Paper - The Organization and Cell-Lineage of the Ascidian Egg 7
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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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VII. The Organization of the Egg
It is interesting to observe how recent studies of development have led to the recognition of morphogenetic differentiations at earlier and earlier stages in the ontogeny; a dozen years ago the germ layers were the earliest differentiations of this sort which were generally recognized. It was in the attempt to determine the cellular origin of the germ havers that it became evident that the cleavage cells themselves were of morphogenetic value. Some of the differentiations of the cleavage cells could be traced back to the ver} r first cleavage or even to the unsegmented egg ; thus the study of cell-lineage led logically and unavoidably to the conclusion that the cleavage cells and even the unsegmented egg must be organized with reference to the parts and axes of the future animal.
For our present purposes the organization of the germ cells lias reference only to such differentiations as are of direct value in the building of the embryo, in other words, such as are morphogenetic, and it may be held to include phenomena of polarity, symmetry and localization ; it obviously includes other things also, such as regeneration and regulation, which are not. however, objects of investigation in this work.
A. Polarity
Fifty years ago Remak showed that the pigmented hemisphere of the frog's egg gave rise to the cells of Von Baer's animal germ-layer," while the white hemisphere gave rise to the 'vegetative germ-layer." The middle of the ectodermal hemisphere hasever since been known as the animal pole, the middle of the endodermal hemisphere as the vegetative (vegetal) pole. It is a remarkable fact that with a few possible exceptions, which arc by no means well established, the polar bodies arc formed at the animal pole of the egg in all cases. This is a fact of the most general occurrence and of the highest significance : it indicates that before or during the maturation of the egg there occurs a polar differentiation or localization of the egg substance of such a kind that in all cases the future ectoderm is formed at the maturation pole and the endoderm at the opposite pole.
- 1 A more complete discussion of this suhjert, especially that portion of it which relates to experimental work, is reserved for a subsequent paper, only such matters being treated hen- as are the outgrowth of the observations recorded in the preceding pages.
The apparent exceptions to this rule are few in number and may be examined in some detail ; they are limited to the eggs of certain insects, Petromyzon, copepods, Ascaris, echinoderms and aseidians. The only reason for supposing, as Korschelt and Heider (1903, pp. 545, 546) do, that the polar bodies are not formed at the animal pole in insects and in Petromyzon is that they here lie to one side of the pointed end of the egg; there is no proof that they do not lie at the middle of the ectodermal area. Hacker (1899) says that in the larger species of Cyclops "neither the place of formation of the polar bodies, the place of entrance of the sperm nor the position of the first cleavage spindle are preformed in the egg, but are secondarily determined by the position of the egg in the egg sack" (pp. 193, 194). However, this egg is one which is not easy to orient, and it has by no means been proven that the polar bodies do not form in this case at the middle of the ectodermal area. Even if the justice of all of Hacker's statements bo admitted it has not been shown that the cleavage spindle may not rotate so as to cause the first and second cleavage furrows to pass through the maturation pole, as is usually the case. Such a rotation of the first cleavage spindle takes place in nematodes, and a somewhat similar rotation of the entire egg, after the formation of the first cleavage spindle, has been described by Bigelow (1902) in the case of Lepas, where it had previously been held that the first cleavage was equatorial. Hacker's observations do not show that the chief axis of the egg is not predetermined, and they certainly do not prove that the maturation pole and the ectodermal pole do not coincide.
In Ascaris megalocephala, Boveri (1887) observed that the second polar body is usually formed at some distance from the first "whether through wandering in the protoplasm or through a turning of the entire egg I could not determine" (p. 32). His figures (1888, pi. IV) show that the first cleavage furrow frequently passes through the point of attachment of the second polar body. The study of the cell lineage of Ascaris has shown that most of the ectoderm is segregated in one of the first two cleavage cells (the "primary ectoderm cell" of Zur Strassen, 1896). This would seem to indicate that in this animal the polar bodies-do not lie at tin' middle of the ectodermal pole; however the relations of the maturation pole to the ectodermal pole and to the first cleavage arc not clear in this case, and it may not be impossible that Ascaris may yet be found to conform to the general rule.
As for the echinoderms. Wilson (1895) supposed from indirect evidence that the maturation pole and the future animal pole did not usually coincide in Toropneustes, and further that the chief axis of the egg was established only after fertilization. However, the evidence in favor of this is not conclusive as Wilson admits. On the other hand. Boveri i L901 > has shown in the most convincing manner that in Strongylocentrotus the polarity of the egg may be traced hack to the ovocyte, and that this polarity determines the gastrular axis. It is. therefore, possible that in all echinoderms the polaritj of the egg is predetermined in the ovary, and not after the maturation and fertilization, and that in all cases the maturation and ectodermal poles coincide. 1
The most remarkable ami apparently well established of these exceptions to the rule that the polar bodies are formed at the animal pole is that of the ascidians studied by Castle (1894, 1896), where the polar bodies were said to he formed at the vegetal or endodermal pole of the egg. However, this conclusion rests upon erroneous orientation, as I have shown in the preceding pages ; in ascidians as in other animals the polar bodies are formed at the ectodermal pole. There are, therefore, no well established exceptions to this general law. 2
In many cases it is known that the polar differentiation of the egg may he recognized while the egg is still in the ovary. Reference has just been made to the condition in Strongylocentrotus in which the pole of attachment to the ovarian wall becomes the maturation pole of the egg and the ectodermal pole of the larva. Boveri says that in all known cases the pole toward which the germinal vesicle is eccentric becomes the animal pole. In Unto, Lillie (1900) has demonstrated that it is the free pole of the egg which becomes the maturation and ectodermal pole, while the pole of attachment becomes the vegetal pole. In a number of gasteropoda (Lzmn&a, Succinea. Polygyra, Limax, Phvsa. Planorbis. Ancylus) I have found that there is a marked polar differentiation of the vfi'j: in the ovary, the germinal vesicle being eccentric toward the tree pole of the ovocyte. 1 have elsewhere (1903) shown reason for believing that in dextral snails the polar bodies are formed at the free pole and in sinistral snails at the attached pole of the ovocyte. In his work on Cerebratiihis, Wilson ( 1 9 >> > , found that the polar bodies were formed at the live pole of the ovocyte, and again in his recent paper on Dentalimn (1904), he finds the side of attachment in the ovary represents the lower or vegetal hemisphere. We find then that the chief axis of the egg is very generally present in the ovocyte, and that the free side usually uives rise to the maturation and ectodermal pole, while the attached side becomes the vegetal pole; but in echinoderms and probably also in sinistral gasteropods these conditions are reversed, the side of attachment becoming the ectodermal pole.
In the gasteropods named above, 1 found it possible to recognize this polarity of the ovocyte at a very early stage ; in general it coincides with the "organic axis" (Van Beneden), or the cell axis" (Heidenhain) i.e.. the axis passing through the centrosome or sphere, and the center of the nucleus. This cell axis is a general characteristic of many, if not of all cells, and as it is present in all the cells of the cleaving egg, where it is preserved from one cell generation to another (v. Conklin, 1902), it may be considered to be a differentiation which is continuous from generation to generation. But while the cell axis determines the egg axis and this the gastrular axis, it is not necessary to suppose that in the early ovarian history of the egg one pole is composed of ectodermal substance and the other of endodermal. On the contrary, this is probably not the case. My observations on the living eggs of ascidians and snails leads to the view that it is not the extrusion of the polar bodies at one pole which causes that pole to become the ectodermal one, but rather that it is the movement of the germinal vesicle with its contained clear protoplasm to o?ie pole, and the spreading of this pro/op/asm at this pole, which is the determining factor. In short it is the localization of ectodermal substance at the maturation pole which causes that pole to give rise to ectoderm. I shall return to this subject in the section on localization.
- 1 However, Garbowski (1904) affirms that in Asterias (jlaeialis the polarity of the egg is net determined even in the 8-eelI and 16-cell stages, and thai the blastomeres are equipotential up to the 500-cell stage
- Wheeler 1897, p. 4 1 4t> has discussed in an admirable manner the apparent exceptions to
this law ni' polar differentiation and concludes that these exceptions are by no means well established.
Whether other axes of the egg are predetermined before cleavage is in most instances unknown. In a lew cases all the axes of the future animal are marked out before fertilization ; for example, among insects and cephalopods, as is well known, it is possible to identify anterior and posterior, right and left, dorsal and ventral axes of the egg while it is yet in the ovary. In most cases, however, the only axis which is recognizable before fertilization is the chief axis of the egg. This is true of the ascidians. but here there are certain evidences, which will be presented in the next section, that the other axes are already established, though not directly recognizable until after fertilization.
B. Symmetry
Van Beneden and Neyt (1887) suggested that bilateral symmetry may be characteristic of all cells of bilateral animals, and Lillie (1901) has expressed a similar view regarding the eggs of such animals. This hypothesis, if true, would materially simplify the problem of the earliest differentiations and localizations of the egg, but it is supported by little direct evidence ; in fact, it is surprising that in most bilateral animals bilaterality appears so late in development, In most annelids and mollusks the egg and early cleavage stages are to all appearances radially symmetrical, and in many cases bilateral symmetry first appears with the formation of the mesentoblast cell, 4d. In echinoderms bilaterality is said to appear first in the gastrula stage ; in Amphioxus during cleavage ; in ascidians it appears immediately after fertilization and before the first cleavage ; while in cephalopod and insect eggs it appears during the growth of the ovocyte in the ovary. Wilson has repeatedly expressed the view that characteristic differentiations, such as bilateral symmetry, arise at different periods of development in different cases, and it cannot be denied that the ocular evidence is in favor of this view. On the other hand, there are certain considerations which lead to the conclusion that bilateral organization may be present in the developing egg or embryo long before it is directly visible. For example, in Neritina there are two groups of granules in the protoplasm of the unsegmented egg, one on each side of the polar bodies. Blochmann (1882) observed that these granules were ultimately localized during cleavage in the right and left " Urvelarzellen." They therefore mark out a bilateral organization of the unsegmented egg, although the cleavage up to the time of the formation of the Urvelarzellen" is typically spiral and radially symmetrical. In other gasteropod eggs, where these granules are lacking, not a trace of bilateral organization is visible before the formation of the mesentoblast cell ; yet it can scarcely be supposed that the eggs of these gasteropods are so unlike those of Neritina as to be actually radially symmetrical as they appear to be. Rather it seems probable that the bilateral organization which appears in this one respect in the Neritina egg is characteristic of other gasteropod eggs also, though it does not usually become apparent until a later stage.
Crampton (1894) discovered that the cleavage of the egg in sinistral snails is reversed as compared with that of dextral forms. I have shown elsewhere (1903) that the inverse symmetry of sinistral snails is traceable to the inverse organization of the unsegmented 'egg. Of this fact there can be no doubt, though it is not yet certain how this inverse organization may have been produced. But an inverse organization of the egg, such as would produce inverse symmetry of the embryo and adult, implies of necessity a bilateral organization to begin with ; it must be, therefore, that the eggs of these gasteropods are bilateral, though this fact is not directly evident.
In the aseidian egg the first appearance of bilaterality which I have been able to detect occurs soon after fertilization when the sperm nucleus moves toward one side of the egg which later becomes the posterior pole. One might, therefore, lie inclined to consider that in this case the egg before fertilization was radially symmetrical, and that the chance movement of the sperm into one meridian determined the median plane of the embryo, were it not for the fact that all the movements of the sperm within the egg seem to be directed by the organization of the cytoplasm. The sperm always enters the egg near the vegetal pole, but the fact that the point of entrance is nearer that pole in some instances than in others shows that that point is not a fixed and constant one. After the sperm has penetrated the peripheral layer of protoplasm, and has turned so that its centrosome is directed forward in its movement through the egg it moves up to the equator of the egg in a path nearly parallel with the surface. Arrived at the equator, the upward movement ceases and the sperm nucleus and centrosome. after meeting the egg nucleus, turn in toward the center of the egg. These movements are of such a constant character that they cannot be the result of chance; they must be directed and probably by the cytoplasm of the egg. Furthermore, it seems probable from the evidence of such cases as figures 81 and 85 that the sperm nucleus does not always take the shortest path to the equator as it should do if the egg were radially symmetrical and the median plane were really determined by the path <>f the spermatozoon . On the other hand, it sometimes appare)itly takes the longest path as if it must needs move in a certain meridian. This seems to indicate that the median plane of tlie embryo is not determined by tlie chance path of the spermatozoon within the egg, but rather that both the median plane and the path of the spermatozoon are determined by the structure of the cytoplasm.
Finally, in cases of normal or artificial parthenogenesis the median plane cannot be determined by the path of the spermatozoon. In eggs of this kind the establishment of bilateral symmetry must be held to be due to the structure of the egg itself or to environment, and whichever of these views may be accepted it follows that the path of the spermatozoon cannot be regarded as a general factor in determining the median plane of the embryo.
These and other similar considerations lead to the view that bilateral organization is frequently present in the egg before it becomes visibly manifest, and they lend support to the hypothesis of Driesch (189G) that the eggs of all bilateral animals are bilaterally organized, there being a "polar bilateral direction of particles" in the "intimate structure of the egg." // this be true, the eggs, the cleavage stages and the blastulce of annelids and mollusks, of echinoderms and Amphioxus are as truly bilateral as they are in the ascidians, thohgh this bilaterality may be masked by a radial form of cleavage and by an apparently radial organization of the egg.
I cannot pass over this subject without referring to the extensive work of Roux (1883, 1885, 1887, 1902, 1903) on the determination of the median plane in the frog's egg. This work is too widely known to require more than passing notice. By means of " localized fertilization," i. e., the application of spermatozoa to any meridian of the egg, Roux has determined that the first cleavage plane passes through the entrance point of the spermatozoon and that the median plane of the embryo usually coincides with the first cleavage plane. He therefore considers that the median plane is in typical conditions, determined by the path of the spermatozoon. Moskowski (1902), on the other hand, holds that the first cleavage plane and the median plane of the embryo are determined by definite movements of the egg substance and not by the path of the spermatozoon. Castle (1896) believed that the plane of the first cleavage and the median plane of the embryo were determined, in the ascidians studied by him, by the place of entrance of the spermatozoon, the point of entrance marking the posterior pole ; I mt since the point of entrance is near the vegetal pole, while the posterior pole lies near the equator, it is evident that the point of entrance cannot mark that pole. It is true that the protoplasm which gathers around the head of the sperm as soon as it enters the egg moves with the sperm to the posterior pole and there remains permanently, but the location of this protoplasm at this pole is evidently due to something other than the point of entrance of the spermatozoon. There is no question whatever that, in the ascidians, the path of the sperm within tlie egg coincides with the plane of the first cleavage and with the median plane of the embryo, but there is evidence, as I have shown, that this path is itself determined by the structure of the e^'i.
C. Cytoplasmic Localization
1. Localization in Cleavage Stages
That there is a specification and localization of those portions of the protoplasm of the egg which are destined in development to give rise to definite organs has been repeatedly affirmed and denied since His first propounded the doctrine of "organ forming germ regions" in 1 874. At first this doctrine .took the form of a mental projection of the early embryonic organs back upon the unsegmented egg. Later the study of cell-lineage showed that definite organs of the larva or adult ai'ose from definite blastomeres, which in turn came from definite portions of the unsegmented egg. But although it was thus possible to map out the cleavage cells ami the unsegmented egg into regions corresponding to certain organs of the embryo. it was not usually possible to show that these regions were visibly different from one .another. Nevertheless the fact that certain blastomeres constantly gave rise to certain parts, and that other blastomeres developed very differently and gave riseto other parts, led students of cell-lineage generally to the view T that there must be some protoplasmic difference between such blastomeres. though it might not be directly visible.
On the other hand were those who maintained that the protoplasm of the early cleavage stages was undifferentiated and that specifications which determined the fate of these cells arose only at a later period and under the influence of environmental or extrinsic conditions, such as mutual interaction between the cells, position in the developing embryo, etc. Such views were maintained on the ground of experimental work, especially that of Driesch, Hertwig, Morgan. Wilson and others, but it should not be forgotten that the experimental work of Roux furnished important evidence in favor of the independent differentiation, " Selbstdifferenzirni/o-" . of different blastomeres.
Thus while the study of cell-lineaue showed conclusively that certain cells were destined in the course of normal development to give rise to certain organs and that the individual blastomeres were more or less differentiated from one another, the results of experimental work showed that in many animals individual cleavage cells were capable of giving rise to an entire embryo, and it was. therefore, affirmed by some investigators that these cells could not lie differentiated for any particular end. Inasmuch as these facts of cell-lineage and of experimental embryology were well established, it was only possible to harmonize these discordant results by some form of interpretation. This was undertaken from two different standpoints: (1) It was affirmed that the early cleavage cells were not really differentiated lor any specific end and that each might develop into any part of the embryo; if in any ea-e certain parts or organs came from certain blastomeres it was due merely to the '*' continuity of development" (Hertwig. ().. 1.892).
(2) On the other hand, it was suggested that these discordant results as to the differentiation of tin/ early cleavage cells might be explained by the fact that the eggs of different animals might differ in the time at which differentiations arise. In the eggs of echinoderms. Amphioxits.. fishes and frogs, which had been chiefly employed in experimental work, the cleavage was not known to be constant and differential in character; whereas in all forms the cell-lineage of which was known, the cleavage was both constant and differential. I therefore suggested i ! "- '. ' 7 1 that for the present it would be advisable to recognize two types of cleavage, a determinate type in which the blastomeres are differentiated from one another and are constant in their manner of origin and development, and an indeterminate type.in which such differentiation and constancy are not known to occur. At the same time I was careful to state that this indeterminateness might be only apparent and not real, and "that the denial of a definite prospective value to each blastomere might rest upon the curious basis that no one had followed a single blastomere through the development" (1897, p. 191). In favor of such a distinction was the experimental work which had been done on the eggs of ctenophores and gasteropods ; the cleavage in these animals is known to be determinate, and it was found that from a part of an egg only a part of an embryo would develop. In all cases constant and differential features appear sooner or later in the course of development, but if in some cases they appear late in the cleavage while in others they appear early this would explain the fact that in some species a whole embryo may he produced from one of the first two or first four blastomeres, whereas in other cases only a partial embryo results. Wilson in particular has defended the view that specifications arise at different times in different eggs, and that these differences in the time of specification may explain the different potencies of blastomeres or portions of the egg.
While it is entirely possible that differentiations may appear in some cases earlier than in others, experiments on the development of parts of eggs are no satisfactory test of the presence or absence of such differentiations as the eggs of echinoderms and ascidians well show. The echinoderms were supposed to present one of the best examples of an indeterminate form of cleavage; fragments of the egg or isolated blastomeres here give rise to entire embryos, and it was concluded that differentiations must appear in these eggs relatively late in development. But Boveri (1901) has shown that in Strongyloceiitrotus, and presumably in other echinoderms also, 1 a remarkable stratification of the egg. corresponding to the primary organs of the larva, appears at the time of the maturation of the egg. These observations have taught us more with regard to the actual differentiations of this egg. as contrasted with the potencies of its parts, than all the experiments which have ever been made. Again, the ascidian egg has one of the most determinate and morphogenetic forms of cleavage known and the differentiations of the various parts of the unsegmented egg are very great, and yet the experiments of Driesch i L895, 1903) and Crampton (1897) have shown that entire embryos may be produced from isolated blastomeres of this egg ; such experiments apparently demonstrate the totipotence of the first four blastomeres of the ascidian egg, 2 but all the
See foot-note, p. 89.
! Since this paper was written I have carefully studied the potency of individual blastomeres of the ascidian egg by the experimental method. My result, which will be published elsewhere, show that nothing resembling a normal embryo or larva is ever produced from any fragment of an egg which experiments in the world could not have shown as satisfactorily as direct observation has done the remarkable cytoplasmic differentiations and localizations of this egg.
It seems, therefore, that this apparent conflict between the results of observation and of experiment on the early development of the egg, between the prospective tendency and the prospective potency of its various parts, can be harmonized
neither by the claim that differentiations do not exist in the early stages of development nor by the assumption that differentiations appear earlier in some eases
than in others.
(3) It seems rather that the true explanation of this discrepancy is the one originally suggested by Roux (1892, 1895), viz., that there is a difference in the regenerative or regulative capacity of different ova and that in the experimental studies referred to we are dealing with indirect development or regeneration, as contrasted with direct or normal development . Just as some adult forms show little capacity tor regeneration or regulation while others of equally complex differentiation show this power in a high degree, so it seems that the capacity for regulation shown by eggs is more or less independent of the degree of their differentiation. To all appearances the ascidian egg is more highly differentiated than those of mollusks or ctenophores, and yet the former has a much higher regulative capacity than the latter. If this view of the relative independence of differentiation and regulation be correct the conflict between the results of cell-lineage and of experimental e/ubryology disappears, for the prospective tendency or the actual differentiation of a blastomere and its prospective potency deal with two distinct things.
2. Localisation before Cleavage
The phenomena of germinal localization have heretofore been studied for the most part during the cleavage and subsequent periods of development ; only within the last few years has this study been extended to the egg before cleavage. Nevertheless the brilliant researches of Driesch, Lillie, Boveri, Fischel, Wilson and Carazzi in this field have already yielded most important results, and are full of promise for future work. In some cases this localization of different kinds of protoplasm or of organ-forming substances has been directly observed, in other cases it has been inferred from the results of experiment, but in many instances both observation and experiment lead to the conclusion that the morphogenetic processes begin before cleavage. The work of Lillie on Unio (1901) and Chestopterus (1902), and especially experiments of Fischel (1897, L898, 1903) on the ctenophore egg, and of Wilson (1903), and Yatsu (1904) on the nemertine egg have shown that definite regions of the unsegmented egg give rise to definite organs or regions of the embryo.
Apart from the early separation of protoplasm and }*olk which occurs in many yolk-laden eggs, localization of visibly different kinds of protoplasm in the unsegmented egg has been observed in relatively few cases. Among the earliest observations of this sort are those of Robin (1875) and Whitman (1878) on the eggs of leeches. Here peculiar aggregations of protoplasm occur at the two poles of the egg, after maturation and fertilization, which have been called " polar rings." Vejdovsky (1888) discovered that these polar rings arise in Rhynchelmis from a peripheral layer of brown protoplasm, which has a great affinity for stains. The substance of this layer collects at the two poles of the egg after maturation and fertilization and thus constitutes the polar rings. During the cleavage most of this substance is segregated into the large posterior macromere of the 4-cell stage, and it ultimately passes into the mesomeres (probably the first and second somatoblasts of Wilson). Nevertheless other portions of this protoplasm go into the micromeres; in fact it forms " the general material for the building of the body, with the exception of the intestinal epithelium" (Vejdovsky. 1888, p. 123). Polar rings have also been observed by Foot (1S04. 1896) in AUolobophora, and their method of formation in this form has been determined in a most careful and satisfactory manner ; this work will be discussed more fully in the next section on the genesis of egg organization.
does not include the whole of the right or lilt half. Individual blastomeres produce rounded masses cf cells but have <> power '" give rise to muscle, chorda, neural plate or senst organs, if they do nut contain thost portions of the egg which normally givt rise to these parts.
One of the most remarkable cases on record of the localization of visibly different kinds of ooplasm is found in Myzostoma glabrum in which Driesch (1S!J0). and
more recently Carazzi (1004), observed two conspicuous zones of protoplasm in the
egg before maturation, an upper one which is of a redish tint and a lower one which
is green. During the maturation of the egg the upper zone differentiates into
two. an upper red zone and an equatorial colorless one. According to Driesch
(1896. p. 120) the red zone gives rise principally to the substance of the micromeres (ectoderm), the clear zone to endoderm. and the green one to the substance
of the somatoblasts (ectoderm and mesoderm).
Another case of visible localization of the substances of the unsegmented egg was observed by Boveri ( L901) in the ovocyte, egg and larva of Strongylocentroius lividus ; here before maturation and fertilization the surface of the egg is covered by a uniformly distributed red pigment; after maturation this gathers into an equatorial zone leaving an area of clear protoplasm at the upper pole and another at the lower one. Later development shows that the upper clear cap gives rise to the ectoderm, the red zone to endoderm and the lower cap to mesenchyme.
A visible localization of differently colored substances in the unsegmented eggalso occurs in fresh water snails belonging to the genera Physa, Planorbis and Limncza. In these animals I have found (Conklin. 1903) that a clear cap of protoplasm appears at the upper pole during maturation and then gradually spreads, over the upper hemisphere of the egg; the upper hemisphere thus becomes milkywhite in the living; egg, while the lower half remains yellow. I have followed these white and yellow substances through the development and find that the white substance gives rise to the ectoderm, the yellow to the mesoderm and endoderm.
Quite recently Wilson ( L904) has observed in Dentalium a localization of unlike substances in the unsegmented egg and by a series of experiments he has shown the part which some of these substances take in the formation of certain organs of the larva. As in the case of Strongylocentroius there is here an accumulation of clear protoplasm at each pole of the egg with a broad pigment band around the equatorial region. The clear polar areas, the lower of which forms a prominent lobe, Wilson regards as comparable with the "polar rings" of leeches and oligochaetes. 1 In the course of development the upper white area is allotted to the three quartets of ectomeres; the middle pigmented zone is mainly allotted to the four basal entomeres, while the lower zone passes mainly into the first somatoblast (2d), and possibly also into the second somatoblast (4d) and the left posterior micromere (3d). This work is the most complete and important which has yet been done on the subject of cytoplasmic localization and it firmly establishes the fact that different substances and areas of the unsegmented egg are causally related to different organs and parts of the larva.
It is doubtful whether any other case of cytoplasmic localization hitherto reported is more remarkable than that which has been described in the preceding pages for the ascidian egg. The most striking features of this localization are the great differences in the substances localized, the manner in which this localization is accomplished and its bilateral character.
(1) The first of these features is the result of the different pigments which are assoeiated with the different kinds of protoplasm, and which mark out as on a map the various germinal areas of the egg. In Cynthia the pigment in the peripheral layer of protoplasm is yellow, the yolk is a blueish gray, while the protoplasm which escapes from the germinal vesicle is colorless. Not the pigment but the protoplasm with which it is associated is of differential value, for the pigment may differ most remarkably in different genera of ascidians. but the organs which arise from similar areas are in all eases similar. What has been said of the pigment may also be said of the yolk; this inert substance is not in itself of differential value, but it lies in a definite region of the egg and probably in a particular kind of protoplasm, which it marks out as the yellow pigment does the peripheral layer.
Of these three kinds of protoplasm the yellow (mesoplasm) goes almost entirely into the muscle and mesenchyme cells, though a small portion of it may be found around the nuclei of other cells, the clear protoplasm (ectoplasm) is chiefly distributed to the ectoderm and the gray yolk-laden protoplasm (endoplasm) 2 to the endoderm, though here also some of these substances are distributed to all the cells. It is not to be supposed that these three kinds of protoplasm are the only ones present in the Q^iX. rather it is probable that others are present which are not visibly distinguishable. In fact, soon after the cleavage begins, it is noticeable that the protoplasm in the dorsal part of the crescent is a fainter yellow than that in the ventral part, while from the time of the fertilization onward the middle of the crescent is marked by a small area of clear protoplasm {v. p. 21) ; the deeply pigmented portion of the crescent gives rise to the muscle cells, the lighter or clearer portions to mesenchyme. Inasmuch as the protoplasm which enters into the muscle cells and mesenchyme is localized with such definiteness in the unsegmented egg it can scarcely be supposed that the substances which are to give rise to the neural plate and notochord are not also definitely localized though they may not be directly visible. 1 If this presumption is correct the visibly different organ-forming substances are by no means the only ones present.
Several years ago I suggested (Conklin 1897, p. 39) that the yolk lobe (" polar lobe," Wilson was comparable to the polar rings of leeches.
- It should lie observed that these names are given with reference to the part which these different portions of the ooplasm play in the development of the animal; the peripheral layer of the ovocyte, which would be called ectophtzm if the ovocyte alone were under consideration, is mesoplasm when regarded from the standpoint of its fate in development.
(2) The striking effect of this cytoplasmic differentiation is heightened by the manner in which localization takes place. The downrush of the peripheral la}'er of yellow protoplasm to meet the entering sperm, the subsequent movement of this protoplasm together with the sperm nucleus to the posterior pole and the formation there of the crescent, the migration of the clear protoplasm to the lower pole, thence to the posterior pole and then to the center of the egg. these phenomena are so evident and they occur so rapidly that they strike the observer with amazement.
(3) Finally the bilateral character of this localization is most notable. In all other recorded cases of cytoplasmic localization the various substances become arranged in zones around the chief axis of the egg and the symmetry is apparently radial; here the early stages of localization are also of this sort, and the gray upper pole, the clear middle zone and the yellow lower pole of the Cynthia egg immediately after fertilization are not unlike the localizations in the eggs of Myzosloma or Strongylocent rotus, but in the ascidian this apparent radial symmetry gives place almost immediately to a marked bilateral symmetry which is brought about by the movement of the protoplasm from the lower hemisphere to the posterior pole and the formation there of the crescent.
Certain fundamental resemblances which run through all these cases of cytoplasmic localization are so striking that the}- scarcely need any emphasis hei'e. The existence in the unsegmented egg of a peripheral layer of protoplasm which is clearly distinguishable from the remainder of the egg is a phenomenon of very wide occurrence. In most of the cases just named this peripheral layer aggregates at one or both poles of the egg after fertilization, and in animals belonging to phyla as far apart as annelids, echinoderms. mollusks and chorda tes the substances at the upper pole give rise to ectoderm, those at the lower pole to mesoderm, while the endoderm arises frotn the region intermediate between these two. Although many differences appear in the later development of these animals they do not detract from the value of these fundamental resemblances which apparently afford a sound basis for a comparative morphology of ova.
1 Since this was written I have heen able to distinguish the chora neural-plate substance as early as the 2-cell stage ; it is the light gray protoplasm at the anterior border of the dorsal hemisphere (figs. 28, 32 et seq.) Photomicrographs of living egg of this stage will be published soon in which this substance is clearly shown.
D. Genesis of the Organization of the Egg
It is probable that the differentiations of egg cells, of blastomeres, and possibly t)f all types of cells, are reducible to two fundamental processes: (1) the genesis of unlike substances, and (2) the localization of these substances in definite parts. Few observations or experiments have been made on the former of these processes and probably no other problem of development would better repay a thorough investigation; the localization problem has been approached from many sides and has yielded results of great interest and importance.
It is a significant fact that localization in the unsegmented egg takes place in so many cases at the time of maturation and fertilization. This is the case in certain ascidians, fresh-water snails, nemerteans and echinoderms ; in Myzostoma and Dentalium the two poles of the egg are dissimilar while the egg is still in the ovary, but here also active localization goes on during maturation. In ascidians and fresh-water snails it is not possible to determine whether the movements which lead to localization are dependent upon the maturation or upon the fertilization of the egg. since as yet it has not been possible to separate experimentally these processes; they certainly seem to be associated with the entrance of the spermatozoon, but since the maturation does not here occur until after the fertilization, it is not possible to determine with certainty the relative importance of these two processes in causing localization. In Strongylocentrotus the movements which lead to the formation of the red pigment zone occur after the extrusion of both polar bodies and before fertilization ; in this case therefore the localization is associated with the maturation.
1 . Role of the Nucleus in Differentiation - Cytoplasmic Organisation and the Nuclear Inheritance Theory
The localization which is effected in the ascidian egg upon the entrance of the spermatozoon is by no means the initial localization in this egg. In the ovocyte 1) 'tore maturation and fertilization the mesoplasm, which later give rise to the mesoderm, exists as a peripheral layer of protoplasm, the ectoplasm, which in later stages is chiefly distributed to the ectoderm, is in large part contained within the germinal vesicle, while the yolk-laden portion of the egg, the endoplasm, which later passes largely into the endoderm, is nearly central in position (figs. 61, 76). At an earlier stage neither the peripheral layer nor the yolk are recognizable as such ; the cell body is composed of granular deeply-staining protoplasm, and around the nucleus is a distinct granular mass, the "yolk matrix" of Crampton (1899). In the very young ovocyte this granular mass is situated chiefly on one side of the nucleus, and frequently contains at its center a large granule, surrounded by a clear aria, which I take to be the centrosome ; the granular mass surrounding this is accordingly sphere material or archoplasm.
In the growth of the ovocyte the sphere material enlarges and spreads around the nucleus, forming the yolk nucleus or matrix; it then begins to disintegrate into granules or larger masses. 1 as described by Crampton, which wander out into the cell body. Crampton has observed that these granules give rise to the yolk spherules which first appear in the protoplasmic ground substance around the nucleus, leaving the peripheral layer of the egg free from yolk.
I am of the opinion that the peripheral layer also contains portions of the archoplasm or sphere material; the staining reactions of this layer are like those of the archoplasm; in the disintegration of the sphere flocculcnt masses of archoplasm pass into this layer; finally, comparison with other forms favors this view. The careful observations of Foot ( 1 S < > ) on the yolk nucleus and polar rings of Allohbophora show that in this animal the polar rings may he traced back step by step to a substance in the vicinity of the nucleus of the very young ovocyte, which Foot identifies with archoplasm. In the later stages this substance becomes distributed throughout the cell and forms a more or less irregular peripheral layer; finally the substance of this layer aggregates at the two poles of the egg to form the polar rings, as previously described.
Among gasteropods the sphere material is largely of nuclear origin, containing nuclear sap and dissolved oxj'chromatin, which have escaped from the nucleus during the period of mitosis (Conklin, 1902) ; if the same be true of the ascidians both the peripheral layer of protoplasm (mesoplasm) and the yolk (endoplasm) contain elements which were ultimately derived from the nucleus at the last ovogonic division.
The clear protoplasm (ectoplasm) which is apparent in the egg after maturation, and which, in the course of development, passes mainly into the ectoderm is largely contained within the nucleus of the ovocyte. In the first maturation division an extremely large quantity of nuclear sap, containing an unusual amount of dissolved oxychromatin, escapes into the cell body where it can be recognized as an area of clear protoplasm. This clear protoplasm can be followed through a large part of the development, both in ascidians and in gasteropods. In the latter particularly this clear nuclear plasm is plainly visible in the living egg. It forms a fusiform or columnar area around the first maturation spindle, and after the formation of the polar bodies it flattens out at the surface of the egg, forming first a cone, then a lenticular mass, and finally a cap of clear protoplasm. This cap extends down over the egg to a region a little below the equator, and finally during cleavage it is largely localized in the three quartets of ectomeres.
In the ascidians the later history of this nuclear plasm is not so easily followed as in the gasteropods, owing to the presence of a peripheral layer of mesoplasm. and to the fact that its movements here are more extensive and complicated. In Cynthia it flows to the lower pole along with the yellow mesoplasm. then it moves with the sperm nucleus to the posterior side of the egg and finally to its center. Here it surrounds the cleavage spindle, and at the close of the fust cleavage moves toward the animal pole so that the larger part of it comes to lie in the upper hemisphere. In subsequent divisions it surrounds all the nuclei though the most of it goes into the ectodermal cells as in the case of gasteropods.
- These fragments of the yolk nucleus are larger and more easily seen in Molgula than in either or Cynthia.
This truly remarkable condition in which considerable portions of the cytoplasm are traceable to the nucleus is of the utmost theoretical importance. From all sides the evidence has been accumulating that the chromosomes are the seat of the inheritance material, until now this theory practically amounts to a demonstration. On the other hand, all students of the early history of the egg have observed that the earliest visible differentiations occur in the cytoplasm, and that the position, size and quality of the cleavage cells and of various organ liases are controlled by the cytoplasm. However, in the escape of large quantities of nuclear material into the cell body and the /or/nation there of specific protoplasmic substances we have a possible mechanism for the nuclear control of the cytoplasm, and when, as in the case of the ascidians and fresh water gastcropods, these substances are definitely localized in the egg, and ca?i be traced throughout the development until they cuter into the formation of particular portions of the embryo, a specific mechanism for the nuclear control of development is at hand, and the manner of harmonizing the pacts of cytoplasmic organization with the nuclear inheritance theory is clearly indicated.
Of course substances which enter the nucleus and contribute to its growth must reach it through the cytoplasm, but this does not signify that the same substances are given hack to the cytoplasm as are taken up from it ; on the contrary we know that some of the substances which escape from the nucleus (e. g., oxychromatin) are not identical with those which enter it. Considering the necessity of the nucleus in assimilation and regeneration, it seems most likely that differentiations of the cytoplasm proceed in the first instance from the nucleus; and, indeed, in the case of the egg cell, some of the important cytoplasmic substances can be actually seen to come from the nucleus. This does not indicate that these substances exist from the beginning in the nucleus; on the contrary there is direct and visible evidence that they arise epigenetically. Such epigenesis, however, does not signify lack of primary organization; on the other hand all the evidence favors the view that back of the organization of the cytoplasm is the organization of the chromosomes, which is definite, determinate and primary.
What has been said with regard to the genesis of the different substances of the cytoplasm applies in the main to their localization. It is evident that this localization is progressive, and that it arises epigenetically. But though we may push back this localization to earlier and earlier stages and to simpler and simpler forms we cannot entirely do away with it, even though it may be traced to polarity and chemotropism. Some basis of localization must be present in the earliest stages of the oogenesis, but this may possibly be little more than is found in the body cells in general. It does not seem improbable that the differentiations and localizations of the ovocyte and of the tissue cells are comparable in their manner of origin. The most remarkable difference between the two is that the tissue cells having reached the limit of their differentiation are incapable of further development whereas the egg cell having reached the limit of its differentiation in the ovary may. under the conditions of a free cell, begin another scries of differentiations which lead to the production of an organism.
2. Factors of Localization
a. Cytoplasmic Movements
Undoubtedly the most important of all the localizing factors so far recognized are cytoplasmic movements. Such movements have been observed in unsegmented eggs as well as in the cleavage cells, and they are generally associated with localization of unlike substances and frequently with cell division. The importance of such movements in the differentiations of the egg I first recognized in Crepidula (1899), where the movements of the cytoplasm during cleavage are very extensive. In the ascidian egg, on the other hand, these movements are most pronounced in the period between the fertilization and the close of the first cleavage. In both the ascidian and gasteropod these movements are definitely directed and bringabout a constant and typical form of localization of the materials of the egg.
The fact that these movements are definitely directed shows that they are dependent upon a constant organization of the cell ; their immediate cause is unknown. So far as I have observed, these movements always begin soon after the disappearance of the nuclear membrane and the consequent escape of nuclear material into the cell body. In the case of the gasteropods, I have suggested (1902) that one of the characteristic movements of the telophase of division is due to the affinity of the sphere material for oxygen. After the formation of this sphere material, during each cell division, it moves to the surface of the cell and as nearly as possible to the animal pole. If, however, the eggs be placed in water, from which the oxygen has been removed by boiling, this movement to the surface does not take place. In the ascidian the entrance of the spermatozoon seems to be the inciting cause of the movement. The peripheral protoplasm (mesoplasm) rushes down to the point of entrance and masses around the spermatozoon ; then when the latter moves toward the posterior pole this protoplasm goes with it and is thus gathered into the crescent; finally, when the sperm nucleus moves in toward the centre of the egg the larger part of this protoplasm remains at the surface, while a small portion of it is drawn in with the sperm toward the center of the egg. In these movements, as well as in the subsequent ones during cleavage, the mesoplasm remains near the surface of the cell and in this respect resembles the sphere substance of the gasteropod egg. The flowing of the protoplasm to meet the entering spermatozoon is a phenomenon of rather general occurrence. In most cases this leads only to the formation of a small protoplasmic field around the sperm and sometimes to the formation of an entrance cone ; in the ascidian practically all the protoplasm of the egg takes part in this movement leaving the maturation spindles with only a trace of protoplasm around them. This withdrawal of the protoplasm from the animalpole may be associated with the fact that there are no centrosome or asters in the maturation spindles, whereas there is a large centrosome and aster in connection with the sperm nucleus. Certainly the clear protoplasm is usually found in the region of the asters. What the exact nature of this attraction between the protoplasm and the spermatozoon is, is not known, but the important point here is that the cause of the remarkable movements of the protoplasm which follow the fertilization of the ascidian egg is not unique, and that the whole movement is peculiar only because of its extent and the definite manner in which it is directed.
The movements which take place during cleavage are in part merely the general movements which accompany cell division and in part they are of a localizing character. In the former class are the vortical movements which probably cause the separtion of the chromosomes and the division of the cell body (Conklin, 1902); in the latter are such movements as that which occurs at the close of the first cleavage by which the clear protoplasm is carried from a central position into the upper hemisphere of the egg. After the cleavage has begun the localizations due to movement are strictly limited to the individual cells, no movements of a localizing character occurring through cell w r alls.
b. Cell Division as a Factor of Localization
This brings us to the much discussed question of the role of cell division in development, and more particularly of the influence of cell division on phenomena of localization. There can he no doubt that in many eggs the localization which begins before cleavage continues during that process.
To a certain extent cleavage may he regarded as a localizing factor, but its importance in this respect is certainly far less than that of the active movements just described. Inasmuch as localizations may take place in the absence of cleavage or before it begins, and since many cleavages are non-differential it is evident that there is no close nor necessary connection between the two. Furthermore the cleavage planes do not always coincide with the lines of localization ; this is shown especially well in the ascidian. where the localization in the unsegmented egg is particularly distinct. Thus the cleavage planes do not follow closely the boundaries of the crescent ; the first and second cleavage planes are placed symmetrically with reference to the crescent, but they do not coincide with any of its boundary lines. The third cleavage plane lies above the upper border of the crescent when first formed; later the crescent extends up to the equatorial plane so that the cleavage plane and the upper boundary of the crescent coincide (fig. 31, 32). The fourth cleavage cuts off the median posterior crescent cells from the lateral ones, but leaves an area of yolk in both of these cells (fig. 37). In the median posterior cells this is a small wedge-shaped mass of yolk which is later covered and obscured by the yellow crescent substance (fig. 39). The neural plate arises on the anterior side of the egg from cells which lie both above and below the equator, or plane of the third cleavage ; these neural plate cells are rich in protoplasm, and correspondingly the area from which they arise is richly protoplasmic. The third cleavage cuts right through this protoplasmic area leaving a portion of it above and a part below the equator. In the 8-cell and 16-cell stages the anterior dorsal cells contain both neural-plate and chorda substance; the portion of each of these cells turned toward the equator is protoplasmic, that turned toward the vegetal pole yolk-laden (A 63 , AH figs. XVII. XIX. 110, 117). At the next cleavage these two portions are separated, the upper protoplasmic part becoming the neural plate cells. A 74 and A rs , while the lower yolk-laden part becomes the chorda cells, A 73 and A 77 . The chorda-neural substances arc thus contained in the same cells until the sixth cleavage, though their substances are distinct at a much earlier period. Still other instances might be cited to show that the planes of localization and the planes of cleavage do not always coincide. This is in part due to the fact that the boundaries of the different kinds of germinal material, e.g.. the yellow protoplasm of the Cynthia egg, are not as sharp as are the boundaries of the cells, and consequently the cleavage furrows cannot precisely separate different kinds of germinal material. Nevertheless the cleavage planes are, under normal conditions, constant in position and character and bear a constant relation to the planes of differentiation. But that this relationship is not a casual one is further indicated by experimental studies on cleavage in which the position of the cleavage furrows may be altered without altering the localization of germinal materials or the typical form of development. Therefore the factors which determine localization and those which determine the form of cleavage are more or less independent.
All of these tacts speak unmistakably for the view that localization is more fundamental than cleavage as Whitman (1893) has so ably maintained, and that such correspondence as may exist between the two is of secondary origin and of minor importance. Nevertheless the extreme constancy of cleavage forms shows that we have here a phenomenon, which if of secondary importance to germinal localization, is still of real significance. I have shown that in Crepidula the cleavage is a localizing factor, though secondary in importance to protoplasmic movement, and it seems probable that Wilson (1003) is right when he argues that the relative isolation produced by cleavage gives opportunity for the increase of any initial differences which may exist in the cells at the time of their formation.
Finally it must be concluded as a result of both observation and experiment that the type of cleavage is less constant and less fundamental than the type of localization, but that cleavage may itself be a factor in the progressive specification of cells {cf. Wilson, Lillie, Conklin, et al.).
E. Types of Germinal Localization - Evolution of Types
The wonderful resemblances in the germinal localization of annelids and mollusks, as shown especially in the cleavage, have been repeatedly commented upon. Furthermore this localization is for shadowed in the egg before cleavage begins, and this suggests the inquiry as to whether the resemblances between types of localization grow closer as one approaches the ovocyte, and whether the manner as well as the results of localization are comparable in the different types. At present our knowledge of the localization in these earliest stages of development is very incomplete, and a comparison can be drawn only between annelids, mollusks, ctenophores, echinoderms, ascidians and possibly nemerteans and nematodes.
In most of these phyla a peripheral layer of protoplasm is present before maturation, which after maturation and fertilization collects at one or both poles of the egg; also with the possible exception of the ctenophores and nematodes, there is remarkable uniformity in the localization of the substances of the germinal layers in all of these groups, the ectodermal substances being located in the upper hemisphere, and the endodermal and mesodermal in the lower hemisphere of the
egg. But in the localization of important organ bases there are many differences between these phyla.
1. Annelid-Mollusk Type
The pattern of localization in annelids and mollusks is very similar during the cleavage stages and, so far as can be judged from present knowledge, it is much the same in the unsegmented eggs of these two phyla. The fact that the ectoderm, mesoderm and endoderm come from cells which are identical in origin, position and number; that the umbrella, prototroch, cerebral ganglion, sub-oesophageal ganglion, mesodermal bands, blastopore, stomodaium and intestine come from corresponding region of the egg in the two groups, these facts speak strongly in favor of the regional homologies of the eggs of these phyla, whatever may be thought of their cell homologies (Conklin, 1897; Child, 1900). But regional homologies as well as cell homologies must be based upon similarities of germinal localization, and we would, therefore, be justified in concluding that the types of localization were similar in the unsegmented eggs of annelids and mollusks even in the absence of any direct knowledge upon that subject. But the experiments of Crampton (1896) on Illyanassa and of Wilson (1904) on Dentalium as well as the observations of Lillie (1899, 1901) on [/mo, and my own observations on localization in the eggs of Crepidula, Pliysa, Planorbis and Limncva furnish considerable information as to the time, the manner and the nature of localization in the molluscan egg during and before cleavage, while numerous works on the cell-lineage of the annelids as well as the observations of Wheeler (1897), Driesch (1890) and Carazzi (1904) on the unsegmented egg of Myzosioma show that the nature of localization is here very similar to that found in the mollusks.
In all of these cases the only formative substances which are directly recognizable before cleavage are those of the future germ layers. In the main the ectodermal substances are located in the upper hemisphere and the endodermal in the lower, though Wilson (1904) has found that the apical organ does not form in the larva of Dentalium when the polar lobe at the vegetal pole is removed. The mesodermal substances are also located in the lower hemisphere, and since the primary mesoderm cell (4d) always come from the left posterior macromere of the 4-cell stage and from the posterior blastomere of the 2-cell stage it may be inferred that immediately before cleavage it lies posterior to the vegetal pole; whether it may be located exactly at the vegetal pole in still earlier stages and then later shift to the posterior side, as in ascidians, cannot be determined at present.
When a polar lobe is present the mesodermal substance is probably located in it. Crampton (1896) found in Illyanassa that the mesoblast cell (4d) did not form when the lobe had been removed; Wilson (1904) holds that in Dentalium the substance of the lobe is allotted to both the first and second somatoblasts'(2d and 4d), and that its size is proportional to the size of those cells and of the parts to which they give rise. He calls particular attention to the fundamental resemblances between the eggs of Dcntalium and of Mysostoma'va the matter of the polar lobe and the " pillar of protoplasm." Furthermore this lobe is comparable to the polar rings of leeches and oligochastes. Such a lobe, although present in some annelids and mollusks, is not present in all of them, and this would at first thought seem to mark some important difference in localization. But the presence or absence of such a lobe probably indicates no fundamental dissimilarity in the localization, but rather variations in the surface tension and fluidity of different eggs. Although there are many interesting differences between various annelids and mollusks in the size of the polar lobe, of the blastomeres and of larval organs, these differences mark variations in the proportions of parts rather than in the type of localization. In all known cases among annelids and mollusks corresponding organs arise from corresponding regions of the egg.
It may be concluded also from the work of Wilson (1903) and Yatsu (1904) on Cerebratulns that the character of the localization in the nemertine egg is essentially like that of the annelid and mollusk, though many of the details of localization are less accurately known in this case than in the others named.
2. Ctenophore Type
If Fischel (1903) is right regarding the localization which he ascribes to the unsegmented ctenophore egg there is one fundamental difference between the ctenophore and other animals whose types of localization are known. On the authority of Metschnikoff he derives the mesoderm (somewhat doubtfully it must be said) from the micromeres at the upper pole of the egg, and consequently in his fig. 21 (p. 708) he localizes the mesodermal material at the upper pole of the unsegmented egg. A zone beloAv this, reaching to the equator or a little lower, represents the ectodermal substance, and in it is located the material for the ciliated plates. At the lower pole and in the central part of the egg is the material substratum of the endoderm. In all other well established cases the ectodermal substances lie near the animal pole, while the mesodermal and endodermal substances lie near the vegetal pole. Inasmuch as an apical sense organ is formed at the animal pole in ctenophores in much the same way as in annelids, mollusks and nemerteans, it is difficult to resist the conclusion that the localization of the ectodermal, mesodermal and endodermal substances in the ctenophore egg will ultimately be found to be similar to that which prevails in other types.
3. Echinoderm Type
The form of localization in the echinoderm egg, as shown by Boveri's (1901) work on Strongylocentrotits, is in many respects similar to the annelidmollusk-nemertean type. In this case, however, the mesoplasm is located at the lower pole of the egg and is sorrounded hy an equatorial zone of endoplasm, whereas in annelids and mollusks, after the first two cleavages, the endoplasm lies at the lower pole, and the mesoplasm on the posterior side of this pole, and in one only of the first four blastomeres. Whether the mesoplasm lies on one .side of the vegetal pole in the unsegmented egg of annelids and mollusks cannot be affirmed on direct evidence, but it seems not unlikely that this is the case. If this be true there is here a difference between echinoderms and annelids or mollusks, in the form of localization, though it is by no means impossible to derive one type from the other.
4. Ascidian Type
Finally, the type of localization in the ascidian egg differs in many respects from that of the other phyla mentioned, though showing certain general resemblances to all of them and particularly to the annelid-mollusk type. Castle has called attention to the fact that there are no important resemblances between ascidians and annelids in their cell-lineage, and with this opinion I entirely agree. Nevertheless, in the localization of ectoplasm, mesoplasm and endoplasm in the unsegmented egg there are many similarities between these phyla, hut in the position of specific organ bases the differences are quite notable.
Among ascidians the ectoplasm which escapes from the germinal vesicle at the animal pole does not remain there, as in the fresh-water snails, but flows rapidly to the lower pole, then to the posterior side of the egg, then into the center and finally into the upper hemisphere of the egg; in other phyla the ectoplasm becomes directly localized at the upper pole, here only indirectly. The mesoplasm is first segregated at the lower pole in a manner which recalls the egg of Strongylocentrotus, and then finally becomes localized on the posterior side, a result which somewhat resembles the condition in annelids and mollusks ; in the ascidians the cells of the mesodermal crescent lie in the posterior lip of the blastopore, in annelids and mollusks the teloblasts and mesodermal bands lie in a similar position in the early gastrula stages, but owing to the closure of the blastopore from behind forward they are ultimately removed some distance from the blastopore lip. The mesoderm and mesodermal organs may therefore be said to arise from corresponding regions of the egg in these two groups of animals (text figs. XXXIX, XL). The endoplasm also is localized in corresponding regions of the egg in these phyla.
When, however, we come to compare the positions in the eggs of these phyla of important organ bases the differences are very marked. For example, in annelids and mollusks the apical plate and cerebral ganglion are formed near the animal pole, the sub-oesophageal ganglia from the ventral plate, which is derived from the cell 2d, lying not far below the equator on the posterior-dorsal side and just above the mesodermal teloblasts (text fig. XL) ; subsequently in the concresence of the posterior lip of the blastopore, the bases of the sub-oesophageal ganglia are carried to the ventral side. The nervous system of annelids and mollusks thus has a double origin, one portion arising from the region of the animal pole, the other from the posterior pole, and these two portions subsequently become connected together by commissures which surround the oesophagus. Jn the ascidian the entire central nervous system is formed as a continuous plate which lies along the anterior side of the egg, stretching from a point about 60 from the animal pole and from the equator to a point a little below the equator (text fig. XXXIX); no portion of the nervous system comes from the region of the animal pole and none from the posterior pole. Furthermore, the mouth, which is here a new formation and has nothing to do with the blastopore, does not open through the nerve plate but lies between the anterior end of the neural plate and the animal pole.
We have here differences of a fundamental order, even in the earliest stages of development, between the vertebrate, or rather chordate, and the invertebrate; the early development throws no light upon the way in which the one may have been derived from the other. It is of course possible to conceive of a condition in which the nervous system surrounded the entire blastopore as a ring, which in the case of the annelids underwent concrescence from behind forward, thus forming the ventral plate and ganglia, but which in the chordates underwent concrescence from in front backwards. But however probable such a theory may be it finds little support in the early development of ascidians. It is true that a nerve ring has been described as surrounding the blastopore in ascidians, but I have not been able to find evidence of its existence. Furthermore, there is no evidence in the development of ascidians that there is any concrescence of the anterior lip of the blastopore ; on the contrary the anterior lip grows backward over the archenteron as rapidly in the mid-line as at the sides, a view in which practically all writers on ascidian embryology agree. Finally, the lack of an apical plate and cerebral ganglion at the animal pole in the ascidian constitutes a notable difference from the condition found in most invertebrates. In his great work on Sa/pa, Brooks (1893) has shown in masterly fashion the weakness of the annelidan hypothesis of the origin of chordates and has adduced much evidence in favor of the view that the great metazoic stems run back to simple and minute pelagic ancestors whose common meeting placemust lie be found in still more recent times. The earliest dift'erentations of the egg seem to me to favor this view.
Figs. XXXIX, XL. Diagramatic representations of the types of germinal localization in ascidians and annelids. Mesodermal substance is shaded by lines, neural substance by fine stipples, and chorda material by coarse stipples. Fig. XXXIX, the ascidian type; egg viewed from right side. The mesoplasm, composed of mesenchyme (m'ch) and muscle substance (msl, is represented in its final position, which it assumes before the first cleavage. The neural plate (n. p.) and chorda (eh.) substances are not distinguishable in the unsegmented egg. but are here shown in the positions in which they appear at the 2-cell stage; the chorda and mesenchyme substances should be shown as meeting on the side of the egg, thus forming a chorda-mesenchyme ring around the endoderm. The ectoplasm (ect.) and endoplasm (end.) are localized, as here represented, at the close of the first cleavage. Fig. XL, the annelid type; egg viewed from left side. The substances of the first and second somatoblasts (the former stippled along one border, the latter shaded by lines) are shown in the positions in which these cells are ultimately formed ; in the unsegmented egg the lobe which contains the substances of these cells lies nearer the lower pole. The substances of the cerebral ganglion (c. g.), ventral ganglia (v. g.) and prototroch (proto.) are not distinguishable in the unsegmented egg, but are shown in the regions to which they may be traced by means of the cell lineage.
In conclusion, then, it seems necessary to recognize several types of cytoplasmic localization. Between annelids and mollusks the similarities of localization
extend to the bases of numerous parts and organs, thus confirming the view of
the phylogenetic relationship of these two phyla based upon the resemblances in their
cleavage stages and larvae. Between the annelid-mollusk type of localization and
tin' types found in the other phyla enumerated there are general agreements in the
localization of the materials of the germinal layers, but few, if any, resemblances
which extend to the bases of particular organs. The annelids do not approach
the chordates nor the echinoderms in the earliest stages of localization any more
closely than in their cleavage stages or later development. In all respects in which
localizations differ in the eggs of these animals they resemble the later differences
in their embryos. In short, there is no convergence toward a co?n)non type of localization as one goes back to earlier and earlier stages in the ontogeny.
Important results flow from this conclusion, for the doctrine that " Ontogeny is a short recapitulation of Phylogeny" assumes that there is such convergence toward a common type of structure in the early stages of development. If there be no such convergence the causes of the resemblances which exist between certain eggs, cleavage stages, embryos, larvas and adults must be sought in some other direction. Students of the cell-lineage of annelids and mollusks have maintained that homologies of cleavage must be due to similarities in the protoplasmic structure of the cleavage cells. The same must also be said of the organization of the egg before cleavage begins. Similarities in the material substance of the egg and in the form of its localization must lie at the bottom of all later appearing similarities. And this fact, upon which all students of cell lineage have insisted, furnishes a possible explanation, as Morgan (1903) has recently pointed out, of the resemblances between the embryos of related forms.
Speculations as to the origin and evolution of tyj3es of germinal organization are likely to be more interesting than valuable in the present state of our knowledge. Wilson (1892) first suggested that the localization of the materials of embryonic parts or organs in certain cleavage cells was an illustration of the principle of "precocious segregation" first propounded by Lankester and afterward elaborated by Hyatt, in its application to palaeontology, under the title of the law of acceleration." Lillie (1895) maintained that "it is parallel precocious segregation which conditions cell homologies." and he further showed (1899) that the size and rate of division of individual cells in every case possesses pi'ospeetive significance; in short, that the cleavage forms are beautifully adapted to produce a given type of adult structure. Recently Wilson (1903, 1904) has expressed the view that the earliest differentiations and localizations of the egg, even before cleavage begins, are examples of this same principle of "precocious segregation."
Although this principle is carefully stated so as not to directly affirm that the
organization of the egg is the result of the organization of the adult, or that the
adaptations of the early development have arisen secondarily after the adult structure was estafTHshed, these ideas are nevertheless plainly implied. The early
appearance of differentiations is usually explained as a " throwing hack of adult
characters upon the egg." The whole life cycle is viewed from the standpoint of
the adult ; the embryo and germ exist for the purpose of producing a certain end ;
the adult is primary, the germ secondary. But do not all such ideas put the cart
before the horse ? What is the evidence that any inherited modification of an adult
structure can arise without an antecedent modification of the germ ? We know that
the adult is moulded upon the egg, that specific modifications of the germ do, in
some cases, produce specific modifications of the adult, but the converse proposition
is certainly not established. "Precocious segregation" represents the backward
rather than the forward look; it is a teleological rather than a causal explanation.
As there can be no transmission of heritable qualities from one generation to another except through the germ cells, so there can be no evolution of adult forms except through the evolution of the germ cells. Any inherited modification of a species implies some modification of the germ cells of the species. Even " acceleration" or " precocity" must be due to a modification of the germ in its earliest stages, a modification of some unknown sort which hastens differentiation.
It cannot be maintained that all those animals in which differentiations and localizations are present in the unsegmented egg are, for that reason, debarred from any further evolution, but if this be not true then it must follow that the type of egg organization must undergo modifications during the course of evolution, and granted this we have no need of the principle of " parallel precocious segregation " for explaining any of the homologies of the earl}' development. If the resemblances between annelids and mollusks are not due primarily to the similarities in the adults or larva? or cleavage stages, but to phylogenetic similarities in the organization of the unsegmented egg, we have in this initial resemblance a sufficient explanation of all later resemblances, whereas if we reverse this procedure and hold that the similarities of the adults or larva? are the causes of the likenesses in the earlier stages we must of necessity resort to some such teleological principle as "precocious segregation" for an explanation.
In view of the fact that there are such definite types of differentiation and localization in the eggs of many animals and that the causes which lead to the evolution of animals must operate through modifications of this organization, the character and manner of such modification become problems of the first importance. If the nuclear inheritance theory is true, such modifications must in the first instance affect the chromosomes ; but how and in what respect is wholly unknown. In the case of the cytoplasm it is evident that such modifications may concern the character, or quality, of the differentiations and the place and manner of their localization. Modification of any of these might be expected to produce modifications in the resulting animal.
Relatively slight modifications of this localization, however produced, may lead
to profound modifications of the resulting embryo and adult. I have elsewhere
(1903) shown reason for believing that the cause of inverse symmetry is to be
found in the inverse organization of the egg, and that this inverse organization may
pjssibly be produced by the maturation of the egg at opposite poles in dextral and
sinistral forms. This ease shows that one of the most remarkable forms of mutation with which we are acquainted may be the result of modifications in the localization of germinal substances in the unsegmented egg.
One of the great difficulties in explaining the origin, on evolutionary principles, of different phyla has been the dissimilar locations of corresponding organs or parts. These difficulties are well illustrated by the theories which attempt to derive the vertebrates from annelids, or from any other invertebrate type. Without assuming to defend any of these theories it may here be pointed out that if evolution takes place through modifications of germinal organization, it is no more difficult to explain the different location of parts than their different qualities. Changes in the relative positions of parts, which would be impossible in the adult, may be readily accomplished in the unsegmented egg, as is shown by cases of inverse symmetry. The question is here raised, whether some similar sudden alteration of germinal organization may not lie at the basis of the origin of new types.
Cite this page: Hill, M.A. (2024, April 19) Embryology Paper - The Organization and Cell-Lineage of the Ascidian Egg 7. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_Organization_and_Cell-Lineage_of_the_Ascidian_Egg_7
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