Chapter III: Introduction: 1 (2)

On Tuesday afternoon, July 18, he and Petrus, with a porter to carry provisions and firing to their sleeping-place on the rocks, set out from Courmayeur, the porter returning the same night. They expected to get back to Courmayeur some time on the Thursday, but the day passed without their appearing. This did not cause any great anxiety because it was supposed that they might have found it more convenient to pass over to the Chamonix side than to return to Courmayeur. When on Friday however telegrams dispatched to Chamonix and Montanvert brought answers that nothing had been seen of them, it became evident that some accident had happened, and an exploring party set out for the hills. It was not until early on the Sunday morning that this search party found the bodies, both partly covered with snow, lying on the Glacier de Fresney, below the impassable icefall which separates the upper basin of the glacier from the lower portion, and at the foot of a _couloir_ which descends by the side of the icefall. Their tracks were visible on the snow at the top of the _couloir_. Balfour's neck was broken, and his skull fractured in three places; Petrus' body was also fractured in many places. The exact manner of their death will never be known, but there can be no doubt that, in Balfour's case at all events, it was instantaneous, and those competent to form a judgment are of opinion that they were killed by a sudden fall through a comparatively small height, slipping on the rocks as they were descending by the side of the ice-fall, and not precipitated from the top of the _couloir_. There is moreover indirect evidence which renders it probable that in the fatal fall Petrus slipped first and carried Balfour with him. Whether they had reached the summit of the Aiguille and were returning home after a successful ascent or whether they were making their way back disheartened and wearied with failure, is not and perhaps never will be known. Since the provisions at the sleeping-place were untouched, the deaths probably took place on Wednesday the 19th. The bringing down the bodies proved to be a task of extreme difficulty, and it was not till Wednesday the 26th that the remains reached Courmayeur, where M. Bertolini, the master of the hotel, and indeed everyone, not least the officers of a small body of Italian troops stationed there, shewed the greatest kindness and sympathy to Balfour's brothers, Gerald and Eustace, who hastened to the spot as soon as the news of the terrible disaster was telegraphed home. Mr Walter Leaf also and Mr Conway, friends of Balfour, the former a very old one, who had made their way to Courmayeur from other parts of Switzerland as soon as they heard of the accident, rendered great assistance. The body was embalmed, brought to England, and buried at Whittinghame on Saturday, Aug. 5, the Fellows of Trinity College holding a service in the College Chapel at the same time.

In person he was tall, being fully six feet in height, well built though somewhat spare. A broad forehead overhanging deeply set dark brown eyes whose light shining from beneath strongly marked eye-brows told all the changes of his moods, slightly prominent cheek-bones, a pale skin, at times inclined to be even sallow, dark brown hair, allowed to grow on the face only as a small moustache, and slight whiskers, made up a countenance which bespoke at once strength of character and delicacy of constitution. It was an open countenance, hiding nothing, giving sign at once, both when his body was weary or weak, and when his mind was gladdened, angered or annoyed.

The record of some of his thoughts and work, all that he had given to the world will be found in the following pages. But who can tell the ideas which had passed into his quick brain, but which as yet were known only to himself, of which he had given no sign up to that sad day on which he made the fatal climb? And who can say whither he might not have reached had he lived, and his bright young life ripened as years went on? This is not the place to attempt any judgment of his work: that may be left to other times, and to other hands; but it may be fitting to place here on record a letter which shews how much the greatest naturalist of this age appreciated his younger brother. Among Balfour's papers was found a letter from Charles Darwin, acknowledging the receipt of Vol. II. of the _Comparative Embryology_ in the following words:

"_July 6, 1881._
DOWN, BECKENHAM, KENT.

MY DEAR BALFOUR,

I thank you heartily for the present of your grand book, and
I congratulate you on its completion. Although I read almost
all of Vol. I, I do not feel that I am worthy of your
present, unless indeed the fullest conviction that it is a
memorable work makes me worthy to receive it.

* * * * *

Once again accept my thanks, for I am proud to receive a
book from you, who, I know, will some day be the chief of
the English Biologists.

Believe me,
Yours sincerely,
CHARLES DARWIN."

The loss of him was a manifold loss. He is mourned, and will long be mourned, for many reasons. Some miss only the brilliant investigator; others feel that their powerful and sympathetic teacher is gone; some look back on his memory and grieve for the charming companion whose kindly courtesy and bright wit made the hours fly swiftly and pleasantly along; and to yet others is left an aching void when they remember that they can never again lean on the friend whose judgment seemed never to fail and whose warm-hearted affection was a constant help. And to some he was all of these. At the news of his death the same lines came to the lips of all of us, so fittingly did Milton's words seem to speak our loss and grief--

"For Lycidas is dead, dead ere his prime,
Young Lycidas, and hath not left his peer."

M. FOSTER.

I. ON SOME POINTS IN THE GEOLOGY OF THE EAST LOTHIAN COAST[3].

Footnote 3: From the _Geological Magazine_, Vol. IX. No. 4.
April, 1872.

By G. W. and F. M. BALFOUR, Trinity College, Cambridge.

The interesting relation between the Porphyrite of Whitberry Point, at the mouth of the Tyne, near Dunbar, and the adjacent sedimentary rocks, was first noticed, we believe, by Professor Geikie, who speaks of it in the _Memoirs of the Geological Survey of East Lothian_, pages 40 and 31, and again in the new edition of Jukes's _Geology_, p. 269. The volcanic mass which forms the point consists of a dark felspathic base with numerous crystals of augite: it is circular in form, and is exposed for two-thirds of its circumference in a vertical precipice facing the sea, about twenty feet in height.

The rock is traversed by numerous joints running both in a horizontal and in a vertical direction. The latter are by far the most conspicuous, and give the face of the cliff, when seen from a distance, a well-marked columnar appearance, though the columns themselves are not very distinct or regular. They are quadrangular in form, and are evidently produced by the intersection at right-angles of the two series of vertical joints.

It is clear that the face of the precipice has been gradually receding in proportion as it yielded to the action of the waves; and that at a former period the volcanic rock extended considerably further than at present over the beds which are seen to dip beneath it. These latter consist of hard fine-grained calcareous sandstones belonging to the Lower Carboniferous formation. Their colour varies from red to white, and their prevailing dip is in a N.W. direction, with an average inclination of 12-20°. If the volcanic mass is a true intrusive rock, we should naturally expect the strata which surround it to dip _away_ in all directions, the amount of their inclination diminishing in proportion to their distance from it. We find, however, that the case is precisely the reverse: as the beds approach the base of the cliff, they dip _towards_ it from every side at perpetually increasing angles, until at the point of junction the inclination amounts in places to as much as 55 degrees. The exact amount of dip in the various positions will be seen on referring to the accompanying map.

_A._ Lava sheet. _B._ Sandstone Beds, dipping from every side towards the lava. _CC._ Line of Section along which Fig. 2 is supposed to be drawn.]

We conceive that the phenomenon is to be explained by supposing the orifice through which the lava rose and overflowed the surface of the sedimentary strata to have been very much smaller in area than the extent of igneous rock at present visible; and that the pressure of the erupted mass on the soft beds beneath, aided perhaps by the abstraction of matter from below, caused them to incline towards the central point at a gradually increasing angle. The diagram, fig. 2, will serve further to illustrate this hypothesis. _A_ is the neck or orifice by which the melted matter is supposed to ascend. _C_ shews the sheet of lava after it has overspread the surface of the sandstone beds _B_, so as to cause them to assume their present inclination. The dotted lines represent the hypothetical extension of the igneous mass and sandstones previous to the denudation which they have suffered from the action of the waves.

Professor Geikie, in his admirable treatise on the Geology of the county[4], adopts a view on this subject which is somewhat different from that which is suggested in this paper. He considers that the whole mass is an intrusive neck of rock with perpendicular sides; and that it once filled up an orifice through the surrounding sedimentary strata, of which it is now the only remnant.

Footnote 4: _Memoirs of Geological Survey of Scotland_, sheet
33, pp. 40, 41.

_A._ Orifice by which the lava ascended. _B._ Sandstone Beds. _B´._ Hypothetical extension of ditto. _C._ Sheet of lava spread over the sandstones _B_. _C´_. Hypothetical extension of ditto.]

He admits that the inclination of the sandstone beds towards the igneous mass in the centre is a phenomenon that is somewhat difficult to explain, and suggests that a subsequent contraction of the column may have tended to produce such a result. To use his own words: "In the case of a solid column of felstone or basalt, the contraction of the melted mass on cooling may have had some effect in dragging down the sides of the orifice[5]."

Footnote 5: Note on p. 41 of _Mem. Geol. Survey of East
Lothian_.

But, apart from other objections, it is scarcely conceivable that this result should have been produced by the contraction of the column.

In his recent edition of Jukes's _Manual of Geology_ (p. 269), in which he also refers to this instance, he states that in other cases of "necks" it is found to be an almost invariable rule, "that strata are bent down so as to dip into the neck all round its margin." We are not aware to what other instances Prof. Geikie may allude; but on referring to his _Memoir on the Geology of East Lothian_, we find that he states in the cases of 'North Berwick Law' and 'Traprain' (which he compares with the igneous mass at Whitberry Point), that the beds at the base of these two necks, where exposed, dip _away_ from them, and that at a high angle.

In support of the hypothesis which we have put forward, the following arguments may be urged:

(1) That in one place at least the sedimentary strata are seen to be actually dipping beneath the superincumbent basalt; and that the impression produced by the general relation of the two rocks is, that they do so everywhere.

(2) Since the columns into which the lava is split are vertical, the cooling surface must have been horizontal: the mass must, therefore, have formed a sheet, and not a dyke; for, in the latter case, the cooling surfaces would have been vertical.

(3) It is difficult to conceive, on the supposition that the volcanic rock is a neck with perpendicular sides, that the marine denudation should have uniformly proceeded only so far as to lay bare the junction between the two formations. We should have expected that in many places the igneous rock itself would have been cut down to the general level, whereas the only signs of such an effect are shown in a few narrow inlets where the rock was manifestly softer than in the surrounding parts.

The last objection is greatly confirmed by the overhanging cliffs and numerous blocks of porphyrite which lie scattered on the beach, as if to attest the former extension of that ancient sheet of which these blocks now form but a small remnant. Indeed, the existence of such remains appears sufficient of itself to condemn any hypothesis which presumes the present face of the cliff to have formed the original boundary of the mass.

It may be fairly objected to our theory, as Prof. Geikie himself has suggested, that the high angle at which the strata dip is difficult to account for. But, in fact, this steep inclination constitutes the very difficulty which any hypothesis on the subject must be framed to explain; and it is a difficulty which is not more easily solved by Prof. Geikie's theory than by our own.

II. THE DEVELOPMENT AND GROWTH OF THE LAYERS OF THE BLASTODERM[6].

Footnote 6: From the _Quarterly Journal of Microscopical
Science_, Vol. XIII., 1873.

With Plate 1, figs. 1-5 and 9-12.

The following paper deals with the changes which take place in the cells of the blastoderm of the hen's egg during the first thirty or forty hours of incubation. The subject is one which has, as a general rule, not been much followed up by embryologists, but is nevertheless of the greatest interest, both in reference to embryology itself, and to the growth and changes of protoplasm exhibited in simple embryonic cells. I am far from having exhausted the subject in this paper, and in some cases I shall be able merely to state facts, without being able to give any explanation of their meaning.

My method of investigation has been the examination of sections and surface views. For hardening the blastoderm I have employed, as usual, chromic acid, and also gold chloride. It is, however, difficult to make sections of blastoderms hardened by this latter reagent, and the sections when made are not in all cases satisfactory. For surface views I have chiefly used silver nitrate, which brings out the outlines of the cells in a manner which leaves nothing to be desired as to clearness. If the outlines only of the cells are to be examined, a very short immersion (half a minute) of the blastoderm in a half per cent. solution of silver nitrate is sufficient, but if the immersion lasts for a longer period the nuclei will be brought out also. For studying the latter, however, I have found it better to employ gold chloride or carmine in conjunction with the silver nitrate.

My observations begin with the blastoderm of a freshly laid egg. The appearances presented by sections of this have been accurately described by Peremeschko, "Ueber die Bildung der Keimblätter im Hühnerei," _Sitzungsberichte der K. Akademie der Wissenschaften in Wien_, 1868. Oellacher, "Untersuchung über die Furchung und Blatterbildung im Hühnerei," _Studien aus dem Institut für Experim. Pathologie in Wien_, 1870 (pp. 54-74), and Dr Klein, lxiii. Bande der _Sitz. der K. Acadamie der Wiss. in Wien_, 1871.

The unincubated blastoderm (Pl. 1, fig. 1) consists of two layers. The upper layer is composed of a single row of columnar cells. Occasionally, however, the layer may be two cells thick. The cells are filled with highly refracting spherules of a very small size, and similar in appearance to the finest white yolk spherules, and each cell also contains a distinct oval nucleus. This membrane rests with its extreme edge on the white yolk, its central portion covering in the segmentation cavity. From the very first it is a distinct coherent membrane, and exhibits with silver nitrate a beautiful hexagonal mosaic of the outlines (Pl. 1, fig. 6) of the cells. The diameter of the cells when viewed from above is from 1/2000 - 1/3000 of an inch. The under layer is very different from this: it is composed of cells which are slightly, if at all, united, and which vary in size and appearance, and in which a nucleus can rarely be seen. The cells of which it is composed fill up irregularly the segmentation cavity, though a distinct space is even at this time occasionally to be found at the bottom of it. Later, when the blastoderm has spread and the white yolk floor has been used as food, a considerable space filled with fluid may generally be found.

The shape of the floor of the cavity varies considerably, but it is usually raised in the middle and depressed near the circumference. In this case the under layer is perhaps only two cells deep at the centre and three or four cells deep near the circumference.

The cells of which this layer is composed vary a good deal in size; the larger cells being, however, more numerous in the lower layers. In addition, there are usually a few very large cells quite at the bottom of the cavity, occasionally separated from the other cells by fluid. They were called _formative cells_ (Bildungselemente) by Peremeschko (_loc. cit._); and, according to Oellacher's observations (_loc. cit._), some of them, at any rate, fall to the bottom of the segmentation cavity during the later stages of segmentation. They do not differ from the general lower layer cells except in size, and even pass into them by insensible gradations. All the cells of the lower layer are granular, and are filled with highly refracting spherules precisely similar to the smaller white yolk spherules which line the bottom of the segmentation cavity.

The size of the ordinary cells of the lower layer varies from 1/2000 - 1/1000 of an inch. The largest of the formative cells come up to 1/300 of an inch. It will be seen from this description that, morphologically speaking, we cannot attach much importance to the formative cells. The fact that they broke off from the blastoderm, towards the end of the segmentation--even if we accept it as a normal occurrence, rather than the result of manipulation--is not of much importance, and, except in size, it is impossible to distinguish these cells from other cells of the lower layer of the blastoderm.

Physiologically, however, as will be afterwards shewn, they are of considerable importance.

The changes which the blastoderm undergoes during the first three or four hours of incubation are not very noticeable. At about the sixth or eighth hour, or in some cases considerably earlier, changes begin to take place very rapidly. These changes result in the formation of a hypoblast and mesoblast, the upper layer of cells remaining comparatively unaltered as the epiblast.

To form the hypoblast a certain number of the cells of the lower layer begin to undergo remarkable changes. From being spherical and, as far as can be seen, non-nucleated, they become (vide fig. 2, _h_) flattened and nucleated, still remaining granular, but with fewer spherules.

Here, then, is a direct change, of which all the stages can be followed, of a cell of one kind into a cell of a totally different character. The new cell is not formed by a destruction of the old one, but directly from it by a process of metamorphosis. These hypoblast cells are formed first at the centre and later at the circumference, so that from the first the cells at the circumference are less flattened and more granular than the cells at the centre. A number of cells of the original lower layer are enclosed between this layer and the epiblast; and, in addition to these, the formative cells (as has been shewn by Peremeschko, Oellacher, and Klein, whose observations I can confirm) begin to travel towards the circumference, and to pass in between the epiblast and hypoblast.

Both the formative cells, and the lower layer cells enclosed between the hypoblast and epiblast, contribute towards the mesoblast, but the mode in which the mesoblast is formed is very different from that in which the hypoblast originates.

It is in this difference of formation that the true distinction between the mesoblast and hypoblast is to be looked for, rather than in the original difference of the cells from which they are derived.

The cells of the mesoblast are formed by a process which seems to be a kind of free cell formation. The whole of the interior of each of the formative cells, and of the other cells which are enclosed between the epiblast and the hypoblast, become converted into new cells. These are the cells of the mesoblast. I have not been able perfectly to satisfy myself as to the exact manner in which this takes place, but I am inclined to think that some or all of the spherules which are contained in the original cells develop into nuclei for the new cells, the protoplasm of the new cells being formed from that of the original cells.

The stages of formation of the mesoblast cells are shewn in the section (Pl. 1, fig. 2), taken from the periphery of a blastoderm of eight hours.

The first formation of the mesoblast cells takes place in the centre of the blastoderm, and the mass of cells so formed produces the opaque line known as the primitive streak. This is shown in Pl. 1, fig. 9.

One statement I have made in the above description in reference to the origin of the mesoblast cells, viz. that they are only partly derived from the formative cells at the bottom of the segmentation cavity, is to a certain extent opposed to the statements of the three investigators above mentioned. They state that the mesoblast is entirely derived from the formative cells. It is not a point to which I attach much importance, considering that I can detect no difference between these cells and any other cells of the original lower layer except that of size; and even this difference is probably to be explained by their proximity to the white yolk, whose spherules they absorb. But my reason for thinking it probable that these cells alone do not form the mesoblast are: 1st. That the mesoblast and hypoblast are formed nearly synchronously, and except at the centre a fairly even sprinkling of lower layer cells is from the first to be distinguished between the epiblast and hypoblast. 2nd. That if some of the lower layer cells are not converted into mesoblast, it is difficult to see what becomes of them, since they appear to be too numerous to be converted into the hypoblast alone. 3rd. That the chief formation of mesoblast at first takes place in the centre, while if the formative cells alone took part in its formation, it would be natural to expect that it would begin to be formed at the periphery.

Oellacher himself has shewn (_Zeitschrift für wissenschaftliche Zoologie_, 1873, "Beiträge zur Entwick. Gesch. der Knochenfische") that in osseous fishes the cells which break away from the blastoderm take no share in the formation of the mesoblast, so that we can derive no argument from the formation of the mesoblast in these animals, for believing that in the chick it is derived only from the formative cells.

In the later stages, however, from the twelfth to the twenty-fifth hour, the growth of the mesoblast depends almost entirely on these cells, and Peremeschko's discovery of the fact is of great value.

Waldeyer (_Henle und v. Pfeufer's Zeitschrift_, xxxiv. Band, für 1869) has given a different account of the origin of the layers. There is no doubt, however, in opposition to his statements and drawings, that from the very first the hypoblast is distinct from the mesoblast, which is, indeed, most conspicuously shewn in good sections; and his drawings of the derivation of the mesoblast from the epiblast are not very correct.

The changes which have been described are also clearly shewn by means of silver nitrate. Whereas, at first this reagent brought out no outline markings of cells in the lower layer, by the eighth to the twelfth hour the markings (Pl. 1, fig. 3) are very plain, and shew that the hypoblast is a distinct coherent membrane.

In section, the cells of the hypoblast appear generally very thin and spindle shaped, but the outlines brought out by the silver nitrate shew that they are much expanded horizontally, but very irregular as to size, varying even within a small area from 1/4000 - 1/400 of an inch in the longest diameter.

At about the twelfth hour they are uniformly smaller a short way from each extremity of its longer axis than over the rest of the blastoderm.

It is, perhaps, fair to conclude from this that growth is most rapid at these parts.

At this time the hypoblast, both in sections and from a surface view after treatment with silver nitrate, appears to end abruptly against the white yolk. The surface view also shews that its cells are still filled with highly refractive globules, making it difficult to see the nucleus. In some cases I thought that I could (fig. 3, _a_) make out that it was hour-glass shaped, and some cells certainly contain two nuclei. Some of the cells (fig. 3, _b_) shew re-entrant curves, which prove that they have undergone division.

The cells of the epiblast, up to the thirteenth hour, have chiefly undergone change in becoming smaller.

In surface views they are about 1/4000 of an inch in diameter over the centre of the pellucid area, and increase to 1/2000 of an inch over the opaque area.

In the centre of the pellucid area the form of the epiblast cells is more elongated vertically and over the opaque area more flattened than was the case with the original upper layer cells. In the centre the epiblast is two or three cells deep.

Before going on to the further changes of the blastodermic cells it will be well to say a few words in reference to the origin of the mesoblast.

From the description given above it will be clear that in the chick the mesoblast has an independent origin; it can be said neither to originate from the epiblast nor from the hypoblast. It is formed coincidently with the latter out of apparently similar segmentation cells. The hypoblast, as has been long known, shews in the chick no trace of its primitive method of formation by involution, neither does the mesoblast shew any signs of its primitive mode of formation. In so excessively highly differentiated a type as birds we could hardly expect to find, and certainly do not find, any traces of the primitive origin of the mesoblast, either from the epiblast or hypoblast, or from both. In the chick the mesoblast cells are formed directly from the ultimate products of segmentation. From having a secondary origin in most invertebrates the mesoblast comes to have, in the chick, a primary origin from the segmentation spheres, precisely as we find to be the case with the nervous layer in osseous fishes. It is true we cannot tell which segmentation-cells will form the mesoblast, and which the hypoblast; but the mesoblast and hypoblast are formed at the same time, and both of them directly from segmentation spheres.

The process of formation of the mesoblast in Loligo, as observed by Mr Ray Lankester (_Annals and Magazine of Natural History_, February, 1873), is still more modified. Here the mesoblast arises independently of the blastoderm, and by a process of free cell-formation in the yolk round the edge of the blastoderm. If Oellacher's observations in reference to the origin of formative cells are correct, then the modes of origin of the mesoblast in Loligo and the chick would have nothing in common; but if the formative cells are in reality derived from the white yolk, and also are alone concerned in the formation of the mesoblast, then the modes of formation of the mesoblast in the chick would be substantially the same as that observed by Mr Ray Lankester in Loligo.

No very important changes take place in the actual forms of the cells during the next few hours. A kind of fusion takes place between the epiblast and the mesoblast along the line of the primitive streak forming the axis-string of His; but the line of junction between the layers is almost always more or less visible in sections. In any case it does not appear that there is any derivation of mesoblast cells from the epiblast; and since the fusion only takes place in the region of the primitive groove, and not in front, where the medullary groove arises (see succeeding paper), it cannot be considered of any importance in reference to the possible origin of the Wolffian duct, &c., from the epiblast (as mooted by Waldeyer, _Eierstock und Ei_, Leipzig, 1870). The primitive groove, as can be seen in sections, begins to appear very early, generally before the twelfth hour. The epiblast spreads rapidly over the white yolk, and the area pellucida also increases in size.

From the mesoblast forming at first only a small mass of cells, which lies below the primitive streak, it soon comes to be the most important layer of the blastoderm. Its growth is effected by means of the formative cells. These cells are generally not very numerous in an unincubated blastoderm, but rapidly increase in numbers, probably by division; at the same time they travel round the edge of, and in some cases through, the hypoblast, and then become converted in the manner described into mesoblast cells. They act as carriers of food from the white yolk to the mesoblast till, after the formation of the vascular area, they are no longer necessary. The numerous cases in which two nucleoli and even two nuclei can be seen in one cell prove that the mesoblast cells also increase by division.

The growth of the hypoblast takes place in a very different way. It occurs by a direct conversion, cell for cell, of the white yolk spheres into hypoblast cells. This interpretation of the appearances, which I will describe presently, was first suggested to me by Dr Foster, from an examination of some of my specimens of about thirty-six hours, prepared with silver nitrate. Where there is no folding at the junction between the pellucid and opaque areas, there seems to be a perfect continuity in the silver markings and a gradual transition in the cells, from what would be undoubtedly called white yolk spheres, to as undoubted hypoblast cells (vide Pl. 1, fig. 5). In passing from the opaque to the pellucid areas the number of white yolk spherules in each cell becomes less, but it is not till some way into the pellucid area that they quite cease to be present. I at first thought that this was merely due to the hypoblast cells feeding on the white yolk sphericles, but the perfect continuity of the cells, and the perfect gradation in passing from the white yolk cells to the hypoblast, proves that the other interpretation is the correct one, viz. that the white yolk spheres become directly converted into the hypoblast cells. This is well shewn in sections (vide Pl. 1, fig. 4) taken from embryos of all ages from the fifteenth to the thirty-sixth hour and onwards. But it is, perhaps, most easily seen in embryos of about twenty hours. In such an embryo there is a most perfect gradation: the cells of the hypoblast become, as they approach the edge of the pellucid area, broader, and are more and more filled with white yolk sphericles, till at the line of junction it is quite impossible to say whether a particular cell is a white-yolk cell (sphere) or a hypoblast cell. The white-yolk cells near the line of junction can frequently be seen to possess nuclei. At first the hypoblast appears to end abruptly against the white yolk; this state of things, however, soon ends, and there supervenes a complete and unbroken continuity between the hypoblast and the white yolk.

Of the mode of increase of the epiblast I have but little to say. The cells undoubtedly increase entirely by division, and the new material is most probably derived directly from the white yolk.

Up to the sixth hour the cells of the upper layer retain their early regular hexagonal pattern, but by the twelfth hour they have generally entirely lost this, and are irregularly shaped and very angular. The cells over the centre of the pellucid area remain the smallest up to the twenty-fifth hour or later, while those over the rest of the pellucid area are uniformly larger.

In the hypoblast the cells under the primitive groove, and on each side as far as the fold which marks off the exterior limit of the protovertebræ are at the eighteenth hour considerably smaller than any other cells of this layer.

In all the embryos between the eighteenth and twenty-third hour which I have examined for the purpose, I have found that at about two-thirds of the distance from the anterior end of the pellucid area, and just external to the side fold, there is a small space on each side in which the cells are considerably larger than anywhere else in the hypoblast. These larger cells, moreover, contain a greater number of highly refractive spherules than any other cells. It is not easy to understand why growth should have been less rapid here than elsewhere, as the position does not seem to correspond to any feature in the embryo. In some specimens the hypoblast cells at the extreme edge of the pellucid area are smaller than the cells immediately internal to them. At about the twenty-third hour these cells begin rapidly to lose the refractive spherules they contained in the earlier stages of incubation, and come to consist of a nucleus surrounded simply by granular protoplasm.

At about this period of incubation the formative cells are especially numerous at the periphery of the blastoderm, and, no doubt, become converted into the mass of mesoblast which is found at about the twenty-fifth hour in the region of the vascular area. Some of them are lobate, and appear as if they were undergoing division. At this time also the greatest number of formative cells are to be found at the bottom of the now large segmentation cavity.

In embryos of from thirty to forty hours the cells of the hypoblast have, over the central portion of the pellucid area, entirely lost their highly refractive spherules, and in the fresh state are composed of the most transparent protoplasm. When treated with reagents they are found to contain an oval nucleus with one or sometimes two nucleoli, imbedded in a considerable mass of protoplasm. The protoplasm appears slightly granular and generally contains one or two small vacuoles. I have already spoken of the gradation of the hypoblast at the edge of the blastoderm into white yolk. I have, therefore, only to mention the variations in the size of its cells in different parts of the pellucid area. The points where the cells are smallest seem generally to coincide with the points of maximum growth. Over the embryo the cells are more regular than elsewhere. They are elongated and arranged transversely to the long axis of the embryo. They are somewhat hexagonal in shape, and not unlike the longer pieces in the dental plate of a Myliobatis (Pl. 1, fig. 10). This regularity, however, is much more marked in some specimens than in others. These cells are about 1/4000th of an inch in breadth, and 1/1000th in length. On each side of the embryo immediately external to the protovertebræ the cells are frequently about the same size as those over the embryo itself. In the neck, however, and near the end of the sinus rhomboidalis, they are considerably smaller, about 1/4000th inch each way. The reason of this small size is not very clear, but probably shews that the greatest growth is taking place at these two points. The cells, again, are very small at the head fold, but are very much larger in front of this--larger, in fact, than any other cells of the hypoblast. Outside the embryo they gradually increase in size towards the edge of the pellucid area. Here they are about 1/1000th of an inch in diameter, irregular in shape and rather angular.

The outlines of the cells of the epiblast at this time are easily distinguished from the cells of the hypoblast by being more elongated and angular; they are further distinguished by the presence of numerous small oval cells, frequently at the meeting point of several cells, at other times at points along the lines of junction of two cells (Pl. 1, fig. 12). These small cells look very like the smaller stomata of endothelial membranes, but are shewn to be cells by possessing a nucleus. There is considerable variation in size in the cells in different parts of the epiblast. Between the front lobes of the brain the cells are very small, 1/4000th inch, rising to 1/2000th on each side. They are about the latter size over the greater part of the embryo. But over the sinus rhomboidalis they fall again to from 1/3000th to 1/4000th inch. This is probably to be explained by the growth of the medullary fold at this point, which pushes back the primitive groove. At the sides of the head the cells are larger than anywhere else in the epiblast, being here about 1/1000th inch in diameter. I at present see no explanation of this fact. At the periphery of the pellucid area and over the vascular area the cells are 1/1500th to 1/2000th inch in diameter, but at the periphery of the opaque area they are smaller again, being about the 1/3000th of an inch. This smaller size at the periphery of the area opaca is remarkable, since in the earlier stages the most peripheral epiblast cells were the largest. It, perhaps, implies that more rapid growth is at this time taking place in that part of the epiblast which is spreading over the yolk sac.

EXPLANATION OF PLATE 1, Figs. 1-5 and 9-12.

Fig. 1. Section through an unincubated blastoderm, shewing the upper layer, composed of a single row of columnar cells, and the lower layer, composed of several rows of rounded cells in which no nucleus is visible. Some of the "formative cells," at the bottom of the segmentation cavity, are seen at (_b_).

Fig. 2. Section through the periphery of an eight hours' blastoderm, shewing the epiblast (_p_), the hypoblast (_h_), and the mesoblast commencing to be formed (_c_), partly by lower-layer cells enclosed between the epiblast and hypoblast, and partly by formative cells. Formative cells at the bottom of the segmentation cavity are seen at _b_. At _s_ is one of the side folds parallel to the primitive groove.

Fig. 3. Portion of the hypoblast of a thirteen hours' blastoderm, treated with silver nitrate, shewing the great variation in the size of the cells at this period. An hour-glass shaped nucleus is seen at _a_.

Fig. 4. Periphery of a twenty-three hours' blastoderm, shewing cell for cell the junction between the hypoblast (_h_) and white-yolk spheres (_w_).

Fig. 5. Junction between the white-yolk spheres and the hypoblast cells at the passage from the area pellucida to the area opaca. The specimen was treated with silver nitrate to bring out the shape of the cells. The line of junction between the opaque and pellucid areas passes diagonally.

Fig. 9. Section through the primitive streak of an eight hours' blastoderm. The specimen shews the mesoblast very much thickened in the immediate neighbourhood of the primitive streak, but hardly formed at all on each side of the streak. It also shews the primitive groove just beginning to be formed (_pr_), and the fusion between the epiblast and the mesoblast under the primitive groove. The hypoblast is completely formed in the central part of the blastoderm. At _f_ is seen one of the side folds parallel to the primitive groove. Its depth has been increased by the action of the chromic acid.

Fig. 10. Hypoblast cells from the hinder end of a thirty-six hours' embryo, treated with silver nitrate, shewing the regularity and elongated shape of the cells over the embryo and the smaller cells on each side.

Fig. 11. Epiblast cells from an unincubated blastoderm, treated with silver nitrate, shewing the regular hexagonal shape of the cells and the small spherules they contain.

Fig. 12. Portion of the epiblast of a thirty-six hours' embryo, treated with silver nitrate, shewing the small rounded cells frequently found at the meeting-points of several larger cells which are characteristic of the upper layer.

III. ON THE DISAPPEARANCE OF THE PRIMITIVE GROOVE IN THE EMBRYO CHICK[7].

Footnote 7: From the _Quarterly Journal of Microscopical
Science_, Vol. XIII, 1873.

With Plate 1, Figs. 6-8 and 13-19.

The investigations of Dursy (_Der Primitivstreif des Hühnchens_, von Dr E. Dursy. Lahr, 1866) on the primitive groove, shewing that it is a temporary structure, and not connected with the development of the neural canal, have in this country either been ignored or rejected. They are, nevertheless, perfectly accurate; and had Dursy made use of sections to support his statements I do not think they would so long have been denied. In Germany, it is true, Waldeyer has accepted them with a few modifications, but I have never seen them even alluded to in any English work. The observations which I have made corroborating Dr Dursy may, perhaps, under these circumstances be worth recording.

After about twelve hours of incubation the pellucid area of a hen's egg has become somewhat oval, with its longer axis at right angles to the long axis of the egg. Rather towards the hinder (narrower) end of this an opaque streak has appeared, with a somewhat lighter line in the centre. A section made at the time shews that the opaque streak is due partly to a thickening of the epiblast, but more especially to a large collection of the rounded mesoblast cells, which along this opaque line form a thick mass between the epiblast and the hypoblast. The mesoblast cells are in contact with both hypoblast and epiblast, and appear to be fused with the latter. The line of junction between them can, however, almost always be made out.

Soon after the formation of this primitive streak a groove is formed along its central line by a pushing inwards of the epiblast. The epiblast is not thinner where it lines the groove, but the mass of mesoblast below the groove is considerably thinner than at its two sides. This it is which produces the peculiar appearance of the primitive groove when the blastoderm is viewed by transmitted light as a transparent line in the middle of an opaque one.

This groove, as I said above, is placed at right angles to the long axis of the egg, and nearer the hind end, that is, the narrower end of the pellucid area. It was called "the primitive groove" by the early embryologists, and they supposed that the neural canal arose from the closure of its edges above. It is always easy to distinguish this groove, in transverse sections, by several well-marked characters. In the first place, the epiblast and mesoblast always appear more or less fused together underneath it; in the second place, the epiblast does not become thinner where it lines the groove; and in the third place, the mesoblast beneath it never shews any signs of being differentiated into any organ.

As Dursy has pointed out, there is frequently to be seen in fresh specimens, examined as transparent objects, a narrow opaque line running down the centre of this groove. I do not know what this line is caused by, as there does not appear to be any structural feature visible in sections to which it can correspond.

From the twelfth to the sixteenth hour the primitive groove grows rapidly, and by the sixteenth hour is both absolutely and considerably longer than it was at the twelfth hour, and also proportionately longer as compared with the length of the pellucid area.

There is a greater interval between its end and that of the pellucid area in front than behind.

At about the sixteenth hour, or a little later, a thickening of the mesoblast takes place in front of the primitive groove, forming an opaque streak, which in fresh specimens looks like a continuation from the anterior extremity of the primitive groove (vide Pl. 1, fig. 8). From hardened specimens, however, it is easy to see that the connection of this streak with the primitive groove is only an apparent one. Again, it is generally possible to see that in the central line of this streak there is a narrow groove. I do not feel certain that there is no period when this groove may not be present, but its very early appearance has not been recognized either by Dursy or by Waldeyer. Moreover, both these authors, as also His, seem to have mistaken the opaque streak spoken of above for the notochord. This, however, is not the case, and the notochord does not make its appearance till somewhat later. The mistake is of very minor importance, and probably arose in Dursy's case from his not sufficiently making use of sections. At about the time the streak in front of the primitive groove makes its appearance a semicircular fold begins to be formed near the anterior extremity of the pellucid area, against which the opaque streak, or as it had, perhaps, better be called, "the medullary streak," ends abruptly.

This fold is the head fold, and the groove along the medullary streak is the medullary groove, which subsequently forms the cavity of the medullary or neural canal.

Everything which I have described above can without difficulty be made out from the examination of fresh and hardened specimens under the simple microscope; but sections bring out still more clearly these points, and also shew other features which could not have been brought to light without their aid. In Pl. 1, figs. 6 and 7, two sections of an embryo of about eighteen hours are shewn. The first of these passes through the medullary groove, and the second of them through the extreme anterior end of the primitive groove. The points of difference in the two sections are very obvious.

From fig. 6 it is clear that a groove has already been formed in the medullary streak, a fact which was not obvious in the fresh specimen. In the second place the mesoblast is thickened both under the groove and also more especially in the medullary folds at the sides of the groove; but shews hardly a sign of the differentiation of the notochord. So that it is clear that the medullary streak is not the notochord, as was thought to be the case by the authors above mentioned. In the third place there is no adhesion between the epiblast and the mesoblast. In all the sections I have cut through the medullary groove I have found this feature to be constant; while (for instance, as in Pl. 1, figs. 7, 9, 17) all sections through the primitive groove shew most clearly an adhesion between the epiblast and mesoblast. This fact is both strongly confirmatory of the separate origins of the medullary and primitive grooves, and is also important in itself, as leaving no loophole for supposing that in the region of embryo there is any separation of the cells from the epiblast to form the mesoblast.

By this time the primitive groove has attained its maximum growth, and from this time begins both absolutely to become smaller, and also gradually to be pushed more and more backwards by the growth of the medullary groove.

The specimen figured in Pl. 1, fig. 18, magnified about ten diameters, shews the appearance presented by an embryo of twenty-three hours. The medullary groove (_mc_) has become much wider and deeper than it was in the earlier stage; the medullary folds (_A_) are also broader and more conspicuous. The medullary groove widens very much posteriorly, and also the medullary folds separate far apart to enclose the anterior end of the primitive groove (_pr_).

All this can easily be seen with a simple microscope, but the sections taken from the specimen figured also fully bear out the interpretations given above, and at the same time shew that the notochord has at this age begun to appear. The sections marked 13-17 pass respectively through the lines with corresponding numbers in fig. 18. Section 1 (fig. 13) passes through the middle of the medullary canal.

In it the following points are to be noted. (1) That the epiblast becomes very much thinner where it lines the medullary canal (_mc_), a feature never found in the epiblast lining the primitive groove. (2) That the mesoblast is very much thickened to form the medullary folds at _A_, _A_, while there is no adherence between it and the epiblast, below the primitive groove. (3) The notochord (_ch_) has begun to be formed, though its separation from the rest of the mesoblast is not as yet very distinct[8].

Footnote 8: In the figure the notochord has been made too
distinct.

In fig. 14 the medullary groove has become wider and the medullary folds broader, the notochord has also become more expanded: the other features are the same as in section 1. In the third section (fig. 15) the notochord is still more expanded; the bottom of the now much expanded medullary groove has become raised to form the ridge which separates the medullary from the primitive groove. The medullary folds are also flatter and broader than in the previous section. Section 4 (fig. 16) passes through the anterior end of the primitive groove. Here the notochord is no longer visible, and the adherence between the mesoblast and epiblast below the primitive groove comes out in marked contrast with the entire separation of the two layers in the previous sections.

The medullary folds (_A_) are still visible outside the raised edges of the primitive groove, and are as distinctly as possible separate and independent formations, having no connection with the folds of the primitive groove. In the last section (fig. 17), which is taken some way behind section 4, no trace of the medullary folds is any longer to be seen, and the primitive groove has become deeper. This series of sections, taken in conjunction with the specimen figured in fig. 18, must remove all possible doubt as to the total and entire independence of the primitive and medullary grooves. They arise in different parts of the blastoderm; the one reaches its maximum growth before the other has commenced to be formed; and finally, they are distinguished by almost every possible feature by which two such grooves could be distinguished.

Soon after the formation of the notochord, the protovertebræ begin to be formed along the sides of the medullary groove (Pl. 1, fig. 19, _pv_). Each new protovertebra (of those which are formed from before backwards) arises just in front of the anterior end of the primitive groove. As growth continues, the primitive groove becomes pushed further and further back, and becomes less and less conspicuous, till at about thirty-six hours only a very small and curved remnant is to be seen behind the sinus rhomboidalis; but even up to the forty-ninth Dursy has been able to distinguish it at the hinder end of the embryo.

The primitive groove in the chick is, then, a structure which appears very early, and soon disappears without entering directly into the formation of any part of the future animal, and without, so far as I can see, any function whatever. It is clear, therefore, that the primitive groove must be the rudiment of some ancestral feature; but whether it is a rudiment of some structure which is to be found in reptiles, or whether of some earlier form, I am unable to decide. It is just possible that it is the last trace of that involution of the epiblast by which the hypoblast is formed in most of the lower animals. The fact that it is formed in the hinder part of the pellucid area perhaps tells slightly in favour of this hypothesis, since the point of involution of the epiblast not unfrequently corresponds with the position of the anus.

EXPLANATION OF PLATE 1, Figs. 6-8 and 13-19.

Figs. 6 and 7 are sections through an embryo rather earlier than the one drawn in fig. 8. Fig. 6 passes through the just commencing medullary groove (_md_), which appears in fresh specimens, as in fig. 8, merely as an opaque streak coming from the end of the primitive groove. The notochord is hardly differentiated, but the _complete_ separation of mesoblast and hypoblast under the primitive groove is clearly shewn. Fig. 7 passes through the anterior end of the primitive groove (_pr_), and shews the fusion between the mesoblast and epiblast, which is always to be found under the primitive groove.