The Works of Francis Balfour 1-1
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Foster M. and Sedgwick A. The Works of Francis Balfour Vol. I. Separate Memoirs (1885) MacMillan and Co., London.
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Foster M. and Sedgwick A. The Works of Francis Balfour Vol. II. A Treatise on Comparative Embryology 1. (1885) MacMillan and Co., London. Foster M. and Sedgwick A. The Works of Francis Balfour Vol. III. A Treatise on Comparative Embryology 2 (1885) MacMillan and Co., London. Foster M. and Sedgwick A. The Works of Francis Balfour Vol. IV. Plates (1885) MacMillan and Co., London.
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Vol I. Separate Memoirs (1885)
I. On Some Points in the Geology of the East Lothian Coast
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 5 5 degrees. The exact amount of dip in the various positions will be seen on referring to the accompanying map.
- 1 From the Geological Magazine, Vol. IX. No. 4. April, 1872.
FIG. i. MAP OF STRATA AT WHITBERRY POINT. Scale, 6 in. to the mile.
A. Lava sheet. B. Sandstone Beds, dipping from every side towards the lava. CC. Line of Section along which Fig. i 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 jB,'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 1 , 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.
FIG. 2. VERTICAL SECTION THROUGH CC. DIAGRAM (FiG. i).
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 2 ."
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
1 Memoirs of Geological Survey of Scotland, sheet 33, pp. 40, 41.
2 Note on p. 41 of Mem. Geol. Survey of East Lothian.
28 GEOLOGY OF THE EAST LOTHIAN COAST.
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 tJte 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 1 .
With Plate I. figs, i 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
1 From the Quarterly Journal of Microscopical Science, Vol. xin., 1873.
30 DEVELOPMENT AND GROWTH OF
Keimblatter im HUhnerei," Sitzungsberichte der K. Akademie der Wissenschaften in Wien, 1868. Oellacher, " Untersuchung iiber die Furchung und Blatterbildung im Hiihnerei," Studien aus dem Institut filr Experim. Pathologie in Wien, 1870 (pp. 54 74), and Dr Klein, Ixiii. Bande der Sitz. der K. Acadamie der Wiss. in Wien, 1871.
The unincubated blastoderm (PI. I, fig. i) 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. Thf 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 (PI. I. fig. 6) of the cells. The diameter of the cells when viewed from above is from -%fa -S^M f 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
THE LAYERS OF THE BLASTODERM. 31
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 gTrmj iwou f an incn - The largest of the formative cells come up to 3^ 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 Ji) 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,
32 DEVELOPMENT AND GROWTH OF
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 (PI. I, 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 PI. I, 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
THE LAYERS OF THE BLASTODERM. 33
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, ist. 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 fur wissenscliaftliche Zoologie, 1873, " Beitrage 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 twentyfifth 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 tmd v. Pfeufer's Zeitschrift, xxxiv. Band, fur 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 (PI. I, 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
B. 3
34 DEVELOPMENT AND GROWTH OF
silver nitrate shew that they are much expanded horizontally, but very irregular as to size, varying even within a small area from ^g. -ffo 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> ^) 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 4^7 of an inch in diameter over the centre of the pellucid area, and increase to ^j^y 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
THE LAYERS OF THE BLASTODERM. 35
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 wjiite yolk, and the area pellucida also increases in size.
32
36 DEVELOPMENT AND GROWTH OF
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 PI. I, 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 PI. I, 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
THE LAYERS OF THE BLASTODERM. 37
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 proto-vertebrae, 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
38 DEVELOPMENT AND GROWTH OF
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 (PI. I, fig. 10). This regularity, however, is much more marked in some specimens than in others. These cells are about ^J^yth of an inch in breadth, and y^V^th in length. On each side of the embryo immediately external to the proto-vertebrae 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 -j^o^ 1 mc ^- eacn wa 7- 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
THE LAYERS OF THE BLASTODERM. 39
in size towards the edge of the pellucid area. Here they are about r^th 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 (PI. I, 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, 4oVo tn mcn > rising to ^^th on eacn s ^ e - They are about the latter size over the greater part of the embryo. But over the sinus rhomboidalis they fall again to from ^nnjth to 4oVo tn 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 j(^th 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 T^th to ^^th inch in diameter, but at the periphery of the opaque area they are smaller again, being about the ^oWth 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.
40 DEVELOPMENT AND GROWTH OF THE BLASTODERM.
EXPLANATION OF PLATE I. Figs. 15 and 912.
Fig. i. 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 (l>).
Fig. 2. Section through the periphery of an eight hours' blastoderm, shewing the epiblast (/), 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 / 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. ii. 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 \
With Plate I, figs. 68 and 1319.
THE investigations of Dursy (Der Primitivstreif des Hiihnchens, 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.
1 From the Qziarterly Journal of Microscopical Science, Vol. Kill, 1873.
42 PRIMITIVE GROOVE IN THE EMBRYO CHICK.
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 PI. I, 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
PRIMITIVE GROOVE IN THE EMBRYO CHICK. 43
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 I, 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 I, figs. 7, 9, 17) all sections through the primitive groove
44 PRIMITIVE GROOVE IN THE EMBRYO CHICK.
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 PI. I, fig. 18, magnified about ten diameters, shews the appearance presented by an embryo of twenty-three hours. The medullary groove (me) 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 I (fig. 13) passes through the middle of the medullary canal.
In it the following points are to be noted, (i) That the epiblast becomes very much thinner where it lines the medullary canal (me), 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 (c/i) has begun to be formed, though its separation from the rest of the mesoblast is not as yet very distinct 1 .
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 I. "In the third section (fig. 15) the notochord is still more expanded;
1 In the figure the notochord has been made too distinct.
PRIMITIVE GROOVE IN THE EMBRYO CHICK. 45
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. 1 8, 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 proto-vertebrse begin to be formed along the sides of the medullary groove (PI. I, fig. 19, pv). Each new proto-vertebra (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 a"nd 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
46 PRIMITIVE GROOVE IN THE EMBRYO CHICK.
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 I. Figs. 68 and 1319.
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.
Fig. 8 is a view from above of a twenty hours' blastoderm, seen as a transparent object. Primitive groove (pr). Medullary groove (md}, which passes off from the anterior end of the primitive groove, and is produced by the thickening of the mesoblast. Headfold (//).
Figs. 13 17 are sections through the blastoderm, drawn in fig. 18 through the lines i, 2, 3, 4, 5 respectively.
The first section (fig. 13) passes through the true medullary groove (me); the two medullary folds (A, A) are seen on each side with the thickened mesoblast, and the mesoblast cells are beginning to form the notochord (nc) under the medullary groove. There is no adherence between the mesoblast cells and the epiblast under the medullary groove.
The second (fig. 14) section passes through the medullary groove where it has become wider. Medullary folds, A, A ; notochord, ch.
In the third section (fig. 15) the notochord (ch) is broader, and the epiblast is raised in the centre, while the medullary folds are seen far apart at A.
In section fig. 16 the medullary folds (A) are still to be seen enclosing the anterior end of the primitive groove (pr). Where the primitive groove appears there is a fusion of the epiblast and mesoblast, and no appearance of the notochord.
In the last section, fig. 1 7, no trace is to be seen of the medullary folds.
Figs. 18 and 19 are magnified views of two hardened blastoderms. Fig. 18 is twenty-three hours old; fig. 19 twenty-five hours. They both shew how the medullary canal arises entirely independently of the primitive groove and in front of it, and also how the primitive groove gets pushed backwards by the growth of the medullary groove, pv, Proto-vertebrae ; other references as above. Fig. 1 8 is the blastoderm from which sections figs. 13 17 were cut.
IV. THE DEVELOPMENT OF THE BLOOD-VESSELS OF . THE CHICK \
With Plate II.
THE development of the first blood-vessels of the yolk-sac of the chick has been investigated by a large number of observers, but with very discordant results. A good historical resume of the subject will be found in a paper of Dr Klein (liii. Band der K. Akad. der Wissensch. Wien], its last investigator.
The subject is an important one in reference to the homologies of the blood-vascular system of the vertebrata. As I shall shew in the sequel (and on this point my observations agree with those of Dr Klein), -the blood-vessels of the chick do not arise as spaces or channels between the cells of the mesoblast ; on the contrary, they arise as a network formed by the united processes of mesoblast-cells, and it is through these processes, and not in the spaces between them, that the blood flows. It is, perhaps, doubtful whether a system of vessels arising in. this way can be considered homologous with any vascular system which takes its origin from channels hollowed out in between the cells of the mesoblast.
My own researches chiefly refer to the development of the blood-vessels in the pellucid area. I have worked but very slightly at their development in the vascular area ; but, as far as my observations go, they tend to prove that the mode of their origin is the same, both for the pellucid and the vascular area.
The method which I have principally pursued has been to examine the blastoderm from the under surface. It is very difficult to obtain exact notions of the mode of development of
1 From the Quarterly Journal of Microscopical Science, Vol. XIII, 1873.
48 DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
the blood-vessels by means of sections, though these come in as a valuable confirmation of the other method.
For the purpose of examination I have employed (i) fresh specimens ; (2) specimens treated with spirit, and then mounted in glycerine ; (3) specimens treated with chloride of gold for about half a minute, and then mounted in glycerine ; and (4) specimens treated with osmic acid.
All these methods bring out the same appearances with varying clearness ; but the successful preparations made by means of the gold chloride are the best, and bring out the appearances with the greatest distinctness.
The first traces of the blood-vessels which I have been able to distinguish in the pellucid area are to be seen at about the thirtieth hour or slightly earlier, at about the time when there are four to five proto-vertebrae on each side.
Fig. i shews the appearance at this time. Immediately above the hypoblast there are certain cells whose protoplasm sends out numerous processes. These processes vary considerably in thickness and size, and quickly come in contact with similar processes from other cells, and unite with them.
I have convinced myself, by the use of the hot stage, that these processes continually undergo alteration, sometimes uniting with other processes, sometimes becoming either more elongated and narrower or broader and shorter. In this way a network of somewhat granular protoplasm is formed with nuclei at the points from which the processes start.
From the first a difference may be observed in the character of this network in different parts of the pellucid area. In the anterior part the processes are less numerous and thicker, the nuclei fewer, and the meshes larger ; while in the posterior part the processes are generally very numerous, and at first thin, the meshes small, and the nuclei more frequent. As soon as this network commences to be formed the nuclei begin to divide. I have watched this take place with the hot stage. It begins by the elongation of the nucleus and division of the nucleolus, the parts of which soon come to occupy the two ends of the nucleus. The nucleus becomes still longer and then narrows in the centre and divides. By this means the nuclei become much more numerous, and are found in almost all the larger
DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK. 49
processes. Whether they are carried out into the processes by the movement of the surrounding protoplasm, or whether they move through the protoplasm, I have been unable to determine ; the former view, however, seems to be the most probable.
It is possible that some nuclei arise spontaneously in the protoplasm, but I am much more inclined to think that they are all formed by the division of pre-existing nuclei a view favoured by the number of nuclei which are seen to possess two nucleoli. Coincidently with the formation of the new nuclei the protoplasm of the processes, as well as that surrounding the nuclei at the starting-points of the processes, begins to increase in quantity.
At these points the nuclei also increase more rapidly than elsewhere, but at first the resulting nuclei seem to be all of the same kind.
In the anterior part of the pellucid area (fig. 4) the increase in the number of nuclei and in the amount of protoplasm at the starting-points of the protoplasm is not very great, but in the posterior part the increase in the amount of the protoplasm at these points is very marked, and coincidently the increase in number of the nuclei is also great. This is shewn in figs. 2 and 3. These are both taken from the tail end of an embryo of about thirty-three hours, with seven or eight proto- vertebrae. Fig. 3 shews the processes beginning to increase in thickness, and also the protoplasm at the starting-points increasing in quantity ; at the same time the nuclei at these points are beginning to become more numerous. Fig. 3 is taken from a slightly higher level, i. e. slightly nearer the epiblast. In it the protoplasm is seen to have increased still more in quantity, and to be filled with nuclei. These nuclei have begun to be slightly coloured, and one of them is seen to possess two nucleoli.
Very soon after this a change in the nuclei begins to be observed, more especially in the hinder part of the embryo. While before this time they were generally elongated, some of them now become more nearly circular. In addition to this, they begin to have a yellowish tinge, and the nuclei, when treated with gold (for in the fresh condition it is not easy to
B. 4
50 DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
see them distinctly), have a more jagged and irregular appearance than the nucleoli of the other nuclei.
This change takes place especially at the starting-points of the processes, so that the appearance presented (fig. 5) is that of spherical masses of yellowish nuclei connected with other similar spherical masses by protoplasmic processes, in which nuclei of the original type are seen imbedded. These masses are surrounded by a thin layer of protoplasm, at the edge of which a normal nucleus may here and there be detected, as at fig. 5 a and a, the latter possessing two nucleoli. Some of these processes are still very delicate, and it is exceedingly probable that they undergo further changes of position before the final capillary system is formed.
These differentiated nuclei are the first stage in the formation of the blood-corpuscles. From their mode of formation it is clear that the blood-corpuscles of the Sauropsida are to be looked upon as nuclei containing nucleoli, rather than as cells containing nuclei ; indeed, they seem to be merely ordinary nuclei with red colouring matter..
This would make them truly instead of only functionally homologous with the red corpuscles of the Mammalia, and would .well agree with the fact that the red corpuscles of Mammalia, in their embryonic condition, possess what have previously been called nuclei, but which might perhaps more properly be called nucleoli.
In the anterior part of the blastoderm the processes, as I have stated, are longer and thinner, and the spaces enclosed between them are larger. This is clearly brought out in PI. 2, fig. 4. But, besides these large spaces, there are other smaller spaces, such as that at v. It is, on account of the transparency of the protoplasm, very difficult to decide whether these are vacuoles or simply spaces enclosed by the processes, but I am inclined to think that they are merely spaces. The difficulty of exactly determining this point is increased by the presence of numerous white-yolk spherules in the hypoblast above, which considerably obscure the view. At about the same time that the blood-corpuscles appear in the posterior end of the pellucid area, or frequently a little later, they begin to be formed in the anterior part also. The
DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
masses of them are, however, far smaller and far fewer than in the posterior part of the embryo. It is at the tail end of the pellucid area that the chief formation of blood-corpuscles takes place.
The part of the pellucid area intermediate in position between the anterior and posterior ends of the embryo is likewise intermediate as regards the number of corpuscles formed and the size of the spaces between the processes ; the spaces being here larger than at the posterior extremity, but smaller than the spaces in front. Close to the sides of the embryo the spaces are, however, smaller than in any other part of the pellucid area. It is, however, in this part that the first formation of blood-corpuscles takes place, and that the first complete capillaries are formed.
We have then somewhat round protoplasmic masses filled with blood-corpuscles and connected by means of processes, a few of which may begin to contain blood-corpuscles, but the majority of which only contain ordinary nuclei. The next changes to be noticed take place in the nuclei which were not converted into blood-corpuscles, but which were to be seen in the protoplasm surrounding the corpuscles. They become more numerous and smaller, and, uniting with the protoplasm in which they were imbedded, become converted into flat cells (spindle-shaped in section), and in a short time form an entire investment for the masses of blood-corpuscles. The same change also occurs in the protoplasmic processes which connect the masses of corpuscles. In the case of those processes which contain no corpuscles the greater part of their protoplasm seems to be converted into the protoplasm of the spindle-shaped cells. The nuclei arrange themselves so as completely to surround the exterior of the protoplasmic processes. In this way each process becomes converted into a hollow tube, completely closed in by cells formed from the investment of the original nuclei by the protoplasm which previously formed the solid processes. The remainder of the protoplasm probably becomes fluid, and afterwards forms the plasma in which the corpuscles float. While these changes are taking place the formation of the blood-corpuscles does not stand still, and by the time a system of vessels, enclosed by cellular walls, is formed out of
42
52 DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
the protoplasmic network, a large number of the connecting processes in this network have become filled with blood-corpuscles. The appearances presented by the network at a slightly later stage than this is shewn in PI. 2, fig. 6, but in this figure all the processes are seen to be filled with bloodcorpuscles.
This investment of the masses of corpuscles by a cellular wall occurs much earlier in some specimens than in others, both in relation to the time of incubation and to the completion of the network. It is generally completed in some parts by the time there are eight or nine proto-vertebrae, and is almost always formed over a great part of the pellucid area by the thirty-sixth hour. The formation of the corpuscles, as was pointed out above, occurs earliest in the central part of the hour-glass shaped pellucid area, and latest in its anterior part. In the hinder part of the pellucid area the processes, as well as their enlarged starting-points, become entirely filled with corpuscles ; this, however, is by no means the case in its anterior part. Here, although the corpuscles are undoubtedly developed in parts as shewn in fig. 7, yet a large number of the processes are entirely without them. Their development, moreover, is in many cases very much later. When the development has reached the stage described, very little is required to complete the capillary system. There are always, of course, a certain number of the processes which end blindly, and others are late in their development, and are not by this time opened ; but, as a general rule, when the cellular investment is formed for the masses of corpuscles, there is completed an open network of tubes with cellular walls, which are more or less filled with corpuscles. These become quickly driven into the opaque area in which at that time more corpuscles may almost always be seen than in the pellucid area.
By the formation of a network of this kind it is clear that there must result spaces enclosed between the walls of the capillaries ; these spaces have under the microscope somewhat the appearance of being vesicles enclosed by walls formed of spindle-shaped cells. In reality they are only spaces enclosed at the sides, and, as a general rule, not above and below. They have been mistaken by some observers for vesicles in
DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK. 53
which the corpuscles were supposed to be developed, and to escape by the rupture of the walls into the capillary spaces between. This mistake has been clearly pointed out by Klein (loc. Y.).
At the time when these spaces are formed, and especially in the hinder two-thirds of the pellucid area, and in the layer of blood-vessels immediately above the hypoblast, a formation takes place which forms in appearance a secondary investment of the capillaries. Dr Klein was the first to give a correct account of this formation. It results from the cells of the mesoblast in the meshes of the capillary system. Certain of these cells become flattened, and send out fine protoplasmic processes. They arrange themselves so as completely to enclose the spaces between the capillaries, forming in this way vesicles.
Where seen on section (vide fig. 6) at the edge of the vesicles these cells lining the vesicles appear spindle-shaped, and look like a secondary investment of the capillaries. This investment is most noticeable in the hinder two-thirds of the pellucid area ; but, though less conspicuous, there is a similar formation in its anterior third, where there would seem to be only veins present. Dr Klein (loc. cit., fig. 12) has also drawn this investment in the anterior third of the pellucid area. He has stated that the. vessels in the mesoblast between the splanchnopleure and the somatopleure, and which are enclosed by prolongations from the former, do not possess this secondary investment ; he has also stated that the same is true for the sinus terminalis ; but I am rather doubtful whether the generalisation will hold, that veins and arteries can from the first be distinguished by the latter possessing this investment. I am also rather doubtful whether the spaces enclosed by the protoplasmic threads between the splanchnopleure and somatopleure are the centres of vessels at all, since I have never seen any blood-corpuscles in them.
It is not easy to learn from sections much about the first stages in the formation of the capillaries, and it is impossible to distinguish between a completely-formed vessel and a mere spherical space. The fine protoplasmic processes which connect the masses of corpuscles can rarely be seen in sections, except when they pass Vertically, as they do occasionally (vide PI. 2, fig. 9) in the opaque area, joining the somatopleure and the
54 DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
splanchnopleure. Dr Klein considers these latter processes to be the walls of the vessels, but they appear rather to be the processes which will eventually become new capillaries.
From sections, however, it is easy to see that the appearances of the capillaries in the vascular area are similar to the appearances in the pellucid area, from which it is fair to conclude that their mode of formation is the same in both. It is also easy to see that the first formation of vessels occurs in the splanchnopleure, and that even up to the forty-fifth hour but few or no vessels are found in the somatopleure. The mesoblast of the somatopleure is continued into the opaque area as a single layer of spindle-shaped cells.
Sections clearly shew in the case of most of the vessels that the secondary investment of Klein is present, even in the case of those vessels which lie immediately under the somatopleure.
In reference to the origin of particular vessels I have not much to say. Dr Klein's account of the origin of the sinus terminalis is quite correct. It arises by a number of the masses of blood-corpuscles, similar to those described above, becoming connected together by protoplasmic processes. The whole is subsequently converted into a continuous vessel in the .usual way.
From the first the sinus terminalis possesses cellular walls, as is clear from its mode of origin. I am inclined to think that Klein is right in saying that the aortae arise in a similar manner, but I have not worked out their mode of origin very fully.
It will be seen from the account given above that, in reference to the first stages in the development of the blood-vessels, my observations differ very considerably from those of Dr Klein ; as to the later stages, however, we are in tolerable agreement. We are in agreement, moreover, as to the fundamental fact that the blood-vessels are formed by a number of cells becoming connected, and by a series of changes converted into a network of vessels, and that they are not in the first instance merely channels between the cells of the mesoblast.
By the forty-fifth hour colourless corpuscles are to be found in the blood whose exact origin I could not determine ; probably they come from the walls of the capillaries.
DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK. 55
In the vessels themselves the coloured corpuscles undergo increase by division, as has already been shewn by Remak. Corpuscles in the various stages of division may easily be found. They do not appear to show very active amoeboid movements in the vessels, though their movements are sometimes very active when removed from the body.
To recapitulate some of the cells of the mesoblast of the splanchnopleure send out processes, these processes unite with the processes from other cells, and in this way a network is formed. The nuclei of the original cells divide, and at the points from which the processes start their division is especially rapid. Some of them acquire especially at these points a red colour, and so become converted into blood-corpuscles ; the others, together with part of the protoplasm in which they are imbedded, become converted into an endothelium both for the processes and the masses of corpuscles ; the remaining protoplasm becomes fluid, and thus the original network of the cells becomes converted into a network of hollow vessels, filled with fluid, in which corpuscles float.
In reference to the development of the heart, my observations are not quite complete. It is, however, easy to prove from sections (vide figs. 10 and 11, PL 2) that the cavity of the heart is produced by a splitting or absorption of central cells of the thickened mesoblast of the splanchnopleure, while its muscular walls are formed from the remaining cells of this thickened portion. It is produced in the following way : When the hypoblast is folded in to form the alimentary canal the mesoblast of the splanchnopleure follows it closely, and where the splanchnopleure turns round to assume its normal direction (fig. 11) its mesoblast becomes thickened. This thickened mass of mesoblast is, as can easily be seen from figs. 10 and n, PL 2, entirely distinct from the mesoblast which forms the outside walls of the alimentary canal. At the point where this thickening occurs an absorption takes place to form the cavity of the heart. The method in which the cavity is formed can easily be seen from figs. 10 and 11. It is in fig. u shewn as it takes place in the mesoblast on each side, the folds of the splanchnopleure not having united in the middle line ; and hence a pair of cavities are formed, one on each side. It
56 DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
is, however, probable that, in the very first formation of the heart, the cavity is single, being formed , after the two ends of the folded mesoblast have united (vide k z, fig. 10). In some cases the two folds of the mesoblast appear not at first to become completely joined in the middle line ; in this case the cavity of the heart is still complete from side to side, but the mesoblast-cells which form its muscular walls are deficient above. By the process of absorption, as I said, a cavity 'is produced in the thickened part of the mesoblast of the splanchnopleure, a cavity which is single in front, but becomes divided further behind, where the folds of the mesoblast have not united, into two cavities, to form the origin of the omphalomeseraic veins. As the folding proceeds backwards the starting-point of the omphalomeseraic veins is also pushed backwards, and the cavities which were before separated become joined together. From its first formation the heart is lined internally by an endothelium ; this is formed of flattened cells, spindleshaped in section. The exact manner of the origin of this lining I have not been able to determine; it is, however, probable that some of the central mesoblast-cells are directly converted into the cells of the endothelium.
I have obtained no evidence enabling me to determine whether Dr Klein is correct in stating that the cells of the mesoblast in the interior of the heart become converted partly into blood-corpuscles and partly into a cellular lining forming the endothelium of the heart, in the same way that the bloodvessels in the rest of the blastoderm are formed. But I should be inclined to think that it is very probable certainly more probable than that the cavity of the heart is formed by a process of splitting taking place. Where I have used the word " absorption " in speaking of the formation of the cavity of the heart, I must be understood as implying that certain of the interior cells become converted into the endothelium, while others either form the plasma or become blood-corpuscles.
The originally double formation of the hinder part of the heart probably explains Dr Afanassiev's statement (Bulletin de rAcadem. Imperiale dc St Petersb., torn, xiii, pp. 321 335), that he finds the endothelium of the heart originally dividing its interior into two halves ; for when the partition of the mesoblast
DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK. 57
which separated at first the two halves of the heart became absorbed, the endothelium lining of each of the originally separate vessels would remain complete, dividing the cavity of the heart into two parts. The partition in the central line is, however, soon absorbed.
The account given above chiefly differs from that of Remak by not supposing that the mesoblast-cells which form the heart are in any way split off from the wall of the alimentary canal.
There can be no doubt that His is wrong in supposing that the heart originates from the mesoblast of the splanchnopleure and somatopleure uniting to form its walls, thus leaving a cavity between them in the centre. The heart is undoubtedly formed out of the mesoblast of the splanchnopleure only.
Afanassiev's observations are nearer to the truth, but there are some points in which he has misinterpreted his sections.
Sections PL 2, figs. 10 and 11, explain what I have just said about the origin of the heart. Immediately around the notochord the mesoblast is not split, but a very little way outside it is seen to be split into two parts so and sp ; the former of these follows the epiblast, and together with it forms the somatopleure, which has hardly begun to be folded at the line where the sections are taken. The latter (sp} forms with the hypoblast (Jiy) the splanchnopleure, and thus has become folded in to form the walls of the alimentary canal (d). In fig. 11 the folds have not united in the central line, but in fig. 10 they have so united. In fig. n, where the mesoblast, still following the hypoblast, turns back to assume its normal direction, it is seen to be thickened and to have become split, so that a cavity (of} (of the omphalomeseraic vein) is formed in it on each side, lined by endothelium.
In the section immediately behind section fig. 11 the mesoblast was thickened, but had not become split.
In fig. 10 the hypoblast folds are seen to have united in the centre, so as to form a completely closed digestive canal (d) ; the folds of the mesoblast have also united, so that there is only a single cavity in the heart (/is), lined, as was the case with the omphalomeseraic veins, by endothelium.
In conclusion, I have to thank Dr Foster for his assistance and suggestions throughout the investigations which have formed
58 DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK.
the subject of these three short papers, and which were well carried on in the apartments used by him as a Physiological Laboratory.
EXPLANATION OF PLATE '2.
Fig. i is taken from the anterior part of the pellucid area of a thirty hours' chick, with four proto-vertebrse. At n is a nucleus with two nucleoli.
Figs. 2 and 3 are taken from the posterior end of the pellucid area of a chick with eight proto-vertebrae. In fig. 3 the nuclei are seen to have considerably increased in number at the points of starting of the protoplasmic processes. At n is seen a nucleus with two nucleoli.
Fig. 4 is taken from the anterior part of the pellucid area of an embryo of thirtysix hours. It shews the narrow processes characteristic of the anterior part of the pellucid area, and the fewer nuclei. Small spaces, which have the appearance of vacuoles, are shewn at v.
Fig. 5 is taken from the posterior part of the pellucid area of a thirty-six hours' embryo. It shews the nuclei, with somewhat irregular nucleoli, which have begun to acquire the red colour of blood-corpuscles ; the protoplasmic processes containing the nuclei ; the nuclei in the protoplasm surrounding the corpuscles, as shewn at a, a'.
Fig. 6 shews fully formed blood-vessels, in part filled with blood-corpuscles and in part empty. The walls of the capillaries, formed of cells, spindle-shaped in section, are shewn, and also the secondary investment of Klein at k, and at b is seen a narrow protoplasmic process filled with blood-corpuscles.
Fig. 7 is taken from the anterior part of the pellucid area of a thirty-six hours' embryo. It shews a collection of nuclei which are beginning to become bloodcorpuscles.
Figs, i 5 are drawn with an \ object-glass. Fig. 6 is on a much smaller scale. Fig. 7 is intermediate.
Fig. 8. A transverse section through the dorsal region of a forty-five hours' embryo ; ao, aorta with a few blood-corpuscles, v, Blood-vessels, all of them being formed in the splanchnopleure, and all of them provided with the secondary investment of Klein ; p, e> pellucid area ; o, p, opaque area.
Fig. 9. Small portion of a section through the opaque area of a thirty-five hours' embryo, showing protoplasmic processes, with nuclei passing from the somatopleure to the splanchnopleure.
Fig. 10. Section through the heart of a thirty-four hours' embryo, a. Alimentary canal ; hb, hind brain ; nc, notochord ; e, epiblast ; s, o, mesoblast of the somatopleure ; sp, mesoblast of the splanchnopleure ; hy, hypobiast ; hz, cavity of the heart.
DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK. 59
Fig. n. Section through the same embryo as fig. 10, and passing through the orifice of the omphalo-meseraic vein, of, O
