Paper - The Organization and Cell-Lineage of the Ascidian Egg 6

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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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VI. Comparisons with Amphioxus and Amphibia

The remarkable differentiations apparent in the egg and early cleavage stages of ascidians. the relatively small number of cells present during gastrulation and organogeny, and the comparative ease and certainty with which the axial relations of the egg and embryo can he determined at all stages. these conditions render the ascidian egg the most favorable in the whole phylum of the chordata for an exact study of the early development. In no other chordate has the cell-lineage been followed in detail up to the formation of definitive organ cases, and no where in the phylum has it been possible to determine with the same degree of certainty as here the relations of the axes of the egg to those of the gastrula and larva.

It is therefore worth while to compare the early development of ascidians with that of other primitive chordates in order to see what light may thereby he thrown on certain disputed problems. It must, of course, be understood from the beginning that such comparison can have the weight only of suggestion ; the problems winch have been raised in the study of any group can be solved only by the further study of that group, but comparisons with other forms may be of great service. If evolution be true, if ascidians are genetically related to other chordates. then it must be true that their modes of development are related. Whether the mode of development of ascidians as compared with Amphioxus and amphibians is palingenetic or coenogenetic is largely a matter of opinion, and need not concern us here so only it be granted that there is a relationship between these classes in the matter of their development as well as in their later structure.

Klaatsch (1896) has attempted to elucidate certain disputed points in the develojnnent of Amphioxus by a comparison with the ascidians, proceeding upon the principle that it is well to reason from the relatively known to the relatively unknown, from conclusions in which all agree to questions upon which there is diversity of opinion. Samassa (1898), on the other hand, holds that the ascidian ontogeny has been so greatly shortened and modified as compared with that of Amphioxus that it would be much better to explain the former by the latter than the reverse. All this might be true without destroying the value of comparison, but when Samassa further proceeds, as he does in the following sentence, to deny that there is any relationship between the two forms except in a single stage, he takes away all basis of comparison except for that single stage. He says, p. 20, " Nun ahnelt aber die Ascidienentwicklung der des Amphioxus nur in dem einen Stadium, wenn der Urmund geschlossen ist, der Chorda nach hinten auswachst und die Organe der Larva die fiir Wirbelthiere characteristische gegenseitige Lagerung ziegen . . . Bis zu diesem Stadium ist aber die Entwicklung des Amphioxus und der Ascidien so verschiedenen wie mdglich." We have here, if I understand Samassa correctly, homologies which are found only in a single stage of the ontogeny, which have had no beginnings in homologous parts or processes, have neither homological antecedents nor consequents and have therefore arisen de novo. This. it seems to me. is the logical conclusion to be drawn from Samassa' s statement, and it is one as indefensible on zoological as on philosophical grounds. There are many points of resemblance in the early development of Amphioxus and ascidians. as is well known, and such differences as exist are explicable on the general principle of evolution through divergent modification.

The study of the cell-lineage and early development of a large number of annelids and mollusks has shown that in such general matters as the relations of the axes of the egg to those of the gastrula and larva, and the origin of the germ Layers and of specific organs from certain blastomere or regions of the egg, there is a high degree of uniformity among members of the same phylum and even among related phyla, ft would certainly be surprising if the development of Amphioxus and the ascidians should he found to be more dissimilar than that of annelids and gasteropods.

1. Axial Relations of Egg and Embryo

In considering the axial relations of egg and embryo one is confronted at once with the difficulty of determining what is meant by the anterior pole, unless it be defined in terms of structure rather than function. The animal pole is a structurally definite point, hut the anterior end of the embryo, who can say what it is? In the early development of Amphioxus and ascidians the point which at one time is most anterior does not continue such for any considerable period, and it is practically impossible to determine the exact point of this rounded anterior portion of the embryo which will become the most anterior part of the body. Not only is the animal pole a structurally definite point but the anterior limit of the neural plate is also, and the relative positions of these two can be determined with considerable accuracy. The result of such a determination shows that there is great similarity among the lower Chordata in that the anterior limit of the neural plate is always some distance retnovedfrom the animal pole. In ascidians the chief axis of the egg is plainly marked out not only by the well differentiated cleavage cells but also by the polar bodies which in some cases remain attached to the egg at the point of their formation until the blastopore has closed. In the early gastrula the animal pole is slightly posterior to the middle of the ventral hemisphere, the vegetal pole marks the middle of the dorsal hemisphere, and the deepest point of the gastrocoel (text fig. XXVIII). In the closure of the blastopore the anterior lip overgrows the archenteron. and the blastopore, being closed from in front backwards, is finally limited to a longitudinal groove in the posterior half of the dorsal face of the gastrula.

The relation of the egg axis to the embryonic axis is not a simple one, i. e., they neither coincide nor is one at right angles to the other. During the overgrowth of the anterior lip the animal pole is shifted nearer to the anterior end of the gastrula. This may be, and probably is in part, due to a shifting of the point of greatest curvature at the anterior end to a point nearer the animal pole. The anterior edge of the neural plate never reaches farther forward than about onethird of the way from the equator to the animal pole, and consequently the animal pole lies on the ventral side of the larva but near the head end. Correspondingly the opposite pole of the extended egg axis lies near the posterior end of the dorsal side and consequently not far from the place where the last trace of the blastopore can be seen.

Previous students of ascidian embryology, and particularly Castle and Samassa, have considered that the egg axis was dorso-ventral and hence perpendicular to the embryonic axis. I at first held the same opinion, but observations on the change of shape of the gastrula and particularly upon the anterior limit of the neural plate during the closure of the blastopore have convinced me of the truth of the position here taken (cf text figs. XX VII-XXIX).

The axial relations are not so evident in Amphioxus and amphibians, since the animal and vegetal poles are not so clearly marked as in the ascidians. Hatschek (lSSl ) supposed that the animal pole of the egg in Amphioxus was ventral to the cephalic pole of the embryo; and this view has been supported by Garbowski (1898); on the other hand. Kowalevsky (1867) and many recent writers on the development of Amphioxus (Lwoff 1894, Klaatsch 1896, Samassa 1898, Morgan and Hazen 1900). have considered that the most highly arched portion of the late gastrula represents the animal pole. Since this point is said to become the anterior end of the embryo it is evident that according to this view the chief axis of the egg coincides with the chief axis of the embryo and is antero-posterior in direction, whereas in ascidians it has heretofore been claimed that the egg axis is dorso-ventral in direction and hence perpendicular to the same axis in Amphioxus.

Sueh diversity in this most fundamental of all axial relations seems very improbable considering the many points of resemblance between these groups, and at least sueh conflicting results should be supported by the best of evidence before being given general credence.

Korschelt and Heider in their excellent text book attempt to harmonize these differences in axial relations between Amphioxus and the ascidians by regarding the anterior pole of the ascidian gastrula as the animal pole, but I agree with Samassa (1894). and Castle (1896), that the animal pole never comes to lie at the anterior end of the embryo, though unlike them I hold that it does move in that direction.

In Amphioxus as in the ascidians the anterior limit of the neural plate is situated some distance behind the most highly arched portion of the gastrula, and even if the latter be regarded as the animal pole it would still be true of Amphioxus as of the ascidians that the neural plate does not reach as far forward as the animal pole. But there are reasons for thinking that the animal pole lies ventral to the most highly arched portion of the Amphioxus gastrula. Many investigators agree that the animal pole lies opposite the blastopore ; Samassa has observed in a small percentage of eggs that the polar body is still attached to the embryo at a time when the blastopore is growing smaller, and in all sueh cases he found it at t be pole opposite the blastopore (although, as he maintains, at the anterior end of the embryo). But the point opposite the blastopore lies ventral to the most highly arched portion of the embryo. Even if it should be assumed that both ventral and dorsal lips grow equally, the animal pole would still be located on the ventral side of the most highly arched portion, owing to the peculiar shape of the embryo; if the dorsal lip grows more rapidly than the ventral, which in the light of what takes place in ascidians and amphibians seems probable, the animal pole must lie still farther toward the ventral side. In any event a considerable space must intervene between the anterior limit of the neural plate and the animal pole.

The work of Garbowski (1898), shows that the longitudinal axis of the larva of Amphioxus forms an angle of about 70 with the gastrular axis. a result which, like that of Eatschek and Sobotta, agrees very closely with my observations on ascidians. and which practically removes the supposed discrepencies in axial relations between these two classes.

On the whole it seems to me that then' is every reason for believing that the relations of the egg axis to the embryonic axis are essentially the same in Amphioxus and ascidians, that in both the egg axis is postero-dorsal and antero-ventral in direction and that in neither does the neural plate extend more than one-third of the way from the equator to the animal pole (cf. text figs. XXVII-XXXII).

It' the same axial relations exist in amphibians as in ascidians, the middle of the pigmented hemisphere of the frog's egg does not correspond to the cephalic pole of the embryo but lies ventral to this pole, while the white hemisphere corresponds in the main to the dorsal side. This is approximately the orientation which has been maintained by Pfiuger, Roux. Morgan, Kopsch and H. V. Wilson. Kopsch | 1 '.'00), in particular, has shown that the anterior margin of the neural plate lies some distance below the animal pole, and judging from his figures the axial relations in the embryo of the frog must be almost identically like those in the ascidian {cf text tigs. XXXlII-XXXYh

2. Entrance of Spermatozoon

Among ascidians the sperm enters the egg near the vegetal pole ; it then moves to the posterior pole where it meets the egg nucleus, and the sperm amphiaster is formed at right angles to the copulation path. The outer pigmented layer of protoplasm collects around the sperm nucleus and moves with it to the posterior pole where the mesodermal crescent is formed.

In Amphioxus the sperm also enters near the vegetal pole according to Sobotta (1897), hut whether it then moves to the posterior pole and whether there is a collection of superficial protoplasm around the sperm nucleus is unknown.

In the frog the sperm enters on the posterior side just below the equator and. according to Roux. the point of entrance determines the posterior pole of the embryo. Schultze, on the other hand, thought that the point of entrance lay at the anterior pole, hut since he also with Roux holds that the entrance occurs at the pole opposite that at which gastrulation begins, it is evident that this difference with regard to the pole of entrance is only part of the larger difference between these authors as t<> the general orientation of the embryo. The conditions which are found in the ascidian ess closely auree with the orientation of Roux as against that of Schultze.

In another important respect Roux's observations find a parallel in the ascidian __ : he observed that after fertilization the pigment cap of the fro.i;'- egg shifts so that its margin lies below the equator on the side of the egg where the sperm enters while at the opposite pole it comes to lie above the equator. I believe that this movement of the pigment is comparable to the movements of the layer of yellow protoplasm in the egg of Cynthia.

3. Cleavage

There are many differences in the cleavage of the egg in these three classes of chordates, hut some fundamental characteristics are essentially similar in all of them. The most important of these is that the cleavage is usually bilaterally symmetrical. The first cleavage always coincides with the median plane among ascidians, and every subsequent cleavage is perfectly bilateral, one-half of the egg being the mirrored image of the other. In the frog's egg the first cleavage usually lies in the plane of symmetry 1 , and although the subsequent cleavages grow more and more irregular, bilaterality is sometimes strongly expressed even in the later stages [cf. M. Schultze, 1863; Rauber. 1882).

In Amphioxus, if I correctly understand Wilson (1893, p. 600), the first cleavage coincides with the median plane. In the subsequent cleavages, both Wilson and Samassa (1898) have been unable to find the remarkably regular alternation of meridional and latitudinal cleavages described by Hatschek. These cleavages are extremely variable in form; among them Wilson recognizes three principal types, one radial and two bilateral. After the 16-cell stage, however, almost all the eggs become bilateral, whereas in the 8-cell stage three-fourths of them are radial. Wilson suggests that variations from the bilateral type may occur among ascidians, but I agree with Castle and Samassa that under normal conditions this is never the case. In the 8-cell, 16-cell and 32-cell stages of the bilateral types there are many striking resemblances to corresponding stages of the ascidian ; this applies particularly to Wilson's bilateral type II (cf. his figs. 13-18, 33. 34, 36, 37-30, 41-43, and Samassa' s figs. 2, 6, 7, 9). In these figures the form of the cleavage is so similar and the position of the cells and even the direction of the spindles within the cells so remarkably like what is found in the ascidians that the individual cleavage cells can be correlated in these two animal classes.

Too little is known of the cell-origin of the germ layers in At>iphio.\us to determine accuratelv how close is the likeness to ascidians in this regard. Wilson holds that the eight animal cells of the 16-cell stage are purely ectodermal and that the "secondary macromeres " (A 2 , B 2 , C 2 , D 2 , ) which surround, and were derived from, the four basal cells at the vegetal pole are of mixed character, giving rise to both endoderm and ectoderm, and perhaps also mesoderm. He does not give the evidence upon which this conclusion rests, but its similarity to the conditions which exist in the ascidians should not be overlooked. Here also the eight animal cells are purely ectodermal, while the " secondary macromeres." and in fact, all the cells of the vegetal hemisphere in the 16-cell stage are of mixed character, the four anterior ones containing endoderm and ectoderm (neural plate substance), and the four posterior ones, endoderm and mesoderm. Wilson expressly states that he uses the terms macromere and micromere "solely for the sake of convenience," and he concludes that the cleavage is very unlike that of annelids ; Samassa also emphasizes this same conclusion.

We may conclude, then, that there are certain fundamental resemblances between Amphioxus and ascidians in the matter of cleavage and that the most notable differences between them are found in the number of cells and the degree of their differentiation at any given embryogenic stage ; in ascidians this number is relatively small ami the degree of differentiation high as compared with Amphioxus ; e.g.. at the stage when invagination begins in Amphioxus there arc according to Wilson about 512 cells, at a corresponding stage in Ciona there are 76 cells. It may be presumed that the relative constancy or variability of cleavage in these two classes depends upon the two features just contrasted, viz.. the number of the cleavage cells and the deirree of their differentiation.

  • 1 In the newt, Diemyctylus, Jordan (1893) found that the first cleavage is perpendicular to the median plane.

In a general way the same kinds of likenesses ami differences exist between ascidians and amphibians in the matter of cleavage as between the former and . Xmphioxus. Among amphibians, however, these differences are further increased by the presence of a relatively large quantity of yolk. Whether the ectoderm comes entirely from the four upper cells of the 8-cell stage in these animals cannot be affirmed, but it is evidently derived in chief part from these cells.

4. Blastula and Gastrula

The firm of the blastula and gastrula is much influenced by the relative amount of yolk in different cases. A large coeloblastula, such as is present in Amphioxus, does not occur in the ascidians or amphibians. In the ascidians this is due not merely, nor largely, to the amount of the yolk but rather to the shape of the cells which are always elongated either at one pole or the other so as to nearly fill the blastocoel ; the latter is small at all stages and the embryo and larva very compact. In the amphibians the relatively small size of the blastocoel is due not only to the quantity of yolk, but also to the many-layered character of the blastula wall.

In all three classes the ectoderm arises from the upper hemisphere of the blastula, the endoderm and mesoderm from the lower hemisphere, but the precise relation of these germ layers to the third cleavage plane is not known in the cases of Amphioxus and the Amphibia.

Most investigators affirm that the gastrula invagination in Amphioxus is at first radially symmetrical, and only in the later stages does it become unsymmetrical. Samassa (1898), on the contrary, finds that the gastrula is bilateral from the beginning and concludes that this bilaterality is the direct outcome of the bilaterality of the cleavage stages. In both ascidians and amphibians it is bilateral from the first, the invagination appearing near the anterior border of the dorsal face and then extending so as to include most of the dorsal area.

5. Closure of Blastopore

In ascidians the closure of the blastopore results largely from the progressive posterior growth of the anterior (dorsal) lip, while the posterior (ventral) lip remains relatively fixed in position. Owing to the peculiar differentiation of the cells of the blastopore lip they can be individually followed through a large part of this process; the number of cell rows between the posterior lip and the animal pole and between the anterior lip and that pole can also be determined with accuracy during the earlier stages of the closure: from both of these facts it is certain that the posterior lip takes only a small part in the closure of the blastopore, except in the final stages of that process. The posterior border of the blastopore is formed of mesodermal cells derived from the crescent ; these cells are larger and more rounded than the cells of the anterior border and are easily distinguishable by their color and texture. In the closure of the blastopore they are rolled in at the lateral margins but not at the hinder end. and owing to the large size of these " myoblasts " the posterior portion of the blastopore is reduced to a longitudinal groove. Finally, this groove is closed by growth from all sides, the posterior lip growing more rapidly than the anterior one in the final stages of the process.

Conklin 1905 fig27-33.jpg

Figs. XXVII XXXVIII. Schematic representations of three stages in the gastrulation of an ascidian, of Amphioxus and of an amphibian to show their supposed resemblances in (1) Axial Relations, (2) Closure of Blastopore, (3l Origin of Neural Plate, (4) of Chorda and (5) of Mesoderm. The position assigned to the polar bodies in Amphioxus and the amphibian is to a certain extent hypothetical. The head of the arrow marks approximately the anterior limit of the neural plate; the tail of the arrow, the median mesenchyme cells in the posterior (ventral) lip of the blastopore. The mesodermal cells or areas are shaded by entire or broken lines.

Figs. XXVII XXIX. Right halves of bisected gastrulae of Cynthia ; the chorda cells are shaded by coarse stipples, the neural plate cells in. p.) by line stipples. The muscle cells (m.s.) lie lateral to the mesenchyme cells (m'cfa l and by the overgrowth of the anterior (dorsal) lip of the blastopore are separated from the mesenchyme cells anteriorly (XXIX),

Figs. XXX XXXII, Right halves of bisected gastrula- of Amphioxus (mainly after Hatschek I. In the two earlier stages the existence and position of the mesoderm is hypothetical, being based upon the conditions found in ascidians.

Figs. XXX I II -XXX V. Right halves of bisected gastrula of the frog (mainly after Kopsch I. The areas shaded by stippled lines represent the supposed position of the mesoderm, here covered in endoderm and yolk.

Figs. XXXVI XXXVIII. Dorsal views of late gastrnte of ascidian (XXXVI), Amphioxus (XXXVII), and frog (XXXVIII), The circles marked 1, 2, 3. 4 indicate successive stages in the closure of the blastopore. The actual position of the mesoderm in the ascidian and its supposed position in Amphioxus and the frog is shown by the radiating lines around the blastopore. The unshaded area (Ch.) anterior to the blastopore and between the halves of the mesoderm represents the plate of chorda cells.

The earliest trace of the anterior lip appears just posterior to the chorda cells, the endodermal cells here becoming depressed (fig. 134); at this stage the chorda cells and the neural plate cells which lie just anterior to them are at the same level, but in the posterior growth of this lip the chorda cells are rolled in so that they form the inner, as the neural plate cells form the outer, layer of the anterior (dorsal) lip. None of the neural plate cells and none of the ectodermal cells are ever inrolled, the only cells which suffer this fate being the chorda cells and the muscle cells (myoblasts).

There has been much controversy as to the part played by the anterior and the posterior lips in the closure of the blastopore in Amphioxus and the amphibians. Kowalevsky supposed that the closure in Amphioxus occurred in a radially symmetrical manner, the entire bolder of the blastopore growing equally ; Hatschek thought that the growth of the anterior (dorsal) lip was the chief factor in the closure; Lwoff, Klaatsch, Samassa, Morgan and Hazen agree in the main with Kowalevsky. MacBride (1898) finds that in the final stages of closure the ventral lip grows more rapidly than the dorsal.

Among the amphibians, observation and experiment show that the overgrowth of the dorsal lip is greater than that of the ventral, but the relative amount of growth of each lip is not certain. In early stages of closure the dorsal lip is alone concerned, as is also the ease with ascidians ; in later stages growth takes place from all sides. According to Pfliiger the dorsal lip sometimes moves through an are of 180 in the case of the frog, according to Roux 170; Morgan estimates this movement at 120, Kopsch at 75. and H. V. Wilson at 722. In the ascidians there is no doubt whatever that the closure is due chiefly to the growth of the dorsal lip, though owing to the changing shape of the embryo it is difficult to estimate the angular amount of that growth.

In Amphioxus and the ascidians the growth of the dorsal lip occurs as rapidly in the middle as at the sides and there is therefore no indication of concrescence of lateral lips. At no stage during the closure of the blastopore in these animals is there any indication whatever of such concrescence, either in the form of a notch at the edge of the dorsal lip or of a seam along the middle of the neural plate. In Cynthia and Ciona I have seen every division of the cells of this lip up to an advanced stage and these divisions take place as rapidly and uniformly along the mid-line as at the sides. Practically all investigators, who have studied the embryology of Amphioxus or the aseidians are in agreement upon this point, and if concrescence occurs among the amphibians, as is claimed by some investigators, though denied by others, it can only be said that in this respect the amphibians are very different from these other classes. The evidence that the amphibians do form such an exception is by no means conclusive, as Ziegler (1902) points out,

The question whether and to what extent there is an actual inrolling of cells from the outer to the inner layer in the closure of the blastopore is one which has been much discussed. In all three of these chordate classes an inrolling of cells at the margin of the blastopore has been repeatedly observed, but the relative number, the origin and the character of such cells are matters of dispute. Lwoff (1894) maintains that the entire dorsal lip of Ampktoxus, inner as well as outer layer, is formed from ectoderm cells which are inrolled. All of these inrolled cells he counts as ectoderm and consequently concludes that the chorda and mesoderm are of ectodermal origin. The invagination of the endoderm is, in his opinion, the real gastrulation. whereas the turning in of the ectodermal cells is a coenogenetic process which has nothing to do with the formation of the enteron but is concerned only with the formation of chorda and mesoderm. This conclusion has been criticised by Samassa (1898). Klaatsch (1896), Morgan and Hazen (1900). et al., on the ground that there is no sufficient evidence that the inrolled cells are ectodermal. With this conclusion, when extended to the aseidians. I heartily agree. Here the cells which are inrolled at the anterior border of the blastopore are chorda cells which are yolk laden and resemble endoderm and not ectoderm. The cells which are inrolled at the posterior lateral borders are mesenchyme and muscle cells and in histological structure are very unlike the ectoderm. While therefore agreeing with Lwoff that the chorda and mesoderm cells are inrolled (though from opposite portions of the blastopore lip in aseidians) I agree with his critics that these cells, judged by their lineage and histological character, are certainly not ectodermal.

6. Neural Plate

In aseidians the neural plate material becomes segregated into six cells at the 44-cell stage ; four of these cells lie in a transverse row at the anterior border of the dorsal hemisphere, just below the third cleavage plane and two of them lie just above this plane and therefore in the ventral hemisphere. The four dorsal cells lie just anterior to the four chorda cells from which they were separated at the sixth cleavage. Both- the dorsal and ventral cells divide transversely, the former sjivimz; rise to an arc of eight cells the latter to one of four cells, and to these a single additional cell is added on each side making an arc of six neural plate cells in the ventral hemisphere. In subsequent divisions the neural plate increases much in length and its anterior portion also increases in breadth, but it is never more than eight cells wide in its posterior part, Soon after gastrulation begins the neural cells overgrow the chorda cells and thereafter cover the dorsal lip to its posterior margin. During all this time the anterior margin of the plate reaches only about one-third of the way from the equator to the animal pole. The posterior margin of the plate reaches nearly to the hinder cud of the embryo, and when the blastopore doses a pair of V-shaped folds runs forward from the region of the blastopore inclosing the neural plate between them. These neural folds then fuse from behind forwards thus converting the plate into a tube. Dorsal to the notochord the neural tube becomes solid ; in the region in front of the notochord it retains its lumen. There is no nerve ring around the blastopore and probably none of the ectoderm cells around the posterior margin of the blastopore are added to the neural plate.

In Amphioxus and amphibians the neural plate is first recognizable about the the time of the closure of the blastopore. As in ascidians it arises in the outer layer of the dorsal lip and extends back as far as the blastopore, but whether its cells arise in close connection with the chorda and from both dorsal and ventral hemispheres as in the ascidians is unknown; furthermore, the distance of the anterior edge of the plate from the animal pole is unknown. The work of Kopsch (1900) indicates that in the frog the anterior margin of the plate is situated less than half the distance from the equator to the animal pole, and H. V. Wilson (1000) in particular has shown that the anterior part of the neural plate is formed from the black hemisphere, the posterior part from the white hemisphere. a result which agrees precisely with my observations on ascidians. As is well known the method of closure of the neural tube in Amphioxus is peculiar, while the solid character of the hinder part of the tube is peculiar to the ascidians, but with these exceptions the later history of the neural plate and tube is essentially similar in all three classes.

7. Chorda

In ascidians the substance of the chorda is segregated into a single transverse row of cells just posterior to the neural cells at the 44-cell stage, before there is a trace of gastrulation. These chorda cells are generally clearer and contain rather less yolk than the endoderm cells which lie immediately posterior to them. These four chorda cells divide transversely forming an arc of eight cells and soon thereafter a depression of the endoderm occurs posterior to this arc. which is the beginning of the gastrulation. These chorda cells are flanked on each side by the most anterior cells of the mesenchyme arc. the two arcs together forming the chorda-mesenchyme ring of Castle. The eight chorda cells then divide anteroposteriorly forming two rows of eight cells each. This plate of cells by shoving, by interdigitation and perhaps to a limited extent by folding, decreases in width and increases in length, the cells finally, in a late larval stage, becoming arranged in a single linear series. When they first arise the chorda cells are superficial in position, but in the overgrowth of the dorsal lip they are inrolled so as to lie in the roof of the gastrocoel. The posterior growth of the dorsal lip carries the entire chorda into the hinder half of the embryo, and it afterwards extends to the tip of the developing tail.

In Amphioxus the earliest stage at which the chorda has been positively identified is one when the blastopore is small and the embryo elongated. According to Hatschek it consists at this stage of a plate, about six cells wide, in the roof of the archenteron and extending along the mid-line of the dorsal lip throughout its entire length. This plate is narrower and longer than it is in the ascidians. but is otherwise much the same in appearance. The later history of the chorda is essentially the same in both forms. With regard to the origin of the chorda cells in Amphioxus, Morgan and Hazen (1900) have shown that the cells which are inrolled in the formation of the dorsal lip and some of which must take part in the formaation of the chorda, are clear and contain less yolk than the endoderm cells. Whether these cells form at this stage a plate which is wider from side to side than it is long, as is true of the ascidian, is not known. Lwoff (1804) has also recognized the fact that the chorda cells are rolled in at the margin of the dorsal lip, and for that reason he regards them as of ectodermal origin.

In Amphioxus and in some amphibians the definitive roof of the enteron arises from cells which lie along each side of the chorda plate, and which finally grow under that structure and thus separate it from the gastric cavity ; in the ascidians the chorda lies ultimately in the posterior part of the body where the gastric cavity is almost entirely lacking and there is no growth of endoderm cells under it to form the roof of the enteron. In most amphibians the chorda does not form a broad plate of cells, but is a narrow rod closely united ventrally with the endoderm. which forms the roof of the enteron, and connected laterally with the mesoderm. In these three groups of chordates the chorda plate is widest in ascidians and narrowest in amphibians. In all three it lies in the dorsal lip and is connected laterally with mesoderm (text figs. XXXVI XXXVIII). The later history of the chorda is essentially the same in all three classes.

The question whether the chorda is of endodermal or of mesodermal origin is. as has been frequently said, one of definition of terms. Castle concludes that it is mesodermal because in Amphioxus and lower vertebrates it " is derived from a common fundament with what is universally regarded as mesoderm" and also because it "comes to occupy a position between the inner and outer layers of the embryo." On the other hand, the histological structure of the chorda cells in Cynthia and Ciona is much more like endoderm than mesoderm, and they are unquestionably derived from cells of the gastric endoderm at the 32-cell stage (fig. 117, 103). I believe that special importance should attach to the structure of the cells which form the chorda, and if this be accepted as a guide the chorda, at least among ascidians. should be regarded as endodermal.

8. Origin of Mesoderm

The exact place and manner of origin of the mesoderm of ascidians can be recognized with the greatest certainty in the gastrula, cleavage stages and even in the unsegmented egg. The crescent, from which most if not all of the mesoderm arises, lies just below the equator of the unsegmented egg, and on the posterior side, its arms extending forward to the second cleavage plane. It occupies this position throughout the whole of the cleavage, its substance becoming localized in a number of large rounded cells. In the gastrulation these cells are inrolled along the lateral-posterior borders of the blastopore, thus reducing the posterior portion of the blastopore to a groove and rendering the whole blastopore pear-shaped. No such appearance is found in Amphioxus or amphibians where the blastopore retains its circular form until a late stage; this may be interpreted as due to the fact that in these animals the mesoderm is not so largely developed at an early stage, but it furnishes no satisfactory reason for supposing that the mesoderm is not formed in corresponding positions in all three classes. We know that the neural plate and the notochord come from similar regions in all three, and it is most unlikely that the mesoderm arises froin wholly different regions.

Hatsehek's account of the origin of the mesoderm of Ampliioxus shows some important resemblances to what occurs among ascidians. He found that running back on each side from the first appearing primitive segments was a mesodermal fold which led to a pair of pole cells in the ventral (posterior) lip of the blastopore. All recent investigators nave denied the existence of these pole cells, and there can be little doubt that Hatschek was mistaken with regard to them. Even in the ascidian there are, strictly speaking, no pole cells in this region, nor anywhere else in the embryo. The cells which in the ascidian occupy the position assigned by Hatschek to the pole cells are the posterior mesenchyme cells. These cells form the middle of the crescent, and from them a band of mesoderm cells runs forward on each side, but these bands were not formed by the teloblastic growth of the posterior cells; on the contrary, their substance was localized in the crescent before cleavage began. However the non-existence of the pole cells of Ampliioxus does not destroy belief in Hatsehek's account of the mesodermal folds which run backward from the primitive segments to the blastopore. Several investigators have recognized such folds or bands, and their existence can scarcely be doubted. These bands have been seen only in older gastrulse, and they here occupy a position which corresponds very closely with the mesenchyme bands in the ascidian gastrula. The separation of the muscle band from the mesenchyme band in the older gastrulse of the ascidian [v. p. 69) is evidently a coenogenetic phenomenon, since nothing of this sort is known to occur elsewhere. If the mesodermal bands of Ampliioxus are present in earlier stages than those in which they have been represented by Hatschek, and if they occupy the same relative position as in the ascidian they would surround the posterior border of the blastopore, and only by overgrowth of the dorsal lip and the narrowing of the whole blastopore would they come to lie alongside of the notochord. That mesodermal cells are present in the posterior lip of the gastrula of Amphioxus at an early stage is made probable by the observations of Lwoff, Klaatsch, Morgan and Hazen. Lwoff has found that the longitudinal musculature of Amphioxus arises along the hinder lateral parts of the blastopore, where it comes from ectodermal cells, as he thinks, which are inrolled. Klaatsch agrees with this and compares the "pole cell bands" of ascidians with these mesodermal folds of Amphioxus. He has observed that in both ascidians and Amphioxus these cells are more rounded than other cells of the gastrula. Like Klaatsch, Morgan and Hazen find that around and within the ventral lip of the blastopore, during the early gastrula stages, there are frequently found small rounded cells which contain little yolk. They affirm that the form of these cells is not the result of cell division, as Samassa had assumed, but that they preserve their rounded form even in the resting stage.

Samassa ( 1S98), however, says that in Amphioxus the origin of the mesoderm has no relation to the blastopore. The fact that the mesoderm has it growth zone at the caudal end of the embryo, in the vicinity of the blastopore, is, he says, a condition which it shares in common with all other organs of the embryo. In the face of the positive evidence adduced by Lwoff, Klaatsch, Morgan and Hazen this negative conclusion of Samassa's seem to me to lose much of its weight.

It seems probable from these accounts that mesoderm cells are present in the ventral lip of the early gastrula of Amphioxus just as in the ascidians, and that they give rise to the longitudinal mesodermal folds of later stages ; it remains to be seen whether these mesoderm cells may not be traced back to a still earlier stage, comparable with the crescent in the ascidian egg (cf. text (ins. XXVIIXXXII).

The origin of the mesoderm in amphibians is a much more difficult and complicated question and one into which I cannot enter full}' here. It is generally believed, however, that in the frog's egg the cells which are to form the mesoderm are present when the dorsal lip first appears, and even prior to that time. They are the deeper layer of cells of the blastoporic ring and, therefore, surround the egg below the equator. Whether at their first appearance they surround the entire blastopore is not plain, but in later stages this is said to be the case. According to this view the notochord is a mesodermal structm*e differentiated out of the continuous ring of mesoderm surrounding the blastopore. There is here resemblance to the chorda-mesenchyme ring which is present in the ascidians and probably also in Amphioxus, but in the amphibians this ring appears to give rise at once to a sheet of mesoderm and not to mesodemal bands such as are found in Amphioxus and ascidians (text figs. XXXIII-XXXVI1 1 ).

On the whole it is probable that there is fundamental agreement between Amphioxus and ascidians in the place and manner of mesoderm formation, and though the amphibians differ in some important respects from the other two classes it is possible to interpret their method of mesoderm formation in the same general terms.

Referring to Raid's (1892) " Theorie des Mesoderms," Samassa (1898), and Garbowski (1898) maintain that there is no peristomal" mesoderm in Amphioxus, but that all the mesoderm is "gastral." If the view here taken is correct, all the mesoderm of this animal is at first peristomal while the gastral mesoderm is later derived from this. This is exactly the conclusion which has been readied by Davidoff (1891), and Castle (1896), with regard to the ascidian, a conclusion which I can fully confirm from my own work. Furthermore, it is not improbable that the same thing is true of the amphibians. This is in confirmation of Rabl's view that the peristomal mesoderm is palingenetic, the gastral coenogenetic, and the suggestion is raised that in all these rases the gastral mesoderm is derived from the peristomal through the manner of overgrowth of the dorsal lip of the blastopore (text figs. XXXY1 XXXVIII).

Although 1 have made no special study of the subject, and cannot therefore speak with assurance. I have seen no evidence in favor of Van Beneden and Julin's view thai enterocoels are present in ascidians as in Amphioxus\ in this respect 1 am in accord with the more recent students of ascidian development (Davidoff, Castle).

From these comparisons 1 think it may be safely concluded that there are many fundamental resemblances in the early development of Tunicata, Amphioxus and Amphibia, and that in consequence of the early differentiation of the ascidian egg and embryo and because of the known cell-lineage of some of its important organs the development of these animals throws light upon the embryology of other chord ate classes.

Cite this page: Hill, M.A. (2020, February 18) Embryology Paper - The Organization and Cell-Lineage of the Ascidian Egg 6. Retrieved from

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