The Origin and Development of the Anterior Lymph-Sacs in the Sea-Turtle (Thalasso- Chelys Caretta)

The Origin and Development of the Anterior Lymph-Sacs in the Sea-Turtle (Thalasso- chelys caretta). By E. R. Van Der Jagt,

University of Iowa.

With 6 Text-figures.

INTRODUCTION. IT requires only a hasty inspection of the literature on the lymphatic system to show the unsatisfactory state of our know- ledge concerning its development. In the first place, the onto- geny and phylogeny stand in apparent conflict. There are, moreover, few features of its development upon which there is any agreement among the various investigators who have con- tributed to this field. As a reworking of the subject, the author was guided in choosing the turtle, not only because of the con- troversy over the origin of the anterior lymph-sacs, but also because in this form the embryonic anlagen of the system are easily distinguished from the blood-vessels. In 1911 G. S. Huntington published a paper on the origin of the lymphatic system in reptilian embryos. Since his results and conclusions did not present fully all the evidence, or agree with the results of later investigators, E. L. Clark (1912), Kampmeier (1912), Stromsten (1912), Professor Stromsten sug- gested to the writer the advantages of repeating his work, but limiting the investigation to the turtle. For the completion of this work, I am indebted to Professor Stromsten for his valuable suggestions and helpful criticism during the investigation. MATERIAL AND TECHNIQUE. The embryos of Thalassochelys caretta used in this work were collected by Dr. Stromsten at the Marine Laboratories of the Carnegie Institution of Washington at the Dry Tortugas, 152 E. R. VAN DER JAGT Florida. They were fixed in a mixture of chromic acid, glacial acetic, and 40 per cent, formaldehyde. In the advanced stages the animals were narcotized before fixation in order that the delicate mesenchymal tissue might not be injured by the move- ments of the embryo when first placed in the fixing solution. Throughout the preparation of sections for final mounting care was taken to prevent shrinking or tearing of the tissues. Sections were cut from 10 to 20 micra in thickness, and were stained with Delafield's haematoxylin, iron haematoxylin, or in to to with alum or borax-carmine. They were counter- stained with orange-G (slightly acidulated), eosine-aurantia- orange-G, and Mallory's connective-tissue stain. The observations reported are based wholly upon the study of sections of embryos and camera lucida drawings, which were made serially of all stages in the region under investigation. The drawings were made on cellophane and the cellophane method of reconstruction was used.1 It is the purpose of this paper to present results of a study of development of the lymph-sacs as well as a determination of their anlagen. By the study of serial sections it is shown that independent lymph-spaces occupy the region of the developing sac, and that these spaces by fusion aid in the formation of a plexus which later becomes transformed into the jugular sac.

OBSERVATION AND DISCUSSION. It is evident from the literature (see bibliography) that there are three views concerning the development of the lymph- hearts : 1. The lymph-hearts arise from the veins at various centres of radiation and by continuous elongation and fusion form the hearts. 2. The lymph-hearts are derived from the embryonic venous system either by a direct transformation of certain of its channels or by the fusion of multiple derivatives which have become detached from it.

i Van Der Jagt, E. R., "An improvement in the technique of reconstruction work by the use of cellophane", 'Science', Dec. 1931. LYMPH-SACS OF TURTLE 158 3. The lymph-hearts arise by the confluence of mesenchymal spaces which invest and communicate with the capillaries. The present observations and studies favour the third view which holds that the lymph-hearts and sacs are initiated in development by the vacuolation of the mesenchyme. They are not a product of the veins either by centrifugal growth or the fusion of detached venous elements. The endothelial lining of the lymph-sacs is a gradual differentiation from the mesenchymal tissue. The jugular lymph-sacs of the turtle present a considerable range of variation both in the advanced structure and in the details of their development. The variations, however, are neither so great nor so numerous as in the higher forms. These variations exist not only in different embryos but even upon opposite sides of the same embryo. Besides these individual variations, one often finds certain variations due to developmental conditions. Not only do certain embryos show advanced development in certain tributaries, and retarded conditions in others, but they differ in even more minor details, as for example, in the number and arrangement of secondary tributaries and their anastomotic conditions. Although a general principle of development can be established, the actual mode of origin of certain of the veno-lymphatic anlagen of the jugular lymph- sacs cannot be stated definitely owing to the variability in development of these veno-lymphatics in conjunction with the main venous channels and their tributaries, as well as the variable manner in which they fuse together. The active period of lymph-sac development is comparatively short. Embryos of seventeen to twenty-five days represent the important developmental stages. We may divide the history of the development of the sacs into three periods. 1. The formation of mesenchymal spaces in the anterior cardinal regions. 2. The enlargement and fusion of spaces with each other and with the venous tributaries to form the veno- lymphatics. 3. The confluence of the veno-lymphatics to form the sac, 154 E. R. VAN DER JAGT with the transformation of the mesenchyme cells to endothelial cells.

1. The Formation of Mesenchymal Spaces. This period of development brings up the point immediately as to the source and formation of lymphatic anlagen. A careful study of the stages belonging to this developmental period points out to the observer three facts of major importance: First, the compactness of the mesenchyme at an early stage, as shown in Text-fig. 1. In the lymph-sac area of embryos at the end of the second week of development the mesenchyme is very compact, showing slight signs of vacuolation. This area is bounded by the anterior cardinal vein and its dorsal branches, the wide-meshed superficial plexus, and the ventrolateral branches of the cardinal vein. The nuclei of the mesenchyme cells in this area still show the characteristic features of mesenchyme structure as compared with the well-defined endothelial cells lining the veins in this region. Embryos in the seventeenth day of development show marked changes. The mesenchyme in the dorsolateral region shows a loosening up of the cells. The vacuolation of the mesenchyme is forcing the cells apart. The intercellular spaces are increasing in size and number. The nuclei become widely separated and the cells are only connected by thin strands of protoplasm (Text- fig. 2). Secondly, the formation of isolated lymphatic spaces as the result of increased vacuolation is an important step in the formation of the lymph-sacs. The rapid formation of tissue spaces cause the thin protoplasmic processes to break or disintegrate. These fine processes or strands may be seen projecting out into the lumen of the cavities thus formed (Text-fig. 3). The cavities are not lined by a definite endothelial wall but by the former mesenchyme cells of the individual spaces. The spaces are always present in the region of the developing lymph-sac and are found in direct connexion with the anterior cardinal tributaries. As these two types of anlagen unite to form the veno-lymphatics, vacuolation continues, forming again small spaces in direct connexion with the established veno-lymphatics. LYMPH-SACS OF TURTLE 1S1

This process continues even after the veno-lymphatics begin to fuse. The continual formation of these small isolated lymph- spaces in connexion with the fusing veno-lymphatics, determines the ultimate lymph-sac. The spaces occurring in the lymph-sac region are small in comparison to the lymph-spaces developed in connexion with other lymph-channels. This may

TEXT-FIG. 1: Loggerhead turtle, fourteen days; series 516, X400. Photomicrograph of a section taken through the region of the anterior cardinal vein to show the compactness of the mesenchyme, which is comparatively free from vacuolation at this stage. The nuclei still show the characteristic feature of mesenchyme structure as compared to the well-defined endothelial cells lining the veins in this region. Ant. Card. V., anterior cardinal vein; Mes., mesenchyme; End. V., endothelium of the vein. TEXT-FIG. 2: Loggerhead turtle, seventeen days; series 563, X400. Photomicrograph of the section through the anterior cardinal vein. The mesenchyme in the dorsal and lateral region begins to appear vacuolated. This vacuolation is forcing the mesenchymal cells apart, causing an increase in size and number of intercellular spaces. Thenuclei are becoming more widely separated and the cells show connexions through thin protoplasmic strands. Ant. Card., anterior cardinal; End. V., endothelium of vein; I.C.S., intercellular spaces; Mes., mesenchyme. be due to the compactness of the mesenchyme or the more rapid reabsorption of the lymph in this region. Thirdly, the appearance of the mesenchyme cells in this region is in marked contrast to that of the cells of an embryo in which vacuolation has not occurred in the lymph-sac region. With the appearance of the spongy mesenchyme the shape of the 156 E. B. VAN DER JAGT cells is markedly altered. The protoplasm becomes drawn out to thin protoplasmic strands and only a limited amount of cytoplasm remains to surround the nuclei. The nuclei themselves do not present the round appearance that was observed in the compact stage, but have taken on a more flattened shape, as represented in Text-fig. 2. This change may possibly be due to the pressure of the lymph collecting in the spongy mesenchyme.

XEXT-KIG. 3, A and B. Loggerhead turtle, seventeen and one-half days; aeries 8, X400. Photomicrograph showing the two types of anlagen of the lymph-sac. (A) This figure shows the increased vacuolation of the mesenehyme forming the larger isolated lymph-spaces. In these larger spaces the protoplasmic strands can be seen projecting out into the lumens. Reference symbols for (A) are the same as those for (B). (B) Here the small isolated lymph-spaces are shown in direct connexion with the venous tributaries. These anlagen unite to form the veno-lymphatics. Ant. Card. V., anterior cardinal vein; I.L.S., isolated lymph-spaces; R.B.C., red-blood cell; P.N.S., perineural space; Vg., vagus; D. Ant. Card. T., dorsal anterior cardinal tributaries.

2. Enlargement and Fusion of Spaces, Investing and Communicating with the Venous Tribu- taries to Form the Veno-lymphatics. In a turtle embryo of sixteen days, no lymphatics could be found in the anterior cardinal region. The partial reconstruction with the use of cellophane shows the veins along which the first lymphatic anlagen are soon to appear. The anterior cardinal vein receives segmental branches in this region. It is LYMPH-SACS .OF TURTLE 157 in connexion with the tributaries of these segmental branches that development of the anterior lymph-sacs is initiated. Embryos of about twenty-days show the enlarged tributaries of the veins in the lymph-sac region, and the lymph-spaces in the direct pathway of the developing veno-lymphatics (Text- fig. 4). The exact relationship between the venous tributaries and the spaces cannot be given at the present time. The facts remain, we have the spaces and we have the tributaries in the area occupied by the future lymph-sac. Development of the sac does not begin in any one localized place; the segmental venous tributaries increase in size all at about the same time, that is, following the vacuolation of the mesenchyme in this region. The beginning of veno-lymphatic organization in general is indicated along the line of the primary dorsal precardinal tributaries. Here the mesenchyme begins to differentiate into a spongy, vacuolated state. Huntington applies the term 'veno-lymphatics' to all the venous anlagen of the jugular sacs, at a time when these anlagen were filled with blood and in free communication with the venous channels. In differentiating them from early stages, he states that the only structural distinction that can be made between the venous anlagen (veno-lymphatics) of the jugular sacs and the fully formed sacs themselves, is that the former, being in communication with the veins, are filled with blood and appear to function as veins, while the latter apparently do not. During the present study it was found that there is more than this one structural characteristic to differentiate these anlagen. A careful study of the lining of these structures of Huntington during the time when they are filled with blood, reveals a marked difference in the cell structure from the cells of the fully formed lymph-sac. The cells are not the typical mesenchyme cells, neither are they the typical endothelial cells, found lining the fully developed sac, but rather represent a transitional stage of the mesenchyme cells to the endothelial cells. The blood-filled stage does not appear until several days after development of the sacs has been initiated, and not until 158 E. R. VAN DER JAGT 31 Si

TEXT-FIG. 4: Loggerhead turtle, twenty days; series 237, X400. Photomicrograph of section through anterior cardinal vein and the veno-lymphatics. The latter are continuing to increase in size by union of the isolated spaces with which they are in direct connexion. The isolated lymph-spaces are constantly being formed in the mesenchyme and are in the pathway of the enlarging veno-lymphatics. Confluence and fusion of some of the veno- lymphatics is beginning to take place as noted from the shape of the cavities. Ant. Card. V., anterior cardinal vein; V.L., veno- lymphatics; I.L.S., isolated lymph-space. TEXT-FIG. 5: Loggerhead turtle, twenty-one days ; series 575, X400. Photomicrographs of sections through the veno-lymphatics to show their increase in size resulting in a stage of confluence. The veno-lymphatic plexus is further condensing by coalescence and fusion of the primary dorsal and ventral divisions, with reduction of the main venous channel connexions. The area lateral to the anterior cardinal vein is well occupied by these cavities. Protoplasmic strands may be observed projecting into the lumens of the cavities. The flattened cells lining the cavities represent a transitional stage to endothelial cells. In (B) can be seen the two types of cells present in the lining of the cavities. In areas where the spaces have just been added the transitional type of LYMPH-SACS OF TURTLE 159 the veno-lymphatics have reached a relatively large size. Subse- quent stages show a decreased blood-cell content. It was found, however, that these veno-lymphatics retain their connexions with the segmental veins. It is not until a relatively late stage of development that they lose connexion with the segmental branches and establish communication with the cardinal vein from the posterior end of the sac. Huntington has characterized the blood-filled stage, with reduction and further definition of the venous channels. One actually has the further definition of the veno-lymphatics. These are surrounded in certain areas by a network of spaces which together with a variable portion of the veno-lymphatics continues to condense into a uniform structure. In view of the double relation which this structure sustains, on the one hand, to the embryonic venous system, and on the other hand, to the general lymphatic system with which it establishes secondary connexions, the fact must not be over- looked that this double relationship is not only revealed in an advanced stage, but that in its development both the venous tributaries and the lymph-spaces are concerned. In using the term ' veno-lymphatic' it is not used in the same sense as it has been used in mammalian forms where it means lymphatics derived from the veins. On the contrary, it is used

mesenchyme cell can be observed, while in other areas of the same cavities typical endothelial cells can be seen. Isolated lymph- spaces are still present: (i) I.L.S., isolated lymph-spaces about to be added; (ii) I.L.8., isolated lymph-spaces which have been added; V.L., veno-lymphatics; Ant. Card. V., anterior cardinal vein; I.L.S., isolated lymph-spaces. TEXT-FIG. 6: Loggerhead turtle, about twenty-four days; series 217, X320. Photomicrograph of fully formed jugular lymph-sac. Confluence and fusion of the veno-lymphatics and spaces has continued to this time. Note how the mesenchyme cells of the veno-lymphatics have transformed into the endothelial type of cell. Reduction of the multiple early connexions between the veno-lymphatie plexus and the permanent veins has taken place. The permanent communication from the posterior region of the sac with the vein has now been established at the jugular subclavian tap. Jug. L.S., jugular lymph-sac; Ant. Card. V., anterior cardinal vein; Vg., vagus; End. Jug. L. Seg. Endothelium of the jugular lymph-sac, End. Ant. Card. V., endothelium anterior cardinal vein. 160 B. E. VAN DER JAGT in the sense that the venous endothelium is only in part the source of the lining of the veno-lymphatics, and that the lymphatic endothelium arises independently of the venous endothelium by the flattening of the original mesenchyme cells. Thus it may be said that the lining of the anterior lymph-sacs is of a mixed origin, partly from the vascular endothelium and partly from the mesenchyme cells. This raises the question that, if this be the case, why is it not possible to observe the difference in the cell structure of spaces added to those of the veins. According to the local origin theory, mesenchyme may transform into vascular tissue in practically any region of the body; and can transform into endothelium or vice versa. With this in mind, and also the fact that certain of the segmental venous tributaries atrophy while others anastomose with each other to form other veins, it appears that in certain of these tributaries the endothelial cells are reverting back to mesenchymal cells. This makes them unrecognizable from the partially transformed mesenchymal cells of the lymphatic spaces.

3. Confluence of the Veno-lymphatics to Form the Sacs, with the Transformation of the Mesenchyme Cells to Endothelial Cells. Text-fig. 5 represents a stage of confluence of the enlarged veno-lymphatics. In this stage the veno-lymphatic plexus is further condensing by coalescence and fusion of the primary dorsal and ventral divisions, with a reduction in the main venous channel connexions. Growth of these structures has continued so that the area lateral to the anterior cardinal veins is well occupied by these cavities. Confluence and fusion of the cavities and spaces continues to the twenty-fourth day when the lymph-sacs are well formed (Text-fig. 6). Up to the twenty- fourth day protoplasmic strands may be observed projecting into the lumens of the cavities due to their enlargement and fusion. These cavities are very irregular in size and shape, but still communicate with the segmental veins. At twenty-two days there is found a further reduction of the multiple early connexions between the veno-lymphatic plexus and the per- LYMPH-SACS OF TURTLE 161 manent veins, and, as a result, a more complete separation of the former from the latter, with the plexuses or sac-like structures assuming a greater degree of independence. This separation and loss of early communicating channels appears to proceed from both extremities of the area toward the jugular subclavian sac where the embryonic connexions are retained longest and where the permanent adult communications between the lymphatics and the veins are established. This is in accord with Huntington and McClure's work on the mammalian lymph- sac. The cells lining the cavities are somewhat flattened repre- senting a transitional stage to endothelial cells. These sacs do not come to lie on the muscle-plates as the lymph-hearts do, nor do they receive striated muscle in their walls in the stages studied. The formation of the lymph-sacs shows the close connexion which these structures have or assume with the cardinal veins. From their first appearance, the cavities around which the mesenchymal cells condense and which precede the formation of the real sacs, are, in the last analysis, nothing but the fusion of spaces with tributaries of the segmental veins which have gradually grown in volume and fused together in like manner.

SUMMARY. 1. In the lymph-sac area of embryos at the end of the second week of development the mesenchyme is very compact, showing slight signs of vacuolation. The area is bounded by the anterior cardinal vein and its dorsal branches, the wide meshed superficial plexus and the ventro-lateral branches of the cardinal vein. Within this area the lymph-sac anlagen first appear, lying in the angle between the dorso-lateral branches and the cardinal vein itself. 2. The dorsal tributaries of the pre-cardinals are concerned in the development of the lymph-sacs. In the mesenchyme surrounding these tributaries isolated spaces develop in direct connexion with the veins. 3. The development of the anterior lymph-sacs in the sea- turtle is initiated by vacuolation of the mesenchyme in the region NO. 297 M 162 B. K. VAN DEB JAGT of the cardinal tributaries during the middle part of the third week of development. 4. A series of channels is formed from the anterior cardinal tributaries and isolated spaces which in their greatest development exist as plexuses of vessels connected with the cardinal veins. 5. These channels enlarge and fuse with each other, the rem- nants of their walls appearing as protoplasmic fibres in the lumen, producing a system of cavities very irregular in size and shape. They inter-communicate and connect at the same time with the dorsal tributaries of the pre-cardinal veins. The mesenchyme cells surrounding these cavities are gradually transformed to the flattened endothelial cells which later line the sacs. 6. The lymph-sac is well developed at twenty-four days. Fusion and enlargement of individual cavities has continued up to this time. 7. As the development of the lymph-sacs proceeds they lose connexion with the cardinal veins except for the opening at the posterior end of the sac. 8. The development of the anterior lymph-sacs in the turtle shows two distinct anlagen, one from the mesenchyme and the other from the venous tributaries, which unite to form the veno-lymphatics. The increase in size and confluence of the veno-lymphatics determines the development of the anterior lymph-sacs. BlBLIOGKAPHY. Olark, E. B. (1911).—"An examination of the methods used in the study of the development of the lymphatic system", 'Anat. Record', vol. 5, p. 395. Clark, Eleanor Linton (1912).—"Injection and reconstruction of the jugular lymph-sac in the chick", ibid., vol. 6, p. 263. (1912).—"General observations on early superficial lymphatics in chick embryos", ibid., p. 249. Evans, H. M. (1909).—"On the development of the aorta, cardinal and umbilical veins, and the other blood-vessels of vertebrate embryos from capillaries", ibid., vol. 3, p. 498. (1909).—"On the earliest blood-vessels in the anterior limb-buds of birds and their relation to the primary subclavian artery", 'Amer. Journ. of Anat.', vol. 9, p. 281. LYMPH-SACS OF TURTLE 163

Huntington, G. S., and McClure, C. F. W. (1906-8).—"Tho development of the main lymph-channels of the cat in their relations to the venous system", 'Anat. Record', vol. 1, p. 36. (1908).—"The anatomy and development of the jugular lymph-sacs in the domestic cat", ibid., vol. 2, p. 1. (1910).—"The anatomy and development of the jugular lymph-sacs in the domestic cat", ' Amer. Journ. of Anat.', vol. 10, p. 177. Huntington, G. S. (1911).—"The development of the lymphatic system in • reptiles", 'Anat. Record', vol. 5, p. 261. (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. Kampmeier, 0. F. (1912).—"The value of the injection method in the study of lymphatic development", 'Anat. Record', vol. 6, p. 223. (1912).—"The development of the thoracic duct in the pig", 'Amer. Journ. of Anat.', vol. 13, p. 401. Lewis, F. T. (1906).—"The development of lymphatic system in rabbits", ibid., vol. 5, p. 95. (1909).—"The first lymph-glands in rabbit and human embryos", 'Anat. Record', vol. 3, p. 341. Sabin, F. R. (1901-2).—"On the origin of the lymphatic system from the veins and the development of the lymph-hearts and thoracic duct in the pig", 'Amer. Journ. of Anat.', vol. 1, p. 367. (1904).—"On the development of the superficial lymphatics in the skin of the pig", ibid., vol. 3, p. 183. (1905).—"The development of the lymphatic nodes in the pig and their relation to the lymph-hearts", ibid., vol. 4, p. 355. (1908).—"Further evidence on the origin of the lymphatic endothelium of the blood vascular system", 'Anat. Record', vol. 2, p. 46. (1909).—"The lymphatic system in human embryo, with a considera- tion of the morphology of the system as a whole", 'Amer. Journ. of Anat.', vol. 9, p. 43. Sala, L. (1899-1900).—"Sullo sviluppo dei cuori linfatici e dei dotti toraciei nell'embrione di polio", 'Ricerche Lab. di Anat. Norm. d. r. Univ. di Roma', vol. 7, p. 263. Stromsten, F. A. (1910).—"A contribution to the anatomy and development of the posterior lymphatic heart of turtles", 'Publication 132 of the Carnegie Institution of Washington', vol. iii, p. 77. (1911).—"On the relations between the mesenchymal spaces and the development of the posterior lymph-hearts of turtles", 'Anat. Record', vol. 5, p. 173. (1912).—"On the development of the prevertebral (thoracic) duct in turtles as indicated by a study of injected and uninjected embryos", ibid., vol. 6, p. 343.