea-urchin, St1'o2z_qyZocenh'o6u8 livirlus (Fig. 92). The axis of the ovarian egg is marked by the exeentric position of the nucleus ; at the point on the surface nearest which it lies the polar bodies are formed, and this point is the animal pole. At this point there is a fine canal (micropyle) in the jelly which surrounds the egg. After the extrusion of the polar bodies a brown pigment which had previously been uniformly distributed through the cytoplasm becomes aggregated in a dense but quite superficial subequatorial zone, the upper somewhat ill-defined border of which may, however, extend into the animal hemisphere. The pigment-free region left about the vegetative pole occupies from 7515 to -115 the volume of the whole ovum. The spermatozoon may, but need not, approach the egg by the mieropyle. The fertilization spindle lies in a plane parallel to the equator of the egg (the FIG. 92.——Norma.l development of the sea.-urchin, Sh-ongyloceulrotus l1'm'dus. (After Boveri, 1901.)

The animal pole is u permost in all cases, and in the first two figures the jelly with the cana.i)(micropyle) is shown.

a, primary oocyte ; the pigment is uniformly eripheral.

b, ovum after extrusion of polar bodies: tiie pigment now forms a. subequatoi-ial band. The nucleus is ex-axial.

c, (1, first division (meridional).

e, 8 cells, the pigment almost wholly in the vegetative bla.ston1o1-vs.

j, formation of mesomeres (animal cells) by meridional division: the vegetative cells have divided into macromeres and micromeres.

g, blastula. h, mesenchyme blastula.

1', j, k, invagination of the pigmented cells to form the urchenteron of the gastrula. In j the primary mesenchyme is separated into two groups, in each of which, in A-, a. spicule has been secreted. In I: the secondary, pigmented mesenchyme is being budded off from the inner end of the archenteron. IV. 1 INITIAL STRUCTURE OF THE GERM 185

‘ karyokinetic plane’) and elongates at right angles to the spermpath, which thus lies in the plane of the first, a meridional, furrow (a similar observation had been previously made by Wilson and Mathews on Towopuezzstzav).

The second furrow is likewise meridional and at right angles to the first, the third equatorial or a little nearer to one or other pole; the animal cells get only a very little of the pigment. In the next phase the animal cells divide meridionally and equally to form the eight mesomeres, while the vegetative cells divide latitudinally and very unequally into four large pigmented macromeres next the equator, and four small quite unpigmented micromeres grouped round the vegetative pole.

In the blastula stage all the cells are of the same size, vacuolated and ciliated, and the polarity of the egg is only determinable by the position of the pigment zone. In the next stage the clear vegetative cells derived (presumably) from the micro

FIG. 93.—Ec7n'nus: segmentation under pressure.

a, preparation for third division (radial); b, preparation for fourth division (tangential); b’, after fourth division; c, another form of the 8-cell stage (third division parallel to first); (I, the same after removal of the pressure. (After Drieseh, 1893.)

mercs wander in to form the primary mesenchyme~from which the first triradiate spicules are developed-—and this is followed by the invagination of the pigmented cells to form the arehenteron. Secondary pigmented mesenchyme cells are budded off from the inner end of the latter. The character and sequence of the divisions in segmentation are the same in other Echinid eggs, but the polarity is not marked by pigment, nor indeed recognizable till the micromeres have been formed, and again when the mesenchyme cells wander into the blastocoel.

By means of press11re——under a coverglass-—Driesch succeeded (E0/12'-mw) in altering the direction of the spindles and consequently of the cell-divisions (Fig. 93). The first and second furrows were at 186 INTERNAL FACTORS IV. I

right angles to one another and to the coverglass, but whether meridional or 11ot does not appear. The third furrows were again at right angles to the coverglass and at 45° to the first two; a flat plate of eight cells was thus formed. The next division resulted in the formation of sixteen equal cells still all lying in the same plane ; the formation of micromeres is thus suppressed. Such eggs gave rise to normal larvae.

In the case just quoted the egg-membrane was intact; but similar results were obtained when it was broken. The second furrow was sometimes at right angles to, sometimes parallel to, the first, the third at right angles to both in the latter case, and the fourth

FIG. 94.—The effect of heat upon the segmentation of the Echinoid egg. a, b, c, d, four successive stages in the segmentation of the same egg ofEchinus; e, f, two successive stages in the division of the same egg of Sphaerechinus. (After Drieseh, 1893.)

parallel to the third ,- but if the pressure was released the blastemeres became rounded olf and the sixteen-celled stage consisted of two plates of eight cells each; one or two micromeres were sometimes formed. The development of these eggs is also quite normal. These results have been confirmed by Ziegler.

In this, as in the similar experiment of Hertwig on the Frog’s egg, the normal distribution and arrangement of the nuclei is interfered with without prejudice to the normality of subsequent development. Nuclear division cannot therefore, Driesch contends, be a qualitative process. Boveri has also shown in similar fashion that the order of segmentation may be disarrang-ed and . IV. 1 INITIAL STRUCTURE OF THE GERM 187

the formation of the micromeres suppressed in Stron_qylocem5rotzw and a normal larva still result. The relation, however, of the egg-axis, as determined by the pigment-ring, to the symmetry of the larva remains the same as in the undisturbed egg. Increased temperature, violent shaking, dilute sea-water, and calcium-free sea-water are all means by which Driesch has suc ceeded in disarranging the blastomeres of E’c/tinus in various ways (Fig. 94). Thus when the temperature was raised from 19° C. (the

FIG. 95.——Va.ria.tions in the segmentatioxi of Echimts mic;-otuberculntus produced by dilution of the sea-water. a, tetrahedral four-cell stage; I), eight cells, three premature micromeres; c, eight cells, two precocious microineres; (I, the same egg after the next division. the precocious niicromeres have divided unequally, two normal micromeres have been formed. (After Driesch, 1895.)

normal) to 31° C. the first two (not subsequent) blastomeres separated, though they sometimes reunited. The next division was normal, b11t with the next phase one or two blastomeres divided in a direction perpendicular to that of the others, or the direction of division was different in each. In the following stage the micromeres were wholly or partly suppressed. So also in dilute sea-vmter (20% fresh water) the third division was unequal 188 INTERNAL FACTORS IV. I

in two or more blastomeres, and in the following an excessive number of small cells was formed (Fig. 95). In aless dilute mixture (16% fresh water) the blastulac divided to form each two Plutei. Again by shaking the eggs in the eight-celled stage the blastomeres were disarranged and made to lie almost in one plane ; micromeres were, however, formed as usual from the vegetative cells, but not, of course, in the normal position (Fig. 96). In spite of these very considerable alterations in the size and positions of their constituent cells all these ova nevertheless gave rise to perfectly normal larvae.

By the use of calcium-free sea.-water either the mesomeres or the macromeres and micromeres were separated into two groups. In the latter case the larva had two guts, in the former a normal larva. resulted, so that, as Driesch points out, the ectoderm at the

FIG. 96.~—-Disarrangement of the blastomeres of Echimcs by shaking, a, eight cells; b, sixteen cells; notice that the micromeres are not close together. (After Driesch, 1896.)

antiblastoporic (animal) pole must in this case have been formed from the macromeres.

The conclusion to be drawn from these experiments seems obvious; not only the nuclei, but the parts of the cytoplasm too are equivalent; within at any rate fairly wide limits their normal arrangement may be disturbed without affecting in the least the normality of subsequent development; or, as Driesch phrased it, the destiny of a nucleus or blastomere is not determined by its original situation in the preformed structure of the egg, rather it is a. function of its position in the whole embryo to which that egg gives rise. Its character is decided not by its origin, but by its final position.

Whether there really is a limit to the rearrangement is a question which must be reserved for future discussion. We are 4 IV. I INITIAL STRUCTURE OF THE GERM 189 A /‘


FIG. 97.—A and 0, formation of ex-ovates in the egg of Arbacia by dilution of the sea.-water; K, nucleus; m, egg-membrane. B and D, blastulae formed from A and C ; B becomes constricted into two blastulae, each of which gives rise to a. Pluteus; Dproduces a. single Pluteus. (After Loeb,from Korachelt and Heider.) 190 INTERNAL FACTClRS IV. I

also indebted to Driesch for a long series of experiments on the behaviour of isolated blastomeres and egg-fragments. In the forms which we have hitherto considered—the Vertebrates and Coelentera.———an isolated cell segments as a whole and gives rise to a total larva. In these Echinoderms the isolated blastomere also gives rise to a total larva, but its segmentation is partial; only after segmentation has been completed does the open blastula. close up and resume the polarity of a whole.

Various methods have been employed for separating the blastomercs. Loeb showed that in dilute sea-water (50 the egg

FIG. 98.-—Development of the isolated «} blastomere of Echinus micromberculatms-. 'l‘wo1n1eromeres,twonmcromeres, four mesomeresz a, §; b 4*

c, Hemiblastula: on the right 1s the remaining blastomere, dead. Driesch, 1892.)

swells, bursts its membrane and protrudes an ex-ovate which may be large and develop independently (Fig. 97). Driesch has used heat, pressure, violent shaking with fragments of coverglasses, and the calcium-free sea-water introduced by Herbst. The isolated 71; blastomere in Ea/ziuus becomes rounded and segments as a half, as though the other blastomere were still present. It forms four mesomeres, two macromeres, and two micromeres, IV. 1 INI'l‘IAL STRUCTURE OF THE GERM 191

and by division of these a hemispherical curved p ate, a halfblastula (Fig. 98). When, as may happen, the other astomere dies without being separated, it is embraced by the open side of the survivor. Only after the 32-celled stage is passed do the edges meet and close. A blastula of half-size is the result, which becomes later a gastrula an(l'a Pluteus of perfectly normal form but of half the normal size.


C :1

FIG. 99.——Segmentation of an isolated } blastomere of Eolmms; :1, -1-, (2), Ag, c,h—145i o9I§e)m1cro1nere, one macromere, two mesomeres, d, 391;. (After l‘leSC , 8 .

7% blastomeres behaved in the same way, segmenting as parts, developingultimately into whole normal Plutei. Similarly -,1 blastomeres segmented partially (Fig. 99), with, in some cases, irregularities : two micromeres, for instance, were formed in the second division. The % blastulae will gastrulate, but progress no further.


FIG. 100.—-A, Gastrula with meaenchymc cells and trimdiate spicules reared from a. 2; animal cell of Sphaerechinus. B, The same from a. 5- cell of Echinus: the gut is tripartite. (After Driesch, 1900.)

In the next stage—-% blastomeres—a diiference becomes noticeable between the behaviour of the animal and vegetative, cells. Both kinds.of cells will gastrulate, secrete triradiate spicules and 192 IN’I‘ERNAL FACTORS IV. 1

develop a normally tripartite gut (Fig. 100). But this only occurs in a small proportion of cases, smaller for the animal than for the vegetative cells. Further, the former frequently give rise to blastulae provided with a row of long cilia (Fig. 101), while the latter are delicate, and many die.

Boveri has pointed out that the ability of animal cells to gastrulate depends, in Stron_r/ylocemfrotus, on whether or not they contain a portion of the pigment-zone, and this on the position of the third furrow. Garbowski, however, denies that the pigment is itself an organ-forming substance. For in the first place there is a variety of S51-ongyloceaztrotus livitlus, in which the pigment remains diffuse and never becomes concentrated to form a band at all ; and secondly, even though, when present, it is usually in the subequatorial position described by Boveri, this is not always so; it may be oblique to the egg-axis, or wholly in the

Era. l01.—Long-eiliatedblastula from animal» 01‘ Wh°11.Y in the a _} animal cell of Echimcs. (After vegetative hemisphere, D"es°h’1900‘) It would appear, therefore, either that it is merely associated with some other substance which has up to the present remained invisible, or that in the cases described by Garbowski animal cells, if they were pigmented, would gastrulate as readily as ordinary vegetative blastomeres, in which case the manner in which the pre-determined material is cut up in segmentation would be a matter of indilference.

The differences in the behaviour of the T15 cells are still more marked. The mieromeres will only divide to form a heap of about ten cells ; the mesomeres give rise to either long-eiliated blastulae or imperfect gastrulae, with or without skeleton and 1V.1 INITIAL STRUCTURE OF THE GERM 193

mesenchyme, and with an undivided archenteron (Fig. 102) ; the macromeres, on the other hand, become gastrulae provided with mesenchyme and spicules (Fig. 103).

Lastly, even 51-; blastomeres will occasionally gastrulate, pro~ babl y only if derived from a macromere. This gastrulation was

Fm. 102.—Echinus. Larvae reared from mesomeres (animal cells) of the 16-celled stage. a and b, mesenchyme gastrulae, a has spicules; r, gastrula without mesenehyme; d, a long-ciliated blastula. (After

Driesch, 1900.)

not, however, observed directly, but the germinal value of the smallest gastrulae found was calculated by a. method which will be described below. 517; cells will not reach the gastrula stage.

9 O (I. b 0

FIG. 103,- Two gastrulae of Erhinus reared from 3;, macromeres; both have mesenchyme cells, one has triradiate spicules. (After Driesch, 1900.)

The difierences between the developmental capacities of animal and vegetative cells may be studied in another way. The four micromeres were removed by Driesch in the 16-celled stage; the remaining twelve meso— and macromeres produced a normal Pluteus (an experiment also performed by Zoja on Stro7zy_I/Zocenlrotvue). The eight (or four in the previous stage) animal cells alone, the eight vegetative cells alone also formed, in some cases, a perfect larva. Segmentation was in all these cases partial.


From the fact that the four animal cells together may form a Pluteus while each individually fails to pass beyond the gastrula stage, Driesch has argued that this failure depends solely on lack of sufficient material, not on the want of any specific gut-forming substance, and has supported his contention by other evidence. By placing the eggs of lL'c/firms in dilute sea-water the third division was made unequal ,- the cells were then isolated in ealeium— free water. The large cells thus formed must contain either more of Boveri’s pigment-ring (or rather of the substance of the vegetative hemisphere) or else more of the animal hemisphere, probably the former, since a larger percentage gastrulate than is usual with animal blastomeres. According to Boveri, however, all ought to gastrulate if all contain the specific substance for the archenteron. Further, these large cells are able to form mesenchyme even when the gut is lacking although they possess the middle region of the egg in any case and may lack the micromere area.

Driesch also points out that a 7} blastomere and a § vegetative blastomere l1ave both the same amount of Boveri’s pigment-ring and mesenchyme area, the latter, however, only half as big a gut and half as many mesenchyme cells as the former, and lastly that »,‘;— and :—l% animal blastomeres will not only'gast1-ulate but form mesenchyme as well.

The limitation of the potentialities of ectoderm and cndoderm in later stages has been investigated also by Driesch—-by observing the development of pieces or fragments of the blastulac and gastrulae. These pieces are obtained by cutting or shaking. Loeb has employed dilute sea-water to make the blastula swell, protrude through its membrane and become constricted into two.

In Ea/dmes, S/1/merec/u'mw, and /18/erius, any piece of a blastula when first cut out is crumpled, but soon becomes rounded, and swims about and eventually gastrulates.

Monsters exhibiting a certain degree of duplicity have been produced by Driesch by shaking the egg of Arte/'ia..e in the two-celled stage (this produces apparently a partial separation of the blastomeres) and by placing the blastulac of 152'//imzs in diluted sea-water. In the latter case the gut is single, the skeleton double; in the former the gut is doubled, though the two may subsequently fuse, or even trebled, or may be IV. I INITIAL STRUCTURE 01*‘ THE GERM 195

merely forked. The two guts may be similarly oriented or turned in opposite directions, and it is interesting to observe that Driesch, who believes the first furrow to coincide with the plane separating the two guts, accounts for the latter case by supposing that one blastomere had been rotated upon the other, so that their vegetative ends—the locus of the gut-forming substance-—faeed in opposite directions.

Neither the ectoderm of the early gastrula alone nor the endoderm is capable of giving rise to a larva, though the former can develope a stomodaeum, a statement confirmed by Morgan.

The vegetative half of a gastrula of AS);/zaerec/ziizzm will give rise to a normal small Pluteus whether the cetoderm only, or together with it the tip of the archenteron, has been removed (Fig. 104) ; the animal half will not. VVhen the spicules, one or


FIG. 104. —»The potentialities of the cells of embryonic organs; (1, I). The vegetative and aniinal portions of a gastrula of S1)/I(IeI'€(‘lIiIl1Is grauulm-is, cut equatori-ally in two; 0. Plut-eus reared from a fragment of a gastrula; (1. Normal tripartite pluteus gut. (After Dricsoh, 1896.)


both, are removed the skeleton is one-sided and the Pluteus eonsequently one-armed.

In As/elrias the arehenteron can form a new terminal vesicle and mesenehyme cells afresh when these have been removed (Fig. 105); but should the eoelom sacs be cut off ata later stage, the (secondary) archenteron can form no more, though it divides into the usual three portions. T

Parallel to the behaviour of isolated blastomeres is the segmentation‘ and development of egg-fragments, obtained by shaking, cutting, or (Loeb) dilute sea—water. The fragments may be taken from already fertilized eggs, or from unfertilized: in the latter case each, whether nucleate or enueleate, must be subsequently fecundated.

In segmentation there are a great many ditfereiices, which appear to depend on the nature of the fragment, that is to say, 196 on the part of the egg from which it


IV. I In

has been removed.

the case of the colourless egg of ]$’¢-/zz'm1.9 it is only possible to

FIG. 105.~-The potentialities of the cells of embryonic organs; a. N0l'1]1i1-l Pluteus of S'ph(wre(.'hiIms; b. Pluteus reared from a fragment of a gustrula; 0, 9. Normal Bipinnaria of A8tP1‘iflS _qlaciali.s-; «I, f. Bipinn-aria from the vegetative half of a gastrula; g. Larva. of Asterias with typical tripartite gut, but no coelom, from the vegetative half of a gastrula. removed after development of the eoeloni sacs. (After Dricscli, 1896.)

In another case wha.t appears to

guess at the nature of the piece by observing the mode of segmentation.

The first and second divisions may both be equal, as also the third and fourth ; in this case (1)1-ieseli supposes) a large fragment is involved. Again, the first, second, and third may be normal, but the fourth and fifth equal: no micromeres, therefore, are formed, and the fragment is probably derived from the animal hemisphere. Or while the first and second are equal, one of the four cells will divide unequally; in the next division a large or small number of micromeres is formed, according, presumably, as a larger or smaller portion of the mieromere region has been included in the fragment (Fig. 106i.

be a. meridional half

divides equally twice, and then two of the four equally (mesomeres), two unequally (macro- and micromeres) (Fig. 107), while a supposed vegetative half is segmented to form four large and

four small cells.


Thus the type of segmentation is determined by the initial IV. I INITIAL S'l‘RUC'l‘URl*] OF THE GER,1\[ 197

structure, including the physical constitution, of the ovum alone, a conclusion in which Boveri concurs.

All these fragments will give rise to normal larvae, provided they are not too small. The least egg-fragment that will gastritlate has the same germinal value as the least blastomcre, namely,


FIG. 106.-~ Segmentation of an egg-lragxiient of E¢-Iu'nu.s- supposed to contain the whole of the mieromere area of the egg, and some of the animal hemisphere. u. Eight cells, three equal pairs and one unequal pair; 1;. sixteen cells, four of the eight having formed mieromercs, the other four divided equally. (After Driesch, 1896.)

31-2- (calculated from the volume of the smallest gastrnlae found). Morgan, however, estimates the smallest egg-fiaginent capable of giving rise to a normal larva at from 315 to -616, the least blastenicre that will gastrnlate only


FIG. 107. — Segmentation of an e;,rg-fmgnient (Ez-Irinats) supposed to be an exact lneritlional half". (I. Eight cells, four l)e1ng im-s0Incre.~', two n'1-.Lcro1neres and two microineres; b. sixteen cells. (After Dneseh, 1896J

In partial blastulae, gastrulae, or larvae the number of cells is proportional to the germinal value ; and this is true not merely of the whole embryo or larva, but of each of its organs, the 198 INTERNAL FACTORS IV. 1

eetoderm, the mescnchyme, the archenteron. This assertion Of Driesch’s is corroborated by Morgan for the whole larvae, but not for the organs ; according to him the number ofeells invaginated to form the archenteron in a partial tends to approach that found in

a whole larva, about fifty. l)riesch has also determined the relation to their germinal value of the dimensions of partial blastulae, gastrulae, and Plutei and their organs (Figs. 108 FIG. 108.— Outlines of -}, 22, 1, 112). As the table (Table XX)

« l bl‘th fS7 ""7' . . . ., '(‘X;.teilf Dr?:‘S;‘11"°19"00_)” W‘ “ ‘””'S shows, the suifaees ale vex)

nearly as the germinal values, the ratios of the radii of the partial a11d total larvae consequently greater, of the volumes less than these values.

Flu. 1O9.——Ea1'ly gastrulae of Ec-hinu.~c: a. }, b. g, c. 1, (Z. 3,. (After Driesch, 1900.) IV. 1 INITIAL STRUCTURE OF THE GERM 199


Relation of dimensions of partial blastulae to their germinal value. Germinal values.

1 1

1 . 1. 2+ Rzulii . . . 2 1-2 I .75 Surfaces . 4 2-2-5 1 -5625 Volumes . . 8 3375 1 -4

FIG. 1lU.—l’lutei of I9'rlu'nM.s-: u. b. .1_,, 4'. 1. The skeleton is shown. (After Drie.~xch, 1900.)

From the proportionality of the number of cells to the germinal value it follows that the cells of the whole and of the partial blastulae are of the same size.

If 1:’, S, and V are the radius, surl"aee, and volume of the whole

. . . 1 '

egg, and rm 3", nu the correspon(l1ng- magnitudes Ill a ; larva, S 1:? I’

then 8n = ——,whenee 1'” = _, and therefore 0” = - ~—_.—. By " 1; ~/ 713

this formula. the germinal value of a larva of unknown origin may be calculated. Finally, although segmentation takes place at the normal rate 200 INTERNAL FACTORS IV. I

in the isolated blastomeres, their subsequent development is progressively retarded as the germinal value decreases (Table XXI).


Showing the rate of development in partial larvae. Germinal value.

5 Day ! } : 7’; 7‘: L 5 A 1 ' Gastrula IEarly Gastrulal Still earlier Blastula f Gastrula ' 2 Pluteus Early Pluteus Gastrula with Blastula and 4 Skeleton Gastrula 3 Pluteus Pluteus Early Pluteus Gastrula 4 Pluteus Pluteus Pluteus Gastrnln

a C

FIG. 111,-SpIum'¢=chi/Hts Plutci, showing the gut. u -}, b. .1_., c. }. (After Driesch, 1900.) It is evident from these experiments that, in the Echinoderms, while the isolated parts of the egg‘, whether blastomeres or egg fragments, are only able to segment as they would have done had they remained in connexion with the whole, they are neverIV. I INITIAL STRUCTURE 014‘ THE GERM 201

theless capable of total development in a very marked degree. This capacity is not, however, unlimited, but diminishes as development proceeds, and it appears highly probable, and is indeed often admitted by Driesch, that the chief cause of this restriction is the lack of some specific substance and not the deficiency of mere material, though this may be a subsidiary factor. The progressive loss of potentialities is exhibited also by the embryonic organs as they are formed later on.


FIG. 112. --Echinu.s~, the tripartite gut of the complete l'luleus: (1. }, b. c. in (After Drieseh, 1900.)

Before concluding this section we must notice an experiment which is the converse of those considered above, namely, the production of one embryo from two eggs.

An unsuccessful attempt was made by Morgan to obtain this result. This author only succeeded in showing that the eggs of AS’/1//ac/‘cc/rim/.9, when (leprived of their membranes by being shaken, tend to fuse together, and that from such pairs double monsters may be produced with two guts and two skeletons. One skeleton may, however, very large] y predominate over the other, which indeed remains rudimentary, and the guts may coalesce. 202 INTERNAL FACTORS IV. 1

Driesch, however, who has employed the same method as Morgan and has corroborated his results, has been able to produce, from the fusion of two ova, a larva single in all its parts—mesenehyme, skeleton, and gut. The organs are in correct proportion but larger than the normal (Fig. 113). The number of mesenehyme cells is about twice the normal.

From this experiment it may of course be concluded that the egg substance possesses a degree of isotropy, but not that that isotropy is absolute. Nothing, it should be remarked, is known of the way in \vhich the eggs which fuse to form one embryo are oriented upon one another. Garbowski, however, has stated

A fl

Fla. 1113. A. Norlnal gastrnla of S_pIz(m'r-('h1'nMs._ B. Single gastrula formed by the fusion of two blastulae. (After Drieseh, from Korsehelt and Heider.)

that fragments of different eggs of ]3'c//iizzts (in segmentation stages) may be grafted on one another, and that the product of their union will give rise to’ a normal embryo, whatever the relative positions of the fragments.

The fusion of blastulae to form giant Planulae has been noticed by Metsehnikoff in the medusa, Jli/roco///a mmae, and in 0])//r_//at/'oc/Ia Korsehelt has observed a similar fusion of (llstinet eggs in the body cavity of the parent when the latter is hermaphrodite. Lastly, Sala and Zur Strassen have observed the fusion in twos, threes, or more of the eggs of ./¢m'.s-, a result experimentally produced by the first author by exposure to a low temperature, while Zur Strassen has found that such double eggs, provided they had united before fertilization and then been fertilized by asingle sperm, will produce perfectly normal embryos. IV. I INI'I‘IAL STRUCTURE OF THE GERM 203


T. BOVERI. Die l’o1m-itilt von Ovocytc, Ei und Larve des SIron_r/,z/Iocentrofus Iiritlus, Zool. Jahrb. (Anat), xiv, 1901.

T. BOVERI. Ueber die PoIa.ritii.t des Seeigelcies, Vwh. d. ph_:/s.-nwd. Gas. cu Wiir:Inu'_q, xxxiv, 1902.

H. DRIESCH. Entwicklungsmechzmische Studien : I, II, Zcilschr. uv's.~:. Zool. Iiii, 1892.

II. DRIESCH. Entwicklungsn1ec1mniscl1e Studieu: II|—VI, Zei!s¢'7u‘., 1z'i.s-s. Zoo]. Iv, 1893.

H. Dlulascll. Entwicklungsmechanische Studien: VII-X, Mir/. Zaol. Slut. Ncapel, xi, 1895.

H. Dnusscn. Zur Analysis der Potenzen embryona.lcr Orgamzcllen, Arch. Eul. Merl). ii, 1896.

H. DRIESCII. Betmchtuxigen fiber die 01'gunisution des Eies und ihrc Genese, An-h. ]v.'m. Mo:-7:. iv, 1897.

H. DRIESCH. Primiire und sckundi'u'e Regulationen in den‘ Entwicklung dcr Echinodermen, Arrh. Eur. Mech. iv, 1897.

H. DRIESCH. Die Versclnnelzung der lndivid112L]it€‘Lt bei Ec11inidenkeimen. Arch. EM. Merh. x, 1900.

H. DRIESCII. Die isolirten Blastolneren des Echinidenkcimes, Arch. EM. Mech. x, 1900.

II. DRIESCH. Neue Ergihizungen zur Entwicklungs]physiologic dos Eel)inidenkeimes, Arch. Em. Illech. xiv. 1902.

H. DRIESCII. DreiApho1'ismen 7.111‘ Entwicklungsphysiologie jiingstcr Stndiell, Arch. 12'/1!. Jllcch. xvii, 1904.

II. DRIESCH. Altes und Neues zur Entwicklnngsplnysiologic dcs

jungun Astcridenkeiines, Ar:-h. Enl. Mc-ch. xx, 1906.

M. '1‘. GARBOWSKI. Ueber B1-ustomerentru.nspIa.ntationen bei Sceigeln, Bull. Iufmwal. do l'Arml. ties Sci. dc (,'r(u'orie, 1904.

M. T. (irARB()\‘\'Sl\' I. Ueber die l’oIzu'itiit des Scoigcleies, Hull. Internal. ((0 IA('mI. (105 Sci. dc ('rm'uvi¢', 1905.

M. '1‘. GARBOWSKI. Ueber Bla.sto1nerent1'ansplamtationen bci Sceigeln, Bull. Internal. Ac. Sv. Crucoric, 1905.

1*}. KORSCHELT. Uebcr Kerntllcilung, I‘}ircifung und Bci'1'ucI1i.ung bci 01)/H'_IjU(I'0(']I(I1)Il(’I'i/(ls, Zeitschr. 'mf.s's. Zoo]. lx, 1895.‘

J. LOEB. Ueber cine eiufaclne Methode, zwei odci-1110111‘ zusu.mn1engewaclisene Fmbryoncn nus oinem Ei hervorzubringen, P_/lii_qer’s Arch. Iv, 1894.

J. LOEB. Beitriige zur Entwicklungsmechanik der nus einem Ei entstchenden Doppelbildungen, Arch. Euf. Mech. i, 1895.

E. ME’l‘SCIINIKOFl~‘. Embryologisclie Studien an Medusen, Wien, 1886.

T. H. MORGAN. EXp€l‘I1HC1IId.I. studies on Echinodcrm eggs. Anat. Ans. ix, 1894.

T. H. MOREAN. Studies of the ‘ pa.rbial ' larvae of Spham'ech1'Ims, Arch. Ent. Mech. ii, 1896. 204 INTERNAL FACTORS IV. I

T. H. MORGAN. A study of a. variation in cleavage, Arch. Em. Mach.

ii, 1896. '1‘. H. M()RGAN. The formation of one embryo from two blastulae,

Arch. Euf. Mcch. ii, 1896. L. SALA. Experimentello Untersuchungen iiber die Reifung und Befruchtung der Eier bei Asc(u'i.~: mcgalocqrhalu, Arch. mi/cr. Anal.

xliv, 1895. 0. L. ZUR STRASSEN. Ueber die Riesenbildung bei Ascm-is-Eiern,

Jrch. Ent. Mech. vii, 1898. H. l. ZIEGLER. Ueber l*‘urc-hung unter Pressung, Verh. Anal. (:'escll.,

1894. R. ZOJA. Sullo sviluppo dei blastomeri isolati dalle uova. di alcunc

medusc (e di altri organisini), Arch. Eur. Mach. ii, 1896.

§ 7. Nimnirrinm.

In the behaviour of its isolated blastomeres the Nemertine egg resembles the Echinoderm very closely; the cells segment as parts but ultimately give rise to wholes. The limit of this capacity is, however, sooner reached, for there is a sharper distinction between the potentialities of animal and vegetative blastomeres.

Experiments have been made on (Jcrcb/'a/zzlzw («alone by VVilson, on Ccrcbraiulus nzaI'_I/‘iizafius by Zeleny ; Zeleuy and Yatsu have further investigated the development of fragments removed from various regions of the egg.

The axis of the egg is marked by the excentricity of the nucleus and the point of extrusion of the polar bodies. 1\Iatura.tion precedes fertilization. The first two furrows are meridional and divide the egg into four equal cells- (A, 1!, C’, and 1)). Subsequent cleavage is, however, on the spiral plan seen in Turbollarian, Annelidan, and holoblastic Molluscan eggs.

In the Nemertine the egg-axis becomes the axis of the Pilidium larva, the animal being the aboral, the vegetative the oral, pole.

In both the § and the % blastomeres cleavage is partial and an open blastula is formed, or sometimes a flat plate of cells merely, sometimes a closed sphere. The 1}; later becomes a larva with an apical organ, mescnehyme and archenteron, but no lappets; the % larva has a solid archenteron but neither apical organ.nor lappets. Development is slightly retarded 3 % blastomeres give rise to a IV.1 INITIAL STRUCTURE OF THE GERM 205

larva. with mesenchyme and apical organ, but the archenteron is solid. After the next phase—eight cells—-the upper quartette

FIG. 114.———Ce;-cbrafulus. Larvae from upper quartettes of the 8-cell stage. A. Larva from whole quartette. B, C. The same, but one cell was injured. J), E, F. Slightly older, f'rom complete quartettc. (After Zeleny, 1904.)

(1 11-1 rl) produces a larva with an apical organ and meseuchyme but devoid of an archenteron ; the larva developed from the lower

quartette (,-l—~—Z)) has an archenteron but no apical organ, while a

/1 FIG. 115.~—('erebralulus. Larvae from lower (vegetative) quartet-tes of 8-cell stage. Note absence of apical organ, presence of large, hollow or solid, archenteron. (After Zeleny, 1904.) meridional half, comprising two macromcres and two micromeres, develops both organs, though it is usually asymmetrical. In the 16-celled stage the isolated apical cells (1 a 1-1 (I 1) will

give rise to a. ciliated blastula with mesenchyme and an apical 206 INTERNAL FACTOllS IV. I

organ, b11t without an archcnteron, while from the remaining twelve is derived a ciliated embryo with a large solid arehenteron, but destitute of an apical organ and lappets.

Wilson asserts that the size of cells in partial is the same as in total larvae ; their number appears to be proportional to their germinal value.

From fragments of blastulae normal, or nearly normal, dwarf Pilidia may arise, but the degree of development again depends on the origin of the fragment. VVhile the lower third becomes

FIG. ll6.——Cerebratulus. Larvae from portions of the 8-cell stage. A, II. Larva from lateral 4-cell group. Note presence of both apical organ and archenteron. C. Similar, but younger. I). Larva from upper quartette plus two cells of lower quartette. Note that there are three apical organs, a large bl-astocoel, a small archenterou, and two inxluggiiiations of eetoclerm at the sides of the archent-eron. (After Zeleny.


a eiliated embryo without an apical organ, a piece of the upper two-thirds developed (in one case) into a larva with a gut, with two apical organs, but no lappets (Zeleny). Wilson states that in Cerebrazfulus lacteus the archenteron is small in animal fragments, the apieal organ frequently, though not apparently always, absent in pieces of the vegetative hemisphere.

Animal fragments of the blastula have a greater total potentiality, a greater regulative capacity, than the animahblastom eres IV. '1 INITIAL STRUCTURE OF THE GERM 207

of the eight-celled stage. This is attributed by Zeleny to the greater time and opportunity afforded them of regaining the polarity of the whole.

The behaviour of egg—fragments depends not merely on the part of the egg from which they are taken but also on the time at which they are removed. '

Pieces taken from the ovum, no matter in what way, before the disappearance of the germinal vesicle segment as wholes and give rise to perfect Pilidia. Only in a small percentage (15 %) of cases is their development defective, the lappcts being absent, or the gut abnormal, or the apical org-an absent or multiplied (Yatsu). The proportion of perfect Pilidia obtained from fragments removed a little later, at the time of formation of the first polar spindle, is,

V however, less, only 52 0 It is during the conjugation of the H pronuclei that the definite localization — 4 H of specific substances seems to begin, V '

for now the fate of the fragment de . . . . 0 pends upon the direction in Wl)l(‘l1 the cut is made. Eggs from which the animal

. . FIG. 117.-— Cerebmtulus. portion——about one-third of the whole Diammn of egg with one

——has been taken away become perfect POW‘ b0d)'. and basal PW - - ' t b .( I: ‘I d.‘ ' Pilidia ; when, however, by removal of t§)neS1(":)(3f-c’],(i:,.ii;(;3;i:,1 Jiffy,

the vegetative third, or by an oblique vertical (V), aiul oblique cut, the fragiiient does not contain (1%4_°)“ts' (After Zelcny’ the whole of the lower two-thirds, the

lappets are defective and the apical organ sometimes absent as well. If, therefore, there is present at this stage a special material for this organ it cannot as yet be located in its ultimate position.

The cleavage of these fragments is always partial (Zeleny). After the first furrow the material for the apical organ has still not reached the animal pole : the animal region may be removed from both blastomcres without inhibiting its formation. bcrfeet Pilidia may also be produced from eggs in the two-celled stage when pa.rt of one blastoinere has been obliquely cut away.

Should, however, the blastomeres be cut apart before their complete


separation, each develops into a Pilidium with one lappet but no apical organ; it seems, therefore, that the material for this structure is at this stage placed in the bridge of plasma connecting the two cells, and is fatally injured by the operation.

Taken together these experiments make it, to say the least, highly probable that there are in the Nemertine egg definite substances connected necessarily, that is causally, with the formation of certain organs.

These specific organ-forming substances may be said to be

preformed, but it is equally clear that they are not prelocalized, but only reach their ultimate destination in the course of development.


E. B. WILSON. Experiments on cleavage and localization in the

Nemertine-egg, Arch. Ent. Mech. xvi, 1903. N. YATSU. Experiments on the development of egg-fragments in

Cerebratulus, Biol. Bull. vi, 1904. C. ZELENY. Experiments on the localization of developmental factors

in the Nemertine egg, Joan-n. E.l'p. Zool. i, 1904.

§ 8. CTE.\'OPIIOR.\.

VVith the Ctenophora we come to a group of animals in which the development of a total larva from a single blastomere is no longer possible, though the missing parts may eventually be regenerated.

The egg consists of a central yoll<—mass surrounded by a superficial layer of granular protoplasm. After fertilization this protoplasm passes wholly to one side—the animal hemisphere-—and the egg divides meridionally into equal parts. The protoplasm then spreads itself over the outer surface of each cell, but returns to the animal side prior to the second cleavage, which is again meridional and at right angles to the first. Once more the protoplasm is distributed over the whole outer surface of each hlastomere, only to be again concentrated before the third division, an unequal one, but nearly meridional. There are new four large cells lying together in a square and four smaller cells lying above them in two groups of two each, one at each end of-the plane of IV. I INITIAL STRUCTURE OF THE GERM 209

the second furrow. This becomes the transverse, while the first furrow marks the sagittal plane of the future organism. Each of these cells contains both granular protoplasm and yolk, but at the succeeding division nearly all of the former material passes into the eight small cells, which are nipped off and lie in an oval ring on the vegetative side of the egg 1 (Fig. 118). These small cells or micromeres will give rise to the ectoderm, the large cells or macromcres to the endoderm and mesoderm, the mesoderm


FIG. 11H. -Normal segmentation ofthe Ulenophorc egg. :4 u, Stomach or sagittal plane ; I» I), funnel or transverse plane. C, E, from mieromere pole; D, F, from the side. (After Ziegler, from Korschelt and I-leider.)

being separated off later on at the vegetative pole, which becomes

the oral pole of the embryo. In normal development, therefore,

the embryonic axes are marked out at quite an early stage. Chun was the first to isolate the % and § blastomeres of

‘ Ziegler's description is that in this (the fourth) division the furrow first appears near the animal pole, but yolk then streams from the larger vegetative into the smaller animal portion, until the latter becomes the macromere, the former the inicromere. Division is then completed and the micronieres are at the vegetative pole. This explains the divergences in the accounts of earlier observers (Agassiz, Kowalewsky, Chun, F01, and Metschnikoff). 210 INTERNAL FACTORS 1v. 1

Euc/(aria by shaking. These gave rise to half larvae, provided with four costae, four meridional canals communicating with two subsagittal and two subtransverse canals in the ordinary way, one subgastric canal, one tentacle, but a whole funnel and a whole stomach, the latter formed by an oblique invagination ; the side turned towards the missing blastomere was covered over by ectoderm.

Later on the missing half was regenerated, first the subgastrie vessel, then the meridional canals, over these the costae, and

finally a tentacle.

FIG. 119.~Development of isolated bl-astomeres in Ctenophora. a, /2. Segmentation of 1% blastomere of Befiio ovatu: a, two large, two small cells; b, each gives off a mieromere; c Larva from a .1, blastomere with four costae; (I, c. Segmentation of 1 blastomere ; f. The resulting larva with two costae; g. Larva. with six costae from ~2~ blastomercs. (After Driesch and Morg.m, 1896.) h. Isolated »1"‘,; blastomeres of liwfie omlu;

j. The resulting larva with four costae, one sense--oi'gai1, and one

stomodaeum ; 2'. Isolated -1'3, blastomeres; A-. The resulting larva with three costae, one sense-organ, and one stomodacum. (After Fischcl, 1898.)

Such half larvae are found in the tow-net after a storm and may become (l}olz'7za) sexually mature.

These experiments have been repeated and confirmed on Barbi‘ by Driesch and Morgan. The authors add that the segmentation of these isolated blastomeres is partial. The 71; larva has two costae only and a large and a small canal, the former passing to the costae, the latter to the opposite side and representing apparently IV. 1 INITIAL S'l‘RUC'l‘URE OF THE GERM 211

the other half of the funnel (Fig. 119 mg). Fischel has carried the analysis of the potentialities of the blastomeres a step further (Fig. 119 /z—zt). Though cleavage is partial, as described by Drieseh and Morgan, there are slight irreg~ulari.ties in the position of the blastomeres. A ,1; blastomere produces one eosta only, -1-55 blastomeres (four macro- and four mieromeres) four eostae, {-3(five macro- and five mieromeres) five, -19%, -1%, and 14;, when separated in the same way, respectively three, six, and two eostae. Similar results were obtained by meridional division of the egg in later stages. Fisehel confirms the statements of the other

_FiG. 120.——u. Beriie oz-am: egg in the 16-cell stage. The mieromeres disarmugecl by pressure ; I». The resulting larva with two sense-organs, and four eostae radiating from each : one stoinodacuni. (After Fischel, 1898'.) 1'. Normal segmentation of the egg of Beriie from \Vl1lCll a small portion of the vegetative hemisphere has been removed; d. Larva produced from the same, with eight eostae, four endoderinal canals, and one (central) stomodaeum. (After Driesch and Morgan, 1896.) investigators as to the structure of the half larvae. In % larvae, however, not three, but four, canals are formed, and the stomodaeum is vertical. The sense-organ is not found in embryos developed from small fragments. Removal of the Inacromeres left the number of eostae unaffected, but displacement of the mieromeres by pressure led to the formation of an abnormal larva with two sense-organs and eight eostae arranged in two groups of four each round each sense-organ (Fig. 120 a, 6), or, if the mieromeres were separated into two lets, a group of four round one organ, a group of three round the other, and some scattered combs. With greater pressure three sense-organs were produced and the


costae were very irregular. In these eggs there was, however, only one stomodaeum, and only the normal number of canals.

It appears, therefore, that the individual eostae are definitely related to the meridional divisions of the segmented egg, and further that the material which is concerned in their formation becomes located in the micromeres of the 16-celled stage.

Driesch and Morgan have also shown that if the vegetative hemisphere be taken away from the undivided egg, the animal portion, though it segments normally, is able to produce neither costae nor stomodaeum; it has, however, cndodermal canals. Loss of a small portion of the vegetative hemisphere, however, does not interfere with subsequent normal development (Fig. 120 0, II).

On the other hand an egg which has been deprived of a lateral portion divides, by three unequal divisions, into eight cells, all of which form mieromeres. Nevertheless, the larva has only four, five, or six costae, and some of these rudimentary, only three or four canals, and the stomodaeum, as in half larvae, is oblique.

As Driesch points out, the whole of the nucleus is here, and the embryo is still defective. Difierentiation thus depends on the cytoplasm, but is quite independent of segmentation, which may be perfectly normal without determining the formation of a normal embryo. l)rieseh further argues that the observed defects are due to lack of suflicient mater-ial.merely, not to lack of any preformed specific substance. \Vhether such substances are already present in the unsegmented as they undoubtedly appear to be in the segmented egg, it is perhaps impossible, on the evidence, to decide, but it may be observed that the development of perfect larvae from those eggs from which a portion of the vegetative hemisphere has been detached does not ncccssaril y support the View advanced by Drieseh. '


C. CHUN. Die Dissogonie, eine neue Form der geschlechtliehen Zeugung: 7. Zur Entwicklungsuieclianik der Ctenophoren, I"('.s'l.s'c7u'. L<'m'7.'m'!, 1892.

II. Dmascu and T. H. MORGAN. Zur Analysis der ersten l*lntwicklungsstadien des Ctenophoreneies, Arch. Eur. Illech. ii, 1896.

H. FISCHEL. Experiinentelle Untersuchungen am Ctenophorenci, 1, .‘.lI'fh. Enl. Zlfech. vi, 1898; II, 111, IV, ibid. vii, 1398.

II. E. ZIEGLER. Experimentelle Studien fiber die Zelltheilung: Die Furehungszellcn von Bertie ovata, Arch. Ent. Mech. vii, 189:4» IV. I INITIAL STRUCTURE OF THE GERM 213

§ 9. Crmnrorom AND MoLLUscA.

In the Chaetopoda and Mollusca (except the Cephalopods) it is possible to trace back almost every organ of the body to some particular cell, or group of cells, of the segmenting ovum, to describe, in fact, its lineage in terms of individual cells. Specific materials for the formation of the vaiious parts seem to be sundered from one another by the process of cleavage, which thus presents the characters of a ‘Mosaic’ work in an exceptional degree. 'l‘o what extent such a preformed structure (lees in reality exist experiment alone can decide, and the answer given by experiment, so far, at least, as the nucleus is concerned, is in the negative.

By means of pressure Wilson succeeded in preventing in Nerei.s~ the normal formation of the first quartette of micromeres ; instead, the egg divided into a flat plate of eight equal cells. The pressure was then removed. All eight cells formed micromeres, and later on eight instead of the usual four macromeres were found in the Troehophore la1'va. Four of these necessarily contained nuclei which would normally be placed in cells of the prototroch, and yet the larva was normal. In this case, then, the causes of differentiation cannot lie in the nucleus; they must be sought for, if anywhere, in the cytoplasm.

In the eggs of these forms segmentation is holoblastic and of the spiral type, with the cleavages alternately dexiotropic and laeotropic, and it is easy not only to determine the lineage of each organ, but to compare the oi-igin and the destiny of individual cells in a large series of forms. The first, second, and third quartettes of micromeres are usually destined for ectoderm formation, the second quartette cell in the D quadrant, 2 «I, being the first somatoblast from which the ectodermal structures of the trunk a1'e derived. The eiliated ring or prototroch is formed, in most cases, from certain derivatives of the first quartettc, namely 1 a 2———I (l 5.’, the primary trochoblasts, each of which divides into a group of four; but it may be reinforced by secondary trochoblasts from the same or the second quartette. The second and third quartettes may provide larval nzesoderm or mesenchyme. The trunk mesoderm is usually derived from 4 (I. The remaining 214 INTERNAL mcwons IV.1

cells of this quartette (4 a—4 c) become with the residual macromeres (A—D) the endoderm (or mesendoderm).

A peculiar structure seen in some forms (Ilyamwsa, Dentalinm,


FIG. 121.-—No1-nml cleavage of I(_I](m((.98(L A, B. Formation of polarlobe or yolk-lobe (til); (Y. ‘Trefoi1‘ stage; I), E, F. The polar-lobe passes into CD; 0. The second polar-lobe protruded in H; it passes into D. (After Cmmpton, from Korschelt and Heidcr.)

0/laclopteru.v, M_y:o.v/oirm) is the yolk-lobe, or polar lobe, as it should properly be called. This is a. mass of cytoplasm p1-oi-ruded at the IV. I INITIAL STRUCTURE OF THE GERM 215

vegetative pole after fertilization. It becomes attached entirely to one of the first two blastomeres, namely to CD, into which it is withdrawn; prior to the next division it is again protruded, attached to D only, and then once more withdrawn : at the time of the next division it is protruded again, for the last time (Figs. 121, 122). '

Crampton has isolated the blastomeres of I/‘//amzwz and watched their development (Figs. 123-6). —;-, §-, % (A, B, and C), 1}, §

FIG. 122:-Normal cleavage of II_:/mmssa. (After Crampton, 1896.) a, 8—celled stage from above; I), 1C-celled stage from above; r, 24-celled

stage, from above; :1, optical section, sliglltly oblique, of a later

embryo, from below. Division of mesoblast pole-cell (rI‘).

(a macromere and a micromere), %, -,1; (a micromere), I53, 11} and 193 all continue to segment as though the missing blastomeres were present, except for certain small irregularities in the positions of the cells ; the A, B, and C blastorneres, separately or together, bud off the usual four generations of mieromeres, but D only the first three ; the division of the mic-romeres continues in nearly normal fashion, The 3,. mic-romerye divides twice only. All die, however, 216 INTERNAL FACTORS IV. 1

without giving rise to larvae. A 13- macromere, on the other hand,

or a. -1-13 or later blastomere, will not segment at all. By lowering the temperature the cleavage may be made to

resemble that of whole ova.

FIG. ]23.—-Il_:/anassa: c-lenvzige of smaller blastoniere (AB). a, Undivided; b, embryo, fr_on1 sule ; (.', »j, embryo, from above; «I, -,'-‘:5 emln-yo, gram ahdove _the clot; lindficattesil the for1ne1t'1centre of the egg; 9% .1_,'3‘«e1}J1.bryo;)

rom si e; , grow 1 o ec 0 erm over ie nmcromeres; 3/, orma ion 0 the nncromeres of the foulth generation, from below; It, ciliated partial

embryo of 46 hours. (After Cranipton.)

The yolk-lobe influences the character of segmentation, for if it be removed CD divides like AB. More important ‘than this is IV. I INITIAL STRUCTURE OF TIIE GERM 217

the relation of the yolk-lobe to the mesoderm. When the former is cut olf the latter is not formed at all. There are four generations of micromeres and four equal macromeres, and the larvae, though eiliated and able to swim, never reach the veli_qer stage (Fig. 127).


1/ I

FIG. 1'24.~Il;/«nus-.5-«I: ¢ of CD. (4, Formation of yolk-lobe (yl) ; b, ‘trel'oil'; v, embryo from above; (1, early 1‘ e1ubr_yo, from above:

~'econ(lary yolk-lobe (yl) ; a, resting 3, embryo from above; f, ,‘E_. embryo

from above; (1, passage to 11,- stage, from above; h, embryo, from side ; i, division of pole-cell (d‘), from below. (AftcrUra.1npt0n.)

The potentialities of the blastomeres are thus strictly limited both in segmentation and difl’e1‘entiation. This result is amply confirmed by Wilson's researches on I/eutulimzz and Patel/a.. 218 IN'l‘l*}RN.\L FACTORS IV. 1

In the latter genus the development of the isolated blastomeres, especially of those resulting from later divisions, has been minutely followed. Each separate first quartette cell (1 a—1 (1) forms (Fig. 128) a closed ectoblastic structure with an apical org-an at

FIG. l25.—IIgmm.~sa: cleavage of ;} blzmstomere (Al. (I, 3, froI_n side: b, ,“:_;, from side; 0, passage to 143, fiist form, from side; (I, rest1ng_-‘g. second form, from side; 0, pttssngo to fl, from side ; f, ;_,7,,«, from s1( e. (After Crauupton.)

“ii,” ‘I

Fm. 126.—I/_:/auassa: aw, cleavage of Q; blnstomercs; (1, ,9, stage, from one 1n1c_romere; 12, ~,*;; stage, from two micromeres; r, —,“,, stage, from three nucromeres. d—-f, cleavage of bliLSt01l1(‘1‘CS; cl, -J,-3 einbryo, from below: P, origin of fourth qunrtefte. from below; f, embryo of 48 hours. (After Urzuupton ) IV. I INITIAL STRUCTURE OF THE GERM 219

/‘B co

FIG. 12"’7.—~]I_1/a12assa: cleavage of the egg after removal of the yolklobe. a, :5; b, 1.-;_ r, first quartette formed; (1, second quxutette formed, first qu2Lrtgtt§fl1v1de(l ; a—¢l, from above; e. from below: fourth quzu-tette formed; _/, cxllutod embryo, from side. (After Cr-.unpton.)

(I // 0

FIG. 128.-- Patcllu: development of isolated ;« micromeres. ((, _?; micromere; I), c, first division; «I, 3"-‘,3 stage, with two trochoblasts (stippled), one rosette-cell, and one p1-imary cross-cell; e, larva}. of 24 hours, from the side, showing trocholnlasls below, apical cells above. (Aft:-1' \Vi1Su11.) I 220 INTERNAL FACTORS IV. I

one end, and four cells (derived from a primary trochoblast) provided with long cilia. at the other. When the primary trochoblasts (1 a 2-1 (Z 2) are isolated each divides into four ciliated cells, the cilia arranged as normally in a single row, but no further (Fig. 129 a—e). If the daughter-cells of these troehoblasts (1 a 2. 1 -1 (Z 2. 1 and 1 a 2. 2--1 (Z 2. 2) are separated they divide once only (Fig. 129/’), while their daughter-cells do not divide at all (Fig. 129 g, /1). The sister-cells to these (1 «L 1—1 rl 1) behave in the same way, forming a closed larva with an apical organ at


(*7 3

0 gr

5; I; 0 ll

FIG. 129. —-Pulellu. Development of isolated primary and secondary troehoblasts. a, Primary troehoblast; b, 0, first division; «I, second division ; 0, after 24 hours ; f; pair of primary t1'ocl1oblasts, the products of division of either 1'“ or 12"’, after isolation; ,1/, h, single prilnary trochoblasts, either 12”, 1*", 1”‘, or 1”"; i, a pair of secondary t1'oehoblasts, the products of 1"; j, Ir, single secondary trochoblasts. (After Wilson.)

one end and secondary trochoblasts at the other, while the isolated apical cells and secondary trochoblasts divide as often as they would in the whole embryo and put out their cilia (Fig. 129 i-,{', Fig. 130 u—e). Finally, the whole first quartette becomes an ectoblastie larva with an apical organ at one end and a prototroch at the other, but with no archenteron (Fig. 130/; 9). The cells of the second quartette (2 a——2 (I) likewise form each a hollow ectoblastic

vesicle containing larval mesenehymc, ciliated secondary trochoblasts, and a few (prcaual) ciliated cells (Fig. 131). IV. I INITIAL STRUCTURE OF THE GERM 221

The cells of the third and fourth quartettes do not develop at all.

A -E1, maeromere (1 A-1 D), however, segments partially, to form a micromere of the second quartette, from which secondary

e f 9

Fm. 130.-—I’uIella. Development of isolated cells of first qualrtet-to and of the whole ql1iL1'i:0tl’;C. (1, lsolzited 1‘; I», its first division; ¢', its second division ; (I, after 12 hours ; 0, after 22 hours, showing apical cells zmd secoinl-My troelioblasts; _f, first division of isolated first quumtette; g, second division. (After Wilson.)


FIG. 131. — l’uleIlu. Isolated cells of second qu-.u'tette. um, Cl0‘¢l.V1LgP,; «I. after 24 hours, showing sec.-ond:u'y trooheblusts and ‘.’ prezuml cells. (After Wilson.) trochoblasts are later developed, and one of the third. Subsequently an are-henteron is invaginated (Fig. 132). So the 116- macromere (2 A-—-2 D) forms a micromere (3 a-~31/) and gives rise to a. larva, with lentomesoblast (D) or entohlast only (A— C). 222 INTERNAL FACTORS IV. I

Isolated 71; (A—D) or is (AB or CD) larvae have an apical organ and a prototroeh complete or semicircular; they may remain open or close up ,- sometimes they gastrulate.

It is evident that each cell has exactly the same potentialities for division and difierentiatiou when isolated as when in connexion with its fellows. Only in the shifting of the cells and closing up of the whole cell-mass is there anything which approaches to total development.




8 u

FIG. 1I)‘2.-—1’utella. Development of isolated basal (inacromere). a, First; 11, second division; 1-, d, -56- and 64-cell stage. In 4; the position of the 2-group is normal, not in (I ; 0, after 24 hours, showing two secondary troehoblasts ()roducts of 2“) and two feebly ciliated cells (preanalcel1s‘.’). (After ilson.)

The dependence of this specification of the blastomercs upon definite substances preformed——tl1ough not prclocalized—in the unsegmented ovum is demonstrated by Wilson’s experiments on Deizlztliuril. The newly laid egg of Ijeuializmz contains a brickred yolk—mass surrounded by a thin hyaline eetoplasm ,- the latter is thickened a little to form an animal polar area and very considerably to form another large area. at the vegetative pole. The nucleus is in the centre (Fig. 133). VVhen the sperm enters the

nucleus breaks down, and its substance becomes confluent with both IV. I INITIAL STRUCTURE OF THE GERM 223

areas. This continuity is soon lost and the egg becomes divided into three regions, a clear layer spread over the animal hemisphere, below this the yolk-mass with the fertilization spindle, and a. large,

_I/ /I 1

FIG. 133.-V 1)mlulimu. Normal cle-.wn;,re. The red pigmented part is represented by stippling. a, Freshly laid egg, polar view ; b, the szunc 20 minutes latter, after shedding the menibrame; c, the smile from the side; (I, one hour after fertilization, showing polar bodies; 0, first division and protrusion of first polar lobe; f. ‘trefoil’ stage; (1, the polar lobe has passed into CD; h, p1‘0t1‘u~siOn of second. polztr lobe (Z2) ; 2', second cleavage. (After Wilson.)

nearly yolk-free area at the vegetative pole, which is 11ow protruded as the polar lobe. This becomes attached, as described above for Ilqazzztssra, to the D blastomere. The first quartette of 224 INTERNAL FACTORS IV. I

micromeres is formed from the clear animal area; after this division the polar lobe of D moves up to meet the upper white area, and so it comes about that a large part of this lobe passes into the second micromere, 211, which is the first somatoblast. In the other quadrants the second mieromeres are formed from the upper white area alone. All the cells of the third quartette and (it is not known certainly whether this is the mesoblast) 4 (I in the fourth are quite white. The larva is spindle-shaped with

FIG. 134.—l)enIalium. Cleavage after removal of the first polar lobe ; a, first division: the isolated polar lobe is seen below; b, 4—cell stage, from animal pole; 1', 8-cell stage, from animal pole; d, forum,tion of second quartette, from vegetative pole; 0, same stage as last, open type; f, same stage, from animal pole. (After Wilson.)

a broad, triple prototroch. Tlierc is an apical organ of long cilia and a tuft of sensory hairs at the opposite end (Fig. 135 (6).

When the polar lobe is removed in the ‘ trefoil ’ (2-celled) stage, segmentation continues, but no polar lobe is formed in any stage, the D cells are not larger than those in the other quadrants, the embryo has no lower white area, the segmentation cavity may be open below, and the larvae are deficient and die (Figs. 134-, 135 6). Post-troclial region and apical organ are both absent and IV. 1 INITIAL STRUCTURE OF THE GERM 225

these missing parts are never regenerated ; the pretrochal region is, however, increased and the three rows of cilia in the prototroch are usually developed.

The polar lobe is thus clearly connected with the formation of the trunk and the preoral sense-organ.

The segmentation of theiisolated blastomeres is partial, as in the types already examined, but their subsequent behaviour depends on the presence or absence of the polar lobe. AB larvae or A, B, or C larvae closely resemble the deficient trochophore just described ; CD or D larvae are normal, though the structures derived from the polar lobe are out of proportion to the rest

FIG. 135.——Dentalium. a, Normal trochophore of 24 hours; b, larva of 24 hours after removal of the first polar lobc. (After Wilson. 1904-)

(Fig. 136 a-d, 1}). The failure of the egg to develop normally is not due to insufficiency of material, for the volumes of CD and D are less than that of a whole but lobeless egg.

The micromeres 1 a, 16, 1 c, when isolated, become actively swimming, ectoblastic embryos provided with a prototroch, but with no apical organ, gut, or post-trochal region; the other micromere, lzl, however, has an apical organ, though it is Still Without the hinder region and incapable of gastrulation (Fig. 136 e,f It appears, therefore, that the material for the apical organ is now placed in this cell, and this is proved by experiment ;

for if the polar lobe is cut away during the second cleavage, or

‘I Jzxxuson Q, 226 INTERNAL morons IV. 1

from the isolated CD blastomere, then the larva has an apical organ, but no post-trochal region. Hence between the first and second divisions the stuff which determines the formation of this organ must migrate out of the polar lobe into the animal hemisphere of the D cell.

In the unsegmented egg the specific material for both apical organ and post-trochal region is situated in the vegetative

FIG. 136.—DentaIimn. Larvae from isolated blastomeres. «, b, Twin larvae from the isolated CD (a) and AB (7)) halves of the same egg, 24 hours; 0, d, twin larvae from the isolated D (c) and C (d) quadrants of the same egg, 24 hours; :2, f, twin larvae from '.the isolated posterior micromere 1 d (e) and the 1 c micromere (f) of the same egg, 24 hours; g, }larva from one of the A, B, or C quadrants, 72 hours. (After Wilson.)

hemisphere. The animal half of the egg, whether removed after fertilization, or before and then fertilized, segments like an egg from which the polar lobe has been removed (Fig. 137 6), and develops neither apical organ 11or post-trochal region (rarely an apical organ if the fragment is large). The vegetative hemisphere, when out off before the entry ofithe sperm and afterwards IV. 1 INITIAL STRUCTURE OF THE GERM 227

fertilized, segments as a whole with the polar lobe in correct proportion and gives rise to a dwarf larva with all its parts complete (Fig. 137 a, c). Many, however, die.

A meridional half-egg is able to develop normally, or nearly so.

The behaviour of the enucleate vegetative fragment of the fertilized egg is interesting. Though deprived of nucleus and centrosome it forms its polar lobe synchronously with the divisions of the animal portion ; three times is the lobe protruded and three times withdrawn: the fourth time it is not taken back; this is the moment when the first somatoblast is given off. In this enucleatc fragment the polar lobe is not in proportion but of the same size as in the whole egg.

Even the isolated polar lobe, or pieces of it, exhibits periodic movements, amoeboid ,or of elongation, or protruding a smaller lobe.


FIG. 137.—Dentalium. Development of egg-fragments, the plane of section being indicated in the small figures. a, b, Twins, after horizontal or oblique section near animal pole; c, trochophore developed from a. fragment resembling that shown in a. (After Wilson.)

The existence in the egg of preformed substances specified for the production of particular organs is thus incontrovertibly established, and as long as an egg-fragment or blastomere contains all these it will give rise to an embryo which is, in form, perfect, though lack of enough material (or the shock of the experiment) may bring its life to an untimely end.

At the same time these substances, though preformed, are not necessarily preloealized; their original is not the same as their ultimate situation, and in their redistribution we have to deal with a process which is, as Wilson remarks, truly epigenetic.

Before bringing this section to a conclusion reference must be made to another case in which it seems probable that definite


organ-forming substances exist, though experimental proof is at present lacking.

In the immature egg of the aberrant parasitic Polychaet .413/zoatoma there is, according to Driesch, at the vegetative pole a mass of greenish cytoplasm, the surface of which is convex towards the reddish brown mass of the rest of_ the egg. When the polar bodies are formed the red substance retreats to the animal pole, leaving a clear equatorial zone.

Before segmentation a polar lobe is formed——as in Ilyauassa

FIG. 138.——Deve1opment of the egg of Jllyzosioma. The green substance on the vegetative side is shaded finely ; this passes into the polar lobe (e), and into the D quadrant (1'). The red substance of the animal hemisphere is coarsely dotted. (After Driesch.)

Fm. 139.—Development of the egg of My/zostoma. Formation of the cells 1 d (d‘ in c) and 2 d or X (d’ in e). The latter cell takes a portion of the substance of the polar lobe. (After Driesch.)

and Deutalz'um.—containing the green and part of the clear substance: it passes first to CD and then to D. When the latter divides it is again protruded and withdrawn. The cell 1 (3, like 1 a, 1 6, 1 c, contains the reddish protoplasm. The second micromere in the D quadrant—2 (Z——-takes part of the green substance, and Driesch suggests that the rest of the latter would eventually pass into the second somatoblast (4 ti = M) (Figs. ],38, 139). IV. I INITIAL STRUCTURE OF THE GERM 229


H. E. CRAMPTON. Experimental studies on Gasteropod development, Arch. Eut. Mech. iii, 1896.

H. DRIESCH. Betrachtungen fiber die Organisation dcs Eies und ihre Genese, Arch. Ent. Mech. iv, 1897.

E. B. WILSON. Cleavage and mosaic work. Appendix to Crampton’s paper on Ilyanassa, Arch. Eut. Mech. iii, 1896.

E. B. WILSON. Experimental studies on germinal localization. I. The germ regions in the egg of Dental imn, Jomw. Ezrp. Zool. i, 1904.

E. B. WILSON. Experimental studies on germinal localization. II. Experiments on the cleavage mosaic in Palcllu and Dcnlulium, Journ. Exp. Zool. i, 1904.

§ 10. ASOIDIA.

As has already been shown, there is in the Vertebrata and zlmp/ziowue no constant relation between the first furrow and the sagittal plane of the embryo, although the symmetry of the embryo does stand in a very definite relation to that of the unsegmented ovum. Further, experiment has proved that the isolated blastomeres, up to a certain point at any rate, capable of giving rise to normal total larvae. It is, perhaps, all the more remarkable that in another group of the Chordata, the Ascidians, the symmetry of the embryo is not only predetermined in the symmetry of the egg but also by the planes of segmentation, and that the potentialities of isolated blastomeres remain strictly what they would have been in the whole ovum.

The earliest experiments are due to Chabry (Figs. 140, 141). According to this author the first furrow in the egg of /1.9ci(h'ella asper.9a is meridional and coincides with the sagittal plane; the second, likewise meridional, coincides with the transverse plane; the third, equatorial, separates oral from aboral regions, and is therefore horizontal.

Isolated g blastomeres or 7"} right or left blastomeres give rise to imperfect larvae with only one fixing papilla, only one atrial invagination and one pigment-spot, the eye; the last is absent in the 3,: left larva. % anterior larvae have one fixing papilla, one sense-spot, a,gut, a cerebral vesicle and a posterior notochord ; 230 INTERNAL FACTORS IV. 1

in %} posterior larvae the ehorda and brain are absent, though the three germ-layers are all present.

In the segmentation of the isolated blastomeres the direction of division is altered, in that a furrow (the fourth) which in the whole egg is meridional and at an angle of 45° to the first two becomes parallel to the plane of separation, and so resembles the third furrow of the whole egg.

A right or left % blastomere or %, anterior or posterior, right

FIG. 140. -—A. Egg of Ascidiella aspersa in the two-cell stage. _One blastomere has been killed (G). B. The survivor (D) divides to form, in (J, a small normal gastrula. (After Chahry, from Koischelt and Heuler.)


FIG. 14l.—Larva. of Ascidiella aspersa produced from 1 blastomeres. F, sucker; At, atrium; No, notochord; En, endoderm. (After Chabry, from Korschelt and Heider.)

or left, blastomeres consist after this division of two tiers of four cells each ; the cleavage of these cells resembles, therefore, that of a whole egg.

It should be noticed that the blastomeres cannot be actually separated; it is only possible to kill one, by a needle, and note the development of the survivor.

The more recent researches of Conklin on Cyu//zz'a have confirmed and extended Chabry’s results.

The immature egg of Qynl/ziu, (Fig. 142) comprises a central grey yolk surrounded by a peripheral layer of yellow pigmented IV. 1 INITIAL STRUCTURE OF THE GERM 231

FIG. 142.——Norma1 development of the egg of C'_1/nthia pm-tim. Maturation and fertilization. (After Conklin.)

A. Unfertilized egg before the fading of the germinal vesicle (clear), showing central mass of grey yolk (lightly dotted), peripheral layer of yellow protoplasm (thickly dotted), test-cells and chorion.

B. After the entrance of the sperniatozoon the yellow protoplasm has streamed to the vegetative pole (v); in it, excentrically, is the sperm nucleus (spat). The clear protoplasm derived from the germinal vesicle partly forms a. layer over the yellow protoplasm, in art remains at the animal pole (a). The grey yolk occupies the anima part of the egg.

C. The yellow and clear protoplasmic substances have both streamed to what will be the posterior side (P). A, anterior, V, ventral (animal pole), D, dorsal (vegetative pole).

D. View of the same egg from behind. The two pronuclei are seen side by side in the clear area. B, right; L, left. 232 INTERNAL FACTORS IV. I

protoplasm ; near one pole (the animal), and so determining the egg-axis, is the large germinal vesicle. Prior to the extrusion of the polar bodies the latter breaks down and provides a. third substance, a clear cytoplasm. On the entrance of the spermatozoon near the vegetative pole a remarkable change takes place in the arrangement of these materials. The yellow protoplasm flows down to the vegetative pole and arranges itself symmetrically to the axis; above it is a zone of clear protoplasm derived from the germinal vesicle; these two areas together occupy about onethird of the egg. The remaining, the animal two-thirds, are occupied by the grey yolk with only a small quantity of clear cytoplasm at the animal pole in which, after the formation of the polar bodies, the female pronucleus is embedded.

The egg is still radially symmetrical, but by further shiftings of these substances it becomes bilateral.

The spermatozoon moves in the clear protoplasm to the equator on one side, the posterior side as it will be, and the yellow protoplasm follows suit; the first is now disposed in a broad equatorial band encircling half the circumference of the egg, the second forms a wide crescentic mass below it and reaches nearly to the vegetative pole. The female pronucleus, with its clear area, now joins the male.

The egg is now bilaterally symmetrical about a plane which includes the axis, and passes through the middle of the clear and yellow substances and between the two closely apposed pronuclei.

It seems doubtful whether the movement of the clear and yellow material to the posterior side is immediately due to the agency of the sperm, for the latter does not always take the shortest route to the equator; it may enter on what becomes ultimately the anterior side ; and further, when two sperms enter flley both travel to the same spot, appearing to be passively carried along by the independent streaming of the egg cytoplasm.

The two pronuclei next move into the axis of the egg in the animal hemisphere, together with their clear protoplasm. The grey yolk is thus largely displaced and shifted into the vegetative region. The egg, therefore, now consists of. an animal IV. 1 INITIAL STRUCTURE OF THE GERM 233

hemisphere composed almost entirely of the clear substance, and a vegetative hemisphere, the anterior portion of which includes grey yolk and nothing else, the posterior portion the yellow protoplasm together with a little yolk near the vegetative pole. The cleavage furrows separate these substances from one another in a perfectly definite way (Fig. 143). The first is

A '5' 1"”

FIG. l43.—Cleavage of the egg of Cynthia pm-tita. (After Conklin.)

A. 8 cells, from the left side.

3. 8 cells, from above. The yellow crescent (thickly dotted) is limited to the posterior dorsal cells: the animal cells are clear, the grey yolk (sparsely dotted) is found in the dorsal cells, both anterior and posterior.

C. 16 cells, from the dorsal side.

D. 74 cells, dorsal view, showing the division of the 4 neural platecells (n. ), and 4 chords cells (ch.). There are 10 endoderm cells (e), 6 muscle-cells (m.s.), 8 posterior (p.m.¢-h.) and 2 anterior ( mesenchyme cells. 234 INTERNAL FACTORS IV. I

g /4

' Fm. 144.— nthia parh'tq. Succe 've stages in the development of the same righ alf embryo, the left stomere having been iniured in IV. 1 INITIAL STRUCTURE OF THE GERM 285

meridional and sagittal, the second meridional and transverse, the third equatorial and horizontal. Since the animal pole will be ventral, the vegetative dorsal, the right and left sides of the future embryo are simultaneously determined.

The four ventral animal cells consist almost entirely of clear protoplasm, the two anterior dorsal entirely of grey yolk, the two posterior dorsal almost exclusively of the yellow substance.

The next furrows are, roughly, meridional and at angles of 45° to the first two; they emphasize the bilateral symmetry, for in the anterior half they meet the first furrow above, the second below, and conversely in the posterior half.

The direction of the divisions in the next phase may be most easily gathered from the figures. In the vegetative hemisphere four anterior ehorda-neural cells are separated ofi ; the six posterior cells include all the yellow substance and will give rise to the posterior mcsenchyme and the muscles of the tail; six endoderm yolk-cells remain, four anterior, two posterior. The next division sees the separation of the ehorda from the neural cells in front, of an outside ring of muscle-cells from an inside ring of mcsenchyme cells behind, and of an anterior mcsenchyme cell on each side from the anterior endoderm.

A few anterior animal cells become associated with the neural cells in the formation of the nervous system; the remainder of this hemisphere is ectodermal.

In gastrulation the endoderm cells first sink inside, followed by the ehorda in front and the anterior and posterior mesenchyme cells and the muscle-cells at the sides and behind. Overgrowth is greatest at the anterior dorsal lip, the blastopore thus becoming posterior.

In normal development, therefore, the axes of the embryo are

the 2-cell stage-. The yellow protoplasm is indicated by coarse stippling. (After Conklin.)

a, 8-cell stage, posterior view. V, ventral; D, dorsal. b, 16-cell stage, anterior view. ' Normally, A 5.1 and a 5.3 lie in front of A 5.2 and a 5.4. c, 16-cell stage, posterior view. In normal eggs the cells B 5.2 and b 5.4 lie behind B 5.1 and b 5.3. d, 30-cell stage, dorsal view. e, j; 34-cell stage; e, posterior, f, dorsal view. In normal e gs the cell B 6.4 lies laterally to B 6.3, and the cell B 6.1 lies between 6.3 and A 6.1. g, 46 cells: posterior view. h, 48 cells: dorsal view. The cells B 7.1 and B 7.2 should lie next the middle line. 236 INTERNAL FACTORS IV. I

predetermined by thearrangement of certain substances in the egg, and segmentation exhibits the character of a ‘ Mosaic ’ work, segregating these specific materials into the rudiments of definite organs. The causal connexion between these materials and the organs to which they are beforehand assigned is demonstrated by experiment.

FIG. 145.— Cynthia purtita. Half and three-quarter embryos. (After Conklin.) a, right half gastrula, dorsal view. The neural plate, chorda, and mcsoderm cells are present only on the right side and in their normal osition and numbers. b, left half of young tadpole, dorsal view. he notochord is normal except for size and number of cells: the muscle and mesenchyme cells are present only on one side: the neural plate (71.1). is abnormal in form but not in position. c, right half of young tadpo e, dorsal view: slightly younger than b. m’ch., mesenchyme, ma, muscle-cells. d, left anterior three-quarter embryo, dorsal view. The anterior half is entirely normal with anterior mesen chyme (m'ch.) on both sides. Posterior mcsenchyrne and muscle-cells upon the left side only. IV. I ‘INITIAL STRUCTURE OF THE GERM 237

‘The segmentation of a 4} or of 7*} right or left blastomeres is partial except for the alteration in the direction of the furrows of the fourth phase already described by Chabry. The blastomeres divide as though the other half of the egg had not been killed (Figs. 144,146, 147). Eventually a half gastrula is formed with exactly half the normal number of neural,chorda,endoderm, mesenchyme and muscle-cells ; the gastrula cavity is open on the side of the dead blastomere. Many are, however, abnormal in that the endoderm and muscle- and mesenchyme cells do not invaginate (exogastrulae) or show invaginations of the ectoderm (pseudogastrulae).

The half gastrula may develop into a half larva, with the notochord, muscles, and mesenchyme lying on one side of the medullary folds. The only signs of the regeneration of the missing half are the overgrowth of the ectoderm on the naked side, and the passing over of a few muscle-cells ; the full number of the latter is, however, never found on both sides (Fig. 145 a—c).

The % larva is especially interesting, particula.rly when a posterior blastomere has been killed, for here the anterior end is complete with anterior mesenchyme on both sides ; the posterior mesenchyme and muscles are, however, missing on either the right or left (Fig.145(l). The chorda and neural plate are stated to be smaller than the normal in these larvae, and there is no sense-vesicle.

An embryo developed from the two anterior blastomeres of the 4-celled stage alone possesses the full number of neural, chorda, anterior endoderm and anterior mesenchyme cells ; its hind end is covered by ectoderm but shows no trace of muscles or posterior endoderm or mesenchyme (Fig. 146 0, (Z).

The two posterior blastomeres produce an embryo devoid of neural plate and notochord, of anterior mesenchyme and endoderm. By overgrowth of the ectoderm a sort of solid gastrula is formed with a central mass of endoderm flanked by musclecells and posterior mesenchyme. No tail appears (Fig. 147).

21,-, %, and I15 blastomeres also segment and develop partially. An anterior % has half the proper number of neural, notoehordal, anterior endoderm and mesenchyme cells, but the neural plate does not become folded and the chorda cells protrude irregularly behind. A posterior quarter may gastrulate in the way described for the posterior half, but exogastrulae and pseudogastrulae occur. 238 INTERNAL FACTORS IV. I

Conklin has also divided the gastrula into anterior and posterior halves ; neither can regenerate the missing parts, though the cut surface of each becomes covered by ectoderm.

Driesch, however, states that in another form, P/zallzma manmzillata, anterior and posterior half gastrulae will, if

FIG. 146.—~CyntIu'a pm-tita. Anterior half and three-quarter embryos.

(After Conklin.) a, b, anterior ventral three-quarter embryo, the posterior dorsal cells (B 4.1, B 4.1) containing all the yellow pigment

having been killed in the 8-cell stage. a, ventral view; b, dorsal view.

c, d, anterior half embryo: a, dorsal view, showing the neural plate (stip led); d, deeper focus showing two rows of chorda cells (the nuclei are s aded), and ectoderm and endoderm.

separated before complete closure of the blastopore, each develop into a. larva lacking only the otolith and the eye. In a later stage of gastrulation this becomes impossible, although the IV. I INITIAL STRUCTURE OF THE GERM 239

posterior portion of a young Ascidian will regenerate a. new head with pharynx and eyes ; the anterior half dies.

Driesoh has also maintained that the isolated blastomeres (72,, 3., %) of the same species give rise ultimately to total gastrulae and these to larvae. The larvae are, however, unable to hatch out and are defective—with rudimentary eyes, otolith and suckers-or devoid of these organs altogether.

FIG. 147.—Cg/nthz'a parfita. Posterior half embryos. (After Conklin.) a, 32-cell stage, dorsal view. Cleavage is quite normal. b, 76-cell stage, dorsal view. Muscle-cells (B 8.8, B 8.7 and B 7.8) and posterior mcsenchymc cells (B 8.6, B 8.5, B 7.7, and B 6.3) and posterior endoderm cells (B 7.1, B 7.2) are seen. c, later stage, deep focus, showing ventral endoderm (0 end.) with a. mesenchyme (m'ch.) on each side. d, same stage as last, showing muscle-cells (ms.).

LITERATURE L. CHABRY. Contribution a 1’embryologie normale et tératologique des Ascidies simples, Journ. de l’Anat. et de la Phys. xxiii, 1887. E. G. CONKLIN. The organization and cell-lineage of the Ascidian egg, Joum. gicad. Nat. Sci. Philadelphia, xiii, 1905. 240 INTERNAL FACTORS IV. I

E. G. CONKLIN. Mosaic development in Ascidian eggs, Joum. Exp.

Zool. ii, 1905. _ H. Dnmscn. Von der Entwicklung einzelner Ascidienblastomeren,

Arch. Ent. Mach. i, 1895. H. DRIESCH. Ueber Aenderungen der Regulationsflihigkeiten im Verlauf der Entwicklung bei Ascidien, Arch. Ent. Mech. xvii, 1904.


From the foregoing experiments certain general conclusions may now be drawn.

( 1) First, thedivision of the nucleus during segmentation at least is not the qualitative process imagined by Roux and Weismann. The pressure experiments of Hertwig, Driesch, and Wilson have clearly demonstrated that the nuclei of the segmenting ovum may be disarranged without prejudice to the subsequent normal development of the embryo; and this result is amply confirmed by the normal behaviour of eggs (of sea-urchins) in which the mutual positions of the blastomeres, and therefore also of the nuclei, are altered, as well as by the very numerous cases in which it has been shown to be possible to rear a perfect embryo or larva from an isolated blastomere or blastomeres.

It is evident that during segmentation at least the nuclei are equipotential, and the hypothesis of self-diiferentiation in the form originally propounded by Roux can no longer be upheld. It has, in fact, been now abandoned by its author.

(2) But though in this direction its labours have ended negatively, modern experimental research is yet able to point to a positive achievement of the greatest value and significance. For the same series of investigations has shown that the cytoplasm of the undeveloped ovum is not the homogeneous and isotropic substance which the experiments of Pfliiger led Roux to consider it, but heterogeneous, containing various specific organ-forming stuffs which are definitely and necessarily connected with the production of certain parts or organs of the developing animal. If the polar lobe of the mollusc Ilyamzssa be removed the larva has no mesoderm; if the polar lobe of Dentalium be taken away the larva has no trunk and no apical organ; the egg of a Ctenophor IV. I INITIAL STRUCTURE OF THE GERM 241

is capable of segmenting when deprived of its vegetative hemisphere, but the larva to which it gives rise has no combs and no sense-organ. The egg of the Ascidian Cynmia exhibits in its cytoplasm various substances, easily distinguishable by their colour ; in segmentation these are distributed to the various blastomeres in a perfectly definite way. Remove one or more of these substances and the embryo will lack the organ or organs normally produced from them. Again, a Nemertine egg from which, at a certain stage, the vegetative portion has been cut away, gives rise to a Pilidium without lappets and without a sense-organ.

In these cases, then, at least, the existence of specific organforming stuffs in the cytoplasm of the egg has been proved; the removal of the stuffs inevitably entails the absence of the corresponding organ later on. But though it may therefore be justly said that such stufis are preformed, it is not invariably true that they are preloealized, present, that is to say, at z'u2'tz'o in what will be their eventual situation. For example, the stuff which determines the formation of an apical organ in the larva of Deutalium is originally in the polar lobe of the vegetative hemisphere : but between the first and the second divisions it migrates into the animal portion of the egg ; and the same may be said of the formation of the sense-organ in Cerebratulus and the Ctenophora.

It seems highly probable, though it cannot be said to have been demonstrated, that such specific organ-forming substances are of universal occurrence.

The behaviour of the isolated blastomeres of the eggs of different forms, as well as of the blastomeres of the same form at different stages, is markedly different. The .1, or ,1; blastomere of an Ascidian egg can never produce more than g- or %; larva; the isolated blastomeres of Palella produce just so much, dividing just so many times, as they would have produced had they remained in connexion with their fellows; a radial group of blastomeres (one or more ,1, blastomeres, or two or four or more 113- blastomeres) of a Ctenophore egg gives rise to a larva. with one, two or more costae and meridional canals, as the case may be. On the other hand, the isolated 13 or 71; blastomeres of a Nemertine ovum will produce a. complete la.rva, while, after the next dii./°ision, the totipotentiality is lost; for an. isolated

muxmsox R 24-2 INTERNAL FACTORS IV. 1

animal cell gives rise to a larva without an archenteron, an isolated vegetative cell to one in which the apical organ is lacking. So in Amp/u'o.m4s % and § blastomeres develop into whole embryos, but g blastom eres, whether of animal or vegetative origin, will not gastrulate. In other cases, however, the potentialities do not become restricted so soon. Either the four animal or the four vegetative cells of a Frog’s egg will give rise to an embryo in which the blastoporic lip and archenteron are developed, while in Echinoderms the least blastomere that will gastrulate is 315. It is not, nevertheless, every cell of the 32—celIed stage that is capable of developing an archenteron, but only those, as Driesch has conceded, of vegetative origin.

But whatever differences there may be in the potentialities of the cells of different ova, or of the same ova at different stages or in different regions, it is clear that sooner or later the capacity of a part to become a whole i lost; and it seems only reasonable to conclude that this loss of totipotentiality is due to the loss of one or more necessary cytoplamic substances, to the lack of specific material, and not merely of material. In the egg of Ampfiiowus, which is telolecithal, there is an obvious difference, in the amount of yolk and size of the granules, between the animal and vegetative hemispheres; a difierence in the capacities of these two regions ha been experimentally demonstrated in the Nemertine egg; and, although it may often appear homogeneous, the ovum of the sea-urchin, in all probability, possesses that ‘stratification’ of potentialities, that is of organ—forming stuifs, at right angles to its axis, on which Bovcri has so strongly insisted, for Driesch has admitted that vegetative cells ‘gastrulate’ more readily than animal cells ; it would appear, then, that a gutforming substance is present, and a mesenchyme-forming substance too, but that the concentration of these substances steadily diminishes from the vegetative to the animal pole. Radially about the axis the distribution of these substances may be taken to be uniform, since meridional fractions of the egg, %, §, % or g or g blastomeres, are invariably totipotential.

The same explanation may be applied to other cases. Hence, speaking generally, the limitation of the potentialities of the parts will depend simply on the original distribution of the IV. I INITIAL STRUCTURE OF THE GERM 243

substances in the unsegmented egg. Should they be already segregated (wholly or in part) in distinct regions, as in Ascidians, Molluscs, and Ctenophors, then even the first blastomeres will to that extent be unable, when isolated, to give rise to more than they would have done in the uninjured egg; should theybe uniformly distributed about the axis, but unequally along the axis (Nemertines, Echinoderms, Amp/u,'oams), then a limitation of potentialities will appear when the third division separates the animal from the vegetative cells‘ ; should the arrangement be

FIG. 148.—Diagran1matic sections through the unsegmented egg of Lolig/o pealii. (From Korschelt and Heider, after Watase.) A is in the transverse, B in the sagittal plane of the future embryo. In B the anterior side (m.) is more convex than the posterior (h.). d, dorsal (animal pole); v., ventral (vegetative pole); 1., left; r., right. The protoplasm is black, the yolk shaded.

uniform about the egg-centre, then animal and vegetative blastomeres will be alike totipotential (Coelenterata).

At the same time it would appear to be true that the capacity of total development may be lost for mere lack of material, for Drieseh found that while all four (or all eight in the next stage) animal cells of the sea-urchin egg might give rise to a Pluteus larva, the isolated cells would not. The isolated cells may, how ‘ We may notice that O. Maas (Arch. Ent. Mech. xxii, 1906) has found that the isolated anterior flagellated and posterior granular cells of the Amphiblastula larva of S3/can are not equipotential. The latter can fix and give rise to a. young sponge, the former cannot. 244 INTERNAL FACTORS IV. I

ever, gastrnlate ; when once the gastrula stage has been reached, therefore, further development may be simply a matter of sufficiency of substance. So again the octants of the Ctenophore egg are alike, but each can produce one costa, and one only. There are then certain preformed organ-producing‘ substances, and the arrangement which they either possess before, or acquire

"‘l.'tnL" FIG. l49.—Three segmentation stages in the blastoderm of Sepia aficiualis; the segmentation is of the bilateral type. l, left; r, right; I—V, first to fifth cleavages. The top sides of the figures are anterior. (After Vialleton, from Korschelt and Heider.)

during, segmentation determines the fixed relation which can be observed in almost all cases between the structure of the egg and the axes of the embryo. Thus in Amphibia the head of the embryo is formed at or near the animal pole, and the plane of egg symmetry becomes the sagittal plane. In Mollusca (not

Cephalopoda) and Annelida the apical sense-organ is developed at the animal pole, while the D quadrant is posterior. A blasteTV. I I-NITIAL STRUCTURE OF THE GERM 245

pore is in very numerous eases formed at the vegetative pole, the bilateral symmetry of the eggs of Cephalopoda (Figs. 148, 14-9), Inseeta, some Crustacea, and Aseidia becomes that of the embryo; in Ascaris the first four cells lie in one plane, which becomes the median plane, the germ-cell is posterior and the animal pole dorsal; and soon.

(3) The instances in which the segregation of the organforming substances into the blastomeres takes place at once are especially interesting, as they appear to fulfil exactly the requirements of the ‘ Mosaik-theorie ’, if that doctiine be transferred from the nucleus to the cytoplasm, for here, undoubtedly, segmentation is a qualitative process, a sundering of potentialities. It must be pointed out, however, that even in these cases the factors which determine segmentation and those which determine difl’erentiation may be as distinct as they are elsewhere.

In the Frog there is a much greater correlation between the symmetry of the egg and the median plane of the embryo than between the former an(l the plane of the first furrow.

A % or % blastomere of a Nemertine or a sea-urchin segments as a part, and yet it eventually develops into a whole larva: and the same is true of egg-fragments. All the isolated blastomeres of a Molluse segment as parts, yet in 1)ental2'um, that one which contains all the necessary stuflfs, CD or D, can give rise to a complete embryo, while its fellows cannot. The converse case is to be found in Ctenophors ; here the egg which has been deprived of its vegetative portion segments as a whole and yet produces an embryo devoid of eostae and sense-organs.

There are also certain forms (/Imp/u'o.'ru8 and V61 tebrates, Coelenterata) where the isolated cell segments as a whole, and this appears to depend on the rapidity with which the part can rearrange its material or resume the polarity of the whole (Wctzel). It is further to be noticed that the capacity of a part for total segmentation,like its power of total differentiation, may differ in the same egg at difi’erent times and under difierent conditions ; the fragment of an immature egg of Care//ratulua divides Iikea whole egg, an isolated blastomere like a part ; again, in Ilyanaaaa the cleavage of a part may be made total by lowering the temperature.

When, further, it is remembered how closely similar the 246 INTERNAL FACTORS IV. I

geometrical pattern of segmentation may be in the eggs of different animals, whether of the same or of diiferent groups (spiral segmentation of the eggs of Molluscs, Chaetopods and Turbellaria, similar segmentation of the eggs of, for example, some Echinoderms, Polyzoa, and Vertebrates), without necessarily involving a similarity in the fate of cells which are identical in origin, it will, I believe, be conceded that the factors which are responsible for cleavage and those which determine difierentiation are distinct, though the two may, as in the socalled ‘Mosaik’ segmentations, coincide: and where, as in Ascidians, the Ctenophora, the Cephalopoda, this coincidence is complete, the symmetry of the egg, the symmetry of segmentation, and the symmetry of the embryo are one.

The former factors must be sought for in the arrangement of yolk and protoplasm, possibly in the relative strengths of the centrosomes, in surface-tensions, in the pressure exerted by eggmembranes and so forth ; the latter in definite cytoplasmic organforming stulfs.

(4) Like the parts of the egg the parts of the elementary organs of the embryo are at first equipotential, but exhibit a gradual limitation of potentialities as their development proceeds. As Minot puts it, there is a ‘ genetic restriction ’ of capacities. Thus the arehenteron of Asterias can form a new terminal vesicle when this has been removed, but only before the outgrowth of the coelom sacs. The anterior half of the newt embryo will develop into a whole embryo when out off before the medullary folds have arisen, but not after. When one vitelline vein of a chick is destroyed its fellow will form a. whole, not a half heart; and Sumner’s experiments quoted above have shown that the margin of the Teleostean blastoderm is isotropic.

(5) There still remains to be noticed a point of great importance. It certainly has not been demonstrated, and it cannot be pretended that there are in the developed egg as many specific substances as there are separately inheritable qualities in the body. On the contrary, the evidence of experiment speaks against such a supposition; for, as Driesch has urged, were every separately inheritable quality to be represented in the germ by a distinct morphological unit, the equipotentiality of IV. I INITIAL STRUCTURE OF THE GERM 247'

the isolated parts would be inconceivable. Although the formation of the larval and elementary organs (germ~layers) may depend upon such stuflfs, yet within each such elementary organ the parts are at first and remain for a time equipotential.

Diiferentiation is progressive. The origin of the primary differentiations has been found; it remains for the experiments of the future to discover how fresh distinctions arise until the organization of the whole is complete. Suggestions as to the importance, in this respect, of certain other internal factors have been made, notably of the structure of the nucleus, and the reactions of the parts on one another. These suggestions, for they are hardly more than that at present, we shall have to discuss. First, however, we must briefly consider the part played by the spermatozoon in determining the structure of the egg and the symmetry of the embryo.

§ 12.

It is of the greatest interest to observe that the definitive distribution of these specific substances in the unsegmented egg, and so the determination of the embryonic axes, may be brought about, partly at least, by the spermatozoon.

In the Frog (Ifamz fusca, It. temporaria, and In’. 1Jalu3lrz's, probably also 1?. esculenta) the egg soon after fertilization becomes bilaterally symmetrical. As first Roux and later Schulze have shown, this is due to the appearance on one side of the border of the pigmented area of a grey crescent, caused by the retreat of the pigment into the interior. The grey crescent subsequently becomes white and added to the white area. The side on which it appears is opposite to that on which the spermatozoon had entered, and later becomes the dorsal side of the embryo ; there is also a considerable correlation between the plane of symmetry‘thus bestowed upon the egg and the future sagittal plane, since the latter lies in a large majority of cases in or close to the middle point of the crescent.

This relation between the point of entry of the sperm and the egg- and embryonic symmetry has also been experimentally demonstrated by Roux, who, by applying the sperm to any arbitrarily selected meridian of the egg (by means of a fine cannulus, _a brush, or a silk thread), was able to show that 248 INTERNAL FACTORS IV. I

fertilization can take_ place from any meridian, and that the point of entry so selected becomes the ventral side later on; in other words, the fertilization meridian becomes the sagittal plane. This, as we have seen, is true, or approximately true. Roux, however, believed that the plane of the first furrow also coincided with the other two, in fact that it was the first furrow which determined the sagittal plane, and he brought forward evidence to show that the first furrow, if it did not pass through the point of entry of the sperm, at least either included, or was parallel to, the inner end of the crooked path of the spermatozoon within the egg, the so-called ‘ copulation ’ path (Fig. 150). As we now know there is very little correlation between the first furrow and the sagittal plane. Nevertheless Roux’s work remains of

FIG. 150. —Roux’ diagrams to show the relation of the sperm-path (Pig/.) to the first furrow in the Frog's egg. In A the furrow includes the sperm-path, in B it is parallel to it, in C it is parallel to the inner portion of the path (copulation path), in D it includes only the very last portion of the copulation path. (From Koischelt and Heider, after Roux.)

the greatest significance, for it seems extremely likely that, while it is the actual point of entrance of the sperm and the first, radially directed, part (‘penetration path’) of its track within the egg, which determines the position of the grey crescent, and so of the sagittal plane, it is the second part which determines the direction of the first furrow, since the centresome will divide at right angles to this ‘copulation’ path, or line of junction of the two pronuclei; the axis of the fertilization spindle is of course given by the line of separation of the two centrosomes, its equator by the plane including the two pronuclei and the egg-axis, and this spindle-equator is the plane of the first furrow.‘

In the egg of the sea-urchin Tomopzzezw/es the definitive eggaxis would appear to be fixed not by the original position of the egg ‘ See, however, Appendix A. IV. I I°NITIAL STRUCTURE OF THE GERM 249

nucleus at all, but by the position of the excentric segmentation nucleus. Both male and female pronuclei move through the egg for a longer or shorter distance till they meet, and the combined nucleus then takes up its definitive excentric position (Fig. 151). The plane of the first furrow is given by the egg-axis and the point of entrance, approximately, of the spermatozoon, which may thus be said to determine the symmetry of segmentation, and so of the embryo, since this plane becomes, it is said (Selenka,

FIG. 151.—D1ag1~ams from successive camera. drawings of the living eggs of Toaropneustes, to show the determination of symmetry during fertilization.

The original position of the eg nucleus is shown by the position of the dotted circle marked 9; its pa.t by the succession of such circles.

E, the entrance-point of the spermatozoon, and cone of entrance. The male pronucleus is rendered in black, its path marked by the line uniting the black dots.

M is the meeting-point of the pronuclei. The cleavage nucleus, c, is shaded; its successive positions are indicated. The axis of the fertilization spindle is shown by the line with a small circle at each end passing through the cleavage nucleus; the arrow passing through this is the definitive egg-axis, its point the animal pole. The cleavage nucleus is slightly excentric, and nearer the animal than the vegetative pole.

F is the meridian of the first furrow.

A and B are opposed in polarity, similar in other respects. 0 and D differ in polarity, and otherwise as well. (After Wilson and Mathews.) 250 INTERNAL FACTORS IV. 1

Garbowski, Driesch), the median plane of the Pluteus. In this case there is practically no distinction between ‘ penetration ’ and ‘copulation’ parts of the sperm-track, and, the centrosome dividing perpendicularly to this path, the point of entrance naturally lies in, or nearly in, the first furrow. The other alternative, as Wilson has pointed out, is to suppose that the definitive polarity of the egg existed before fertilization, but exerted no influence upon the egg-nucleus, although able subsequently to compel the fertilization nucleus to take up a position in its axis.‘ There are other Echinoderms, however (:1a5er2'a.9, Stroizyyloccietrotus), in which the original and the definitive egg-axes coincide.

Another very interesting case is Uynt/zia (Fig. 142). In the immature egg of this Ascidian the cytoplasm is concentrically arranged, since the yellow pigmented protoplasm forms a peripheral investment for the central grey yolk: the egg-axis is determined only by the position of the germinal vesicle. Upon the entrance of the spermatozoon the yellow substance together with the clear plasma of the germinal vesicle, which has in the meantime broken down, streams towards the vegetative pole and becomes radially arrayed about the axis. By a further streaming movement these two substances are carried up with the male pronucleus—— or pronuclei in cases of dispermy—to the equator, so marking the future posterior side of the now bilateral egg. These changes are evidently an eifect of fertilization: it cannot, however, be asserted that the point of entrance of the sperm determines the posterior end, since it may enter on the opposite side and get carried across.

There are a few other instances in which a11 alteration of the egg-structure has been noticed to follow on fertilization. In Ctenophora the peripheral layer of granular cytoplasm becomes aggregated at the animal pole (Agassiz) ; in Polyclads (Lang) and Cirripedes (Groom) the egg becomes telolecithal ; and so in P/rzyew

‘ In Diplogaster longicauda also it appears to be the point of union of the two pronuclei which determines the definitive egg-axis and orientation of the embryo. The polar bodies are always extruded and the female pronucleus formed at that end of the ellipsoid egg which is turned towards the ovary, while the sperm enters at the opposite end. The pronuclei may meet and units at either end; but it is at the end where their union takes place that the smaller of the first two blastemeres is found and, later, the binder end of the embryo (H. E. Ziegler, Zeitschr. wise. Zool. lx, 1890). IV. I INITIAL STRUCTURE OF THE GERM 251

(Kostanecki and Wierzejski): the ‘polar rings ’ appear in Allololzoja/tom, Clepsine, and Rhync/aelmis (K. Foot, Whitman, Vejdovsky), while in Teleostei the periblastic hyaline layer becomes concentrated as the blastodisc at the animal pole. The case of Dentalium has been already referred to (p. 223, Fig. 133).

Although the cases in which the role played by the spermatozoon in determining egg and embryonic structure are, as has been observed, not very numerous, yet it is fully to be expected that renewed investigation will show that some such rearrangement of the materials of the egg is in many, perhaps in all, instances one of the first results of fertilization.


A. AGASSIZ. Embryology of the Ctenophorae, Mcm. Amer. Acad. Arts and Sciences, N.S. x, 1873.

E. G. CONK LIN. The organization and cell-lineage of the Ascidian egg, Jam-n. Ac. Nat. Sci. Philadelphia, xiii, 1905.

K. Foo'r. Preliminary note on the maturation and fertilization of the egg of Allolobophora foetida, Journ. Morph. ix, 1894.

T. T. GROOM. On the early development of Cirripedia, Phil. Trans. Roy. Soc. elxxxv, B. 1894.

K. VON KOSTANECKI and A. WIERZEJSKI. Ueber das Verhalten der sogen. achromatischen Substanzen im befruchteten Ei (Physa fontinalis), Arch. milcr. Anat. xlvii, 1896.

A. LANG. Fauna. und Flora des Golfes von Neapel: XI. Die Polycladen, Leipzig, 1884.

W. Roux. Die Bestimmung der Medianebene des Froschembryo

(lurch die Copulationsrichtung des Eikernes und des Spermakernes, Arch. mikr. Anat. xxix, 1887; also, Ges. Abh. 21, Leipzig, 1885.

F. VEJD0vsK1'V. Entwickelungsgeschichtliche Untersuchungen, Prag, 1888-92.

C. 0. WIIITMAN. The embryology of Clepsine, Quart. Jouru. Micr. Sci. xviii, 1878.

E. B. WILSON and A. P. MATHEWS. Maturation, fertilization and polarity in the Echinoderm egg, Journ. Morph. x, 1895.

§ 13. WIIAT PART DOES THE NUGLEUS PLAY IN DIFFERENTIATION ?1 We have already criticized and rejected the hypothesis that the division of the nucleus during segmentation is a qualitative process. It may still be urged, however, that the nucleus is not

‘insignificant in dilferentiation. In support of this contention

the following arguments have been put forward. 1. It is urged that the nucleus is essential for the life of the cell,

1 See also Appendix B. 252 INTERNAL FACTORS IV. 1

and not only for its metabolism (Figs. 152, 153) but for the production of form (Fig. 154).

2. It is obvious that the germ-cells are the vehicles whereby the inheritable characters of the species are transmitted from one generation to the next; they are the material basis of inheritance. The germ-cells of the two sexes are, however, as unlike as possible in every character except their nuclei, in which, as the study of maturation has abundantly shown, they are exactly alike. In fertilization, the union of the two germ-cells, two distinct processes are involved. The first is the mutual stimulation whereby the lost _ power of cell-division is restored. This ',5 ' is a process independent of the nuclei (in Metazoa). The other is, however, the union of the nuclei (or their chromosomes); and since the offspring are held to inherit equally from the two parents, it has been - - supposed that with the union of the nuclei, the only parts of the cells that are

FIG. 152.——Egg-cell of

Dytiscus mwginalis in its follicle with two nurse cells. From the nurse cells nutritive material passes into the egg-cell, towards which the nucleus

exactly alike, the paternal and maternal contributions are intermingled, and that therefore it is in the chromatin of the nuclei that the vehicle of transmission of hereditary qualities must be looked for.

sends out pointed pseudopodia. (After Korschelt, from Korschelt and Heider.)

3. Thirdly, it has been pointed out (originally by Roux) that karyokinesis appears to be a mechanism expressly adapted to the simultaneous division of a large number of qualitatively different units. From the existence of karyokinesis it is argued that the nucleus is not homogeneous, and that in development the various units of which it is composed are directly concerned in the process of dilferentiation.

The doctrine of the individuality of the chromosomes has been brought forward in support of this belief, as well as the

constancy of their number. 4. The diminution of the chromosomes. Boveri has observed

in Aacmia, in the nuclei of purely somatic cells, apeguliar process IV. I INITIAL STRUCTURE OF THE GERM 253

to which he has given the name of the diminution of the chromatin. The middle parts of the chromosomes in somatic cells become divided transversely into small granules, the ends remain rodshaped. The granules of the middle parts are alone divided and

pass to the daughter nuclei, while the ends are cast out into the cytoplasm and there degenerate. The chromosomes of the germ-cells (or of the parent-cells of the germ-cells) do not undergo this change, but remain intact (Fig. 155).

It might be argued that in such a. case as this the determination of certain characters —-somatic-—is brought about by the expulsion of the chromatin into the cytoplasm.

5. A great deal of stress has been laid on the importance of the experiment, due to Boveri, in which the enucleate fragment of the egg of one species of sea-urchin gives rise, when fertilized with the sperm of another species, to a larva _ _ _ which exhibits the charams i..F’1»‘TI..i§’.3 3?‘.-:...‘3.‘£T"""q~i‘.’.f ‘.”...’l.‘i‘.§f.'.i'“iL'

of the male parent alone. placed at the point of origin. B, the

- same in Cmurbita pepo. C, root-hair 6' Boven has brought for of Cannabis .-atim: the nucleus is at

ward evidence to show that the growing point. (After Haberlandt, the abnormal development of from Korschelt and Ileider.) Echinoderm eggs which follows on polyspermy is in reality due to the irregular distribution of the chromosomes to the daughtercells. Hence it is argued that the chromosomes are qualitatively diflferent.

These are the reasons which are, or have been, brought forward in support of the belief that the nucleus is not insignificant in difierentiation. We may now discuss them in order,


premising only one thing, namely, that both maternal and paternal nuclei are certainly not necessary for the production of a new individual, however beneficial it may be for the elements of two parents to be commingled in the body of one ofispring. In artificial parthenogenesis an egg is stimulated to development by a solution, and produces a normal larva; this creature possesses nuclei of maternal origin. The

_ e converse case is seen in the

W "K "- i ‘ phenomenon of ‘ merogony’.

M An enueleate egg-fragment, fertilized with sperm of the



same species, gives rise to a new organism, provided only with paternal nuclei.

The older view that the union of the pronuclei was the essential act in fertilization ean no longer be held, for, certainly, both nuclei are not necessary. The most, therefore, that can be said will be that a complete set of the chromosomes (or smaller

chromatin units) of the species FIG. 154.——0n the left the protozoan

Stentor, cut into three pieces, each con- ls necessary for d1fierentla't10n'

tainingha piecle of thfi nucleus. On Whether this is so or not we the rig t, eac piece as regenerated - - , the missing parts and become a com— must now mqune'

lete Stentor. (After Gruber, from 1. It will of course be al°“°h°1t “ml Heme") lowed thatthe nucleus is necessary for the life of the cell and to a certain extent for the assumption of form. The numerous and familiar experiments on Protozoa have sufficiently established this point} A nucleate piece will live and regenerate lost parts, an enucleate piece will not (Fig. 154-). 2. It will also be admitted that the maturation processes in the germ-cells of the two sexes are extraordinarily alike and end in the reception by each germ-cell of one-half the normal ‘ See especially M. Verworn, Die physiologisehe Bedeutung des

Zellkerns, 12fl1'1ger's Arch. li, 1892 ; in this paper an account of the work of Gruber and others will also be found. ‘ IV. 1 INITIAL STRUCTURE OF THE GERM 255

FIG. l55.——The process of chromatin diminution as seen in the somatic cells of Ascaris megalocephala. (After Boveri, 1899.?

1. Mitosis in the 2-celled stage. In the first somatic cel (S, or AB), the primary ectoderm, the chromatin undergoes diminution, not in the germ-cell (Pl). la chromosomes being ‘diminished’.

2. 4-cell stage, T-shape. In A and B the discarded masses of chromatini are seen. S, (EM St), second somatoblast or endomesostomodaeal ru iment.

3. 4-cell stage, lozenge-shape. In A and B the next mitosis is beginning, in P, and E M St the nuclei are in the resting stage. A is anterior, 2:] and g are dorsal, all four cells lie in one plane, the sagittal plane of t c em ryo.

4. In EM St the chromatin is being diminished. Division of P, into P3 and S3 (C), the secondary ectoderm. a, b, primary ectoderm of right, a, 8, primary ectoderm of left side.

5. The endoderm (E,, E,) has now been separated from mesoderm and stomodaeum. P3 has just divided into P4 and SԤd)' tertiary ectoderm.

6. Diminution of chromatin in S, (D). The our endoderm cells (E) beginning to be invaginated: on each side two mesoderm cells (M) in which franular chromosomes may be seen, and two stomoclaeal cells (St). Ventra. view. 256 INTERNAL FACTORS IV. 1

number of chromosomes of precisely the same shape and size. In fertilization the two sets of chromosomes—each in half the normal number-are united. It is of course difficult to believe that this extraordinary resemblance of the nuclei, while all the other characters of the cells are unlike, is without significance.

3. With regard to the third point a distinction must be made between two hypotheses. The first is the individuality of the chromatin, the second the individuality of the chromosomes.

For the first, as we shall see, independent evidence exists, and mitosis is certainly a mechanism admirably adapted to the simultaneous division (or separation of already divided halves) of a large number of qualitatively distinct bodies.

But the second hypothesis in no way follows from the first, for the grouping of the unlike units may obviously be different at each successive division without in the least impairing their individuality. There is, indeed, evidence for the persistent individuality of the chromosomes in only one or two cases. Boveri has noticed, in the segmenting egg of A.<:carz'.s-, a constancy in the position of the pockets of the nuclear membranes which lodge the ends of the chromosomes, and a similarity in the arrangement of the chromosomes in the nucleus in successive nuclear divisions. Sutton again has observed that in Bracfi_g/stola each chromosome forms a separate reticulum, situate in a separate pocket of the nucleus. There are other cases in which each chromosome forms its own vesicle (Echinoderm eggs). These instances are, however, few and far between. There are so many nuclei in which nothing can be observed of the chromosomes in the resting stage, often there is nothing but a fine granular mass, and a study of the early and end stages of mitosis seems to show that the chromosomes are precipitated from solution in the nuclear sap and redissolved when the division is over.

In such a solution the chromatin elements would retain their individuality, their qualitative difierences, just as each one of a number of crystals retains its distinctive properties in a mixed solution and exhibits them when recrystallization occur. In such a sense, and such only, is it possible to speak in general of the individuality of the chromatin. '

4. With regard to the diminution of the chromosomes in IV. I INITIAL STRUCTURE OF THE GERM 257

Ascm-is, Boveri has pointed out that the extruded outer ends of the chromosomes are very irregularly distributed to the daughtercells, whereas they should surely, if they are determinants, be equally divided between the two. More than that, however, Boveri has found evidence which shows that not only does the diminished chromatin not determine the characters assumed by the cytoplasm, but that on the contrary it is the cytoplasm which decides which chromosomes shall be diminished, and which remain intact.

Boveri has observed certain cases of dispermy in Ascaria, which are followed by simultaneous division of the ovum into four, consequent, presumably, on a quadripolar mitosis which is due, in turn, to the presence of an extra pair of centrosomes. The total number of chromosomes present in such eggs is 3 n, where n is the reduced number, since each spermatozoon introduces n. This number becomes doubled by division, and the number is then 6n or 12, since 1» in Ascm-2'3 megalocep/zala v. bivalens = 2. These twelve chromosomes have to be distributed over the four cells into which the ovum divides: their distribution is irregular. The next division is described as being tangential in two on? the cells, at angles of 45° to the first divisions in the other two: it leads to the formation of two groups of four cells each, in each of which the cells are arranged in the T shape characteristic of the normal four-celled stage. The normal four-celled stage is reached by (1) an equatorial division, followed by a meridional division in the animal cell (/1 B), and a latitudinal division in the vegetative cell, which separates a cell S2, which like A and B is somatic, from a cell P, at the vegetative pole. The chromosomes in P2 are intact, in A, B, and 82 they are diminished (Fig. 155).

Boveri argues that if the diminution of the chromosomes were an intrinsic property of the chromatin, there should always be 6 (3 7;) chromosomes intact and the rest diminished, just as there are always 4« (2 n) intact in the normal egg, no matter how the chromosomes had become distributed over the first four cells and their descendants. This, however, is not the case. Whole chromosomes are found only in those cells——one in each group of four—which correspond in position to the P2 cell of the


normal egg; the chromosomes in the other cells are all diminished, and no cell contains chromosomes of both kinds. Further, the number of whole chromosomes may be 5 in one cell and 3 in the other, or 4 and 3, or 3 and 3, or 4 and 2, or 3 and 2.

Boveri concludes, it seems with perfect justice, that it is the (vegetative) cytoplasm which has determined that the chromosomes it contains shall remain whole, while, contrariwise, the animal cytoplasm of the other cells brings about the process of diminution.

In Ascaria lumbricoizles there is a diminution of the chromosomes very similar to that seen in A. megalocep/mla, and, to judge from

FIG. 156.—Pluteus of_Sphaerechz'nus granularis from in front and from the side. (After Boveri, 1896.)

certain abnormalities similar to those just mentioned, brought about by the same means (Bonnevie). In Dytiacus there is something like a diminution of the chromatin in the nurse-cells, while the oocyte retains all the chromatic material intact (Giardina). The distinction seen here may, however, only be that observed in all ova between the nutritive and reproductive functions of the nucleus (trophochrornatin and idiochromatin).

5. Boveri’s hybridization experiment, on which rests the assertion that the nucleus alone conveys the inheritable characters of the species, was as follows.

The Plutei of Sp/laerec/ainua _qranuZarz's and of Ecfiinus microtuberculatue were found (at Naples) to be markedly difierent-. IV. I INITIAL STRUCTURE OF THE GERM 259

The larva of the former (Fig. 156,) is short and squat, the oral lappet not divided into lobes, and the skeleton provided with a fenestrated anal arm—produced by three long parallel bars united by numerous cross-bars--an apical branch to the oral arm, and, at the apex, a square ‘frame ’ formed by the union of twigs from the last mentioned and from the apical arms.

The larva of Ea/limzs, on the other hand, is long and lank, the oral lappet is deeply cleft, and in the skeleton the anal arm is not fenestrated ; there is no apical branch of the oral arm, and the ex

FIG. 157.-—Pluteus of Echinus mhromberculatus from in front and from the side. (After Boveri, 1896.)

tremity of the apical arm is thickened and club-shaped (Fig. 157). Boveri first fertilized the ova of Spfiaerecfiimw with the sperm of Ea/zz'mw, and so produced a hybrid larva whose characters were intermediate between those of the two parents in the following respects—in form, being shorter and broader than that of Ea/ainus, longer and narrower than that of Spfiaerec/zinus, in having the oral lappet slightly divided, and in the skeleton, the extremity of the apical arm being swollen and branched, the anal arms being double but not fenestrated, and the oral arms being (sometimes) provided Vvith an apical branch (Fig. 158). s 2 260 INTERNAL FACTORS IV. I

A mass culture of egg-fragments of Sp/Eaerec/2imw—containing presumably whole eggs, and nucleate and enucleate fragmentswas then made, and this was fertilized with the sperm of Ea/tinua. Amongst the larvae developed in such a culture Boveri found a small number (twelve) of dwarf individuals which possessed the paternal (Ec/zimw) characters alone (Fig. 159 6), and he suggested that these had come from enucleate fragments of eggs. He was further strengthened in this opinion by the fact that the nuclei were small, which he attributed to their containing only one-half the normal number of chromosomes.

This conclusion has been adversely criticized by Seeliger. Seeliger, working at Trieste and subsequently at Naples, has

FIG. 158.—Pluteus of the_ cross Sphaerechinus granularis 9 x Echinus microtuberculatus c7’, from in front and from the side. (After Boveri,

1896) pointed out that the diiferences between the Plutei of these two sea-urchins are not as great as Boveri asserted them to be—-the extremity of the apical arm in I]c/ainus is, for instance, not always club-shaped—and that therefore it cannot be so confidently asserted that the hybrid is intermediate. Secondly, he asserts that in an ordinary hybrid culture (whole eggs of Sp/zaerecfiz'nus fertilized by sperm of Ea/timw) there are to be found together with variable larvae of more or less an intermediate type, individuals with purely paternal characters, though the pure Sp/merecfiz'nu.s type never occurs, and that the nuclei are variable in size. This contention has been upheld by Morgan, and, after a good deal of controversy, has finally been admitted by Boveri himself. No conclusion can therefore be drawn from the original experiment. IV. I INITIAL STRUCTURE OF THE GERM 261

An explanation of the discrepancies in the results of the difierent observers has been offered by Vernon. Vernon has found that Stranyylocentrotus livirlus (which has a Pluteus almost exactly like that of Ea/ainzw) is at a minimum of sexual maturity in the summer months, but that from that time onwards the power of the male to transmit its characters to the hybrid larva, when crossed with Sp/aaerec/mms 9 , increases, and that it is possible to obtain a culture consisting entirely of larvae of the paternal type, in respect, that is to say, of the skeleton. Vernon was inclined to look upon this difference in maturity as a seasonal variation, but Doneaster has since attributed it to the effect of temperature alone.

The transmission of the characters of the skeleton may therefore be the function of the nucleus, though, as just pointed out, there is no stringent proof of this. There are other larval characters, however, which, as Driesch has justly urged, must depend on the cytoplasm of the ovum, the colour, for example, if the egg is pigmented, and the size of the larva, so far as this depends on the size of the egg. From a series of hybridization experiments between Fm. 159"”, Fully

E0/ainus, :5):/uwrec/zi7ms, Stroizgz/loce7zt1'otu3, fonned platens of E-ch,-_

and /lrbacia, Driesch indeed concludes gut; m1'c(21'otubefr1culatusf.‘ that the rate of cell-division up to the tile °1i,1,:l,§, E’},i,-,,,:,“;:;e formation of the primary mesenchyme, r<leared(?from fan enu - _ - e eatc )c<rg— raoment the vacuolation of the ectodeim cells (in of Sphamf,=,h,.,m;* ,.er_

S};/merec/zines), the number of the primary tilized with the sperm mesenchyme cells, are all characters which five (After depend on the ovum, and the ovum alone, i i

and therefore on its cytoplasm. With regard to the skeleton Driesch admits that this, and hence, indirectly, the shape of the larva, may be influenced by the sperm, at least when the egg of Sp/taercc/¢z'nu8 is fertilized either by Ea/Linus or Strongyl0centrotus. .Fischel has made similar experiments and arrived

at a like conclusion. 262 INTERNAL FACTORS IV. I

Boveri has traversed some of these statements of Driesch’s. The number of primary mesenchyme cells is, he maintains, a mean between the paternal and maternal number, and the form of the larva may be intermediate before the skeleton is developed. Between these contradictory opinions it is not easy to decide, but it may be mentioned that the number of mesenchyme cells is peculiarly liable to variation, as Driesch has himself found.

Before concluding this section we should mention some most interesting experiments on heterogeneous cross-fertilization.

Loeb was the first to show that by the addition of a small quantity of calcium chloride and sodium hydrate to sea-water, it was possible to fertilize the eggs of Stron_9gylocent1-otu.s- [)mp1tmine with the sperm of Arte)-ias ockracea. The matter has since been more completely investigated by Godlewski, who has succeeded in rearing Plutei from the eggs of sea-urchins fertilized with the spermatozoa of the Crinoid Amfedon. The necessary condition of success is the prior treatment of both eggs and sperm with an abnormally alkaline sea-water (about 1'75 c.c.

T73 NaOH per 100 c.c. of sea-water).

The sea-urchins employed were Sp/merec/«mus, 8trou_qylocentrotua, and Ecfiimzs. The eggs of the first reached the gastrula stage, of the two last the Pluteus. Fertilization was found to be perfectly normal, with production of a vitelline membrane, rotation of the sperm-head, and so on. Cleavage was of the Echinoid type (maternal) with formation of micromeres at the fourth division; no such micromeres occur in Antetlon, the fourth division being meridional in both hemispheres. Primary mesenchyme was formed (this again is absent in Anterlou), but the number of cells was slightly in excess of the normal: and after gastrulation the skeleton of the Pluteus was developed (except in 15'];/(ae7'ec/tinzts); the larva of Antedon of course has no skeleton.

The Antedon chromosomes persisted and the nuclei of the hybrids were intermediate in size between those of the parent forms.

When an enucleate fragment of the egg of Ecfiimw was fertilized by Antezlon all the processes mentioned above were normal, except that segmentation was irregular, and that development ceased after gastrulation. The death-rate of these larvae was high. IV. I INITIAL STRUCTURE OF THE GERM 263

6. It remains for us now to consider the conclusions which Boveri has drawn from the behaviour of eggs in which owing to pathological mitosis the distribution of the chromatic elements was irregular.

The investigations of O. and R. Hertwig had previously shown that if the eggs of sea-urchins were weakened by treatment with poisons (strychnine, quinine, and others), then the vitelline membrane which normally prevents the entrance of more than one spermatozoon was not formed, that consequently several

FIG. 160.——Diagram of one case of irregular chromosome distribution in adoubly fertilized egg; an b. , c,, and d, ; a,, be, c,, and d2,and as, b,, c3; and d, are the three complete, specific sets of unlike chromosomes. (After Boveri, 1904.) '

spermatozoa entered, with the resulting formation of multipolar mitoses and irregular cleavage.

Boveri has succeeded in inducing dispermy in the eggs of sea-urchins without the use of the poisonous reagent, by simply adding an excess of sperm to the eggs. The number of abnormal larvae found in such a culture is greater than that observed when the quantity of spermatozoa used is less, and in proportion to the number of dispermic fertilizations.

In dispermic eggs there is an extra pair of centrosomes, and these two usually form with the other two a quadripolar figure with four spindles occupying the four sides of the square(Fig. 160). In the equators of these four spindles are placed the chromosomes, 264 INTERNAL FACTORS IV. 1

the number of which is 39: x 2, where n is the reduced number (in E'c/limzs 9), since there are two male and one female pronuclei and all the chromosomes have divided. Cell-division occurs across each spindle and the whole breaks up simultaneously into four. To these four cells the 37: x 2 chromosomes are distributed, and, as Boveri points out, the chance of each cell obtaining a complete set of the n chromosomes of the species (supposing these to be diflferent from one another) is

C (1

FIG. 16l.——La.rvae from doubly fertilized eggs; (I, of SphaerechimI.s_4/r_anu?qris; b—d, of Echimts micrombcrt-ulatus; a, r, and (Z are from tripartite, b from a quadripartite egg. (Aft-er Boveri, 1904.) small. Such eggs, as a matter of fact, practically always give rise to abnormal larvae. Out of 1,170 examined not a single one was normal.

The chance, however, that one cell of the four will receive a complete set of chromosomes is far greater, and when the =5 blastomeres were isolated it was found that 25 % gastrulated, though none gave rise to Plutei. Moreover, all four developed differently. IV. I INITIAL STRUCTURE OF THE GERM 265

Again, by shaking the eggs it is possible to prevent the division of one centre. A tripolar mitosis is the result and a simultaneous division into three. Out of 695 such eggs 58 became normal Plutei. This is also in accordance with the theoretical expectation, for the probability of each of three receiving a complete set of chromosomes when there are 3 u x 2 to be distributed is much greater than when these chromosomes have to be distributed amongst four cells.

The abnormal larvae are of various types, some showing no trace of the Pluteus organization, some a small trace, such as a skeleton on one side. Others have no gut, or some of the pigment cells are missing (Fig. 161).

The distortion of development cannot be attributed to the

deficiency in the number of chromosomes. Each of the cells 31; x 2 3 n

4 = 72- chromosomes, for the larvae reared from them may have all their nuclei of the same size (containing the same number of chromosomes), and it is known (artificial parthenogtnesis and merogony) that a larva will develop quite well with only 11. Nor is it needful that all the parts should have the same number of chromosomes, for normal larvae may be experimentally produced, with 71 chromosomes in the nuclei of one region of the body and 2 n in the rest (see below). At the same time dispermy does not necessarily involve abnormality : in such eggs the two pairs of centres may remain apart and no quadripolar figure be produced: these develop into ordinary Plutei (Fig. 162 B). Conversely, quadripolar mitoses may be found in monosperm eggs, but these produce pathological blastulae. No explanation of the abnormality, therefore, is possible, so Boveri concludes, except that it is due to the irregularity in the distribution of the chromosomes. For normal development it is essential that each cell should receive not a definite number but a definite combination of chromosomes, a complete set of the chromosomes of the species. It is the lack of this complete combination in one or more cells that is the direct cause of the derangement of development.

Hence the chromosomes are not alike, but qualitatively difierent. _

Their division is, however, never (except in reduction) a

of a quadripartite ovum may have 266 INTERNAL FACTORS IV. I

qualitative but always a quantitative process. The combination is handed on in its entirety to every cell of the body. The chromatin has in this sense an individuality, for however much the chromosomes may disappear while the nucleus is at rest, the set of qualitatively distinct units always re-emerges (though not necessarily in the same order) to be quantitatively divided between the two daughter—cells. We reach, therefore, a conception of the chromatin akin to Roux’s idea of the ‘ Reserve-idioplasson ’, the germ-plasm which is passed on entire to all the tissues of the body.

And now Boveri proceeds to develop a theory already suggested by Driesch of the part played by the chromatin in differentiation. The cytoplasm is heterogeneous, anisotropic; and these difierenees in the cytoplasm are sufficient to account for the earliest diiferentiations, the pattern of segmentation, the determination of the embryonic axes and possibly some few others. But then the chromatic elements come into play—not the chromosomes, which are too large and too few to be regarded as determinants—but far smaller subdivisions of these. Under the influence of the cytoplasm, diiferent in the various cells, some of the units become patent and active, while others remain latent, and by the reaction of the former upon the cytoplasm new difierentiations arise which will in their turn call forth into activity new nuclear elements.

Whatever value may be attached to these speculations, there can be no doubt of the importance of the experiments on which they are founded. Provisionally we have good ground for believing in the dissimilarity of the chromatic elements and of their necessity for differentiation. It only remains to be seen whether further investigations on other forms will bear out the interpre— tation which Boveri has placed upon his observations.

There is one more point worthy of notice. An ordinary sexually produced individual possesses in its nuclei 2n chromosomes, that is, two complete sets. In maturation one division of the chromosomes is transverse, and Boveri suggests that in this transverse division homologous units are separated from one another, or, to use the expression made familiar by current Mendelian theory, members of pairs of allelomorphs. c

Before bringing this section to a close a brief reference must IV. I INITIAL STRUCTURE OF THE GERM 267

be made to the relation established by Boveri, as a result of the examination of sea-urchin larvae (Ecfiimw), between the number of chromosomes on the one hand and the size of the nucleus and the size of the cell on the other.

The number of chromosomes in a larva, or part of a larva, may be varied in the following ways.

1. In merogony the number is half the normal number, i.e.

'71. (The larva is termed by Boveri hemikaryotic arrhenokaryotie.)

2. In artificially parthenogenetic larvae it is also 12. (Hemikaryotic thelykaryotie.)

3. In ‘ monaster’ eggs the number is twice the normal, i. e. 4 oz. (Diplokai-yotic.)

This condition is brought about by shaking the eggs, and so preventing the division of the centrosome. The pronuelei unite and enter a resting stage. From this the chromosomes emerge in the full number and split, but since the centrosome remains single, there is no cell-division, and the chromosomes once more form a resting nucleus, but now in twice the normal number.

Later the centrosome divides and the egg develops normally.

4. If the eggs are kept for twenty-four hours in sea-water and then fertilized with sperm which has been weakened in dilute potash the male pronucleus lags behind its centre. The latter divides about the female pronucleus, and thus in the two-celled stage one blavstomere has a male and a female nucleus, and therefore 21; chromosomes (amphikaryotie), while the other has only a female (thelykaryotic). In this stage, or subsequently, the male unites with the female pronucleus, and so one-half, or onequarter, possibly only one—eighth, of the larva has nuclei with 2 72, the remainder nuclei with n chromosom_es.

5. It sometimes happens in dispermy that the two pairs of centrosomes remain apart without forming a quadripolar figure (Fig. 162). Such eggs, as we have seen, develop normally. Sooner or later the cells become divided into two sets, one with nuclei containing 2 71 chromosomes (amphikaryotie), derived from the female and one of the male pronuelei, the other having n chromosomes from the remaining male (arrhenokaryotic).

A superficial examination shows that the nuclei of hemikaryotic embryos and larvae are smaller but more numerous than in 268 INTERNAL FACTORS IV. I

normal (amphikaryotic) embryos or larvae of the same stage. In diplokaryotie larvae, on the other hand, they are larger and fewer; while in larvae in which one portion is amphikaryotic, the other hemikaryotic, the two regions are distinctly marked ofl by the difierence in the size and number of their nuclei (Fig. 163).

The exact relation between the number of chromosomes originally entering into the composition of the nucleus and the dimensions of the nucleus and the cell has been ascertained by Boveri to be as follows :—

I. The surface-area of the nuclei is directly proportional to the number of chromosomes. The chromatin, it is worth noticing, is often placed beneath the nuclear membrane.

FIG. l62.—Dispermic eggs of Strongylocentrotus Iividus. A, The four centres have united to form a tetraster. B, The two pairs of centres have remained apart to form two independent spindles, one of which contains the female and one of the male nuclei, the other the remaining male nucleus. (After Boveri, 1905.)

II. The number of nuclei is inversely proportional to the number of chromosomes ,- but III. Cell-volume is inversely proportional to the number of

cells in larvae of the same stage developed from whole eggs _

of the same size; hence

IV. Cell-volume varies directly with the number of chromosomes and with the surface area of the nucleus. This Boveri terms the fixed relation between nucleus and cytoplasm.

V. It follows from I and IV that nuclear-volume increases more rapidly thon cell-volume.

VI. Conversely, the law holds good of egg-fragments of different sizes but containing the same number of chromosomes.

Since, in the larvae developed from such fragments, the IV. I INITIAL STRUCTURE OF THE GERM 269

thickness of the cell-layers is the same, the surface may be taken as an index of the volume and is proportional to the original volume of the fragments. Since the number of chromosomes is the same, the cell-volumes should be the same, and the number of cells ought to be proportional to the surface. This was, as a matter of fact, found to,be true in the few instances examined.

This result is merely a restatement of Driesch’s rule, that in larvae developed from isolated blastomeres the surface of the larvae and the number of cells are both proportional to the germinal value.

It appears, therefore, that the moment at which segmentation shall end may be determined by the moment at which this constant relation between nucleus and cytoplasm is reached.


J” .6.


FIG. 163.—— a, Gastrula reared from an eggof Slrougylocenfrotus lim'du.s-, in which the sperm nucleus had passed entirely into one of the first two blastomeres. Optical section of a stained preparation. (After Boveri, 1905.)

b, Ectoderm in the neighbourhood of the blastopore of J. dispcrmic gastrula of .S'trong_r/locentrotus lividus. (After Boveri, 1905.)

Driesch has, however, recently reported certain exceptions to his rule, amongst the mesenchyme cells in particular. These may be more or less numerous than they should be, may differ in size and in the size of their nuclei. It is possible, as suggested, that the division of the centrosome has been suppressed (monaster) with consequent reduplication of the chromosomes. Such an explanation may account for the variability in the size of the nuclei observed by Seeliger in his hybrids.

In the Alga /5:piro_qym Gerassimow has observed a close relation between the size of the nucleus and that of the cell; here, however, it is the nuclear volume which is directly proportional to the cell-surface. 270 INTERNAL FACTORS IV. 1


K. BONNEVIE. Ueber Chromatindiminution bei Nematoden, Jen. Zeitschr. xxxvi, 1902. J

K. BONNEVIE. Abnormitiiten in der Furchung von Ascaris lumbricoides, Jen. Zeitschr. xxxvii, 1903.

M. Bovmu. Ueber Mitosen bei einaeifiiger Chromosomenbildung, Jen. Zeitschr. xxxvii, 1903.

T. BOVERI. Ueber die Befruchtungs- und Entwicklungsfiihigkeit kernloser Seeigeleier und fiber die Moglichkeit ihrer Bastardirung, Arch. Ent. Mech. ii, 1896.

T. Bovmu. Zur Physiologie der Kern- und Zelltheilung, S’.-B. ph_1/ Gee. W1212bz¢2'g, 1896.

T. BOVERI. Die Entwicklung von Ascaris megalocephala, mit besonderer Riicksicht auf die Kernverhiiltnisse, Festschr. Kuppfer. J ena, 1899.

T. Bovmu. Ueber mehrpolige Mitosen als Mittel zur Analyse des Zellkerns, Verh. phys.-med. Ges. Wzlnzburg, xxxv, 1903. '

T. Bovnm. Ueber da.s Verhalten des Protopla.sma.s bei monocentrischen Mitosen, S’.-B. phys.-med. Ges. Wdrzburg, 1903.

T. Bovmu. Ueber den Einfluss der Samenzelle auf die La.rvencha.raktere der Echiniden, Arch. Ent. Mach. xvi, 1903.

T. BOVERI. Noch ein Wort iiber Seeigelbastarde, Arch. Ent. Mech. xvii, 1904.

T. BOVERI. Protoplasmadifferenzierung als ausléisender Faktor fiir Kernverschiedenheit, S.—B. phys.-med. Ges. Wfirzbu;-g, 1904.

T. BOVERI. Ergebniase fiber die Konstitution der chromatischen Substanz des Zellkerns, Jena, 1904.

T. Bovmu. Ueber die Abhfingigkeit der Kerngrosse und Zellenzahl der Seeigel-Larven von der Chromosomenzahl der Ausgangszellen, Jena, 1905.

L. DONCASTER. Experiments in hybridization with especial reference to the effect of conditions on dominance, Phil. Trans. Roy. Soc. cxcvi, B. 1904.

H. Dnmscn. Ueber rein-miitterliche Charaktere an Bastardlai-ven von Echiniden, Arch. Ent. Mech. vii, 1898.

H. Dnmscn. Ueber Seeigelbastarde, Arch. Eut. Mech. xvi, 1903.

H. DRIESCH. Zur Cytologie parthenogenetischer Larven von Strongylocentrotus, Amh. Ent. Mech. xix, 1905.

A. FISCHEL. Ueber Bastardirungsversuche bei Echinodermen, Arch. Ent. Mech. xxii, 1906.

J. J. GERASSIMOW. Ueber den Einfluss des Kerns auf das Wa.chsthum der Zelle, Bull. Soc. Imp. Nat. Moscou, 1901.

J. J. GERASSIMOW. Die Abhfingigkeit der Grosse der Zelle von der Menge ihrer Kernmasse, Zeitschr. allg. Phys. i, 1902.

A. GIARDINA. Origine dell'oocite e delle cellule nutrici nel Dytiscus, Internat. Monatachr. Anat. u. Phys. xviii, 1901. ' IV. I INTERACTIONS OF THE PARTS 271

E. GODLEWSKI. Untersuchungen fiber die Bastardierung der Echiniden- und Crinoidenfamilie, Arch. Ent. Mech. xx, 1906.

M. KRAHELSKA. Sur le développement mérogoniquc des ueufs de Psammechinus, Bull. Internai. Acarl. Sci. Cmcovie, 1905.

J. LOEB. Ueber die Befruchtung von Seeigeleiern durch Seestem samen, P_flflger's Arch. xcix, 1903. J. LOEB. Ueber die R.eaction‘des Seewassers und die Rollo der Hydro xylionen bei der Befruchtung der Seeigeleier, Pfliiger-’s Arch. xcix, 1903. J. LOEB. Weitere Versuche fiber heterogene Hybridisation bei Echino dermen, Ifflfiger-‘s Arch. civ, 1904. A. P. MATHEWS. The so-called cross-fertilization of Asterias by

Ar-bacia, Amer. Journ. Phys. vi, 1901-2. T. H. MORGAN. The fertilization of non-nucleated fragments of

Echinoderm eggs, Arch. Eut. Mech. ii, 1896.

W. ROUX. Ueber die Bedeutung der Kerntheilungsfiguren, Leipzig, 1883, Ges. Abh. 17. Leipzig, 1895. O. SEELIGER. Giebt es gesehlechtlich erzeugte Organismen ohne miitterliche Eigenschaften ? Arch. Em. Mech. i, 1895.

O. SEELIGER. Bemerkungen fiber Baatardlarven der Seeigel, Arch.

Ent. Mech. iii, 1896.

E. TEICHMANN. Ueber Furchung befruchteter Seeigeleier ohne Beteiligung des Spermakerns, Jen. Zeitschr. xxxvii, 1903.

H. M. VERNON. The relations between the hybrid and parent forms

of Echinoid larvae, Phil. Trans. Roy. Soc. cxc, B. 1898. H. M. VERNON. Cross-fertilization among Echinoids, Arch. Eut.

Mech. ix, 1900.


Up to the present we have discussed only the original structure of the fertilized ovum as an internal factor in difierentiation. We have now to consider to what extent the development of each part may be due to influences exerted upon it by other portions of the embryo.

One of the first suggestions of such an interaction was put forward by Oscar Hertwig. ‘We recognize’, he says in his Zez't- mul Streiyfrage, ‘apart from the nature of the egg-substance itself, the conditions of development, first in those perpetually changing mutual relations in which the cells of an organism are placed to one another, and secondly in the influences exerted upon the organism by the external world ’ ; and again, ‘physiologically expressed, the dissimilar diiferentiation of cells consists in the reaction of organic substance to dissimilar stimuli,’ and 272 INTERNAL FACTORS IV. 2

he proceeds to illustrate his meaning by referring to the influence of light in determining whether the sexual organs shall be formed on the upper or under side of a fern prothallus, to the dependence of the fate of indifierent buds of plants upon external conditions, to the formation of stems on the upper, roots on the lower side of a horizontal stolon of Amfennularia, as demonstrated by Loeb, and to certain alterations in the character of epithelia and other tissues which are brought about by pathological conditions.

For a more detailed elaboration of this very fruitful idea we are indebted to Herbst. Herbst classifies organic reactions to stimuli as either directive or formative. The former are of two kinds, tactic when the response is some locomotion of a freer body, tropic when the movement exhibited in response is a movement of growth. The reactions may be further classified according to the nature of the stimulus, gravity, light, heat, electricity, chemical substances, currents of water, the contact of a solid surface. Thus we have positive and negative heliotropism, galvanotaxis, geotropism, galvanotropism, thigmotaxis, and so on, and many examples of all of these are cited. These are mostly familiar facts, but some, for their special interest, may be quoted here. Thus the hydroid Sertularellw in one-sided illumination produces stolons instead of hydranths, the tubes of the Polychaet Sergmla grow upwards towards the light (or against gravity), an inverted stock of Sertularia produces a polyp sympodium at the upper—originally lower——end, the stems of Aulemuluria are negatively, the stolons positively geotropic, spermatozoa swim to solid bodies, and stolons of Hydroids attach themselves to some foreign object.

Speaking generally, a specific reaction depends not merely on the nature of the stimulus, but on the structure of the reacting body itself. Further, the degree of sensitiveness to the stimulus is not constant. It varies at different periods of life, under the influence of external conditions such as temperature, and with the strength of an already existing stimulus (Weber’s Law).

Now many of the events of ontogeny, as we have seen, resolve themselves into movements of the parts of the organismmovements of free parts, locomotion, and growth movementsIV.2 INTERACTIONS OF THE PARTS 273

and the suggestion is made by Herbst that these movements are to be interpreted as so many responses to stimuli, which are then not merely directive but formative, the stimuli being either external or exerted by other parts of the body. Thus the spreading of the blastoderm over the yolk may be a case of oxygenotaxis, the movement of cells of the Frog’s blastula towards one another described by Rouxl as Cytotropismus may be due to a mutual chemotactie stimulus, the migration of cells to the surface of the Arthropod ovum is perhaps oxygenotactic, the vitellophags are ehemotactic, the application of mesoderm cells to the outgrowing axis cylinders of nerve fibres, of connective tissue and muscle-cells to blood-vessels, of the choroidal cells to the optic cup (they are absent ventrally when the choroid fissure fails to close), the reunion of separated blastomeres in Triclads and others, may all be tactic phenomena of a positive kind, while the dispersal of the elements of a complex may be negative. Similarly, the outgrowth and anastomoses of nerves, glands, ducts, the concrescence of layers may be tropisms of various sorts.

The suggested stimuli in some cases proceed from the external environment, in others they are exerted by one part on another. The former have already been discussed, but it may be pointed out again that the effect called forth depends both on the stimulus and on the reacting organism or organ. Neither in these cases nor in any others is the production of specific form due to the action of an external agency upon a homogeneous material. For even in those instances where the fate of some apparently indifferent tissue seems to be decided by the action of such an agency-—as in the production of stems from whichever side of an Anlemmlaria stock happens to be uppermost (Loeb)—it is evident that all that the external agent can do is to decide between a limited number of definite alternatives in respect of each of which the parts of the organism are equipotential. Responses to stimuli of the second kind possibly play an important part in ontogeny. Unfortunately, the experimental evidence for this is at present somewhat meagre.

In the fish Funrlulus the yolk-sac is deeply pigmented, the pigment-cells being especially closely aggregated round the

‘ See above, Ch. II. 1, p. 55.

unumrson T 274 INTERNAL FACTORS ' IV. 2

blood-vessels. When the creature is deprived of oxygen the pigment disappears, and it is supposed that under normal conditions the pigment cells are chemotactically attracted by the oxygen in the blood (Loeb).

Two other cases come from Echinoid larvae. Driesch has been able to displace, by shaking, the two groups of primary mesenchyme cells, those destined to secrete the skeletal spicules of the

FIG. 164.-Diagrams of Driesch's experiment into the responsiveness to stimuli of mesenchyme cells in Echinus. A, Normal arrangement of mesenchyme cells. B, The cells deranged by shaking, returning in C to their original position. (From Korschelt and Hcider, after Driesch.)

larva. The cells, displaced towards the animal pole, return to their original position after six hours, and subsequent development is normal 164). Driesch has attributed this movement of the cells to a stimulus exerted by the endoderm.

Again Herbst has found that when the larvae of sea—urchins are placed in solutions of lithium salts or in sea-water deprived of the sulph-ion, or devoid of the carbon—ion, the skeleton of the IV. 2 INTERACTIONS OF THE PARTS 275

Pluteus is either distorted, in the first two cases, or absent, as in the last. The distortion takes the form of a multiplication of the triradiate spicules, and the arms are correspondingly multiplied. In water devoid of CO2 there are no spicules and no arms} Herbst has therefore urged that normally the outgrowth of the ciliated ring into the arms is due to a stimulus—thigmotropic, perhaps——exe1-ted by the tip of the spicule. The converse of this is seen later on when the arms diminish in length as the calcium carbonate of the Pluteus skeleton is made use of by the developing urchin.

The development of the lens of the vertebrate eye has also been asserted to be due to a contact_stimulus exerted by the optic cup upon the overlying ectoderm. Spemann was the first to show that when the medullary plate of the Frog (Rana fusca) was injured in front of the optic vesicle, the optic cup (if developed at all) may or may not come in contact with the ectoderm, the latter being, at this point, uninjured. In the first case a lens is formed, in the second not. Similar experiments have been carried out by Lewis on other species of Frog and on Amblystoma, with similar results. Lewis has also shown that a lens will be formed from any patch of ectoderm taken from some other part of the body and grafted over the optic cup. Schaper’s evidence in support of this conclusion is doubtful. This author removed the whole of the nervous system from a 4 mm. tadpole by a horizontal cut with the exception alone of the downwardly growing fore-brain and optic vesicles. The wound healed and development continued. The optic cup was solid, being filled with a mass of degenerating cells. A lens was formed opposite the upper edge of the optic cup. It did not, however, separate from the ectoderm, but difierentiated in silu, with development of lens fibres. Hence Schaper argues that the formation of the lens is a process of self-differentiation. The contact of the optic cup is, however, as he admits, in this case not excluded.

A more serious objection is raised by Miss King, who has found (in the Frog) that even when, owing to a prior injury, the optic cup is far away from the skin, or in some cases absent altogether,

‘ See above, Ch. III. 8, Figs. 73, 74. T 2 276 INTERNAL FACTORS IV. 2

the lens may still be formed. It may also be mentioned that Mencl has described a double-headed monster of Salmo salar in which one head has two well-formed lenses, though the other parts of the eye are absent. The lenses, however, lie quite close to the fore-brain, which may have exerted the stimulus, supposing a stimulus to be necessary.

It will be seen that the evidence is extremely contradictory, and forbids any definite conclusion.‘

It is curious that in tadpoles grown in certain solutions (sodium chloride, bromide, and nitrate) the optic cup is often at some distance from the ectoderm, and that in these cases the lens is absent, though it may happen that the upper edge of the optic cup is sharply bent down in front of the aperture, as though to form a lens from the edge of the iris, as occurs in the regeneration of the lens, after extirpation, in the Newt.”

Spemann and Lewis have also found that in the absence of contact between the optic cup and the ectoderm the cornea is not developed, that is to say, the overlying ectoderm does not ‘clear’ (lose its pigment), it does not thin out, and Descemet’s membrane is not formed. The more extensive series of experiments is due to Lewis, who turned back a flap of skin and wholly or partially removed the optic vesicle, the skin being then returned to its place. In the first case there was no eye, no lens, no cornea, though on the other side of the body all were present. In the second case the lens and cornea were both formed, provided that enough was left of the vesicle for the optic cup to come into contact with the ectoderm. As a control experiment the flap of skin was merely removed and replaced; the development of the eye was normal. If at a later stage, when corneal changes had already begun, both optic cup and lens were taken out, the cornea was not completed. The pigment indeed disappeared, but the ectoderm did not diminish in thickness and Descemet’s membrane was not formed.

Removal of the lens alone did not interfere with the production

1 Stockard has recently shown that when the egg of Fzmdulus is placed in MgCl, the two optic cu s wholly or partly fuse while a single or double median lens is deve oped (Arch. Ent. Mech. xxiii, 1907). This would seem to support the view advanced by Lewis.

2 See above, Ch. III. 8, p. 134.


of the cornea provided that the optic cup could touch the ectoderm, and if this same condition is fulfilled, the cornea will be differentiated in the ectoderm that grows in after the layer that originally lay over the eye had been destroyed, but if the cup and lens are taken away no new cornea is formed even after four weeks. The size of the cornea is in proportion to the area of contact. Finally, if, at a later stage still, the optic cup and lens are taken away, the already formed cornea degenerates. Should the results of these extremely interesting investigations be confirmed, the development of some parts at least of the vertebrate eye would be processes of ‘ dependent diiferentiation ’ 1, dependent, that is, on causes outside themselves, on stimuli exerted by other parts, though not, of course, wholly dependent, since the structure of the reacting, aswell as that of the stimulating, organ is contributory to the quality of the eifect. How far such an action of the parts upon one another is of general occurrence as a factor in differentiation future research alone will show. It seems probable, however, that it will in any case be found to be limited to the first moments in the formation of the organs 2, for we know from other evidence that as development proceeds the system which was ‘ equipotential ’, to use Driesch’s phrase, becomes ‘ inequipotential ’, that the parts lose their totipotence, that they gain independence and the power of self-differentiation, that the value of the correlations between them diminishes. This is what Minot has called the law of genetic restriction. But in early stages there are correlations between the‘ organs, the differentiation of one does depend upon that of another, and it is this mutual stimulation of the parts that figures so prominently in the theory of development worked out by Driesch. This theory we shall have to examine in the following chapter.

' 1 Although the expression ‘dependent differentiation‘ is due to Roux, and although Roux expressed the opinion that certain developmental processes—the beginning of parthenogenetic development, the determination of the position of the grey crescent and first furrow by the entrance-point of the spermatozoon, the ‘post-generation’ of the dead blastomere by the living one in the Frog's egg, for instance—-were of the nature of responses to stimuli, still the role assigned by the ‘Mosa.ikTheorie’ to the influences of the parts upon one another in differentiation was subordinate, and restricted to the later stages of development.

2 Except, presumably, in cases of ‘composition’ where elements of different origin unite in the formation of a. single organ. 278 INTERNAL FACTORS IV. 2


H. Dnmscn. Die taktische Reizbarkeit der Mesenchymzellen von Echinus microtuberculatus, Arch. Ent. Mech. iii, 1896.

C. HERBST. Ueber die Bedeutung der Reizphysiologie fiir die kausale Auffassung von Vorgfingen in der tierischen Ontogenese, Biol. Centralbl. xiv, xv, 1894, 1895.

C. HERBST. Experimentelle Untersuchungen iiber den Einfluss der

veriindert-en chemischen Zusammensetzung des umgebenden Mediums

ztuf die Entwickelung der Thiere: II. Mitt. Zool. Stat. Neapel, xi, 1895.

C. HERBST. Ueber die zur Entwickelung der Seeigellarven nothwendigen anorganischen Stofi‘e, ihre Rolle und ihre Vertretbarkeit. I. Die zur Entwiclrelung nothwendigen anorganischen Stoffe, Arch. Ent. Mach. v, 1897. 11. Die Rolle der nothwendigen anorganischen Stofi'e, ibid. xvii, 1904.

0. HERTWIG. Zeit- und Streitfragen der Biologie, Jena, 1894.

H. D. KING. Experimental studies on the eye of the Frog embryo, Arch. Ent. Meek. xix, 1905.

W. H. LEWIS. Experiinental studies on the development of the eye in Amphibia. I. On the origin of the lens, Amer. Journ. Anal. iii, 1904.

W. H. LEWIS. Experimental studies on the development of the eye in Amphibia. II. On the cornea, Journ. Exp. Zool. ii, 1905.

J. LOEB. Untersuchungen zur physiologischen Morphologie der Thiere: II. Wfirzburg, 1892.

J. LOEB. A contribution to the physiology of coloration in, Journ. Mmph. viii, 1893.

E. MENCL. Ein Fall von beiderseitigen Augenlinsenbildung wiihrend der Abwesenheit von Augenblasen, Arch. Ent. Mech. xvi, 1903.

W. Roux. Ueber den Antheil von ‘Ausltssungcn ’ an der individuellen Entwicklung, Arch. Ent. Mech. iv, 1897.

A. SCHAPER. Ueber einige Fiille atypischer Linsenentwicklung unter abnormen Bedingungen, Auat. Am. xxiv, 1904.

H. SPEMANN. Ueber Linsenbildung bei defekter Augcnblase, Anat.

Anz. xxiii, 1903.

Jenkinson (1909): 1 Introductory | 2 Cell-Division and Growth | 3 External Factors | 4 Internal Factors | 5 Driesch’s Theories - General Conclusions | 6 Appendices

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