The Works of Francis Balfour 1-4
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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)
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
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
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, Omphalo-meseraic vein ; other references as above.
These two sections shew that the heart is entirely formed from the mesoblast of the splanchnopleure, and that it is formed by the splitting of that part of the mesoblast which has turned to assume its normal direction after being folded in to form the muscular wall of the alimentary canal. In fig. 1 1 the cavities so formed on each side have not yet united, but in fig. 10 they have united. When the folding becomes more complete the cavities (of, of) in fig. 1 1 will unite, and in this way the origin of the omphalo-meseraic veins will be carried further backwards. In the section immediately behind section 1 1 the mesoblast had become thickened, but had not split.