Paper - The Organization and Cell-Lineage of the Ascidian Egg 3

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Conklin EG. The Organization and Cell-Lineage of the Ascidian Egg (1905) J. Acad., Nat. Sci. Phila. 13, 1.

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III. Orientation of Egg and Embryo

As a preparation to the study of the cell-lineage and later development of the ascidian vixix it is necessary to consider at once the orientation of the egg and early cleavage stages. This is the more necessary since the utmost possible diversity of opinion has been expressed with regard to this matter.

1. Van Beneden and Julins System of Orientation

Van Beneden and Julin (1884) were the first to undertake to relate the early stages of development of the ascidian egg to the later stages. Their work was in fact one of the earliest and most admirable contributions to the subject of cell-lineage. They followed the cleavage, cell by cell, as far as the 44-cell stage and pointed out what they supposed to be the relations of each of these cells to the germ Layers. They determined the relations of the axes of the egg and early cleavage stages to those of the gastrula and larva and, for the first time in the history of embryology, established the fact that the principal axes of the larva may be identified in the nnseginented egg. The evidence's upon which they based their conclusions as to the axial relations of egg and embryo and as to the fate of the cleavage cells are not fully stated in their brief paper of only fifteen pages; but their statements of fact are perfectly clear and explicit. In brief these are as follows :

(1) The (list cleavage spindle is eccentric toward the posterior pule of the egg, and the median plane of the future embryo is marked out by the bilateral symmetry of the unsegmented egg (p. ti).

(2) The plane of the lirst cleavage coincides with the plane of bilateral symmetry, and therefore divides the egg into right and left halves (p. G).

(3) The second cleavage plane is transverse to the long axis of the embryo and separates two large anterior cells from two small posterior ones (p. 7).

(4) The intersection of these two planes marks the vertical axis of the egg; one end of this axis corresponds to the middle of the dorsal, the other to the middle of the ventral face of the gastrula (p. 7).

(5) The third cleavage separates 4 larger dorsal cells from 4 smaller ventral ones (p. i); the latter are ectodermal, the former mixed."

(ti) At the fourth cleavage these 8 cells give rise to l(i; X ventral cells, all ectodermal, and S dorsal cells, 6 of which are mixed, and 2, which are smaller than any of the others and lie at the posterior pole, ectodermal (p. 8).

(7) By division these 1 li cells give rise to 32; l(i ventral cells, all ectodermal and Id dorsal cells. 4 ectodermal derived from the 2 posterior ectoderm cells of the previous stage. 6 ectodermal derived from the 6 mixed cells. 4 endodermal and 2 still mixed. With regard to the identification of the dorsal and ventral faces at this stage they say: "'On bien les cellules ectodermique torment ensemble une calotte appliquee par sa concavite" contra les globes endodermiques et mixtes (comme dans fig. 10, c), ou bien e'est le contraire qui a lieu, les globes endodermiques et mixtes s'etalent en surface de facon a constituer ensemble une calotte moulee sur l'ectoderme (fig, '*. c)."

(8) At the next stage there are 44 cells; 32 ectodermal, easily recognized by their transparency, and 12 other cells very much larger. The ectodermal cap is notably extended and tends to envelope the endoderm.

From this stage onward there is no question as to the identification of the dorsal and ventral faces or the anterior or posterior ends. As will presently appear, my work, like that of Chabry (1887), entirely confirms the orientation adopted by Van Beneden and Julin, though I cannot agree with them as to the fate of certain individual cells.

2. Seeliger's System

Seeliger's (1885) later work was much less detailed and satisfactory with regard to the orientation of the early cleavage stages, as Castle has shown. His principal conclusions as to orientation are :

(1) The first cleavage plane coincides with the median plane of the embryo, hut neither anterior nor posterior, dorsal nor ventral can he recognized at this stage (p. 48).

(2) The second cleavage divides the egg into two smaller anterior cells and two larger posterior ones (p. 48).

(3) The third cleavage separates4 dorsal endodermal cells from 4 ventral ectodermal ones; the two posterior ventral cells are larger than any of the others. Structurally all these cells are alike (p. 49).

(4) In the 16-cell stage the 8 dorsal endodermal cells are yellow and have small nuclei ; the 8 ventral ectodermal ones are clear (p. 50).

In the identification of individual cells and their axial relations Seeliger was much at fault. The small cells of the 4-cell and later stages are certainly not anterior in position but posterior, as has been shown by Van Beneden and Julin, Chahry. Samassa, and Castle; while the two larger cells of the 8-cell stage are not ventral but dorsal in position, not posterior hut anterior, as their relations to the two small posterior cells show. Seeliger therefore mistook anterior for posterior, dorsal tor ventral and consequently right for left; in short, he committed all the mistakes possible in orientation.

3. Samassd's System

Ten years after the publication of Van Beneden and Julin' s work, Samassa (1894) working on Ciona and Clavellina reached very different conclusions from those set forth by the first named authors. With the first four conclusions of Van Beneden and Julin mentioned above he agrees, save that in the unsegmented egg he claims that only the median plane and the anterior and posterior, but not the dorsal and ventral, poles can he recognized. With regard to the identification of the dorsal and ventral sides he held that Van Beneden and Julin were completely in error and that they had mistaken the dorsal for the ventral, the endodermal for the ectodermal pole in all stages up to the 44-cell stage. As the most important evidence of this false orientation Samassa cites Van Beneden and Julin' s figures '.' c and 10 c, which represent optical sections in the sagittal plane of a 32-cell and a 44-cell stage respectively. In the first of these the ectoderm cells are shown as columnar, the endoderm cells as flattened; whereas in the second, figure 10c, the ectoderm cells are flattened and the endoderm columnar. '"The figures of these two authors." says Samassa, "are sufficient to show that figure 10c is properly and figure c falsely oriented ; in both cases the cylindrical cells belong to the endoderm and are dorsal in position." The words of Samassa directed against Van Beneden and .Julin apply with equal or even greater force to himself : "Van Beneden and Julin have not once sought," he says, "to bring forward one fact in support of this remarkable transformation." With the exception of the worthless a priori argument that cells which have once been cylindrical must always remain so Samassa has not produced a single argument or fact in favor of his contention.

4. Castle's System

In the same year Castle (1894), in a preliminary paper and again in his final paper (1896) on the early embryology of Ciona intestinalis, reversed the orientation maintained by Van Beneden and Julin and held with Samassa that in all stages preceding the 44-cell stage the Belgian investigators had mistaken dorsal for ventral and vice versa. Furthermore, after having studied the formation of the polar bodies, he was lead to the truly remarkable conclusion that these bodies in ascidians are formed at the endodermal pole, whereas in all other animals, so far as known, they are formed at the ectodermal pole of the egg. His conclusions were stated in the most positive manner and have been widely accepted, notwithstanding that such an orientation is absolutel} unique, and for this very reason should have been received with caution. Inasmuch as Castle's work is the most thorough and extensive treatment of the early development of ascidians since the appearance of Van Beneden and Julin's paper, and since his conclusions are diametrically opposed to my own. it seems desirable to give with some fulness his conclusions as to orientation as well as the evidences upon which these conclusions are based. In speaking of Van Beneden anil Julin's work he says ( 1894, p. 200): . . . 'It is my purpose to show that by yielding themselves to conjecture in so small a matter as these three cell divisions, the eminent authors fell into an error which invalidates the most important conclusions of their otherwise excellent work. For in correlating the 44-cell stage with the 32-cell stage they have changed the orientation so that they have identified the dorsal side of one with the ventral side of the other, the endodermal half of one with the ectodermal half of the other. Their orientation of all the stages prior to the jj-ce/l stage is accordingly wrong. Their terms ectodermal and endodermaL ventral and dorsal, as employed up to this stage, must be interchanged." Again with regard to the point at which the polar bodies form he says (1894, p. 211) : "1 have repeatedly seen the polar bodies and observed continuously the cleavage stages following their formation. These observations lead to the surprising but unavoidable conclusion that the point on the surface of the egg at which the polar bodies form becomes later the center of the dorsal or endodermal half of the eps." Again in his later work (1896, p. 22b) he says with regard to this matter: . . '-The form changes accompanying maturation occur, in Ciona at least, and presumably in ascidians in general, at the pole of the egg opposite to that at which they occur in Amphioxus, and, so far as known, in all other animals producing eggs with polar differentiation; for the changes connected with maturation are uniformly reported to take place at the animal, i.e., at the more richly protoplasmic pole, whereas in Ciona they take place at the vegetative pole. . . . The statement made in the preceding paragraph presents a condition of affairs so directly contrary to that found in other groups of animals, as well as to what has been assumed by all previous writers to he the case in ascidians, that it requires the presentation of unmistakable evidence in its support. Such evidence I have to offer, both from the study of the living egg and from that of preparations."

Fig. VII. Four-cell stuni' of Ciona iniestinalis viewed from the animal pole ; the creuated line represents the boundary between the protoplasm and yolk; the dotted line marks the anterior limit of the crescent at the vegetal pole ; the four cells are approximately equal in size.

Fig. VIII. Four-cell stage of Cynthia partita seen from the animal pole ; the limits of protoplasm and crescent are represented as in the preceding figure; the two posterior cells are a little smaller than the anterior ones.

What is this evidence? So far as it relates to the origin of the polar bodies at the vegetal pole it is twofold; (a) the polar bodies are formed at the yolk-rich pole, (b) this pole becomes the endodermal pole of the gastrula. As to the first of these propositions I have already shown that the germinal vesicle fades and the first maturation spindle appeal's at the protoplasmic pole (tigs. 77, 78, 172). Only later, after the entrance of the spermatozoon, does the protoplasm now away from this pole. leaving the maturation spindle closely surrounded by yolk; still later, during the first cleavage, the protoplasm Hows back again to near the center of the egg and at the (dose of this cleavage it moves still nearer to the pole at which the polar bodies lie (figs. 100, 102. 10(1. 107. 178); thereafter this pole is always the more richly protoplasmic. Therefore, except for a brief period after the fertilization and before the first cleavage, when the protoplasm is temporarily withdrawn from the maturation pole through the influence of the spermatozoon, the maturation or animal pole and the more richly protoplasmic pole are one and the same in ascidians as in other animals.

As to the statement that the polar bodies are formed at a point which corresponds to the center of the dorsal or endodermal pole of the gastrula it is evident that unless the polar bodies have been actually followed through the development to a stage when the ectodermal and endodermal poles are unmistakable, this statement must rest upon indirect evidence furnished by a study of the cleavage stages. As a matter of fact, Castle has not figured nor described the polar bodies in any egg later than the 1G to 24-cell stage, whereas there is no trace of gastrulation in Ciona before the 76-cell stage (fig. 200). Undoubtedly therefore Castle's evidence that the polar bodies are formed at the endodermal pole must be indirect rather than direct, and must he derived from the study and orientation of the cleavage stages. We may therefore turn at once to the evidences which led him to reverse Van Beneden and Julin's orientation of these stages. So far as 1 am able to discover there are, in addition to several minor considerations which could at best be considered only as confirmatory, two and only two general lines of evidence which he brings forward in favor of his contention. They are the following:

(1) The hemisphere in which division is earliest as the egg passes from the L6cell stage to the 32-cell stage, and from the latter to the 46-cell stage becomes later the ventral or ectodermal hemisphere of the embryo ( 1S94, p. 206 ; 1896, pp. 229 and 235). The second paper refers to this proposition as having been demonstrated in the first. What is this demonstration'.' So far as 1 can ascertain it consists merely in the assumption that the cells, which in the lG-cell and 32-cell stages divide earlier than the others, must continue to divide more rapidly and thus give rise to the more numerous ectoderm cells of the gastrular stage. So far from there being any demonstration of this proposition there is actually no evidence offered in support of it. Furthermore, I can affirm from my own studies that it is not true. The cells which lag behind in division up to the 64-cell stage, thereafter divide much more rapidly than the others and give rise to the ectoderm of the gastrula [cf. figs. 130134 and 196-204).

(2) Castle's second reason for rejecting the orientation of Van Beneden and Julin is the same as Samassa's, viz., the peculiar shape of the cells at the two poles. In the 32-cell stage and even earlier the cells at the maturation pole are long and columnar while those at the opposite pole are thin and superficially lame. "They [the columnar cells] retain this columnar form up to and throughout gastrulation " (1896, p. 237). They thus give rise directly to the columnar endoderm cells which are ultimately invaginated. On the other hand. Van Beneden and Julin maintained that the flattened cells of the 32-cell stage became the columnar cells of the 1 44-cell stage and that the columnar cells of the earlier stage became the flattened ones of the latter stage. Castle says that their figures show at a glance the absurdity of such an interpretation (1894, p. 208; 1896, p. 237). Since the whole orientation which he adopts as opposed to that of Van Beneden and Julin rests upon the establishment of this one point, it passes belief that hi', as well as Samassa, should not have taken the most evident and direct step to prove it. Van Beneden and Julin figure optical sections in the sagittal plane of an egg in the 32-cell stage showing the columnar cells at the ventral pole, and of one in the 44-cell stage showing them at the dorsal pole. Castle figures actual sections of a 32-cell stage and of a 76-cell stage, but none between these two. A study of actual or of optical sections of eggs transitional between the 32-cell and the 76-cell stages would have shown conclusively that the columnar cells of the former are graduall} transformed into the flattened (ells of the hitter, and the flattened cells of the one into the columnar cells of the other, and would thus have completely established Van Beneden and Julius orientation. Such a series of optical sections of the Ciona egg, viewed from the left side and also from the posterior pole, is shown in text figures IX to XVI, and the various stages in this change of shape can there he clearly followed. A similar series of actual sections of the egg of Cynthia is shown in text figures XVII to XXIV. I do not find that this transformation is quite as rapid in Cynthia and Ciona as is indicated by Van Beneden and Julin's figures 9 c and 10 c for Clavellina. At the 44-cell stage the cells at both poles are columnar and of nearly equal height (text figs. XIII, XIV), and not until the 64-cell or even the 76-cell stage is this transformation complete. It must not he supposed, however, that this change in shape of the cells at the two poles is a continually progressive one. since all the cells hecome more superficial during division and more columnar during rest. Consequently every cell changes shape more or less during each cycle of division; this is well shown in figures XVIII and XX.

Other details which Castle regards as confirmatory of his view will lie taken up Inter, hut enough lias now been said, in my opinion, to show the untrust worthiness of his principal evidence against Van Beneden and Julin's system of orientation and in favor of his own.

5. Evidences in favor of Van Beneden and Julias System

While it is evident from these many and serious differences of opinion that it is easy to make mistakes in the orientation of the ascidian egg, it is not true that the egg is an unusually difficult one to orient. In fact there are few eggs, except those in which the cleavage is markedly unequal, in which this can be so easily done. All the embryonic axes are (dearly distinguishable in the unsegmented egg, and at every stage in development there are numerous landmarks by which the different poles of the egg may be recognized. With the exception of Seeliger, all students of the early development of ascidians have recognized that from the 16cell stage onward, the posterior pole is marked by two cells much smaller than any others in the entire egg. The chief difficultv has been, as evidenced by the work of Seeliger, Samassa, and Castle, in distinguishing the dorsal and ventral faces in the pregastrular stages. This is due to the fact that the cells at these two poles are of somewhat similar shape, size and arrangement, as may he seen by referring to figures 117. 118, 120, L24, 13(1 and 131 of this paper. However, the differences between these poles are so marked that there never need he any confusion regarding them.

Figs. IX-XII. Camera drawings of entire stained eggs of Ciona iiitesthuiUs viewed as transparent objects. Figs. IX and XI are seen from the posterior pole; Figs. X and XII from the left side. Figs. IX and X represent a 16-cell stage passing into a 24-cell stage ; Figs. XI and XII a 32 -cell passing into a 44-cell stage. The head and tail of the arrow mark the position of the equator (third cleavage plane) at the anterior and posterior poles. The cells of the crescent (mesoderm) are B 6 -3, B 6 -> and B 6 = ; all the other cells of the lower hemisphere are yolk laden and the boundary between protoplasm and yolk is indicated by a crenated line; the stippled areas adjoining the median plane in the cells Bs- 2 and B 6 -3 represent caps of deeply staining protoplasm (clear in life). The segmentation cavity is shaded by vertical lines and the cells bordering it are seen in median optical section ; the cells at the upper pole are columnar, those at the lower pole flattened. The polar bodies, although shaded diagramatically, are present exactly where they appear in the drawings.

(1) The most striking difference between the two poles is found in the fact that at all stages of the cleavage and gastrulation one pole is rich in protoplasm, the other rich in yolk. This is particularly noticeable in Cynthia and Molgula, but is also true of Ciona, though in this genus the differences between the two poles are not quite so marked as in the other genera named. This difference is so great that in properly stained eggs one can always tell at a glance which is the yolk pole and which the protoplasmic. In eggs stained in picro-haematoxylin the protoplasm is red or light purple, the yolk yellow and the two poles are so unlike that there can be no excuse for mistaking them. In Cyntliia, indeed, these differences can be easily recognized in the living egg, the yolk being slate-gray and the protoplasm colorless or yellow (figs. 28, 32, 37, 38). The yolk spherules are not scattered through the cytoplasm, but the limits of the yolk and cytoplasm are sharp and distinct. In the cells at the yolk pole the cytoplasm is limited to a small area around the nucleus; at the protoplasmic pole the cytoplasm occupies a large part of the cell, the yolk being limited to the inner ends of the cells. This is seen especially well in actual sections taken in the vj:'s axis XVII to XXIV, and it can there be seen that the cytoplasm is largely found in those cells which lie on that side of the egg where the polar bodies are found, while the cells at the opposite pole are almost entirely filled with yolk. These yolk-laden cells are ultimately tnvaginated and form endoderm (text figs. XXI to XXIV) and arc therefore dorsal in position, while the protoplasmic cells at the opposite pole form ectoderm and are ventral in position. The cells which form the posterior boundary of the yolk-rich hemisphere contain the small spherules, already described, which are characteristic of the yellow protoplasm of Cynthia. The distribution of the yolk shows conclusively, therefore, that the cells of the animal or ventral hemisphere contain most of the clear protoplasm and give rise to the ectoderm, while the cells of the vegetal or dorsal hemisphere contain most of the yolk and yellow protoplasm and give rise to the endoderm and mesoderm.

Figs. XIII-XVI. Camera drawings of eutire eggs of Ciona intestinalu viewed as transparent objects ;

Figs. XIII and XV are seen from the posterior pole ; Figs. XIV and XVI from the left side. Figs. XIII and XIV represent a 44-cell stage passing into a 62-cell stage; Figs. XV and XVI a 76-cell passing into a 110-cell stage. The position of the equator, the boundary between protoplasm and yolk and the segmentation cavity are represented as in the preceding figures. In Fig. XIV the cells A7-4 and A? 8 are neural plate cells, A?-3 and A" " chorda cells; in Fig. XVI the neural plate cells are A 8 ?, A 8 - 8 , A 8 ."5, A 8 -' 6 , while the chorda cells are A 8 -s, A 8 - 6 , A 8 -'3, A 8 - 1 '!. The crescent cells (mesoderm) are B 6 -3, BM, B?-t, B?-3 (Figs. XIII and XIVl and their dirivatives B7-5, &<>, B7 7, B?- 8 , B 8 -5, B 8 - 6 , BK B 8 - 8 (Fig. XV and XVI). The other cells of the lower hemisphere are endodermal. In Figs. XIII and XIV the cells bordering the segmentation cavity (seen in optical section) are of about equal height at the two poles; in Figs. XV and XVI the cells at the loVer pole are columnar, those at the upper pole flattened. The polar bodies are present exactly where they appear in the drawings.

Figs. XVII-XX. Actual sections of eggs of Cynthia partita; Figs. XVII anil XIX in the median plane, Figs. XVIII and XX in a transverse plane. Figs. XVII and XVIII represent a 20-24 cell stage; Figs. XIX and XX a 32-44 cell stage. Unshaded portions of cells represent clear protoplasm ; closely crowded spheres, the yolk ; minute spherules, the yellow protoplasm of the crescent. The clear protoplasm is located chiefly in the cells of the animal half of the egg (ectoderm) ; it is also found in the crescent cells (mesoderm) 1 15; =, B 6! , B 6 '3) and neural plate cells (A7-4, upper half of A 6 -=0 of the lower hemisphere. The remaining cells of the lower hemisphere, (endoderm B5- 1 , B 6 - 1 , A 6,1 , A fc -3, and chorda, A<-3 and lower half of A 6-2 ) are filled with yolk ; yolk is also found in the central ends of all the other cells. The yellow protoplasm is limited to the crescent cells and to a single pair of cells of the upper hemisphere (bs-3). In Fig. XVII the cells at the two poles are approximately equal in height ; in Fig. XVIII the cells at the animal pole are flatter, probably owing to the fact that they are dividing ; in Figs. XIX and XX the cells at the animal pole are columnar, those at the vegetal pole flattened. The polar bodies are actually present where they are represented.

such sections are shown in text figures

Figs. XX1-XXIV. Sections of eggs of Cynthia partita; Figs. XXI and XXIV in the median plane. Fig. XXIII a little to one side of the median plane at the posterior end, Fig. XXII in a transverse plane. Fig. XXI represents a 64-cell stage, Fig. XXII a 64-76 cell stage. Fig.XXIII a 76-110-cell stage, and Fig. XXIV a 110-cell stage. The clear protoplasm, the yellow protoplasm and the yolk are represented as in the preceding figures. The clear protoplasm is localized chiefly in the ectoderm and neural plate cells, the yellow protoplasm in the crescent cells (mesoderm) and the yolk in the endoderm and chorda cells; yolk is also present in the inner ends of the ectoderm and mesoderm cells. The polar hodies shown in Fig. XXIII in dotted outline do not lie in the plaueof the section drawn, but in that of the next section of ilie series. It is probable that the neural plate and chorda cells of this figure (k~-* and A>3) have already divided in a transverse plane {v. fig. 131), and that these cells should therefore be labelled A 8 *? and \ a> in Fig. XXIV. In Fig. XXII the mesoderm cell B 6 - 2 (Fig. XVIII.i has divided into a mesenchyme cell (B"-3) and a muscle cell IB'"*), the former containing little and the latter much of the yellow protoplasm.

(2) This orientation is further confirmed by a study of the yellow crescent of the Cynthia egg and of the cells which develop from it. As has been shown, the yelkvw protoplasm of this egg collects at the Lower (vegetal) pole and then moves up to a position just below the equator on the posterior side where it forms a yellow crescent. At the first cleavage this crescent is divided in the middle into right and left halves: at the second cleavage it passes into the two posterior cells of the 4-cell stage; at. the third cleavage it goes into the two posterior vegetal cells of the 8-cell stage. In two subsequent divisions the yellow protoplasm is separated from the yolk with which it is associated and thereafter forms a crescent of yellow cells which surrounds the posterior side of the egg just below the equator (figs. 37, 39, 41, 42). At all stages of development this crescent or. the cells which arise from it, lies in the posterior half of the vegetal hemisphere, and the yellow cells are never separated from the mid-dorsal line by more than a single row of yolk cells (figs. 44-48). On the other hand these yellow cells are separated from the mid-ventral line by an ever increasing number of clear protoplasmic cells (figs. 43, 45, 122, 129, 137, et seq.). The single row of yolk cells mentioned above as lying between the yellow cells and the dorsal mid-line invaginates during: gastrulation and aives rise to the ventral cord of endoderm in the tail of the larva, while the yellow cells, which are also invaginated, trive rise to the mesoderm. A study of this yellow crescent and of the cells which develop from it shows conclusively that it always lies on the posterior border of the yolk-rich or dorsal hemisphere, that at the 16-cell and 32-cell stages, it is separated from cells which give rise to the ventral endoderm, and that it is invaginated with the endoderm and forms the muscle cells and mesenchyme of the tadpole.

(3) Wholly similar results as to the orientation of the egg and embryo follow from a study of the lineage of all the other cells of the embryo. I believe that I have seen every division of every cell up to the 21 8-cell stage, and in the critical period between the 32-cell and 7(>-cell stages I have seen these divisions in hundreds of cases. The evidence from this source as to the orientation cannot here be presented in detail but must be deferred to that portion of this paper which deals particularly with the cell-lineage ; however, it can be said that in not a single instance have I found any evidence against the orientation according to Van Beneden and Julin, while every observation which I have made on the cell-lineage speaks in favor of that orientation.

(4) Finally a most direct and convincing evidence in favor of this system of orientation is found in the position of the polar bodies throughout development. In preparations of the eggs of the three genera of ascidians which I have studied, the polar bodies are easily distinguishable from the test cells by their deeper stain ; in Ciona they are also larger than the test cells. In the last named genus I have seen the polar bodies attached to the egg or imbedded in it at every stage from the unsegmented egg to the gastrula (Plates XI and XII). In every single instance they have been found at a point on the ectodermal (ventral) hemisphere which a study of the cell-lineage shows to correspond to the animal pole of the unsegmented egg. I have not observed the polar bodies in every egg of Cynthia which I have studied or drawn, possibly because they do not in this genus remain attached to the egg so persistently as in Ciona. but wherever I have been able to identify them they have been found at the same polo of the egg as in Ciona (figs. 87. 92, 96 3 102, 106, HIT. ins. 1 It). I ir,. I hi. L30, L33, L39, I 13). In stages later

than figures L39 and I !"> the protoplasm of the polar cells becomes vesicular and stains so faintly that they can no longer be identified with certainty.

Castle maintained from indirect evidence, as 1 have already shown, that the polar bodies of Ciona are formed at the middle of the endodermal or dorsal half of the egg. I have never in a simile instance observed anything which might he mistaken for a polar body at this pole, whereas I have found the most positive and oft repeated evidence that the polar bodies lie at the ectodermal or ventral pole from the time of their formation to the gastrular stage. These ascidians therefore form no exception to the genera] rule that the polar bodies are formed at the middle of the ectodermal hemisphere of the egg.

It is not necessary in this place to point out in more detail than has been given already the sources of error in the work of Seeliger and of Samassa, especially as their work does not undertake to follow the exact cell-lineage of every cell up to the gastrular stage or later. With Castle's work, however, the case is quite different, for while the considerations already mentioned probably explain the sources of his error of orientation, they do not explain the way in which he has incorporated this error in the cell-lineage which he has followed to an advanced stage. In brief, I find almost all of Castle's figures correctly drawn, and I can without difficulty correlate his drawings cell for cell with my own. The most important exceptions to this statement are found in his figures 53 and 54, hut even here the differences are not great. His gastrular stages are of course correctly oriented since the dorsal and ventral faces of the embryo are unmistakably marked out as soon as the invagination begins. All of his pregastrular stages, however, with the exception of a 48-cell and a 64-cell stage, shown in his figures 57 to GO. are erroneously oriented, dorsal being mistaken for ventral and ventral for dorsal. With the lineage which he gives of every cell up to the 4G-cell stage (his fig. 56), I entirely agree, but in passing to the 48-cell stage (his figs. 57 and 58) he inverts the egg and shifts the equator one cell-row r nearer the vegetal pole than it should be, consequently all of the lineage of the later stages is wrong. While therefore the stages from 48 cells on are correctly orientated, the lineage of the individual cells is incorrect; before the 48-cell stage the lineage is correct but the orientation wrong. The evidence for this grows in part out of the general considerations already mentioned, but it is also founded upon a detailed study of the cell-lineage, to which we now turn.