The Works of Francis Balfour 1-2
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Foster M. and Sedgwick A. The Works of Francis Balfour Vol. I. Separate Memoirs (1885) MacMillan and Co., London.
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)
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 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.
- 1 From the Quarterly Journal of Microscopical Science, Vol. xin., 1873.
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 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, 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 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 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 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.
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 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.