Paper - Histogenesis and morphogenesis of the thoracic duct in the chick (1913): Difference between revisions

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=Histogenesis and Morphogenesis of the Thoracic Duct in the Chick=
==Development of blood cells and their passage to the blood stream via the thoracic duct==
Adaai M. Miller
The Analomical Laboratory of Columbia Univernily
Twentty-Eight Figures (Seventeen Plates)
==I. Introduction==
The study of the development of the jugular lymph sac in the chick, the result of which was published in this Journal/ (1) led to the investigation also of the developing thoracic duct and the means whereby its communication with the lymph sac is established. This investigation has been carried on with the advice and under supervision of Dr. Huntington and in the light of his recent work on reptiles (2) and the cat (3).
Within the past few years different investigators have shown that in the frog (4), (5), the chick (1), (8), the rabbit (6), the cat (9), and in man (7) each jugular l>Tnph sac develops directl}^ from a venous capillar}^ network adjacent to the junction of the early precardinal with the postcardinal vein to form the duct of Cuvier. It has also been pointed out by Huntington (14) that the jugular lymph sacs, regarded as of venous origin, constitute the connecting links between the hemal vascular system and the general system of lymphatic vessels.
The origin of the systemic h'mphatic vessels is a problem on which investigators are sharply divided. A summary of the different views, including the bibliography, can be found on pages 10-13 of Huntington's monograph in the Memoirs of The Wistar Institute (3).
^ References, by number, will be found on page 162.
As regards the particular case of the thoracic duct, Sala (10) in 1900 published the results of his work on the chick in which he holds that it develops by canalization of solid mesenchymal cords. In 1902 Sabin (11), after working with the injection method, published her account of the development of the lymphatic system in pig embryos. In this she reinforces and extends the view of Langer (12) and Ranvier (13), that lymphatics arise from veins by a process of sprouting and centrifugal growth, maintaining that the system as a whole is developed by blind ducts that 'bud off' from the veins of the cervical and inguinal regions, widen out to form sacs from which lymphatics grow to the skin, stating also that "at the same time a growth of ducts occurs along the dorsal line following the aorta to make a thoracic duct from which lymphatics grow to the various organs." The two views expressed by Sala and Sabin are thus diametrically opposed, the one being that the lymphatics arise in the mesenchyme independently of the veins, the other that the lymphatics are outgrowths from the veins.
In 1905 Lewis (6) expressed the view that in the rabbit the lymphatic system is derived directly from the embryonal veins, multiple detached portions of these becoming confluent to form the permanent systemic lymphatics, stating that the thoracic duct "arises from a plexus of lymphatics surrounding the aorta" (p. 109).
In 1907 Huntington and McClure (25), studying the development of lymphatic vessels in their relation to the veins in embryos of the cat, found that "the lymphatics begin as extra-intimal spaces along the course of the primitive embryonal veins. They* subsequently become confluent and form continuous vascular channels" (p. 42).
Huntington, in 1908 (14), while retaining this view of the genesis of the systemic lymphatic vessels, as distinguished from the jugular lymph sacs, defined the latter as the connecting links between the hemal vascular system and the general system of the lymphatic vessels, which "arise, not by transfonnation of veins, but by the formation of spaces lying outside the intimal lining of the veins, which spaces, becoming confluent, form the general lymphatic channels of the body" (p. 25).
In the same year (1908) McClure (21) abandoned the view previously held jointly by him and Huntington as regards the development of the thoracic and right lymphatic ducts in cat embryos, and states:
The anlages of the thoracic and right lymphatic ducts consist of a series of independent outgrowths which first appear along the common jugular and innominate and then along the azygos veins exactly in the line subsequently followed by these ducts; these outgrowths are subsequently split off from the veins, by a process of fenestration, in the form of a series of isolated, more or less spindle-shaped spaces which later become confluent with one another and with a process of the jugular lymph sac to form a continuous system disconnected from the veins, .... (p. 542).
In 1909 Sabin (7) reiterates her original view, based on the study of injected pig embryos, stating that the "presumption seems to lie on the side that the thoracic duct develops in the same manner as all other ducts," namely, "from endothelial sprouts from the sacs" (p. 58).
McClure in 1910 (23), after further studies of critical stages in lymphatic development, retracted his former view and stated :
The venous line .... along which the cat's thoracic duct develops is topographically replaced by the lymphatic channel, not directly, as assumed by me (in 1908), but secondarily by extra-intimal lymphatic space development, .... the 'extra-intimal theory,' as originally outlined by Huntington and myself (in 1906), establishes a fundamental principle of development for the main systemic lymph channels in mammals (p. 105).
Sabin, in a later article ('11) (22), states that "the thoracic duct develops in part as a down growth of the jugular sac and in part, especially its dilated portion or cisterna chyli, as a direct transformation of the branches of the azygos veins" (p. 424) . This expression of opinion seems to be a correction of her earlier statements and a partial adoption of Lewis's view. In place of her former concept of an uninterrupted centrifugal lymphatic growth from the sacs, she now appears to hold that in addition a portion of the thoracic duct develops as the result of direct transformation of azygos venous tributaries into lymphatics.
Huntington again in 1911 confirmed and elaborated his former view by extensive observations on reptiles (2) and the cat, stating specifically on page 13 of the first number of the Memoirs of The Wistar Institute (3) that "the entire extensive system of lymphatic vessels proper of the adult animal, including the thoracic and right l}miphatic ducts and their tributaries, is formed by the confluenceof the extravenous intercellular mesodermal spaces," and that "these spaces are lined by a lymphatic vascular endothelium which is Jiot derived from the hemal vascular endothelium, but develops independently of the same," and giving his summary and conclusions in remarkably clear terms on pages 153-171 of the same publication. He also points out that the systemic lymphatic development in the mammalian embryo is "by no means confined to the immediate environment of degenerating embryonic veins. The same field, which shows the above described histogenetic processes in the development of extra-intimal lymphatic spaces surrounding and replacing a decadent venule, will at the same time contain numerous equivalent lymphatic mesenchymal clefts and spaces which continue to develop independently of any association with retrograding veins" (p. 49).
Sabin in 1912 (24) still maintains that "the thoracic duct (in the pig) arises in part as a downgrowth from the left jugular sac and in part from a plexus of lymphatics which buds off from the veins of the Wolffian body" (p. 336).
Recently Kampmeier (15), after studying serial sections of both uninjected pig embryos and one of Sabin's injected specimens, concluded that
the actual genesis of the thoracic duct is initiated by the appearance of blind mesenchymal Ij^mphatic spaces either around or not immediately in contact with the venous derivatives, or veno-lymphatics, which become detached from their venous trunks and break up into degenerating segments .... During their inception and growth the walls of the discontinuous thoracic duct anlagen are composed of mesenchymal cells .... Injected specimens of the early lymphatic stages certify the reality of blind uninjectible anlagen beyond the farthest points to which the injecta have penetrated, demonstrating that discontinuities in a developing lymphatic channel are not 'appearances' found only by the study of uninjected embryos (pp. 463-464).
Stromsten (16) in his account of the development of the thoracic duct in turtles arrived at a like conclusion, stating that "the development of the peri-aortic lymphatic plexus in the loggerhead turtle is immediately preceded by the formation of isolated, independent spaces. They (the spaces) cannot be injected . . . ." (p. 354). "The intercellular spaces thus formed enlarge and fuse together to form lymphatic lacunae. At a later stage the lacunae acquire an endothelial lining and become the isolated anlagen of the thoracic duct" (p. 356).
As regards the thoracic duct in the chick, it^will be my object in this article (1) to demonstrate its origin by confluence of intercellular spaces in the mesenchyme, independent of the veins, and to reconsider the significance of the mesenchymal cords described by Sala in their relation to the developing lymphatics; (2) to discuss the establishment of the morphological drainage line of the thoracic duct and the means by which this duct and the jugular lymph sacs communicate; and (3) to show that the organization of the avian thoracic duct corresponds in type with that established in reptiles and mammals.
==II. Material==
Embryos of the domestic fowl (Gallus gallus) have been used chiefly on account of the certainty in procuring the critical stages. Some embryos of the English sparrow (Passer domesticus) have also been used. Of this material I have examined thirty-two individual embryos in serial sections, comprising twenty-seven chicks and five sparrows. Four of the chicks were injected with India ink through the umbilical vein (table 1).
Unfortunately the sparrow embryos, of which series nos. 123, 124, 126, 154 and 509 were examined, were not measured and could be judged as to stages of development only by comparison with the chicks.
The embryos were fixed in vom Rath's, Bouin's, or Zenker's fluid (some in Zenker-formol), Bouin's fluid giving the best results with the least shrinkage. The sections were cut in paraffin and stained on the slide by one or the other of the following methods. After fixation in vom Rath's mixture, the staining was
TABLE 1
CHICK EMBRYOS SERIES NO.
TIME OF INCUBATION IN HOURS
GREATEST LENGTH IN MM AFTER FIXATION
415
96
7.0
371
108
6.75
336
120
9.0
355
120
11.5
356
120
12.0
326
130
12.5
411
132
9.0
410
132
9.25
370
132
9.5
357
132
10.75
412
145
10.75
340
145
11.0
428
145
11.0
414
145
11.25
339
145
13.5
426
160
13.5
465
165
14.0
463
166
16.0
464
166
16.5
520
171
inj
ected
521
192
inj
ected
522
192
inj
ected
519
206
523
216
inj
ected
320
230
524
240
483
260
done with a much diluted Delafield's hematoxylin, followed by a weak solution of picric acid in alcohol. After all the other fixatives the sections were overstained in Weigert's hematoxylin, decolorized in water acidulated with HCl, and counterstained in a weak solution of Orange G in distilled water! The blood cells' are clearly differentiated by either method; the cytoplasm of those containing even a trace of hemoglobin shows some tinge of yellow. Developing muscle tissue and nerve fibers also are yellow. Other elements are stained by the hematoxylin, the delicate processes of the irregular mesenchymal cells showing especially well.
==III. Histogenesis==
Sala, in his account of the development of the thoracic duct in the chick (10), states that the anlagen of this lymph channel appear as isolated mesenchymal spaces which become clothed with endothelial cells derived from the mesenchyme, and which subsequently coalesce to form continuous vessels. He thus implies a denial that the lymphatics in this region arise from veins and that the lymphatic endothelium is derived from hemal vascular endothelium. He also calls attention to accumulations of mesenchymal cells which contrast clearly with the surrounding tissue. They appear to consist in large part of elements exhibiting all the characteristics of young connective tissue cells, with roundish forms, no processes and large intensely colorable nuclei. Among these elements appear red corpuscles in larger or smaller numbers. The accumulations or clumps of cells develop first on the mesial aspect of the superior vena cava, and then extend caudad to the level of the celiac artery. Without describing the histogenesis of these masses of cells, Sala states further that within them in large part the anlagen of the thoracic duct are excavated or 'hollowed out.' The details of the 'hollowing' process are not given.
In the main I can confirm the results of Sala's observations so far as he has carried them. As for the isolated mesenchymal spaces which he describes as the anlagen of the thoracic duct my study of their histogenesis in the closely graded series of chick embryos leads to the conclusion that they do arise independently of the veins and that their endothelial lining is derived from the indifferent mesenchymal cells bordering upon them. The details of the process I shall attempt to demonstrate in the following pages. After tracing the development of the accumulations of cells in the mesenchyme and their subsequent history in relation to the developing thoracic duct, it seems to the writer that a different and greater significance can be attached to them than was given by Sala. Since the cells composing them correspond so closely in their history to the developing blood cells as described by Dantschakoff (17) in the extraembryonic area of the chick, undoubtedly they should be considered as collections of intraembryonic developing blood cells.
These clumps of cells, as noted by Sala, develop in the vicinity of the aortic arches, especially the sixth, and along the dorsal aortic roots and the aorta as far as the exit of the superior mesenteric artery. The early stages in the formation can be clearly seen in embryos of 100 to 110 hours.
Some of the stellate elements of the mesenchymal reticulum (syncytium) become differentiated from their neighbors. Their processes are retracted and separated from the general reticulum, the cells thus becoming rounded (figs. 1 and 2). The cytoplasm acquires a more strongly basophilic character, increases in amount and becomes more homogeneous than that of the true mesenchyme. The nuclei contain a relatively small amount of chromatin and one or two, usually two, distinct nucleoli. Mitotic figures are occasionally seen (fig. 2). While the cell contours are generally regular, there are sufficient irregularities to denote an ameboid character. These cells appear to be identical with the large mononuclear cells which Dantschakoff describes as differentiating from the blood islands in the area vasculosa of the blastoderm and later in the capillaries of the yolk sac, and which she calls lymphocytes.
The cells here under consideration increase in number not only by their own proliferation but also by continued differentiation from the mesenchymal syncytium (fig. 2). For the most part they lie in compact groups, which gradually increase in size as the cells increase in number, but some cells usually appear in the vicinity of the groups (figs. 4, 5, 7, 8 and 10, 16) and not infrequently at some distance from them. After their differentif,tion and separation from the mesenchymal syncytium all the cells, both members of the groups and isolated, lie free in the spaces among the stellate elements of the mesenchymal tissue (figs. 3 and 4). No blood vessels or lymphatics are present in the immediate vicinity of the cell groups when the latter first develop and consequently the cells are extravascular (figs, 1, 2, 3 and 4). It will be shown later that after the lymphatics develop in this region the cells for the most part are included within them and thus become intravascular elements.
The application of the term 'blood islands' to the accumulations of cells has been avoided because there is no real condensation of the mesenchyme whereby the differentiating elements fuse to form a solid protoplasmic mass. Each stellate element differentiates individually (figs. 1 and 2) and condensation occurs only as a subsequent aggregation of the differentiated cells. The resulting large mononuclear cells, however, so closely correspond to the first lymphocytes that develop in the area vasculosa of the blastoderm, the subsequent histories of the cells arising in the two localities being identical, the conclusion is warranted that the differentiated mesenchymal elements in question serve as an intraembryonic source of blood cells.
While the present investigation has not been of such a nature as to determine whether the mononuclear basophilic cells which have been described give rise to granular leucocytes, they certainly give rise to red blood cells.. The cytoplasm of some of the cells gradually loses its basophilic character and acquires a stronger affinity for the plasma dyes, at the same time becoming quite homogeneous. These changes are concomitant with the addition of hemoglobin. The nuclear changes comprise an increase in the amount of chromatin, its arrangement into a rather heavy reticulum and the disappearance of the nucleoli (fig. 2).
Cells thus modified and containing a moderate amount of hemoglobin, as indicated by the reaction to plasma stains, are erythrobalsts. Without further visible structural changes these acquire more hemoglobin until eventually they are indistinguishable from the red blood cells in the general circulation. They have therefore become erythrocytes. It is probable also that they are definitive rather than the larger primitive erythrocytes described by Dantschakoff. The changes described above occur both in the cells composing the groups and in the isolated cells, sd that fully developed red blood cells as well as the earlier de\'elopmental stages are seen not only in the groups but also in the mesenchymal spaces more or less remote from the groups.
Among the developing erythi'oblasts, especially in the later stages, are many small cells which apparently go through the same processes of differentiation as the red blood cells themselves and resemble the latter in every respect except size. These are undoubtedly microcytes, the earlier stages being microblasts.
After the middle of the fifth day the masses of developing blood cells increase rapidly in size by proliferation of the component cells and continued differentiation of the branching elements of the mesenchymal syncytium. They increase in number also by the same processes of differentiation in other localities. Up to about the beginning of the sixth day all the developing blood cells in question remain extravascular, that is, free in the intercellular mesenchymal spaces which are not lined by endothelium.
By the end of the seventh day the aggregations of cells reach the height of their development. At this time they extend in two main lines from the level of the aortic arches along the dorsal aortic roots to the confluence of the latter to form the aorta (figs. 6, 20 and 22, 16). At this level, or a little further caudal, the two main lines unite to form a single line which extends about to the level of the superior mesenteric artery (figs. 7, 8, 10, 20 and 22, 16). At their greatest development the larger groups together form an almost continuous mass of the cells which in places is greater in diameter than the aorta. Usually there are also numerous smaller outlying groups which belong to the same general line (figs. 7 and 8).
While the main lines above described are established in all the embryos examined, yet there is a wide range of variability in the form of the groups and their arrangement in the lines. In most of the embryos examined a large mass of cells or a collection of smaller groups had developed in the region dorsal to the aortic roots and esophagus (figs. 6 and 22, 16a). One of the most interesting and one of the most important features of the masses of developing blood cells is the fact that the main lines in their general arrangement correspond with the lines of the thoracic duct (figs. 20, 22 and 24, 16, 17 ,17a).
To anticipate, it may be stated here that as the multiple anlagen of the thoracic duct develop the masses of developing blood cells are in a large part included within them, and thus become strictly intravascular (see fig. 10, 16, 17). The details of this process will be considered in the subsequent description of the formation of the lymphatic spaces and channels. After the seventh day, as the lymphatic channels in this region develop and establish communication with the jugular lymph sacs, the intravascular masses of blood cells decrease and, by about the eleventh da}^ are reduced to a few groups scattered through the lymphatic plexus (cf. figs. 24, 27 and 28). A considerable number of extra vascular groups persist, however, until the fourteenth or fifteenth day, or even later. In the later stages the vast majority of the cells in these groups are practically mature erythrocytes lyiiig free in the mesenchymal spaces in the vicinity of the lymphatics (fig. 11).
Inasmuch as the reduction in the masses of blood cells in the lymphatics follows the coalescence of the lymphatic spaces to form continuous channels and the establishment of communication between the latter and the jugular lymph sacs, and since the lymph sacs open into the great veins, the blood cells in question eventually reach the general circulation by way of the thoracic duct and jugular lymph sacs. The thoracic duct at one period acts, therefore, as conveyor of the erythrocyte series of hemal cellular elements which have developed from the indifferent mesenchyme along the line of the duct.
A consideration of the histogenesis of the lymph channels constituting the anlagen of the thoracic duct leads on to controversial ground. As stated earlier in this paper, the controversy hinges upon the question whether the lymphatics, exclusive of the lymph sacs or hearts, arise as sprouts or outgrowths from pre-existing vascular channels or de novo from the intercellular mesenchymal spaces.
It is the opinion of the writer that in the chick the lymph channels which constitute the anlagen of the thoracic duct arise through enlargement and coalescence of intercellular spaces in the mesenchymal tissue, and that the endothelial lining of these channels is derived directly from indifferent mesenchymal cells^ that chance to border upon the spaces. In the material studied there is no evidence of any growing out, budding or sprouting from the endothelium of pre-existing blood vessels, the tissue in which the thoracic duct develops being non-vascular. The lymphatics in question, and their endothelial lining, arise independently of pre-existing vascular channels.
* It should be understood that when 'mesenchymal cells' are spoken of, they are considered as the irregularly stellate masses of protoplasm the processes of which anastomose to form the mesenchymal syncytium, or reticulum.
Prior to the appearance of any specialized spaces or channels in the mesenchyme in the region of the future thoracic duct, the mesenchymal syncytium consists of irregularly stellate protoplasmic elements the slender processes of which anastomose freely with like processes of neighboring elements. The cytoplasm is finely granular and the nuclei, while relatively large and vesicular, contain little chromatin and one or two distinct nucleoli. Among the protoplasmic components of this tissue are the correspondingly irregular interstices or spaces which also are continuous with one another. These are called the mesenchymal intercellular spaces. The tissue as a whole might be compared with a sponge, the anastomosing protoplasmic parts representing the parenchyma of the sponge and the intercellular spaces the pores. While some of the protoplasmic elements during this time differentiate into blood cells, as previously described, our conception of the general syncytium and its spaces is in no way invalidated.
The first changes in the mesenchyme leading toward the formation of definite channels occur during the latter half of the sixth day of incubation. These changes, instead of involving the mesenchyme generally, begin in several localities. In one of the localities, for example, the intercellular spaces increase in size and coalesce, most of the protoplasmic elements of the syncytium being pushed farther apart or broken. In this manner a considerably larger space is formed out of a number of the original smaller mesenchymal spaces (fig. 9, 17). For the most part the smaller spaces of the surrounding tissue open freely into the larger space, although in places along the edge of the latter the protoplasmic elements lose some of their stellate character and become flattened on the side toward the larger space.
The phenomena in general indicate the accumulation of the fluid filling the mesenchymal spaces, the cells and their processes being subjected to pressure and friction incident to the flow of the interstitial substance. Thus there arises in the mesenchyme a space larger than the original interstitial spaces but derived directly from them by their enlargement and coalescence (cf. figs. 12, 13 and 14). So far as there is any definite lining for the new space, it is formed by the partiall}^ or wholly flattened mesenchymal cells upon which the fluid in the space impinges (fig. 13).
The further changes in one of these larger spaces consists in the main of its elongation through the enlargement and addition to it of other intercellular mesench3aiial spaces, a progressive flattening of the cells along its sides, and an approximation of the edges of the flattened cells to form a definite lining of endothelium. There is thus formed a distinct channel in the mesenchyme. For the most part it is lined and its lumen is separated from the surrounding interstitial mesenchymal spaces by a layer of endothelial cells which represent metamorphosed stellate elements of the mesenchyme (figs, 15 and 16). At or near its ends the channel opens freely into the adjoining mesenchymal tissue spaces which in the further course of development are added to the lumen of the channel and thus become a part of it (figs. 15, 16, 17 and 18). The endothelial lining already formed merges near the ends of the channel with the mesenchymal syncytium which in turn, as the channel elongates, gives rise to more endothelium by differentiation of certain of its elements (figs. 15, 16, 17 and 18).
As stated in a previous paragraph, the larger spaces appear in several localities in the mesenchyme. Consequently the channels resulting from their further development are for a time isolated; that is, they are not directly connected with one another or with the jugular lymph sacs or any part of the hemal vascular system. These isolated spaces and channels constitute the multiple anlagen of the thoracic duct (fig. 21),
In succeeding stages each of the channels in question increases in size, especially in length, until it meets and coalesces with its neighbors. The increase in length is due in the main to the addition of more mesenchymal tissue spaces to its ends and the concomitant transformation of more stellate mesenchymal cells into endothelial cells. There is probably also some proliferation of the endothelial cells, although in the study of the sections mitotic figures were not seen.
The coalescence of the originally unconnected lymphatics results in a network or plexus of channels (cf. figs. 21, 22 and 24). This is constantly being augmented by the coalescence of other independently formed spaces and channels with one another and with the previouly established plexus. Most of the vessels composing the plexus lie longitudinally in the embryo along the line of the aorta and dorsal aortic roots, and out of this plexus is e\'entually crystallized the main drainage lines of the thoracic duct. The establishment of these lines is best considered, however, in the section on morphogenesis.
The correlation of groups of developing blood cells in this region, the formation of which has already been described, and the lymph spaces and channels constituting the anlagen of the thoracic duct now remains to be discussed. As stated earlier in this paper, the blood cells that are differentiated from the mesenchymal syncytium, whether they are arranged in groups or isolated, lie free in the tissue spaces. In case the tissue spaces enlarge and coalesce in the region where the blood cells are situated the latter are then allowed to become free also in the larger space resulting from the enlargement and coalescence. The larger space become^ lined with endothelium, in the manner previously described, to form a definite vessel or channel. The blood cells, therefore, which were originally free in the tissue spaces are included in an}" of the lymphatics developing in that particular locality (fig. 10, 16, 17).
In that manner some of the blood cells become intravascular elements during the earliest stages of lymphatic development. Occasionally an entire group of cells is included in a lymph channel; in other cases only part of a group or a few scattered cells. It is true also that a great many lymphatics develop in the mesenchyme quite apart from the blood cells (figs. 15, 16 and 17).
In the earlier stages of lymph vessel formation there are great numbers of blood cells, in various degrees of differentiation, in the mesenchymal tissue spaces in the vicinity of or more or less remote from the lymph spaces and channels. In part at least these cells become intravascular when new lymphatics are formed out of the tissue spaces in which they lie and join the general plexus of previously formed vessels.
Thus far, therefore, the admission of the blood cells to the lymph vessels depends merely upon the topographical relationship in the development of the two sets of structures. There are, however, other factors which in all probability enter into this process. Two are of especial interest and importance in the case under consideration.
It has been pointed out by Dantschakoff that the primitive blood cells in the area vasculosa of the blastoderm and the large mononuclear cells derived from them are capable of ameboid movement. The developing blood cells in the region of the thoracic duct anlagen are demonstrably of the same type as those in the area vasculosa. Hence it is reasonable to conclude that some of the developing blood cells pass from the tissue spaces into the vessels by virtue of their ameboid character.
The other factor has hitherto, so far as the writer is aware, been considered only in the study of living tissues. It seems justifiable, however, to extend the conclusions drawn therefrom to fixed tissues. In their study of chick blastoderms in vitro, McWhorter and Whipple (18) have observed the to and fro movement, synchronous with the heart-beat, of blood cells not only in the isolated, endothelium-lined spaces which eventually coalesce to form blood vessels but also in the tissue spaces. Furthermore, they have observed the entrance of blood cells into the general circulation following then- to and fro movements in the tissue spaces. These phenomena certify the pulsation of the fluid substance in the tissue spaces in response to the heart-beat. It is not uiu-easonable, therefore, to conclude that some of the blood cells in the region of the developing thoracic duct are driven or sucked into the lymph spaces or channels which, as pointed before, open freely into the mesenchymal tissue spaces.
As a corollary to the phenomena mentioned in the preceding paragraph, an additional factor in the formation of endothelium might be suggested. Granting that the blood cells lying free in the tissue spaces move to and fro in response to the heart-beat, as has been clearly observed in the living chick blastoderm, the assumption is justifiable that their movement, with the resultant friction and pressure upon the adjacent protoplasmic elements of mesenchymal tissue, assists in the flattening of these elements and the consequent formation of endothelial cells.
The hydrodynamic factors of pressure and friction of the interstitial fluid substance upon the protoplasmic elements of the mesenchyme, which were first discussed by Thoma (19) in connection with blood vessel formation and have already been noted in this particular case of development of lymphatics, together with the additional factor of pressure and friction of oscillating blood cells would, in the opinion of the writer, afford adequate mechanical means of changing the irregular plastic elements of the mesenchymal syncytium into the endothelial cells of the vessel wall.
==IV. Morphogenesis==
Up to this point we have been considering the histogenetic changes which occur in the mesenchj^mal tissue, resulting in the formation of lymph channels and their endothelial lining and of blood cells. It has been demonstrated that aggregations of the developing blood cells, identical with the mesenchymal 'cords' described by Sala, appear along the lines of the future thoracic duct. It has been shown also that the rudiments of the thoracic duct develop as isolated spaces and channels in the mesenchyme, that the endothelial lining of the channels is derived directl}^ from the mesenchymal cells forming their borders and that the channels coalesce to form a plexus. It is our object, under the head of morphogenesis, to trace the subsequent history of the isolated channels and the plexus formed therefrom.
Chick embryo of six days and sixteen hours, 13.5 mm. {Columbia Embryological Collection, series no. 4^6). Reconstruction, ventral view. Figure 20. The incipient stages of thoracic duct development are shown about this time. The lymph spaces {17) are three in number, two on the right side and one on the left, situated in the mesenchyme ventro-lateral to the aorta {I) about midway between the exits of the celiac (-5) and superior mesenteric arteries. They are isolated, for absolutely not any divect connection with any other vessel can be traced even with high powers of magnification. They open freely into the surrounding intercellular spaces, as show in figures 12, 13 and 14, which are photographic reproductions of three successive sections of the embryo.
The spaces fit the previous description, given in the section on histogenesis, of accumulations of an intercellular fluid in the mesenchyme. The objection that they may be shrinkage spaces is nullified by the fact that the preservation of the tissue is practically perfect, and that in this and in other stages the more nearly perfect the preservation the more clearly defined are the spaces. Furthermore, in cases of poorer preservation where there are obvious shrinkage spaces in the mesenchyme the boundaries of such spaces are almost invariably ragged and irregular and do not anywhere exhibit a smooth endothelial lining.
The masses of developing blood cells in this particular embryo (six days and sixteen hours) are perhaps unusually extensive (fig. 20, 16). They extend in irregular groups from about the level of the superior mesenteric artery forward along the ventral and ventro-lateral aspect of the aorta (1) to the level of the celiac artery (5), with a tendency to cluster around the last named vessel (cf. fig. 7, 16). They then divide into two general lines, one on each side, which bend laterad and extend forward along the mesial aspect of the ducts of Cuvier (12) and precardial veins (10), ending rather abruptly in a large mass which hes at the level of the sixth aortic arches (3) and extends across the mesial line ventral to the dorsal aortic roots (2).
The three spaces representing the first anlagen of the thoracic duct (17) bear no particular relation to the large groups of developing blood cells (16), although a few isolated blood cells lie in the tissue spaces around the larger rudimentary lymph spaces.
Chick embryo of six days and twenty-one hours, llf. 7nm. (Columbia Collection, series no. ^65). Reconstruction, dextro-ventral view. Figure 21. In this embryo there is a considerable increase in the size and number of lymph spaces and channels which constitute the early anlagen of the thoracic duct (17). They are situated for the most part in the mesenchymal tissue ventral and ventrolateral to the aorta (i) near the exit of the celiac artery (5). Other spaces and channels of a similar nature are seen along the dorsal aortic root (2), thus continuing the same general line of lymphatics toward the jugular lymph sac (15) with its thoracic duct 'approach' {15a). Some of these early lymphatics are of considerable length and have acquired a distinct endothelial lining. Others are simply enlarged spaces in the mesenchyme exactly like those described in the preceding stage (figs. 13 and 14). There is not yet any plexiform arrangement of the channels.
The principle underlying the formation of the longer channels is in the main the enlargement and coalescence of two or more of the original smaller spaces or channels. For instance, two isolated spaces or channels lying near each other in the mesenchyme increase in size until eventually they flow together to form a single space or channel. Or, the same process occurs in several spaces or channels in a discontinuous series until all the members of the series flow together and thus form a continuous channel.
There is also in all probability some proliferation of the endothelium lining the channels. In fact there is no valid reason for not assuming that, after the inception of a given vascular channel, either hemal or lymphatic, along with its increase in size there is a concomitant proliferation of its endothelial cells. The crucial point, however, is the origin of the channels. The evidence at hand points clearly to the origin of the lymphatics constituting the rudiments of the thoracic duct directly from the mesenchymal interstitial spaces in the manner previously described, the endothelium of the lymphatics representing mesenchymal cells which are modified in accordance with the new conditions of pressure and friction.
In this embryo (series no. 465) the rudiments of the thoracic duct (fig. 21, 17) are isolated. There is no connection between these and the hemal vascular system; nor on the other hand is there any communication with the jugular lymph sacs (15). The thoracic duct 'approach' of the lymph sac, which the duct eventually joins, is well developed at this stage (15a) but a considerable distance intervenes between the 'approach' and the rudimentary duct.
The masses of developing blood cells in this embryo are unusually scarce. A few small groups (16) are situated in the mesenchymal tissue ventral to the aorta (1), and three larger groups (also marked 16) are seen between the dorsal aortic root (2) and the jugular vein (10).
Chick embryo of six days and twenty-two hours, 16 mm. (Columbia Collection, series no. 463). Reconstruction, ventral view. Figure 22. In this embryo the lymphatics (17) are in approximately the same stage of development as in the preceding embryo. They are situated for the most part along the ventral aspect of the aorta (1). A few spaces are situated ventro-lateral to the dorsal aortic roots (2).
The masses of developing blood cells (16) are much more extensive than in the preceding embryos of about the same stage, thus exhibiting the variability of the structures. The groups associated with the rudimentary thoracic duct (17) lie ventrolateral to the aorta (1), with some tendency to cluster around the celiac artery (5) . The rest of the groups extend in a continuous mass on each side along the ventro-lateral aspect of the dorsal aortic roots (2) and along the mesial aspect of the jugular veins (10) (cf. fig. 6, 16) as far forward as the fourth aortic arch (4)A large aggregation of developing blood cells (16a) also lies between and somewhat dorsal to the aortic roots (2) and the arches (3, 4) (cf. fig. 6, 16a), being connected with the left lateral groups by cords extending ventral and dorsal to the roots and arches. Associated with this large mass are a few lymphatics (19) (cf. fig. 6, 19) which subsequently join the thoracic duct and may also communicate with the cephalic end of the jugular lymph sac.
One of the most interesting features of this particular stage is the well developed thoracic duct ^approach' of the jugular lymph sac (fig. 22, 15a). A reconstruction to show this structure was made on a larger scale and is illustrated in figure 23. The lymph sac iself (13) lies dorsal to the jugular vein (10) and fits into the angle between the latter and the subclavian vein (11). The thoracic duct 'approach' (15a) is situated on the mesial aspect of the j ugular-subclavian angle and thence extends a short distance caudad along the mesial side of the precaval vein. It lies for the most part between a mass of developing blood cells {16) and the vein [10) and ends blindly (see also fig. 19, 16, 15a). A short distance farther caudad are two spaces in the mesenchyme which represent the extreme cephalic end of the rudimentary thoracic duct (fig. 23, 17). These spaces are isolated, for with high magnification there cannot be discerned any connection between them and the thoracic duct 'approach' {15a) or the other rudiments of the thoracic duct lying still farther caudad.
The thoracic duct 'approach' of the jugular lymph sac in the chick is without doubt the homologue of a similar structure" described by Huntington and McClure (9) in the cat. Like the lymph sac itself, it is of venous origin in the chick as in the mammal, and forms an integral part of the sac. It arises from some of the more mesially and caudally situated components of the early venous plexus in the region of the jugulo-subclavian angle. When fully formed it extends caudad and mesad for some distance along the mesial aspect of the precaval vein, and terminates blindly (figs. 19 and 23, 15a). Subsequently when it is joined by the thoracic duct, it serves as the portal of entry of this duct into the lymph sac.
Chick embryo of seven days {Columbia Collection, series no. 512). Reconstruction, ventral view in figure 24-; view from left side, figure 25. In this embryo there is considerable advance in the development of the rudiments of the thoracic duct. The spaces and channels, which in the preceding stage were unconnected, have here coalesced to form an extensive plexus {17) of channels extending from the level of the junction of the dorsal aortic roots {2) nearly to the exit of the superior mesenteric artery. The plexus lies for the most part ventral to the aorta {!), but a few of its components extend around on the lateral aspect of this vessel. Most of the channels in the network are much larger than the original spaces and channels, leaving but small areas of mesenchymal tissue among them; in part they have even fused to form irregular sinuses. The celiac artery (5) in most of its longitudinal course penetrates the plexus {17). The plexus as a whole is still isolated, being connected neither with any part of the hemal vascular sj^stem nor with the thoracic duct 'approach' of the jugular lymph sac {15a).
Along the dorso-lateral aspect of the aorta (1) a number of spaces and channels have also developed (fig. 25, 20). In addition, a few smaller lymphatics have appeared along the lateral aspect of the aorta. The dorsal aorta is thus almost completely encircled by a group of spaces and channels comprising the large ventral lymphatic plexus described in the preceding paragraph and the dorsal and lateral sets of lymphatics.
These encircling lymphatics are in the aggregate homologous with the dorsal set of peri-aortic sinuses in reptiles, as described by Huntington (2). They are also homologous, in all probability, with the azygos portion of the thoracic duct in the cat, as described by Huntington (3), and in Tragulus, as described by Tilney (20). A comparison with Stromsten's (16) description and figures indicates, too, that the lymphatics associated topographically with the dorsal aorta in the chick are collectively the homologue of the peri-aortic network of h'mph vessels in the loggerhead turtle, or, more specifically, with that portion of the network surrounding the dorsal aorta. In addition, the lymphatics around the dorsal aorta in the chick, as previously described, may be placed in the same phylogenetic line as the postcardinal and supracardinal divisions of the thoracic duct in the pig, as recently worked out by Kampmeier (15).
Returning to a further consideration of conditions in the chick at this stage (seven days, fig. 24), it is seen that a few small Ijnmph spaces {17a), also isolated, have appeared along the ventrolateral aspect of the dorsal aortic roots (2) in the interval between the plexus previously described {17) and the thoracic duct 'approach' of the jugular l}Tnph sac {15a). These small spaces represent the beginning of the connection between the thoracic duct 'approach,' on each side, and the unpaired portion of the thoracic duct itself, here composed of the large plexus {17).
The conditions in the chick thus correspond so closely to those in other forms that it is possible to draw a clear homology between the lymphatics joining the thoracic duct 'approach' to the unpaired portion of the duct itself in the chick and the preazygos segment of the thoracic duct in the cat (Huntington) and Tragulus (Tilney), with the cephalic portion of the peri-aortic lymph plexus in the loggerhead turtle (Stromsten), and with the precardinal division of the thoracic duct in the pig (Kampmeier).
In the mesenchyme between and dorsal to the aortic roots and arches in this embryo (fig. 24) a number of lymph spaces have developed. Some of these lie within the masses of developing blood cells in this region {16a) while others are situated at some distance from them. A few have coalesced to form a distinct channel (19). The subsequent history of these lymphatics will be given in the discussion of later stages.
A few isolated lymph spaces are also found in and near the root of the dorsal mesentery at the level where the celiac artery enters the mesentery (figs. 24 and 25, 21). These belong to the category of mesenteric lymphatics, but do not yet communicate with the developing thoracic duct.
The masses of developing blood cells in this embryo are extensive (figs. 24 and 25, 16, 16a). Those associated with the main portion of the developing thoracic duct lie either in the meshes of the lymph plexus (17) or are already included within the vessels composing the plexus. A plate-like mass extends over the mesial aspect of the pleural cavity on the right side. The blood cells still, as in preceding stages, tend to cluster about the celiac artery (5). There is no connection in this stage between the masses just mentioned and those which lie farther forward along the mesial aspect of the great veins (10, 12), and which are associated with the thoracic duct 'approach' of the jugular lymph sac (15a). In the region between and dorsal to the dorsal aortic roots (2) and aortic arches (3) there is seen a large mass (16a) with which certain lymphatics (19), previously referred to, are associated. Situated farther caudad are also several masses associated with the lymphatics which lie along the dorso-lateral aspect of the aorta (fig. 25, 16a).
A feature shown in this reconstruction, and not in any other, is a portion of the splanchnic plexus of veins (figs. 24 and 25, IJf).^
' Splanchnic venous plexus is the name given by Dr. A. J. Brown, in his yet unpublished work on the development of the pulmonary veins, to the network of venous capillaries in the wall of the alimentary tube in the earlier stages of development.
This is seen near the cephalic end of the large ventral lymph plexus {17) but has no connection with the latter.
At this point it may be well to state that in the chicks of the Columbia Collection I have found no evidence of extra-intimal or perivenous origin of the lymphatics that make up the thoracic duct, such as described by Huntington in the cat and Kampmeier in the pig. This mode of development appears to be a strictly mammalian specialization. In fact Huntington (3), speaking of the extra-intimal replacement of veins by developing lymphatics, states (p. 155) :
The association of these (Ij^mphatic) channels, in the mammalian embryo, with certain embryonal venous lines is purely a secondarj^ mechanical and topographical relationship, expressed by the condensed term of 'extra-intimal' development of mammalian systemic lymphatic vessels, and absolutely devoid of genetic significance. This is, without reference to other vertebrate classes, proved by the development within restricted areas in the mammalian embryo of systemic lymphatic channels through the direct confluence of intercellular mesenchymal clefts, not related topographically or in any other sense to the embryonal veins. It is true that in the mammal this independent lymphatic genesis is extremely limited, and that the majority of the lymphatic vessels develop in close association with embryonal veins, as products of the confluence of perivenous extra-intimal spaces. But this is merely, as shown by comparison with other amniote embryos, the expression of the peculiar relations obtaining in the mammal between the venous and lymphatic circuits of the vascular system, developed independently of each other.
Further, Huntington (2), describing the independently formed system lymphatic channels of the reptilian embryo, by confluence of intercellular mesenchymal spaces, states concerning the latter:
They are not complicated by close topographical relations to adjacent temporary embryonic venous plexuses, as in the mammal, but develop independently by themselves in mesenchymal territory not occupied by hemal vascular elements In both lacertilian and chelonian embryos the greater part of the enormously enlarged systemic lymphatic channels develop without any reference whatever to embryonic veins, in mesenchymal areas where the latter are extremely scanty or entirely wanting (pp. 271-273).
Stromsten (16) has reached similar conclusions in studying the development of the prevertebral peri-aortic sinuses in chelonian embryos.
These finding in reptiles, and my own results in the bird, warrant the conclusion that the sauropsids agree absolutely genetically with the mammals in the development of the main axial lymphatic lines bj^ confluence of independently formed intercellular mesenchymal spaces, but that the latter are characterized by close topographical association of these spaces with temporary embryonic veins which they in large part replace, while the former present no such association. In them the progress of thoracic duct development is not, as is the case in the mammal, complicated by the presence of an extensive azygos venous system, and axial lymphatic development, especially in the reptile, occurs chiefly by confluence of mesenchymal spaces surrounding the main arterial trunks.
Chick embryo of eight days {Columbia Collection, series no. 513). Reconstruction, ventral view. Figure 26. The next important change in the lymphatics constituting the thoracic duct comprises the further development of certain isolated spaces situated along the ventro-lateral aspect of the dorsal aortic roots in the interval between the cephalic end of the large ventral plexus and the thoracic duct 'approach' of the jugular lymph sac. The incipient stage in the formation of these spaces was illustrated in flgure 24, 17a. In the embryo now under consideration they have enlarged and coalesced to form a continuous channel (figure 26, 17a). This in turn has united with the ventral unpaired portion of the thoracic duct {17) and with the thoracic duct 'approach' of the lymph sac {15a). There is thus established a direct and free communication between the ventral lymph plexus {17), which had arisen as an independent and isolated structure, and the jugular lymph sac {15). Therefore, as clearly shown in figure 26, the thoracic duct 'approach' {15a), which was previously described as an integral part of the jugular Ijanph sac (see fig. 23, 15a), serves as the portal of entry of the thoracic duct into the lymph sac (fig. 26, 15).
The writer has in a previous article (1) shown and will here again point out in a later stage that in the chick, as in reptiles and mammals, a communication is established between the jugular lymph sac and the great veins in this region through one or more taps. And inasmuch as the thoracic duct opens into the lymph sac, the latter serves as the portal of entry of the systemic lymphatics into the venous system, a point upon which emphasis has already been laid by Huntington (2) (3) in his work on reptiles and the cat.
In the embryo of eight daj's the large ventral lymph plexus of the preceding stage (cf. fig. 24, 17) has undergone further coalescence of its component channels to form a large irregular sinus with a few fenestrae (fig. 26, 17). The sinus lies ventral to the aorta (1) and extends from the junction of the dorsal aortic roots {2) to the level of the exit of the superior mesenteric artery {9). At its cephalic end it branches off into the two slender trunks, one on each side, which extend cephalad and laterad to join the 'approaches' {15a) of the lymph sacs {15). These trunks, which are the last components of the Ijrmphatic drainage line to develop, constitute in the bird, according to the anatomical terminology, the right and left thoracic ducts.
The lymphatics (19) situated between and dorsal to the aortic roots {2) and arches {3, Jf) are here seen to comprise a plexus and a few outlying isolated spaces and channels (cf. fig. 24 and 26, 19). This plexus now communicates with the chain of lymphatics {20) lying dorso-lateral to the aorta ( / ) , which in* turn communicate with the large ventral plexus through channels formed by coalescence of spaces lateral to the aorta (cf. fig. 25, 20, 17). The entire group of lymphatics in the region of the dorsal aortic roots and the dorsal aorta as far back as the superior mesenteric artery, with the exception of the mesenteric lymphatics (fig. 26, 21), which have not yet joined the thoracic duct, drain into the jugular lymph sacs.
The masses of developing blood cells were omitted from the reconstruction of the eight day stage (fig. 26). A careful study of the serial sections showed that they were fewer and smaller than in the preceding stage. This may be due to the variability characteristic of the masses, or it may be due to actual reduction of the masses since the blood cells, as stated in in the section on histogenesis, now have access to the jugular lymph sacs through the recent connection established between these structures and the chain of lymphatics with which the blood cells have for the most part been associated.
Chick embryo of nine days and fourteen hours {Columbia Collection, series no. 320). Reconstruction, ventral view and viewfrorn left side. Figures 27 and 28. At this stage the main features of the adult thoracic duct have been established. The large plexus {17) ventral to the aorta {1) now drains through the right and left thoracic ducts {17a) into the 'approaches' {15a) of the jugular lymph sacs {15) and then through the sacs into the great veins {10, 12).
The ventral plexus {17), the isolated anlagen of which were the first lympathics to develop in the thoracic duct line, is relatively smaller and the component channels less dilated than in the preceding stages. This is probably due to the outflow of the contents into the lymph sacs and veins through the more recently formed channels which have been called the right and left thoracic ducts {1 7a) . The mesenteric lymphatics {21 ) have increased in size and for the most part coalesced to form sinus-like channels. Between these and the thoracic duct chain no connection can be detected at this stage, although little tissue intervenes. Farther cephalad another isolated group of lymphatics {22) is associated with the esophagus.
The right and left thoracic ducts {17a) are longer than in the preceding stage (cf. fig. 26, 17a). The left is considerably greater in diameter than the right. Each opens into the corresponding 'approach' {15a) of the jugular lymph sac. The 'approach' on each side is still patent as a branch of the main portion of the sac.
There is now free communication between the lymph sacs {15) and the great veins through recently formed taps, of which two are present on the left side of the embryo and one on the right. Of the two on the left side, one is situated on the dorsomesial side of the superior vena cava {12) just below the level of the jugulo-subclavian junction {10, 11); the other is situated farther forward on the mesial side of the jugular vein {10). The tap on the right side of the embryo is located on the dorso-mesial side of the superior vena cava at the level of the jugulo-subclavian junction.
In a previous article on the development of the jugular lymph sac in birds (1) the writer stated, in the description of tap formation, that "it is not improbable that a study of later stages will reveal a homologue of the common jugular tap in the mammal" (loc. cit,, p. 486). There is little doubt that the tap on the mesial side of the left jugular vein, referred to in the preceding paragraph, fulfils the requirement. Moreover, the other tap on each side near the jugular-subclavian junction in the chick is in all probability homologous with the jugulo-subclavian tap in the mammal (26), although it is slightly different in position.
The previously described lymphatics situated dorsal to the aortic roots and arches and dorso-lateral to the aorta here constitute a long chain of continuous channels reaching from the level of the jugular lymph sac to the level of the celiac artery (figs. 27 and 28, 19, 20; cf. figs. 24, 25 and 26, 19, 20). At the extreme cephalic end one of the channels of this series opens into the right lymph sac. The probable significance of this opening will be considered in the subsequent discussion of the masses of developing blood cells. The plexus [19) in the region of the aortic arches is considerably reduced as compared with preceding stages. The portion of the dorsal plexus {20) associated with the dorsal aorta communicates with the large ventral plexus {17) through small channels which curve around the lateral aspect of the aorta. It is seen, therefore, that the entire group of lymphatics associated with the aorta and dorsal aortic roots, with the exception of a few still isolated spaces and channels, can now discharge the contents of the channels into the great veins.
The most interesting and, in fact, from the standpoint of the hemophoric* function of the thoracic duct, the most important feature of this stage is the great reduction in size and number of the masses of developing blood cells. It has been shown in the foregoing pages that the differentiating blood cells associated with the developing lymphatics are admitted or gain access to the lymph spaces and channels and that when the communication is established between the thoracic duct and the jugular lymph sacs the blood cells can thus reach the sacs. Now since the taps between the lymph sacs and great veins have been formed, the blood cells are admitted to the hemal vascular system.
Hemophoric — blood bearing or carrying — is a term suggested by Dr. Schulte as a coordinate with hemopoietic — blood producing.
In this connection there are two aspects of the question which are especially worthy of consideration. In the first place there are few blood cells within any of the channels forming a part of the thoracic duct system, while in earlier stages, before the thoracic duct had open communication with the lymph sacs and great veins, many of the lymph channels were filled with the hemal cellular elements. In view of this, and the fact that there are no other means of egress for the blood cells formerly contained in the lymphatics, it must be concluded that the blood cells reach the hemal vascular system via the thoracic duct, the jugular lymph sacs and the recently formed taps between the sacs and the great veins.
The other feature is the great reduction in the size and number of the extravascular masses of developing blood cells. The diminished masses are shown in yellow in figures 27 and 28, the conditions in which should be compared with those in figures 22 and 24. A few fairly extensive groups are found in the mesenchymal tissue among the channels composing the large ventral plexus [17). These are for the most part quite closely associated with the lymphatics. A few small groups still are found in the region of the thoracic duct 'approach' (fig. 28, 15a). The extensive masses associated in earlier stages with the lymph plexus dorsal to the aortic arches have almost wholly disappeared. This plexus itself is considerably reduced, and it is not unreasonable to assume that the connection between the cephalic end of the plexus and the jugular lymph sac, previously alluded to, is in some way associated with the discharge of the numerous blood cells differentiated in this region into the lymph sac.
In view, therefore, of the intimate relationship between the developing thoracic duct and blood cells in the same general region, and of the sudden and marked reduction in number of these blood cells, both intravascular and extravascular, following the establishment of communication between the duct and the jugular lymph sacs and great veins, the importance of the thoracic duct as a carrier of hemal cellular elements for a period of embryonic life in the bird can scarcely be doubted.
==V. Summary==
Prior to the appearance of lymphatics in the region of the future thoracic duct, namely, along the aorta and dorsal aortic roots, the mesenchyme comprises a syncytium of irregular strands with correspondingly irregular interstitial spaces. The tissue is nonvascular.
The initial change in conditions is manifested in the appearance of distinct lacunae in the mesenchymal tissue along the ventro-lateral aspect of the aorta at the level of the celiac artery. The lacunae are bounded by unmodified protoplasmic elements of the mesenchymal syncytium, and open freely into the adjacent intercellular — interstitial — spaces. Obviously the lacunae represent enlarged intercellular spaces, and the inference is justifiable that they are filled with the intercellular fluid.
In a slightly advanced stage of development in general there is a greater number of lacunae in the same region in the embryo and also an increase in size and a difference in the appearance of some of the lacunae. The increase in size certainly depends in part upon actual dilatation and in part upon addition of more of the adjacent mesenchymal intercellular spaces, for every possible gradation can be seen between the smallest and the largest. The difference in appearance is observed to be due to the presence of flat cells which form a distinct boundary or wall, although not usually complete. Morphologically these cells are equivalent to endothelial cells. Inasmuch as they shade by invisible gradations into the unmodified mesenchymal cells bordering upon the rest of the lacuna, we conclude that they are derived directly from the indifferent mesenchymal cells. The differentiation, we may also infer, is due in part to pressure and friction incident to the flow of the tissue fluid. Another factor in the differentiation may also be pressure and friction incident to the to and fro motion of blood cells in the tissue spaces and lacunae in response to the heart-beat, a phenomenon observed in living blastoderms.
Studies of later stages show these isolated lacunae to be the rudiments of the thoracic duct, and the conclusions that they are direct derivatives of the mesenchymal intercellular spaces and
160 ADAM M. MILLER
that the flat cells forming their endothelial walls are differentiated in situ from the mesenchymal cells are based upon prolonged and thorough studies, with high magnification, of serial sections of chick embryos in practically perfect states of preservation. The writer is therefore forced to ally himself unequivocally with the advocates of the view that the thoracic duct originates independently of the veins and lymph sacs.
Studies of subsequent stages also show that the numerous isolated lacunae, or rudiments of the thoracic duct, enlarge still further, principally in a longitudinal direction, and coalesce with one another to form a plexus of lymph channels which lies ventral to the aorta. Other similar isolated lymphatics develop along the dorso-lateral aspect of the aorta and in the region dorsal to the aortic roots and arches, and then coalesce to fonii plexuses. Eventually all the plexuses intercommunicate.
In the meantime a connection is established between the large ventral plexus and a branch of each jugular lymph sac known as the thoracic duct 'approach,' All the components of the thoracic duct system thus drain into the jugular lymph sacs. Communications, or taps, are established between the lymph sacs and the great veins, and the thoracic duct then drains into the hemal vascular system, the lymph sacs serving as portals of entry.
In the region of the developing thoracic duct, namely, along the aorta and dorsal aortic roots, and also in the region dorsal to the aortic arches, a great number of blood cells arise. The genesis of these cells is indicated by certain changes in some of the irregular elements of the mesenchymal syncytium, comprising a marked increase in the basophilia of the cytoplasm, a rounding of the cell body and a separation from the general mesenchymal reticulum. The resulting cells thus lie free in the interstitial spaces, and structurally are similar to the large mononuclear cells (lymphocytes of Dantschakoff) in the area vasculosa of the blastoderm. They increase in number both by mitosis and by constant differentiation from the mesenchymal syncytium.
Many, at least, of these basophilic cells are transformed into erythrocytes through the addition of hemoglobin to the cytoplasm and certain nuclear modifications comprising the disappearance of the nucleoli and the rearrangement of the chromatin into a heavy reticulum.
These developing blood cells, at first scattered in the mesen-chymal intercellular spaces, become aggregated, following the increase in their number, into extensive masses which lie along the line of the thoracic duct and also in the region dorsal to the aortic arches.
When the lymphatics comprising the rudiments of the thoracic duct develop, some of the developing blood cells, even some of the smaller groups, are seen to be contained within them, while others are still free in the mesenchymal intercellular spaces, that is, extravascular. Subsequently more and more of the cells are observed to be intravascular. It may be concluded that the developing blood cells become intravascular by simple inclusion as the lymph channels develop, or by virtue of their ameboid character, or as a result of their motion to and fro in the tissue fluid in response to the heart-beat.
The blood cells that are admitted to the lymph channels constituting the thoracic duct system during its development rapidly diminish in number after communication is established between the thoracic duct and the jugular lymph sacs and between the latter and the great veins. It can be inferred then that the blood cells which develop along the line of the thoracic duct reach the blood stream via this duct and the lymph sacs. Considering the vast number of hemal cellular elements, especially erythrocytes, arising in this region and the probability that they reach the general circulation via the thoracic duct, this duct assumes an additional phase of importance in the chick in that it performs a hemophoric, or blood carrying, function.
In conclusion, I wish to thank Dr. Huntington and Dr. Schulte for their valuable criticism and suggestions. Dr. McWhorter for his painstaking work in making the photomicrographs, and Mr. Petersen for his careful execution of the color plates.
==References==
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the main lymph channels of the cat in their relations to the venous system. Anat. Rec, vol. 1, no. 3.
(26) McClure, C. F. W., and Silvester, C. F. 1909 A comparative study of
the lymphatico-venous communications in adult mammals. Anat. Rec, vol. 3.
==Plates==
PLATE 1
EXPLAXATIOX OF FIGURES
1 From a section of a chick embryo of 108 hours, 6.75 mm. (series no. 371; slide II, section 80). Outlines drawn under Edinger projection apparatus, X 1500; reduced to XlOOO. In the upper left corner is a portion of the aorta with a few erythrocytes. Outside of the aortic wall is an area of mesenchyme in which some elements show stages of differentiation leading toward large basophilic cells, two of the latter lying free in the tissue spaces at the lower left corner of the figure. These are apparently identical with the developing blood cells (lymphocytes) in the area vasculosa of the blastoderm as described by Dantschakoff.
2 From the same embryo as figvu-e 1 (slide II, section 97). Outlines drawn under Edinger i^rojection apparatus, X 1500; reduced to X 1000. A small part of the aorta is shown in the upper right corner. Some of the protoplasmic elements of the mesenchymal syncytium are shown in various stages of differentiation leading toward the free basophilic cells (lymphocytes of Dantschakoff). In the lower left corner is seen one of these cells in the anaphase of mitosis. At the right of this is a practically mature erythrocyte lying free in a mesenchymal intercellular space.
3 From a section of a chick embryo of 132 hours, 9 mm. (series no. 411; slide IV, section 16). Outlines drawn under Edinger projection apparatus, X 1500; reduced to X 1000. This figure, taken just ventral to the aorta, shows a number of the rounded basophilic cells lying free in the mesenchymal intercellular spaces.
PLATE 2
EXPLANATiOX OF FIGURES
4 From a section of a chick embryo of o (lays, 12 mm. (series no. 336; slide IV, section?). Photomicrosi'aph, X 166.
1, aorta 10, preeardinal vein
3, aortic arch VI 16, grouj) of developing blood cells
4, aortic arch 1\
5 From a section of a cfiick embryo of 6 days, 13.5 mm. (series no. 339; slide VIII, section 35). Photomicrojirajjh, X 166.
2, dorsal aortic root 16, groups of developing blood cells
3, aortic arch VI 27. vagus nerve
PLATE 3
EXPLANATION OF FIGURES
6 From a section of a chick embryo of 7 days (series no. 512; slide III, section 15). Photomicrograph, X SO.
2, dorsal aortic root 19, lymphatics dorsal to aortic arches
3a, pulmonary artery and esophagus
10, precardinal vein 27, vagus nerve
16, groups of developing hhjod cells esophagus in center of figure
along precardinal vein bronchi at lower border of figure
16a, groups of developing blood cells
dorsal to esophagus
7 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm (series no. 426; slide XI, section 21). Photomicrograph, X 133.
1, aorta
5, celiac artery
8, dorsal somatic artery
16, groups of developing blood cells
23, celom (pleural cavity)
25, mesonephros
26, lung anlage
PLATE 4
EXPLANATION OF FIGURES
8 From a section of a chick embryo of 6 daj's and 22 hours, 16.5 mm. (series no. 464; slide XXII, section 22). Photomicrograph, X 133.
1, aorta 16, groups of developing blood cells
5, celiac artery 23, celom (pleural cavity)
8, dorsal somatic artery 24, sympathetic nerves
9 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm. (series no. 426; slide XVIII, section 3). Photomicrograph, X 233.
1, aorta 17, lymph spaces, rudiments of thoracic
16, developing blood cells duct
23, celom (abdominal cavity)
PLATE 5
EXPLANATION OF FIGURES
10 From a section of a chick embryo of 7 days (series no. 512; slide VII, section 1). Photomicrograph, X 233.
1, aorta 17, lymphatics, rudiments of thoracic
5, celiac artery duct
16, groups of developing blood cells 23, celom (abdominal cavity)
11 From a section of a chick embryo of 14 days (series no. 518; slide XIII, section 9). Photomicrograph, X 133.
1, aorta 17, lymphatics (part of thoracic duct
16, blood cells, mostly mature erythro- system)
cytes, in tissue spaces
PLATE 6
EYPLAXATIOX OF FIGURES
12, 13, 14 From tliree saccessive .sections of a chick einbryo of (i days and 16 hours, 13.5 mm. (series no. 42(i: slide XII. sections 9, 10 and 11). Photomicrographs, X 350.
In figure 13 the arrow (/?) points to a lymph space in the mesenchyme. At its right side the lymph space is seen clearly to open into the adjacent mesenchymal intercellular spaces. The preceding section in the series (fig. 12) shows no space (the arrow points to the same locality as in fig. 13). In figure 14 the arrow points to a light area in the mesenchyme which represents the opening of the space of figure 13 into the adjacent mesenchymal intercellular spaces; this can be seen much more clearly with the microscope by changing the focus. The lymph space shown in figure 13 is the same one represented in figure 20. 17 at the right of the aorta. Aorta. /; celom (abdominal cavity), 23.
PLATE 7
EXPLANATION OF FIGURES
15, 16, 17 and 18 From four successive sections of a chick embryo of 7 days (series no. 512; slide VII, sections 21 and 22; slide VIII, sections 1 and 2). Photomicrographs, X 250.
In figure 15 four lymph spaces, in part lined with endothelium, are seen at the left of the aorta (1). The one nearest the aorta, while in part lined with endothelium, opens freely into the adjacent mesenchymal intercellular spaces. The largest space also opens below in a similar manner. In figure 16, from the succeeding section in this series, only one of the spaces appears, opening above into tissue spaces. The others have simply become continuous with tissue spaces and therefore do not appear as distinct lacunae; this can be clearly demonstrated with the microscope by changing the focus on the rather thick sections (20 micra). In figure 17 the arrow points to the termination of the distinct lacuna of figure 16; the free communication with the tissue spaces is quite obvious. In figure 18 there are no lacunae, all those of the preceding sections having opened into the mesenchymal intercellular spaces. Aorta, 1; part of sympathetic nervous system, 24-
PLATE S
EXPLAXATIOX OF FIGURE
Ul From a section of a chick embi\vo of 6 days and 22 hours, 16 mm. (series no. 463; sliiU' X\'I, section 9). Photomicrograph, X 160.
£, dorsal aortic root 15, jugular lymph sac
3(1. pulmonary artery 15a, thoracic duct 'approach'
12. precaval vein 27, vagus nerve
13, vertebral vein
PLATE 9
EXPLANATION OF FIGURE
20 Drawn from a reconstruction of a chick embryo of 6 days and 10 liOLirs, 13.5 mm. (series no. 426). Ventral view.
1, aorta
2, dorsal aortic roots
3, aortic arch VI
3(1, pulmonary artery
4, aortic arch IV
5, celiac artery
10, precardinal vein
11, subclavian vein
12, duct of Cuvier
13, vertebral vein
15, jugular lymph sac
16, groups of developing blood cells
17, first rudiments of thoracic duct
PLATK 10
EXPLANATIOX OF FIGURE
21 Dniwn from a reconstruction of a chick cnihrvo of (i days and '_M hours, 14 mm. (series no. 465). Ventro-mesial view.
1, aorta ^^. duct of Cuvier
2, dorsal aortic roots ^.?, vertebral vein
3, aortic arch VI 15, jugular lymjih sac
3n, pulmonary artery 15a, thoracic duct 'approach' of jugular
5, celiac artery lymph sac
6, notocord 16, groups of develo))ing blood cells
10, precardinal vein 17, rudiments of thoracic duct
11, subclavian vein
PLATE 11
EXPLANATION OF FIGURE
22 Drawn from a reconstruction of a chick embryo of 6 days and 22 hours, 16 mm. (series no. 463). Ventral view.
I , aorta
£, dorsal aortic root
3, aortic arch VI
3a, pulmonary artery
4, aortic arch IV
5, celiac artery
10, precardinal vein
II, subclavian vein 1°2, duct of Cuvier 13, vertebral vein
15, jugular lymph sac
15a, thoracic duct 'approach' of jugular lymph sac
16, groups of developing blood cells 16a, groups of blood cells dorsal to
aortic roots and arches
17, rudiments of thoracic duct
18, lymphatics along aortic arches
19, lymphatics dorsal to aortic roots and arches
21, mesenteric lymphatics
PLATE 12
EXPLANATION OF FIGURE
23 Drawn from a reconsti-uftion of a chick embryo of 6 days and 22 hours, 16 mm. (series 463). Mesial view.
10, prccardiiial vein 15a, thoracic duct 'apiiroach' of jugular
11, suhclavian vein lymph sac
12, duct ofCuvicr 16, groups of developing blood cells 15. jugular lymi)h sac 17, rudiments of thoracic; duct, extreme cephalic end cuse.
PLATE 13
EXPLANATION OF FIGURE
24 Drawn from a reconstruction of a chick embryo of 7 days (series no. 512) Ventral view.
1, aorta
2, dorsal aortic roots
3, aortic arch VI
3n, pulmonary artery 5, celiac artery 7, carotid artery
10, precardinal vein
11, subclavian vein
12, duct of Cuvier
14, part of splanchnic plexus of veins
15, jugular lymph sac
15a, thoracic duct 'approach' of jugular lymph sa:'
16, groups of developing blood cells 16a, blood cells dorsal to aortic roots
and arches
17, thoracic duct, ventral plexus (homologue of azygos segment)
17a, thoracic duct (homologue of i)reazygos segment)
18, lymphatics along aortic arches
19, lymphatics dorsal to aortic roots and arches
21, mesenteric lymphatics
PLATE 14
EXPLANATION OF FIGURE
25 From same reeonstnu-tioii as in fig. 24. View from left side.
I, aorta
3, aortic arch VI 5, celiac artery
7, carotid artery
8, dorsal somatic arteries 10, ])recardinal vein
II, subclavian vein 12, duct of Cuvier IS, vertebral vein
14, part of splanchnic plexus of veins
15, jugular lymph sac
16, groups of developing blood cells 16a, blood cells dorsal to aortic roots
17, thoracic duct, ventral plexus
18, lymphatics along aortic arches 20, lymphatics dorso-lateral to aorta ?/, mesenteric lyniphatic^s
190
PLATE 15
EXPLANATION OF FIGURE
26 Drawn from a reconstruction of a chick embryo of 8 days (series no. 513). Ventral view.
1, aorta
2, dorsal aortic roots
3, aortic arch VI
3a, pulmonary artery
4, aortic arch IV
5, celiac artery
6, notocord
7, carotid artery
9, superior mesenteric artery
10, precardinal (jugular) vein
11, subclavian vein
12, duct of Cuvier 15, jugular lymph sac
15a, thoracic duct 'approach' of jugular lymph sac
17, thoracic duct, ventral plexus (homologue of azygos segment
17a, thoracic duct (homologue of preazygos segment)
18, lymphatics along aortic arches and duct of Cuvier
19, lymphatics dorsal to aortic roots and arches
20, lymphatics dorso-Iateral to aorta
21, mesenteric lymphatics
PLATE 16
EXPLANATION OF FIGURE
27 Drawn from a reconstruction of a chick embryo of days and 14 hours (series no. 320). Ventral view.
1, aorta
^, dorsal aortic roots
3, aortic arch VI
3a, puhnonary artery
4, aortic arch IV
5, celiac artery 7, carotid artery
9, superior mesenteric artery
10, p ecardinal (juguhir) vciii //, subchivian vein
12, duct of Cuvier 15, juguhir lymph sac 15(1, thoracic duct 'approach' of jugular lymph sac
16, groups of developing blood cells
17, thoracic duct, ventral plexus (honiologue of azygos segment.
17a, thoracic duct (homologue of preazygos segment.
18, lymphatics along aortic arches
19, lymphatics dorsal to aortic roots and arches
£1, mesenteric lymphatics ^^, lymphatics associated with esophagus
PLATE 17
EXPLAXATION OF FIGURE
28 From same reconstruction as fijjuro 27. View from left side.
I, aorta
3, dorsal aortic roots 3, aortic arch VI
7, carotid artery
8, dorsal somatic arteries
.9, superior mesenteric artery 10, precardinal (jugular) vein
II, subclavian vein
12, duct of Cuvier
13, vertebral vein
15, jugular lymph sac
15a, thoracic duct 'approach' of jugular lymph sac
16, groups of developing blood cells
17, thoracic duct
18, lympathics along aortic arches
19, lymphatics dorsal to aortic roots and arches
20, lymphatics dorso-lateral to aorta £1, mesenteric lymphatics
£2, lymphatics associated with esophagus
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Miller AM. Histogenesis and morphogenesis of the thoracic duct in the chick; development of blood cells and their passage to the blood stream via the thoracic duct. (1913) Amer. J Anat. 15(1): 130-.

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This historic 1913 paper by Miller describes chicken thoracic duct development.



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Histogenesis and Morphogenesis of the Thoracic Duct in the Chick

Development of blood cells and their passage to the blood stream via the thoracic duct

Adaai M. Miller

The Analomical Laboratory of Columbia Univernily

Twentty-Eight Figures (Seventeen Plates)

I. Introduction

The study of the development of the jugular lymph sac in the chick, the result of which was published in this Journal/ (1) led to the investigation also of the developing thoracic duct and the means whereby its communication with the lymph sac is established. This investigation has been carried on with the advice and under supervision of Dr. Huntington and in the light of his recent work on reptiles (2) and the cat (3).

Within the past few years different investigators have shown that in the frog (4), (5), the chick (1), (8), the rabbit (6), the cat (9), and in man (7) each jugular l>Tnph sac develops directl}^ from a venous capillar}^ network adjacent to the junction of the early precardinal with the postcardinal vein to form the duct of Cuvier. It has also been pointed out by Huntington (14) that the jugular lymph sacs, regarded as of venous origin, constitute the connecting links between the hemal vascular system and the general system of lymphatic vessels.

The origin of the systemic h'mphatic vessels is a problem on which investigators are sharply divided. A summary of the different views, including the bibliography, can be found on pages 10-13 of Huntington's monograph in the Memoirs of The Wistar Institute (3).

^ References, by number, will be found on page 162.


As regards the particular case of the thoracic duct, Sala (10) in 1900 published the results of his work on the chick in which he holds that it develops by canalization of solid mesenchymal cords. In 1902 Sabin (11), after working with the injection method, published her account of the development of the lymphatic system in pig embryos. In this she reinforces and extends the view of Langer (12) and Ranvier (13), that lymphatics arise from veins by a process of sprouting and centrifugal growth, maintaining that the system as a whole is developed by blind ducts that 'bud off' from the veins of the cervical and inguinal regions, widen out to form sacs from which lymphatics grow to the skin, stating also that "at the same time a growth of ducts occurs along the dorsal line following the aorta to make a thoracic duct from which lymphatics grow to the various organs." The two views expressed by Sala and Sabin are thus diametrically opposed, the one being that the lymphatics arise in the mesenchyme independently of the veins, the other that the lymphatics are outgrowths from the veins.

In 1905 Lewis (6) expressed the view that in the rabbit the lymphatic system is derived directly from the embryonal veins, multiple detached portions of these becoming confluent to form the permanent systemic lymphatics, stating that the thoracic duct "arises from a plexus of lymphatics surrounding the aorta" (p. 109).

In 1907 Huntington and McClure (25), studying the development of lymphatic vessels in their relation to the veins in embryos of the cat, found that "the lymphatics begin as extra-intimal spaces along the course of the primitive embryonal veins. They* subsequently become confluent and form continuous vascular channels" (p. 42).

Huntington, in 1908 (14), while retaining this view of the genesis of the systemic lymphatic vessels, as distinguished from the jugular lymph sacs, defined the latter as the connecting links between the hemal vascular system and the general system of the lymphatic vessels, which "arise, not by transfonnation of veins, but by the formation of spaces lying outside the intimal lining of the veins, which spaces, becoming confluent, form the general lymphatic channels of the body" (p. 25).


In the same year (1908) McClure (21) abandoned the view previously held jointly by him and Huntington as regards the development of the thoracic and right lymphatic ducts in cat embryos, and states:

The anlages of the thoracic and right lymphatic ducts consist of a series of independent outgrowths which first appear along the common jugular and innominate and then along the azygos veins exactly in the line subsequently followed by these ducts; these outgrowths are subsequently split off from the veins, by a process of fenestration, in the form of a series of isolated, more or less spindle-shaped spaces which later become confluent with one another and with a process of the jugular lymph sac to form a continuous system disconnected from the veins, .... (p. 542).

In 1909 Sabin (7) reiterates her original view, based on the study of injected pig embryos, stating that the "presumption seems to lie on the side that the thoracic duct develops in the same manner as all other ducts," namely, "from endothelial sprouts from the sacs" (p. 58).

McClure in 1910 (23), after further studies of critical stages in lymphatic development, retracted his former view and stated :

The venous line .... along which the cat's thoracic duct develops is topographically replaced by the lymphatic channel, not directly, as assumed by me (in 1908), but secondarily by extra-intimal lymphatic space development, .... the 'extra-intimal theory,' as originally outlined by Huntington and myself (in 1906), establishes a fundamental principle of development for the main systemic lymph channels in mammals (p. 105).

Sabin, in a later article ('11) (22), states that "the thoracic duct develops in part as a down growth of the jugular sac and in part, especially its dilated portion or cisterna chyli, as a direct transformation of the branches of the azygos veins" (p. 424) . This expression of opinion seems to be a correction of her earlier statements and a partial adoption of Lewis's view. In place of her former concept of an uninterrupted centrifugal lymphatic growth from the sacs, she now appears to hold that in addition a portion of the thoracic duct develops as the result of direct transformation of azygos venous tributaries into lymphatics.


Huntington again in 1911 confirmed and elaborated his former view by extensive observations on reptiles (2) and the cat, stating specifically on page 13 of the first number of the Memoirs of The Wistar Institute (3) that "the entire extensive system of lymphatic vessels proper of the adult animal, including the thoracic and right l}miphatic ducts and their tributaries, is formed by the confluenceof the extravenous intercellular mesodermal spaces," and that "these spaces are lined by a lymphatic vascular endothelium which is Jiot derived from the hemal vascular endothelium, but develops independently of the same," and giving his summary and conclusions in remarkably clear terms on pages 153-171 of the same publication. He also points out that the systemic lymphatic development in the mammalian embryo is "by no means confined to the immediate environment of degenerating embryonic veins. The same field, which shows the above described histogenetic processes in the development of extra-intimal lymphatic spaces surrounding and replacing a decadent venule, will at the same time contain numerous equivalent lymphatic mesenchymal clefts and spaces which continue to develop independently of any association with retrograding veins" (p. 49).

Sabin in 1912 (24) still maintains that "the thoracic duct (in the pig) arises in part as a downgrowth from the left jugular sac and in part from a plexus of lymphatics which buds off from the veins of the Wolffian body" (p. 336).

Recently Kampmeier (15), after studying serial sections of both uninjected pig embryos and one of Sabin's injected specimens, concluded that

the actual genesis of the thoracic duct is initiated by the appearance of blind mesenchymal Ij^mphatic spaces either around or not immediately in contact with the venous derivatives, or veno-lymphatics, which become detached from their venous trunks and break up into degenerating segments .... During their inception and growth the walls of the discontinuous thoracic duct anlagen are composed of mesenchymal cells .... Injected specimens of the early lymphatic stages certify the reality of blind uninjectible anlagen beyond the farthest points to which the injecta have penetrated, demonstrating that discontinuities in a developing lymphatic channel are not 'appearances' found only by the study of uninjected embryos (pp. 463-464).


Stromsten (16) in his account of the development of the thoracic duct in turtles arrived at a like conclusion, stating that "the development of the peri-aortic lymphatic plexus in the loggerhead turtle is immediately preceded by the formation of isolated, independent spaces. They (the spaces) cannot be injected . . . ." (p. 354). "The intercellular spaces thus formed enlarge and fuse together to form lymphatic lacunae. At a later stage the lacunae acquire an endothelial lining and become the isolated anlagen of the thoracic duct" (p. 356).

As regards the thoracic duct in the chick, it^will be my object in this article (1) to demonstrate its origin by confluence of intercellular spaces in the mesenchyme, independent of the veins, and to reconsider the significance of the mesenchymal cords described by Sala in their relation to the developing lymphatics; (2) to discuss the establishment of the morphological drainage line of the thoracic duct and the means by which this duct and the jugular lymph sacs communicate; and (3) to show that the organization of the avian thoracic duct corresponds in type with that established in reptiles and mammals.

II. Material

Embryos of the domestic fowl (Gallus gallus) have been used chiefly on account of the certainty in procuring the critical stages. Some embryos of the English sparrow (Passer domesticus) have also been used. Of this material I have examined thirty-two individual embryos in serial sections, comprising twenty-seven chicks and five sparrows. Four of the chicks were injected with India ink through the umbilical vein (table 1).

Unfortunately the sparrow embryos, of which series nos. 123, 124, 126, 154 and 509 were examined, were not measured and could be judged as to stages of development only by comparison with the chicks.

The embryos were fixed in vom Rath's, Bouin's, or Zenker's fluid (some in Zenker-formol), Bouin's fluid giving the best results with the least shrinkage. The sections were cut in paraffin and stained on the slide by one or the other of the following methods. After fixation in vom Rath's mixture, the staining was


TABLE 1


CHICK EMBRYOS SERIES NO.


TIME OF INCUBATION IN HOURS


GREATEST LENGTH IN MM AFTER FIXATION


415


96




7.0


371


108




6.75


336


120




9.0


355


120




11.5


356


120




12.0


326


130




12.5


411


132




9.0


410


132




9.25


370


132




9.5


357


132




10.75


412


145




10.75


340


145




11.0


428


145




11.0


414


145




11.25


339


145




13.5


426


160




13.5


465


165




14.0


463


166




16.0


464


166




16.5


520


171


inj


ected



521


192


inj


ected



522


192


inj


ected



519


206





523


216


inj


ected



320


230





524


240





483


260





done with a much diluted Delafield's hematoxylin, followed by a weak solution of picric acid in alcohol. After all the other fixatives the sections were overstained in Weigert's hematoxylin, decolorized in water acidulated with HCl, and counterstained in a weak solution of Orange G in distilled water! The blood cells' are clearly differentiated by either method; the cytoplasm of those containing even a trace of hemoglobin shows some tinge of yellow. Developing muscle tissue and nerve fibers also are yellow. Other elements are stained by the hematoxylin, the delicate processes of the irregular mesenchymal cells showing especially well.


III. Histogenesis

Sala, in his account of the development of the thoracic duct in the chick (10), states that the anlagen of this lymph channel appear as isolated mesenchymal spaces which become clothed with endothelial cells derived from the mesenchyme, and which subsequently coalesce to form continuous vessels. He thus implies a denial that the lymphatics in this region arise from veins and that the lymphatic endothelium is derived from hemal vascular endothelium. He also calls attention to accumulations of mesenchymal cells which contrast clearly with the surrounding tissue. They appear to consist in large part of elements exhibiting all the characteristics of young connective tissue cells, with roundish forms, no processes and large intensely colorable nuclei. Among these elements appear red corpuscles in larger or smaller numbers. The accumulations or clumps of cells develop first on the mesial aspect of the superior vena cava, and then extend caudad to the level of the celiac artery. Without describing the histogenesis of these masses of cells, Sala states further that within them in large part the anlagen of the thoracic duct are excavated or 'hollowed out.' The details of the 'hollowing' process are not given.

In the main I can confirm the results of Sala's observations so far as he has carried them. As for the isolated mesenchymal spaces which he describes as the anlagen of the thoracic duct my study of their histogenesis in the closely graded series of chick embryos leads to the conclusion that they do arise independently of the veins and that their endothelial lining is derived from the indifferent mesenchymal cells bordering upon them. The details of the process I shall attempt to demonstrate in the following pages. After tracing the development of the accumulations of cells in the mesenchyme and their subsequent history in relation to the developing thoracic duct, it seems to the writer that a different and greater significance can be attached to them than was given by Sala. Since the cells composing them correspond so closely in their history to the developing blood cells as described by Dantschakoff (17) in the extraembryonic area of the chick, undoubtedly they should be considered as collections of intraembryonic developing blood cells.

These clumps of cells, as noted by Sala, develop in the vicinity of the aortic arches, especially the sixth, and along the dorsal aortic roots and the aorta as far as the exit of the superior mesenteric artery. The early stages in the formation can be clearly seen in embryos of 100 to 110 hours.

Some of the stellate elements of the mesenchymal reticulum (syncytium) become differentiated from their neighbors. Their processes are retracted and separated from the general reticulum, the cells thus becoming rounded (figs. 1 and 2). The cytoplasm acquires a more strongly basophilic character, increases in amount and becomes more homogeneous than that of the true mesenchyme. The nuclei contain a relatively small amount of chromatin and one or two, usually two, distinct nucleoli. Mitotic figures are occasionally seen (fig. 2). While the cell contours are generally regular, there are sufficient irregularities to denote an ameboid character. These cells appear to be identical with the large mononuclear cells which Dantschakoff describes as differentiating from the blood islands in the area vasculosa of the blastoderm and later in the capillaries of the yolk sac, and which she calls lymphocytes.

The cells here under consideration increase in number not only by their own proliferation but also by continued differentiation from the mesenchymal syncytium (fig. 2). For the most part they lie in compact groups, which gradually increase in size as the cells increase in number, but some cells usually appear in the vicinity of the groups (figs. 4, 5, 7, 8 and 10, 16) and not infrequently at some distance from them. After their differentif,tion and separation from the mesenchymal syncytium all the cells, both members of the groups and isolated, lie free in the spaces among the stellate elements of the mesenchymal tissue (figs. 3 and 4). No blood vessels or lymphatics are present in the immediate vicinity of the cell groups when the latter first develop and consequently the cells are extravascular (figs, 1, 2, 3 and 4). It will be shown later that after the lymphatics develop in this region the cells for the most part are included within them and thus become intravascular elements.


The application of the term 'blood islands' to the accumulations of cells has been avoided because there is no real condensation of the mesenchyme whereby the differentiating elements fuse to form a solid protoplasmic mass. Each stellate element differentiates individually (figs. 1 and 2) and condensation occurs only as a subsequent aggregation of the differentiated cells. The resulting large mononuclear cells, however, so closely correspond to the first lymphocytes that develop in the area vasculosa of the blastoderm, the subsequent histories of the cells arising in the two localities being identical, the conclusion is warranted that the differentiated mesenchymal elements in question serve as an intraembryonic source of blood cells.

While the present investigation has not been of such a nature as to determine whether the mononuclear basophilic cells which have been described give rise to granular leucocytes, they certainly give rise to red blood cells.. The cytoplasm of some of the cells gradually loses its basophilic character and acquires a stronger affinity for the plasma dyes, at the same time becoming quite homogeneous. These changes are concomitant with the addition of hemoglobin. The nuclear changes comprise an increase in the amount of chromatin, its arrangement into a rather heavy reticulum and the disappearance of the nucleoli (fig. 2).

Cells thus modified and containing a moderate amount of hemoglobin, as indicated by the reaction to plasma stains, are erythrobalsts. Without further visible structural changes these acquire more hemoglobin until eventually they are indistinguishable from the red blood cells in the general circulation. They have therefore become erythrocytes. It is probable also that they are definitive rather than the larger primitive erythrocytes described by Dantschakoff. The changes described above occur both in the cells composing the groups and in the isolated cells, sd that fully developed red blood cells as well as the earlier de\'elopmental stages are seen not only in the groups but also in the mesenchymal spaces more or less remote from the groups.

Among the developing erythi'oblasts, especially in the later stages, are many small cells which apparently go through the same processes of differentiation as the red blood cells themselves and resemble the latter in every respect except size. These are undoubtedly microcytes, the earlier stages being microblasts.

After the middle of the fifth day the masses of developing blood cells increase rapidly in size by proliferation of the component cells and continued differentiation of the branching elements of the mesenchymal syncytium. They increase in number also by the same processes of differentiation in other localities. Up to about the beginning of the sixth day all the developing blood cells in question remain extravascular, that is, free in the intercellular mesenchymal spaces which are not lined by endothelium.

By the end of the seventh day the aggregations of cells reach the height of their development. At this time they extend in two main lines from the level of the aortic arches along the dorsal aortic roots to the confluence of the latter to form the aorta (figs. 6, 20 and 22, 16). At this level, or a little further caudal, the two main lines unite to form a single line which extends about to the level of the superior mesenteric artery (figs. 7, 8, 10, 20 and 22, 16). At their greatest development the larger groups together form an almost continuous mass of the cells which in places is greater in diameter than the aorta. Usually there are also numerous smaller outlying groups which belong to the same general line (figs. 7 and 8).

While the main lines above described are established in all the embryos examined, yet there is a wide range of variability in the form of the groups and their arrangement in the lines. In most of the embryos examined a large mass of cells or a collection of smaller groups had developed in the region dorsal to the aortic roots and esophagus (figs. 6 and 22, 16a). One of the most interesting and one of the most important features of the masses of developing blood cells is the fact that the main lines in their general arrangement correspond with the lines of the thoracic duct (figs. 20, 22 and 24, 16, 17 ,17a).

To anticipate, it may be stated here that as the multiple anlagen of the thoracic duct develop the masses of developing blood cells are in a large part included within them, and thus become strictly intravascular (see fig. 10, 16, 17). The details of this process will be considered in the subsequent description of the formation of the lymphatic spaces and channels. After the seventh day, as the lymphatic channels in this region develop and establish communication with the jugular lymph sacs, the intravascular masses of blood cells decrease and, by about the eleventh da}^ are reduced to a few groups scattered through the lymphatic plexus (cf. figs. 24, 27 and 28). A considerable number of extra vascular groups persist, however, until the fourteenth or fifteenth day, or even later. In the later stages the vast majority of the cells in these groups are practically mature erythrocytes lyiiig free in the mesenchymal spaces in the vicinity of the lymphatics (fig. 11).

Inasmuch as the reduction in the masses of blood cells in the lymphatics follows the coalescence of the lymphatic spaces to form continuous channels and the establishment of communication between the latter and the jugular lymph sacs, and since the lymph sacs open into the great veins, the blood cells in question eventually reach the general circulation by way of the thoracic duct and jugular lymph sacs. The thoracic duct at one period acts, therefore, as conveyor of the erythrocyte series of hemal cellular elements which have developed from the indifferent mesenchyme along the line of the duct.

A consideration of the histogenesis of the lymph channels constituting the anlagen of the thoracic duct leads on to controversial ground. As stated earlier in this paper, the controversy hinges upon the question whether the lymphatics, exclusive of the lymph sacs or hearts, arise as sprouts or outgrowths from pre-existing vascular channels or de novo from the intercellular mesenchymal spaces.

It is the opinion of the writer that in the chick the lymph channels which constitute the anlagen of the thoracic duct arise through enlargement and coalescence of intercellular spaces in the mesenchymal tissue, and that the endothelial lining of these channels is derived directly from indifferent mesenchymal cells^ that chance to border upon the spaces. In the material studied there is no evidence of any growing out, budding or sprouting from the endothelium of pre-existing blood vessels, the tissue in which the thoracic duct develops being non-vascular. The lymphatics in question, and their endothelial lining, arise independently of pre-existing vascular channels.


  • It should be understood that when 'mesenchymal cells' are spoken of, they are considered as the irregularly stellate masses of protoplasm the processes of which anastomose to form the mesenchymal syncytium, or reticulum.



Prior to the appearance of any specialized spaces or channels in the mesenchyme in the region of the future thoracic duct, the mesenchymal syncytium consists of irregularly stellate protoplasmic elements the slender processes of which anastomose freely with like processes of neighboring elements. The cytoplasm is finely granular and the nuclei, while relatively large and vesicular, contain little chromatin and one or two distinct nucleoli. Among the protoplasmic components of this tissue are the correspondingly irregular interstices or spaces which also are continuous with one another. These are called the mesenchymal intercellular spaces. The tissue as a whole might be compared with a sponge, the anastomosing protoplasmic parts representing the parenchyma of the sponge and the intercellular spaces the pores. While some of the protoplasmic elements during this time differentiate into blood cells, as previously described, our conception of the general syncytium and its spaces is in no way invalidated.

The first changes in the mesenchyme leading toward the formation of definite channels occur during the latter half of the sixth day of incubation. These changes, instead of involving the mesenchyme generally, begin in several localities. In one of the localities, for example, the intercellular spaces increase in size and coalesce, most of the protoplasmic elements of the syncytium being pushed farther apart or broken. In this manner a considerably larger space is formed out of a number of the original smaller mesenchymal spaces (fig. 9, 17). For the most part the smaller spaces of the surrounding tissue open freely into the larger space, although in places along the edge of the latter the protoplasmic elements lose some of their stellate character and become flattened on the side toward the larger space.

The phenomena in general indicate the accumulation of the fluid filling the mesenchymal spaces, the cells and their processes being subjected to pressure and friction incident to the flow of the interstitial substance. Thus there arises in the mesenchyme a space larger than the original interstitial spaces but derived directly from them by their enlargement and coalescence (cf. figs. 12, 13 and 14). So far as there is any definite lining for the new space, it is formed by the partiall}^ or wholly flattened mesenchymal cells upon which the fluid in the space impinges (fig. 13).

The further changes in one of these larger spaces consists in the main of its elongation through the enlargement and addition to it of other intercellular mesench3aiial spaces, a progressive flattening of the cells along its sides, and an approximation of the edges of the flattened cells to form a definite lining of endothelium. There is thus formed a distinct channel in the mesenchyme. For the most part it is lined and its lumen is separated from the surrounding interstitial mesenchymal spaces by a layer of endothelial cells which represent metamorphosed stellate elements of the mesenchyme (figs, 15 and 16). At or near its ends the channel opens freely into the adjoining mesenchymal tissue spaces which in the further course of development are added to the lumen of the channel and thus become a part of it (figs. 15, 16, 17 and 18). The endothelial lining already formed merges near the ends of the channel with the mesenchymal syncytium which in turn, as the channel elongates, gives rise to more endothelium by differentiation of certain of its elements (figs. 15, 16, 17 and 18).

As stated in a previous paragraph, the larger spaces appear in several localities in the mesenchyme. Consequently the channels resulting from their further development are for a time isolated; that is, they are not directly connected with one another or with the jugular lymph sacs or any part of the hemal vascular system. These isolated spaces and channels constitute the multiple anlagen of the thoracic duct (fig. 21),

In succeeding stages each of the channels in question increases in size, especially in length, until it meets and coalesces with its neighbors. The increase in length is due in the main to the addition of more mesenchymal tissue spaces to its ends and the concomitant transformation of more stellate mesenchymal cells into endothelial cells. There is probably also some proliferation of the endothelial cells, although in the study of the sections mitotic figures were not seen.

The coalescence of the originally unconnected lymphatics results in a network or plexus of channels (cf. figs. 21, 22 and 24). This is constantly being augmented by the coalescence of other independently formed spaces and channels with one another and with the previouly established plexus. Most of the vessels composing the plexus lie longitudinally in the embryo along the line of the aorta and dorsal aortic roots, and out of this plexus is e\'entually crystallized the main drainage lines of the thoracic duct. The establishment of these lines is best considered, however, in the section on morphogenesis.

The correlation of groups of developing blood cells in this region, the formation of which has already been described, and the lymph spaces and channels constituting the anlagen of the thoracic duct now remains to be discussed. As stated earlier in this paper, the blood cells that are differentiated from the mesenchymal syncytium, whether they are arranged in groups or isolated, lie free in the tissue spaces. In case the tissue spaces enlarge and coalesce in the region where the blood cells are situated the latter are then allowed to become free also in the larger space resulting from the enlargement and coalescence. The larger space become^ lined with endothelium, in the manner previously described, to form a definite vessel or channel. The blood cells, therefore, which were originally free in the tissue spaces are included in an}" of the lymphatics developing in that particular locality (fig. 10, 16, 17).

In that manner some of the blood cells become intravascular elements during the earliest stages of lymphatic development. Occasionally an entire group of cells is included in a lymph channel; in other cases only part of a group or a few scattered cells. It is true also that a great many lymphatics develop in the mesenchyme quite apart from the blood cells (figs. 15, 16 and 17).

In the earlier stages of lymph vessel formation there are great numbers of blood cells, in various degrees of differentiation, in the mesenchymal tissue spaces in the vicinity of or more or less remote from the lymph spaces and channels. In part at least these cells become intravascular when new lymphatics are formed out of the tissue spaces in which they lie and join the general plexus of previously formed vessels.

Thus far, therefore, the admission of the blood cells to the lymph vessels depends merely upon the topographical relationship in the development of the two sets of structures. There are, however, other factors which in all probability enter into this process. Two are of especial interest and importance in the case under consideration.

It has been pointed out by Dantschakoff that the primitive blood cells in the area vasculosa of the blastoderm and the large mononuclear cells derived from them are capable of ameboid movement. The developing blood cells in the region of the thoracic duct anlagen are demonstrably of the same type as those in the area vasculosa. Hence it is reasonable to conclude that some of the developing blood cells pass from the tissue spaces into the vessels by virtue of their ameboid character.

The other factor has hitherto, so far as the writer is aware, been considered only in the study of living tissues. It seems justifiable, however, to extend the conclusions drawn therefrom to fixed tissues. In their study of chick blastoderms in vitro, McWhorter and Whipple (18) have observed the to and fro movement, synchronous with the heart-beat, of blood cells not only in the isolated, endothelium-lined spaces which eventually coalesce to form blood vessels but also in the tissue spaces. Furthermore, they have observed the entrance of blood cells into the general circulation following then- to and fro movements in the tissue spaces. These phenomena certify the pulsation of the fluid substance in the tissue spaces in response to the heart-beat. It is not uiu-easonable, therefore, to conclude that some of the blood cells in the region of the developing thoracic duct are driven or sucked into the lymph spaces or channels which, as pointed before, open freely into the mesenchymal tissue spaces.

As a corollary to the phenomena mentioned in the preceding paragraph, an additional factor in the formation of endothelium might be suggested. Granting that the blood cells lying free in the tissue spaces move to and fro in response to the heart-beat, as has been clearly observed in the living chick blastoderm, the assumption is justifiable that their movement, with the resultant friction and pressure upon the adjacent protoplasmic elements of mesenchymal tissue, assists in the flattening of these elements and the consequent formation of endothelial cells.

The hydrodynamic factors of pressure and friction of the interstitial fluid substance upon the protoplasmic elements of the mesenchyme, which were first discussed by Thoma (19) in connection with blood vessel formation and have already been noted in this particular case of development of lymphatics, together with the additional factor of pressure and friction of oscillating blood cells would, in the opinion of the writer, afford adequate mechanical means of changing the irregular plastic elements of the mesenchymal syncytium into the endothelial cells of the vessel wall.

IV. Morphogenesis

Up to this point we have been considering the histogenetic changes which occur in the mesenchj^mal tissue, resulting in the formation of lymph channels and their endothelial lining and of blood cells. It has been demonstrated that aggregations of the developing blood cells, identical with the mesenchymal 'cords' described by Sala, appear along the lines of the future thoracic duct. It has been shown also that the rudiments of the thoracic duct develop as isolated spaces and channels in the mesenchyme, that the endothelial lining of the channels is derived directl}^ from the mesenchymal cells forming their borders and that the channels coalesce to form a plexus. It is our object, under the head of morphogenesis, to trace the subsequent history of the isolated channels and the plexus formed therefrom.

Chick embryo of six days and sixteen hours, 13.5 mm. {Columbia Embryological Collection, series no. 4^6). Reconstruction, ventral view. Figure 20. The incipient stages of thoracic duct development are shown about this time. The lymph spaces {17) are three in number, two on the right side and one on the left, situated in the mesenchyme ventro-lateral to the aorta {I) about midway between the exits of the celiac (-5) and superior mesenteric arteries. They are isolated, for absolutely not any divect connection with any other vessel can be traced even with high powers of magnification. They open freely into the surrounding intercellular spaces, as show in figures 12, 13 and 14, which are photographic reproductions of three successive sections of the embryo.

The spaces fit the previous description, given in the section on histogenesis, of accumulations of an intercellular fluid in the mesenchyme. The objection that they may be shrinkage spaces is nullified by the fact that the preservation of the tissue is practically perfect, and that in this and in other stages the more nearly perfect the preservation the more clearly defined are the spaces. Furthermore, in cases of poorer preservation where there are obvious shrinkage spaces in the mesenchyme the boundaries of such spaces are almost invariably ragged and irregular and do not anywhere exhibit a smooth endothelial lining.

The masses of developing blood cells in this particular embryo (six days and sixteen hours) are perhaps unusually extensive (fig. 20, 16). They extend in irregular groups from about the level of the superior mesenteric artery forward along the ventral and ventro-lateral aspect of the aorta (1) to the level of the celiac artery (5), with a tendency to cluster around the last named vessel (cf. fig. 7, 16). They then divide into two general lines, one on each side, which bend laterad and extend forward along the mesial aspect of the ducts of Cuvier (12) and precardial veins (10), ending rather abruptly in a large mass which hes at the level of the sixth aortic arches (3) and extends across the mesial line ventral to the dorsal aortic roots (2).

The three spaces representing the first anlagen of the thoracic duct (17) bear no particular relation to the large groups of developing blood cells (16), although a few isolated blood cells lie in the tissue spaces around the larger rudimentary lymph spaces.

Chick embryo of six days and twenty-one hours, llf. 7nm. (Columbia Collection, series no. ^65). Reconstruction, dextro-ventral view. Figure 21. In this embryo there is a considerable increase in the size and number of lymph spaces and channels which constitute the early anlagen of the thoracic duct (17). They are situated for the most part in the mesenchymal tissue ventral and ventrolateral to the aorta (i) near the exit of the celiac artery (5). Other spaces and channels of a similar nature are seen along the dorsal aortic root (2), thus continuing the same general line of lymphatics toward the jugular lymph sac (15) with its thoracic duct 'approach' {15a). Some of these early lymphatics are of considerable length and have acquired a distinct endothelial lining. Others are simply enlarged spaces in the mesenchyme exactly like those described in the preceding stage (figs. 13 and 14). There is not yet any plexiform arrangement of the channels.

The principle underlying the formation of the longer channels is in the main the enlargement and coalescence of two or more of the original smaller spaces or channels. For instance, two isolated spaces or channels lying near each other in the mesenchyme increase in size until eventually they flow together to form a single space or channel. Or, the same process occurs in several spaces or channels in a discontinuous series until all the members of the series flow together and thus form a continuous channel.

There is also in all probability some proliferation of the endothelium lining the channels. In fact there is no valid reason for not assuming that, after the inception of a given vascular channel, either hemal or lymphatic, along with its increase in size there is a concomitant proliferation of its endothelial cells. The crucial point, however, is the origin of the channels. The evidence at hand points clearly to the origin of the lymphatics constituting the rudiments of the thoracic duct directly from the mesenchymal interstitial spaces in the manner previously described, the endothelium of the lymphatics representing mesenchymal cells which are modified in accordance with the new conditions of pressure and friction.

In this embryo (series no. 465) the rudiments of the thoracic duct (fig. 21, 17) are isolated. There is no connection between these and the hemal vascular system; nor on the other hand is there any communication with the jugular lymph sacs (15). The thoracic duct 'approach' of the lymph sac, which the duct eventually joins, is well developed at this stage (15a) but a considerable distance intervenes between the 'approach' and the rudimentary duct.


The masses of developing blood cells in this embryo are unusually scarce. A few small groups (16) are situated in the mesenchymal tissue ventral to the aorta (1), and three larger groups (also marked 16) are seen between the dorsal aortic root (2) and the jugular vein (10).

Chick embryo of six days and twenty-two hours, 16 mm. (Columbia Collection, series no. 463). Reconstruction, ventral view. Figure 22. In this embryo the lymphatics (17) are in approximately the same stage of development as in the preceding embryo. They are situated for the most part along the ventral aspect of the aorta (1). A few spaces are situated ventro-lateral to the dorsal aortic roots (2).

The masses of developing blood cells (16) are much more extensive than in the preceding embryos of about the same stage, thus exhibiting the variability of the structures. The groups associated with the rudimentary thoracic duct (17) lie ventrolateral to the aorta (1), with some tendency to cluster around the celiac artery (5) . The rest of the groups extend in a continuous mass on each side along the ventro-lateral aspect of the dorsal aortic roots (2) and along the mesial aspect of the jugular veins (10) (cf. fig. 6, 16) as far forward as the fourth aortic arch (4)A large aggregation of developing blood cells (16a) also lies between and somewhat dorsal to the aortic roots (2) and the arches (3, 4) (cf. fig. 6, 16a), being connected with the left lateral groups by cords extending ventral and dorsal to the roots and arches. Associated with this large mass are a few lymphatics (19) (cf. fig. 6, 19) which subsequently join the thoracic duct and may also communicate with the cephalic end of the jugular lymph sac.

One of the most interesting features of this particular stage is the well developed thoracic duct ^approach' of the jugular lymph sac (fig. 22, 15a). A reconstruction to show this structure was made on a larger scale and is illustrated in figure 23. The lymph sac iself (13) lies dorsal to the jugular vein (10) and fits into the angle between the latter and the subclavian vein (11). The thoracic duct 'approach' (15a) is situated on the mesial aspect of the j ugular-subclavian angle and thence extends a short distance caudad along the mesial side of the precaval vein. It lies for the most part between a mass of developing blood cells {16) and the vein [10) and ends blindly (see also fig. 19, 16, 15a). A short distance farther caudad are two spaces in the mesenchyme which represent the extreme cephalic end of the rudimentary thoracic duct (fig. 23, 17). These spaces are isolated, for with high magnification there cannot be discerned any connection between them and the thoracic duct 'approach' {15a) or the other rudiments of the thoracic duct lying still farther caudad.

The thoracic duct 'approach' of the jugular lymph sac in the chick is without doubt the homologue of a similar structure" described by Huntington and McClure (9) in the cat. Like the lymph sac itself, it is of venous origin in the chick as in the mammal, and forms an integral part of the sac. It arises from some of the more mesially and caudally situated components of the early venous plexus in the region of the jugulo-subclavian angle. When fully formed it extends caudad and mesad for some distance along the mesial aspect of the precaval vein, and terminates blindly (figs. 19 and 23, 15a). Subsequently when it is joined by the thoracic duct, it serves as the portal of entry of this duct into the lymph sac.

Chick embryo of seven days {Columbia Collection, series no. 512). Reconstruction, ventral view in figure 24-; view from left side, figure 25. In this embryo there is considerable advance in the development of the rudiments of the thoracic duct. The spaces and channels, which in the preceding stage were unconnected, have here coalesced to form an extensive plexus {17) of channels extending from the level of the junction of the dorsal aortic roots {2) nearly to the exit of the superior mesenteric artery. The plexus lies for the most part ventral to the aorta {!), but a few of its components extend around on the lateral aspect of this vessel. Most of the channels in the network are much larger than the original spaces and channels, leaving but small areas of mesenchymal tissue among them; in part they have even fused to form irregular sinuses. The celiac artery (5) in most of its longitudinal course penetrates the plexus {17). The plexus as a whole is still isolated, being connected neither with any part of the hemal vascular sj^stem nor with the thoracic duct 'approach' of the jugular lymph sac {15a).


Along the dorso-lateral aspect of the aorta (1) a number of spaces and channels have also developed (fig. 25, 20). In addition, a few smaller lymphatics have appeared along the lateral aspect of the aorta. The dorsal aorta is thus almost completely encircled by a group of spaces and channels comprising the large ventral lymphatic plexus described in the preceding paragraph and the dorsal and lateral sets of lymphatics.

These encircling lymphatics are in the aggregate homologous with the dorsal set of peri-aortic sinuses in reptiles, as described by Huntington (2). They are also homologous, in all probability, with the azygos portion of the thoracic duct in the cat, as described by Huntington (3), and in Tragulus, as described by Tilney (20). A comparison with Stromsten's (16) description and figures indicates, too, that the lymphatics associated topographically with the dorsal aorta in the chick are collectively the homologue of the peri-aortic network of h'mph vessels in the loggerhead turtle, or, more specifically, with that portion of the network surrounding the dorsal aorta. In addition, the lymphatics around the dorsal aorta in the chick, as previously described, may be placed in the same phylogenetic line as the postcardinal and supracardinal divisions of the thoracic duct in the pig, as recently worked out by Kampmeier (15).

Returning to a further consideration of conditions in the chick at this stage (seven days, fig. 24), it is seen that a few small Ijnmph spaces {17a), also isolated, have appeared along the ventrolateral aspect of the dorsal aortic roots (2) in the interval between the plexus previously described {17) and the thoracic duct 'approach' of the jugular l}Tnph sac {15a). These small spaces represent the beginning of the connection between the thoracic duct 'approach,' on each side, and the unpaired portion of the thoracic duct itself, here composed of the large plexus {17).

The conditions in the chick thus correspond so closely to those in other forms that it is possible to draw a clear homology between the lymphatics joining the thoracic duct 'approach' to the unpaired portion of the duct itself in the chick and the preazygos segment of the thoracic duct in the cat (Huntington) and Tragulus (Tilney), with the cephalic portion of the peri-aortic lymph plexus in the loggerhead turtle (Stromsten), and with the precardinal division of the thoracic duct in the pig (Kampmeier).

In the mesenchyme between and dorsal to the aortic roots and arches in this embryo (fig. 24) a number of lymph spaces have developed. Some of these lie within the masses of developing blood cells in this region {16a) while others are situated at some distance from them. A few have coalesced to form a distinct channel (19). The subsequent history of these lymphatics will be given in the discussion of later stages.

A few isolated lymph spaces are also found in and near the root of the dorsal mesentery at the level where the celiac artery enters the mesentery (figs. 24 and 25, 21). These belong to the category of mesenteric lymphatics, but do not yet communicate with the developing thoracic duct.

The masses of developing blood cells in this embryo are extensive (figs. 24 and 25, 16, 16a). Those associated with the main portion of the developing thoracic duct lie either in the meshes of the lymph plexus (17) or are already included within the vessels composing the plexus. A plate-like mass extends over the mesial aspect of the pleural cavity on the right side. The blood cells still, as in preceding stages, tend to cluster about the celiac artery (5). There is no connection in this stage between the masses just mentioned and those which lie farther forward along the mesial aspect of the great veins (10, 12), and which are associated with the thoracic duct 'approach' of the jugular lymph sac (15a). In the region between and dorsal to the dorsal aortic roots (2) and aortic arches (3) there is seen a large mass (16a) with which certain lymphatics (19), previously referred to, are associated. Situated farther caudad are also several masses associated with the lymphatics which lie along the dorso-lateral aspect of the aorta (fig. 25, 16a).

A feature shown in this reconstruction, and not in any other, is a portion of the splanchnic plexus of veins (figs. 24 and 25, IJf).^

' Splanchnic venous plexus is the name given by Dr. A. J. Brown, in his yet unpublished work on the development of the pulmonary veins, to the network of venous capillaries in the wall of the alimentary tube in the earlier stages of development.


This is seen near the cephalic end of the large ventral lymph plexus {17) but has no connection with the latter.

At this point it may be well to state that in the chicks of the Columbia Collection I have found no evidence of extra-intimal or perivenous origin of the lymphatics that make up the thoracic duct, such as described by Huntington in the cat and Kampmeier in the pig. This mode of development appears to be a strictly mammalian specialization. In fact Huntington (3), speaking of the extra-intimal replacement of veins by developing lymphatics, states (p. 155) :

The association of these (Ij^mphatic) channels, in the mammalian embryo, with certain embryonal venous lines is purely a secondarj^ mechanical and topographical relationship, expressed by the condensed term of 'extra-intimal' development of mammalian systemic lymphatic vessels, and absolutely devoid of genetic significance. This is, without reference to other vertebrate classes, proved by the development within restricted areas in the mammalian embryo of systemic lymphatic channels through the direct confluence of intercellular mesenchymal clefts, not related topographically or in any other sense to the embryonal veins. It is true that in the mammal this independent lymphatic genesis is extremely limited, and that the majority of the lymphatic vessels develop in close association with embryonal veins, as products of the confluence of perivenous extra-intimal spaces. But this is merely, as shown by comparison with other amniote embryos, the expression of the peculiar relations obtaining in the mammal between the venous and lymphatic circuits of the vascular system, developed independently of each other.

Further, Huntington (2), describing the independently formed system lymphatic channels of the reptilian embryo, by confluence of intercellular mesenchymal spaces, states concerning the latter:

They are not complicated by close topographical relations to adjacent temporary embryonic venous plexuses, as in the mammal, but develop independently by themselves in mesenchymal territory not occupied by hemal vascular elements In both lacertilian and chelonian embryos the greater part of the enormously enlarged systemic lymphatic channels develop without any reference whatever to embryonic veins, in mesenchymal areas where the latter are extremely scanty or entirely wanting (pp. 271-273).

Stromsten (16) has reached similar conclusions in studying the development of the prevertebral peri-aortic sinuses in chelonian embryos.


These finding in reptiles, and my own results in the bird, warrant the conclusion that the sauropsids agree absolutely genetically with the mammals in the development of the main axial lymphatic lines bj^ confluence of independently formed intercellular mesenchymal spaces, but that the latter are characterized by close topographical association of these spaces with temporary embryonic veins which they in large part replace, while the former present no such association. In them the progress of thoracic duct development is not, as is the case in the mammal, complicated by the presence of an extensive azygos venous system, and axial lymphatic development, especially in the reptile, occurs chiefly by confluence of mesenchymal spaces surrounding the main arterial trunks.

Chick embryo of eight days {Columbia Collection, series no. 513). Reconstruction, ventral view. Figure 26. The next important change in the lymphatics constituting the thoracic duct comprises the further development of certain isolated spaces situated along the ventro-lateral aspect of the dorsal aortic roots in the interval between the cephalic end of the large ventral plexus and the thoracic duct 'approach' of the jugular lymph sac. The incipient stage in the formation of these spaces was illustrated in flgure 24, 17a. In the embryo now under consideration they have enlarged and coalesced to form a continuous channel (figure 26, 17a). This in turn has united with the ventral unpaired portion of the thoracic duct {17) and with the thoracic duct 'approach' of the lymph sac {15a). There is thus established a direct and free communication between the ventral lymph plexus {17), which had arisen as an independent and isolated structure, and the jugular lymph sac {15). Therefore, as clearly shown in figure 26, the thoracic duct 'approach' {15a), which was previously described as an integral part of the jugular Ijanph sac (see fig. 23, 15a), serves as the portal of entry of the thoracic duct into the lymph sac (fig. 26, 15).

The writer has in a previous article (1) shown and will here again point out in a later stage that in the chick, as in reptiles and mammals, a communication is established between the jugular lymph sac and the great veins in this region through one or more taps. And inasmuch as the thoracic duct opens into the lymph sac, the latter serves as the portal of entry of the systemic lymphatics into the venous system, a point upon which emphasis has already been laid by Huntington (2) (3) in his work on reptiles and the cat.

In the embryo of eight daj's the large ventral lymph plexus of the preceding stage (cf. fig. 24, 17) has undergone further coalescence of its component channels to form a large irregular sinus with a few fenestrae (fig. 26, 17). The sinus lies ventral to the aorta (1) and extends from the junction of the dorsal aortic roots {2) to the level of the exit of the superior mesenteric artery {9). At its cephalic end it branches off into the two slender trunks, one on each side, which extend cephalad and laterad to join the 'approaches' {15a) of the lymph sacs {15). These trunks, which are the last components of the Ijrmphatic drainage line to develop, constitute in the bird, according to the anatomical terminology, the right and left thoracic ducts.

The lymphatics (19) situated between and dorsal to the aortic roots {2) and arches {3, Jf) are here seen to comprise a plexus and a few outlying isolated spaces and channels (cf. fig. 24 and 26, 19). This plexus now communicates with the chain of lymphatics {20) lying dorso-lateral to the aorta ( / ) , which in* turn communicate with the large ventral plexus through channels formed by coalescence of spaces lateral to the aorta (cf. fig. 25, 20, 17). The entire group of lymphatics in the region of the dorsal aortic roots and the dorsal aorta as far back as the superior mesenteric artery, with the exception of the mesenteric lymphatics (fig. 26, 21), which have not yet joined the thoracic duct, drain into the jugular lymph sacs.

The masses of developing blood cells were omitted from the reconstruction of the eight day stage (fig. 26). A careful study of the serial sections showed that they were fewer and smaller than in the preceding stage. This may be due to the variability characteristic of the masses, or it may be due to actual reduction of the masses since the blood cells, as stated in in the section on histogenesis, now have access to the jugular lymph sacs through the recent connection established between these structures and the chain of lymphatics with which the blood cells have for the most part been associated.

Chick embryo of nine days and fourteen hours {Columbia Collection, series no. 320). Reconstruction, ventral view and viewfrorn left side. Figures 27 and 28. At this stage the main features of the adult thoracic duct have been established. The large plexus {17) ventral to the aorta {1) now drains through the right and left thoracic ducts {17a) into the 'approaches' {15a) of the jugular lymph sacs {15) and then through the sacs into the great veins {10, 12).

The ventral plexus {17), the isolated anlagen of which were the first lympathics to develop in the thoracic duct line, is relatively smaller and the component channels less dilated than in the preceding stages. This is probably due to the outflow of the contents into the lymph sacs and veins through the more recently formed channels which have been called the right and left thoracic ducts {1 7a) . The mesenteric lymphatics {21 ) have increased in size and for the most part coalesced to form sinus-like channels. Between these and the thoracic duct chain no connection can be detected at this stage, although little tissue intervenes. Farther cephalad another isolated group of lymphatics {22) is associated with the esophagus.

The right and left thoracic ducts {17a) are longer than in the preceding stage (cf. fig. 26, 17a). The left is considerably greater in diameter than the right. Each opens into the corresponding 'approach' {15a) of the jugular lymph sac. The 'approach' on each side is still patent as a branch of the main portion of the sac.

There is now free communication between the lymph sacs {15) and the great veins through recently formed taps, of which two are present on the left side of the embryo and one on the right. Of the two on the left side, one is situated on the dorsomesial side of the superior vena cava {12) just below the level of the jugulo-subclavian junction {10, 11); the other is situated farther forward on the mesial side of the jugular vein {10). The tap on the right side of the embryo is located on the dorso-mesial side of the superior vena cava at the level of the jugulo-subclavian junction.


In a previous article on the development of the jugular lymph sac in birds (1) the writer stated, in the description of tap formation, that "it is not improbable that a study of later stages will reveal a homologue of the common jugular tap in the mammal" (loc. cit,, p. 486). There is little doubt that the tap on the mesial side of the left jugular vein, referred to in the preceding paragraph, fulfils the requirement. Moreover, the other tap on each side near the jugular-subclavian junction in the chick is in all probability homologous with the jugulo-subclavian tap in the mammal (26), although it is slightly different in position.

The previously described lymphatics situated dorsal to the aortic roots and arches and dorso-lateral to the aorta here constitute a long chain of continuous channels reaching from the level of the jugular lymph sac to the level of the celiac artery (figs. 27 and 28, 19, 20; cf. figs. 24, 25 and 26, 19, 20). At the extreme cephalic end one of the channels of this series opens into the right lymph sac. The probable significance of this opening will be considered in the subsequent discussion of the masses of developing blood cells. The plexus [19) in the region of the aortic arches is considerably reduced as compared with preceding stages. The portion of the dorsal plexus {20) associated with the dorsal aorta communicates with the large ventral plexus {17) through small channels which curve around the lateral aspect of the aorta. It is seen, therefore, that the entire group of lymphatics associated with the aorta and dorsal aortic roots, with the exception of a few still isolated spaces and channels, can now discharge the contents of the channels into the great veins.

The most interesting and, in fact, from the standpoint of the hemophoric* function of the thoracic duct, the most important feature of this stage is the great reduction in size and number of the masses of developing blood cells. It has been shown in the foregoing pages that the differentiating blood cells associated with the developing lymphatics are admitted or gain access to the lymph spaces and channels and that when the communication is established between the thoracic duct and the jugular lymph sacs the blood cells can thus reach the sacs. Now since the taps between the lymph sacs and great veins have been formed, the blood cells are admitted to the hemal vascular system.


Hemophoric — blood bearing or carrying — is a term suggested by Dr. Schulte as a coordinate with hemopoietic — blood producing.


In this connection there are two aspects of the question which are especially worthy of consideration. In the first place there are few blood cells within any of the channels forming a part of the thoracic duct system, while in earlier stages, before the thoracic duct had open communication with the lymph sacs and great veins, many of the lymph channels were filled with the hemal cellular elements. In view of this, and the fact that there are no other means of egress for the blood cells formerly contained in the lymphatics, it must be concluded that the blood cells reach the hemal vascular system via the thoracic duct, the jugular lymph sacs and the recently formed taps between the sacs and the great veins.

The other feature is the great reduction in the size and number of the extravascular masses of developing blood cells. The diminished masses are shown in yellow in figures 27 and 28, the conditions in which should be compared with those in figures 22 and 24. A few fairly extensive groups are found in the mesenchymal tissue among the channels composing the large ventral plexus [17). These are for the most part quite closely associated with the lymphatics. A few small groups still are found in the region of the thoracic duct 'approach' (fig. 28, 15a). The extensive masses associated in earlier stages with the lymph plexus dorsal to the aortic arches have almost wholly disappeared. This plexus itself is considerably reduced, and it is not unreasonable to assume that the connection between the cephalic end of the plexus and the jugular lymph sac, previously alluded to, is in some way associated with the discharge of the numerous blood cells differentiated in this region into the lymph sac.

In view, therefore, of the intimate relationship between the developing thoracic duct and blood cells in the same general region, and of the sudden and marked reduction in number of these blood cells, both intravascular and extravascular, following the establishment of communication between the duct and the jugular lymph sacs and great veins, the importance of the thoracic duct as a carrier of hemal cellular elements for a period of embryonic life in the bird can scarcely be doubted.


V. Summary

Prior to the appearance of lymphatics in the region of the future thoracic duct, namely, along the aorta and dorsal aortic roots, the mesenchyme comprises a syncytium of irregular strands with correspondingly irregular interstitial spaces. The tissue is nonvascular.

The initial change in conditions is manifested in the appearance of distinct lacunae in the mesenchymal tissue along the ventro-lateral aspect of the aorta at the level of the celiac artery. The lacunae are bounded by unmodified protoplasmic elements of the mesenchymal syncytium, and open freely into the adjacent intercellular — interstitial — spaces. Obviously the lacunae represent enlarged intercellular spaces, and the inference is justifiable that they are filled with the intercellular fluid.

In a slightly advanced stage of development in general there is a greater number of lacunae in the same region in the embryo and also an increase in size and a difference in the appearance of some of the lacunae. The increase in size certainly depends in part upon actual dilatation and in part upon addition of more of the adjacent mesenchymal intercellular spaces, for every possible gradation can be seen between the smallest and the largest. The difference in appearance is observed to be due to the presence of flat cells which form a distinct boundary or wall, although not usually complete. Morphologically these cells are equivalent to endothelial cells. Inasmuch as they shade by invisible gradations into the unmodified mesenchymal cells bordering upon the rest of the lacuna, we conclude that they are derived directly from the indifferent mesenchymal cells. The differentiation, we may also infer, is due in part to pressure and friction incident to the flow of the tissue fluid. Another factor in the differentiation may also be pressure and friction incident to the to and fro motion of blood cells in the tissue spaces and lacunae in response to the heart-beat, a phenomenon observed in living blastoderms.

Studies of later stages show these isolated lacunae to be the rudiments of the thoracic duct, and the conclusions that they are direct derivatives of the mesenchymal intercellular spaces and


160 ADAM M. MILLER

that the flat cells forming their endothelial walls are differentiated in situ from the mesenchymal cells are based upon prolonged and thorough studies, with high magnification, of serial sections of chick embryos in practically perfect states of preservation. The writer is therefore forced to ally himself unequivocally with the advocates of the view that the thoracic duct originates independently of the veins and lymph sacs.

Studies of subsequent stages also show that the numerous isolated lacunae, or rudiments of the thoracic duct, enlarge still further, principally in a longitudinal direction, and coalesce with one another to form a plexus of lymph channels which lies ventral to the aorta. Other similar isolated lymphatics develop along the dorso-lateral aspect of the aorta and in the region dorsal to the aortic roots and arches, and then coalesce to fonii plexuses. Eventually all the plexuses intercommunicate.

In the meantime a connection is established between the large ventral plexus and a branch of each jugular lymph sac known as the thoracic duct 'approach,' All the components of the thoracic duct system thus drain into the jugular lymph sacs. Communications, or taps, are established between the lymph sacs and the great veins, and the thoracic duct then drains into the hemal vascular system, the lymph sacs serving as portals of entry.

In the region of the developing thoracic duct, namely, along the aorta and dorsal aortic roots, and also in the region dorsal to the aortic arches, a great number of blood cells arise. The genesis of these cells is indicated by certain changes in some of the irregular elements of the mesenchymal syncytium, comprising a marked increase in the basophilia of the cytoplasm, a rounding of the cell body and a separation from the general mesenchymal reticulum. The resulting cells thus lie free in the interstitial spaces, and structurally are similar to the large mononuclear cells (lymphocytes of Dantschakoff) in the area vasculosa of the blastoderm. They increase in number both by mitosis and by constant differentiation from the mesenchymal syncytium.

Many, at least, of these basophilic cells are transformed into erythrocytes through the addition of hemoglobin to the cytoplasm and certain nuclear modifications comprising the disappearance of the nucleoli and the rearrangement of the chromatin into a heavy reticulum.

These developing blood cells, at first scattered in the mesen-chymal intercellular spaces, become aggregated, following the increase in their number, into extensive masses which lie along the line of the thoracic duct and also in the region dorsal to the aortic arches.

When the lymphatics comprising the rudiments of the thoracic duct develop, some of the developing blood cells, even some of the smaller groups, are seen to be contained within them, while others are still free in the mesenchymal intercellular spaces, that is, extravascular. Subsequently more and more of the cells are observed to be intravascular. It may be concluded that the developing blood cells become intravascular by simple inclusion as the lymph channels develop, or by virtue of their ameboid character, or as a result of their motion to and fro in the tissue fluid in response to the heart-beat.

The blood cells that are admitted to the lymph channels constituting the thoracic duct system during its development rapidly diminish in number after communication is established between the thoracic duct and the jugular lymph sacs and between the latter and the great veins. It can be inferred then that the blood cells which develop along the line of the thoracic duct reach the blood stream via this duct and the lymph sacs. Considering the vast number of hemal cellular elements, especially erythrocytes, arising in this region and the probability that they reach the general circulation via the thoracic duct, this duct assumes an additional phase of importance in the chick in that it performs a hemophoric, or blood carrying, function.


In conclusion, I wish to thank Dr. Huntington and Dr. Schulte for their valuable criticism and suggestions. Dr. McWhorter for his painstaking work in making the photomicrographs, and Mr. Petersen for his careful execution of the color plates.


References

1) Miller, A. M. 1912 The development of the jugular lymph sac in birds. Amer. Jour. Anat., vol. 12.

2) Huntington, George S. 1911 The development of the lymphatic system in reptiles. Anat. Rec, vol. 5.

3) 1911 The anatomy and development of the systemic lymphatic vessels in the domestic cat. Memoirs of The Wistar Institute of Anatomy and Biology, no. 1, May.

4) HoYER, H. 1905-1908 Untersuchungen iiber das Lymphgefasssystems der Froschlarven. Bull, de I'acad. des sciences de Cracovie, classe des sciences math, et nat., I Theil, 1905; II Theil, 1908.

5) Knower, H. McE. 1908 The origin and development of the anterior lymph hearts and the subcutaneous lymph sacs in the frog. Anat. Rec, vol. 2.

6) Lewis, F. T. 1905 The development of the lymphatic system in rabbits. Amer. Jour. Anat., vol. 5.

7) Sarin, Florence R. 1909 The lymphatic system in human embryos, with a consideration of the morphology of the system. Amer. Jour. Anat., vol. 9.

8) MiERZEjEWSKY, L. 1909 Beitrag zurEntwicklung desLymphgefiisssystems der Vogel (vorlaufige Mitteilung). E.xtrait du bull, de I'acad. des sciences de Cracovie.

9) Huntington, G. S., andMcClure, C. F. W. 1910 The anatomy and devel opment of the jugular lymph sac in the domestic cat. Amer. Jour. Anat., vol. 10.

(10) Sala, L. 1900 Sullo sviluppo dei cuori limfatici e dei dotti toracici nell'

embryone di polio. Ricerche fatta nel Laboratorio di Anatomia Normale della R. Univ. di Roma, vol. 7.

(11) Sarin, Florence R. 1902 On the origin of the lymphatic system from

veins, and the development of the lymph hearts and thoracic duct in the pig. Amer. Jour. Anat., vol. 1.

(12) Langer, C. 1868 tJber d^s Lymphgefiisssystem des Frosches. Sitz. d.

Akad. d. Wissensch., Bd. 57.

(13) Ranvier, L. 1897 Morphologie et developpement des vaissaux lympha tiques chez les mammiferes. Arch, d'anatomie microscopique, tome 1.

(14) Huntington, George S. 1908 The genetic interpretation of the develop ment of the mammalian lymphatic system. Anat. Rec, vol. 2.

(15) Kempmeier, Otto F. 1912 .The development of the thoracic duct in the

pig. Amer. Jour. Anat., vol. 13.

(16) Stromsten, Frank A. 1912 On the development of the prevertebral (tho racic) duct in turtles as indicated by a study of injected and uninjected embryos. Anat. Rec, vol. 6.

(17) Dantschakoff, W. 1908 Untersuchungen iiber die Entwickelung des Blutes und Bindegewebes bei den Vogeln. I. Die erste Entstehung der Blutzellen beim Hiihnerembryo und der Dottersack als blutbildenea Organ. Anatomische Hefte, 113 Heft (37. Band, Heft 3).


(18) McWhorter, J. E., AND Whipple, A. (). 1912 The development of the blastoderm of the chick in vitro. Anat. Rec, vol. 6.

(1^9) Thoma, R. 1893 Untersuchungen iiber die Histogenesc vind Histomechanik des Blutgefjisssystems. Stuttgart.

(20) TiLXEY, F. 1912 The development of the veins and lymphatics in Tragu lus meminna. Amer. Jour. Anat., vol. 13.

(21) ^IcClure, C. F. W. 1908 The development of the thoracic and right lym phatic ducts in the domestic cat. Anat. Anz., Bd. 32, nos. 21 and 22.

(22) Sabix, Florence R. 1911 A critical study of the evidence presented in

several recent articles on the development of the lymphatic system. Anat. Rec, vol. 5. .(23) ]\IcClure, C. F. W. 1910 The extra-intimal theory and tlie development of the mesenteric lymphatics in the domestic cat (Felis domestica). Anat. Anz., Erganz. z. Bd. 37.

(24) Sabix, Florexce R. 1912 On the origin of the abdominal lymphatics in

mammals from the vena cava and the renal veins. Anat. Rec, vol. 6.

(25) HuxTixGTON, Geo. S., and McClure, C. F. W. 1907 The development of

the main lymph channels of the cat in their relations to the venous system. Anat. Rec, vol. 1, no. 3.

(26) McClure, C. F. W., and Silvester, C. F. 1909 A comparative study of

the lymphatico-venous communications in adult mammals. Anat. Rec, vol. 3.


Plates

PLATE 1


EXPLAXATIOX OF FIGURES


1 From a section of a chick embryo of 108 hours, 6.75 mm. (series no. 371; slide II, section 80). Outlines drawn under Edinger projection apparatus, X 1500; reduced to XlOOO. In the upper left corner is a portion of the aorta with a few erythrocytes. Outside of the aortic wall is an area of mesenchyme in which some elements show stages of differentiation leading toward large basophilic cells, two of the latter lying free in the tissue spaces at the lower left corner of the figure. These are apparently identical with the developing blood cells (lymphocytes) in the area vasculosa of the blastoderm as described by Dantschakoff.

2 From the same embryo as figvu-e 1 (slide II, section 97). Outlines drawn under Edinger i^rojection apparatus, X 1500; reduced to X 1000. A small part of the aorta is shown in the upper right corner. Some of the protoplasmic elements of the mesenchymal syncytium are shown in various stages of differentiation leading toward the free basophilic cells (lymphocytes of Dantschakoff). In the lower left corner is seen one of these cells in the anaphase of mitosis. At the right of this is a practically mature erythrocyte lying free in a mesenchymal intercellular space.

3 From a section of a chick embryo of 132 hours, 9 mm. (series no. 411; slide IV, section 16). Outlines drawn under Edinger projection apparatus, X 1500; reduced to X 1000. This figure, taken just ventral to the aorta, shows a number of the rounded basophilic cells lying free in the mesenchymal intercellular spaces.


PLATE 2

EXPLANATiOX OF FIGURES


4 From a section of a chick embryo of o (lays, 12 mm. (series no. 336; slide IV, section?). Photomicrosi'aph, X 166.

1, aorta 10, preeardinal vein

3, aortic arch VI 16, grouj) of developing blood cells

4, aortic arch 1\

5 From a section of a cfiick embryo of 6 days, 13.5 mm. (series no. 339; slide VIII, section 35). Photomicrojirajjh, X 166.

2, dorsal aortic root 16, groups of developing blood cells

3, aortic arch VI 27. vagus nerve


PLATE 3


EXPLANATION OF FIGURES


6 From a section of a chick embryo of 7 days (series no. 512; slide III, section 15). Photomicrograph, X SO.

2, dorsal aortic root 19, lymphatics dorsal to aortic arches

3a, pulmonary artery and esophagus

10, precardinal vein 27, vagus nerve

16, groups of developing hhjod cells esophagus in center of figure

along precardinal vein bronchi at lower border of figure

16a, groups of developing blood cells

dorsal to esophagus

7 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm (series no. 426; slide XI, section 21). Photomicrograph, X 133.


1, aorta

5, celiac artery

8, dorsal somatic artery

16, groups of developing blood cells


23, celom (pleural cavity)

25, mesonephros

26, lung anlage


PLATE 4


EXPLANATION OF FIGURES


8 From a section of a chick embryo of 6 daj's and 22 hours, 16.5 mm. (series no. 464; slide XXII, section 22). Photomicrograph, X 133.

1, aorta 16, groups of developing blood cells

5, celiac artery 23, celom (pleural cavity)

8, dorsal somatic artery 24, sympathetic nerves

9 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm. (series no. 426; slide XVIII, section 3). Photomicrograph, X 233.

1, aorta 17, lymph spaces, rudiments of thoracic

16, developing blood cells duct

23, celom (abdominal cavity)



PLATE 5


EXPLANATION OF FIGURES


10 From a section of a chick embryo of 7 days (series no. 512; slide VII, section 1). Photomicrograph, X 233.

1, aorta 17, lymphatics, rudiments of thoracic

5, celiac artery duct

16, groups of developing blood cells 23, celom (abdominal cavity)

11 From a section of a chick embryo of 14 days (series no. 518; slide XIII, section 9). Photomicrograph, X 133.

1, aorta 17, lymphatics (part of thoracic duct

16, blood cells, mostly mature erythro- system)

cytes, in tissue spaces


PLATE 6


EYPLAXATIOX OF FIGURES


12, 13, 14 From tliree saccessive .sections of a chick einbryo of (i days and 16 hours, 13.5 mm. (series no. 42(i: slide XII. sections 9, 10 and 11). Photomicrographs, X 350.

In figure 13 the arrow (/?) points to a lymph space in the mesenchyme. At its right side the lymph space is seen clearly to open into the adjacent mesenchymal intercellular spaces. The preceding section in the series (fig. 12) shows no space (the arrow points to the same locality as in fig. 13). In figure 14 the arrow points to a light area in the mesenchyme which represents the opening of the space of figure 13 into the adjacent mesenchymal intercellular spaces; this can be seen much more clearly with the microscope by changing the focus. The lymph space shown in figure 13 is the same one represented in figure 20. 17 at the right of the aorta. Aorta. /; celom (abdominal cavity), 23.


PLATE 7

EXPLANATION OF FIGURES

15, 16, 17 and 18 From four successive sections of a chick embryo of 7 days (series no. 512; slide VII, sections 21 and 22; slide VIII, sections 1 and 2). Photomicrographs, X 250.

In figure 15 four lymph spaces, in part lined with endothelium, are seen at the left of the aorta (1). The one nearest the aorta, while in part lined with endothelium, opens freely into the adjacent mesenchymal intercellular spaces. The largest space also opens below in a similar manner. In figure 16, from the succeeding section in this series, only one of the spaces appears, opening above into tissue spaces. The others have simply become continuous with tissue spaces and therefore do not appear as distinct lacunae; this can be clearly demonstrated with the microscope by changing the focus on the rather thick sections (20 micra). In figure 17 the arrow points to the termination of the distinct lacuna of figure 16; the free communication with the tissue spaces is quite obvious. In figure 18 there are no lacunae, all those of the preceding sections having opened into the mesenchymal intercellular spaces. Aorta, 1; part of sympathetic nervous system, 24-



PLATE S

EXPLAXATIOX OF FIGURE

Ul From a section of a chick embi\vo of 6 days and 22 hours, 16 mm. (series no. 463; sliiU' X\'I, section 9). Photomicrograph, X 160.

£, dorsal aortic root 15, jugular lymph sac

3(1. pulmonary artery 15a, thoracic duct 'approach'

12. precaval vein 27, vagus nerve

13, vertebral vein


PLATE 9


EXPLANATION OF FIGURE


20 Drawn from a reconstruction of a chick embryo of 6 days and 10 liOLirs, 13.5 mm. (series no. 426). Ventral view.


1, aorta

2, dorsal aortic roots

3, aortic arch VI

3(1, pulmonary artery

4, aortic arch IV

5, celiac artery

10, precardinal vein


11, subclavian vein

12, duct of Cuvier

13, vertebral vein

15, jugular lymph sac

16, groups of developing blood cells

17, first rudiments of thoracic duct



PLATK 10


EXPLANATIOX OF FIGURE


21 Dniwn from a reconstruction of a chick cnihrvo of (i days and '_M hours, 14 mm. (series no. 465). Ventro-mesial view.

1, aorta ^^. duct of Cuvier

2, dorsal aortic roots ^.?, vertebral vein

3, aortic arch VI 15, jugular lymjih sac

3n, pulmonary artery 15a, thoracic duct 'approach' of jugular

5, celiac artery lymph sac

6, notocord 16, groups of develo))ing blood cells

10, precardinal vein 17, rudiments of thoracic duct

11, subclavian vein


PLATE 11


EXPLANATION OF FIGURE


22 Drawn from a reconstruction of a chick embryo of 6 days and 22 hours, 16 mm. (series no. 463). Ventral view.


I , aorta

£, dorsal aortic root

3, aortic arch VI

3a, pulmonary artery

4, aortic arch IV

5, celiac artery

10, precardinal vein

II, subclavian vein 1°2, duct of Cuvier 13, vertebral vein

15, jugular lymph sac


15a, thoracic duct 'approach' of jugular lymph sac

16, groups of developing blood cells 16a, groups of blood cells dorsal to

aortic roots and arches

17, rudiments of thoracic duct

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots and arches

21, mesenteric lymphatics


PLATE 12


EXPLANATION OF FIGURE


23 Drawn from a reconsti-uftion of a chick embryo of 6 days and 22 hours, 16 mm. (series 463). Mesial view.

10, prccardiiial vein 15a, thoracic duct 'apiiroach' of jugular

11, suhclavian vein lymph sac

12, duct ofCuvicr 16, groups of developing blood cells 15. jugular lymi)h sac 17, rudiments of thoracic; duct, extreme cephalic end cuse.




PLATE 13


EXPLANATION OF FIGURE


24 Drawn from a reconstruction of a chick embryo of 7 days (series no. 512) Ventral view.


1, aorta

2, dorsal aortic roots

3, aortic arch VI

3n, pulmonary artery 5, celiac artery 7, carotid artery

10, precardinal vein

11, subclavian vein

12, duct of Cuvier

14, part of splanchnic plexus of veins

15, jugular lymph sac

15a, thoracic duct 'approach' of jugular lymph sa:'


16, groups of developing blood cells 16a, blood cells dorsal to aortic roots

and arches

17, thoracic duct, ventral plexus (homologue of azygos segment)

17a, thoracic duct (homologue of i)reazygos segment)

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots and arches

21, mesenteric lymphatics


PLATE 14


EXPLANATION OF FIGURE


25 From same reeonstnu-tioii as in fig. 24. View from left side.


I, aorta

3, aortic arch VI 5, celiac artery

7, carotid artery

8, dorsal somatic arteries 10, ])recardinal vein

II, subclavian vein 12, duct of Cuvier IS, vertebral vein


14, part of splanchnic plexus of veins

15, jugular lymph sac

16, groups of developing blood cells 16a, blood cells dorsal to aortic roots

17, thoracic duct, ventral plexus

18, lymphatics along aortic arches 20, lymphatics dorso-lateral to aorta ?/, mesenteric lyniphatic^s


190


PLATE 15


EXPLANATION OF FIGURE


26 Drawn from a reconstruction of a chick embryo of 8 days (series no. 513). Ventral view.


1, aorta

2, dorsal aortic roots

3, aortic arch VI

3a, pulmonary artery

4, aortic arch IV

5, celiac artery

6, notocord

7, carotid artery

9, superior mesenteric artery

10, precardinal (jugular) vein

11, subclavian vein

12, duct of Cuvier 15, jugular lymph sac


15a, thoracic duct 'approach' of jugular lymph sac

17, thoracic duct, ventral plexus (homologue of azygos segment

17a, thoracic duct (homologue of preazygos segment)

18, lymphatics along aortic arches and duct of Cuvier

19, lymphatics dorsal to aortic roots and arches

20, lymphatics dorso-Iateral to aorta

21, mesenteric lymphatics


PLATE 16


EXPLANATION OF FIGURE


27 Drawn from a reconstruction of a chick embryo of days and 14 hours (series no. 320). Ventral view.


1, aorta

^, dorsal aortic roots

3, aortic arch VI

3a, puhnonary artery

4, aortic arch IV

5, celiac artery 7, carotid artery

9, superior mesenteric artery

10, p ecardinal (juguhir) vciii //, subchivian vein

12, duct of Cuvier 15, juguhir lymph sac 15(1, thoracic duct 'approach' of jugular lymph sac


16, groups of developing blood cells

17, thoracic duct, ventral plexus (honiologue of azygos segment.

17a, thoracic duct (homologue of preazygos segment.

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots and arches

£1, mesenteric lymphatics ^^, lymphatics associated with esophagus



PLATE 17


EXPLAXATION OF FIGURE

28 From same reconstruction as fijjuro 27. View from left side.


I, aorta

3, dorsal aortic roots 3, aortic arch VI

7, carotid artery

8, dorsal somatic arteries

.9, superior mesenteric artery 10, precardinal (jugular) vein

II, subclavian vein

12, duct of Cuvier

13, vertebral vein

15, jugular lymph sac


15a, thoracic duct 'approach' of jugular lymph sac

16, groups of developing blood cells

17, thoracic duct

18, lympathics along aortic arches

19, lymphatics dorsal to aortic roots and arches

20, lymphatics dorso-lateral to aorta £1, mesenteric lymphatics

£2, lymphatics associated with esophagus

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