Book - Contributions to Embryology Carnegie Institution No.45
|Embryology - 16 Feb 2019 Expand to Translate|
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| This 1920 paper by Clark and clark on the chicken lymphatic system.
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- 1 On The Origin And Early Development Of The Lymphatic System Of The Chick
- 1.1 Introduction
- 1.2 I. The History of the Earliest Lymphatics in Chick Embryos
- 1.3 Method
- 1.4 Lymphatics of Posterior Lymph-Heart Region in Chicks of 5 to 6 Days
- 1.5 Lymphatics of Posterior Lymph-Heart Region in Chicks of 4 to 5 Days
- 1.6 II. Operations on Chick Embryos
- 1.7 Method of Operation
- 1.8 Summary of the Results Obtained from Operations on Chick Embryos
- 1.9 General Conclusions
- 1.10 Bibliography
- 1.11 Description of Plates
- 1.12 Glossary Links
On The Origin And Early Development Of The Lymphatic System Of The Chick
By Eliot R. Clark and Eleanor Linton Clark,
Of the Anatomical Laboratory, University of Missouri.
With seven plates and fifteen text-figures.
- Links: Carnegie Institution of Washington - Contributions to Embryology | Immune System Development | Chicken Development
These studies were begun and partly carried out in Professor Mall's laboratory in Baltimore, and have derived much from the inspiration of his enthusiastic and kindly interest. We believe it safe to say that few problem is in anatomy were of as great interest to Professor Mall as that of the origin and mode of growth of the lymphatic system.
The numerous investigations and discoveries in this field during the past twenty years were started with the epoch-making studies of Sabin in 1901, begun at the instigation of Professor Mall and, almost without exception, can be attributed indirectly to his guiding genius. It is an interesting illustration of his method of achieving results that not one of the papers on the development of the lymphatic system, published from his laboratory during this period, carries his name on the title page; although, he might, with justification, have insisted that many of these publications include his name as joint author. That he did not is evidence of his pure unselfishness and his reahzadon of the importance of developing early the sense of independence, responsibihty, and achievement in his students, if they, in turn, were to become sturdy, independent anatomists.
It is surprising to re^dew the many changes in and additions to our knowledge of the lymphatic system which have taken place since 1900, when Professor Sabin began her studies. Previously, the conception of adult anatomy of the lymphatic system was in a very- hazy state. In fact, with the exception of the lymph-glands, the thoracic duct, and other large h'mphatics, it could scarcely be designated as a real system, since it was supposed to merge, in obscure and devious ways, with the peritoneal cavity, with the serous cavities in general, and with "spaces" everywhere in the tissues. Even the discovery of von Recklinghausen (1862) that silver nitrate stained the endothelium of lymph-vessels in a definite manner was obscured by the faulty interpretation, which explained these silver markings as revealing a sieve-like structure of the lymphatic wall, whereas they are now known to represent cell-boundaries in a definite membrane. As for the embryology of the lymphatic system, it was practically a virgin wilderness lighted only by the uncertain torch of Budge's work, in which his injection of the extra-embryonic coelom was interpreted as a picture of the primitive lymphatic system.
To-day, less than twenty years after the beginning of the investigations in Dr. Mall's laboratory — years fraught with strenuous controversy at times presenting seemingly irreconcilable %dews — we possess an adequate knowledge of the structure of the lymphatic system in the adult; a definite idea of the manner of growth of the lymphatic capillary and a few hints as to some of its reactive powers; a picture of the development of some of the component parts of the system, such as the ducts, sacs, and Ijonph-hearts, and, in some instances, a correlation of their morphology with their function; a history of its development in many organs of the mammalian embryo and of its differences in pattern and extent in various animals ; and a fairly complete idea of its connections with the venous system and of the persistence of such communications in the adult. A more and more complete picture of a definite, independent, and important system has emerged from all these studies, together with a history of the manner in which this system has developed from the earliest beginnings — ^where, however, the outlines of the picture once more become hazy. Since most of this work has been recently reviewed by Sabin (1913 and 1916), it seems unnecessary to enter into a detailed discussion of literature. The work done since that time, and those investigations which bear directly upon the particular problems considered here, will be referred to in the course of the descriptions.
In the present investigations we have attempted to carry the study back to the first appearance of the lymphatics in the embryo, since this part of the history seemed to be still lacking in clearness.. The study involved the consideration of the following unsolved problems: (1) The nature of the earliest lymphatics. (2) The tissue from which they originate. (3) The manner in which the differentiation is accomplished. (4) The question as to how soon the lymphatics form a specific, independent system, whose growth proceeds by the sprouting of pre-existing endothelium in the manner proved for the stage of development of the vessels in the tail of the frog larva (Clark, 1909). (5) The question of the points of origin of lymphatic endothelium in the embryo; whether there are strictly limited areas in which the first lymphatic endothelium arises, or whether the differentiation is diffuse.
For convenience the description of these investigations and the results obtained have been divided into two parts. Part I deals with the history of the earliest lymphatics in chick embryos and is concerned with the problems of their character and origin. Part II is a report of the operations on chick embryos and is concerned with the question of the points of origin of the first lymphatics.
I. The History of the Earliest Lymphatics in Chick Embryos
In approaching this phase of the problem we decided to select some one region of the embryo and to study it intensively^ by all available methods, assuming that the manner of formation of the earliest lymphatics follows the same laws in other regions. The posterior lymph-heart region was chosen because of its accessibility for observation in the living and for direct injection, and because previous work had made us familiar with it.
The posterior lymph-heart of the chick was discovered in 1882 by Budge, and various stages in its development have since been described by 8ala (1900), Mierzejewski (1911), E. R. and E. L. Clark (1912), and West (1915). The region of the posterior lymph-heart is situated on either side of the tail, beginning at the angle formed by the posterior border of the pelvis and the tail, and superficial and lateral to the myotomes. In chicks of 8 days the l>Tnph-hearts in this region are clear, almond-shaped bodies, easily visible in the living embryo (as stated by Sala and by Budge), and their pulsations can be noted. Sala found that the lymph-hearts communicate with the intersegmental coccygeal veins, connecting at first with five of these veins and later with three. By the tenth day the hearts are rounded in form and surrounded by a layer of fat. Budge injected the IjTnph-hearts from the allantoic lymph-vessels in chicks of 9 daj's. He found that they were present in the chick until after the time of hatching, but were absent in adult chickens. Stannius (1843) and Panizza (1830) had previously shown that lymph-hearts are present in adult aquatic birds as well as in the ostrich and cassowary.
In studying earlier stages of the Iymph-heart Sala made use of cross sections of chicks of 6| to 7 days old. In these he describes a series of "spaces" connected with the intersegmental coccygeal veins and concludes that the IjTnph-heart is formed by the coalescence of these spaces; at one time he refers to them as mesenchymal spaces and at another as continuations of the veins them.selves.
In 1901 Sabin published her paper describing the embryonic h^mphatic system of the pig, which, in the earliest stages, consisted of paired jugular and iliac sacs and the thoracic duct connecting them. These sacs were connected with veins and seemed to be the homologues of the Ijonph-hearts of lower animals, and Sabin concluded at the time that they were outgrowths from the veins and were the primary points of development for the lymphatic system, from which all other vessels were derived by a process of outgrowth. This view that the lymphatics grow by sprouting had been advocated by Ranvier (1895) from studies made on much older embryos.
In 1906 F. T. Lewis carried the history of the lymphatic system back a step farther by his studies of rabbit embryos. In the region of the jugular lymph-sac, and before the appearance of that structure, he found a discontinuous plexus of capillaries lined with endothelium. From the study of fixed material he suggested the possibility that these were "veno-lymphatics" or blood-vessels, formerly part of the general circulatory sj'stem, which had become cut off and were about to be transformed into a lymph-sac. Huntington and McClure (1910) described a stage of veno-lymphatic and pre-lxinphatic vessels as preceding the appearance of the jugular sac in cat embryos, and Miller (1912) figured a similar discontinuous preIjinphatic plexus in the region of the jugular lymph-sac of chicks.
In 1909 Mierzejewski studied the superficial lymphatics of chick embryos by the injection method of Hoyer, and showed that there is a plexus of capillaries present in the region of the posterior lymph-heart in chicks of 5^ days, 24 hours earlier than the first appearance of the IjTnph-heart rudiment, as described by Sala. Mierzejewski considered this to be a lymphatic plexus which had grown out from the coccygeal veins.
In 1911 we began our studies of this region in chick embryos and portions of these have been published from time to time. We observed that the lymph-heart begins to pulsate in chicks of 6 to G| days, and that the early contractions occur only at the time of the periodic movements of the embryo. When granules of India ink were injected into this translucent, pulsating area, a rich plexus of lymphatic capillaries was revealed, thus showing that the lymph-heart of the chick is a functioning heart before it is a sac (Clark and Clark, 1912, 1914). Similarly, a continuous plexus of lymphatic capillaries was injected in the jugular region at a stage in wliich IMiller had found the discontinuous " pre-lymphatic " anlage (E. L. Clark, 1912).
While studying this posterior lymph-heart region in living chicks of 5 to 6 days, a stage before a beating lymph-heart is present, we found in this and adjacent areas a richly anastomosing plexus of capillaries, independent of the surrounding blood-capillaries and easily distinguishable from them, chiefly by means of the stagnant blood which they normally contained. Preliminary reports of studies of these early IjTnphatics, made on living chicks, have been pubUshed (Clark and Clark, 1912). The discovery that early lymphatics contain stagnant blood enabled us to directly inject lymphatic capillaries with much greater facihty and precision than before, and by this method to study these early vessels and their relation to the blood- vascular system (E. L. Clark, 19126). A preUminary report of microscopic studies of these early lymphatics has also been published (E. R. Clark, 1914), in which it is stated that the early lymphatic endothUum possesses definite morphological characteristics which distinguish it from the mesenchyme cells.
In later studies made on pig embryos Sabin (1912, 1913) reported the finding of lymph-capillaries, filled with stagnant blood, which were connected with the jugular vein and with the abdominal veins in stages before sacs are present in these regions. Also, West (1915) published a study of the posterior lymph-heart region in the chick, in cross-sections of which he found a continuous plexus of lymphatic capillaries before the formation of a lymph-heart.
These various studies apparently demonstrate the incorrectness of the view that lymph-sacs and lymph-hearts are the jorimary structures of the lymphatic system, and also that they arc; derived from veno-lymphatics which at one time functioned as blood-vessels containing circulating blood. They point instead to the existence of a continuous plexus of lymphatic capillaries which subsequently becomes converted into lymph-hearts and lymph-sacs.
Although the origin of this primarj' lymphatic plexus has not yet been determined for birds and mammals, studies have been made on what appear to be the first stages of lymphatic development in amphibians. Fedorowicz (1913) studied the origin of the posterior lymph-heart in frogs, and described the first lymphatic vessels as solid strands of cells derived from the endothelium of the lateral caudal vein. These subsequently acquire lumina, unite with one another, and later coalesce to form the lymph-heart.
Kampmeier (1915) studied the early stages in the development of the anterior Iymph sinus in Bufo. His discovery that the endothelial cells in this space retain the yolk-granules longer than do the connective-tissue cells made possible a careful cytological study of the early stages with high powers of the microscope. Kampmeier came to the conclusion that the first lymphatic vessels are derived from the veins by a process of outgrowth in a number of places. These outgrowths, many of which are at first sohd, unite with one another to form the plexus which precedes the formation of the lymph-sinus.
Beginning with this work in 1912, the authors have spent several years in a careful study, by various methods, of these early lymphatic capillaries in the region of the posterior lymph-heart of the chick in an effort to discover their origin.
Our method of studying Hving chicks has previously been described (Clark and Clark, 1912, 1914). The essentials are a binocular microscope inclosed in a warm chamber kejit at incubator temperature, Ringer's solution for keeping the chick moist, and a bright fight for direct illumination (sunlight or a desk-arc). A large window was made in the egg-shell, and the amnion opened and pulled aside. In this way chicks were kept alive and active for 7 or 8 hours.
For the hinphatic injections we used India ink, diluted to one-half with tap water, and fine glass cannulae (10 to 15 ix at the tip) attached to a rubber tube. For double injections we used various materials, India ink for the lymphatics and Berlin blue with 5 per cent gelatin for the blood-vessels; or India ink in the bloodvessels and Berfin blue in the l3^Ilphatics. We also injected lymphatics wdth silver nitrate (0.5 per cent) and the blood-vessels with India ink. Such injected specimens were fixed in Carno.y's fluid, dehydrated in absolute alcohol, and cleared in benzol and oil of wintergreen. Or, if we desired to section such embrj-os, they were fixed in Bouin's fluid.
For our studies of sections the blood-vessels were injected completely- with India ink. Good injections w-ere obtained either through the allantoic artery or through one of the vitelline veins. In either case the heart was allowed to pump the injection mass around through the body. The umbilical cord was tied and the embryo taken from the yolk and dropped into the fixing solution. For this purpose we obtained the best results by the use of Clung's modification of Bouin's fluid: Saturated aqueous solution of picric acid, 75 per cent; formalin (40%), 20 per cent; glacial acetic, 5 per cent.
The embryos were carefully dehydrated to avoid shrinkage, and then embedded in paraffin. In some cases cross-sections of the tail region were made, but in most instances the sections were cut parallel to the surface. By this means the greater part of the lymphatic plexus was contained in a few sections and parts could be reconstructed which would be completely lost in cross-sections. The sections were cut 10 and 15 fx in thickness, stained on the slide with Ehrlich's hematoxj'lin, and counterstained with a mixture of eosin, orange G, and aurantia. By this staining method the nucleoli of the Iymphatic endothehal cells are reddish in color, wliich is an aid in distinguishing them from the mesenchyme cells whose nucleoli are bluish-purple. This same contrast was obtained by the use of IMann's methyl-blue eosin stain.
Such sections were studied by means of oil-immersion reconstruction. The blood-vessels could be identified by the presence of India ink, and the other vessels were all carefully drawn, every strand of endothelium, every nucleus and nucleolus being recorded. The structures were not studied in this manner until extensive investigations of the lymphatics and blood-vessels of this region had been carried out in living and injected embryos. Familiarity with the region as a preparation, and the modification of parallel sections, the constant use of the oil-immersion lens, and a criterion for distinguishing endothelial cells enabled us to detect definite vessels and even a plexus at stages in which our earlier studies and those of other investigators gave no hint of the existence of lymphatics. Needless to say this method is extremely tedious. A drawing of all the endothelial cells, including nuclei and nucleoli, was made of each section, and the drawings of successive sections were combined in graphic paper reconstructions.
Lymphatics of Posterior Lymph-Heart Region in Chicks of 5 to 6 Days
As has been stated, the pulsation of the posterior lymph-heart can be seen in chicks of 6 to 6| days, a stage when injection shows that the heart is still in the form of a plexus. This lymph-heart plexus connects medially with the first five intersegmental coccj^geal veins, and ventrally with a superficial lymphatic plexus which spreads out over the pelvis and anterior body-wall, and is continuous from the tail to the axilla. Cleared, injected specimens show that this superficial plexus has connections through the axillary region with the deep lymphatic plexus located dorsal to the anterior and posterior cardinal veins, near their junction at the duct of Cuvier, with which veins it communicates at a number of places. (A drawing of this plexus has been published in an earlier article — E. L. Clark, 1912.) In a chick of 5 days and 18 hours this continuous superficial plexus can be injected and most of it can be seen in the living chick because of the stagnant blood present in its lumen. The lymph-heart has not started to beat at this stage.
In younger embryos (5 to 5.5 days) blood-filled Iymphatics are also visible, but a continuous plexus over the surface of the body can not be seen in the living or injected specimens. Instead, a plexus is found anteriorly, near the region of the thoraco-epigastric vein, and connected through the axillary region with the deep jugular plexus and with the veins, as in the older specimen; also another j^osterior j)lexus connected with the intersegmental coccygeal veins and confined to the region of the posterior lymph-heart and to the neighboring area over the posterior tij) of the polvis. In these younger embryos the lymphatic plexus, as seen in the living or injected specimens, is less luxuriant than in the chicks of 5^ to 6 days, and the vessels composing it are much finer.
The appearance of the posterior blood-filled lymphatic plexus in the living chick is illustrated in plate 1, figure 17. The more superficial portion, over the tip of the pelvis, connects with the deeper plexus which later forms the lymph-heart. This superficial plexus is easily distinguishable from the blood-capillaries of the region in a number of ways. For the most part it lies beneath the superficial bloodcapillaries. The pattern of the two sets of ves.sels is different; the Ij-mphatic capillaries are more irregular in form than the blood-vessels. The dark-red color of the plexus containing stagnant blood contrasts with the lighter, more yellowish tinge of the blood-capillaries. The most striking feature is the contrast between the rapid motion of the blood-corpuscles in the blood-capillaries and the blood in the Iymphatic plexus which remains stagnant. Observation of the two plexuses shows clearly that this plexus of capillaries filled with stagnant blood is a distinct and independent system of vessels.
Various tests were also made in order to learn more of the character of this early plexus and its relation to the blood vascular system. The plexus containing stagnant blood was injected by direct puncture of a selected hTnphatic capillary. The near-by circulating blood-vessels remained undisturbed after such an injection, thus showing the independence of the two systems. Figure 21, plate 2, illustrates such an injection in the exact location occupied by the beating IjTnph-heart of older embryos and a few delicate superficial vessels continuous with it. The injection often reveals a few fine connections which had not been observed in the living, but in general the extent of the plexus, as demonstrated by the presence of the blood and by injection, is the same.
When a small amount of ink was injected into one of the vessels of this lymphheart plexus without disturbing any of the superficial blood-capillaries, the granules could be seen to enter the intersegmental coccygeal veins and to move along in the main caudal vein. Cleared specimens with injected lymphatics enabled us to study this relationship of the veins still further. Figure 1 shows the lymph-heart plexus and its venous connections of both sides. The view is a dorsal one, for ^ith the chick in its natural position, lying on one side, the lymph-heart plexus lies directly over the intersegmental veins of the tail, thus concealing the points of connection from the observer.
Complete blood-vessel injections were obtained, and such an injection left the lymphatic plexus filled with stagnant blood, while all the surrounding blood capillaries became fixlled with the injection mass. Figure 18, plate 1, is a drawing of a fresh specimen made immediately after such a blood-vessel injection.
Double injections were obtained with the blood-vessels completely injected with India ink and the lymphatics filled with silver nitrate or Berlin blue. Plate 3, figure 24, shows such an embryo with some early Iymphatics in the IjTnph-heart region injected with silver nitrate. A thick cross-section made from the same specimen (plate 3, fig. 25) shows the location of some of these little vessels with regard to one of the intersegmental coccygeal veins and to the superficial bloodcapillaries. When this blood-filled plexus was injected directly with silver nitrate (0.5 per cent) the characteristic endothelial markings appeared, demonstrating that these vessels can not be simple mesenchymal spaces, as might be inferred from Sala's descriptions.
When the large arteries and veins of the yolk-sac and allantois were opened and the embryo was allowed to bleed freely, the l)lood could be seen to fade out of the superficial blood-capillaries, but the lymphatic plexus remained undisturbed and still filled with stagnant blood. An embryo thus bled makes a rather striking picture, with the bright-red lymphatic plexus standing out against the white background. Figure 22, plate 2, illustrates the superficial lymphatic plexus in a 6-day chick that had been bled in this manner.
Examination of the superficial plexus in sections of chicks of 5 to 6 days in which the blood-vessels had been completely injected confirmed the observations, made by the other methods, that there is a continuous plexus which is independent of the surrounding blood-vessels except for connections with branches of the intersegmental coccygeal veins. In addition, the sections showed that this plexus possesses a definite endothelial lining, and that the endothelial nuclei of the Ij^mphatics have certain morphological characteristics which distinguish them from the nuclei of the adjacent connective-tissue cells. These nuclei have a pale, fairly homogeneous, granular appearance, and contain a single nucleolus or a pair of nucleoli, which are definite, discoid bodies, sharply marked off from the remainder of the nuclear material by clear-cut, rounded outlines. The definiteness of this endothelial nucleolus was shown in a striking manner in cases in which an endothelial nucleus had been cut into during the process of sectioning. In many such instances the nucleolus had been dragged out of the nucleus l)ut still retained its characteristic shape. The single nucleolus varies in form according to the shape of the nucleus and to the plane in which the cell has been cut. With the method of staining used these nucleoli have a distinctly reddish color.
Fig. 1. Dorsal view of tail region of an injected embryo of 5 days 23 hours, showing the lymph-heart plexuses on both sides and the coccygeal veins with which they .are connected, i.c.v. — intersegmental coccygeal vein. X 24.
The nucleus of the mesenchyme cell differs in all the particulars mentioned. It contains two or more nucleoli which are not sharply differentiated from the remainder of the chromatin material of the nucleus, but which extend out into prongs and threads, and these do not have a characteristic shape. These nucleoli take a distinctly bluish stain. The remainder of the nucleus of the connective-tissue cells is darker in appearance than that of the endothelial cells and frequently contains small clumps of chromatin material. In chicks of this stage the blood-vessel endothehal nuclei are slightly smaller than those of the lymphatics, more regular in shape, and are apt to contain two nucleoU instead of a single large one. In earUer stages these distinctions are frequently absent, and without the injection material present in the blood capillaries it would be practically impossible to distinguish the two types of nuclei. Figures 26 and 27, plate 4, show microscopic drawings of a younger embryo (4 days and 23 hours) and illustrate the difference between the endothelial nucleus and the nucleus of the mesenchyme cell.
An examination of sections confirmed the observations based upon injections and the study of the living; i. e., that in chicks of 5 to 6 days a primitive plexus of lymphatics is present in this posterior part of the pelvis and in the tail region and precedes the formation of the lymph-ducts and the beating lymph-heart which occupy the same area in older embryos. The plexus is indifferent and irregular in character; comparatively large bulbous nodes alternate with very fine, even solid connections and delicate processes. The connections of this plexus with the first five intersegmental coccygeal veins or their branches were easily seen in sections, but aside from these the plexus was found to be independent of the blood vascular system. A number of mitotic figures were seen in the endothelium of this lymphatic plexus. The presence of these mitoses, scattered here and there in endothelial cells, as well as the general character of the plexus, gives the impression of a wild and rapid growth.
In studying sections, cut parallel to the surface, of chicks of 5 to 5^ days in which the blood-vessels had been completely injected, with the aid of the oilimmersion lens a more extensive plexus of lymphatics could be made out than we had been able to discover with other methods. For example, figure 3 is from a reconstruction of a portion of the plexus over the pelvis of a chick of 5 days 7.5 hours. In this specimen the blood-filled lymphatics of the posterior part of the pelvis and of the lymph-heart region were injected with India ink and the bloodvessels subsequently filled with Berlin blue gelatin (5 per cent) . The drawing shows a part of the rich plexus of fine, delicate vessels which we were able to reconstruct in an area well beyond the injected part. The reason why the injection failed to reveal this is obvious from the character of the plexus. Although the endothelium is continuous the lumen is not. Many of the connections are exceedinglj' narrow and others are entirely solid. It is interesting to note the large number of mitoses present in this small part of the plexus.
As we stated in 1912 and 1915, the stagnant blood normally present in the early lymphatics of this stage is due merely to pressure conditions. It is easy to show by injection experiments on living chicks, by cleared injected specimens, and by microscopic sections, that the early lymphatic system in chicks of this stage is connected in certain regions with the venous system. No valves are present at this time. Although there is evidence from study of parallel sections that an absorptive function has already started in these vessels, such function is comparatively slight and not sufficient to overcome the lateral pressure of the veins. That the blood enters the lymphatic system from the veins can be demonstrated in a number of ways.
(1) The amount of blood present in the superficial lymphatic system can be increased in the living chick by changing the position of the embryo so as to allow the force of gravity to affect its various parts. For example, the posterior lymphatics, which spread out over the surface of the tail and over the posterior border of the pelvis, lie, for the most part, anterior to the coccygeal veins with which they are connected. By pulling on shreds of the amnion the tail can be raised so that this region is uppermost, and the embryo can be held in this position for some minutes, at the expiration of which more blood enters the lymphatics and the anterior (lower) portions of the plexus become much redder and more distended.
Fio. 2. — Chick of 5 days 7\ hours, measuring 14.5 mm. before fixation. The lymphheart plexus was injected with India ink. The small square marked Y indicates the region reconstructed and shown in figure 3. X 3.7.
Fig. 3. — Drawing of a portion of the superficial plexus from the region beyond the injection in the same embryo shown in figure 2 (the region marked with a small square). The plexus was reconstructed with the aid of the oil-immersion lens from sections cut parallel to the surface. X indicates mitotic figure in lymphatic endothelial cells. X 300. (Magnification of original drawing, 1 to 800.)
(2) Again, the amount of blood in the early lymphatic plexus can be increased noticeably and quickly by interfering with the blood circulation. When the heart becomes embarrassed from any cause (such as the addition of strong chloretone or too high temperature of the warm chamber) there occurs a back pulsation in the veins, which can be observed to the best advantage in the large vessels of the allantois and yolk-sac. Within a few seconds after the beginning of such a circulatory disturbance the superficial lymphatics become markedly redder and more congested, owing to the increased amount of blood which enters them as a result of this increased venous pressure.
(3) Further evidence for this backing up of the blood from the veins was found in our studies (1915) of the beginning IjTnph flow in chicks of 6 to 6| days. In these we traced the steps in the process by which the blood is washed out of the lymphatics. In the stages of its first pulsation, while the flow is relatively feeble, the lymph-heart was observed to fill up with blood in the interval between beats. These observations also showed that there is a stage in the development of the superficial lymphatics in which the pressure in the lymphatics overcomes that of the veins.
(4) In chicks of 7 days, a stage in which there is normally no stagnant blood present in the lymphatics, it was found that the flow of lymph is more rapid and the lymph-heart contractions are stronger. If the action of the lymph-heart is paralyzed by chloretone, a drug which also increases shghtly the back pressure in the veins, the lymph-heart and adjoining vessels soon fill up with stagnant blood and become visible once more in the living embryo.
(5) The pictures obtained from sections of cliicks of 5 days also bear out this point, for the newer portions of the plexus (those portions which do not possess a continuous lumen) are quite empty of blood-cells, and hence invisible in the living; and the blood is present only in the portion next to the veins where the lumen is continuous.
All these observations, therefore, show that the stagnant blood present in the lymphatic plexus of chicks of 5 to 6 days has backed up from the veins. Its presence is merely a transitory pressure phenomenon.
Some writers have claimed that chick lymphatics have a "hemophoric" function, meaning that they carry newly-formed blood-cells from the tissues to the blood-vessels (Miller, 1913; West, 1915). That IjTnph-capillaries have the power of picking up blood-cells that have become extruded into the tissues was shown by one of the authors in 1909. In sections of some of the earUer chicks of this series (4 to 5 days) pecuUar, large cells were found to be present in the mesenchyme, which might be interpreted as blood-forming cells or as large phagoc}i;es; but for the stages in which the blood-cells are present in such quantity as to render a large plexus visible under the binocular microscope or even to the naked eye, all the evidence points to the backing-up of this blood from the veins as the true interpretation.
Lymphatics of Posterior Lymph-Heart Region in Chicks of 4 to 5 Days
As stated above, the plexus of blood-filled lymphatics in the region of the posterior lymph-heart, and in the more superficial regions ventral and anterior to it, is easily visible in living chicks of 51 days. Pushing the study of this region back to still earlier stages, we found the first evidence of lymphatics in the living in embryos of about 12 mm. greatest length (measured in the fresh) and about 4 days 20 hours to 5 days old. These first lymphatics were visible as a number of se])arate knobs of stagnant blood in the region just lateral to several of the dorsal intersegmental coccygeal veins. At this stage these little dots of blood give the region of the posterior lymphheart a characteristic speckled appearance. Since the knobs he between the observer and the veins their connections can not be seen, but ink granules injected directly into them can be observed to pass into the nearest intersegmental coccygeal vein.
In many cases we studied this region in the living 4 to 8 hours before the appearance of these knobs, and were satisfied that no circulating-blood vessels are present in the exact area where these vessels make their appearance. About an hour after the first knobs become visible new ones can be seen near them, and connected by narrow vessels. Injection of these structures shows small, discrete clusters, somewhat resembling bunches of grapes, connected, as were the earlier knobs, with the intersegmental veins of the tail. The steady and rapid extension of these blood-filled structures to form a plexus was observed in many embryos. They extend toward the surface and spread out in the region superficial and ventral to the Iymph-heart region. Two or three delicate projections from the plexus can first be seen, connections between these make their appearance, then certain parts of the plexus enlarge and become more densely packed with blood-cells. New sprouts appear in advance, and the same process of extension, accompanied by plexus formation, is repeated. The several parts of the plexus are irregular in size; many of the lymphatics are several times as wide as a blood-capillary, while some of the connections and processes are as small as and often much smaller than a blood-capillary. All of these successive stages were tested by pressure over the parts filled with blood, in order to see if the blood could be forced farther, and also tested by injection. By both of these methods it was found that, in these early stages, practically all of the injectible lymphatics are normally filled with stagnant blood. This blood, which backs up from the veins with which the early lymphatics connect, forms a vital injection mass which is constantly being forced into the developing plexus, and by this means reveals the extension of the continuous lumen of the lymphatics.
As already stated, in chicks of 5 days, sectioned parallel to the surface, a plexus of delicate vessels could be reconstructed, by use of oil-immersion, well beyond the limit of the injectible or blood-filled lymphatics. We next studied still younger embryos by this method of paper reconstruction, with the aid of the oil-immersion, in an effort to trace back the picture of the developing lymphatics.
Plate 5 is a reconstruction of the region of the posterior lymph-heart in a chick of 4 days 23 hours, measuring 12 mm. in the fresh state. In this specimen, examined in the living, the first knobs filled with blood were visible. They were not injected, but from other specimens of the same stage it may safely be inferred that injections would have shown us small clusters connected with the intersegmental coccygeal veins. When the sections of the embn-o were studied with the higher magnificat'ons a continuous plexus was reconstructed in this region. Some of the vessels are as large as blood-capillaries and contain blood-cells; these undoubtedly represent the blood-filled knobs seen in the living; others are much narrower, and still others are solid. Long narrow processes extend superficially and ventrally from this plexus in the exact position in which the blood-filled sprouts make their appearance in living chicks a few hours later. The plexus connects at several points with branches of the intersegmental coccygeal veins in each segment (X in the drawing) . Some of these connections are fairly large and others are solid.
This lymphatic plexus is composed at this early stage of continuous endothelium wliich has the characteristic endothelial nuclei already described. Figures 26 and 27, plate 4, were drawn from vessels composing this same plexus and show the differences in form and color between the nuclei and their nucleoli and those of adjacent connective-tissue cells. Figure 29, plate 4, is also from this plexus and shows a lymphatic endothelial cell in mitosis. Figure 28, plate 4, is from another specimen of the same stage and shows one of the early blood-filled knobs in section, with its connections with the veins. One of these connections is open and contains a blood-cell, while the other is solid and thread-like.
Just as the study of the Uving blood-filled lymphatic plexus in chicks of 5 to 6 days showed that the reconstruction from cross-sections gave only a very incomplete account of the true conditions (F. T. Lewis, 1906, and Miller, 1912), so the picture obtained from these studies, made with the oil-immersion, shows that our earlier account of the blood-filled "bunches" seen in the living and injected at this stage (Clark and Clark, 1912) was extremely fragmentary. This is also ihe stage which West (1915) describes as his first stage (a chick of the same age as this specimen, and one measuring 10.5 m-m. after fixation and dehydration), in which only a few "mesenchymal spaces" could be reconstructed in this region. Figure 31, plate 6, represents the condition in a part of the region in a still younger embryo. This chick was 4 days and 9 hours old and measured 11.75 mm. in the fresh state. No blood-filled lymphatics could be seen in the living chick. The blood-vessels were completely injected with India ink and the embryo fixed, sectioned, and stained in the usual manner. The oil-immersion reconstruction showed us that even at this stage a plexus of vessels, with the characteristic form of an early lymphatic plexus and the characteristic endothelial nuclei, is present in the same region which is later occupied by the blood-filled lymphatic plexus, and still later by the beating lymphheart. The plexus at tlais stage is not so luxuriant as that of the next older stage shown in figure 30, plate 5, and the vessels composing it are relatively smaller. The fine and soUd connections between different parts of the plexus are relatively more numerous. A number of venous connections are also present at this early stage. Finally, figure 32, plate 6, is taken from an oil-immersion reconstruction of a portion of this region in a chick of 4 days and 7 hours, measuring 9 mm. in the living. Here, besides the injected blood-vessels and the indifferent mesenchme, there were suspicious-looking vessels with characteristic endothelial nuclei in a part of the region chosen for intensive study. As in the other sectioned specimens, all of these nuclei were drawn, with the characteristic nucleoli, as was also the endothelium with wliidi they were connected. The vessels thus reconstructed were found to be decidedly fewer in number than in the succeeding stages, and it is evident that no continuous plexus was present. It was noticeal)le that practically all of these vessels were connected with veins (fig. 32, plate 6).
In studying the region over the pelvis in chicks of 4 days 23 hours, in sections cut parallel to the surface, a very interesting condition was noted. This is the same region as that shown in figure 3, in a chick of 5 days 7| hours. In the exact position of this delicate, continuous plexus of the older embryo we found a number of very fine vessels which did not contain any injection mass. A great many of these vessels were connected with blood-vessels. It might be inferred that such vessels are all new-forming blood-cajnllaries were it not for the difference in their appearance and location (for the most part beneath the superficial blood-vessels) and the fact that similar pictures were not found in the adjacent areas in which new formation of blood-vessels is also taking place. The character of these vessels is strikingly like that of those present in the earliest stage of lymphatic formation in the region of the posterior lymph-heart, recorded in figure 32, plate 6, and appears to show that this region over the pelvis also gives rise to new lymphatic vessels, and that here their origin occurs several hours later than the first formation in the tail region. Evidence obtained from the operations confirm this view and will be taken up later. The presence of numerous connections with blood-vessels appears to favor the view that the earliest lymphatics arise as outgrowths from the blood-vessels. Such communications with blood-vessels are not found in the later stages in which a continuous lymphatic plexus is present in this region.
And here we are forced to abandon the history of the first lymphatics of the chick, for the stage shown in plate 6, figure 32, is the youngest in which we were able to discover anything resembling a lymphatic. A chick of 4 days, measuring 9 mm., was sectioned parallel to the surface and this region was studied with the oil-immersion for a trace of possible lymphatic endothelium, but none was found. The intersegmental branches of the coccygeal veins have developed in the 4-day chick, but in the region of the later lymphatic plexus we could find no endothelium and no endothcUal nucleus— nothing but a uniformly dense mesenchyme without the spaces present in later stages.
With regard to the much discussed question as to whether these first identifiable lymphatics arise from mesenchyme cells which differentiate into endotheUal cells and send back processes to the veins, or whether they arc outgrowths from the venous endothelium, we are unabk; to give a final and conclusive answer. The fact that, in the earliest stages in wliich the characteristic lymphatic endothehum can be identified, these first lymphatics are found to connect with veins in many jjlaces, while in later stages the number of such connections diminishes instead of increasing, appears to favor the view of outgrowth from the veins. But these studies do not exclude the possibility that for a very brief period mesenchyme cells may change into endothelial cells wliich form angioblasts and quickly acquire, first, a connection with the vein and then a lumen. It is to be hoped that some transpareiit region will eventually be found that will make it pos.sible to watch the tlilTerentiation of these first lymphatics in the living and settle this question definitely.
Although the possibility that the first Ijonphatics may arise from mesench>Tne cells is not excluded by these studies, their differentiation from mesenchyme spaces appears to be clearly untenable. These vessels appear at a time when the surrounding tissue is uniformly dense. The more open tissue directly adjoining the muscle, which in clucks of 4§ to 5 days contains spaces, is not invaded by the developing lymphatics. The stages showing the loose tissue containing the spaces formerly thought by some writers to be "pre-lymphatics," were found to be stages in which there is already a luxuriant plexus of lymphatics whose continuous endothchal lining and total independence of the spaces were demonstrated by all the methods used for these investigations. The isolated "pre-lymphatic" spaces, some of which were endothelial-lined and others apparently not, are quite evidently the result of incomplete reconstruction, inevitable when uninjected material and low powers of the microscope are used.
The first lymphatics occur in a dense region in which spaces are conspicuously absent, and the primitive plexus spreads out regardless of the character of the tissue which it invades. Everywhere this earliest plexus has the same form, consisting of deUcate, string-like processes, minute thread-like connections, and larger, rounded, nodal points, the latter probably representing the result of beginning absorption. The plexus appears to be only secondarily influenced by the character of the tissue in which it is situated, and in chicks of 5 days and over those portions situated in a looser area, such as the lymph-heart region and the region anterior to the hind-hmb, have larger vessels than those located in dense regions, such as the posterior tip of the pelvis and the axilla.
It is, of course, a great disappointment that we were unable, even bj^ the use of such a painstaking method, to carry the liistorj^ of the first lymphatics to a point at which the mode of origin of the lymphatic system could be estabhshed beyond a doubt; but we believe that the present studies have yielded valuable information with regard to the nature of the earliest lymphatics in stages in which the existence of such vessels had not been suspected. It has been possible to show that these vessels have definite morphological characteristics which distinguish them from other tissues. Also, the form of the beginning Ijrmphatic plexus, with its many solid processes and its numerous mitoses, makes it appear that the method of growth of lymphatic capillaries, described by Clark (1909, 1912) for the transparent tails of amphibian larvae, is also the method of growth of the primitive lymphatic system. In other words, the specificity of lymphatic endothehum has been traced back to a very early stage.
The history of the lymphatics of the chick in the region of the posterior IjTnphheart has been traced back from the 8-day stage described by Budge (1882) and Sala (1900), in which a definite pulsating lymph-sac is present, to the stage of 6^ days in which the pulsations begin, although the heart is still in the form of a plexus; then to the stages of 5 to 6 days, in which a luxuriant blood-filled l>Tnphatic plexus is present; to a still earlier stage in which a continuous plexus of finer vessels, without a continuous lumen, occupies this region; and finally, to a stage in which can be found a few very delicate vessels, most of which are connected with bloodvessels, and which have not yet formed a plexus.
The subsequent history of the superficial lymphatic system in chicks of 5§ to 9 days had previously been studied (E. L. Clark, 1915). By the method of injecting a very few granules of India ink directly into single lymphatic capillaries at various stages, it was possil:)le to study the consecutive stages in the wasliingout of the stagnant blood from the early superficial ])lexus with the establishment of hTnph-flow, and also, simultaneously, the associated morphological changes. In this series of studies we found that the beginning Ijonph-flow in the various parts of the primitive superficial lymphatic system is accompanied by the differentiation of chaimels or honph-ducts from the indifferent plexus, and that an increase in the flow is associated with an increase in the size and straightness of such ducts. The study also showed that in older chicks (8 days) lymph-sacs develop at a point where there are two conflicting pressures, and that such sacs are formed by the enlargement of a single lymph-channel or by the enlargement and coalescence of adjacent lymph- vessels. (Ranvier, 1896, 1897.)
Thus a continuous history has been obtained of the superficial l3Tnphatic region in chicks of 5| to 9 days, while in one typical region (that of the posterior lymphheart) we have been able to complete the jjicture of the developing lymphatics from their first appearance as endothelium-hned vessels in chicks of 4 days and a few hours, up to the 9-day stage, when a heart with valves has developed. This study has shown that the important earlier investigations of the embryology of the l^Tnphatic system, in which lymph-sacs connected with veins were thought to be the primary structures, the later studies in which the "veno-lymphatics" of these regions were pictured as forming an incomplete plexus, and the seemingly isolated vessels occurring elsewhere in the body, as well as the reconstruction of "spaces" in the path of developing lymphatics, were all mere fragments of the history of this system and subject to various interpretations. Again, the finding of a continuous plexus of independent lymph-capillaries connected with veins and filled with stagnant blood left a part of the history untold. The study with the oilimmersion lens of sections cut parallel to the surface and hence to the plane in which the lymphatics develop, and stained so as to bring out the contrasts between endothelial nuclei and the nuclei of mesenchyme cells, together with the injection of the blood-vessels, has enabled us to add another chapter to the history of the early Iymphatics. This is a stage in which there is present a continuous plexus of vessels (some of them bulbous in shajie, and others very narrow), which does not have a continuous lumen and which connects with the venous system in a number of places. Back of this we find a still earlier stage, the earliest so far described in birds or mammals, in which only a few vessels, most of them very delicate, are present in this region. Connections between these and the veins were found in practically every instance. These vessels do not form a continuous plexus. It is to be hoped that some future method will reveal the cells which are the direct ancestors of these earliest vessels with the characteristics of lym])hatics, and thus com]ilete the history.'
We now come to the second question wliich we attempted to solve with regard to the development of lymphatics — viz, the points of origin of the first lymphatics.
- The picture of the earliest lymphatic vessels of the chick, obtained from these studies, are essentially similar tfl the
earliest vessels of amphibians, as described by Fedorowicz (1913) and by Kampmeier (1915) and, like the results obtained by those authors, our observations favor the view that these earliest vessels arise as outgrowths from the endothelium of the veins.
II. Operations on Chick Embryos
Earlier investigations (E. R. Clark, 1909, 1912) have shown that growth of lymphatic capillaries is by a process of sprouting from pre-existing endothelium, while the present study shows that the primitive Ijinphatic plexus, in the earliest stages of its development, increases rapidly in richness and by this process of growth undoubtedly invades near-by regions.
Many investigators have considered that the points of origin for the first IjTnphatics are limited to certain definite regions associated with veins. Thus Sabin (1901) originally described two points of origin for the IjTnphatics in pigs — the jugular l\Tnph-sac and the ihac sacs (both paired). These were later increased by the addition of the retroperitoneal sac (single) discovered by F. T. Lewis (1906). All of the lymphatics of the body were thought to be derived from these points by the process of outgrowth. Other investigators cUffered from this view, considering that lymphatics have a diffuse origin. Huntington and McClure (1906, 1910) advocated the view that the origin of lymph-sacs is in certain definite regions associated with veins, while beUeving that other "peripheral" hinphatics arise diffusely from the mesenchyme. F. T. Lewis (1906) suggested that IjTnphatics arise diffusely from cut-off blood-vessels. Kampmeier (1915) describes the origin of lymphatic endothelium in J5t//o as occurring diffusely by outgrowths from venous endothelium at many places.
We attacked the problem by the experimental method. By operating on chick embryos at a stage before any lymphatics had developed we removed various regions and then studied the modifications produced on the development of lymphatics. The questions we attempted to solve were the following:
1. Are the points of origin for the lymphatic system normally limited or diffuse?
2. If limited, where are these points of origin? Do they constitute only those regions in which the lymphatics are found to maintain connections with veins (the regions of the "primary lymph-sacs")?
3. If the points of origin are normally limited, is it possible for lymphatics to develop in situ in other parts of the body when such points of origin have been removed? This would also answer the questions: Is the tissue (blood-vessel endothelium or mesenchyme) from which the first lymphatics differentiate equipotential, or is there some specLal quality of this tissue in one region which is not found in another?
In seeking the answers we began by removing the regions from which all of the superficial lymphatics have been thought to arise (Mierzejewski, 1909) — the region of the posterior IjTivph-heart in the tail, and the anterior region near the duct of Cuvier. These are the two regions where IjTniDhatics connecting with veins had been demonstrated in cliicks of 5 days and where these venous connections are maintained throughout embryonic life. The experiments performed were not of a kind to throw any light on the question as to the origin of IjTiiphatics from mesenchjTne or from blood-vessel endothelium, since both of these tissues were present before the development of lymphatics in all of the regions studied.
'The operations on chick embryos were begun in the spring and summer of 1913, in the laboratory of Professor H. Hoyer, at the University of Cracow, Poland. The work for operation 1 . and a part of the experiments recorded under operation 2, were completed at that time. It is a pleasure to have this opportunity to thank Professor Hoyer for his cordial hospitality, and for the extremely courteous, friendly, and stimulating interest which he displayed in our work during the very enjoyable months which we spent in hit laboratory.
Method of Operation
Description of the details of the operations must be left for the account of the individual experiments, since a number of modifications were adopted in each case. All of the operations were carried out under the binocular microscope inclosed in a warm chamber heated to incubator temperature. The operations were performed on chicks taken from the incubator to the warm box, where the shell was swabbed with cotton or cloth saturated in alcohol. As soon as the alcohol had evaporated a hole was pricked in the shell with a sterile needle. Into this opening the point of a pair of sharp forceps was inserted and the shell carefully picked off over an area about 6 mm. in diameter. A little warm sterile Ringer's solution was then dropped on the shell membrane, thus making the dissecting away of this membrane from the yolk much easier. Immediately after removing the shell the yolk sags away from the opening and it then becomes necessary to add, gradually, enough Ringer's solution to bring the embryo up to a level with the opening. If it is added too rapidly it may cause the embiyo to rise out of the opening and result in a tear in the yolk membrane. If the region of operation is covered by amnion this membrane must be opened with forceps before proceeding. After the operation is finished the amnion can be "sutured" by pinching the edges together, whereupon it heals and continues to develop in a normal manner.'
The instruments used differed according to the type of operation performed; small iridectomy scissors were used when a portion such as the tail or wing bud was removed, while well-sharpened needles or a small knife with a triangular blade proved most useful in cases where it was necessary to dissect away somites or body-wall. In all cases sharp edges and fine points to the instruments are essential. Moderate aseptic precautions were used. The Ringer's solution was boiled and allowed to cool to a temperature of 37 to 39° C. before using. Needles and mica for the window were flamed, wliile more deUcate instruments, such as the small forceps, knives, and scissors, were simply dipped in alcohol and allowed to dry.
After completing the operation more Ringer's solution was dropped into the opening. This helped to exclude air and to prevent sticking of the yolk-membrane to the ragged edges of the shell. A thin piece of mica was then placed over the opening and the edges sealed with a warmed mixture of beeswax (4 parts) and resin (6 parts). The egg was then returned to the incubator. The sticking of the yolk-membrane to the shell can be prevented if the egg is kept with the window side down, so that the embryo will float to the opposite side. It is also advisable to rotate the egg gently several times during the first few hours after the operation. The condition of the embryo (whether alive or dead), its stage of development, and hints as to the success of the operation can be observed from day to day by rotating the egg so as to bring the window on top, and waiting for a few moments until the embryo moves around under the opening. The proper time at wliich to reopen the egg can also be determined in this way.
' A decided improvement in the technic of operatinj? has been developed since this work was done. It w.n found that a confiidcrable mortality is caused by the fact that, when the hole is made in the shell, the air is gradually forced out of the airchamber until the chamber is obliterated. As a result a .slight increase in the temperature of the egs after clo.sing causes such an increase in pressure as to stop the heart-beat. If this is prevented by the simple expedient of immersing the e^2 during the operation in water at incubator temperature to a dc|>th sufficient to cover tin- air-rh unbiT. the air remains in the chamber, the ciiick doe.s not sink away from the opening, and the mortality during the first 4>. hours after operation is reduced nearly to zero. It is well to roll the egg slowly before operation, in order to make sure that the embryo U freely movable, and also to candle it in order to control the mobility and to determine the line of latitude on which to make the opening.
At the desired time, the mica was removed and the embryo again exposed. The presence of lymphatics was then tested by injections with India ink. In many of the experiments injection of the left side served as a control for the operated side. In some specimens the blood-vessels were subsequently injected with Berlin blue gelatin (5 per cent). The embryos were fixed in Carnoy's solution, dehydrated in three changes of absolute alcohol and cleared in benzol and oil of wintergreen. In cases where it was desired to section, the specimen was fixed in Bouin's fluid and stained by the same method described in Part I.
Operation 1. Removal of the Posterior Lymph-Heart Region.
The first operation consisted in the removal of the posterior lymph-heart region before the development of any posterior hnnphatics. Since our other studies had made us familiar with the normal appearance of the lymphatic plexus which develops in this region, and since we had studied the invasion of the neighboring region (the posterior tip of the pelvis) by Ijonphatics connected with this plexus, this appeared to be the logical place for starting such an experiment. We therefore removed the tail in chicks ranging in age from 2 days 14 hours to 3 days, a stage at which 37 to 40 somites have chfferentiated. All of the tail region posterior to the thirty-third or thirtj'-foiu-th segment was snipped off with iridectomy scissors. The operation is comparatively simple, although a good deal of bleeding frequently follows the cutting. The total mortality amounted to 50 per cent, when precautions were developed for preventing the embryo from sticking to the shell. The eggs were returned to the incubator and development allowed to continue. They were reopened at intervals of 3 to 5 days after the operation, the IjTnphatics injected on both sides of the embr^'o, and their character and extent compared with those of normal chicks of a corresponding stage. Since healthy operated chicks were found, in most cases, to be delayed about 12 hours in their development, the injected embryos corresponded to chicks of 5 days 20 hours, up to 7 days 20 hours. These chicks developed with well-rounded stumps instead of tails, and the posterior IjTnph-hearts were absent.
In a normal chick of 5 days 20 hours l.ymphatics can be injected in the region of the thoraco-epigastric vein between the two limbs, where they form a plexus which connects through the axillary region with the deep jugular plexus, which in turn connects with the anterior and posterior cardinal veins in the region of the duct of Cuvier. Posteriorly, the lymph-heart plexus, with its venous connections and its superficial extensions over the posterior tip of the pelvis, can be injected (fig. 5). In operated cliicks of this stage (6da5's) the side plexus wasreadil3^injectible and entirely normal in appearance ; but diligent search by means of many careful injection tests failed to reveal any posterior l3'mphatic plexus over the pelvis or stump. In embryos allowed to develop longer and examined when 6| to 7 days old (or the stage corresponding to normal 6 to 6^ day chicks), a few ink granules injected into one of tlie lymphatics of the anterior side plexus in the living chick moved anteriorly and disaiipeared in the axillary region, thus showing that lymphflow had begun. Complete injection of these lymphatics showed that a plexus was present, normal in appearance for a cliick of 6^ days, with a channel differentiated from the primitive plexus in the position of the path taken by the injected ink granules. More posteriorly an extensive plexus, continuous with the side plexus, was injected over the pelvis. At the posterior tip of the pelvis, however, where in a normal chick with a beating lymph-heart a rich plexus is present with one or more definite lymph-ducts differentiated, the operated chicks ' showed an indifferent, net-Uke plexus, composed of very fine capillaries with delicate, blind VC V-a't'- Fia. 4— Chick of 3 days; 37
' ... -Ill v^X V somites. The stage of
endings characteristic of the terminal border *°'<^^c V operations i and 2. a.t.b..
. ■. ^^_3\V_- \>\ anterior limb-bud; p.l.b.,
01 a plexus. \AV r^ posterior Umb-bud. For
In operated chicks which developed still " " ff V ,^ Xlt'rnUrt^so!
farther, this most posterior portion of the super- joUav l For operation 2,somites 17
' . , \ .,,.... V\k to 20 were dissected away
ficial lymphatic plexus, while still primitive m >«s-:^ and the wing-bud cut off.
character, was more extensive, and frequently
the plexuses from the two sides were found to anastamose over the stump. No connections between the lymphatics and veins of this posterior region were injected in the operated specimens, no channels differentiated from the pelvic plexus, and in no case did a new lymph-heart differentiate.
In tailless chicks of the early stage of development, in which the early anterior plexus alone could be injected, stagnant blood was frequently found in the vessels of this region. No blood was ever found in the lymphatic plexus which appears over the pelvis in older embryos. By the time a continuous plexus from axilla to stump is present, the circulation has started in the side ])lexus and has washed the blood out of these lymphatics; wliile the posterior part of the plexus, having no connections with veins, does not receive any stagnant blood.
The results obtained from this operation are as follows :
When a point of origin for lymphatics (in this case the posterior lymph-heart region) is removed at a stage before any lymphatics have developed, the neighboring region (in this case the posterior tip of the pelvis), normally sui:)plied from this point of origin, receives its lymphatic supply by ingrowth from another point of origin (more anterior lymphatics). This conclusion appears to follow from the delaj'ed appearance of lymphatics over the posterior part of the pelvis and from the difference in their character shown by the deUcate, bUndly-ending tips. Although the region removed is one in wliich connections between lymphatics and veins are present normally from the earliest stage and maintaintMl throughout embryonic life, the lymphatics wliich eventually develop over the stump in these tailless chicks do not show any connections with veins in injectetl specimens.
An interesting side result came from studying the conditions of lymph-flow in this series of operated chicks in comparison with the conditions present in normal embryos. When the posterior lymph-heart fails to dev^elop, the IjTTiph-flow in the pelvic IjTiiphatics does not start in chicks of 6 to 7 days and, coincidentally, definite lymph-channels do not appear in this region. On the other hand, in the anterior part of the superficial lymphatic plexus the circulation begins and the earliest ducts are formed in a normal manner.
Operation 2. Removal of Anterior Point of Origin for the Superficl\l Lymphatics.
As already stated, a plexus of l^Tnphatics, situated beneath the shoulder and connecting with the veins in the region of the duct of Cuvier, was injected in chicks of 5 to 7 days. Connections from this plexus with the superficial side plexus through the axilla can always be injected in embryos of 5 da3's and older. This plexus, on account of its situation and relation to the venous system, has been thought to be homologous with the jugular hmiph-sac of mammals (Sabin) and has been considered to be the point of origin for the IjTiiphatics of the anterior part of the body, including those of the side region.
In starting experiments to remove this region of origin we first attempted to destroy the jugular (anterior cardinal) vein before the development of any \yniphatics. This proved to be a rather difficult procedure and various methods were tried out and abandoned — the electric cautery and radium, because of the difficulty of localizing the burn; suturing of the vein with minute shreds of cloth, because of excessive injury to the delicate tissues, etc. We finallj' succeeded in removing the vein by the following method: Boiled and filtered aqueous Berlin blue was injected into the jugular vein in the head region. this material clumps on contact with the blood and sticks to the vessel wall, thereby plugging the vessel and at the same time rendering the wall visible. The injection was stopped at the proper moment so as to prevent the entrance of the blue granules into the heart. By this means the jugular vein, the duct of Cuvier, and a plexus of capillaries in the region of the posterior cardinal vein were injected and their circulation stopped. All of these injected vessels were then dissected awa}^ with sharp needles, and some of the tissue around them, as far anteriorly as the ear vesicle, was also removed.
This operation was performed on chicks of 42 to 48 hours. On examination 4 to 6 days later it was found that in every case a well-developed jugular vein was present on the operated side. In one case this was larger than the corresponding vein on the unoperated side. In other chicks it was somewhat smaller, but in all of them it was unquestionably present. It would seem that we were justified in concluding that conditions are present in the neck region wliich favor development of a large vein. This result, although of interest in connection with the problem of the development of blood-vessels, did not carry us any farther in the solution of the problems connected with the lymphatic system. In all of these specimens a normal plexus of lymphatics could be injected in the region of the thoraco-epigastric vein, which connected in a normal manner with a deep lymph-plexus, which, in turn, connected with the newly-regenerated jugular vein.
We next tried to isolate the side region, between the wing and leg, from the region beneath the shoulder, from which its Ij-mphatics were supposed to be derived.
We first attempted to ]M-event the down-growth of lymiihaties by inserting the tip of a porcupine (luill into the angle formed by the duct of Cuvier and posterior cardinal veins in an embryo of 3 days, a stage at winch this region is situated anterior to the shoulder. However, when such embryos were examined two or three days later it was found that this wedge had in no way interfered with the normal development, for the veins were present in their normal position beneath the shoulder, while the plug had remained in the neck region anterior to the shoulder. As might be expected, normal lym])hatics were present over the side and connected with the deeper plexus in a normal manner.
Our next operation consisted in making a gap which would isolate the side region from the region associated with the veins beneath the shoulder. This was effected by removing the wing-bud and destroying a number of adjacent segments.
Fig. 5. — Normal chick of 55 days, with superficial lymphatics injected with India ink. Compare witli figure 6. X 12.
Fio. 6. — Chick from operation 2, with superficial lymphatics injected. Embryo was operated on at 2 days 23 hours. Somites
17 t<) 20 were dissected away and the wing-bud removed. Three days later (when the chick was 5 days and
18 hours old) the egg was reopened and the lymphatics injected. Comparison with figure 10, which shows the superficial lymphatics of a normal embryo of the same stage of development, shows clearly that the side region of the operated chick has been effectually isolated from the deep anterior point of origin. In sjiite of this fact the lymphatics of the side region have developed normally. X 12.
Fio. 7. — Chick of 2 days 1.S hours which died soon after operation (operation 3). Six somites (22 to 27) wore dissected away together with the body-wall opposite them. The tail-bud posterior to somite 32 was cut off. X 12.
The wing-bud was snipped off with iridectomy scissors and the segments dissected away with fine needles and forceps. In the first operations of this series, in which only three of the somites were injured, the gap produced was not large enough, and the normal lymphatics of the side plexus, in chicks of 6 to 7 days, were found to connect along the dorsal side of the hole with the deep cervical lymphatics connecting with the veins. When four to seven segments, in addition to the wing-bud, were removed, the result was a large "chasm." In these embryos, allowed to develop two to three days after the operation, the jugular lymphatic plexus connected with the veins was present, but no vessels running ]wsteriorly from it through the axilla could be injected. In fact, the hole was of such a nature that there was no skin or subcutaneous tissue present for vessels to grow in. However, when the body-wall, between the gap and the posterior Umb, was tested in such an embryo, a normal lymphatic plexus was iyijected in every instance. This extended anteriorly as far as the edge of the gap but could not be injected any farther. In such specimens (chicks of 5 to 5| days) the posterior lymphatics were normal and were present in the lymph-heart region and near it; no lymphatics were injected over the pelvis. This condition is shown in figure 6 and can be compared with figure 5, showing the injected lymphatics of a normal chick of the same stage. Injection of lymphatics on the opposite (unoperated) side showed a similar condition, both in character and extent of the lymphatic plexus, except for the fact that axillary IjTnphatics, connecting the side lymphatics with the deep plexus, were present.
The fact that the side plexus in such operated specimens develops in the same region and at the same time as in normal embryos,- and at a period before any growth from the posterior point of origin could have taken place, coupled with the fact of its completely normal appearance, speaks for the existence of another normal point of origin for the superficial lymphatics of the chick.
Observation of normal chicks at early stages (5 days and under) showed a stage in which a number of knobs of stagnant blood are present in that part of the lymphaticplexus of the side region just anterior to the leg. Bj^ injection these knobs were found to connect with a number of veins lying parallel to each other, which at this stage flow ventrally, encircUhg the abdomen. These veins are transitory; in chicks of 5| days they disappear and the drainage for this region is all anterior, through the newly developed thoraco-epigastric vein — a stage at which a definite hTtiphatic plexus can be injected for the length of the side, with deep connections through the axilla with the jugular IjTnph-plexus. At this 5|-da3^ stage and later no connections can be injected between these IjTnphatics of the side plexus and the neighboring blood-vessels.
It is also evident from these experiments that the regions in which Ij'mphatic vessels retain their venous connections during all or part of embryonic life are not the only points of origin for the Ijonphatic system.
Operation 3. Isolation of the Leg and Pelvis.
The foregoing experiments gave only a partial answer to the problems in which we were interested. We therefore tried to develop an operation for isolating completely a region supposed normally to be supphed by ingrowi^h from some point or region of origin. For this purpose we operated on chicks toward the second half of the third day of incubation (embryos averaging 2 days and 17 hours), removed the tail region as in operation 1, and also dissected out the five or six somites posterior to the omphalo-mesenteric vein and the adjacent body-wall on the right side. By this means we succeeded in obtaining embryos without tails (with the posterior point of origin removed) in which a gap had been made on one side, isolating the leg and pehis from the anterior Ijonphatics, and in which the probable point or region of origin anterior to the leg had been removed. More than a dozen chicks survived this double operation.
On examining an embryo of this kind which had been allowed to develop until it was 7 days old we found a gap in the body-wall anterior to the leg which extended dorsally as far as the dense tissue around the spinal cord (where lymjjhatics are never found at this stage), and which exposed th(> abdominal viscera. The amnion was often attached along the borders of the gap.
Injection of the left side of the embryo (the unoperated side excejjt for the removal of the tail) showed a continuous lymphatic plexus which extended from the deep jugular plexus connected with veins, through the axilla, down the side, over
Fig. 8. — Left side of a chick of 6 days. This side is normal except for the absence of the tail. Continuous superficial lymphatic plexus injected with India ink. This serves as a control for the right side of the same embryo shown in figure 9, which is an example of operation 3. X 12.
Fig. 9. — Right side of same embryo shown in figure 8. Chick of 6 days. Example of operation 3. At 2 days and 12 hours the six segments anterior to the posterior limbbud of this embryo were dissected away, together with the body wall opposite. The tail was then cut off. Lj-mphatics injected with India ink. The vessels anterior to the gap aie normal in appearance and extent. Over the posterior part of the pelvis a lymphatic plexus, normal in appearance but less extensive than usual, was injected. No lymphatics were injected on the anterior portion of the pelvis or over the stump. Compare with the lymphatics of the opposite side shown in figure 8. X 12.
the pelvis and to the posterior stump, where the vessels were fine and delicate. Lymphatics were also present in the region doreal to the shoulder connecting, anterior thereto, with the deep jugular plexus, but not yet posteriorly with the side IjTiiphatics. In other words, this left side possessed a lymphaticplexus of the type described in operation 1, normal cverj-w^here except for the absence posteriorly of the beating lymph-heart and the consequent primitive character of the vessels over the posterior tip of the pelvis (fig. 8).
On the right side our injections showed anteriorly the presence of normal lymphatics in the deep jugular region, in the region dorsal to the shoulder, in the axilla, and also in that portion of the side region anterior to the gap. Here, at the edge of the gap, the lymphatic injection ended in a number of delicate points. Repeated injections over the anterior part of the i^elvis and leg in operated chicks of this stage failed to show any Ij'mphatics corresijonthng to those present in this region in normal embryos or to the jjIcxus already injected on the opposite side. But by ijlungirig the cannula into the tissue more posteriorly, over the pro.ximal part of the pelvis, a plexus of lymphatic capillaries of normal appearance, richness, and location was injected with ease (fig. 9).
This operation was repeated a number of times, and by allowing the chicks to incubate for varying periods, we obtained an interesting series of embryos.
In an operated embryo of 5 days and 15 hours, corresponding to a normal chick of about 5| days, injection revealed a normal anterior plexus on the left side extending deep into the axilla. As in normal chicks, the lymphatics dorsal to the shoulder had not yet developed. Posteriorly, the lymph-heart region was absent and no lymphatics could be injected over the pelvis. In other words, the condition of lymphatics was identical with that found in embryos after operation 1.
On the right side, where the double operation had been performed, the Ijonphatic plexus anterior to the gap was present and normal in appearance. It extended from the deep jugular plexus connecting with the veins, posteriorly through the
Fig. 10. — Left side of anerabrj-o of 6 days and 15 hours. Normal except for the absence of the tail. SuperBcial lymphatic plexus injected. Control for right (operated) side shown in figures 11 and 12. X 12.
Fig. 11. — Right (operated) side of a chick of 6 days and 15 hours. Same embryo as in figure 9. L.arge gap present in bodywall extending to mid-line of the back, leaving spinaLcord exposed. Embryo twisted toward left side. Lj-mphatic plexus injected dorsal to shoulder and in axillary region normal. Posteriorly, lymphatic plexus injected on posterior half of pelvis and around the stump (fig. 12). Normal in character. No lymphatics injected over anterior part of pelvis. X 12.
Fig. 12. — Posterior view of the same embryo shown in figures 10 and 11, showing the lymphatic plexus extending around the tail-stump from both sides. X 12.
axilla, and ended just anterior to the gap in fine delicate points, smaller than the vessels usually found in this region. Posterior to the gap — i. e., over the leg and pelvis as on the opposite side — no lymphatics could be injected.
The next stage found in this experiment is that which has already been described— the chick pictured in figures 8 and 9.
In the next stage (an embryo of 6 days and 15 hours) the left side showed a luxuriant lymphatic plexus from the axilla, over the side, the pehds and almost around the stump (fig. 10). The anterior portion of this plexus was entirely normal, as was also the posterior jwrtion, except for the absence of large channels over the posterior tip of the pelvis and the presence in their place of a more primitive plexus with fine delicate endings.
On the right side there was a very large gap which bordered dorsally on the spinal column and left the entire Wolffian body and most of the hver exposed. The lymphatic injection showed a normal plexus dorsal to the shoulder, connected anteriorly, as on the left side, with the deep cervical plexus, and posteriorly with the side Ij-mphatics. This ])lexus was not extensive, owing to the large size of the gaii. It connected anteriorly through the axilla with the deep jugular plexus, and posteriorly ended blindly in numerous fine points at the edge of the gap. On the anterior border of the pelvis (in the region just posterior to the gap), as in the younger chicks just described, no Ijinphatics were injected. Farther posteriorly on the pelvis, however, a large plexus of lymphatic capillaries was injected. This was normal in appearance and more extensive than that shown in fig. 9. It extended over the tip of the pelvis, ending, Uke the plexus of the opposite side, near the middle of the stump in numerous finely-pointed, bUnd tips (figs. 11 and 12).
A still older stage is shown in figure 13, a cliick of 7 days. The lymphatics of the left side resembled those shown in figure 10, except for the presence of some larger ducts in the side region between the limbs. On the right side the lymphatics of the shoulder region, and of the side region anterior to the gap, were normal for chicks of this stage. Over the pelvis, from the region just posterior to the gap and extending well around the stump, was a luxuriant plexus of lymphatics resembling those of the opposite side in character and extent. No connections between this posterior lym})hatic pk^xus and the veins could be found in any sjxH'imen after this type of operation. It should be emphasized, however, that no microscopic studies were made on the operated chicks at the younger stages in which probable venous connections were found in this region in normal chicks.
this series of specimens shows that there is apparently still another point of origin for the superficial lymphatics of cliick embryos — i. e., a region over the posterior jiart of the pelvis. The lymphatics which develop here make their ai^pearance somewhat later than tho.se in the jugular region, the side region anterior to the leg, or those which differentiate in the posterior lymjjh-hcart region of the tail.
'iG. 13. — Ripht side of an operated embryo of 6 days IShour.s, showing a later stage of development of the superficial lymphatics after operation .3. Operative procedure same as that described under fiRure 12. Injected lymphatics in the suprascapular and a.xillary regions normal in appearance. The lymphatics posterior to the gap, in this specimen, cover the whole pelvis and stump just as on the left (unoperated) side. X 12.
This is evident from injections of normal embryos, in which the delicate plexus along the side, the deep plexus near the duct of Cuvier, and the h^mph-heart plexus, all can be demonstrated before any lymphatics over the pelvis can be injected.
Our studies of oil-immersion reconstructions also gave evidence of the later differentiation of these lymphatics. For example, at the stage shown in plate 5 the lymph-heart plexus and a few extensions (probably outgrowths from it) are shown as a continuous plexus. More anteriorly, in the region where the pehdc plexus is found in older embryos, we were able to reconstruct a number of fine vessels, some of them connected with blood-vessels but differing in appearance from blood-vessel sprouts, and others apparently isolated. About eight hours later, as shown in figure 3, a continuous lymphatic plexus is present throughout the region over the posterior half of the pelvis, which connects with the lymphatic plexus in the tail. This continues to grow rapidly in extent and richness, as evidenced by the many soUd processes at the edge of the plexus and the numerous mitoses in the endothelial cells. The connections with blood-vessels, found in these sections in the earliest stages, evidently are soon lost, since none can be injected in the later stages in which a continuous lumen is present.
These observations support the conclusion drawn from the results of the operation just referred to, that there is a separate point of origin for the superficial lymphatics over the posterior part of the pelvis.
Operation 4. Isolation of the Anterior Part of the Leg and Pelvis.
Although operations 2 and 3 had yielded interesting results in regard to the points of origin for the superficial lymphatics, a conclusive answer had not j^et been given to the other problem, i. e., whether lymphatics can develop in a region which has been completely isolated from its normal source of lymphatic supply. The evidence obtained from injections of normal embryos, and from experiment 3, apparently showed that the anterior portion of the leg and pelvis receives its lymphatic supply from the anterior lymphatic plexus of the body-wall in the case of normal chicks, or by invasion from the posterior part of the pelvis when this anterior source of supply has been removed. We therefore attempted a still more radical operation — the effective isolation of this region. This entailed the removal of several segments (three to se\'en) and the body- wall adjacent to them in the region just anterior to the right hip. The procedure was a repetition of that used in operation 3. Then, the anterior part of the posterior limb-bud, together with the somites opposite, being left intact, the remainder of the leg and the adjacent somites on that side were dissected away. The operation was completed, as in operation 3 by snipping off the tail, the aim being to completely isolate the anterior part of the leg and pelvis of the right side from the area of Ij'mphatic origin of that side, and also from that over the posterior part of the pelvis. The left side of the embryo was allowed to develop in a normal manner except for the absence of the tail, including the lymph-heart region.
In the first two experiments of this kind three somites opposite the anterior half of the right leg were allowed to remain intact. These embryos developed without tails, with a large gap in the body-wall on the right side anterior to the leg, and with the right leg smaller than usual and possessing two toes. On the left side a continuous plexus of superficial Ijinphatics was injected over the side, pelvis, and stumj). On the right side the j^lexus anterior to the gap in the axillarj^ region was normal in appearance, and over the posterior part of the pelvis and stump a normal lymphatic plexus was injected with ease. In other words, the result was identical with that obtained in experiment 3. The operation was repeated in the same manner except that in this case only two somites were left untouched in the region opposite the anterior portion of the right leg, and the leg was bisected, the posterior half and the three remaining segments opposite it being removed.
In this operation 6 embryos were opened at varying incubation ages. Plate 7 and text-figures 14 and 15 illustrate their appearance at 7 and 7| days respectively.
Fig. 14. — Left side of a chick of 7 days and 12 hours. Normal except for absence of tail. Superficial lymphatics injected. Compare with right (operated) side shown in figure 15. X 5.
Fig. 15. — Right side of the same embryo shown in figure 14. Example of operation 4. At 2 days 18 hours incubation the three segments anterior to the posterior limb-bud of the right side were dissected away, together with the body- wall opposite them; and, in addition, the two segments opposite the anterior part of the leg; the next two segments, with the limbbud opposite, were left intact, and the next three, opposite the posterior part of the leg, were dissected away, the limb-bud bisected and the posterior half removed. The operation was completed by removing the tail. Compare appearance of embryo with chick shown in plate 7, a younger specimen from the same type of operation. The injected lymphatics of the shoulder region and axilla were present and normal in appearance. Over the pelvis and tail stump in this and other embryos from operation 4 no lymphatics could be injected. X 5.
The gap anterior to the leg and the rounded stump characteristic of operation 3 are noticeable, and in addition the right leg may be seen to be deformed and to possess only one toe.
Four of the embryos were opened at 6^ to 7^ days (the stage illustrated in plate 7, figure 33) and the lymphatics injected. The left side was tested first and the injection showed the presence of the continuous jilexus described in operations 1 and 3 (left side). On the right side normal h'mphatics were injected in the suprascapular region, in the region beneath the shoulder, in the axilla, and as far posterior as the gap in the body-wall. However, when the region over the pelvis was tested, repeated careful injection /aj7cd to show any sign of a lymphatic plexus. The injected ink, instead of remaining in blebs, as is usual in the case of extravasations in the subcutaneous tissues, spread out rapidly through the tissue in finger-like projections which finally ran together and made a continuous black sheet. That the failure to inject the lymphatics in the right side was not due wholly to the condition of the tissue is evidenced by the fact that in man}^ of these embryos the tissue over the pelvis of the left side was more edematous than that of the right side, and yet h^mphatics were always injected on the left side at the first insertion of the cannula.
The results of the injection tests in operation 4 made it appear as if the answer to this part of our problem had at last been found. One of these embryos (shown in plate 7, fig. 33) was selected for sectioning. As may be seen, the gap in the bodywall anterior to the leg was sufficiently large to isolate the leg and pelvis from its anterior source of supply, while the rounded stump and deformed leg, with only one digit, exactly resembled the condition present in the four other embryos just described. In this specimen we refrained from testing for lymphatics the region over the pelvis of the right side, since we feared that the extravasations thus produced might obscure the cytological picture. On the left side we injected a small amount of Berlin blue, enough to demonstrate the presence of a normal Ijonphatic plexus. The blood-vessels were then injected completely, the embryo was fixed in Bouin's fluid, dehydrated, cleared, and sectioned according to the method described for the studies in Part I.
In studying the sections of the pelvic region we found a number of irregular vessels which had not received the injection mass. In reconstruction it was found that these vessels were located, for the most part, in the layer beneath the superficial blood-capillaries, some of them coming to the surface in the interstices between blood-vessels. From their location and general appearance there appeared to be no doubt that these vessels were indeed lymphatics. They differed considerably in character from normal lymphatics of this stage and were not so large or nearly so numerous as the lymphatics of the opposite side. Although these vessels formed a plexus in places, this resembled the more primitive type formed by the earliest lymphatics described in Part I, except for the greater size of some of the component vessels. Many of the connections were very narrow and others were thread-Uke and solid. The absence of any extensive lymphaticplexus over the pelvis after operation 4, and the lack of any continuous lumen in the scanty plexus which was present, undoubtedl}' accounted for our failure to inject IjTnphatics in other chicks of this series. It is significant that these are the same factors which prevented injections of the earliest lymphatics described in Part I.
This straggling lymphatic plexus, pecuUarlj^ primitive in many respects and much less luxuriant than usual, was for the most part independent of the surrounding blood-vessels. However, certain of these irregular vessels possessed an undoubted connection with an injected blood-vessel. They were in no wise distinguishable from the other vessels of the plexus and differed markedly from the straight, regular, and narrow blood-vessel sprouts. Moreover, no such "sprouts" were found on the left side of this embryo, where, as has been indicated, a normal and extensive lymphatic plexus interlaced with the blood-capillaries without communicating with them. From the earher studies, reported in this paper, of the characteristics of the earliest lymphatics and their relation to blood-vessels, there seemed to be some evidence in this specimen that these Ij-mphatic vessels (clearly recently formed structures) were in process of differentiation from blood-vessels; but, as was the case in the study of the differentiation of the lymphatics of the IjTnph-heart region, we can state only that such a theory can not be settled definitely from a study of fixed material.
The convincing points obtained from the study of sections of an embryo operated on in this manner are :
(1) That undoubted lymphatics are present in a region which had been effectively isolated from all known points of origin for its lymphatics.
(2) That these h-mphatics are abnormal in appearance for this stage, and, in comparison with those of the unoperated side, resemble primitive lymphatics to such a degree that it would seem highly probable that they were undergoing the early development described for the first lymphatics of the posterior lymph-heart region. The greater size of some of the vessels of this plexus is probably attributable to the greater looseness of the surrounding tissue and to the consequently greater expansion of these newly-formed vessels, for it will be remembered that the earliest lymphatics normally- differentiate in a comparatively dense tissue.
Thus, in operation 4 it was shown that lymphatics will develop in situ in a region that has been completely isolated from the points of origin from which, presumably, it would normally receive its lymphatic supply. These vessels are not normal for this stage of development and do not resemble those of the unoperated side. From the fact that, unlike the normally appearing plexus found in embryos after operation 3, no plexus of vessels was injected, and also from the appearance of these lymphatics in sections, they appear to be primitive vessels which develop in situ in a manner comparable with that described for the earhest lymphatics of the posterior lymph-heart region. The development of such lymphatics is evidently delayed in this case, since only these primitive vessels are present at a stage in which the opposite side possesses an abundant lymphatic plexus.
Summary of the Results Obtained from Operations on Chick Embryos
The experiments just recorded have given the following answers to questions of the points of origin for the superficial lymphatics of chick embryos :
- Regions of origin for the superficial lymphatics of chicks are not limited to
those areas in which venous connections are retained during embryonic life — i. e., to the regions of the so-call(>d primary lymph-sacs which, in the chick, are beneath the shoulder and are associated with the veins near the duct of Cuvier, and the tail region associated with the first five coccygeal veins.
- There are at least four such regions of origin for the superficial lymphatics
of chick embryos: the two mentioned above, where the venous connections are retained, and two others, one in the side region, probably that portion just anterior to the leg, and another on the posterior part of the pelvis. In the latter two no venous connections can be demonstrated after the early lymphatic plexus has become continuous over the surface of the body (operations 2 and 3).
- Such points of origin are not restricted in size; in fact the areas in which
differentiation of superficial lymphatics takes place appear to be fully as large as the regions which are supplied by ingrowth.
- When a region of origin is removed by operation, at a stage before Ij'mphatics
have started to develop, the adjacent region usually supplied by extension of lymphatics from this point of origin receives its lymphaticsupply by ingrowth from another source (operation 1).
- When the point of origin removed is one of those in which venous connections are normalh' retained (such as the posterior Ij-mph-heart region) no new permanent connections with veins develop. In this case a beating lymph-heart never develops elsewhere to replace the one whose anlage has been removed (operation 1).
- When a region is effectively isolated from all its usual sources of origin, lymphatics will eventually develop in situ (operation 4) .
In a word, the results of the operations justify the general conclusion that, while the formation of permanent connections between the lymphatic system and the veins is apparently restricted to certain definite regions, the differentiation of lymphatic endothelium is not so restricted, but occurs at man}' places in the embryo.
The modern work initiated by the studies of Ranvier, Sabin, and MacCallum, has shown that the lymphatic sj^stem is composed of definite vessels wliich are everywhere separated from the spaces and cells of the connective tissue by an endotheUal membrane. The discovery that this membrane is present throughout the lymphatic system has made necessary a revision of the older terms for certain fluids of the body, all of which had been known as lymph, and a new di^'ision into (1) plasma, (2) tissue fluid, and (3) h'mph, designating respectively the fluid inside the blood vessels, the fluid of the tissue spaces (including the cerebro-spinal fluid and the fluid of the serous cavities) and the fluid inside the lymphatics (Sabin, 1916) . The basis for this new conception of the lymphatic system, with the emphasis which it throws upon the importance of endothelium, rests chiefly on the embryological studies begun by Sabin and carried on by numerous other investigators. These morj^hological studies have shown that l3'mph-^'essels invade the different regions and organs of the embr^vo by a process of ingrowth, and observations on the transparent tails of Hving tadpoles have estabhshed the fact that lymphatic capillaries remain completely independent of the surrounding cells and tissues and grow by a process of sprouting from preexisting lymphatic endothelium. It has also been shown, in studies of chick embryos, that the primitive form of the lymphatic system is an indifferent, net-like plexus, out of which ducts and sacs develop secondarily in response to pressure conditions inside and outside the vessels. A beginning has been made toward finding out some of the reactive powers of lymphatic endothelium : it is known to be phagocj'tic and to be capable of responding to certain substances (by growing toward them) which do not stimulate a similar response on the part of the blood-capillaries.
The point in the development of the lymphatic system at wliich this lymphatic endothelium becomes the specific tissue present in the stage represented by the lymiihatics of the tadpole's tail, and the manner in which this tissue originates, are questions which have been investigated extensively but never completely settled.
In these observations it has been found that in chick embryos the lymphatic capillaries may be identified as definite endothehum-lined vessels in embryos of 4| days, a much eariier stage than any previously described. These early vessels possess endothelial nuclei with morphological characteristics which distinguish them from the nuclei of the mesenchyme cells. The earUest lymphatics are tubes and strands of endothelium, most of them connected with blood-vessels. They have a marked tendency to plexus formation and the resulting plexus in its primitive form is made up of vessels of irregular shape and size, bulbous portions alternating with fine sprouts and soUd connections of extreme delicacy. The continuous endothelium precedes the formation of a continuous lumen in the primitive lymphatic plexus. Mitotic figures are numerous in these lymphatics, thus showing that the method of growth by sprouting is acquired very early.
The present studies exclude the possibiUty that lymphatics are formed from spaces in the connective tissue. The numerous connections between the earhest IjTuphatics and blood-vessels, the majority of which are lost within the first 30 hours of lymphatic development, make it appear probable that the first lymphatic vessels have arisen from the endothelium of blood-vessels. However, the possibiUty that the differentiation of lymphatic endothelium may occur in the manner recently described for the first origin of blood-vessel endothelium (Stockard, Sabin) has not been excluded by the methods of study used in this investigation.
Our studies have also shown that the points of origin for lymphatics are by no means confined to those regions in which lymph-sacs connected with veins are present in older embryos. The operations on chick embryos have demonstrated at least four regions of origin for the superficial lymphatics. These experiments further show that the character of the blood-vessel endothelium (or mesenchyme) is not specialized in certain Umited regions as regards its ability to give rise to lymphatic endothelium; for when a region, normally suppUed by ingrowth, is effectually isolated from its sources of supply, lymphatics will eventually develop in situ, but whether from blood-vessel endothelium or mesenchyme cells was not determined.
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thoracic duct in the chick. Arner. Jour. Anat., vol. 15. Panizza, 1830. Osservazionni antropo-zoatomico fis iologiche. Pavia. (Ref. Sabin, 1913). Ran\'ier, L., 1895. Morphologic du systSme lymphatique. De I'origine des Ij'mphatiques dans la peau de la grenouille. C. R. Acad, des Sc, t. 120, p. 1.32.
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, 1897. Morphologie et d^veloppement des vais seaux IjTnphatiques chez les mammiferes. Arch, d' anat. micr., t. 1, p. 69-137. Sabin, F. R., 1902. On the origin and development of the lymphatic system from the veins, and the development of the lymph-hearts and thoracic duct in the pig. Amer. Jour. Anat., vol. 1, p. 367-389.
, 1004. On the development of the superficial
lymphatics in the skin of the pig. Amer. Jour. Anat., vol. 3, p. 18.3-195.
, 1911. A critical study of the evidence presented
in several recent articles on the development of the Ivmphatic system. Anat. Rec, vol. 5, p. 417-446.
— , 1912. On the origin of the abdominal lymphatics
m mammals from the vena cava and the renal veins. Anat. Rec, vol. 6, pp. 335-342. , 1913. The origin and development of the lymphatic system. Johns Hopkins Hosp. Reports, Monographs, New Series 5.
, 1916. The method of growth of the lymphatic
system. Science, N. S., vol. 44, p. 145-158.
— -, 1917. Origin and development of the primitive
vessels of the chick and the pig. Contributions to embryology, vol. 6; Carnegie Inst. Wash. Pub. 226. S.ALA, L., 1900. Sullo Sviluppo dei cuore Umfatici e dei dotti toracici neU' embrione di polio. Richerche Lab. Anat. normale, Univ. di Roma, vol. 7, p. 263. Stock.ard, C. R., 1915. The origin of blood and vascular endothelium in embryoS without a circulation of the blood, and in the normal embryo. Amer. Jour. Anat., vol. 18, p. 227-327. West, Randolph, 1915. The origin and early development of the posterior lymph-heart in the chick. Amer. Jour. Anat., vol. 17, p. 403-436.
Description of Plates
Fio. 16. A living chick of 5 days 16 hours. Moasurement 16.5 mm. (fresh specimen). X 5. The square indicates the region shown in figure 17.
Fig. 17. Posterior part of pelvis and tail region of same embryo, showing the two sets of superficial vessels visible in living specimen. Blood-capillaries orange-red in color, lymphatics deep red. The arrow indicates direction of blood-flow in the blood-capillaries. The blood in the lymi^hatics is stagnant. X 24.
Fio. 18. Chick of 5 days 12 hours. X 3. The portion posterior to the dotted line is the region illustrated in figure 19.
Fio. 19. Tail and posterior pelvic region of the same embryo. The blood-vessels wore injected through one of the vitelline veins. The lymphatic ple.\us remained filled with stagnant blood. The drawing was made from the fresh specimen immediately after injection. The deeper lymphatics of the posterior lymph-heart plexus are shown in paler red, the more superficial capillaries in brighter red. X 18.
Fig. 20. Chick of 5 days 22 hours. The lymphatic plexus in the posterior lymph-heart region and over the posterior tip of the pelvis, is injected with India ink. X 24. The square indicates the region shown in figure 21.
Fig. 21. Tail and posterior pelvic region of the same embryo. The lymphatic plexus in the posterior lymph-heart region, and the more superficial plexus connected with it, were injected directly with India ink without disturbing the neighboring blood-vessels. A — superficial lymphatic plexus; B and C — lymph-heart plexus; D — points at which cannula was inserted to make injections. X 36.
Fig. 22. Posterior half (left side) of a chick 5 days 15 hours old. Measurement before fixation 16 mm.; blood-filled lymphatic plexus present in the tail region and over the pelvis and hip. The embryo was bled and the drawing of the blood-filled lymphatic plexus made from the fresh specimen. X 36.
Fig. 23. Chick of 5 daj's 18 hours. X 2.5. The portion posterior to the lines is the region shown in figure 24.
Fig. 24. Tail and posterior pelvic region of the same embryo. Double injection. Blood-vessels completely injected with India inli (black in the drawing). liymphatics injected with silver nitrate 0.5 per cent (white in the drawing). The lines A and B indicate the plane of the section shown in figure 25.
Fig. 25. Thick cross-section through the tail region of the same specimen; i.e. a. — intersegmental coccygeal artery; i.c.v. — intersegmental coccygeal vein. The superficial blood-vessels are dark in the drawing and the lymphatics white as in figure 24. X 36.
Fig. 26. Microscopic drawing (oil immersion) of a portion of the lymph-heart region of a chick of 4 days 23 hours, measuring V2}4, mm. before fixation. The blood-vessels were completely injected with India ink. The specimen was sectioned parallel to the surface and the sections stained with EhrUch's hematoxylin, counterstained with eosin, orange G. and aurantia. The figure shows a portion of a lymphatic capillary and of a blood-capillary, containing ink granules, and the adjacent mesenchyme cells, lym. — lymphatic; l.n. — lymphatic nucleus; b.v.n. — blood-vessel nucleus; m.n. — mesenchyme cell nucleus. X 1,200.
Fig. 27. Higher power drawing of the nucleus of a lymphatic endothelial cell (1. n.), and of the nucleus of a mesenchyme cell (m.n.). X 1800.
Fig. 28. Microscopic drawing of a chick of 5 days J hour, showing an early lymph-vessel of the posterior lymphheart region and its connections with an injected blood-vessel; lym. — lymphatic; b.v. — blood-vessel; con. — connections between lymphatic and blood-vessel; l.n. — lymphatic nucleus; r.b.c. — red blood-cells inside of the lymphatic vessel. X 800. (Magnification of original drawing 1 to 1200.)
Fig. 29. Microscopic drawing of a chick of 4 days 23 hours, showing an early lymph-vessel of the lymph-heart plexus with an endothehal nucleus undergoing mitotic division. X 800. (Magnification of original drawing 1 to 1200.)
Fig. 30. Oil immersion reconstruction of the vessels of the lymph-heart, region in a chick of 4 days 23 hours; measurement 12i mm. before fixation. The early lymphatic plexus is shown in blue. The injected blood-vessels are shown in fines. X, connections between lymphatics and blood-vessels. X 320. (Magnification of original drawing 1 to 800.)
Fig. 31. Oil immersion reconstruction of the vessels of the lymph-heart region in a chick of 4 days 9| hours; measurement 11.75 mm. before fixation. Probable lymphatics in blue. Injected blood-vessels in lines. X, connections between lymphatics and blood-vessels. X 320. (Magnification of original drawing 1 to 800.)
Fig. 32. Oil immersion reconstruction of the earliest lymph-vessels in the region of the posterior lymph-heart of a chick of 4 days 7 J hours. Measurement before fixation 9 mm. The probable lymph- vessels are blue. Injected blood-vessels indicated in lines. X 320. (Magnification of original drawing 1 to 800.)
Fig. 33. Operated chick of 7 days. At 2 days 14 hours incubation five segments anterior to the posterior limb-bud of the right side were dissected away. The next two segments and the anterior half of the limb were left intact, and the next succeeding three segments, opposite the posterior part of the leg, w^ere removed, together with the posterior half of the leg. The tail was then removed. This is an example of the type of embryo obtained from ojieration 4. X indicates an exposed portion of the spinal cord located at about the mi<lline of the back. X 20.
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