CHAPTER II.

THE SEGMENTATION.

I have not been fortunate enough to obtain an absolutely complete series of eggs during segmentation.

In the cases of Pristiurus and Scyllium only have I had any considerable number of eggs in this condition, though one or two eggs of Raja in which the process was not completed have come into my hands.

In the youngest impregnated Pristiurus eggs, which I have obtained, the germinal disc was already divided into four segments.

The external appearance of the blastoderm, which remains nearly constant during segmentation, has been already well described by Leydig[79].

Footnote 79: _Rochen und Haie._

The yolk has a pale greenish tinge which, on exposure to the air, acquires a yellower hue. The true germinal disc appears as a circular spot of a bright orange colour, and is, according to Leydig's measurements, 1-1/2m. in diameter. Its colour renders it very conspicuous, a feature which is further increased by its being surrounded by a narrow dark line (Pl. 6, fig. 2), the indication of a shallow groove. Surrounding this line is a concentric space which is lighter in colour than the remainder of the yolk, but whose outer border passes by insensible gradations into the yolk. As was mentioned in my preliminary paper (_loc. cit._), and as Leydig (_loc. cit._) had before noticed, the germinal disc is always situated at the pole of the yolk which is near the rounded end of the Pristiurus egg. It occupies a corresponding position in the eggs of both species of Scyllium (stellare and canicula) near the narrower end of the egg to which the shorter pair of strings is attached. The germinal disc in the youngest egg examined, exhibited two furrows which crossed each other at right angles in the centre of the disc, but neither of which reached its edge. These furrows accordingly divided the disc into four segments, completely separated from each other at the centre of the disc, but united near its circumference.

I made sections, though not very satisfactorily, of this germinal disc. The sections shewed that the disc was composed of a protoplasmic basis, in which were imbedded innumerable minute spherical yolk-globules so closely packed as to constitute nearly the whole mass of the germinal disc.

In passing from the coarsest yolk-spheres to the fine spherules of the germinal disc, three bands of different-sized yolk-particles have to be traversed. These bands graduate into one another and are without sharp lines of demarcation. The outer of the three is composed of the largest-sized yolk-spherules which constitute the greater part of the ovum. The middle band forms a concentric layer around the germinal disc, and is composed of yolk-spheres considerably smaller than those outside it. Where it cuts the surface it forms the zone of lighter colour immediately surrounding the germinal disc. The innermost band is formed by the germinal disc itself and is composed of spherules of the smallest size. These features are shewn in Pl. 6, fig. 6, which is the section of a germinal disc with twenty-one segments; in it however the outermost band of spherules is not present.

From this description it is clear, as has already been mentioned in the description of the ripe unimpregnated ovum, that the germinal disc is not to be looked upon as a body entirely distinct from the remainder of the ovum, but merely as a part of the ovum in which the protoplasm is more concentrated and the yolk-spherules smaller than elsewhere. Sections shew that the furrows visible on the surface end below, as indeed they do on the surface, before they reach the external limit of the finely granular matter of the germinal disc. There are therefore at this stage no distinct segments: the otherwise intact germinal disc is merely grooved by two furrows.

I failed to observe any nuclei in the germinal disc just described, but it by no means follows that they were not present.

In the next youngest of the eggs[80] examined the germinal disc was already divided into twenty-one segments. When viewed from the surface (Pl. 6, fig. 3), the segments appeared divided into two distinct groups--an inner group of eleven smaller segments, and an outer group of segments surrounding the former. The segments of both the inner and the outer group were very irregular in shape and varied considerably in size. The amount of irregularity is far from constant and many germinal discs are more regular than the one figured.

Footnote 80: The germinal disc figured was from the egg of a Scyllium stellare and not Pristiurus, but I have also sections of a Pristiurus egg of the same age, which do not differ materially from the Scyllium sections.

In this case the situation of the germinal disc and its relations to the yolk were precisely the same as in the earlier stage.

In sections of this germinal disc (Pl. 6, fig. 6), the groove which separates it from the yolk is well marked on one side, but hardly visible at the other extremity of the section.

Passing from the external features of this stage to those which are displayed by sections, the striking point to be noticed is the persisting continuity of the segments, marked out on the surface, with the floor of the germinal disc.

The furrows which are visible on the surface merely form a pattern, but do not isolate a series of distinct segments. They do not even extend to the limit of the finely granular matter of the germinal disc.

The section represented, Pl. 6, fig. 6, bears out the statements about the segments as seen on the surface. There are three smaller segments in the middle of the section, and two larger at the two ends. These latter are continuous with the coarser yolk-spheres surrounding the germinal disc and are not separated from them by a segmentation furrow.

In a slightly older embryo than the one figured I met with a few completely isolated segments at the surface. These segments were formed by the apparent bifurcation of furrows as they neared the surface of the germinal disc. The segments thus produced are triangular in form. They probably owe their origin to the meeting of two oblique furrows. The last-formed of these furrows apparently ceases to be prolonged after meeting the first-formed furrow. I have not in any case observed an example of two furrows crossing one another at this stage.

The furrows themselves for the most part are by no means simple slits with parallel sides. They exhibit a beaded structure, shewn imperfectly in Pl. 6, fig. 6, but better in Pl. 6, fig. 6_a_, which is executed on a larger scale. They present intervals of dilatations where the protoplasms of the segments on the two sides of the furrow are widely separated, alternating with intervals where the protoplasms of the two segments are almost in contact and are only separated from one another by a very narrow space.

A closer study of the germinal disc at this period shews that the cavities which cause the beaded structure of the furrows are not only present along the lines of the furrows but are also found scattered generally through the germinal disc, though far more thickly in the neighbourhood of the furrows. Their appearance is that of vacuoles, and with these they are probably to be compared. There can be little question that in the living germinal disc they are filled with fluid. In some cases, they are collected in very large numbers in the region of a furrow. Such a case as this is shewn in Pl. 6, fig. 6_b_. In numerous other cases they occur, roughly speaking, alternately on each side of a furrow. Some furrows, though not many, are entirely destitute of these structures. The character of their distribution renders it impossible to overlook the fact that these vacuole-like bodies have important relations with the formation of the segmentation furrows.

Lining the two sides of the segmentation furrows there is present in sections a layer which stains deeply with colouring reagents; and the surface of the blastoderm is stained in the same manner. In neither case is it permissible to suppose that any membrane-like structure is present. In many cases a similar very delicate, but deeply-stained line, invests the vacuolar cavities, but the fluid filling these remains quite unstained. When distinct segments are formed, each of these is surrounded by a similarly stained line.

The yolk-spherules are so numerous, and render even the thinnest section so opaque, that I have failed to make satisfactory observations on the behaviour of the nucleus. I find nuclei in many of the segments, though it is very difficult even to see them, and only in very favourable specimens can their structure be studied. In some cases, two of them lie one on each side of a furrow; and in one case at the extreme end of a furrow I could see two peculiar aggregations of yolk-spherules united by a band through which the furrow, had it been continued, would have passed. The connection (if any exists) between this appearance and the formation of the fresh nuclei in the segments, I have been unable to elucidate.

The peculiar appearances attending the formation of fresh nuclei in connection with cell-division, which have recently been described by so many observers, have hitherto escaped my observation at this stage of the segmentation, though I shall describe them in a later stage. A nucleus of this stage is shewn on Pl. 6, fig. 6_c_. It is lobate in form and is divided by lines into areas in each of which a deeply-stained granule is situated.

The succeeding stages of segmentation present from the surface no fresh features of great interest. The somewhat irregular (Pl. 6, figs. 4 and 5) circular line, which divides the peripheral larger from the central smaller segments, remains for a long time conspicuous. It appears to be the representative of the horizontal furrow which, in the Batrachian ovum, separates the smaller pigmented spheres from the larger spheres of the lower pole of the egg.

As the segments become smaller and smaller, the distinction between the peripheral and the central segments becomes less and less marked; but it has not disappeared by the time that the segments become too small to be seen with the simple lens. When the spheres become smaller than in the germinal disc represented on Pl. 6, fig. 5, the features of segmentation can be more easily and more satisfactorily studied by means of sections.

To the features presented in sections, both of the latter and of the earlier blastoderms, I now return. A section of one of the earlier germinal discs, of about the age of the one represented on Pl. 6, fig. 4, is shewn in Pl. 6, fig. 7.

It is clear at a glance that we are now dealing with true segments completely circumscribed on all sides. The peripheral segments are, as a rule, larger than the more central ones, though in this respect there is considerable irregularity. The segments are becoming smaller by repeated division; but, in addition to this mode of increase, there is now going on outside the germinal disc a segmentation of the yolk, by which fresh segments are being formed from the yolk and added to those which already exist in the germinal disc. One or two such segments are seen in the act of being formed (Pl. 6, fig. 7, _f_); and it is to be noticed that the furrows which will eventually mark out the segments, do so at first in a partial manner only, and do not circumscribe the whole circumference of the segment in the act of being formed. These fresh furrows are thus repetitions on a small scale of the earliest segmentation furrows.

It deserves to be noticed that the portion of the germinal disc which has already undergone segmentation, is still surrounded by a broad band of small-sized yolk-spherules. It appears to me probable that owing to changes taking place in the spherules of the yolk, which result in the formation of fresh spherules of a small size, this band undergoes a continuous renovation.

The uppermost row of segmentation spheres is now commencing to be distinguished from the remainder as a separate layer which becomes progressively more distinct as segmentation proceeds.

The largest segments in this section measure about the 1/100th of an inch in diameter, and the smallest about 1/300th of an inch.

The nuclei at this stage present points of rather a special interest. In the first place, though visible in many, and certainly present in all the segments[81], they are not confined to these: they are also to be seen, in small numbers, in the band of fine spherules which surrounds the already segmented part of the germinal disc. Those found outside the germinal disc are not confined to the spots where fresh segments are appearing, but are also to be seen in places where there are no traces of fresh segments.

Footnote 81: In the figure of this stage, I have inserted nuclei in all the segments. In the section from which the figure was taken, nuclei were not to be seen in many of the segments, but I have not a question that they were present in all of them. The difficulty of seeing them is, in part, due to the yolk-spherules and in part to the thinness of the section as compared with the diameter of a segmentation sphere.

This fact, especially when taken in connection with the formation of fresh segments outside the germinal disc and with other facts which I shall mention hereafter, is of great morphological interest as bearing upon the nature and homologies of the food-yolk. It also throws light upon the behaviour and mode of increase of the nuclei. All the nuclei, both those of the segments and those of the yolk, have the peculiar structure I described in the last stage.

In specimens of this stage I have been able to observe certain points which have an important bearing upon the behaviour of the nucleus during cell-division.

Three figures, illustrating the behaviour of the nucleus, as I have seen it in sections of blastoderms hardened in chromic acid, are shewn in Pl. 6, figs. 7_a_, 7_b_ and 7_c_.

In the place of the nucleus is to be seen a sharply defined figure (Fig. 7_a_) stained in the same way as the nucleus or more deeply. It has the shape of two cones placed base to base. From the apex of each cone there diverge towards the base a series of excessively fine striæ. At the junction between the two cones is an irregular linear series of small deeply stained granules which form an apparent break between the two. The line of this break is continued very indistinctly beyond the edge of the figure on each side.

From the apex of each cone there diverge outwards into the protoplasm of the cell a series of indistinct markings. They are rendered obscure by the presence of yolk-spherules, which completely surround the body just described, but which are not arranged with any reference to these markings. These latter striæ, diverging from the apex of the cone, are more distinctly seen when the apex points to the observer (Fig. 7_b_), than when a side of the cone is in view.

The striæ diverging outwards from the apices of the cones must be carefully distinguished from the striæ of the cones themselves. The cones are bodies quite as distinctly differentiated from the protoplasm of the cell as nuclei, while the striæ which diverge from their apices are merely structures in the general protoplasm of the cell.

In some cells, which contain these bodies, no trace of a commencing line of division is visible. In other cases (Fig. 7_c_), such a line of division does appear and passes through the junction of the two cones. In one case of this kind I fancied I could see (and have represented) a coloured circular body in each cone. I do not feel any confidence that these two bodies are constantly present; and even where visible they are very indistinct.

Instead of an ordinary nucleus a very indistinctly marked vesicular body sometimes appears in a segment; but whether it is to be looked on as a nucleus not satisfactorily stained, or as a nucleus in the act of being formed, I cannot decide.

With reference to the situation of the cone-like bodies I have described I have made an observation which appears to me to be of some interest. I find that bodies of this kind are found in the yolk _completely outside_ the germinal disc. I have made this observation, in at least two cases which admitted of no doubt (vide Fig. 7, _nx´_).

We have therefore the remarkable fact, that whatever connection these bodies may have with cell-division, they can occur in cases where this is altogether out of the question and where an increase in the number of nuclei can be their only product.

These are the main facts which I have been able to determine with reference to the nuclei of this stage; but it will conduce to clearness if I now finish what I have to say upon this subject.

At a still later stage of segmentation the same peculiar bodies are to be seen as during the stage just described, but they are rarer; and, in addition to them, other bodies are to be seen of a character intermediate between ordinary nuclei and the former bodies.

Three such are represented in Pl. 6, figs. 8_a_, 8_b_, 8_c_. In all of these there can be traced out the two cones, which are however very irregular. The striation of the cones is still present, but is not nearly so clear as it was in the earlier stage.

In addition to this, there are numerous deeply stained granules scattered about the two figures which resemble exactly the granules of typical nuclei.

All these bodies occupy the place of an ordinary nucleus, they stain like an ordinary nucleus and are as sharply defined as an ordinary nucleus.

There is present around some of these, especially those situated in the yolk, the network of lines of the yolk described by me in a preliminary paper[82], and I feel satisfied that there is in some cases an actual connection between the network and the nuclei. This network I shall describe more fully hereafter.

Footnote 82: _Loc. cit._

Further points about these figures and the nuclei of this stage I should like to have been able to observe more completely than I have done, but they are so small that with the highest powers I possess (Zeiss, Immersion No. 2 = 1/15 in.) their complete and satisfactory investigation is not possible.

Most of the true nuclei of the cells of the germinal disc are regularly rounded; those however of the yolk are frequently irregular in shape and often provided with knob-like processes. The gradations are so complete between typical nuclei and bodies like that shewn (Pl. 6, fig. 8_c_) that it is impossible to refuse the name of nucleus to the latter.

In many cases _two nuclei_ are present in one cell.

In later stages knob-like nuclei of various sizes are scattered in very great numbers in the yolk around the blastoderm (vide Pl. 7). In some cases it appears to me that several of these are in close juxtaposition, as if they had been produced by the division of one primitive nucleus. I do not feel absolutely confident that this is the case, owing to the fact that in the investigation of a knobbed body there is great difficulty in ascertaining that the knobs, which appear separate in one plane, are not in reality united in another.

I have, in spite of careful search, hitherto failed to find amongst these later nuclei cone-like figures, similar to those I found in the yolk during segmentation. This is the more remarkable since in the early stages of segmentation, when very few nuclei are present in the yolk, the cone-like figures are not uncommon; whereas, in the latter stages of development when the nuclei of the yolk are very common and obviously increasing rapidly, such figures are not to be met with.

In no case have I been able to see a distinct membrane round any of the nuclei.

I have hitherto attempted to describe the appearances bearing on the behaviour of the nuclei in as objective a manner as possible.

My observations are not as complete as could be desired; but, taken in conjunction with those of other investigators, they appear to me to point towards certain definite conclusions with reference to the behaviour of the nucleus in cell-division.

The most important of these conclusions may be stated as follows. In the act of cell-division the nuclei of the resulting cells are formed from the nucleus of the primitive cell.

This may occur:--

(1) By the complete solution of the old nucleus within the protoplasm of the mother cell and the subsequent reaggregation of its matter to form the nuclei of the freshly formed daughter cells,

(2) By the simple division of the nucleus,

(3) Or by a process intermediate between these two where part of the old nucleus passes into the general protoplasm and part remains always distinguishable and divides; the fresh nucleus being in this case formed from the divided parts as well as from the dissolved parts of the old nucleus.

Included in this third process it is permissible to suppose that we may have a series of all possible gradations between the extreme processes 1 and 2. If it be admitted, and the evidence we have is certainly in favour of it, that in some cases, both in animal and vegetable cells, the nucleus itself divides during cell division, and in others the nucleus completely vanishes during the cell-division, it is more reasonable to suspect the existence of some connection between the two processes, than to suppose that they are entirely different in kind. Such a connection is given by the hypothesis I have just proposed.

The evidence for this view, derived both from my own observations and those of other investigators, may be put as follows.

The absolute division of the nucleus has been stated to occur in animal cells, but the number of instances where the evidence is quite conclusive are not very numerous. Recently F. E. Schultze[83] appears to have observed it in the case of an Amoeba in an altogether satisfactory manner. The instance is quoted by Flemming[84]. Schultze saw the nucleus assume a dumb-bell shape, divide, and the two halves collect themselves together. The whole process occupied a minute and a half and was shortly followed by the division of the Amoeba, which occupied eight minutes. Amongst vegetable cells the division of the nucleus seems to be still rarer than with animal cells. Sachs[85] admits the division of the nucleus in the case of the parenchyma cells of certain Dicotyledons (Sambucus, Helianthus, Lysimachia, Polygonum, Silene) on the authority of Hanstein.

Footnote 83: _Archiv f. Micr. Anat._ XI. p. 592.

Footnote 84: "Entwicklungsgeschicte der Najaden," LXXI. Bd. _der Sitz. der k. Acad. Wien_, 1875.

Footnote 85: _Text-Book of Botany_, English trans. p. 19.

The division of the nucleus during cell-division, though seemingly not very common, must therefore be considered as a thoroughly well authenticated occurrence.

The frequent disappearance of the nucleus during cell-division is now so thoroughly recognised, both for animal and vegetable cells, as to require no further mention.

In many cases the partial or complete disappearance of the nucleus is accompanied by the formation of two peculiar star-like figures. Appearances of the kind have been described by Fol[86], Flemming[87], Auerbach[88] and possibly also Oellacher[89] as well as other observers.

Footnote 86: "Entw. d. Geryonideneies." _Jenaische Zeitschrift_, Bd. VII.

Footnote 87: _Loc. cit._

Footnote 88: _Organologische Studien_, Zweites Heft.

Footnote 89: "Beiträge z. Entwicklungsgeschichte der Knochenfischen." _Zeit. für Wiss. Zoologie_. Bd. XXII. 1872.

These figures[90] are possibly due to the streaming out of the protoplasm of the nucleus into that of the cell[91]. The appearance of striation may on this hypothesis be explained as due to the presence of granules in the protoplasm. When the streaming out of the protoplasm of a nucleus into that of a cell takes place, any large granule which cannot be moved by the stream will leave behind it a slack area where there is no movement of the fluid. Any granules which are carried into this area will remain there, and by the continuation of a process of this kind a row of granules may be formed, and a series of such rows would produce an appearance of striation. In many cases, _e.g._ Anodon, vide Flemming[92], even the larger yolk-spherules are arranged in this fashion.

Footnote 90: The memoirs of Auerbach and Strasburger (_Zellbildung u. Zelltheilung_) have unfortunately come into my hands too late for me to take advantage of them. Especially in the magnificent monograph of Strasburger I find drawings precisely resembling those from my specimens already in the hands of the engraver. Strasburger comes to the conclusion from his investigations that the modified nucleus always divides and never vanishes as is usually stated. If his views on this point are correct part of the hypothesis I have suggested above is rendered unnecessary. The striæ of the protoplasm, which in accordance with Auerbach's view I have considered as being due to a streaming out of the matter of the nucleus, he regards as resulting from a polarity of the particles in the cell and the attraction of the nucleus. My own investigations though, as far as they go, quite in accordance with those of Strasburger, do not supply any grounds for deciding on the meaning of these striæ; and in some respects they support Strasburger's views against those of other observers, since they demonstrate that in Elasmobranchii the modified nucleus does actually divide.

Footnote 91: This is the view which has been taken by Auerbach (_Organologische Studien_).

Footnote 92: _Loc. cit._

On the supposition that the striation of these figures is due to the outflow from the nucleus, the appearances presented in Elasmobranchii admit of the following explanation.

The central body consisting of two cones (figs. 7_a_, 7_c_) is almost without question the remnant of the primitive nucleus. This is shewn by its occupying the same position as the primitive nucleus, staining in the same way, and by there being a series of insensible gradations between it and a typical nucleus. The contents must be supposed to be streaming out from the two apices of the cones, as appears from the striæ in the body converging on each side towards the apex, and then diverging again from it. In my specimens the yolk-spherules are not arranged with any reference to the radiating striation.

It is very likely that in the cases of the disappearance of the nucleus, its protoplasm streams out in two directions, towards the two parts of the cell which will eventually become separated from each other; and probably, after the division, the matter of the old nucleus is again collected to form two fresh nuclei.

In some cases of cell-division a remnant of the old nucleus is stated to be visible after the fresh nuclei have appeared. These cases, of which I have not seen full accounts, are perhaps analogous to what occasionally happens with the germinal vesicle of an ovum. The whole of the contents of the germinal vesicle become at its disappearance mingled with the protoplasm of the ovum, but the resistant membrane remains and is eventually ejected from the egg, vide p. 215 _et seq_. If the remnant of the old nucleus in the cases described is nothing more than its membrane, no difficulty is offered to the view that the constituents of the old nucleus may help to form the new ones.

In many cases the total bulk of the new nuclei is greater than that of the old one; in such instances part of the protoplasm of the cell necessarily has a share in forming the new nuclei.

Although, in instances where the nucleus vanishes, an absolute demonstration of the formation of the fresh nuclei from the matter of the old one is not possible; yet, if cases of the division of the old nucleus to form the new ones be admitted to exist, the derivation in the first process of the fresh nuclei from the old ones must be postulated in order to maintain a continuity between the two processes of formation; and, as I have attempted to shew, all the circumstantial evidence is in favour of it.

Admitting the existence of the two extreme processes of nuclear formation, I wish to shew that my results in Elasmobranchii tend to demonstrate the existence of intermediate steps between them. The first figures I described of two opposed cones, appear to me almost certainly to represent nuclei in the act of dissolution; but though a portion of the nucleus may stream out into the yolk, I think it impossible that the whole of it does[93].

Footnote 93: After Strasburger's observation it must be considered very doubtful whether the streaming out of the contents of the nucleus, in the manner implied in the text, really takes place.

I described these bodies in two states. An earlier one, in which the two cones were separated by an irregular row of deeply stained granules; and a later one in which a furrow had already appeared dividing the cones as well as the cell. In neither of these conditions could I see any signs of the body vanishing completely. It was as clearly defined and as deeply stained as an ordinary nucleus, and in its later condition the signs of the streaming out of material from its pointed extremities were less marked than in the earlier stage.

All these facts, to my mind, point to the view that these cone-like bodies do not disappear, but form the basis for the new nuclei. Possibly the body visible in each cone in the later stage, was the commencement of this new nucleus. Götte[94] has figured structures somewhat similar to these bodies, but I hardly understand either his figure or his account sufficiently clearly to be able to pronounce upon the identity of the two. In case they are identical, Götte gives a very different explanation of them from my own[95].

Footnote 94: _Entwicklungsgeschite der Unke_, Pl. 1, fig. 18.

Footnote 95: As I before mentioned, Strasburger (_Zellbildung u. Zelltheilung_) has represented bodies precisely similar to those I have described, which appear during the segmentation in the egg of _Phallusia mammillata_ as well as similar figures observed by Butschli in eggs of _Cucullanus elegans_ and _Blatta Germanica_. The figures in this monograph are the only ones I have seen, which are identical with my own.

A second of my results, which points to a series of intermediate steps between division and solution of the nucleus, is the distribution in time of the peculiar cone-like bodies. These are present in fair abundance at an early period of segmentation, when there are but few nuclei either in the blastoderm or the yolk. But at later periods, when there are both more nuclei, especially in the yolk, and they are also increasing in numbers more rapidly than before, no bodies of this kind are to be seen. This fact becomes the more striking from the lobate appearance of the later nuclei of the yolk, an appearance which exactly suits the hypothesis of the rapid budding off of fresh nuclei.

The observations of R. Hertwig[96] on the gemmation of _Podophrya gemmipara_, support my interpretation of the knobbed condition of the nuclei. Hertwig finds (p. 47) that

The horse-shoe shaped nucleus grows out into numerous anastomosing projections. Over the free ends of the projections little knobs appear on the surface of the body, into which the lengthening ends of the processes of the nucleus grow up. Here they bend themselves into a horse-shoe form. The newly-formed nucleus then separates from the original nucleus, and afterwards the bud containing it from the body.

Footnote 96: _Morphologisches Jahrbuch_, Bd. 1. pp. 46, 47.

From the peculiar arrangement of the net-work of lines of the yolk around these knobbed nuclei, it is reasonable to conclude that interchange of material between the protoplasm of the yolk and the nuclei is still taking place, even during the later periods.

These facts about the distribution in time of the cone-like bodies afford a strong presumptive evidence of a change in the manner of nuclear increase.

The last argument I propose urging on this head is derived from the bodies (Pl. 6, fig. 8_a_, _b_, _c_) which I have described as intermediate between the true cone-like bodies and typical nuclei. They appear to afford evidence of less and less of the matter of the nucleus streaming out into the yolk and of a large proportion of it becoming divided.

The conclusion to be derived from all these facts is that for Elasmobranchii in the earlier stages of segmentation, and during the formation of fresh segments, a partial solution of the old nucleus takes place, but all its constituents serve for the reconstruction of the fresh nuclei.

In later periods of development a still smaller part of the nucleus becomes dissolved, and the rest divides; but the two fresh nuclei are still derived from the two sources. After the close of segmentation the fresh nuclei are formed by a simple division of the older ones.

The appearance of the cone-like bodies in the yolk outside the germinal disc is a point of some interest. It demonstrates in a conclusive manner that whatever influence (if any) the nucleus may have in ordinary cases of cell division, yet it may undergo changes of a precisely similar character to those which it experiences during cell division, without exerting any influence on the surrounding protoplasm[97]. If the lobate nuclei are also nuclei undergoing division, we have in the egg of an Elasmobranch examples of all the known forms of nuclear increase unaccompanied by cell division.

Footnote 97: Strasburger's (_loc. cit._) arguments about the influence of the nucleus in cell division are not to my mind conclusive; though not without importance. It is difficult to reconcile his views with the facts of cell division observable during the Elasmobranch segmentation; but even if their truth be admitted they do not bring us much nearer to a satisfactory understanding of cell division, unless accompanied (and at present they are not so) by a rational explanation of the forces which produce the division of the nucleus.

The next stage in the segmentation does not present so many features of interest as the last one. The segments are now so small, as to be barely visible from the surface with a simple lens. A section of an embryo of this stage is represented in Pl. 6, fig. 8. The section, which is drawn on the same scale as the section belonging to the last stage, serves to shew the relative size of the segments in the two cases.

The epiblast is now more distinct than it was. The segments composing it are markedly smaller than the remainder of the cells of the germinal disc, but possess nuclei of an absolutely larger size than do the other cells. They are irregular in shape, with a slight tendency to be columnar. An average segment of this layer measures about 1/700 inch.

The cells of the lower layer are more polygonal than those of the epiblast, and are decidedly larger. An average specimen of the larger cells of the lower layer measures about 1/400 in. in diameter, and is therefore considerably smaller than one of the smallest cells of the last stage. The formation of fresh segments from the yolk still continues with fair rapidity, but nearly comes to an end shortly after this.

Of the nuclei of the lower layer cells, there is not much to add to what has already been said. Not infrequently two nuclei may be observed in a single cell.

The nuclei in the yolk which surrounds the germinal disc are more numerous than in the earlier periods, and are now to be met with in fair numbers in every section (fig. 8, _n´_).

These are the main features which characterise the present stage, they are in all essential points similar to those of the last stage, and the two germinal discs hardly differ except in the size of the segments of which they are composed.

In the last stage which I consider as belonging to the segmentation, the cells of the whole blastoderm have become smaller (Pl. 6, fig. 9).

The epiblast (_ep_) now consists of a very marked layer of columnar cells. It is, as far as I have been able to observe, never more than one cell deep. The cells of the lower layer are of an approximately uniform size, though a few of those at the circumference of the blastoderm considerably exceed the remainder in the bulk.

There are two fresh features of importance in germinal discs of this age.

Instead of being but indistinctly separated from the surrounding yolk, the blastoderm has now very clearly defined limits.

This is an especially marked feature of preparations made with osmic acid. In these there may frequently be seen a deeply stained doubly contoured line, which forms the limit of the yolk, where it surrounds the germinal disc. Lines of this kind are often to be seen on the surface of the yolk, or even of the blastoderm, but are probably to be regarded as products of reagents, rather than as organised structures. The outline of the germinal disc is well rounded, though it is occasionally broken, from the presence of a larger cell in the act of being formed from the yolk.

It is not probable that any great importance is to be attached to the comparative distinctness of the outline of the germinal disc at this stage, which is in a great measure due to a cessation in the formation of fresh cells in the surrounding yolk, and in part to the small and comparatively uniform size of the cells of the germinal disc.

The formation of fresh cells from the yolk nearly comes to an end during this period, but it still continues on a small scale.

The number of the nuclei around the germinal disc has increased.

Another feature of interest which first becomes apparent during this stage is the asymmetry of the germinal disc. If a section were made through the germinal disc, as it lay _in situ_ in the egg capsule, parallel or nearly so to the long axis of the capsule, one end of the section would be found to be much thicker than the other. There would in fact be a far larger collection of cells at one extremity of the germinal disc than at the other. The end at which this collection of cells is formed points towards the end of the egg capsule opposite to that near which the yolk is situated. This collection of cells is the first trace of the embryo; and with its appearance the segmentation may be supposed to terminate.

The section I have represented, though not quite parallel to the long axis of the egg, is sufficiently nearly so to shew the greater mass of cells at the embryonic end of the germinal disc.

This very early appearance of a distinction in the germinal disc between the extremity at which the embryo appears and the non-embryonic part of the disc, besides its inherent interest, has a further importance from the fact that in Osseous Fishes a similar occurrence takes place. Oellacher[98] and Götte[99] both agree as to the very early period at which a thickening of one extremity of the blastoderm in Osseous Fishes is formed, which serves to indicate the position at which the embryo will appear. There are many details of development in which Osseous Fish and Elasmobranchii agree, which, although if taken individually are without any great importance, yet serve to shew how long even insignificant features in development may be retained.

Footnote 98: _Zeitschrift für Wiss. Zoologie_, Bd. XXIII. 1873.

Footnote 99: _Archiv für Micr. Anat._ Bd. IX. 1873.

* * * * *

The segmentation of the Elasmobranch egg presents in most of its features great regularity, and exhibits in its mode of occurrence the closest resemblance to that in other meroblastic vertebrate ova.

There is, nevertheless, one point with reference to which a slight irregularity may be observed. In almost all eggs segmentation commences by, what for convenience may be called, a vertical furrow which is followed by a second vertical furrow at right angles to the first. The third furrow however is a horizontal one, and cuts the other two at right angles. This method of segmentation must be looked on as the normal one, in almost all the important groups of the animal kingdom, both for the so-called holoblastic and meroblastic eggs, and the gradations intermediate between the two. The Frog amongst vertebrates exhibits a most typical instance of this form of segmentation.

In Elasmobranchii the first two furrows are formed in a perfectly normal manner, but though I have not observed the actual formation of the next furrow, yet from the later stages, which I have observed, I conclude that it is parallel to one of the first formed furrows; and it is fairly certain that, not till a considerably later period, is a furrow homologous with the horizontal furrow of the Batrachian egg formed. This furrow appears to be represented in the Elasmobranch segmentation by the irregular circumscription of a body of central smaller spheres from a ring of peripheral larger ones (vide Pl. 6, figs. 3, 4 and 5).

In the Bird the representative of the horizontal furrow appears relatively much earlier. It is formed when there are eight segments marked out on the surface of the germinal disc[100]. From Oellacher's[101] account of the segmentation in the fowl[102] it seems certain, as might be anticipated, that this furrow is nearly parallel to the surface of the disc, so that it cuts the earlier formed vertical furrows and causes the segments of the germinal disc to be completely circumscribed below as well as at the surface. In the Elasmobranch egg this is not the case; so that, even after the smaller central segments have become separated from the outer ring of larger ones, none of the segments of the disc are completely circumscribed, and only appear to be so in surface views (vide Pl. 6, fig. 6). Segmentation in the Elasmobranch egg differs in the following particulars from that in the Bird's egg:

(1) The equivalent of the horizontal furrow of the Batrachian egg appears much later than in the Bird.

(2) When it has appeared it travels inwards much more slowly.

Footnote 100: Vide _Elements of Embryology_, p. 23.

Footnote 101: _Stricker's Studien_, 1869, Pt. I, Pl. II. fig. 4.

Footnote 102: Unfortunately Professor Oellacher gives no account of the surface appearance of the germinal discs of which he describes the sections. It is therefore uncertain to what period his sections belong.

As a result of these differences, the segments of the germinal disc of the Birds' eggs are much earlier circumscribed on all sides than those of the Elasmobranch egg.

As might be expected, the segmentation of the Elasmobranch egg resembles in many points that of Osseous Fishes (vide Oellacher[103] and Klein[104]). It may be noticed, that with Osseous as with Elasmobranch Fishes, the furrow corresponding with the horizontal furrow of the Amphibian's egg does not appear at as early a period as is normal. The third furrow of an Osseous Fish egg is parallel to one of the first formed pair.

Footnote 103: _Zeitschrift für Wiss. Zool._ Bd. XXII. 1872.

Footnote 104: _Monthly Microscopical Journal_, March, 1872.

In Oellacher's[105] figures, Pl. 23, figs. 19-21, peculiar beadings of the sides of the earlier formed furrows are distinctly shewn. No mention of these is made in the text, but they are unquestionably similar to those I have described in the Elasmobranch furrows. In the case of Elasmobranchii I pointed out that not only were the sides of the furrow beaded, but that there appeared in the protoplasm, close to the furrows, peculiar vacuole-like cavities, precisely similar to the cavities which were the cause of the beadings of the furrows.

Footnote 105: _Loc. cit._

The presence of these seems to shew that the molecular cohesion of the protoplasm becomes, as compared with other parts, much diminished in the region where a furrow is about to appear, so that before the protoplasm finally gives way along a particular line to form a furrow, its cohesion is broken at numerous points in this region, and thus a series of vacuole-like spaces is formed.

If this is the true explanation of the formation of these spaces, their presence gives considerable support to the views of Dr Kleinenberg upon the causes of segmentation, so clearly and precisely stated in his monograph upon Hydra; and is opposed to any view which regards the forces which come into play during segmentation as resident in the nucleus.

I have not observed the peculiar threads of protoplasm which Oellacher[106] describes as crossing the commencing segmentation furrows. I have also failed to discover any signs of a concentration of the yolk-spherules, round one or two centres, in the segmentation spheres, similar to that observed by Oellacher in the segmenting eggs of Osseous Fish. The appearances observed by him are probably connected with the behaviour of the nucleus during segmentation, and are related to the curious bodies I have already described.

Footnote 106: _Loc. cit._

With reference to the nuclei which Oellacher[107] has described as occurring in the eggs of Osseous Fish during segmentation, there can, I think, be little doubt that they are identical with the peculiar nuclei in the Elasmobranch eggs.

Footnote 107: _Loc. cit._

He[108] says:

In an unsegmented germ there occurred at a certain point in the section ... a small aggregation of round bodies. I do not feel satisfied whether these aggregations represent one or more nuclei.

Fig. 29 shews such aggregation; by focusing at its optical section eleven unequally large rounded bodies measuring from 0.004 - 0.009 mm. may be distinguished. They lay as if in a multilocular gap in the germ mass, which however they did not quite fill. In each of these bodies there appeared another but far smaller body. These aggregations were distinguished from the germ by an especially beautiful intense violet gold chloride colouration of their elements. The smaller elements contained in the larger were still more intensely coloured than the larger.

Footnote 108: _Loc. cit._ pp. 410, 411, &c.

He further states that these aggregations equal the segments in number, and that the small bodies within the elements are not always to be seen with the same distinctness.

Oellacher's description as well as his figures of these bodies leaves no doubt in my mind that they are exactly similar bodies to those which I have already spoken of as nuclei, and the characteristic features of which I have shortly mentioned, and shall describe more fully at a later stage. A moderately full description of them is to be found in my preliminary paper[109].

Footnote 109: _Loc. cit._ p. 415. [This Edition, p. 64.]

Their division into a series of separate areas each with a deeply-stained body, as well as the staining of the whole of them, exactly corresponds to what I have found. That each is a single nucleus is quite certain, though their knobbed form might occasionally lead to the view of their being divided. This knobbed condition, observed by Oellacher as well as myself, certainly supports the view, that they are in the act of budding off fresh nuclei. Oellacher conceives, that the areas into which these nuclei are divided represent a series of separate bodies--this according to my observations is not the case. Nuclei of the same form have already been described in Nephelis, and are probably not very rare. They pass by insensible gradations into ordinary nuclei with numerous granules.

One marked feature of the segmentation of the Elasmobranch egg is the continuous advance of the process of segmentation into the yolk and the assimilation of this into the germ by the direct formation of fresh segments out of it. Into the significance of this feature I intend to enter fully hereafter; but it is interesting to notice that Oellacher's descriptions point to a similar feature in the segmentation of Osseous Fish. This however consists chiefly in the formation of fresh segments from the lower parts of the germinal disc which in Osseous Fish is more distinctly marked off from the food-yolk than in Elasmobranchii.

I conclude my description of the segmentation by a short account of what other investigators have written about its features in these fishes. One of the earliest descriptions of this process was given by Leydig[110]. To his description of the germinal disc, I have already done full justice.

Footnote 110: _Rochen u. Haie._ It is here mentioned that Coste observed the segmentation in these fishes.

In the first stage of segmentation which he observed 20-30 segments were already visible on the surface. In each of these he recognized a nucleus but no nucleolus.

He rightly states that the segments have no membrane, and describes the yolk-spherules which fill them.

The next investigator is Gerbe[111]. I have unfortunately been unable to refer to this elaborate paper, but I gather from an abstract that M. Gerbe has given a careful description of the external features of segmentation.

Footnote 111: "Recherches sur la segmentation des products adventifs de l'oeuf des Plagiostomes et particulièrement des Raies." Robin, _Journal de l'Anatomie et de la Physiologie_, p. 609, 1872.

Schenk[112] has also made important investigations on the subject. He considers that the ovum is invested with a very delicate membrane. This membrane I have failed to find a trace of, and agree with Leydig[113] in denying its existence. Schenk further found that after impregnation, but before segmentation, the germinal disc divided itself into two layers, an upper and a lower. Between the two a cavity made its appearance which Schenk looks upon as the segmentation cavity. Segmentation commences in the upper of the two layers, but Schenk does not give a precise account of the fate of the lower. I have had no opportunity of investigating the impregnated ovum before the commencement of segmentation, but my observations upon the early stages of this process render it clear that no division of the germinal disc exists subsequently to the commencement of segmentation, and that the cavity discovered by Schenk can have no connection whatever with the segmentation cavity. I am indeed inclined to look upon this cavity as an artificial product. I have myself met with somewhat similar appearances, after the completion of segmentation, which were caused by the non-penetration of my hardening reagent beyond a certain point.

Footnote 112: "Die Eier von Raja quadrimaculata innerhalb der Eileiter." _Sitz. der k. Akad. Wien._ Vol. LXXIII. 1873.

Footnote 113: _Loc. cit._ My denial of the existence of this membrane naturally applies only to the egg after impregnation, and to the genera Scyllium and Pristiurus.

Without attempting absolutely to explain the appearances described by Professor Schenk, I think that his observations ought to be repeated, either by himself or some other competent observer.

Several further facts are recorded by Professor Schenk in his interesting paper. He states that immediately after impregnation, the germinal disc presents towards the yolk a strongly convex surface, and that at a later period, but still before the commencement of segmentation, this becomes flattened out. He has further detected amoeboid movements in the disc at the same period. As to the changes of the germinal disc during segmentation, his paper contains no facts of importance.

Next in point of time to the paper of Schenk, is my own preliminary account of the development of the Elasmobranch Fishes[114]. In this a large number of the facts here described in full are briefly alluded to.

Footnote 114: _Loc. cit._

The last author who has investigated the segmentation in Elasmobranchii, is Dr Alexander Schultz[115]. He merely states that he has observed the segmentation, and confirms Professor Schenk's statements about the amoeboid movements of the germinal disc.

Footnote 115: "Die Embryonal Anlage der Selachier. Vorläufige Mittheilung," _Centralblatt f. Med. Wiss._ No. 33, 1875.

EXPLANATION OF PLATE 6.

Fig. 1. Section through the germinal disc of a ripe ovarian ovum of the Skate. _gv._ germinal vesicle.

Fig. 2. Surface-view of a germinal disc with two furrows.

Figs. 3, 4, 5. Surface-views of three germinal discs in different stages of segmentation.

Fig. 6. Section through the germinal disc represented in fig 3. _n._ nucleus; _x._ edge of germinal disc. The engraver has not accurately copied my original drawings in respect to the structure of the segmentation furrows.

Figs. 6_a_ and 6_b_. Two furrows of the same germinal disc more highly magnified.

Fig. 6_c_. A nucleus from the same germinal disc highly magnified.

Fig. 7. Section through a germinal disc of the same age as that represented in fig. 4. _n._ nucleus; _nx._ modified nucleus; _nx´._ modified nucleus of the yolk; _f._ furrow appearing in the yolk around the germinal disc.

Figs. 7_a_, 7_b_, 7_c_. Three segments with modified nuclei from the same germinal disc.

Fig. 8. Section through a somewhat older germinal disc. _ep._ epiblast; _n´._ nuclei of yolk.

Figs. 8_a_, 8_b_, 8_c_. Modified nuclei from the yolk from the same germinal disc.

Fig. 8_d_. Segment in the act of division from the same germinal disc.

Fig. 9. Section through a germinal disc in which the segmentation is completed. It shews the larger collection of cells at the embryonic end of the germinal disc than at the non-embryonic. _ep._ epiblast.