Chapter VI (2)

Turning to the actual development of these parts, the discrepancy between theory and fact becomes very remarkable. From the somatic layer of the mesoblast, part of the voluntary muscular system and the dermis is no doubt derived, but the splanchnic layer supplies the material, not only for the muscular wall of the digestive canal and the vascular system, but also for the whole of the axial skeleton _and a great part of the voluntary muscular system of the body, including the first-formed muscles_. Though remarkable, it is nevertheless not inconceivable, that the skeleton might be derived from the splanchnic mesoblast, but it is very difficult to understand how there could be formed from it a part of the voluntary muscular system of the body indistinguishably fused with part of the muscular system derived from the somatopleure. No fact in my investigations comes out more clearly than that a great part of the voluntary muscular system is formed from the splanchnic layer of the mesoblast, yet this fact presents a most serious difficulty to the view that the somatic and splanchnic layers of the mesoblast in Vertebrates are respectively derived from the epiblast and hypoblast.

In spite, therefore, of general _à priori_ considerations of a very convincing kind which tell in favour of the double origin of the mesoblast, this view is supported by so few objective facts, and there exists so powerful an array of facts against it, that at present, at least, it seems impossible to maintain it. The full strength of the facts against it will appear more fully in a review of the present state of our knowledge as to the development of the mesoblast in the different groups.

To this I now pass.

In a paper on the "Early stages of Development in Vertebrates[215]" a short _resumé_ was given of the development of the mesoblast throughout the animal kingdom, which it may be worth while repeating here with a few additions. So far as we know at present, the mesoblast is derived from the hypoblast in the following groups:

Echinoderms (Hensen, Agassiz, Metschnikoff, Selenka, Götte), Nematodes (Bütschli), Sagitta (Kowalevsky, Bütschli), Lumbricus and probably other Annelids (Kowalevsky), Brachiopoda (Kowalevsky), Crustaceans (Bobretzky), Insects (Kowalevsky, Ulianin, Dohrn), Myriapods (Metschnikoff), Tunicates (Kowalevsky, Kuppfer), Petromyzon (Owsjanikoff), Osseous fishes (Oellacher, Götte, Kowalevsky), Elasmobranchii (Self), Amphibians (Remak, Stricker, Götte).

Footnote 215: _Quart. Jl. of Micros. Science_, July, 1875.
[This Edition, No. VI.]

The list includes members from the greater number of the groups of the animal kingdom; the most striking omissions being the Coelenterates, Mollusks, and the Amniotic Vertebrates. The absence of the Coelenterates has been already explained and my grounds for regarding the Amniotic Vertebrates as apparent rather than real exceptions have also been pointed out. The Mollusks, however, remain as a large group, in which we as yet know very little as to the formation of the mesoblast.

Dr Rabl[216], who seems recently to have studied the development of Lymnæus by means of sections, gives some figures shewing the origin of the mesoblast; they are, however, too diagrammatic to be of much service in settling the present question, and the memoirs of Professor Lankester[217] and Dr Fol[218] are equally inconclusive for this purpose, for, though they contain figures of elongated and branched mesoblast cells passing from the epiblast to the hypoblast, no satisfactory representations are given of the origin of these cells. I have myself observed in embryos of Turbo or Trochus similar elongated cells to those figured by Lankester and Fol, but was unable clearly to determine whence they arose. The most accurate observations which we have on this question are those of Professor Bobretzky[219]. In Nassa he finds that the three embryonic layers are all established during segmentation. The outermost and smallest cells form the epiblast, somewhat larger cells adjoining these the mesoblast, and the large yolk-cells the hypoblast. These observations do not, however, demonstrate from which of the primary layers the mesoblast is derived.

Footnote 216: _Jenaische Zeitschrift_, Vol. IX.

Footnote 217: _Quart. Jl. of Micros. Science_, Vol. XXV. 1874,
and _Phil. Trans._ 1875.

Footnote 218: _Archives de Zoologie_, Vol. IV.

Footnote 219: _Archiv f. Micr. Anat._ Vol. XIII.

The evidence at present existing is clearly in favour of the mesoblast being, in almost all groups of animals, developed from the hypoblast, but strong as this evidence is, it has not its full weight unless the actual manner in which the mesoblast is in many groups derived from the hypoblast, is taken into consideration. The most important of these are the Echinoderms, Brachiopods and Sagitta.

In the Echinoderms the mesoblast is in part formed by cells budded off from the hypoblast, _the remainder, however, arises as one or more diverticula of the alimentary tract_. From the separate cells first budded off there are formed the cutis, part of the connective tissue and the calcareous skeleton[220]. The diverticula from the alimentary cavity form the water-vascular system and the somatic and splanchnic layers of mesoblast. _The cavity of the diverticula after the separation of the water-vascular system, forms the body-cavity. The outer lining layer of the cavity forms the somatic layer of mesoblast and the voluntary muscles; the inner lining layer the splanchnic mesoblast which unites with the epithelium of the alimentary tract._ Though this fundamental arrangement would seem to be universal amongst Echinoderms, considerable variations of it are exhibited in different groups.

Footnote 220: The recent researches of Selenka, _Zeitschrift
f. Wiss. Zoologie_, Vol. XXVII. 1876, demonstrate that in
Echinoderms the muscles are derived from the cells first split
off from the hypoblast, and that the diverticula only form the
water-vascular system and the epithelial lining of the
body-cavity.

There is _one_ outgrowth from the alimentary tract in Synapta; _two_ in Echinoids, Asteroids and Ophiura; _three_ in Comatula, and four (?) in Amphiura. The cavity of the outgrowth usually forms the body-cavity, but sometimes in Ophiura and Amphiura (Metschnikoff) the outgrowths are from the first or soon become solid, and only secondarily acquire a cavity, which is however homologous with the body-cavity of the other groups.

In Sagitta[221] the formation of the mesoblast and the alimentary tract takes place in nearly the same fashion as in the Echinoderms. The simple invaginate alimentary cavity becomes divided into three lobes, a central and two lateral. The two lateral lobes are gradually more and more constricted off from the central one, and become eventually quite separated from it; their cavities remain independent, _and form in the adult the body-cavity_, divided by a mesentery into two distinct lateral sections. _The inner layer of each of the two lateral lobes forms the mesoblast of the splanchnopleure, the outer layer the mesoblast of the somatopleure._ The central division of the primitive gastræa cavity remains as the alimentary tract of the adult.

Footnote 221: Kowalevsky, "Würmer u. Arthropoden," _Mém. Acad.
Pétersbourg_, 1871.

The remarkable observations of Kowalevsky[222] on the development of the Brachiopoda have brought to light the unexpected fact that in two genera at least (Argiope and Terebratula) the mesoblast and body-cavity develop as paired constrictions from the alimentary tract in a manner almost identically the same as in Sagitta.

Footnote 222: "Zur Entwicklungsgeschichte d. Brachiopoden",
Protokoll d. ersten Session der Versammlung Russischer
Naturforscher in Kasan, 1873. Published in _Kaiserliche
Gesellschaft Moskau_, 1874 (Russian). Abstracted in Hoffmann
and Schwalbe, _Jahresbericht f._ 1873.

It thus appears that, so far as can be determined from the facts at our disposal, the mesoblast in almost all cases is derived from the hypoblast, and in three widely separated groups it arises as a pair of diverticula from the alimentary tract, each diverticulum containing a cavity which eventually becomes the body-cavity. I have elsewhere suggested[223] that the origin of the mesoblast from alimentary diverticula is to be regarded as primitive for all higher animals, and that the more general cases in which the mesoblast becomes split off, as an undivided layer, from the hypoblast, are in reality derivates from this. The chief obstacle in the way of this view arises from the difficulty of understanding how the whole voluntary muscular system can have been derived at first from the alimentary tract. That part of a voluntary system of muscles might be derived from the contractile diverticula of the alimentary canal attached to the body-wall is not difficult to understand, but it is not easy to believe that the secondary system so formed could completely replace the primitive muscular system, derived, as it must have been, from the epiblast. In my paper above quoted will be found various speculative suggestions for removing this difficulty, which I do not repeat here. If it be granted, however, that in Sagitta, Brachiopods, and Echinoderms we have genuine examples of the formation of the whole mesoblast from alimentary diverticula, it is easy to see how the formation of the mesoblast in Vertebrates may be a secondary derivate from an origin of this nature.

Footnote 223: Comparison of Early Stages, _Quart. Jl. Micros.
Science_, July, 1875. [This Edition, No. VI.]

An attempt has been already made to shew that the mesoblast in Elasmobranchii is formed in a very primitive fashion, and for this reason the Elasmobranchii appear to be especially adapted for determining whether any signs are exhibited of a derivation of the mesoblast as paired diverticula of the alimentary tract. There are, it appears to me, several such features. In the first place, the mesoblast is split off from the hypoblast not as a single mass but as a pair of distinct masses, comparable with the paired diverticula already alluded to. Secondly, the body-cavity when it appears in the mesoblast plates, _does not arise as a single cavity, but as a pair of cavities, one for each plate of mesoblast_, and these cavities remain permanently distinct in some parts of the body, and nowhere unite till a comparatively late period. Thirdly, the primitive body-cavity of the embryo is not confined to the region in which a body-cavity exists in the adult, _but extends to the summit of the muscle-plates_, at first separating parts which become completely fused in the adult to form the great lateral muscles of the body. It is difficult to understand how the body-cavity could have such an extension as this, on the supposition that it represents a primitive split in the mesoblast between the wall of the gut and the body-wall; but its extension to this part is quite intelligible, on the supposition that it represents the cavities of two diverticula of the alimentary tract, from whose muscular walls the voluntary muscular system has been derived. Lastly, I would point out that the derivation of part of the muscular system from what appears as the splanchnopleure is quite intelligible on the assumed hypothesis, but, as far as I see, on no other.

Such are the main features presented by the mesoblast in Elasmobranchii, which favour the view of its having originally formed the walls of the alimentary diverticula. Against this view of its nature are the facts (1) of the mesoblast plates being at first solid, and (2), as a consequence of this, of the body-cavity never communicating with the alimentary canal. These points, in view of our knowledge of embryological modifications, cannot be regarded as great difficulties to my view. We have many examples of organs, which, though in most cases arising as involutions, yet appear in other cases as solid ingrowths. Such examples are afforded by the optic vesicle, auditory vesicle, and probably also by the central nervous system, of Osseous Fish. In most Vertebrates these organs are formed as hollow involutions from the exterior; in Osseous Fish, however, as solid involutions, in which a cavity secondarily appears.

The segmental duct of Elasmobranchii or the Wolffian duct (segmental duct) of Birds are cases of a similar kind, being organs which must originally have been formed as hollow involutions, but which now arise as solid bodies.

Only one more instance of this kind need be cited, taken from the Echinoderms.

The body-cavity and the mesoblast investing it arise in the case of most Echinoderms as hollow involutions of the alimentary tract, but in some exceptional groups, Ophiura and Amphiura, are stated to be solid at first and only subsequently to become hollow. Should the accuracy of Metschnikoff's account of this point be confirmed, an almost exact parallel to what has been supposed by me to have occurred with the mesoblast in Elasmobranchii, and other groups, will be supplied.

The tendency of our present knowledge appears to be in favour of regarding the body-cavity in Vertebrates as having been primitively the cavity of alimentary diverticula, and the mesoblast as having formed the walls of the diverticula.

This view, to say the least of it, suits the facts which we know far better than any other theory which has been proposed, and though no doubt the _à priori_ difficulties in its way are very great, yet it appears to me to be sufficiently strongly supported to deserve the attention of investigators. In the meantime, however, our knowledge of invertebrate embryology is so new and imperfect that no certainty on a question like that which has just been discussed can be obtained; and any generalizations made at present are not unlikely to be upset by the discovery of fresh facts.

The only other point in connection with the mesoblast which I would call attention to is the formation of the vertebral bodies.

My observations confirm those of Remak and Gegenbaur, shewing that there is a primary segmentation of the vertebral bodies corresponding to that of the muscle-plates, followed by a secondary segmentation in which the central lines of the vertebral bodies are opposite the partitions between the muscle-plates.

The explanation of these changes is not difficult to find. The primary segmentation of the body is that of the muscle-plates, which must have been present at a time when the vertebral bodies had no existence. As soon however as the notochordal sheath was required to be strong as well as flexible, it necessarily became divided into a series of segments.

The conditions under which the lateral muscles can cause the flexure of the vertebral column are clearly that each muscle-segment shall be capable of acting on two vertebræ; and this condition can only be fulfilled when the muscle-segments are opposite the intervals between the vertebræ. Owing to this necessity, when the vertebral segments became formed, their centres corresponded, not with the centres of the muscle-plates, but with the inter-muscular septa.

These considerations fully explain the secondary segmentation of the vertebræ by which they become opposite the inter-muscular septa. On the other hand, the primary segmentation is clearly a remnant of the time when no vertebral bodies were present, and has no greater morphological significance than the fact that the cells to form the unsegmented investment of the notochord were derived from the segmented muscle-plates, and only secondarily became fused into a continuous tube.

_The Urinogenital System._

The first traces of the urinary system become visible at about the time of the appearance of the third visceral cleft. At about this period the somatopleure and splanchnopleure become more or less fused together at the level of the dorsal aorta, and thus, as has been already mentioned, each of the original plates of mesoblast becomes divided into a vertebral plate and lateral plate (Pl. 11, fig. 6). The mass of cells resulting from this fusion corresponds with Waldeyer's intermediate cell-mass in the Fowl.

At about the level of the fifth protovertebra the first trace of the urinary system appears.

From the intermediate cell-mass a solid knob grows outwards towards the epiblast (woodcut, fig. 4, _pd_). This knob consists at first of 20-30 cells, which agree in character with the neighbouring cells of the intermediate cell-mass, and are at this period rounded. It is mainly, if not entirely, derived from the somatic layer of the mesoblast.

From this knob there grows backwards a solid rod of cells which keeps in very close contact with the epiblast, and rapidly diminishes in size towards its posterior extremity. Its hindermost part consists in section of at most one or two cells. It keeps so close to the epiblast that it might be supposed to be derived from that layer were it not for the sections shewing its origin from the knob above mentioned. We have in this rod the commencement of what I have elsewhere[224] called the segmental duct.

Footnote 224: "Urinogenital Organs of Vertebrates," _Journ. of
Anat. and Phys._ Vol. X. [This Edition, No. VII.]

The sections are to shew the development of the segmental duct (_pd_) or primitive duct of the kidneys. In _A_ (the anterior of the two sections) this appears as a solid knob projecting towards the epiblast. In _B_ is seen a section of the column which has grown backwards from the knob in _A_.

_spn._ rudiment of a spinal nerve; _mc._ medullary canal; _ch._ notochord; _X._ string of cells below the notochord; _mp._ muscle-plate; _mp´._ specially developed portion of muscle-plate; _ao._ dorsal aorta; _pd._ segmental duct; _so._ somatopleura; _sp._ splanchnopleura; _pp._ pleuro-peritoneal or body-cavity; _ep._ epiblast; _al._ alimentary canal.]

My observations shew that the segmental duct is developed in the way just described in both Pristiurus and Torpedo. Its origin in Pristiurus is shewn in the adjoining woodcut, and in Torpedo in Pl. 11, fig. 7, _sd_.

At a stage somewhat older than I, the condition of the segmental duct has not very materially altered. It has increased considerably in length, and the knob at its front end is both absolutely smaller, and also consists of fewer cells than before (Pl. 11, fig. 7, _sd_). These cells have become more columnar, and have begun to arrange themselves radially; thus indicating the early appearance of the lumen of the duct. The cells forming the front part of the rod, as well as those of the knob, commence to exhibit a columnar character, but in the hinder part of the rod the cells are still rounded. In no part of it has a lumen appeared.

At this period also the knob, partly owing to the commencing separation of the muscle-plate from the remainder of the mesoblast, begins to pass inwards and approach the pleuro-peritoneal cavity.

At the same stage the first not very distinct traces of the remainder of the urinary system become developed. These appear in the form of solid outgrowths from the intermediate cell-mass just at the most dorsal part of the body-cavity.

The outgrowths correspond in numbers with the vertebral segments, and are at first quite disconnected with the segmental duct. At this stage they are only distinctly visible in the first few segments behind the front end of the segmental duct. A full description of them will come more conveniently in the next stage.

By a stage somewhat earlier than K important changes have taken place in the urinary system.

The segmental duct has acquired a lumen in its anterior portion, which opens at its front end into the body-cavity. (Pl. 11, fig. 9, _sd._) The lumen is formed by the columnar cells spoken of in the last stage, acquiring a radiating arrangement round a central point, at which a small hole appears. After the lumen has once become formed, it rapidly increases in size.

The duct has also grown considerably in length, but its hind extremity is still as thin, and lies as close to the epiblast, as at first. The segmental involutions which commenced to be formed in the last stage, have now appeared for every vertebral segment along the whole length of the segmental duct, and even for two or three segments behind this.

They are simple independent outgrowths arising from the outer and uppermost angle of the body-cavity, and are at first almost without a trace of a lumen; though their cells are arranged as two layers. They grow in such a way as to encircle the oviduct on its inner and upper side (Pl. 11, fig. 8 and Pl. 12, fig. 14_b_, _st_). When the hindermost ones are formed, a slight trace of a lumen is perhaps visible in the front ones. At a stage slightly subsequent to this, in Scyllium canicula, I noticed 29 of them; the first of them arising in the segment immediately behind the front end of the oviduct (Pl. 12, fig. 17, _st_), and two of them being formed in segments just posterior to the hinder extremity of the oviduct.

Pl. 12, figs. 16 and 18 represent two longitudinal sections shewing the segmental nature of the involutions and their relation to the segmental duct.

Many of the points which have been mentioned can be seen by referring to Pl. 11 and 12. Anteriorly the segmental duct opens into the pleuro-peritoneal cavity. In the sections behind this there may be seen the segmental duct with a distinct lumen, and also a pair of segmental involutions (Pl. 12, fig. 14_a_). In the still posterior sections the segmental duct would be quite without a lumen, and would closely adjoin the epiblast.

It seems not out of place to point out that the modes of the development of the segmental duct and of the segmental involutions are strikingly similar. Both arise as solid involutions, from homologous parts of the mesoblast. The segmental duct arises in the vertebral segment immediately in front of that in which the first segmental involution appears; _so that the segmental duct appears to be equivalent to a single segmental involution_.

The next stage corresponds with the first appearance of the external gills. The segmental duct now communicates by a wide opening with the body-cavity (Pl. 11, fig. 9, _sd_). It possesses a lumen along its whole length up to the extreme hind end (Pl. 11, fig. 9_a_). It is, however, at this hinder extremity that the most important change has taken place. This end has grown downwards towards that part of the alimentary canal which still lies behind the anus. This downgrowth is beginning to shew distinct traces of a lumen, and will appear in the next stage as one of the horns by which the segmental ducts communicate with the cloaca (Pl. 11, fig. 9_b_). All the anterior segmental involutions have now acquired a lumen. But this is still absent in the posterior ones (Pl. 11, fig. 9_a_).

Owing to the disappearance of the body-cavity in the region behind the anus, the primitive involutions there remain as simple masses of cells still disconnected with the segmental duct (Pl. 11, figs. 9_b_, 9_c_ and 9_d_).

_Primitive Ova._ The true generative products make their first appearance as the _primitive ova_ between stages I and K.

In the sections of one of my embryos of this stage they are especially well shewn, and the following description is taken from those displayed in that embryo.

They are confined to the region which extends posteriorly nearly to the end of the small intestine and anteriorly to the abdominal opening of the segmental duct.

Their situation in this region is peculiar. There is no trace of a distinct genital ridge, but the ova mainly lie in the dorsal portion of the mesentery, and therefore in a part of the mesoblast which distinctly belongs to the splanchnopleure (Pl. 12, fig. 14_a_). Some are situated external to the segmental involutions; and others again, though this is not common, in a part of the mesoblast which distinctly belongs to the body-wall (Pl. 12, fig. 14_b_).

The portion of mesentery, in which the primitive ova are most densely aggregated, corresponds to the future position of the genital ridge, but the other positions occupied by ova are quite outside this. Some ova are in fact situated on the outside of the segmental duct and segmented tubes, and must therefore effect a considerable migration before reaching their final positions in the genital ridge on the inner side of the segmental duct (Pl. 12, fig. 14_b_).

The condition of the tissue in which the ova appear may at once be gathered from an examination of the figures given. It consists of an irregular epithelium of cells partly belonging to the somatopleure and partly to the splanchnopleure, but passing uninterruptedly from one layer to the other. The cells which compose it are irregular in shape, but frequently columnar (Pl. 12, figs. 14_a_ and 14_b_).

They are formed of a nucleus which stains deeply, invested by a _very delicate_ layer of protoplasm. At the junction of somatopleure and splanchnopleure they are more rounded than elsewhere. Very few loose connective-tissue cells are present. The cells just described vary from .008 Mm. to .01 Mm. in diameter.

The primitive ova are situated amongst them and stand out with extraordinary clearness, to which justice is hardly done in my figures.

The normal full-sized ova exhibit the following structure. They consist of a mass of somewhat granular protoplasm of irregular, but more or less rounded, form. Their size varies from .016 - .036 Mm. In their interior a nucleus is present, which varies from .012 - .016 Mm., but its size as a rule bears _no_ relation to the size of the containing cell.

This is illustrated by the subjoined list of measurements.

Size of Primitive ova in Size of nucleus of Primitive
degrees of micrometer scale ova in degrees of micrometer
with F. ocul 2. scale with F. ocul 2.

10 8
13 8
13 8
14 7
15 7
13 7-1/2
11 8
16 5-1/2
12 7
10 7
15 6
13 6
12 7

The numbers given refer to degrees on my micrometer scale.

Since it is the ratio alone which it is necessary to call attention to, the numbers are not reduced to decimals of a millimeter. Each degree of my scale is equal, however, with the object glass employed, to .002 Mm.

This series brings out the result I have just mentioned with great clearness.

In one case we find a cell has three times the diameter of the nucleus 16 : 5-1/2; in another case 10 : 8, the nucleus has only a slightly smaller diameter than the cell. The irrationality of the ratio is fairly shewn in some of my figures, though none of the largest cells with very small nuclei have been represented.

The nuclei are granular, and stain fairly well with hæmatoxylin. They usually contain a single deeply stained nucleolus, but in many cases, especially where large (and this independently of the size of the cell), they contain two nucleoli (Pl. 12, figs. 14_c_ and 14_d_), and are at times so lobed as to give an apparent indication of commencing division.

A multi-nucleolar condition of the nuclei, like that figured by Götte[225], does not appear till near the close of embryonic life, and is then found equally in the large ova and in those not larger than the ova which exist at this early date.

Footnote 225: _Entwicklungsgeschichte der Unke_, Pl. 1, fig. 8.

As regards the relation of the primitive ova to each other and the neighbouring cells, there are a few points which deserve attention. In the first place, the ova are, as a rule, collected in masses at particular points, and not distributed uniformly (fig. 14_a_). The masses in some cases appear as if they had resulted from the division of one primitive ovum, but can hardly be adduced as instances of a commencing coalescence; since if the ova thus aggregated were to coalesce, an ovum would be produced of a very much greater size than any which is found during the early stages. Though at this stage no indication is present of such a coalescence of cells to form ova as is believed to take place by Götte, still the origin of the primitive ova is not quite clear. One would naturally expect to find a great number of cells intermediate between primitive ova and ordinary columnar cells. Cells which may be intermediate are no doubt found, but not nearly so frequently as might have been anticipated. One or two cells are shewn in Pl. 12, fig. 14_a_, _x_, which are perhaps of an intermediate character; but in most sections it is not possible to satisfy oneself that any such intermediate cells are present.

In one case what appeared to be an intermediate cell was measured, and presented a diameter of .012 Mm. while its nucleus was .008 Mm. Apart from certain features of the nucleus, which at this stage are hardly very marked, the easiest method of distinguishing a primitive ovum from an adjacent cell is the presence of a large quantity of protoplasm around the nucleus. The nucleus of one of the smallest primitive ova is not larger than the nucleus of an ordinary cell (being about .008 Mm. in both). It is perhaps the similarity in the size of the nuclei which renders it difficult at first to distinguish developing primitive ova from ordinary cells. Except with the very thinnest sections a small extra quantity of protoplasm around a nucleus might easily escape detection, and the developing cell might only become visible when it had attained to the size of a small typical primitive ovum.

It deserves to be noticed that the nuclei even of some of the largest primitive ova scarcely exceed the surrounding nuclei in size. This appears to me to be an argument of some weight in shewing that the great size of primitive ova is not due to the fact of their having been formed by a coalescence of different cells (in which case the nucleus would have increased in the same proportion as the cell); but to an increase by a normal method of growth in the protoplasm around the nucleus.

It appears to me to be a point of great importance certainly to determine whether the primitive ova arise by a metamorphosis of adjoining cells, or may not be introduced from elsewhere. In some of the lower animals, _e.g._ Hydrozoa, there is no question that the ova are derived from the epiblast; we might therefore expect to find that they had the same origin in Vertebrates. Further than this, ova are frequently capable in a young state of executing amoeboid movements, and accordingly of migrating from one layer to another. In the Elasmobranchii the primitive ova exhibit in a hardened state an irregular form which might appear to indicate that they possess a power of altering their shape, a view which is further supported by some of them being at the present stage situated in a position very different from that which they eventually occupy, and which they can only reach by migration. If it could be shewn that there were no intermediate stages between the primitive ova and the adjoining cells (their migratory powers being admitted) a strong presumption would be offered in favour of their having migrated from elsewhere to their present position. In view of this possibility I have made some special investigations, which have however led to no very satisfactory results. There are to be seen in the stages immediately preceding the present one, numerous cells in a corresponding position to that of the primitive ova, which might very well be intermediate between the primitive ova and ordinary cells, but which offer no sufficiently well marked features for a certain determination of their true nature.

In the particular embryo whose primitive ova have been described these bodies were more conspicuous than in the majority of cases, but at the same time they presented no special or peculiar characters.

In a somewhat older embryo of Scyllium the cells amongst which the primitive ova lay had become very distinctly differentiated as an epithelium (the germinal epithelium of Waldeyer) well separated by what might almost be called a basement membrane from the adjoining connective-tissue cells. Hardly any indication of a germinal ridge had appeared, but the ova were more definitely confined than in previous embryos to the restricted area which eventually forms this. The ova on the average were somewhat smaller than in the previous cases.

In several embryos intermediate in age between the embryo whose primitive ova were described at the commencement of this section and the embryo last described, the primitive ova presented some peculiarities, about the meaning of which I am not quite clear, but which may perhaps throw some light on the origin of these bodies.

Instead of the protoplasm around the nucleus being clear or slightly granular, as in the cases just described, it was filled in the most typical instances with numerous highly refracting bodies resembling yolk-spherules. In osmic acid specimens (Pl. 12, fig. 15) these stain very darkly, and it is then as a rule very difficult to see the nucleus; in specimens hardened in picric acid and stained with hæmatoxylin these bodies are stained of a deep purple colour, but the nucleus can in most cases be distinctly seen. In addition to the instances in which the protoplasm of the ova is quite filled with these bodies, there are others in which they only occupy a small area adjoining the nucleus (Pl. 12, fig. 15_a_), and finally some in which only one or two of these bodies are present. The protoplasm of the primitive ova appears in fact to present a series of gradations between a state in which it is completely filled with highly refracting spherules and one in which these are completely absent.

This state of things naturally leads to the view that the primitive ova, when they are first formed, are filled with these spherules, which are probably yolk-spherules, but that they gradually lose them in the course of development. Against this interpretation is the fact that the primitive ova in the younger embryo first described are completely without these bodies; this embryo however unquestionably presented an abnormally early development of the ova; and I am satisfied that embryos present considerable variations in this respect.

If the primitive ova are in reality in the first instance filled with yolk-spherules, the question arises as to whether, considering that they are the only mesoblast cells filled at this period with yolk-spherules, we must not suppose that they have migrated from some peripheral part of the blastoderm into their present position. To this question I can give no satisfactory answer. Against a view which would regard the spherules in the protoplasm as bodies which appear subsequently to the first formation of the ova, is the fact that hitherto no instances in which these spherules were present have been met with in the late stages of development; and they seem therefore to be confined to the first stages.

_Notochord._

The changes undergone by the notochord during this period present considerable differences according to the genus examined. One type of development is characteristic of Scyllium and Pristiurus; a second type, of Torpedo.

My observations being far more complete for Scyllium and Pristiurus than for Torpedo, it is to the two former genera only that the following account applies, unless the contrary is expressly stated. Only the development of the parts of the notochord in the trunk are here dealt with; the cephalic section of the notochord is treated of in a subsequent section.

During stage G the notochord is composed of flattened cells arranged vertically, rendering the histological characters of the notochord difficult to determine in transverse sections. In longitudinal sections, however, the form and arrangement of the cells can be recognised with great ease. At the beginning of stage G each cell is composed of a nucleus invested by granular protoplasm frequently vacuolated and containing in suspension numerous yolk-spherules. It is difficult to determine whether there is only one vacuole for each cell, or whether in some cases there may not be more than one.

Round the exterior of the notochord there is present a distinct though delicate cuticular sheath.

The vacuoles are at first small, but during stage G rapidly increase in size, while at the same time the yolk-spherules completely vanish from the notochord.

As a result of the rapid growth of the vacuoles, the nuclei, surrounded in each case by a small amount of protoplasm, become pushed to the centre of the notochord, the remainder of the protoplasm being carried to the edge. The notochord thus becomes composed during stages H and I (Pl. 11, fig. 4-6) of a central area mainly formed of nuclei with a small quantity of protoplasm around them, and of a thin peripheral layer of protoplasm without nuclei, the widish space between the two being filled with clear fluid. The exterior of the cells is indurated, so that they may be said to be invested by a membrane[226]; the cells themselves have a flattened form, and each extends from the edge to the centre of the notochord, the long axis of each being rather greater than half the diameter of the cord.

Footnote 226: This membrane is better looked upon, as is done
by Gegenbaur and Götte, as intercellular matter.

The nuclei of the notochord are elliptical vesicles, consisting of a membrane filled with granular contents, amongst which is situated a distinct nucleolus. They stain deeply with hæmatoxylin. Their long diameter in Scyllium is about 0.02 Mm.

The diameter of the whole notochord in Pristiurus during stage I is about 0.1 Mm. in the region of the back, and about 0.08 Mm. near the posterior end of the body.

Owing to the form of its constituent cells, the notochord presents in transverse sections a dark central area surrounded by a lighter peripheral one, but its true structure cannot be unravelled without the assistance of longitudinal sections. In these (Pl. 12, fig. 10) the nuclei form an irregular double row in the centre of the cord. Their outlines are very clear, but those of the individual cells cannot for certain be made out. It is, however, easy to see that the cells have a flattened and wedge-shaped form, with the narrow ends overlapping and interlocking at the centre of the notochord.

By the close of stage I the cuticular sheath of the notochord has greatly increased in thickness.

During the period intermediate between stages I and K the notochord undergoes considerable transformations. Its cells cease to be flattened, and become irregularly polygonal, and appear but slightly more compressed in longitudinal sections than in transverse ones. The vacuolation of the cells proceeds rapidly, and there is left in each cell only a very thin layer of protoplasm around the nucleus. Each cell, as in the earlier stages, is bounded by a membrane-like wall.

Accompanying these general changes special alterations take place in the distribution of the nuclei and the protoplasm. The nuclei, accompanied by protoplasm, gradually leave the centre and migrate towards the periphery of the notochord. At the same time the protoplasm of the cells forms a special layer in contact with the investing sheath.

The changes by which this takes place can easily be followed in longitudinal sections. In Pl. 12, fig. 11 the migration of the nuclei has commenced. They are still, however, more or less aggregated at the centre, and very little protoplasm is present at the edges of the notochord. The cells, though more or less irregularly polygonal, are still somewhat flattened. In Pl. 12, fig. 12 the notochord has made a further progress. The nuclei now mainly lie at the side of the notochord, where they exist in a somewhat shrivelled state, though still invested by a layer of protoplasm.

A large portion of the protoplasm of the cord forms an almost continuous layer in close contact with the sheath, which is more distinctly visible in some cases than in others.

While the changes above described are taking place the notochord increases in size. At the age of fig. 11 it is in the anterior part of the body of Pristiurus about 0.11 Mm. At the age of fig. 12 it is in the same species 0.12 Mm., while in Scyllium stellare it reaches about 0.17 Mm.

During stage K (Pl. 11, fig. 8) the vacuolation of the cells of the notochord becomes even more complete than during the earlier stages, and in the central cells hardly any protoplasm is present, though a starved nucleus surrounded by a little protoplasm may be found in an occasional corner.

The whole notochord becomes very delicate, and can with great difficulty be conserved whole in transverse sections.

The layer of protoplasm which appeared during the last stage on the inner side of the cuticular membrane of the notochord becomes during the present stage a far thicker and more definite structure. It forms a continuous layer with irregular prominences on its inner surface; and contains numerous nuclei. The layer sometimes presents in transverse sections hardly any indication of a division into a number of separate cells, but in longitudinal sections this is generally very obvious. The cells are directed very obliquely forwards, and consist of an oblong nucleus invested by protoplasm. The layer formed by them is very delicate and very easily destroyed. In one example its thickness varied from .004 to .006 Mm., in another it reached .012 Mm. The thickness of the cuticular membrane is about .002 Mm. or rather less.

The diameter of a notochord in the anterior part of the body of a Pristiurus embryo of this stage is about 0.21 Mm. Round the exterior of the notochord the mesoblast cells are commencing to arrange themselves as a special sheath.

In Torpedo the notochord at first presents the same structure as in Pristiurus, _i.e._ it forms a cylindrical rod of flattened cells.

The vacuolation of these cells does not however commence till a relatively very much later period than in Pristiurus, and also presents a very different character (Pl. 11, fig. 7).

The vacuoles are smaller, more numerous, and more rounded than in the other genera, and there can be no question that in many cases there is more than one vacuole in a cell. The most striking point in which the notochord of Torpedo differs from that of Pristiurus consists in the fact that in Torpedo there is never any aggregation of the nuclei at the centre of the cord, but the nuclei are always distributed uniformly through it. As the vacuolation proceeds the differences between Torpedo and the other genera become less and less marked. The vacuoles become angular in form, and the cells of the cord cease to be flattened, and become polygonal.

At my final stage for Torpedo (slightly younger than K) the only important feature distinguishing the notochord from that of Pristiurus, is the absence of any signs of nuclei or protoplasm passing to the periphery. Around the exterior of the cord there is early found in Torpedo a special investment of mesoblastic cells.

EXPLANATION OF PLATES 11 AND 12.

COMPLETE LIST OF REFERENCE LETTERS.

_al._ Alimentary tract. _an._ Point where anus will be formed. _ao._ Dorsal aorta. _ar._ Rudiment of anterior root of spinal nerve. _b._ Anterior fin. _c._ Connective-tissue cells. _cav._ Cardinal vein. _ch._ Notochord. _df._ Dorsal fin. _ep._ Epiblast. _ge._ Germinal epithelium. _ht._ Heart. _l._ Liver. _mp._ Muscle-plate. _mp´._ Early formed band of muscles from the splanchnic layer of the muscle-plates. _nc._ Neural canal. _p._ Protoplasm from yolk in the alimentary tract. _pc._ Pericardial cavity. _po._ Primitive ovum. _pp._ body-cavity. _pr._ Rudiment of posterior root of spinal nerve. _sd._ Segmental duct. _sh._ Cuticular sheath of notochord. _so._ Somatic layer of mesoblast. _sp._ Splanchnic layer of mesoblast. _spc._ Spinal cord. _sp.v._ Spiral valve. _sr._ Interrenal body. _st._ Segmental tube. _sv._ Sinus venosus. _ua._ Umbilical artery. _um._ Umbilical cord. _uv._ Umbilical vein. _V._ Splanchnic vein. _v._ Blood-vessel. _vc._ Visceral cleft. _Vr._ Vertebral rudiment. _W._ White matter of spinal cord. _x._ Subnotochordal rod (except in fig. 14_a_). _y._ Passage connecting the neural and alimentary canals.

PLATE 11.

Fig. 1. Section from the caudal region of a Pristiurus embryo belonging to stage H. Zeiss C, ocul. 1. Osmic acid specimen.

It shews (1) the constriction of the subnotochordal rod (_x_) from the summit of the alimentary canal. (2) The formation of the body-cavity in the muscle-plate and the ventral thickening of the parietal plate.

Fig. 1_a_. Portion of alimentary wall of the same embryo, shewing the formation of the subnotochord rod (_x_).

Fig. 2. Section through the caudal vesicle of a Pristiurus embryo belonging to stage H. Zeiss C, ocul. 1.

It shews the bilobed condition of the alimentary vesicle and the fusion of the mesoblast and hypoblast at the caudal vesicle.

Fig. 3_a_. Sections from the caudal region of a Pristiurus embryo belonging to stage H. Zeiss C, ocul. 1. Picric acid specimen.

It shews the communication which exists posteriorly between the neural and alimentary canals, and also by comparison with 3_b_ it exhibits the dilatation undergone by the alimentary canal in the caudal vesicle.

Fig. 3_b_. Section from the caudal region of an embryo slightly younger than 3_a_. Zeiss C, ocul. 1. Osmic acid specimen.

Fig. 4. Section from the cardiac region of a Pristiurus embryo belonging to stage H. Zeiss C, ocul. 1. Osmic acid specimen.

It shews the formation of the heart (_ht_) as a cavity between the splanchnopleure and the wall of the throat.

Fig. 5. Section from the posterior dorsal region of a Scyllium embryo, belonging to stage H. Zeiss C, ocul. 1. Osmic acid specimen.

It shews the general features of an embryo of stage H, more especially the relations of the body-cavity in the parietal and vertebral portions of the lateral plate, and the early-formed band of muscle (_mp´_) in the splanchnic layer of the vertebral plate.

Fig. 6. Section from the oesophageal region of Scyllium embryo belonging to stage I. Zeiss C, ocul. 1. Chromic acid specimen.

It shews the formation of the rudiments of the posterior nerve-roots (_pr_) and of the vertebral rudiments (_Vr_).

Fig. 7. Section of a Torpedo embryo belonging to stage slightly later than I. Zeiss C, ocul. 1, reduced 1/3. Osmic acid specimen.

It shews (1) the formation of the anterior and posterior nerve-roots. (2) The solid knob from which the segmental duct (_sd_) originates.

Fig. 8. Section from the dorsal region of a Scyllium embryo belonging to a stage intermediate between I and K. Zeiss C, ocul. 1. Chromic acid specimen.

It illustrates the structure of the primitive ova, segmental tubes, notochord, etc.

Fig. 8_a_. Section from the caudal region of an embryo of the same age as 8. Zeiss A, ocul. 1.

It shews (1) the solid oesophagus. (2) The narrow passage connecting the pericardial (_pc_) and body cavities (_pp_).

Fig. 9. Section of a Pristiurus embryo belonging to stage K. Zeiss A, ocul. 1. Osmic acid specimen.

It shews the formation of the liver (_l_), the structure of the anterior fins (_b_), and the anterior opening of the segmental duct into the body-cavity (_sd_).

Figs. 9_a_, 9_b_, 9_c_, 9_d_. Four sections through the anterior region of the same embryo as 9. Osmic acid specimens.

The sections shew (1) the atrophy of the post-anal section of the alimentary tract (9_b_, 9_c_, 9_d_). (2) The existence of the segmental tubes behind the anus (9_b_, 9_c_, 9_d_). With reference to these it deserves to be noted that the segmental tubes behind the anus are quite disconnected, as is proved by the fact that a tube is absent on one side in 9_c_ but reappears in 9_d_. (3) The downward prolongation of the segmental duct to join the posterior or cloacal extremity of the alimentary tract (9_b_).

PLATE 12.

Fig. 10. Longitudinal and horizontal section of a Scyllium embryo of stage H. Zeiss C, ocul. 1. Reduced by 1/3. Picric acid specimen.

It shews (1) the structure of the notochord; (2) the appearance of the early formed band of muscles (_mp´_) in the splanchnic layer of the protovertebra.

Fig. 11. Longitudinal and horizontal sections of an embryo belonging to stage I. Zeiss C, ocul. 1. Chromic acid specimen. It illustrates the same points as the previous section, but in addition shews the formation of the rudiments of the vertebral bodies (_Vr_) which are seen to have the same segmentation as the muscle-plates.

Fig. 12.[227] Longitudinal and horizontal section of an embryo belonging to the stage intermediate between I and K. Zeiss C, ocul. 1. Osmic acid specimen illustrating the same points as the previous section.

Footnote 227: The apparent structure in the sheath of the
notochord in this and the succeeding figure is merely the
result of an attempt on the part of the engraver to represent
the dark colour of the sheath in the original figure.

Fig. 13. Longitudinal and horizontal section of an embryo belonging to stage K. Zeiss C, ocul. 1, and illustrating same points as previous section.

Figs. 14_a_, 14_b_, 14_c_, 14_d_. Figures taken from preparations of an embryo of an age intermediate between I and K, and illustrating the structure of the primitive ova. Figs. 14_a_ and 14_b_ are portions of transverse sections. Zeiss C, ocul. 3 reduced 1/3. Figs. 14_c_ and 14_d_ are individual ova, shewing the lobate form of nucleus. Zeiss F, ocul. 2.

Fig. 15. Osmic acid preparation of primitive ova belonging to stage K. Zeiss immersion No. 2, ocul. 1. The protoplasm of the ova is seen to be nearly filled with bodies resembling yolk-spherules: and one ovum is apparently undergoing division.

Fig. 15_a_. Picric acid preparation shewing a primitive ovum partially filled with bodies resembling yolk-spherules.

Fig. 16. Horizontal and longitudinal section of Scyllium embryo belonging to stage K. Zeiss A, ocul. 1. Picric acid preparation. The connective-tissue cells are omitted.

The section shews that there is one segmental tube to each vertebral segment.

Fig. 17. Portion of a Scyllium embryo belonging to stage K, viewed as a transparent object.

It shews the segmental duct and the segmental involutions--two of which are seen to belong to segments behind the end of the alimentary tract.

Fig. 18. Vertical longitudinal section of a Scyllium embryo belonging to stage K. Zeiss A, ocul. 1. Hardened in a mixture of osmic and chromic acid. It shews

(1) the commissures connecting together the posterior roots of the spinal nerves;

(2) the junction of the anterior and posterior roots;

(3) the relations of the segmental ducts to the segmental involutions and the alternation of calibre in the segmental tube;

(4) the germinal epithelium lining the body-cavity.