Difference between revisions of "American Journal of Anatomy 5 (1906)"

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{{Amer. J Anat. Volumes}}
 
{{Amer. J Anat. Volumes}}
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=THE AMERICAN JOURNAL OF ANATOMY=
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EDITORIAL BOARD
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CHARLES R. BAUDEEN, University nf Wisco>ni)i.
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HENRY H. DONALDSON, Wistar Institute of Aiiatotiiy
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THOMAS DWIGHT, Harvard University
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JOSEPH MARSHALL FLINT, University of California.
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SIMON H. GAGE, Cornell University
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G. CARL HUBER, University of Michigan.
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GEORGE S. HUNTINGTON, Columbia University.
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FRANKLIN P. MALL, Johns Hopkins University
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J. PLAYFAIR McMURRICH,
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University of Jfic/iiyaii.
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CHARLES S. MINOT, Harvard University.
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GEORGE A. PIERSOL, University of Pennsylvania.
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HENRY McE. KNOWER, Secuetary, Johns Hopkins University.
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VOLUME V 1906
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THE AMERICAN JOURNAL OF ANATOMY BALTIMORE, MD., U. S. A.
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BALTIMORE, MD., V- 8. A.
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==CONTENTS OF VOL V==
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===No. 1. December 1, 1905===
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I. John Warken. The Development of the Paraj^hysis and the Pineal liegion in Nectiinis Maculatus = 1 With 23 text figures.
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II. {{Ref-Bell1905}} With 3 plates and 5 text figures.
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III. Jeremiah 8. Ferguson. The Veins of the Adrenal.  G3 With 3 text figures.
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IV. George Walker. The Blood Vessels of the Prostate Gland 73 With 3 colored plates.
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V. {{Ref-Allen1905}} 9 With 1 double plate and G text figures.
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VI. {{Ref-Lewis1905a}} With 8 text figures.
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VII. {{Ref-Lewis1905b}} With 1 text figure.
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===No. 2. May 31, 1906===
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{{Ref-Harrison1906}} With 5 figures.
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IX. Ai.i!i:i;t C. Eyclesiiymi:!; .-iiid James Meredith Wilson. The (lastnilation and I'iinbrvo Formation in Amia Calva 13;i With I (lonl.lc plalcs.
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{{Ref-McClure1906}} With 1 single and 4 double plates and 27 text figures.
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LIST OF MEMBERS OF ASSOCIATION OF AMERICAN ANATOMISTS XXII-XXXII
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===No. 3. July 25, 1906===
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XI. {{Ref-Mall1906liver}} With 74 figures and 7 tables.
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XII. Albert C. Eycleshy'mer. The Development of Clnoma tophores in Necturus 309 With 7 figures.
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XIII. Sidney Klein, S. M., ]\L D. On the Nature of the Granule Cells of Paneth in the Intestinal Glands of Mammals 315 With 5 figures.
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XIV. {{Ref-EdwardsHahn1906}} With 15 text figures.
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===No. 4. September 1, 1906===
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XV. Robert Bennett Bean. Some Racial Peculiarities of the Negro Brain ... 353 With 16 fig-ures, 12 charts, and 7 tables.
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XVI. {{Ref-Mall1906bone}} With (1 text figures and 7 tables.
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XVII. {{Ref-Bremer1906}} With 16 text figures.
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XVIII. Charles R. Stockard. The Development of the Mouth and Gills in Bdellostoma Stouti 481-517 With 36 figures.
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XIX. PROCEEDIXGS OF THE AS80CIATI0X OF AMERICAN ANATOMISTS. NINETEENTH SESSION, August 6-10, 1905. TWENTIETH SESSION, Decemher 27, 28, and 29, 1905 I-XX
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==The Development Of The Paraphysis And The Pineal Region In Necturus Maculatus==
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By
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John Warren.
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Demonstrator of Anatomy, the Anatomical Laboratory, Harvard Medical
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School.
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With 23 Text Figures.
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The presence of the paraphysis in Necturns was noted by Prof. C. S. Minot in his article " On the Morphology of the Pineal Region, based on its Development in x'Vcanthias " (38), and a brief description of certain stages given. C. L. Herrick (15, PI. YIII, Fig. 1, 3, 4) gives a brief account of the adult paraphj'sis, and shows it in the above figures, vvlicre it is named " Preparaphysis." Osbom (31, PI. IV) shows th-e paraphysis in an adult brain in comparison with the brains of other amphibia. Kingsbury (21) describes briefly the adult paraphysis as well as a few of the earlier stages, and also gives an account of the epi))hysis and the plexuses. I have found, however, no detailed account of all the stages in the development of the paraphysis and the pineal region. This term is used here in the same sense as in Minot's article, quoted above.
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The greater part of the specimens studied for this article were taken from the Embryological Collection of the Harvard Medical School, and the numbers of each section used are given. Other specimens were prepared specially for this purpose. In some cases where the plane of section was uneven, two or more sections were used in drawing the figures ill order to show all the structures, which should appear in the uK^dian line.
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Fig. 1 is a median sagittal section through the brain of an einl)ryo of 8-9 mm. I am indebted to a colleague for the drawing of this section, as this stage is wanting in the collection. In the roof of the fore brain three arches are seen. From before backward these are the j)aia|)liysal arch, P. A., the post- velar arch, P. V. A., and the epiphysal arch, Ep. A. The first two are separated by the velum transversum. V, which marks the limit between the two subdivisions .of the fore brain. Hence the paraphysal arch belongs to the telencephalon, the other two to the
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2 Paraphysis and the Pineal Ees^ion in Necturns Maenlatus
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dieneephalon. The epiphysal are]i is homided l)y two angles, which represent the position of the future supra and posterior commissures. The velum transversum is a simple infolding of the brain roof, and consists of two distinct layers, one caudad and one cephalad, the space between them being filled by a loose mesenchymal tissue, which later contains numerous blood vessels. This figure is practically identical with Minot's figure of acanthias of the same stage (28, Fig. 1), and is, therefore, of great importance in showing the homologies of these parts in elasmobranchs and amphibians. It is probable, as Minot states, that these arches occur in most of the vertebrate series.
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The term post-velar arch, introduced by Minot (28), is much better for purposes of description than the terms " zirbelpolster " of German writers, and the " dorsal sack " or " postparaphysis " of American authors.
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Fig. 1. Embryo of 8-9 mm. Sagittal section, X 63 diams.
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Fig. 2. Embryo of 10 mm. Harvard Embryological Collection, Sagittal Series, No. 269, Section 39, X 63 diams.
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Fig. 2 represents the roof of the dieneephalon and telencephalon of an embryo of 10 mm. The two layers of the velum are nearer together and in the region of the epiphysal arch are seen the first signs of the epiphysis, E. This structure is a small rounded diverticulum, which arises from the cephalic end of the arch. It is hollow and opens into the cavity of the fore brain.
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Fig. 3 is a similar section of an embryo of 12 mm. The velum is a trifle longer and the epiphysis a little larger than in the preceding figure. Immediately cephalad to the velum a very small evagination in the paraphysal arch can be seen, P. This is the first sign of the paraphysis, and it appears distinctly later than the epiphysis. The latter overlaps its short stalk both caudad and cephalad, and at this stage the stalk is still hollow, though its cavity was obliterated in this section.
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Fig. 4 is a section of an embryo of 13 mm. The velum is again a little longer and its caudal layer is now distinctly thinner than its cephalic layer. The paraphysis is now a well-marked narrow diverticulum extending dorsad from the paraphysal arch parallel to the velum. The paraphysal arch just cephalad to the opening of the paraphysis has been
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Fig. 3. Embryo of 12 mm. Harvard Embryological Collection, Sagittal Series, No. 49, Section 58, X 63 diams.
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Fig. 4. Embryo of 13 mm. Harvard Embryological Collection, Sagittal Series, No. 598, Sections 71 and 75, X 63 diams.
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forced downward to a slight degree, as there is relatively more space between it and the ectoderm than in the previous figures. The epiphysis is about the same size as in Fig. 3, and its opening into the brain is clearly seen.
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Fig. 5 is a section of an embryo of 12A mm., which is, however, further advanced than that of Fig. 4. The velum, the post-velar arch, and the epiphy.sis are about the same, but the paraphysis is distinctly longer, and
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Fig. 5. Embryo of 12.4 mm. Harvard Embryological Collection, Sagittal Series, No. 675, Section 57, X 63 diams.
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Fig. 6. Same as Fig. 5, X about 120 diams.
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has become a narrow tube. The brain roof cephalad to it has descended still more into the cavity of the telencephalon and the opening of the paraphysis is much nearer the tip of the velum. Fig. 6 is the same section as Fig. 5, only drawn on a higher scale to show the histological details. The walls of the paraphysis and "velum consist of a single layer of cells, with large oval nuclei and without very distinct cell boundaries.
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These cells are, of course, continuous with tliose which form the brain wall in this region. The same is true of the epiphysis, but the walls seem thicker, as the organ has been cut somewhat obliquely. Close to the paraphysis two vessels can be seen, a larger one cephalad and a much smaller one caudad, _ F<?6'. The vessels lie in intimate relation to this structure, and it is important to note their relation at this early stage, because as development progresses the relation between paraphysis and blood vessels becomes more and more intimate.
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Fig. 7 is a section of an embryo of 15 mm. The most striking feature here is the increase in size of the paraphysis. which has become a long tube with a lumen extending its entire length, and at its distal end a lateral diverticulum has appeared. The roof of the fore brain has now descended to such a degree that the opening of the paraphysis is on a level with the tip of the velum. The velum itself has lost its cephalic layer, and consists of one .layer only, which, however, is much longer than the velum in Fig. 5. If Figs 4, 5, and 7 are compared it will be seen that
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Fig. 7. Embryo of 15 mm. Harvard Embryological Collection, Sagittal Series. No. 79, Sections 85 and 89, X 63 diams.
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the distal end of the paraphysis is practically at the same distance from the ectoderm in each case. As the paraphysis has developed during those stages into a long tube, its growth must have occurred by a downward extension of the neighboring parts into the cavity of the fore brain. This is practically the same process described by Minot in Acanthias. It is also shown by the great increase in distance between the roof of the telencephalon and the ectoderm from Fig. 4 to Fig. 7. The opening of the paraphysis in Fig. 3 is nearly on a level with tlie base of the velum, and as the down growth of the parts takes place the opening of the paraphysis and the paraphysal arch descend, apparently pushing the cephalic layer of the velum ahead of them. Therefore the single layer of the velum in Fig. 7 really corresponds to the original caudal layer, plus the cephalic layer, which has been forced down ahead of the opening of the paraphysis.
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In stud5ing Fig. 7 it might seem as if the posterior wall of the paraphysis corresponded to the cephalic layer of the velum. This, however, is
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not tlie case, as can be seen in a wax reconstruction of the parts, Fig. S. This is a reconstruction of the brain of an embryo of 14.5 mm. The tops of the hemispheres, H, have been removed to give a clear view of the paraphysis, P, which otherwise would be more or less covered in by them. The paraphysis appears as a straight tube in the median line and caudad to it is seen a broad partition, V , extending the whole width of the diencephalon. This is the velum, consisting of one layer only, which represents the tw^o originally distinct cephalic and caudal layers. The down grow^th of the parts in order to provide room for the development of the paraphysis has formed a deep angle in the roof of the fore brain. This
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Fig. 8. Wax model of brain of embryo of 14.5 mm. Harvard Embryological Collection, Sagittal Series, X 120 times.
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angle is bounded caudad by the velum and cephalad or ventrad by the narrow roof of the telencephalon (paraphysal arch) immediately cephalad to the paraphysis. As the hemispheres develop, they grow at first in a dorsal direction and occupy the space left by the formation of this angle, so that the paraphysis is practically buried between the hemispheres in front and the velum behind. Fig. 10. The growth of the paraph3^sis must, therefore, be regarded as liaving an important effect on the development of the fore brain at this stage.
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Up to this stage the development of the velum has been in a ventral direction towards the floor of the fore brain, but now it begins to grow in quite a ditferent direction. In Fig. 7 a distinct bulging of the velum is
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seen, which is extending candad at ncarl}- a right angle to its previous line of growth. If the roof of the telencephalon be closely examined a slight bulging will be seen just cephalad to the opening of the paraphysis. These two outgrowths into the fore brain mark the beginning of the choroid plexuses, which, therefore, have in their origin a very intimate and definite relation to the opening of the paraphysis, one arising caudad and the other cephalad to it. The epiphysis at this stage has increased considerably in size, and the cavity in its stalk is now permanently obliterated. The body of the organ overlaps the stalk a little behind, and is beginning to grow well forward of it. The posterior commissure, P. C, appears here for the first time, a distinct interval in the roof of the brain lying between it and the stalk of the epiphysis.
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Fig. 9. Embryo of 17.5 mm. Harvard Embryological Collection, Series, No. 540, Sections 113-115, X 63 diams.
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Fig. 9 is a section through the brain of an embryo of 17.5 mm. The paraphysis has increased in length, and from its distal end, which is somewhat enlarged, small tubules are given off. The whole tube is tipped somewhat forward. The choroid plexus is now well developed, and consists of two distinct parts, one dorsal and one ventral. The dorsal part corresponds to the velum, which has grown caudad as far as the mid brain and has absorbed a large part of the post-velar arch. The ventral part is developed from the original paraphysal arch, and is growing towards the floor of the fore brain. Burckhardt (3) refers to these plexuses as " plexus medius " and " plexus inferioris," respectively, and Mrs. Gage (13), who studied them in Diemyctylus, where the anatomical conditions closely resemble those of JSTecturus, names them the " diaplexus " and " prosoplexus." Prof. Minot has suggested the terms diencephalic
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plexus for the dorsal part, and tel encephalic plexus for the ventral part, and I shall use these terms, as they express more clearly the exact origin of each plexus.
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The diencephalic plexus, V. Plx., appears as a large wedge-shaped mass covered by a thin layer of cells, and consisting of a loose connective tissue in the interstices of which numerous blood corpuscles can be seen. The telencephalic plexus, Tel. Plx., has the same general characteristics as the diencephalic. The epiphysis has become flattened and more elongated, and is attached by a narrow stalk to the brain wall. The supra commissure, S. C, is seen just cephalad to the stalk of the epiphysis, which is prolonged forward above it. I was unable to obtain any sagittal series between 15 and 17.5 mm., but in a transverse series of 16.5 mm. the first traces of this commissure can just be made out. and therefore it
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Fig. 10. Wax model of brain of embryo of 18 mm. Harvard Embryological Collection, Frontal Series, No. 850, X about 75 diams.
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probably appears between 16 mm. and 17 mm. as a rule, but at these early stages there is a good deal of variation in the development of all these parts. The posterior commissure is rather larger than in the previous stage.
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Fig. 10 is the drawing of the model of the brain of an embryo of 18 ram. This model is intended to show the circulation of the paraphysis at this stage. The distal end of the paraphysis, P, is surrounded by a venous circle, from either side of which veins, Ves., run outward and backward just caudad to the hemispheres, H, to terminate in the internal jugular vein, I. J. V. This vein is passing backward external to the fifth, V, and seventh, VII, cranial nerves. Fig. 6 showed the intimate relation of the paraphysis to these vessels at 12.4 mm., and when the sections of this series were followed out it was found that here the vessels surrounded the tip of the paraphysis. It seems that as the paraphysis develops it forces its way into the veins lying over this part of the fore brain, and the tnbnles, as they are given off at la.ter stages, force their way into these veins, Fig. 15, forming the sinusoidal type of circulation described by Minot (29) and Lewis (25). From the venous circle shown in Fig. 10 smaller vessels run down along the sides of the paraphysis and anastomose with the vessels of the choroid plexuses. A vessel also runs back to the epiphysis, and a larger one forward between the hemispheres. The circulation of this region appears at this stage to be mostly venous, as I could trace the arteries only to their point of entrance in the anlage of the skull, and the return circulation probably occurs by means of a minute capillary network over the surface of tlie brain.
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Fig. 11 is a section through the brain of an embryo of 26 mm. The
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Fig. 11. Embryo of 26 mm. Harvard Embryological Collection, Sagittal Series, No. 377, Sections 125 and 126, X 63 diams.
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paraphysis here is much more developed. It inclines somewliat forward, and from its wide central lumen a number of tubules are given oif in every direction. The epiphysis and the commissures show but little change. The striking feature of this figure is the great development of the plexuses. The diencephalic plexus, D. Pl.r.. has grown through the mid-brain nearly to the hind-brain, and the telencephalic plexus, Tel. Fix., has grown downwards into the depths of the cavity of the fore brain towards the infundibular recess.
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Fig. 12 is a transverse section of an embryo of 26 mm., corresponding approximately to the line A-B, Fig. 11. The section passes through the epiphysis, E, and the supra commissure, S. C, just beneath it. Then through the diencephalic plexus, D. Fix., and that part of the cavity of the diencephalon between this plexus and the roof, T>ien. The section then passes through the paraphysis at a point where two small tubules are given off, then through the telencephalic plexus, Te2. Plx.. the telen
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FiG. 12. Embryo of 26 mm. Harvard Embryological Collection, Transverse Series, No. 376, Section 89, X 63 diams. (See line A-B, Fig. 11.)
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cephaloii, Teh. the lateral ventricles, L. V., and the foramina of Munro, F. M. In this section the plexuses of tlie hemispheres, L. Plx., are seen. They arise on either side of the origin of the telencephalic plexus, and
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Fig. 13. Embryo of 26 mm. Harvard Embryological Collection, Frontal Series, No. 378, Section 138, X 63 diams. (See line C-D, Fig. 11.)
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pass outward at right angles to it through the foramina o£ Munro into the lateral ventricles.
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Fig. 13 is a frontal section through an embryo of 2Q mm., corresponding closely to the line C-D, Fig. 11. The section passes through the paraphyses, P, and a large lateral tubule, and then through the entire length
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Fig. 14. Same Series as Fig. 13. Section 108. (See line E-F, Fig. 11.)
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of the diencephalic plexus, D. Fix., the distal end of which is here enlarged and has reached to the hind brain, H. B. Fig. 14 is of the same
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Fig. 15. Same as Fig. 11. X about 150 diams.
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series as Fig. 13, and corresponds approximately to the line E-F, Fig. 11. It passes through the telencephalic plexus, Tel. Fix., the plexus of the
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hemispheres. L. Plx., and the lateral ventricles, L. V. It shows clearly how the plexuses of the hemispheres arise from the telencephalic plexus and pass at first outward and then forward through the foramina of Munro towards the cephalic extremity of the lateral ventricles.
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Fig.- 15 is a high power drawing of Fig. 11, magnified 150 diams. The wall of the paraphysis consists of a single layer of cells with large oval nuclei, and these cells are continuous with the cells covering the choroid plexuses, but the latter are flatter and form a thinner layer. On either side of the paraphysis two large vessels are seen, ves., the epithelial cells of which lie directly against the wall of the paraphysis. The little tubules seem to be forcing their way into these vessels, which are branches of the vessels seen in Fig. 6 and Fig. 10. The vessels also pass down into the choroid plexuses. Fig. 16 is a section of an embryo of 31.4 mm.
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Fig. 16. Embryo of 31.4 mm. Harvard Embryological Collection, Sagittal Series, No. 537, Sections 119-122, X 63 dlams.
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The general arrangement is practically the same as in Fig. 11, except that all the parts have progressed somewhat in their development. The distal end of the paraphysis has begun to grow distinctly more cephalad, and the whole structure is much larger than at 26 mm. The choroid plexuses are more extensive, and from the diencephalic plexus a prolongation is extending downwards towards the telencephalic plexus. This latter has pretty well filled up the depths of the third ventricle, and from it prolongations dip down into the recesses in the floor of the fore brain. The two commissures are practically the same as they were at 26 mm., and
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12 Paraph vsis and the I'iiieal Heo-ion in Noetiiriis Maeulatus
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though the epiphysis is a little larger it has been displaced considerably candad, as this part of these sections was unluckily somewhat injured.
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Fig. IT is a section through the brain of an adult necturus. This drawing is magnified 38 diams. only, as it was too large to draw on the same scale as the preceding figures. The paraphysis, P. forms a very complex structure extending far forward above and between the hemispheres. It consists in a general Avay of a proximal and a distal part. The former is broad and thick, and oxtends forward and upward. It then turns forward at quite a marked angle to form the distal part, which is narrow and. tapering. The central canal in the proximal part is very large and irregiilar, but in the distal portion much narrower. From all parts of this canal a large number of tubules are given off, which extend in every direction, and between which lies a confused mass of bloodvessels. One sees here a large vessel ventrad to the organ, and a smaller dorsad to it. the same relations as appear at 12.4 mm., Fig. 6. From these vessels branches pass into the choroid plexuses.
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Fig. 18 is a transverse . section of an adult brain corresponding approximately to the line A-B, Fig. 17. This is drawn on the same scale as most of the preceding figures, 63 diams. The paraphysis, P, is seen in the median line between the hemispheres. It shows a distinct central eavit}', with many tubules running out in every direction, between which is a mass of blood-vessels of all sizes. Below a portion of the telencephalic plexus aiu1 the ])l(\\-us of the hemispheres are seen.
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Via. 17. Brain of adult necturus. Sag-ittal Section, x 38 diams.
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Fig. is. Brain of adult necturus. Transverse section, X 63 diams. (See line A-B. Fig. 17.)
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14 Paraphysis and the Pineal Eegion in Nectunis Maciilatus
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Fig. 19 is from a wax recoustruction of tlie adult paraphysis on a scale of 120 diams., made from the same series as Fig. 18. The angle between the proximal and distal parts is quite striking, and is much more marked in Ichthyophis (Burckhardt, 4, Fig. 1), but of course this division into proximal and distal parts is really a purely arbitrary one. This model gives a good idea of the complex structure of the organ. The tubules are of all shapes and sizes, often convoluted and anastomosing with each other. The spaces between them, which the vessels occupy, are quite large and striking.
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Fig. 19. Wax model of paraphysis of adult necturus, same series as Fig. 18, X about 120 diams.
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Pig, 20 represents a small portion of the paraphysis of Fig. 17, magnified 560 diams., and shows clearly the relation of the tubules to the vessels. In the centre of the figure is a tubule, T, dividing into two branches, T^ T-. Surrounding these tubules on every side are sinusoids, si., whose flat endothelial cells are seen lying directly against the epithelial wall of the tubules with no connective tissue between them. We find here in order first a sinusoid, then a tubulcj then another sinusoid and another tubule, and finally a sinusoid. The wall of the tubules consists of a single layer of cells with large oval nuclei and very indistinct cell boundaries. The nuclei contain masses of granules arranged very irregu
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Joljn Warren
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larly. There can be no question abont the glandular nature of the paraphysis, and its circulation is evidently sinusoidal.
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The choroid plexus, Ch. Fix., Fig. 17, appears as a confused mass of
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Fig. 20. Small portion of adult paraphysls, same section as Fig. 17, X 560 diams.
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vessels covered by a thin layer of cells. This mass completely fills up the cavity of the fore and mid brains, and may in some cases appear in the hind brain, Fig. 23, though there seems to be considerable variation
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Fig. 21. Wax model of epiphysis of adult necturus. X 280 diams.
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in the caudad development of this part of the ple.xus. Tlie two parts of the plexus overlap each other, and are also closely interlaced.
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The epiphysis, E. is still attached to the brain by a very narrow stalk. The body overlaps the stalk somewhat behind, and then is prolonged forward as an oval flattened body above the roof of the diencephalon, and its cavity seems to be divided more or less into compartments. Fig. :H represents a wax reconstrnction of an adult epiphysis seen from ^above. It is irregularly triangular in shape with a broad base and a blunt apex. Its surface is grooved more or less by vessels which lie against its Avails. Fig. 22 is the same model with the top removed. The interior is more or less subdivided by incomplete septa. At its apex there is a small cavity
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Fig. 22. Same as Fig. 21, with top of epiphysis removed.
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1)0unded behind by a partial septum, then comes a large chamber, which divides into two passages running back towards the angles at the base. Between these two passages appears a comparatively solid area, interrupted, hoAvever. to some extent by small spaces, aa^IucIi communicaio Avith each other and the larger chambers. This solid area lies over tbe stalk of the organ. The sujDra commissure appears to be comparatively small,
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Fig. 23. Brain of adult necturus. Viewed from above. X 7 diams.
 +
 +
while the posterior is large and forms a deep groove in the roof of tlie brain. Fig. 23 is a vioAv of the brain of an adult necturus shoAving the relative positions of paraphysis and epiphysis. The tufted extremity of the diencephalic plexus can be seen in the fourth ventricle.
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If Fig. 1, the embryo of 8-9 mm., is compared Avith Fig. 17, the adult, one sees that the paraphysal arch has been Avholly taken up in the formation of the telencephalic plexus, the plexus of the hemispheres, and
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John Warren 17
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the paraphysis. The velum and the greater part of the post-velar arch have been absorbed in the formation of the diencephalic plexus. A portion of this arch, however, persists and forms that part of the roof of the diencephalon between the diencephalic plexus and the supra commissure. The epiphysal arch has formed the epiphysis.
 +
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The paraphysis is a structure common to all vertebrates either in the adult or embryonic condition (Selenka, 34, Francotte, 11), but previous observations on mammals leave much to be desired. It always arises from the telencephalon cephalad to the velum transversum, and its opening is placed between and dorsad to the foramina of Munro as emphasized by Dexter (5).
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In the cyclostomes, Ammocoetes (Kupffer, 24), and Petromyzon (Burckhardt, 3), the paraphysis appears as a small sac-like diverticulum lying ventrad and close to the enlarged distal end of the epiphysis. In elasmobranchs, Minot (28) and Locy (27) found that the paraphysis in Acanthias appears at quite a late stage as a small outgrowth from the paraphysal arch and, owing to the small size of the post-velar arch and the compression of the velum, it comes to lie immediately cephalad to the epiphysis. In ganoids, Kupffer found in Accipenser that the paraphysis appears first as a small outgrowth which later becomes a somewhat sacculated vesicle (23, Fig. 19). Hill (18) and Eycleshymer and Davis (9) studied the paraphysis in Amia. Here it begins as a simple vesicle, which increases rapidly in size and gives off diverticuli from its central cavity. In teleosts (Burckhardt, 3) the paraphysis appears late and remains in a rudimentary condition. In the dipnoi, Burckhardt (3) describes the paraphysis in Protopterus as a wide outgrowth giving off small . diverticuli.
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In amphibia the organ becomes highly differentiated and its appearance in the adult brain is very striking. It appears as an elongated body lying above and between the hemispheres, and extending cephalad for a varjdng distance in various forms. Fig. 23. Osborn (31, PI. 4) shows a view of the brains of Siredon, Necturus, Proteus, and Siren. The paraphysis has the same general form in each of these, but it is somewhat larger in Necturus. The paraphysis of Triton and Ichthyophis (Burckhardt, 4) has the same characteristics. In the latter the paraphysis appears in sagittal section as a hammer-shaped organ extending forward above the hemispheres. (4, Fig. 1). In Rana the paraphysis has the same position as in Necturus, but is smaller. On removing the top of the skull in Necturus the paraphysis is seen lying beneath the pia surrounded by the blood-vessels which cover this part of the brain. It appears to the naked eye so vascular and also in sections so intimately related to the 2
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18 Paraphysis and the Pineal Pegion in Necturns Maculatus
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choroid plexus that it is not astonishing that it was at first regarded as a portion of this plexus. According to Minot (28) the paraphysis of Eana is characterized by the character of its epithelium, its tubular structure and its apparently sinusoidal circulation. This is practically similar to the conditions found in Necturus.
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In ^lenopoma Sorenscn (36) describes the paraph3'sis as a solid vascular mass, and in Ichthyophis Burckhardt (3) describes it as an elaborately folded structure of a glandular character. In Amblystoma, Eycleshymer (8) shows that the organ gives off tubules and has a digitated appearance. In Diemyctylus (Mrs. Gage, 13), in an embryo of 10 mm. the paraphysis closely resembles that of Necturus at 18 mm. (Minot, 28, Fig. 13), and in the adult it is a long tube giving off many tubules in close relation to vessels.
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Herrick (15) calls the paraphysis of an adult Necturus the " preparaphysis " and says it consists of an irregular central chamber with complicated diverticuli in close relation to vessels. This description corresponds closely with Fig. 17. The model of the adult paraphysis, Fig. 19, shows the complicated arrangement of the tubules, many of which anastamose with each other, and the spaces between the tubules are filled by blood-vessels.
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Fig. 6 shows how the paraphysis at 12.5 mm. is beginning to invade a large vessel lying over it on the surface of the brain. This vessel at this point is much enlarged. Fig. 10 shows this relation much clearer and also that these vessels in relation to the paraphysis are tributaries of the internal jugular vein. Fig. 15 shows the relation of the paraphysis to these vessels at 26 mm. and that the vessels pass into the choroid plexuses both dorsad and ventrad to the organ. The little tubules can be seen growing out into the vessels. Fig. 17 shows how these relations between vessels and tubules in the adult become much more intimate, and the vessels corresponding to those in Fig. 15 are seen passing into the choroid plexuses dorsad as well as ventrad to the paraphysis. Schobel (33), who studied the circulation in the brain of certain amphibia, of which Necturus was one, shows that in the adult two large vessels pass outward just caudad to the hemispheres to empt}'^ eventually in the internal jugular vein. These vessels surround the paraphysis and anastomose with two or three large vessels running forward between the hemispheres. This is practically the same arrangement shown in the model in Fig. 10. He does not mention the paraphysis but refers to it as a large venous plexus. Eex (32) also has studied the veins in the amphibian brain, and his preparations are practically the same as Schobel's. He refers to the paraphysis as the " nodus chorioideus " and says that it is a sort
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John Warren 19
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of meeting point for the veins of the fore and mid brains. He shows beautifully in his injections how veins pass both dorsad and ventrad to the paraphj'sis to enter the plexuses, and how closely these vessels are related to the tubules of this structure. Mrs. Gage shows practically the same arrangement in Diemyctylus
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As regards the arteries Schobel shows that they are much smaller than the veins, and describes a small vessel passing caudad to the hemispheres to pass eventually into the plexuses. The intercrescence of the tubules of the adult paraphysis and the veins is shown clearly in Fig. 30, each vessel and tubule lying back to back with no connective tissue between them. In view of all these facts it seems evident that the circulation of the paraphysis is sinusoidal. According to the above descriptions, the development of the paraphysis into a complicated, glandular organ, which is also very vascular, seems to be a striking characteristic of the amphibia.
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In lacertilia the paraphysis of Anguis fragilis has been studied by Francotte and of Lacerta vivipara by Francotte (10, 11, 12) and Burckhardt (3). The latter shows the paraphysis in an embryo of 13 mm. as a narrow tube with a slightly expanded distal extremity, much as that of ISTecturus of 15 mm. Francotte describes the paraphysis of Lacerta vivipara as a long tube giving off a mass of tubules which lies under the parietal eye, and resembles the epiphysis of birds (11, Fig. 14; 12, Fig. 24). In Anguis (10, Figs. 15 and 19) the paraphysis forms a long narrow sack, with somewhat convoluted walls, which curves back over the post-velar arch to end in close relation to the parietal eye.
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The conditions in the lizard are essentially the same (10, Fig, 31). In Phrynosoma coronata Sorensen (35, Fig. 2) describes the paraphysis as a long, narrow tube, immediately cephalad to the epiphysis.
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In the ophidia Leydig (26, Fig. 6) shows the paraphysis of an embryo of Vivipara urcini near birth as a large, wide tube with no convolutions or diverticuli and practically the same conditons in a young " Eingelnatter" and Tropidonatus natrix (26, Figs. 5 and 2).
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Among the chelonia Voelzkow (39) has described the paraphysis in Chelone imbricata as at first a wide tube much convoluted, which later decreases somewhat in size. (Figs. 21 and 22.) Its distal end inclines caudad close to the epiphysis. In Chelone mydas Humphrey (20, Fig. 7, PI. II) shows the paraphysis as a long tubular structure, giving off small tubules, and in an embryo of Chelydra it appears as a large, wide sack, from which tubules arise. It is in closer relation to the epiphysis than in Chelone mydas or imbricata.
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In Cistudo Herrick shows a model of Sorensen (15, Fig. 5, PI. VI) of the paraphysis, which is a wide tube with convoluted walls and tubules.
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20 Paraphysis and the Pineal Region in Necturus Maculatus
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In the crocodilia Voelzkow (39) has described the paraphysis of Crocodilus madagascarensis grand and Caiman niger spix. In the former the paraphysis is at first a wide tube which becomes convoluted and much longer and narrower. In the latter the paraphysis forms a larger tube and the convolutions and tubules are more complicated. In both cases the organ reaches its greatest development in embryonic life and retrogrades later, though more so in the crocodile. He was unable, however, to follow the development in the caiman as far as in the crocodile. Owing to the thickenings in the brain wall the organ is crowded somewhat caudad against the post-velar arch.
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In birds the paraphysis is relatively rudimentary. Burckhardt (3) shows the paraphysis in an embryo of the crow as a small diverticulum not unlike that of Petromyzon. Dexter (5) worked out in detail the development of the organ in the common fowl, and showed that it appeared at first as a small diverticulum. The walls become much thickened and in a chicken of 10 days it is a small, oval structure, about 150 fx in its greatest diameter, with very thick walls (5, Fig. 5). Selenka (34) has described the paraphysis in the oppossum, but as far as I am aware little is known of the development of the paraphysis in mammals, though Francotte (11) has observed it in human embryo of twelve weeks.
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From the cyclostomes to the amphibia the paraphysis shows a steadily progressive development, and the various forms through which it passes, from the simple diverticulum of Petromyzon to the elaborate gland of the urodela, are illustrated in a general way by the stages of its development in Necturus. In the vertebrates above the amphibia the paraphysis retrogrades and practically retraces its steps through the reptilia and birds to mammals, reaching in the chick essentially the same form in which it started in Petromyzon. Its development, therefore, may be indicated by a curve, which ascends steadily from the cyclostomes, reaches its height in urodela, and descends through the reptilia and birds to mammals.
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The epiphysis is present in nearly all vertebrates. It is stated to be absent in the alligator (Sorensen, 36 and 37), and in the caiman and crocodile (Voelzkow, 39) and in Torpedo (d'Erchia, 7).
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The epiphysis of Necturus as compared with the paraphysis is relatively poorly developed and in this respect resembles the epiphysis of other urodela (Mrs. Gage, 13). In Diemyctylus Mrs. Gage found that the epiphysis was entirely cut off from the brain and that its cavity was nearly obliterated. In Ichthyophis (Burckhardt, 1 and 4) the epiphysis is a small, pear-shaped organ attached to the brain by a narrow solid stalk. Herrick (15) in Menopoma, describes the epiphysis as an irreg
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John Warren 21
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ular number of vesicles attached to the brain by a narrow opening. According to Kingsbury (21) the structure in Necturus consists of an aggregation of closed vesicles, forming an oval, flattened body, and there is no connection with the brain. The cavity of the epiphysis communicates through its stalk with the cavity of the diencephalon up to 15 mm., when the cavity in the stalk becomes obliterated. The stalk persists and was present in all the adult brains which I examined, but in some cases it was so small that it could easily be overlooked. The reconstruction of an adult epiphysis. Fig. 22, shows that the cavity of the organ forms a large chamber subdivided to a certain extent by incomplete septa. A much more solid area is seen towards the caudal extremity, which is placed just over the stalk. The same characteristics I have observed in another model made from a different brain. One gets the idea that the epiphysis consists of a series of vesicles in studying sagittal sections a little to one side of the median line, as for instance in Fig. 17, where the epiphysis was displaced a little to one side.
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. There has been such a vast amount written on the origin of the epiphysis and the pineal or parietal eye and their homologies that it seems superfluous to add anything more here. In a very general way, however, there seems to be some sort of proportion in the relative development of the paraphysis, epiphysis, and the parietal eye. In urodela where there is no parietal eye and a small epiphysis, the paraphysis reaches its highest degree of development. In those forms where the paraphysis is rudimentary or relatively slightly developed the parietal eye is present or else the epiphysis is relatively highly developed. Compare, for example, the figures of Burckhardt (3) of Petromyzon, Minot (28) of Acanthias, Burckhardt (3) of Trout, Leydig (26) and Voelzkow (38) of reptilia, and Dexter (5) of the fowl. Rana, however, seems to be a marked exception to this statement, as there the paraphysis, epiphysis, and pineal eye are all present and well developed, and the same may be said for Lacerta (Francotte, 10 and 11) and Sphenodon (Dendy, 6). As the paraphysis and epiphysis are glandular structures they have probably some sort of compensatory function and where one is highly developed the other is relatively rudimentary or even absent. Compare in this respect also Torpedo with Acanthias and the crocodile and alligator with the chick.
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As a rule the stalk of the epiphysis is placed immediately caudad to the supra commissure, in all cases I believe, except in the toad, where there is a distinct interval between it and this commissure (Sorenson, 36). In Necturus there is an interval in the roof of the brain between the stalk and the posterior commissure. This portion of the roof of the brain was
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22 Paraphysis and the Pineal Eegion in Necturus Macnlatus
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described by Kupffer (23) as the " schaltstlick," and according to him it is best developed in amphibia. Burckhardt (3) maintains that it occurs in all vertebrates from Pctromyzon np, but according to Kupffer it is absent in Accipenser (23, Fig. 19), and it is also wanting in Acanthias (Minot, 28, Pig. 10) and in the fowl (Dexter, 5, Fig. 9).
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The velum transversum is probably characteristic of all vertebrates. Minot (28). In Petromyzon the velum appears as a small transverse fold, and the post-velar arch is well marked. The plexus development is, however, very slight. In elasmobranchs the velum of Acanthias forms a long, narrow, transverse fold, and the post-velar arch is so small that the origin of the velum seems to be close to the supra commissure. The caudal layer of the velum is distinctly thinner than the cephalic (Minot, 28, Fig. 6). This is also seen in Torpedo (d'Erchia, 7, Fig. 12), and in Necturus, Fig. 6. The velum later on has the character of a choroid plexus, but the plexus of the hemispheres is very rudimentary (Minot, 28). In Notidanus Burckhardt (3) shows a long, narrow velum, a short post-velar arch, and a small telencephalic plexus. The plexus of the hemispheres, however, is absent.
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In Accipenser (Kupffer, 23, Fig. 19) the velum is long, well developed and folded to a certain extent, and the post-velar arch is quite extensive. In ganoids (Studnicka, 38) the membranous roof of the brain serves as the tela choroidea of higher types. In this class of vertebrates according to Burckhardt (3) the plexus of the hemispheres is lacking, but the telencephalic plexus is well developed, and in teleosts the former is also wanting, but the latter present in a reduced form.
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In amphibia all the plexuses are highly developed, and in J^ecturus they are of marked extent (Kingsbury, 21). The velum in Necturus appears at first as a transverse fold in the roof of the brain separating the diencephalon from the telencephalon. This fold develops at first ventrad and then caudad through the mid brain as far as the hind brain. This great growth of the velum forms the diencephalic plexus. The post-velar arch, which at first is wide and well marked, is practically absorbed by the overgrowth of the velum, and a small portion only persists in the roof of the diencephalon between the origin of the diencephalic plexus and the supra commissure. Fig. 17. The telencephalic plexus develops from the paraphysal arch immediately cephalad to the paraphysis, the opening of which therefore is surrounded by these two plexuses. They fill up the cavity of the third ventricle and mid brain, and the diencephalic plexus may appear in the hind brain (Osl>orn, 29). This seems to vary in different cases, and in the majority of brains which I was able to examine the extremity of this plexus did not actually extend
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John Warren 23
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into the hind brain. In- Fig. 33, however, this extremity appears as a marked tuft in the fourth ventricle. The plexuses of the hemispheres arise on either side from the origin of the telencephalie plexus and pass into the lateral ventricles, extending nearly to their cephalic extremities.
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In Lacerta vivipara (Francotte, 12, Fig. 24) the post- velar arch has been much com})ressed from before backward so as to form a deep narrow angle. At the apex of the angle the folds of the diencephalic plexus are seen. The velum is smooth and apparently is not included in the formation of the plexus. In Anguis fragilis (Francotte, 10, Figs. 19 and 15), the post-velar arch does not seem to be so much compressed, and the plexus formation somewhat greater. As he says, however, the development of those parts in Lacerta is practically the same as in Anguis. According to Burckhardt the telencephalie plexus is much reduced in size, consisting merely of small folds, but the plexus of the hemispheres is well developed (Burckhardt, 3).
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In the turtles Humphrey (20) found that the velum of Chelydra is but slightly developed, and no diencephalic plexus is formed. All the other plexuses are telencephalie in origin.
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Herrick (15, Fig. 5, PI. VI), shows in Cistudo a well-developed telencephalie plexus and a diencephalic plexus represented by many folds in the caudal layer of the velum and the post-velar arch. In Chelone imbricata Yoelzkow (39, Figs. 19 and 22) shows at first a well marked velum and a wide post-velar arch. In later stages the velum and practically all the arch are thrown into folds to form the diencephalic plexus. The telencephalie plexus is also well developed. In the serpents much the same arrangement can be seen. The velum (Leydig, 26, Figs. 2, 5, and 6) forms a prominent fold, and it and the post-velar arch form a very vascular plexus. In the crocodilia (Voelzkow, 39, Figs. 7, 11, 13, 15), the velum and the post-velar arch are at first well marked, but the parts later become so compressed from before backward that the arch forms a deep acute angle in the depths of which plexus foldings are seen. The caudal layers of the velum, however, takes no part in the plexus formation. In birds, Dexter (5) found that the velum of the fowl is small and the post-velar arch broad at first. This becomes compressed so as to form an acute angle much as in the crocodilia. The cephalic limb of this angle and all the velum is converted into the choroid plexus. In birds the plexus of the hemispheres is very well developed, but the telencephalie plexus is practically absent.
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In fishes the plexus development is quite simple, in many cases being merely the thin membranous roof of the third ventricle; in others, however, this is much folded and vascular (Sorensen, 35). In certain forms
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24 Paraphysis and tfio Pineal Eegion in Necturus Maculatus
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there is a telenccphalic plexus, but the plexus of the hemispheres is absent or rudimentary (Burckhardt, 3). In amphibia there is a great overgrowth of all the plexuses, especially of the diencephalic plexus, which here reaches its highest development. In reptilia the plexus of the hemispheres is well developed, but the telencephalic plexus is reduced in size (Burckhardt, 3), and the diencephalic much more so. In birds the plexus of the hemispheres is highly developed, the telencephalic plexus practically absent, and the diencephalic plexus, while very similar to that of reptilia, approaches nearer to the tela choroidea of higher mammalia.
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Osborn first named the supra-commissure and worked out its homologies. According to him (30) the urodela are distinguished from the anura by the frequent extensive development of this commissure, which is large in Amphiuma, smaller in Necturus, and much reduced in Eana. It appears in Necturus a little later than the posterior commissure, as is usual in most cases, as far as I am aware, except in Ammocoetes, where it appears shortly before the posterior commissure (Kupffer, 24, Fig. 5). It is found in all the chief types of vertebrates, and is usually smaller than the posterior (Minot, 28). It is developed from the diencephalon, while the posterior belongs to the cephalic limit of the mid brain.
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Conclusions.
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1. The paraphysis appears first in an embryo of 12 mm. It is developed from the telencephalon immediately cephalad to the velum transversum as a small diverticulum, which becomes eventually a complicated gland with anastomosing tubules. The gland is very vascular, and has a sinusoidal circulation.
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2. The epiphysis appears first in an embryo of 9-10 mm., and is developed from the diencephalon. It is always attached to the brain by a small solid stalk, and the cavity is partially subdivided by incomplete septa.
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3. The velum transversum grows at first ventrad and then caudad as far as the hind brain, forming in this way the diencephalic portion of the choroid plexus. The post-velar arch, which is at first quite extensive, is almost entirely absorbed in this extensive growth of the v.elum.
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4. The telencephalic plexus arises from the roof of the telencephalon, and fills up the depths of the cavity of the third ventricle. The opening of the paraphysis is surrounded by these two plexuses.
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5. The plexus of the hemispheres arises at a right angle from the telencephalic plexiis just cephalad and ventrad to the opening of the paraphysis.
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John Warren 25
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6. The supra-commissure appears first at 16-17 mm. It lies immediately cephalad to the stalk of the epiphysis and is comparatively small.
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7. The posterior commissure appears first at 15 mm., and there is a marked interval in the roof of the diencephalon between it and the epiphysis.
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I wish in conclusion to express my acknowledgments to Prof. Minot for his kind advise and interest in the preparation of this article.
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BIBLIOGRAPHY.
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The following are the principal articles consulted, but of course do not form a complete bibliography of this subject:
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1. BuRCKHAKDT, R. — Die Zirbel von Ichthyophis Glutinosus und Protopterus
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Annectens. Anat. Anz., Bd. VI.
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2. Die Homologien des Zwischenhirndaches bei Reptilien und Vogeln.
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Anat. Anz., Bd. IX, 320-324.
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3. Der Bauplan des Wirbeltiergehirns. Morpholog. Arbeiten, IV
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Bd., 2 Heft, 131.
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4. Untersuchungen am Gehirn und Geruchsorgan von Triton und
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Ichthyophis. Zeitschr. f. Wiss. Zoologie, Bd. 52.
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5. Dexter, F. — The Development of the Paraphysis in the Common Fowl.
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American Journ. Anat., Vol. II, No. 1, 13-24.
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6. Dendy, a. — On the Development of the Pineal Eye and Adjacent Organs
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in Sphenodon (Hatteria). Quart. Journal Micros. Soc, Vol. 42, 111.
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7. D'Ebchia, F. — Contributo alio studio della volta del cervello intermedio
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e della regione parafisaria in embrioni di Pesci e di Mammiferi. Monitore Zoologico, VII, 118 e 201.
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8. Eycleshymer, a. C. — Paraphysis and Epiphysis in Amblystoma. Anat.
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Anz., Bd. VII.
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9. Eycleshymer, A. C, and Davis, B. M. — The Early Development of the
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Paraphysis and Epiphysis in Amia. Journal of Comp. Neurology, Vol. 7.
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10. Francotte, p. — Recherches sur le developpement de L'epiphyse. Arch.
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de biologie, T. VIII.
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11. Note sur I'ceil parietal, l'epiphyse, la paraphyse et les plexus
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choroides du troisieme Ventricule. Bull, de I'acad, royale, etc., d. Belg., 3 Serie, T. 27.
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12. Contribution a I'tHude de I'oeil parietal, de l'epiphyse chez les
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Lacertiliens.
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13. Gage, S. P. — The Brain of Diemyctylus Viridescens. Wilder Quart. Cent.
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Book, 1898.
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14. Gaupp, E. — Zirbel, Parietalorgan und Paraphysis. Ergebn. Anat. Entwick. Ges., VII, 208-285.
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15. Hebbick, C. L. — Topography and Histology of the Brain of certain Rep tiles. Journ. of Comp. Neurology, Vol. I, 37; Vol. Ill, 77-104, 119-138.
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16. Topography and Histology of certain Ganoid Fishes. Journ. of
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Comp. Neurology, Vol. I, 162.
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26 Paraphysis and the Pineal Kegion in Nectun;s Macnlatus
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17. Hkkuick, C. L. — Embryological Notes on the Brain of a Snake. Journ.
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Neurology, Vol. I, lGO-176. IS. Hill, C. L. — The Epiphysis in Teleosts and Amia. Journ. of Morphology.
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Vol. IX, 237-268.
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19. His, W. — Zur allgemeinen Morphologie des Gehirns. His. Archiv, 1892,
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346-383.
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20. Humphrey, O. D. — On the Brain of the Snapping Turtle. Journ. of Comp.
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Neurology, Vol. IV, 73-108.
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21. Kingsbury, B. F. — The Brain of Necturus Maculatus. Journ. of Comp.
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Neurology, Vol. V.
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22. The Encephalic Evaginations in Ganoids. Journ. of Comp. Neurology, Vol. VII.
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23. KxiPFFER, C. V. — Studien zur vergleichenden Entwicklungsgeschichte des
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Kopfes der Kranioten. Hefte I.
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24. Derselbe. Hefte II.
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25. Lewis, F. T. — The Question of Sinusoids. Anat. Anx., Bd. XXV, No. 11.
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26. Leydig, F. — Zirbel und Jacobsonsche Organe einiger Reptilien. Archiv f.
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Mikrosk. Anatomie, Bd. 50.
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27. LocY, W. A. — Contribution to the Structure of the Vertebrate Head.
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Journ. of Morphology, XI.
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28. MiNOT, C. S. — On the Morphology of the Pineal Region, based on its
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Development In Acanthias. American Journ. of Anatomy, Vol. I, No. 1, 81-98.
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29. On a Hitherto Unrecognized Form of Blood Circulation without
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Capillaries in Organs of Vertebrates. Pro. Boston Soc. Nat. Hist., Vol. 29, No. 10, S. 185-215.
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30. OsBORN, H. F. — Preliminary Observations on the Brain of Menopoma.
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Proceed. Phil. Acad., 1884.
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31. Contribution to the Internal Structure of the Amphibian Brain.
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 +
Journ. of Morphology, Vol. II, 51-86.
 +
 +
32. Rex, H. — Beitrage zur Morphologie der Hirnvenen der Amphibien. Morph.
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Jahrb., XIX, 295-311.
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33. ScHOBEL, Jos. — Ueber die Blutgefasse des Cerebrospinalen Nervensystems
 +
 +
der Urodelen. Archiv f. Wissen. Mikros., Bd. XX, 87-91.
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34. Selenka, E. — Das Stirnorgan der Wirbelthiere. Biolog. Centralbl., Bd. X,
 +
 +
323-326.
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35. Sorensen, a. D. — The Roof of the Diencephalon. Journ. of Comp. Neu rology, III, 50-53.
 +
 +
36. Comparative Study of the Epiphysis and the Roof of the Diencephalon. Journal Comp. Neurology, IV, 153-170.
 +
 +
37. Continuation of above. Vol. IV, 153-170.
 +
 +
38. Studenicka, F. Cii. — Beitrage zur Anatomie und Entwicklungsgeschichte
 +
 +
des Vorderhirns der Cranioten.
 +
 +
39. VoELZKOw, A. — Epiphysis und Paraphysis bei Krokodilien und Schild krot>en. Abhand. der SenchenbiXrgischen Naturforschenden Gesellschaft, Bd. XXVII, Heft. II.
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John Warren
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27
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ABBREVIATIONS.
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A. C. — Anterior commissure. L. Y. Ch. Fix. — Choroid plexus. M. B Dien. — Diencephalon. 0. C D. Flex. — Diencephalic plexus. Tel. Plx. i?.— Epiphysis. Tel. Ep. A. — Epiphysal arch. F. C F. B. — Fore-brain. P. T- A. —
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F. M. — Foramen of Munro. ■ P. A. H. — Hemisphere. F.
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H-JB.— Hind brain. S.C.
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Hyp. — Hypophysis. Si.
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I. J. V. — Internal jugular vein. T.
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L. Fix. — Choroid plexus of lateral Tes.
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ventricle. ^'
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Lateral ventricle. Mid-brain. Optic commissure. -Telencephalic plexus. -Telencephalon. -Posterior commissure. Post-velar arch. Paraphysal arch. Paraphysis. -Superior commissure. Sinusoid. •Tubule. ■Vessel. -Velum transversum.
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THE DEVELOPMENT OF THE THYMUS.
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BY
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E. T. BELL, B. S., M. D.
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Instructor in Anatomy, University of Missouri.
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With 3 Plates and 5 Text Figures.
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This paper is intended mainly as a contribution to our knowledge of the histogenesis of the thymus in mammals. Special attention is given to the origin and development of the corpuscles of Hassall, since their mode of formation has never been satisfactorily described in mammals and their significance in all forms is in dispute. An attempt is also made to show in detail the changes that occur during the transformation of the thymus from the epithelial to the lymphoid condition.
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This work was begun at the suggestion of Dr. D. D. Lewis at the University of Chicago. The greater part of it has been done at the University of Missouri. Special acknowledgments are due Dr. C. M. Jackson for valuable criticism and suggestions. T wish also to thank Mr. Charles H. Miller of the University of Chicago for his kindness in sending me material.
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Material and Methods.
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As material for the greater part of my work, I. have used pig embryos from 8 mm. to full term (26 cm. to 30 cm.). These are especially suitable for such work since they may be procured in abundance from the large packing houses at almost any stage of development. For special purposes I have studied a few specimens from human foetuses, and from the cat, rat, and guinea pig. The smaller pigs used (8 mm. to 27 mm.') belong to the collection in the anatomical laboratory at the University of Missouri. These were stained in bulk with alum-cochineal and mounted in serial sections. In the later stages, which were prepared specially for this work, tbe ventral half of the cervical and anterior thoracic regions was usually out out and embedded from pigs from 3 cm. to 8 cm. On specimens from 8 cm. to 30 cm.. I dissected out the thymus and used sucb parts as were desired.
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^ The crown-rump measurement is used in all cases.
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AMERICAN .TOCTRNAL OF ANATOMY. VOI,. V.
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30 Tlie Development of the Thymus
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All pig material was fixed in Zenker's fluid, embedded in paraffin, and mounted in serial sections from 3 ju, to 10 /x thick. Except those of the young stages (8 mm. to 27 mm.) the sections were stained on the slide. Most of them were stained with hsematoxylin or iron-hgematoxylin and counterstained with Congo red. For special purposes many other stains were used.
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To demonstrate the delicate protoplasmic threads of the syncytium during the later stages of the lymphoid transformation, I stained by the iron-haematoxylin method but omitted the final decolorization in ironalum. Protoplasm is stained deep black; nuclear structure is poorly shown, but the finest cytoplasmic processes may be seen.
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For the demonstration of connective tissue fibrillae in the syncytium, I found the method recommended by Jackson (13, S. 39) most satisfactory.
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Text Fig. 2.
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Text Figure 1. Cranial view of third gill pouch (thymic anlage) ; X 33; pig embryo, 11 mm.; ec, ectoderm; I, lumen; nt, nodulus thymicus; ph, connection to pharynx; s p, sinus prsecervicalis.
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Text Figure 2. Ventral view of thymic anlage; X 33; pig embryo, 15 mm.; ec, ectoderm; I, lumen; n t, nodulus thymicus; ph, connection to pharynx; s p, sinus prsecervicalis.
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To determine the relation of the blood-vessels to the corpuscles of Hassall, I put a young kitten under deep anesthesia and injected a large quantity of a strong aqueous solution of Prussian blue into the aorta through the common carotid artery. The heart continues to beat even after an amount of fluid twice as great as the total volume of the blood has been injected. An injection made in this way is under a slightly increased blood pressure and easily reaches the finest capillaries. There is therefore a thorough injection with little danger of rupturing delicate blood-vessels.
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Organogenesis. My observations on this phase of development are not sufficiently complete to warrant a full discussion. A brief survey may however prepare the way for a better understanding of the histogenesis. The text figures show the shape in outline of the third gill pouch and thymic anlage
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E. T. Bell 31
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from 11 mm. to 27 mm. . They are graphic reconstructions. Since this pouch is nearly all converted into thymus it may be regarded as the thymic anlage from a very early stage.
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At 11 mm. (Text Figure 1), the pouch is a hollow epithelial tube directed from without ventrally and mesially. The lumen (l) is large and communicates freely with the pharynx. On the dorso-lateral aspect of the pouch is a solid epithelial mass (n t) distinctly different in structure from the rest of the pouch. This is the nodulus thymicus (Kastschenko, 14) and will be referred to by that term. This structure has been described by Stieda (26), Prenant ' (22), and others as the anlage of the carotid gland.
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It was evidently mistaken by Minot ' in a 12-mm. pig for the anlage of the entire thymus. It may be seen as early as the 8 mm. stage budding off from the cranio-lateral aspect of the pouch. Immediately behind the nodulus thymicus, but not connected to it at this stage is the inner blind extremity of the sinus prsecervicalis (s p). These become fused at 12 mm. or 13 mm.
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At 15 mm. (Text Figure 2) the thymic anlage is more elongated. It now projects ventrally and medianwards, its free end lying immediately caudad to the median thyroid anlage and just craniad to the pericardium. Its lumen (Z) is still in communication with the pharynx. The sinus prsecervicalis {s p) is drawn out, its lumen being smaller and longer. It is now fused to the outer two-thirds of the posterior aspect of the nodulus thymicus (n t).
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At 18 mm. (Text Figure 3) the anlage is growing rapidly in a caudal direction and just entering the thoracic cavity. It is connected to the pharynx by a delicate epithelial cord. There is still a lumen in its caudal part. The sinus praecervicalis has lost its connection to the nodulus thymicus." The outer part of its lumen has disappeared and it seems about to lose its connection with the ectoderm.
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At 20 mm. (Text Figure 4) the thymus extends well into the thoracic cavity. Its thoracic segment (t h) has increased considerably in size and is united to the gland of the opposite side.
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The nodulus thymicus still forms the greater part of the head. The anlage has no connection with the pharynx or the epidermis. There is
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"Prenant is said to have since abandoned this idea and accepted Kastschenko's view. (v. Ebner in Kolliker's Gewebelehre des Menschen. Aufl. 6, Bd. 3, 1, S. 340.)
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^Laboratory Text of Embryology, p. 191; also p. 209 and Fig. 124.
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On the opposite side in this specimen, .these structures were fused over a
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very small area.
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32
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The Development of the Thymus
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now an elongated mass of thymic tissue extending upwards behind the nodiilus thymicus, and fused with it. Its upper pointed extremity curves outwards around the hypoglossal nerve. This is the " thymus superficialis " (t s) of Kastschenko and is regarded by him as being formed from the sinus praecervicalis. Kastschenko describes this elongated portion as being always separate from the rest of the head, being connected only by connective tissue. In my preparations it is clearly continuous with the rest of the anlage, and seems to have formed by growing out from it. The presence of a lumen in its lower end favors Kast
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.ec
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Text Fig. 3.
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Text Fig. 4.
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Text Figure 3. Ventral view of thymic anlage; X 33; pig embryo, 18 mm.; ec, ectoderm; I, lumen; nt, nodulus thymicus; ph, connection to pharynx; s p, sinus praecervicalis.
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Text Figure 4. Ventral view of thymic anlage; X 33; pig embryo, 20 mm.; f, area fused with gland of opposite side; I, lumen; nt, nodulus thymicus; th, thoracic segment; t s, thymus superficialis.
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schenko's view, for there is no lumen in the head at 18 mm. in my preparations. On the other hand the separation from the head at the 18 mm. stage favors the idea that the sinus prsecervicalis degenerates. I have not studied a sufficient number of specimens at this transition stage to enable me to decide this point, though I believe the ectoderm takes no part in the formation of the thymic anlage. Kastschenko's results are opposed by nearly all other students of this problem, but his work should not be discarded before the development of the thymus superficialis in the pig has been accurately determined.
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At 27 mm. (Text Figure 5) the thymus is much longer and extends
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E. T. Bell
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33
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well down into the thoracic cavity in relation to the base of the heart where it has fused with the gland of the opposite side. Buds are now beginning to form through the greater part of its extent. Traces of the original lumen (I) may still be seen in several places. The "thymus superficialis " is bent around the twelfth nerve. A delicate cord of thymic
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Text Fig. 5.
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Text Figxjre 5. Ventral view of thymic anlage; X 33; pig embryo, 27 mm.; /. area fused with gland of opposite side; 1, lumen; nt, nodulus thymicus; th, thoracic segment; t s, thymus superficialis.
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tissue fused with the l)ack of the nodulus thymicus connects the thymus superficialis to the rest of the head.
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From about 3 cm. until toward the end of fcetal life the thymus shows the two constrictions, described by Prenant (22) (for the sheep) as the intermediary and the cervico-thoracic cords. These cords connect three 3
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3-i Tlic Development of the Tliynius
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enlargements which we may call the head, the luid-t-ervical segment, and the thoracic segment. I will consider each part separately. The thoracic segment develops rapidly, spreading out ahove and in front of the heart. The glands of the two sides fuse completely in this region. The lymphoid transformation is noticeable at 3.5 cm. and well advanced at 4.5 cm. The medulla begins to form at 8 cm. The cervico-thoracic cord is at first very narrow but soon thickens and joins the cord of the opposite side. At full term they form a sharp constriction, 3 mm. to 4 mm. wide and 5 mm. to 6 mm. long, situated at the superior aperture of the thorax, and connecting the mid-cervical and thoracic segments. The histological changes take place here later than in the enlargements.
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The mid-cervical segment develops like the thoracic segment but somewhat more slowly. Budding, lymphoid transformation, and formation of the medulla all begin here a little later than in the head and thoracic segment. Its caudal end is slightly in advance of its cranial end. The intermediary cord is well marked at 4 cm. It soon becomes very attenuated, having at 6 cm. in many places a diameter of only 15 fx. Prenant suggests that this drawing out of the gland is caused by the rapid growth of the neck. Later it increases in size and at full term is noticeable only as a very slight constriction between the head and the mid-cervical segment. The histological changes are much later here than in other parts of the gland.
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The greater part of the head in the early stages is formed by the nodulus thymicus. This body grows slowly, attaining a diameter in crosssection of .5 mm. at 8 cm. Its cross-sectional area at 8 cm. is about onethird that of the rest of the head of the thymus. At this stage the nodulus thymicus is a rounded body lying on the inner aspect of the head in relation to the carotid artery. A small area of its outer surface is fused with the lymphoid tissue of the thymus. Its histological structure has been fully described by Prenant (22). From the earliest stages, it consists of cords of epithelial cells separated by blood capillaries. At 8 cm. the thymus superficialis (Kastschenko) is a large body lying craniad and dorsal to the rest of the head but connected to it at its caudal extremity.
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I have no observations on the head of the thymus between 8 cm. and full term, having overlooked it in collecting my material. In a full term pig (30 cm.), the cervical part of the thymus is in two distinct parts. The postero-ventral part, representing part of the head, the intermediary cord, and the mid-cervical segment, is about 3 cm. long and 1 cm. wide. It extends from the upper border of the thyroid cartilage to the thorax, and with the corresponding part of the opposite gland encloses the
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E. T. Bell 35
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trachea, thyroid, and lower ]iait of the larynx. A slight narrowing at the junction of the upper and middle thirds indicates the position of the intermediary cord. The antero-dorsal part^ the thymus superficialis, is rounded in cross-section, tapei-rrfg- to a point behind. Its anterior end is about 7 mm. in diameter and loops around the twelfth nerve as in the earliest stages. A lobule hangs over ventral to the nerve, a thin cord being dorsal to it. The posterior end of the thymus superficialis extends to the cricoid cartilage dorsal to the postero-ventral part of the gland. It is united to this part of the gland by a very delicate band of thymic tissue. I did not find the nodulus thymicus at full term. It has either moved away from the head or degenerated.
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The duct of the thymus is the lumen of the third gill pouch. A glance at the Text Figures will show its development. It is broken up into segments and finally obliterated. I could find no traces of it at 3.7 cm. or later. On the theory of the exclusively endodermal origin of the thymus. I cannot explain the absence of a lumen in the head at IS mm. and its presence at 20 mm. and 27 mm. unless it be due to individual variation in different specimens.
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It appears from the foregoing that in the pig the exclusively endodermal origin of the thymus from the third gill [loueh is probable, but a slight participation by the ectoderm has not been satisfactorily excluded. Kastschenko's conclusions, however, as to the ectodermal origin of the thymus are imwarranted by his recorded observations. Prenant, after his careful work (on the sheep), was not sure that a small mass of ectoderm did not enter into the formation of the head. Practically all other investigators of this problem maintain that tlie ectoderm takes no part in the formation of the thymus. The epithelial l)ody (nodulus thymicus) developing in connection witli tlie head of the thymus from the third gill pouch does not form the carotid gland. Kastschenko's description of the origin of the carotid gland in mammals from the adventitia of the internal carotid is now accepted by the majority of anatomists, and it therefore has nothing to do with the thymus in oi'igin.
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TiiK Hist<)L()(;y of tiik FrLLY-F()i;.Mi:i) Thymus.
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Before taking u\) tlic histogenesis, I shall bi'iclly (.-(Uisider the histology of the gland as shown in a 24-cni. embryo. At this stage ihe gland nuiy be regarded as fully formed. As is well known, tlie thymic lobule consists of a cortical and a medidlary portion, — the medulla of all the lobules being united by the medullary cord. The cortex consists of a delicate reticulum with its spaces well filled by cells, usually lymphocytes. The reticulum may be regarded as composed of small branched anastom
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36 The Development of the Thymus
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osing cells, though of eourse no cell houndaries are distinguishable. The nuclei are poor in chromatin, rounded, and usually 4.5 /a to 5.5 /x in diameter. The amount of cytoplasm around the nuclei and connecting them is usually very small. At some nodes there is a greater amount of cytoplasm giving the appearance of a large reticulum cell. In connection with the lilood-vessels, which are numerous in the cortex, are often found branched cells with pale nuclei and cytoplasm that stains more intensely than that of the rest of the reticulum. By a modification of Mallory's method used by Jackson (13, S. 39), I have been able to demonstrate numerous fibrillEe in the reticulum. In some cortical areas at this stage there are a great many erythroblasts. Masses of free erythrocytes are often found, usually near a comparatively large vessel, but such cells occur singly anywhere in the cortex.
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In the medulla, the syncytial character of the stroma is much more pronounced. The cytoplasm is much more abundant than in the cortex, and the spaces are smaller and not so numerous. There is not so much room for lymphocytes as in the cortex, hence the lighter color of the medulla in stained preparations. The nuclei of the syncytium are either pale or dark, both types showing wide variations in size. By Jackson's method (13, S. 39), fibrilla3 may be readily demonstrated in the syncytium. In Plate I, Fig. 6, is shown the arrangement of these fibrillse (s f) in the medulla at 24 cm. They often may be traced into the areas which are forming the concentric corpuscles. In some parts of the medulla the fibrillae are very numerous ; in a few places, entirely absent. In both cortex and medulla eosinophile cells are often found. These occur in groups in the interlobular tissue around the blood-vessels, around some of the corpuscles of Hassall, and singly in the reticulum. These have been described by Schaffer (24). Free erythrocytes a.re rarely found in the medulla. In the medulla are also found the corpuscles of Hassall. Since the structure of these bodies depends largely upon their age, it may be better understood from the consideration of their development.
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The Lymphoid Transformation.'
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Kolliker, in 79, first advanced the idea that the leucocytes are formed directly from the epithelial cells of the thymic anlage. According to Minot (in human embryology, p. 878), "he records for the rabbit that between the twentieth and twenty-third days the cells of the thymus become smaller and their outlines disappear, so that the organ appears to
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° This term will be used to include those changes that occur in the thymus during its passage from an epithelial to a characteristic lymphoid structure.
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,E. T. Bell 37
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bo ail atiuiiiulaticni of small round nuclei. At about the same period blood-vessels and eonneetive tissue grow into the epithelial anlage."
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His {I'i 1)). 80, and Stieda (26), 81, claimed that the corpuscles of Hassall are the only remnants of the epithelial anlage, that the lymphocytes, reticulum, etc., are of mesenchymal origin.
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Maurer (17 a), 86, described the leucocytes as arising directly from the cells of the epithelial anlage in the thymus of teleosts. In the amphibian thymus (17 b), 88, he thinks that the leucocytes are probably of mesenchymal origin. He was unwilling to conclude that they arose from the epithelium because he could not find transition forms. In lizards (17 c), 99, he records that even before the separation of the epithelial anlage of the thymus from the pharynx, changes begin. The peripheral cells are closely crowded together and show many mitoses. There arises between the central cells, or is formed in vacuoles in their protoplasm, a fluid which accumulates until the nuclei surrounded by a thin zone of protoplasm are connected only by protoplasmic threads. A loose medulla is thus formed which looks like a cellular reticulum. The cortex is still solid. The lymphocytes are formed from the epithelial cells; none come from without. Later, blood-vessels and connective tissue grow in. He believes that the reticulum is of mesenchymal origin in all forms. Maurer (17 d), 02, still holds that in amphibians the lymphocytes are probably of mesenchymal origin.
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Plermann et Tourneux (11), 87, find that in man and other mammals the epithelial anlage of the thymus is gradually converted into leucocytes and reticulum cells. Vacuoles appear during the transformation which seem to be formed by the absorption of large cells. In a sheep embryo of 130 mm., the clear epithelial cells have all disappeared, giving rise to small round cells and reticulum cells. Prolongations of connective tissue, each containing a blood-vessel, grow into the anlage during the transformation. They are not sure that all the thymic elements are epithelial in origin, being especially in doubt about the origin of some of the reticulum cells.
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Gulland (8), 91, describes the development of the tonsil in the rabbit. Leucocytes first appear in the connective tissue around the thymus. Later they appear in the connective tissue around the tonsil. They infiltrate the tonsillar epithelium. Fo leucocytes are of epithelial origin. After studying the tonsil he examined the thymus in a few specimens and concluded that the same process of leucocyte infiltration occurred there. He does not give the details of their infiltration, and did not see any of the transition forms of nuclei in the thymus at that period.
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Prenant (22), 94, made a careful study of the development of the
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38 The Drvi'lopincnt ol' the 'riiynius
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thynuis in sheep embrvos. His results are as follows. At 25 mm. the gland is composed of distinct polyhedral cells with nuclei of only one kind. A few mitoses and an occasional direct division are to be seen. At 20 mm. mitoses are numerous (oi^e nucleus in fifty). jSTuclei are regularly rounded or elliptical and some small nuclei occur juxtaposed to large nuclei. At 28 mm. many mitoses are present and irregular spaces have appeared. These spaces are not blood-vessels nor parts of the thymic duet but vacuoles. Some nuclei, noticeably small and darkly colored, lie close to the large, clear nuclei and seem to be budded off from them. Some nuclei (rare) are broken into three or four chromatic bodies. Embryos of 40 mm. have undergone in great part the lymphoid transformation. All transitions are found between the large, pale elliptical nuclei of clear reticular structure and the small, deeply colored rounded nuclei whose sap is strongly stained. These last are certainly lymphocytes and constitute an immense majority of the cellular elements. Large, clear nuclei are found Joined to small dark ones — nuclear couples.
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At 85 mm. the medulla appears; the cortex corresponds to the entire thymic mass of preceding stages. The cortex contains a great many lymphocytes separated by islands and rows of pale nuclei. There are about thirty lymphocytes to one pale nucleus. Mitoses are numerous in the cortex. In the medulla at this stage, the large clear, and small dark, nuclei are about equal in number, and mitoses are rarer than in the cortex. In later embryonic stages a clear peripheral zone is present where cell proliferation takes place. Mitoses are now more numerous in the medulla than in the cortex. It is probable that a certain number of the epithelial cells persist as reticulum cells in the fully-formed organ.
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J. Beard (3 a), 94, (3 b), 99, thinks that the function of the thymus is to form the first leucocytes. He finds that in the skate the epithelial cells are converted early into lymphocytes which emigrate into the blood. There are many breaks in the gland where the lymphocytes escape in masses. The thymus is the only source of leucocytes until the other lymphoid organs are formed.
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Ver Eecke (28), 99, finds that in the frog the epithelial thymus is invaded by lymphocytes and connective tissue. The epithelial cells are not destroyed but merely dispersed by the mesenchymal elements. He calls the resulting tissue lympho-epithelial. This idea of the commingling of the two tissues had already been advanced by Eettcrer.
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Xusbaum and Prymak (20), 01, on teleosts, agree with j\Iaurer that the lymphocytes are of epithelial origin but disagree on the details of their formation. They find that the epithelial anlage is at first composed of cells with distinct lioundaries. It is not different from the epithelium
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E. T. Bell 39
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of the pharynx. Before any blood-vessels or connective tissue have invaded the organ, changes begin in. the central part. These changes consist in the breaking up of the cytoplasm so that the cells become branched and connected by delicate processes. These processes finally break apart leaving a nucleus surrounded by a thin layer of protoplasm — a lymphocyte. The peripheral epithelial layer multiplies rapidly, forming nuclei somewhat smaller and darker than their own. These nuclei become gradually changed into the nuclei of lymphocytes and break away from the other cells. All transitions are present between the large, clear epithelial nuclei and the lymphocytes. Blood-vessels and connective tissue grow in from the outside.
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It appears from a survey of the literature that, of those who have studied the origin of lymphocytes in the mamalian thymus. His, Stieda, and Gulland have advocated the idea that they invade the gland from without, and that the original epithelial anlage persists only as remnants, the corpuscles of Hassall. They also consider the stroma of mesenchymal origin. On the other hand, Kolliker, Hermann and Tourneux, and Prenant, have described the lymphocytes as derived directly from the epithelial cells of the anlage. Hermann and Tourneux and Prenant ascribed a similar origin to part of the reticulum.
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Xeither His, Stieda, nor Gulland made a detailed histological study of the changes that take place in the thymus during the transformation. They did not see the vacuolization of the cytoplasm, the changes in the epithelial nuclei, etc. — processes wdiich undoubtedly occur. Gulland made nearly all his observations on the tonsil and then from a superficial examination of the thymus concluded that the process is the same there. The conclusions of these men are therefore not to be compared on this point with those obtained by the thorough and careful work of Prenant.
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On amphibians, Maurer hesitatingly agrees with His, and Yer Eecke accepts the mesenchymal origin of the leucocytes ; while on fishes Maurer, Beard, and Nusbaum and Prymak believe in the epithelial origin of lymphocytes. Maurer's work on reptiles is in agreement with his work on teleosts.
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As to the origin of leucocytes in the lymphoid organs of tlie alimentary canal, opinion is divided. Eetterer, v. Davidoff, Rudinger, Klaatsch, and others have described the leucocytes as arising from epithelium and being invaded by mesenchymal elements forming adenoid tissue. Stohr, Gulland, Tomarkin, and others describe them as penetrating the epithelium from without.
 +
 +
I shall now discuss my own observations on the lymphoid transformation in the thymus of the pig. From a very early stage (11 mm.), the
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40 The Development of the Thymus
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 +
t'pithelium of the third gill pouch is a syncytium. No cell boundaries exist. The nuclei, large and irregular in shape, are embedded in a common mass of cytoplasm. In the thymus at 20 mm. I find a syncytium of dense cytoplasm embedded in which are large nuclei of irregular shape and size. No distinct types of nuclei are present yet; all stain with medium intensity. A few mitoses are to be seen.
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 +
In a section of the mid-cervical segment at 3.7 cm. (Plate I, Fig. 1), I find evidence that the lymphoid transformation has begun. The syncytium is composed of coarsely reticulated cytoplasm much looser in texture than that of the preceding stage. It contains a few irregular spaces (s s) which are evidently of the nature of vacuoles. These may be formed, as Maurer suggests, by liquefaction of the cytoplasm. There is no reason to suppose that cells degenerate and form them as Hermann and Tourneux believed. Three types of nuclei may be distinguished; large pale nuclei (Ipn) large dark nuclei (I dn), and small dark nuclei (lymphoblasts) (Ih). Transition forms occur between these types. The large dark nuclei are intermediate forms between the pale nuclei and the lymphoblasts. A few mitoses (m) occur. No blood-vessels are present inside the anlage but they may be seen between the buds just outside. At this stage, the head and the thoracic segment have areas that are somewhat farther advanced than this. The intermediary and cervico-thoracic cords show no changes.
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At a later stage than the above (Plate I, Fig. 2), in the thoracic segment of a 4.5-cm. pig, jthe spaces of the syncytium (s s) have increased greatly in number and size. The anlage is now a cellular reticulum. The large pale nuclei are somewhat less numerous than the dark nuclei and many have become angular, adapting themselves to the nodes of the syncytium. They contain less chromatin than in the preceding stage. Large dark nuclei and lymphoblasts are present; the lymphoblasts are much more numerous than in the preceding stage. A very few small dark nuclei are completely separated from the syncytium. These are lymphocytes. There are no lymphocytes in the connective tissue around the thymus or in the blood at this stage. I did not examine the tonsil or spleen at any stage. A few small blood-vessels are to be seen; their walls consist of endothelium only. There are more mitoses than at the preceding stage, but none happened to be present in the area shown in the figure. During mitosis, at all stages of development, except the early epithelial condition, the chromosomes are so closely packed that it is very difficult to distinguish them individually.
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 +
In a section through the mid-cervical segment of a 7-cm. pig (Plate I, Fig. 5), we see a stage somewhat later than the one shown in
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E. T. Bell 41
 +
 +
Fig. 2. In various jiarts of the section lymphocytes (I) are completely formed. Tlie great majority of the small round nuclei are in the lymphoblast (l h) condition, i. e.,thcy are not yet completely separated from the syncytium. There are a few lymphocytes outside the thymus in the interlobular tissue in this region; around the head and the thoracic segment at 7 cm. they are numerous, these parts being in a later stage of transformation. I have never seen lymphocytes outside the thyfnus, where there were none inside it ; but they appear outside shortly after they are formed here. Those formed next the interlobular septa seem to pass out very early. Of course the lymphoblasts, which are distinguishable from the lymphocytes only by being imbedded in the syncytium, are to be seen in the thymus long before any appear outside.
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 +
At the stage shown in Fig. 5, a great many nuclei are in mitosis. I have not seen at any stage, the amitoses and nuclear couples described by Prenant for the sheep. In some parts of the section comparatively large solid epithelial areas occur. These are found as often in the central as in the peripheral part. Many of the pale nuclei are smaller than those shown in Fig. 2. The blood-vessels are somewhat larger and more numerous than those at 4.5 cm.
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 +
It is to be noted that the epithelial anlage does not at any stage become converted entirely into small round cells as many observers have stated. Distinctly pale angular reticular nuclei can always be seen.
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 +
In the mid-cervical segment at 8.5 cm. (Plate I, Fig. 4), a great many lymphocytes (/) are formed. These lie between the persisting epithelial cells which are now arranged in irregular cords and islands. In these epithelial masses, lymphoblasts may still be seen indicating that the formation of lymphocytes is still in progTCss. Many of the pale nuclei are now small. The heavy hsematoxylin stain in this case makes the nuclei darker than they would appear with an ordinary stain. A few nuclei are in mitosis.
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This figure shows also the first appearance of the medulla {ind). The medulla is formed directly, as shown in the figure, from persisting parts of the epithelial syncytium. Certain centrally situated masses of this syncytium undergo changes of such a nature that they stain readily with cytoplasmic stains such as Congo red. In sections stained with hematoxylin and Congo red, the medulla is first recognized as a l)rightly colored area situated usually about the center of the lobule. Tlicse epithelial masses that give rise to the medulla seem to increase in size about the time of the change in staining capacity. The first differentiation of the medulla is chemical rather than morphological, for there are other persisting epithelial masses even larger than it in the same section
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42 The Development of the Thymus
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that do not react in the same way with the cytophismic stains. The meduna appears in the head and the thoracic segment at 7.5 cm. to 8 cm. All the gland except this small central area forms the cortex. Bloodvessels now reach 'all parts of the gland, but are still few in number. I cannot distinguish any wall except the endothelium on those actually inside the gland.
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In a 9.5-cm. pig, the medulla is larger. It contains pale nuclei of various sizes, large dark nuclei, and lymphoblasts. Its spaces are smaller than those of the cortex. The early stages in the formation of the corpuscles of Hassall appear as soon as the medulla begins to form. The epithelial cords in the cortex have become less conspicuous, but are still forming lymphocytes. A few nuclei are in mitosis. Many blood capillaries may now be seen penetrating the gland from the periphery. These vessels run in the epithelial masses and have a wall of large endothelial cells which gives them the appearance of radiating cords. When these vessels first appear, as at this stage, they have only an endothelial wall. The blood-vessels grow in as small capillaries which, after their entrance, increase in size and branch ; they do not break in as large vessels surrounded by mesenchymal tissue. I am fairly sure that aside from the endothelial cells few or no mesenchymal cells come into the thymus. Around the greater part of the periphery of the gland is a solid zone of syncytium two or three nuclei deep which is in transformation like the epithelial cords inside. This zone, described by Prenant as a zone of proliferation, grows rapidly, as the frequent mitoses indicate. Its inner boundary is forming lymphocytes and reticulum cells.
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 +
In a 14-cm. pig, the lymphoid transformation is practically at an end except in the medulla. The peripheral zone of proliferation has disappeared. The cortex has about the same structure as at 24 cm., as previously described. In the medulla, lymphoblasts, large pale nuclei, and the large dark intermediate types are still present. There are a few mitoses here. It is very probable therefore that the formation of lymphocytes is still in progress in the medulla. The medulla at 2-i cm. shows a similar structure except that there are fewer lymphoblasts. These facts persuade me to regard the medulla as a center for lymphocyte formation at least as late as birth. Connective tissue fibrillae begin to appear in the gland along the large blood-vessels and the interlolmlar septa as early as 10.5 cm. They are only a little farther in at 16 cm. ; but near full term they are present in nearly all parts of the stroma. (See Plate I, Fig. 6.)
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The above account may be summarized as follows: In the pig the epithelial syncytium of the thymic anlage becomes loosened up by the
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E. T. Bell 43
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formation of vacuoles in it. These vacuoles increase in number and size, converting the anlage into a cellular reticulum. While this vacuolization is in progress, the nuclei, which at first are of one kind with a medium amount of chromatin, ditferentiate into large clear, large dark, and small dark (lymphoblast) forms. The large dark nuclei probably divide by mitosis and form the lymphoblasts. The lymphoblasts gradually break loose from the syncytium, passing into its spaces and becoming lymphocytes. Shortly after lymphocytes begin to be formed, some of them pass out of the gland into the surrounding connective tissue. The lymphoid transformation begins in embryos of 2.5 cm. to 3 cm. and continues in the cortex until 13 cm. or 13 cm. In the medulla it is not complete at birth. Since the thymus increases greatly in size during this period the epithelial syncytium must grow rapidly. Lymphocytes are constantly being formed at the expense of the growing syncytium. A peripheral zone of proliferation is present from about 8 cm. to 13 cm. The medulla is formed as a chemical differentiation of certain centrally situated areas of the epithelial syncytium. The histological changes occur earlier in the head and thoracic segment than in the mid-cervical segment and very much earlier than in the cords. The reticulum of both cortex and medulla is practically all of e])ithelial origin. Some branched cells around the blood-vessels in the cortex may be of mesenchymal origin.
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My reasons for regarding tlie lymphocytes as of epithelial origin are as follows :
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A. The lympholilasts are true epithelial nuclei, because (1) there are numerous transition forms between them and the large dark nuclei which later cannot l)e regarded as invading lymphocytes; (3) they are closely embedded in the syncytium and show no evidence of having eaten their way through the protoplasm; (3) they are present from a very early stage and increase in number as development proceeds; (4) they are present before blood-vessels invade the gland and have no constant relation to blood-vessels or to the surface of the gland that indicates an invasion from either of these directions; (5) they are present before lymphocytes appear in the connective-tissue around the thymus.
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B. Some observers admit tliat the small dark nuclei (lymphoblasts) are of epithelial origin but do not admit that tlicy form lym])hocytes. The considerations that lead me to bciicNc tliat the lymphoblasts do form the lymphocytes are: (1) the small dark nuclei (lymphoblasts) show every possible relation to the syncytium from being completely embedded in it to lying free in the syncytial s]")aces. A comparison with later stages shows tb.at tliis appearance is not due to poor fixation oi- to the
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44 'V\\v ncvclopnioiit of tlio M'hymns
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adherence of the nuclei to the reticuhini ; (?) the first free niiclci often appear in the center of the gland whoi tlicrc are no other free nuclei in the periphery at that level; (3) there is good evidence that lymphocytes emigrate from the thymns in large numbers. If we examine the thymus of a 7-cm. pig in serial sections we find that the lymphoid transformation is less advanced in the mid-cervical segment than in the head. In the mid-cervical segment there are a few lymphocytes in the interlobular tissue. In the lower end of the head where there are more lymphocytes inside the gland, lymphocytes pack the interlobular tissue and form a thin zone around the periphery of the gland. In the middle of the head where the transformation is far advanced, lymphocytes pack the interlobular tissue and form a thick zone around the entire gland. Indeed, in some sections, there are more l3T3iphocytes in the zone outside than are present inside the gland. If this zone of lymphocytes be passing into the gland, it is not easy to understand why it is formed from Avithin outwards, and why it is thickest where the greatest number of lymphoc3ies are already present inside. No satisfactory suggestion has yet been made as to why lymphocytes should thus suddenly pour into the thymus at a time when if present at all elsewhere they are rare. They do not come to break up the thymic epithelium, for that is already a reticulum before free cells are present (Fig. 3, Plate I). Where lymphocytes invade intestinal epithelium as in the tonsil they eat paths through it leaving spaces. The epithelial reticulum of the thymus is not formed in that way. On the other hand it is not difficult to believe that this zone of lymphocytes is formed l^y cells passing out the periphery of the thymus and that the gland thus contributes a great number of lymphocytes to the organism; (4) I have not been able to find lymphocytes in the connectivetissue around the th}Tuus before they are present inside. An invasion by way of the blood-vessels may be excluded, since the thick zone of lymphocytes formed around the gland shows that these cells either enter or leave it through the preiphery.
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The Corpuscles of Hassall.
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These bodies were first mentioned by Hassall ( 10 ) in 46, He speaks of them as being composed of mother cells which enclose the newlyformed daughter cells and nuclei. He thought the central mass was formed by the outer enclosing layers. He found bodies which lie regarded of the same nature in fibrous coagulations in the heart.
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Virchow (29), 51, in a discussion of endogenous cell formation, compares Hassall's corpuscles to carcinoma pearls. He had about the same
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E. T. Bell 45
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conception of the nature of the corpuscles as Hassall. This oft-quoted comparison was therefore not based upon a deep insight into their nature.
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Giinzburg (9), 57, did not advance beyond Hassall's conception that the central mass is formed by the peripheral layers.
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Paulitzky (21), 63, described the center of the corpuscles as homogeneous or granular. They sometimes contain an elliptical nucleus, sometimes fat droplets. The larger ones have in the center several nuclei' or cell-like forms. The central part is formed from masses of epithelial cells. Connective tissue cells grow around them and are transformed into epithelial cells forming the peripheral part of the corpuscle.
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The term "concentric corpuscles" was introduced by Ecker (6), who described them as arising directly from gland cells by fatty metamorphosis. He distinguished (1) simple corpuscles, round vesicles with thick concentric hulls, containing inside a fatty opalescent mass, and (2) compound corpuscles, which consist of several vesicles with a common hull. The peripheral layers of a corpuscle consist of flattened cells.
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His (13 a, 12 b), 60, 80, described the corpuscles as consisting of an outer striated shell, probably composed of nucleated cells, and containing lymphocyte-like cells inside. He supposed them to be the original cells of the epithelial anlage which become entangled in the reticulum in some way. Their rapid growth in their narrow confines causes the concentric form.
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Cornil et Eanvier (5), 69, considered the corpuscles as arising from the endothelium of blood-vessels and compared them to the spheres of their " Sarcome angiolithique."
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This suggestion of a vascular origin, made by Cornil et Eanvier, was elaborated by Afanassiew (la), 77.
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Afanassiew held that the corpuscles of Hassall arise from the endothelium of the smaller veins and capillaries. The endothelial cells increase in size, become cubical, and later fill the lumen of the vessel. During the proliferation of the endothelium, the blood-vessels break up into segments which are now nearly solid cords. These cords are at first connected to each other and to blood-vessels, but they soon break apart. The surest proof that the corpuscles are of vascular origin is that erythrocytes may be found inside them. Vascular injections, however, do not go into a corpuscle except in a very early stage, since the lumen is soon obliterated by the endothelial plugs. The corpuscles are formed entirely by the endothelial cells. Afanassiew worked on embryos of man, the rabbit, and the calf.
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Stieda (26), 81, in sheep embryos, describes the epithelial mass of the
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4G Tlie Dcv('lf)|)tn(Mit of the Tli\nms
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thymic aulage as being broken up by ingrowing adenoid tissue. From 50 mm. to 60 mm., there arc no hirge epithelial ceils; but later at 100 mm. ho iinds in the adenoid tissue large cells 9 /a to 15 /x in diameter, isolated or united in groups, Avhose protoplasm colors light-red with carmine. These large cells have a concentric structure. Some of them are enclosed bv large cells whose cytoplasm does not color with carmine, giving rise to a yellowish mass of irregular form and stratified appearance. In older embryos (250 mm.), the cellular masses are numerous but the large colored cells are rare. The yellowish masses are groups of the large cells which have undergone a transformation like that of the stratum corneum of the epidermis. Stieda considers the large colored cells which form the corpuscles as remnants of the epithelial anlage, although he admits that for a long period during development he found no trace of them. He explains the formation of the corpuscles in accordance with Cohnheim's hypothesis that most tumors arise from unused tissue remnants.
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Ammann (2) 82, made most of his observations on human foetuses. He describes the corpuscles as arising from connective tissue. The corpuscles are cellular in structure and are formed of one, two, or three central cells around which a variable number of cells, increasing with age, are arranged like the coats of an onion. The corpuscles are formed from reticulum cells and leucocytes. Growth consists in the apposition of cells from without. The life of a corpuscle consists usually of four stages : (1) Stadium der Transparenz ; (3) Stadium der colloidcn Entartung; (3) Stadium der Verkalkung; (4) Stadium des Zerfalls. The nucleus of a reticulum cell or leucocyte increases in size at the expense of the cell body. Its increase in size establishes the concentric form. The corpuscle undergoes colloid and usually calcareous degeneration. Fat droplets, cholesterin crystals, and colloid granules are found together in the degenerating corpuscles. Breaking up in this way makes absorption possible. No epithelial remnants are to be observed. Xo erythrocytes are found in the corpuscles.
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In four cases of atrophic thymus gland wliich yet contained lymphoid tissue Ammann found corpuscles in all stages of development. He also found that the corpuscles are formed most rapidly when the thymus is at the height of its development. From these facts he concluded that they are not connected with the involution of the thymus as Afanassiew thought. He thought that their formation is due to a physiological decrease in the intensity of growth of the medulla, due to the rapid growth of the cortex.
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Watney (31), 83, agreed with Ammann that the corpuscles arise from connective tissue cells.
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E. T. Bell 47
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Monguidi (18), 85, distinguished true and false concentric corpuscles — • the latter being onl}- sections of blood-vessels.
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Hermann et Tourneux (11), 87, gave a description of the structure and formation of the concentric corpuscles about like that given by Ammann except that they regard the reticulum cells from which the corpuscles develop as of epithelial origin.
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Gulland (8), 91, regarded the corpuscles as epithelial remnants and compared them to the epithelial pearls of the tonsil.
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Maurer (17 c), 99, described the corpuscles as epithelial in origin. His description of their formation is however different from that of His. All the cells of the epithelial anlage at first assume a lymphoid character. Later, some of these cells reassume their epithelial nature and then form the corpuscles. His conclusions for teleosts and amphibians are similar to the above results which he obtained from the lizard.
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Yer Eecke (28), 99, for the frog, describes the leucocytes and connective tissue cells as invading the thymic anlage and separating the epithelial cells. The epithelial cells, separated by the mesenchymal elements, lie at first in groups or singly. They go through a cycle of two phases, a stage of growth, and a stage of involution. In the former stage, they increase to three or four times their original size and their cytoplasm differentiates into circular layers like the coats of an onion. The majority are monocellular. Some cells grow together making a more complex multicellular type. There are some intermediate forms, cells with a dense dark protoplasmic body, indistinct striations, and a nucleus partly or completely hidden in a precocious degeneration. In the stage of involution, which sets in early, the cytoplasm degenerates by the formation of vacuoles containing a hyaline liquid. The liquefaction may be in the form of a diffuse vacuolization, a large central vacuole, or a peripheral vacuole circular in section. The nucleus loses its affinity for stains, becomes deformed, breaks up, and finally disappears. The corpuscles are finally absorbed. They never contain erythrocytes. The cells do not degenerate to form a corpuscle. The liquefaction forms an internal secretion which is forced out by the muscle tissue in the reticulum.
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Entirely different results on amphibians are reported by Nusbaum and Machowski (19), 02. These investigators revive the old idea of Afanassiew, accepting his results except that they think the adventitia as well as the endothelium of the blood-vessels takes part in the formation of the corpuscles. They find erythrocytes in the corpuscles. These erythrocytes either gradually shrivel and disappear, or they are absorbed by leucocytes or endothelial cells. The leucocytes after digesting the hemoglobin of the erythrocytes become eosinophile cells which are numerous in the thymus.
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48 The Development of the Thymus
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Wallisch (30), 03, measured the volume of the human thymus and of the corpuscles of Hassali at various stages. He finds that the total volume of the corpuscles of a 7-mo. embryo is 4.6 mm./ and of those of a 6-mo. child, 174.6 mm." The total volume of the thymus of a 78-mm. embryo, when it has already been partly transformed into adenoid tissue is only 6.8 mm.^ Since there is no evidence that the cells of the corpuscles multiply, he concludes that they cannot be regarded merely as remnants of the original epithelial anlage.
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Disregarding the crude observations of the earliest investigators, there remain three distinct theories of the formation of the corpuscles of Hassali.
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1. The epithelial anlage of the thymus is broken up by the invading mesenchymal elements. The separated masses of epithelial cells undergo further changes mainly of a degenerative nature to form the corpuscles. This was the belief of His and KoUiker. According to this interpretation, the corpuscles are to be regarded as remnants that have nothing further to do with the gland. Stieda, Maurer, and Ver Eecke held this view in a modified form. Stieda regarded the cells forming the corpuscles as epithelial remnants but admitted that they go through a stage in which, for a time, they lose their epithelial character. This is substantially the same as ]\Iaurer's view. He thinks that the cells of the epithelial anlage all become lymphoid, and that some of them afterwards reassume their epithelial nature and form the corpuscles. Yer Eecke regards the corpuscles as epithelial remnants but thinks that they are glandular in nature, not mere useless remains.
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2. The corpuscles are formed from the proliferating walls of bloodvessels. This idea was suggested by Cornil and Eanvier and elaborated by Afanassiew. Nusbaum and Machowski accept Afanassiew's view except that they believe the adventitia of the blood-vessels as well as their endothelium takes part in the formation of a corpuscle. These investigators thought that the formation of the corpuscles is connected with the involution of the thymus.
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3. The corpuscles are formed from reticulum cells of the medulla and grow by apposition of the surrounding cells. This view was advanced by Ammann. Ammann thought that the reticulum is of connective tissue origin. He also believed that leucocytes formed the central part at least of some corpuscles. Hermann and Tourneux accepted Ammann's results, except that they ascribed an epithelial origin to the reticulum. (I do not know whether they accepted the origin from leucocytes described by Ammann.) Ammann thought that the corpuscles formed because of a physiological decrease in the rate of growth in the medulla.
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E. T. Bell 49
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l\Iy own observations on tlie development of tlie eorpuseles of Hassall in pi_o- embrvos. will now be considered. The medulla, as previouslv described, begins to form from the epithelial syncvtium usually near the center of the lobule. It is first recognized by its more marked reaction with cytoplasmic stains such as Congo red. Shortly after the medulla begins to form, the earliest stages of the corpuscles may be observed. - A few corpuscles have appeared at 9.5 cm. I did not find them earlier. They are all formed from the epithelial syncytium of the medulla.
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Before beginning this discussion I will explain the use of ray terms. By a corpuscle of Hassall, I mean a modified area of the epithelial syncytium of the medulla, containing at some period of its development, one or more nuclei, and whose cytoplasm has been in part or entirely transformed into colloid. The term colloid is applied to various substances probably of widely different chemical nature, but is fairly adapted to our imperfect knowledge. I shall use the term here in the restricted sense employed by Ziegler," i. e., hyaline substances of epithelial origin, that do not give the reactions of mucin.
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Colloid in the corpuscles of Hassall does not usually appear as solid masses in its early formation, but as fibers, granules, or sheets which are separated by more or less cytoplasm that is not yet changed. This stage I have called, "colloid in formation" (c f). It later assumes a more solid homogeneous appearance which I call solid colloid (c s). Often the solid colloid stains intensely with cyto])lasmic stains. I call this kind solid deeply-staining colloid (c .*? d). In later stages, the colloid often loses its affinity for cytoplasmic stains, staining a very pale color or not staining at all. I call this variety old colloid (o c).
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According to their mode of development, the corpuscles of Hassall may be classified as follows :
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A. Concentric Corpuscles.
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a. Simple.
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1. Ordinary type.
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2. Epithelioid type.
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3. Cystic ty.pe.
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b. Compound.
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B. Irregular Corpuscles.
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a. Compact type.
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b. Eeticular type.
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Gen. Pathology, 10th ed., Warthin's translation, p. 205.
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50 Tlie Development of the Thymus
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A. The concentric corpuscles inchule those that from their earliest appearance are concentric in structure. Adopting Ecker's classification, I distinguis^i simple concentric corpuscles and compound concentric corpuscles.
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(a) Three types of simple concentric corpuscles are to be considered. (1) The ordinary type is far more numerous than any other. The earliest recognizable stage is shown in Plate II, Fig. 11. A nucleus (n) of the syncytium of the medulla has enlarged to perhaps twice its ordinary volume and has lost the ability to stain in the characteristic way with ha?matoxylin. Its sap is clear and a few reddish stained granules represent its chromatin. x\round it in the cytoplasm is an indistinct uneven layer of colloid (c /). The colloid is not yet solid and is being formed in concentric fibers or sheets. A slightly later stage is shown in Plate II, Fig. 14 and Fig. 15 (left side of figure). Some of the colloid (c s) next to the nucleus is now solid. The next stage is shown in Plate II, Fig. 15 (right side of figure). These corpuscles show a thick layer of colloid (c s d) that stains intensely with Congo red. Just outside the deeply staining colloid, colloid in formation may be seen. The nuclei are clear, and have become smaller and irregular in outline. The colloid seems to be pressing upon them and obliterating them. The colloid transformation gradually involves the adjacent c3^toplasm of the syncytium until other nuclei are involved. The corpuscle has now reached the condition shown in Plate II, Fig. 12. The central area (o c) is solid, the nucleus having disappeared entirely. Another (n') is nearly obliterated by the colloid. Part of the central area {o c) no longer stains intensely, and it is breaking loose by the formation of a concentric space. Several nuclei are surrounded by colloid in formation. Their long axes are nearly in a tangential direction. These nuclei are clear but only moderately swollen.
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In the further development of the corpuscle (Plate III, Fig. 17 and Plate II, Fig. 7), the central area {c s d) increases in size. The nuclei involved in this area become obliterated probably by the pressure of the colloid and are no longer distinguishable. This central area usually splits off and may break up into many smaller masses. The peripheral part of the corpuscle increases by extension of the colloid formation into the adjacent part of the syncytium. This extension takes place in the early stages by direct progressive involvement of the immediately adjacent cytoplasm; in later stages (Fig. 7), by the formation of concentric lamellae which cut ofl' unchanged areas of cytoplasm. The lamelhp increase in size and number, the cytoplasm included between them is changed into colloid. They finally become closely packed, giving the characteristic and
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E. T. Bell 51
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well-kncnvn onion-like structure found in the fully-formed eorpusele. The nuclei that are enclosed between the lamells! gradually lose their chromatin and become flattened out. They do not swell and are not obliterated. It seems that swelling occurs only in nuclei that are surrounded by deeply staining colloid, and that this change is preparatory to their obliteration by or transformation into colloid. The amount of the corpuscle that breaks up to form the softer center is very variable. The size of the center usually seems to increase with the age of the corpuscle.
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Plate III, Fig. 21, shows a variation from the ordinary concentric type. The central nucleus (n) stains reddish but is not enlarged. Most of the other nuclei are unchanged. All the colloid (c /") is in the early fibrous and granular stage.
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From 20 cm. to full term nmny corpuscles show masses of calcareous material in or near the center. This material rarely appears in younger corpuscles (Plate III, Fig. 17, cl). It stains a violet blue with Delafield's ha?matoxylin.
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The majority of the corpuscles of Hassall belong to the ordinary type of simple concentric cor])uscles described above. It is very clear that they have nothing to do with blood-vessels. They never contain erythrocytes nor anything resembling them. Earely a lymphoblast or leucocyte is found inside the corpuscle. These seem to be usually involved in the corpuscle like ordinary stronui nuclei during the formation of the lamellae. (Their occurrence in other types will be discussed later.) It is also clear that these corpuscles arise from the syncytium of the medulla. They are epithelial in origin, since the entire stroma of the gland is derived from epithelium, but they are certainly not remnants of the original epithelial anlage. Xeither are they forjned from lymphoidlike elements that reassume their epithelial nature as Maurer described for the lizard.
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Some of Ammann's observations are in accord with my results. The swelling of the nucleus was noted by Ammann as tlu; first step toward the formation of the corpuscle. It should he noted, however, that rarely a corpuscle begins to form as a mass of colloid out in the cytoplasm and involves nuclei secondarily. I cannot distiiiguish his " Stadium der Transparenz " for I cannot be sure that a corpuscle is beginning to form until some colloid is present. The formation of the colloid is associated with the swelling of the nucleus. His other three stages, "Stadium der coUoiden Entartung," " Stadium der Yerkalkung," and " Stadium des Zerfalls " are easily seen. I have never seen corpuscles begin in leucocytes as x\mmann described. His statement thnt the corpuscle grows by apposition of reticulum cells is true in a modified sense. He thought that
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52 Tln' I )L'V('lo[)iiu'iit of []]{' 'I'liymus
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the outer ])art of a {•or])nscle is formed of reticuhim cells that have moved up and thittened themselves out around it. The description just given shows ihat tlie coi'puscles are iieNcr composed of distinct cells, and that the increase in size is due to an extension outward of tlie colloid foi'mation and not to a moving in of the adjacent tissue.
 +
 +
The concentric form of this type of corpuscle is due at first to its l)eing formed around a spherical or ellipsoidal nucleus. The swelling of this nucleus creates a centrifugal pressure in the adjacent cytoplasm. Before or during its transformation into colloid, the cytoplasm also increases in quantity\ That the cytoplasm increases in quantity is shown hy the fact that the nuclei are fewer in the corpuscle than in any adjacent area of the syncytium of equal size. This centrifugal pressure presses the newly formed colloid into concentric lamellae. It at first turns the long axes of the nuclei tangentially. and later flattens them and makes them concave toward the center.
 +
 +
3. The epithelioid type of corpuscle is characterized by large areas of cytoplasm so marked off by colloid lamella? as to give the appearance of a mass of large epithelial cells. They may contain only one nucleus embedded in a well-defined area of cytoplasm (Plate III, Figs. 18 and 20). These correspond to the monocellular corpuscles that have been described for lizards and amphibians. They are rare in the pig. I have not been able to trace these very far, as they soon become indistinguishable from other forms. The only difference I have noted is that the outer colloid lamellae begin to form early, causing the peculiar appearance of a large epithelial cell. Again the epithelioid type may present an appearance such as shown in Plate I, Fig. 3. These do not seem to be formed around any special nucleus. The outer colloid lamella? form before any center has been established, niarking off large cytoplasmic areas that may look like large cells. The centrifugal pressure of expansion caused by the great increase of cytoplasm in this area determines the concentric form in these corpuscles. Pure epithelioid corpuscles are very rare, but epithelioid areas in other corpuscles are not uncommon. The occurrence of epithelioid areas in corpuscles of the ordinary type shows that it is due to variations in a fundamentally similar process.
 +
 +
3. In the cystic type of corpuscle, the central part, instead of becoming transformed into colloid, undergoes early liquefaction, forming vacuoles. The central nucleus does not increase in size as in the ordinary type, but shrivels up and disappears. The corpuscle begins by the formation of outer colloid lamellae-^the central mass is not changed into colloid. In Plate II, Fig. 10, the central area (/; m) is undergoing a diffuse liquefaction. The nucleus (iv) is colorless and shrunken. In Plate TI.
 +
 +
 +
 +
E. T. Bell 53
 +
 +
Fig. 8 (right side of figure), the central area has fonned two lai-ge vacuoles (v). On the left side of the same figure, a concentric vacuole (v) has formed, separating off a central spherical nucleated mass of protoplasm. The nucleus of this mass of protoplasm is shrunken and the cytoplasm shows many small vacuoles. The corpuscle shown in Plate II, Fig. 9, is probably a later stage of the form just described. The central protoplasmic mass has become converted into an ellipsoidal pale bo'dy (pm). The small circular body in this shriveled mass is probably the nucleus. Some corpuscles like the one shown in Fig. 9 are found in wdiich the central mass has entirely disappeared. The further growth of corpuscles of this type seems to be by formation of colloid lamella? as in the ordinary type. They soon become indistinguishable from other forms.
 +
 +
The cystic type of corpuscle is rare in the pig. This evidently corresponds to the form in amphibia that misled Nusbaum and Machowski into reviving Afanassiew's theory. The central masses, in Figs. 8 and 9, might readily be mistaken for red corpuscles in animals in which these cells are nucleated. But the red cells of the blood of the pig are not nucleated at this stage. I have traced a number of these corpuscles (as well as those of other types) in serial sections and have never seen any indications of a connection to blood-vessels. Nusbaum and Machowski (19), (Fig. 1, c, S. 116) show a corpuscle which is similar to jny Fig. 9. It will 1)0 noted that the central space in neither of these figures is lined by endothelium. The early form of corpuscle shown by Nusbaum and Machowski (Fig. 1, d, S. 116) is very probably a normal bloodvessel with cubical endothelium. I have often found such vessels with cubical endothelium in the interlobular tissue of the pig's thymus at 10 cm. to 12 cm. They probably may be found at other stages also. In the thymus of a kitten, injected l)y the intra-vitam Prussian blue method previously described, the majority of the corpuscles were found to be in early stages. The injection did not penetrate any corpuscle. I had a somewhat better opportunity to study the relations of the corpuscles to the blood-vessels in a 14 cm. human embryo. Here the vessels of the thymus were all very much distended with blood and the corpuscles were in early stages. No blood cells were found in the corpuscles.
 +
 +
(b) Compound concentric corpuscles are formed whenever two or more simple concentric corpuscles begin to form so close together that they come in contact during their later growth. An early stage of such a corpuscle is shown in Plate II, Fig. 15. The colloid lamellae are formed around each center until they come in contact; they are tlien formed around both centers. In Plate III, Fig. 22, a compound concentric cor
 +
 +
 +
5-t Tlio D(>\('l()|)in('iit of the Tliyiinis
 +
 +
piisclc is shown. 'I'lici'c aiT three siiiiph' coiu'cntric corpuscles in it — one of them (the h>\vcst in the liyiii-c) in a very early stage. Several lamella' ai'c common lo the older cor|)uscles, and one is common to all three. This ari'an<i'ement of the lamella' is a mechanical efTect of the tension in the cytoplasm, due to the ccntrifug^al pressure from the two centers. The size of the separate centers in a compound corpuscle depends upon the stage they have reached when they come in contact. If a compound coi'puscle he formed hy the union of two simple corpuscles in an early stage, as in Plate 111, Fig. 19, all indications of its compound nature are soon lost. A corpuscle originally compound may, then, in later stages, hecome indistinguishal)Ie fi'om simple corjniscles. The simple corpuscles uniting to form a compound concentric cor]niscle may be of any of the types previously described.
 +
 +
B. Irregular Corpuscles.
 +
 +
This grou]) includes those corpuscles which are not at first concentric. Concentric areas may appear later. According to the classification previously given, I distinguish a compact type and a reticular type.
 +
 +
(a) The compact type (Plate III, Fig. 16) first appears as a compact area of syncytium of irregular shape. It is recognizable l)y the colloid it contains. The nuclei are not noticeably increased in size and have no regular arrangement. Their chromatin still stains dark with nuclear stains. The colloid (c f) is not yet solid. The corpuscle has no distinct center. These corpuscles grow by direct colloid transformation of the adjacent syncytium. No distinct lamella^ are formed. The colloid may remain in the fibrous condition shown in the figure (c f) or it may become solid, but it never reaches the deeply staining condition unless a concentric area be established.
 +
 +
A later stage of this tyi)e is shown in Plate III, Fig. 23. The corpuscle is sharply marked off from the syncytium. Some of its colloid is solid. A concentric area (cs) is beginning to form. The nuclei are not markedly different from those of the adjacent syncytium. These corpuscles may become large and branched. Often one or more concentric areas are developed after the corpuscle has attained considerable size. By the growth of these concentric areas, irregidar corpuscles may become converted into concentric corpuscles.
 +
 +
(b) The reticular type is produced by colloid formation in the ordinary reticulum of the medulla. In the types previously described, the spaces of the reticulum are \isually obliterated as the colloid formation advances; but in this form the spaces persist as a part of the corpuscle. Pure reticular corpuscles vary greatly in size, sometimes involving only
 +
 +
 +
 +
E. 'W Bell ■ 55
 +
 +
one node of the syncytium. Thev are never concentric, and never form lamellae. Eeticnlar areas often occur in other forms of corpuscles. In tiiis way leucocytes are often involved in tlic corpuscle, since they lie in the spaces of the reticulum. Lymphocytes often get into a corpuscle in the lymphohlast condition, heing cut off hy tlio formation of lamellae outside tliem (Plate III, Fig. 22). The Icui'Dcytes shut in the cor])uscle in this way during development uuiy not degenerate. They probably persist and help to remove the corpuscle in its final stages of degeneration.
 +
 +
The amount of expansion of the cyt()i)lasm before or during the colloid transformation is probably small in the irregular reticular corpuscles, since it does not obliterate the spaces of the syncytium. In the compact type, the spaces of the syncytium are obliterated and there is evidence of some expansive force (note the arrangenunit of the nuclei in the upper part of Fig. 23, Plate III). In the figure referred to, the number of nuclei in any part of the corpuscle is less than in an equal area of the adjacent reticulum. These facts indicate that there is an expansion of the cytoplasm. That this expansive force does not produce a concentric form is due primarily to the fact that there is no expansion of a nucleus and distinct center of formation as is present in concentric corpuscles of the ordinary type. The absence of the onion-like structure in irregular corpuscles is due to the fact that the colloid is not laid down in lanielUw
 +
 +
Significance of the corpuscles of Hassall. It has been shown in the preceding pages that the corpuscles of Hassall in the pig are not epithelial remnants, and also that they are not formed from blood-vessels. There is no evidence connecting their development with the involution of the thymus, for they begin to form before the lymphoid transformation is complete and are most numerous when the thymus is at the height of its development. I have not been al)le to see the decrease in the rate of growth of the medulla described by Amnumn, and even if such did occur it is difficult to understand how it could cause the formation of a corpuscle.
 +
 +
The above theories are, therefore, inconsistent with the facts of development in the pig. It seems to me that the formation of a corpuscle is not to be regarded as a ]u-ocess of degeneration. The fact that the formation of colloid is an essential feature in the development of every corpuscle is a strong argument that it is a form of secretion such as occurs in its neighl)oring branchial derivative, the thyroid. The fact that the corpuscles differentiate in an aj^parently uniform syncytium is further evidence airainst a theory of degeneration. Since the lymphocyte-forming fuiu-tion of tbc tliymus is probaI)ly secondary, it is not
 +
 +
 +
 +
56 The Dov(^lopin(<Tit of tlio Thymus
 +
 +
unreasonable to suppose that its primitive fimetion was the formation of a colloid secretion such as occurs in the thyroid, and that the corpuscles are abortive expressions of this primitive function/
 +
 +
Giant Cells.
 +
 +
Polykaryocytes may often 1)0 seen in the medulla. Tliese bodies develop froui the syncylium of the uieilulla. They are first noticeable as groups of small s])herieal nuclei in a solid area of the syncytium. Tliese nuclei stain with merlium intensity and are all very similar in size and color. The area containing this group of nuclei becomes a well-defined node of the reticulum and persists as such. A polykaryocyte is, therefore, a large node of the reticulum containing a number of small nuclei very similar in appearance. These cells often occur in groups. They are entirely distinct from the corpuscles. They are evidently similar to the polykaryocytes found in bone marrow and other lymphoid tissues.
 +
 +
Summary.
 +
 +
The following is a resume of the development of the thymus in the pig :
 +
 +
The thymus of the pig is probably developed entirely from the endoderm of the third gill pouch.
 +
 +
By a gradual process of vacuolization and liquefaction of the cytoplasm, the epithelial syncytium of the thymic anlage is converted into a cellular reticulum.
 +
 +
From the first appearance of vacuolization, three types of nuclei are present: large pale nuclei; small dark nuclei (lymphoblasts), and large dark intermediate forms.
 +
 +
The lymphoblasts gradually break loose from the cellular reticulum, moving into its spaces and forming lymphocytes. Mitoses are most numerous at the period of the most rapid formation of lymphocytes. The medulla continues to form lymphocytes at least as late as birth.
 +
 +
Lymphocytes appear in the connective tissue aromul tlu> th.ynuis shortly after they are formed ; and lymphoblasts, wdiich are distinguishable from lymphocytes only by being embedded in the syncytium, arc present in the thymus a long period before lymphocytes are found anywhere in the neighborhood of the thymus.
 +
 +
The celhdar reticulum of the earlier stages persists in a modified form as the reticulum of both cortex and medulla. It retains more cytoplasm
 +
 +
' Ver Eecke (28) believes that the corpuscles in amphibians are of a glandular nature.
 +
 +
 +
 +
E. T. Bell 57
 +
 +
in tlio inedulla. Practically all the reticnhira of both cortex and nie(liilla. as well as the lymphocytes, are, therefore, of epithelial origin.
 +
 +
The {■oi-])iisclos of Hassall develop from the syncytium and arc, therefore, epithelial in origin. They are, however, not to be considered as remnants of the original epithelial anlage.
 +
 +
In development various types of corpuscles are distinguished. Tlie ordinary typo of concentric corpuscles first appears as an enlarged clear nucleus around which colloid is being formed. Before or during the formation of colloid, the cytoplasm increases in quantity, filling the spaces of the reticulum and producing a centrifugal pressure which shapes the newly-formed colloid into concentric lamellse and flattens the neighboring nuclei, making them concave toward the center. The central nuclei usually become obliterated.
 +
 +
The epithelioid type is distinguished by its resemblance to large epithelial cells, this appearance being due to the formation of colloid lamella" around largo areas of clear cytoplasm. The central part of the corpuscle usually remains unchanged until after some of the colloid lamellfE are formed.
 +
 +
The cystic type differs from the ordinary type only in that the central part undergoes vacuolization instead of colloid transformation. Those with concentric vacuoles may simulate blood-vessels containing nucleated red cells. Corpuscles never contain erythrocytes; neither can they be injected at any stage of development. Serial sections also show tliat there is no connection to blood-vessels at any stage.
 +
 +
Compound concentric corpuscles are formed by the union of two or more simple concentric corpuscles during development.
 +
 +
Irregular corpuscles are not concentric ' in arrangement, and are formed in the syncytium in an irregular manner. In the compact type of irregular corpuscles, concentric areas may form.
 +
 +
The formation of colloid is an essential feature in the development of every corpuscle, and is not to be considered as a process of degeneration.
 +
 +
Since the conclusion of my work and after my manuscript was given to the publishers, two articles dealing with the thymus have appeared.
 +
 +
Ph. Stohr (Ueber die Thymus, Sitzungsberichte der phys.-med. Gesellschaft zu Wiirzburg, June 8, 1905) believes that the thymus first epithelial in nature becomes converted entirely into small cells of lymphoid appearance. Later the large reticulum cells are formed from these by enlargement. The corpuscles of Hassall are formed by the massing together and enlargement of these lymphoid-like cells. The small round cells of the gland are epithelial in origin but are to be regarded not as lymphocytes but as epithelial cells. The thymus is not a source of lymphocytes.
 +
 +
 +
 +
58 J'lu^ Devolopmont nf the Tliymiis
 +
 +
The author apparently believes that none of the small rounrt cells leave the gland though he admits that lymphocytes enter. But as mentioned above the zone of connective tissue immediately around the head at 7 cm. may contain even more lymphocytes than are present inside the gland at that time If these are all entering the gland then it is probable that most of the small round cells are really lymphocytes. This conception then does not simplify the problem but is only a theoretical compromise between the two views a'i to the origin of the lymphocytes.
 +
 +
J. Aug. Hammar (Zur Histogenese und Involution der Thymusdriise, Anat. Anz. Bd. XXVII, June 17, 1905) regards the reticulum as formed from the epithelial anlage but thinks the evidence at hand insufficient to decide the question as to the origin of the lymphocytes. He finds lymphocytes outside the thymus in many animals (man, cat, chick, frog) before any are present inside the gland. The corpuscles of Hassall develop from the epithelial reticulum and undergo hyaline (colloid?) degeneration.
 +
 +
My description of the formation of the corpuscles of Hassall differs essentially from Hammar's, in that I believe the formation of the corpuscle consists in the expansion of the cytoplasm of the syncytium and its conversion into colloid. Hammar did not recognize " colloid in formation," though he speaks of the coarse fibrillar structure of the protoplasm. He -did not describe such corpuscles as are shown in Fig. 7, Plate II.
 +
 +
The considerations presented above in favor of the epithelial origin of the lymphocytes seem to me much stronger than those given by Hammar. His statements as to the presence of lymphocytes around the thymus before they are present inside are to be taken with some reservation inasmuch as he mentions small round cells separate from the syncytium earlier, but regards ihem as degenerating epithelial cells (S. 65). His figure from the human foetus (Fig. 18, S. 66) does not seem to be strong support for his statement. Certainly many lymphocytes are present in the pig thymus when the reticulum is broken up as much as shown in the figure referred to. It is also to be borne in mind that the different parts of the thymus undergo the lymphoid transformation at different times and that a single section may therefore be misleading.
 +
 +
LITERATURE.
 +
 +
la. Afanassiew, B. — Ueber die concentrischen Korper der Thymus. Archiv
 +
 +
f. mikr. Anat., Bd. XIV, 1877. lb. Weitere Untersuchungen iiber den Bau und die Entwickelung
 +
 +
der Thymus und der Wintcrschlafdriise der Saugethiere. Archiv f.
 +
 +
mikr. Anat., Bd. XIV, 1877. 2. Ammann, a. — Beitrage zur Anatomic der Thymusdriise. Basel, 1882. 3a. Beard. — The development and probable function of the thymus. Anat.
 +
 +
Anz., Bd. IX, 1894. 3b. The true function of the thymus. Lancet, 1899.
 +
 +
4. Born, G.— Ueber die Derivate der embryonalen Schlundbogen und
 +
 +
Schlundspalten bei Saugethieren. Archiv f. mikr. Anat., Bd. XXII, 1883.
 +
 +
5. CoRNiL et Ranvier. — Manuel d'histologie pathologique. Paris, 1869, p.
 +
 +
135 (cited from Ammann).
 +
 +
 +
 +
E. T. Bell 59
 +
 +
6. EcKER. — Art. " Blutgefassdrusen," Wagner's Handw. der Phys., Ill (cited
 +
 +
from Ammann).
 +
 +
7. Priedleben, a. — Die Physiol, der Thymusdriise. Frankfurt, 1858.
 +
 +
8. GULLAND. — The Development of adenoid tissue with special reference
 +
 +
to the tonsil and thymus. Laboratory Reports issued by the Royal College Phys., Edinburgh, Vol. Ill, 1891.
 +
 +
9. GiJNZBURG. — Ueber die geschichteten Korper der Thymus. Zeitschr. f.
 +
 +
klin. Med., Bd. VI, 1857, S. 456 (cited from Henle und Meissner. Bericht iiber die Fortschritte der Anat. u. Physiol.).
 +
 +
10. Hassall. — The microscropical anatomy of the human body in health and
 +
 +
disease. London, 1846 (cited from Ammann).
 +
 +
11. Hermann et Tourneux. — Article thymus, Diet, encycl. des Sciences Medi cales. Troisieme Serie, 17, 1887. 12a. His, W.— Zeitschrift f. wiss. Zoologie, Bd. X, S. 348. Leipzig, 1860. 12b. Anatomic menschlicher Embryonen. Leipzig, 1880, S. 56.
 +
 +
13. Jackson, C. M. — Zur Histologie und Histogenese des Knochenmarkes.
 +
 +
Archiv f. Anat. und Physiol., Anat. Abth., 1904.
 +
 +
14. Kastschenko. — Das Schicksal der embryonalen Schlundspalten bei
 +
 +
Saugethieren. Archiv f. mikr. Anat., Bd. XXX, 1887.
 +
 +
15. Klein. — Neuere Arbeiten iiber die Glandula Thymus. Centralbl. f. allg.
 +
 +
Pathol, u. pathol. Anat., 1898.
 +
 +
16. Langerhans und Savei.iew. — Beitrage zur Physiologic der Brustdriise.
 +
 +
Virchow's Archiv, Bd. 134, 1S93. 17a. Maurer. — Schilddriise und Thymus der Teleostier. Morph. Jahrb.. Bd.
 +
 +
XI, 1886. 17b. Schilddriise, Thymus, und Kiemenreste bei Amphibien. Morph.
 +
 +
Jahrb., Bd. XIII, 1888. 17c. Schilddriise, Thymus, und andere Schlundspaltenderivate bei der
 +
 +
Eidechse. Morph. Jahrb., Bd. XXVII, 1899. 17d. In Hertwig's Handbuch der Entwickelungslehre der Wirbelthiere,
 +
 +
Lief. 6-8, S. 131 ff., 1902.
 +
 +
18. MoNGUiui. — Sulla glandula timo. Parma, 1885 (cited from Prenant).
 +
 +
19. NusBAUM, J., und Maciiowski. — Die Bildung der concentrischen Korper chen und die phagocytotischen Vorgange bei der Involution der Amphibienthymus, etc. Anat. Anz., Bd. XXI, 1902.
 +
 +
20. NusBAUM, J., und Prymak, T.— Zur Entwickelungsgeschichte der lym phoiden Elemente der Thymus bei den Knochenfischen. Anat. Anz., Bd. XIX, 1901.
 +
 +
21. Paulitzky. — Disquis. de stratis glandulse thymi corpusculis. Habilita tionsschr., Halis, 1S63 (cited from Henle und Meissner's Bericht iiber die Fortschritte der Anat. und Physiol.).
 +
 +
22. Prenant. — Developpement organique et histologique du thymus, de la
 +
 +
glande thyroide, et de la glande carotidienne. La Cellule, Tome X, 1894.
 +
 +
23. Prymak. T. — Beitrage zur Kenntnis des feineren Baues und der Involu tion der Thymusdriise bei den Teleostieren. Anat. Anz., Bd. XXI, 1902.
 +
 +
 +
 +
60 'I'lio Dovclnpmont of the T'hynms
 +
 +
24. SciiAKFER, J. — Ueber den feirieren Bau der Thymus und deren Beziehung^
 +
 +
zur Blutbildung. Sitzungsber. d. K. Acad. d. Wissensch. Math.naturw. Kl. Wien., Bd. CII, Abt. Ill, 1893.
 +
 +
25. SciiEnEL, J. — Zellvermehrung in der Thymusdriise. Archiv f. mikr.
 +
 +
Anat., Bd. XXIV.
 +
 +
26. Stieda, L. — Untersuchungen uber die Entwickelung der Glandula Thy mus, Glandula Thyroidea, und Glandula Carotica. Leipzig, 1881 (cited from Hermann et Tourneux).
 +
 +
27. Sx'LTAN.^Beitrag zur Involution der Thymusdriise. Virchow's Archiv,
 +
 +
Bd. 144, 189G.
 +
 +
28. Vek Eecke. — Structure et modifications fonctionelles du thymus de la
 +
 +
grenouille. Bulletin de I'Acadc'mie royale de MC'dicine de Belgique, 1899.
 +
 +
29. ViKciiow, R. — Kritisches iiber den oberschlesischen Typhus. Archiv, Bd.
 +
 +
3, 1851, S. 222.
 +
 +
30. Wallisch. — Zur Bedeutung der Hassall'schen Korperchen. Archiv f.
 +
 +
mikr. Anat., 1903.
 +
 +
31. Watxey. — The minute anatomy of the thymus. Philos. Transact, of the
 +
 +
Royal Society of London, Vol. 173, Part III, 1883 (cited from Prenant).
 +
 +
EXPLANATION OF PLATES.
 +
 +
All the figures were drawn with Leitz obj. 1/12, oc. 4, and camera lucida. The magnification after the reduction of the plates is about 1060 diameters. All drawings were made from transverse sections of the mid-cervical segment of the thymus unless they are otherwise indicated.
 +
 +
The following abbreviations designate the structures indicated in all the figures :
 +
 +
c f — colloid in formation. I p n — -large pale nucleus.
 +
 +
cl — -calcareous deposit. m — nucleus in mitosis,
 +
 +
c s — solid colloid. vid — beginning of medulla,
 +
 +
c s d — solid colloid that stains n — nucleus.
 +
 +
deeply. o c — old colloid.
 +
 +
e — erythrocyte. p m — protoplasmic mass.
 +
 +
end — -endothelial nucleus. sf — fibril in syncytium.
 +
 +
I — lymphocyte. ss — space in syncytium.
 +
 +
Z& — lymphoblast. v — vacuole. I d n — large dark nucleus.
 +
 +
Plate I.
 +
 +
Fig. 1. From a 3.7-cm. pig embryo. Stained with iron-lijematoxylin and Congo red. Vacuolization of the cytoplasm and differentiation of the nuclei have begun.
 +
 +
Fig. 2. From the thoracic segment of a 4.5-cm. pig embryo. Stained with iron-haematoxylin and Congo red. A cellular reticulum is now formed. Large pale nuclei, lymphoblasts, and the large dark intermediate forms are present.
 +
 +
Fig. 3. Epithelioid type of concentric corpuscle. From a 16-cm. pig embryo. Stained with hajmatoxylin and Congo red. Colloid lamellae (c s d)
 +
 +
 +
 +
E. T. Bell 61
 +
 +
separate large areas of cleai- cytoplasm, causing the appearance of large epithelial cells. Colloid is being formed between the lamellae and around several nuclei.
 +
 +
Fit;. 4. From a 8.5-cm. pig embryo. Stained with iron-hsematoxylin (not decolorized). The medulla has appeared. Lymphocytes are present between the epithelial cords.
 +
 +
Fig. 5. From a 7-cm. pig embryo. Stained with iron-htematoxylin and Congo red. A few lymphocytes have been formed. In the cellular reticulum are large pale nuclei, lymphoblasts, and large dark intermediate nuclei. The nuclei in mitosis are very compact.
 +
 +
Fig. 6. From the medulla of a 24-cm. pig embryo. Stained with Jackson's modification of Mallory's method (ref. in text). Many fibrilte are seen in the syncytium.
 +
 +
Plate II.
 +
 +
Fig. 7. Ordinary type of simple concentric corpuscle. From a 16.5-cm. pig embryo. Stained with hsematoxylin and Congo red. The corpuscle is well advanced in development. Concentric lamellag of colloid have been formed. The cytoplasm between the lamellse is in an early stage of colloid transformation. Colloid fibers cut transversely appear as dots. The nuclei are becoming flattened by the pressure of expansion. The central mass stains irregularly and all traces of the nuclei in that region are gone.
 +
 +
Fig. 8. Two cystic concentric corpuscles. From a 16-cm. pig embryo. Stained with iron-hgematoxylin and Congo red. On the left, a nucleated mass of protoplasm has been separated off by the formation of a vacuole annular in section. This might be mistaken for a blood-vessel containing a nucleated red cell. In this central protoplasmic mass the nucleus is shrunken and the cytoplasm vacuolated. In the small corpuscle on the right, two large vacuoles have formed.
 +
 +
Fig. 9. Cystic concentric corpuscle. From a 14-cm. pig embryo. Stained with hsematoxylin and Congo red. The central protoplasmic mass is pale and shrunken. The small circular body in it probably is the remains of the nucleus. Colloid lamellae are forming. Colloid fibers cut transversely appear as dots.
 +
 +
Fig. 10. Cystic concentric corpuscle. From a 10.5-cm. pig embryo. Stained with hsematoxylin and Congo red. The center contains no colloid and seems to be softening. The nucleus is shrunken.
 +
 +
Fig. 11. Ordinary concentric corpuscle in a very early stage. From a l{j.5-cm. pig embryo. Stained with iron-ha'matoxylin and Congo red. The nucleus is enlarged and colloid is forming around it. A few colloid fibers may be seen in the cytoplasm for some distance from the central nucleus.
 +
 +
Fig. 12. Ordinary concentric corpuscle. Several nuclei are involved. From a 10.5-cm. pig embryo. Stained with hsematoxylin and Congo red. The deeply-staining colloid has completely obliterated the central nucleus (in the region o c), and nearly obliterated another («'). Some of the colloid now stains pale (o c).
 +
 +
Fig. 13. Ordinary concentric corpuscle. . From a 10.5-cm. pig. Stained with hiematoxylin and Congo red. The central nucleus is being obliterated
 +
 +
 +
 +
(52 The I)i'V('l()})iiHiit nf the Tlivnius
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by the deeply-staiuing colloid. The neighboring nuclei are beginning to show the effect of the centrifugal pressure.
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Fig. 14. Ordinary concentric corpuscle in an early stage. From a 10.5-cm. pig. Stained with haematoxylin and Congo red. A band of deeply-staining colloid has been formed. Just outside this is colloid in formation.
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Fig. 15. Two simple concentric corpuscles which would have formed a compound concentric corpuscle. From a 10.5-cm. pig. Stained with haematoxylin and Congo red. The left corpuscle is a little more advanced than Fig. 11. The right corpuscle shows a large area of deeply-staining colloid which has pressed the nucleus into a small Irregular shape.
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Plate III.
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Fig. 16. Compact irregular corpuscle In an early stage. From a 14-cm. pig embryo. Stained with haematoxylin and Congo red. The colloid is not yet solid. The nuclei are not essentially different from those of the adjacent syncytium.
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Fig. 17. Ordinary concentric corpuscle. From a 12-cm. pig embryo. Stained with haematoxylin and Congo red. There is a large, central, deeplystaining colloid mass in which calcareous deposits (c?) have been made. The neighboring nuclei show the effects of the centrifugal pressui-e.
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Fig. is. Epithelioid concentric corpuscle in an early stage. From a 10.5cm. pig embryo. Stained with haematoxylin and Congo red. The outer colloid lamella marks off a nucleated mass of cytoplasm resembling a large cell. The nucleus is undergoing the same changes as occur in the central nucleus of an ordinary concentric corpuscle.
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Fig. 19. Compound concentric corpuscle. From a 10.5-cm. pig embryo. Stained with haematoxylin and Congo red. This would have soon lost all evidence of its compound nature.
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Fig. 20. Epithelioid concentric corpuscle. From a 10.5-cm. pig embryo. Stained with hematoxylin and Congo red. Some colloid is forming outside the circular area. Solid deeply-staining colloid is forming.
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Fig. 21. Ordinary concentric corpuscle, showing a variation from the usual type. From a 16.5-cm. pig embryo. Stained with iron-haematoxylin and Congo red. The central nucleus is reddish but not enlarged. No solid colloid has been formed.
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Fig. 22. Compound concentric corpuscle. From a lG.5-cm. pig embryo. Three centers are present. The pale colloid in the upper part is probably older than the deeply-staining variety. In the lower part of the figure, a young corpuscle is shown.
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Fig. 23. Compact irregular corpuscle. From a 16-cm. pig embryo. Stained with haematoxylin and Congo red. Some of the colloid is solid. No definite center is present but one is beginning to form (c s) . The nuclei are not markedly different from those of the adjacent syncytium.
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THE DEVELOPMENT OF THE THYMUS. E. T. BELL.
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Idn
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AMERICAN JOURNAL OF ANATOMY--VOL V.
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E. T. BELL, DEL.
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THE DEVELOPMENT OF THE THYMUS. E. T. BELL.
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12
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^^®\r
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AMERICAN JOURNAL OF ANATOMY--VOL V.
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E. T. BELL, DEL.
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THE DEVELOPMENT OF THE THYMUS. E. T. BELL.
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rsil
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rsil .,
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AMERICAN JOURNAL OF ANATOMY--VOL V.
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E. T. BELL, DEL.
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THE YEIXS OF THE ADEE^AL.
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BY
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JEREMIAH S. FERGUSON, M. Sc, M. D.
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From the Histological Laboratory of Cornell University Medical College.
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Neio York, N. Y.
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With 3 Text Figures.
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Within the past decade our knowledge of the functions of the adrenal glands, and of their relations to the rest of the economy, has been greatly enhanced l)y many careful cliomical and physiological researches. Tlie recent studies of Aichel (1), Wiesel (2, 3), Soulie (4), and others have placed the early development of the organ upon a fairly certain basis. These advances in the physiology and embryology of the organ have not as yet been accompanied by corresponding advances in our appreciation of its minute anatomy. Hence this branch of the subject is, at the present time, one of unusual interest.
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The intimate relation of the parenchyma of the adrenal to its bloodvessels, as shown by the general tendency to regard the organ as a true gland whose secretion enters its blood-vessels and leaves the organ through its efferent veins, makes it specially important that these vessels should be carefully studied and their structure and distribution accurately recorded.
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The exhaustive study of Flint (5), on the course of the adrenal vessels, based as it was upon carefully prepared reconstructions, leaves little to be desired along this line. The writer is, however, unable to find in the literature any reference to the minute structure of the veins of the adrenal, with the notable exception of Minot's (6) article on sinusoids.
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To be sure Pfaundler (7) mentioned the occurrence, in the medulla of the adrenal, of venous vessels whose only wall consisted of endothelium. Gottschau (8) also, though omitting their description, has figured similar vessels in his Plate XVIII, Fig. 1. But as to the structure of the larger blood-vessels of the adrenal glands the literature seems to be entirely barren.
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The architecture of the arterial walls does not appear to offer any distinctive peculiarities, the tissues of which they consist being arranged in a manner precisely similar to that which characterizes the arteries of American Journal of Anatomy. — Vol. V.
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64 The N'ein.s oi' the Adrenal
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similar size occnrriiiii- in olliei' or<ians. 'I'lie veins, liowever, j^resent distinct and remarkable peeidiarilies which it is the iiur))()se oi' the present |>a|)ci- to describe.
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Methods (iiid Mafcr'ud. — The tissue used for this stud}^ has included specimens of the adrenal from twenty-one human adults, together with the casual examination of fetal adrenals of the pig and of man. The adrenals of other mammals, e. g., nionke}^ dog, cat, rabbit, and guineapig, have also been more or less carefiilly studied.
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These tissues have been fixed and hardened with many reagents, among which are Zenker's solution, formol. Miiller-formol, alcohol, corrosive acetic mixture, Tellyesniczky's fluid, and Flemming's solution. The stains used were hematein by various methods, acid hematein, iron hematein, etc., and for counter stains eosin, orange. Van Clieson's picrofuchsin, Weigert's elastic tissue stain, Mann's methyl blue-eosin mixture, Congo-red, and Ehrlich's triacid mixture. A combination of Mann's hematein, Weigert's elastic tissue stain and A'an Gieson's picro-fuchsin, gave the best results for the differentiation of the muscular and connective tissues. This method was applied as follows, and may be used after any of the above fixatives.
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1. Stain 10-12 minutes in Mann's hematein or in Bohmer's hematoxylin, until somewhat overstained. 2. Wash well in water. 3. Stain 10-20 minutes in recently prepared resorcin-fuchsin solution after the method of Weigert (9). -1. Wash in water. 5. Stain 1-3 minutes in the freshly prepared picric acid-acid fuchsin solution of A^an Gieson (10). 6. Wash and dehydrate in 95 per cent, or in absolute alcohol. 7. Clear, and mount.
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Types of Adrenal Veins. — The efferent veins of the adrenals arise in the medulla of the organ by the union of the broad capillaries of the medulla and the adjacent zone of the cortex. These capillaries form broad thin-walled vessels which have been described by Minot (6) as sinnsoids. They converge toward the middle of the medulla, where they pass into somewhat larger vessels, which, for convenience, may be termed small central veins. These veins tend toward the hilum, are relatively short, and by union with one another soon form thicker-walled vessels which may be described as large central veins. These large veins pass toward the hilum, near which, they unite to form a large efferent vessel, the suprarenal vein. This last vein makes its exit from the hilum of the organ and enters either the vena cava inferior, as is the rule on the right side, or the renal vein, as frequently occurs on the left.'
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From this brief review of the course of these vessels it will l)e seen thai four distinct venous types have been enumerated, and it is the purpose
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Jeremiah S. Ferguson 65
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of the writer to show that these types exhibit well-defined structural peculiarities.
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Observations. — The sinusoids, after the careful description by Minot (6), will require but brief mention. These vessels possess the wall of a capillary and the lumen of a venule. A number of such vessels may be seen in Fig. 1, in the central portion of the medulla, on either side of the group of central veins. Their wall consists of nucleated endothelial plates which rest directly upon the parenchymal cells. Their lumen is several times the diameter of the- medullary capillaries. They are dis > >
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Fig. 1. A group of vessels from the central portion of tlie medulla of the human suprarenal gland, a, sinusoids; h. small central veins. Fixation, 5 per cent formalin; stain, Mayer's hematein; thickness, 8// ; photomicrograph, X 100.
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tinguished from the small central veins by the absence of connective tissue from the wall of the sinusoids.
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The small central veins are of the type shown in Fig. 1. The wall of these vessels consists of two coats, endothelial and connective tissue. The latter is always relatively thin, though the vessels possess a very considerable lumen. Venules of this type of structure. Fig. 1, collect the blood from the sinusoids of the medulla. Frequently, however, the sinusoids open directly into the small central veins and venules, the connective tissue of the venous wall being occasionally continued for a very short distance upon the endothelium of the sinusoid. 5
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66
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The Veins of the Adrenal
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The connective tissue of the small central veins is richly supplied with elastic fibers, which are disposed in oblique nnd circular directions,
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Fig. 2. The medulla ol a suprarenal gland of man, showing a group of large central veins. The middle and lowermost veins are in transection, the uppermost vessel in longitudinal section. The series of sections shows that this last vessel is a branch of that in the middle of the figure. Fixation, Zenker's fluid; stain, hematein and methyl-blue, Mann's method; thickness, 10 // ; photomicrograph, X GO.
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occasional elastic fibers are also longitudinal. The typical small central veins contain no muscle. As they ap]iroach tlieir termination in the
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JciTiniah S. i^\'rgiL-'on 67
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large central veins a few smooth nnisole fibers are found, but these are always disposed in a longitudinal direction. As soon as longitudinal muscle fibers appear in apprcciahh' uiiinl)ors the venous wall acquires the type of the succeeding variety, ihc large central vein.
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In the large central veins, as in the small, but two coats can be readily distino-uished. The inner coat, or intima, in these vessels consists of a lining endothelium, which rests upon a very thin membrane of delicate connective tissue, containing numy elastic fibers.
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The outer coat, or adventitia, is also a vei-y thin m(!ml)rane of fibroelastic tissue, but its fibrous Ijiindlos are coarser than those of the intima, and its clastic fibers form a very close network. The outer portion of this coat contains a few longitudinal smooth muscle tibers. The great majority of these fibers, however, ai'c arranged in the form of longitudinal ridges which project into the adjacent medullary tissue. From one to live of these muscular ridges occur in the circumference of the vein (Fig. 2 and 3). Except at those points at which the muscle occurs, the venous wall is extremely thin (Fig. 3). The muscular ridges are frequently so large as to materially obstruct the lumen of the vessel (e. g., the middle vessel in Fig. 2, also the uppermost vessel, which is cut in v(!ry neirly longitudinal section), and they form so noticcuible a peculiarity that their presence may be considered charactei-istic of this type of vessel. The writer has never failed to find these peculiar muscular ridges more or less highly developed in each of the human adrenals which he has examined : he believes them to be constantly present. They are less highly developed in the suprarenal vessels of the lower mammals, but even there they may frequently be demonstrated.
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The muscle fibers of the larger ridges are an-anged in bundh^s which are enveloped in fibro-elastic septa of connective tissue. All of the muscle fibers in these bundles are longitudinally disposed. This arrangement is well shown in Fig. 3, in which a hii'ge central vein is seen in transection at a point near the entrance of a large branch. p]xamination of sections somewhat higher in the series shoAvs the union of these two vessels.
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In the section photographed, tlu; l)raiicli has been longitudinally cut. The fine dai'k lines shown in the figure are bauds of elastic fibers which are enveloped in delicate white fibrous tissue inclosing the cut ends of the bundles of smooth muscle. The tendency to form longitudinal ridges is shown in this figure by the irregular disti-ibufion of the muscle, one side of the vessel, in both the parent stem and the branch, beirig almost devoid of muscle fibers. The muscular character' of these ridges is beyond doubt.
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G8
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The Veins of the Adrenal
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Fig 3 Large central veins from the medulla of the human suprarenal gland The figure shows the distribution of the elastic tissue and the bundles of smooth muscle which are seen in transection in the larger vein and m longitudinal section in the smaller ones below. The series shows thes. latter vessels to be branches of the former, the section being selected to show a plane near the point of division. The smaller vessels are jeiy obliquely cut and the muscle is distinctly longitudinal. Fixation Zfnker s fluid; stain, Mann's hematein, Weigert's elastic tissue, and Van Gieson s picro-fuchsin; thickness, 10 a; photomicrograph, X 37.
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Jeremiah S, Eerguson 69
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The writer has observed that the formation of such heavy ridges as those shown in Fig. 3, nearly always occurs at those points where the vessel branches. It is possible that, as in the case of the somewhat similar ridges in the veins of the erectile tissues (see Kolliker's Handbuch der Gewebelehre, 6te Aufl., 1902, pages 486 and 487), these muscular protuberances may to some extent serve the purpose of valves.
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As the large central veins approach the hilum of the organ they form still larger vessels which partake of the structure of the suprarenal vein. The point of transition from the one type to the other is variable, occasionally the type of the large central veins is continued to the exit of the suprarenal vein at the hilum of the organ. More frequently the primary branches of the suprarenal vein may be traced for a considerable distance into the medulla of the organ, still retaining the type of structure found in the larger vessel.
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The suprarenal vein presents three coats, intima, media, and adventitia. The tunica intima, in addition to its endothelial lining, possesses a thin membrane of very delicate connective tissue in which occasional branched connective tissue cells may be distinguished; such cells are, however, very scanty. This coat also contains a delicate network of elastic fibers.
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The tunica media of the suprarenal vein is extremely thin, rarely ever does it exceed in thickness the tunica intima. It consists chiefly of fibro-elastic tissue, the elastic fibers forming quite a dense network. Few muscle fibers occur in this coat, nowhere are they found in sufficient numbers to form a definite layer, as in veins of similar size in other organs. Some of the muscle fibers are circularly disposed, but many of them are longitudinal.
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The tunica adventitia is by far the thickest of. the three coats and forms two-thirds to five-sixths of the entire vascular wall. It consists chiefly of smooth muscle fibers, all of which are longitudinally disposed. These smooth muscle fibers form characteristic coarse bundles which are distributed around the entire circumference of the vessel. The largest of these bundles may occasionally form projecting ridges as in the smaller veins, but as a rule the muscular tissue is more evenly distributed than in the central veins. Each of the muscle bundles is enveloped in a perimysial sheath of connective tissue, which blends with that of the tunica media. These adventitial sheaths possess a dense network of elastic fibers, in fact the greater part of the elastic tissue in the vascular wall is frequently found in the adventitia. On its outer surface the tunica adventitia is continuous with the capsule of the adrenal or with the adjacent connective tissue.
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This peculiar type of vessel is not strictly confined to the suprarenal
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•70 'JMu' W'ins of tlie Adiviial
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gland, but occurs, more or less typically developed, in many of the large abdominal veins, notably in the renal veins and vena cava, into which the suprarenal veins empty. But UQwhere is this peculiar venous type more strikingly developed, nowhere is the adventitia relatively so much thicker than the media, nowhere is a greater proportion of the smooth muscle of the venous wall longitudinally disposed, nowhere is there relatively less circular muscle, than in the suprarenal vein. Eealizing the intimate relation of the parenchyma of the organ to its blood-vessels, and adopting, if we may, the accepted physiological function of the adrenal — the formation of an internal secretion, a powerful vaso-constrictor which is poured into the blood within the capillaries and veins of the organ — ^the peculiar longitudinal arrangement of the muscular tissue, the valve-like protuberances at the junctions of the venous vessels, the absence of circular muscle from the walls of the veins of all sizes, and the general appearance of these vessels which are so remarkably different from the veins of most other organs, become, to say the least, extremely significant of a close structural relation, physiologically speaking, to the presence of an astringent secretion in the outflowing blood current.
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In this connection, one further observation is of importance. In the periadrenal connective tissue are numbers of small veins which return the abundant blood supply of the tissues of this region, most of them emptying into the phrenic veins. Many of these veins do not differ from the similar veins of other parts, but in many others the writer has observed that the muscle tissue is almost entirely disposed in a longitudinal direction, a condition which is quite the reverse of that found in the adipose and areolar tissues of other portions of the body.
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The writer also finds that many of the small veins of the adrenal, instead of opening into the central veins as is usually the case, pursue a less frequent course, penetrating the cortex and capsule of the organ, and emptying into the small veins of the surrounding connective tissue. The frequency with which this condition was associated with the occurrence of longitudinal muscle fibers in the periadrenal veins, suggests a more than casual relationship between the two conditions.
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Summary.
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In conclusion, the above facts may be summarized as follows:
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1. The efferent blood-vessels of the adrenals form four successive vascular types, the sinusoids, the small central vein, the large central vein, and the suprarenal vein.
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2. Each of these types presents distinctive characteristics.
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Jeremiah S. Ferguson 71
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3. In all four tA^pes circular muscle is either absent or noticably deficient.
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4. In the large central veins prominent and characteristic muscular ridges are constantly present, and are frequently in relation with those points at which the branches of these vessels enter.
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5. These peculiarities of structure may possibly bear a close physiological relation to the function of the adrenal as a gland that forms an internal secretion which has been shown to be a powerful vaso-constrictor and stimulant of smooth or involuntary muscle.
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BIBLIOGRAPHY.
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1. AicHEL.— Munch, med. Wochenschr., 1900, XLVII, 1228; and Arch. f. mik.
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Anat, 1900, LVI, 1.
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2. WiESEL.— Anat. Hefte. 1901, XVI, 115.
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3. Ibid., 1902, XIX, 481.
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4. SouLiE.— J. de I'anat. et de la physiol., 1903, XXXIX, 197, 390, 634.
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5. Flint. — Contrib. -dedicated to W. H. Welch, Baltimore, 1900, 153; also in
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Johns Hop. Hosp. Rep., 1900, IX, 153.
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6. MiNOT.— Proc. Post. Soc. Nat. Hist., 1900, XXIX, 185.
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7. Pfaundler. — Sitz. d. Akad. d. Wissensch., Wien, 1892, CI, 515.
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8. GoTTSCHAU.— Arch. f. Anat., 1883, 412.
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9. Weigert.— Centralbl. f. allg. Path. u. path. Anat., 1898, IX, 289. 10. Freeborn.— Proc. N. Y. Path. Soc, 1893, 73.
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THE BLOOD VESSELS OF THE PEOSTATE GLAND.
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BY
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GEORGE WALKER, M. D.
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Associate m Surgery, Johns Hopkins University. From the Anatomical Laboratory, Johns Hopkins University. With 2 Colored Plates.
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As the structures of the body are being more and more carefully investigated it is found that organs are composed of like structural units, which when repeated a number of times form the whole organ. In general these units are formed by the glandular structures, the blood-vessels, or by both, as is the case in the liver.
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Some eight years ago, at the suggestion of Dr. Mall, I undertook the study of the prostate gland, with the hope of finding structural units in it. In this search I was successful. Since then my work has been continued in the laboratories of Professor Born ^ of Breslau and Professor Spalteholz ^ of Leipzig, and although this communication is several years late in appearing, it should in reality have preceded those that were published in 1899.
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In the present study for the most part the prostate glands of dogs were used. Several cadavers were injected and the gross blood supply was studied in part from these. After the animals had been killed by chloroform, the aorta was exposed just above the bifurcation and injected with various substances. A preliminary washing out of the blood-vessels with salt solution was practised in a few of the first injections, but this was soon discarded as it did not seem to enhance the value of the method.
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Carmine gelatine, followed by ultramarine-blue gelatine, as an injecting mass, gave the most satisfactory results. About 250 cc. of the carmine gelatine were injected first, the injection being stopped as soon as all of the tissues had acquired a maximum carmine hue. This was followed by the injection of ultramarine-blue gelatine, which was kept up until no more of the material would pass in. The carmine gelatine
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^Walker. George: Ueber die Lymphgefasse der Prostata beim Hunde. Arch, fiir Anatomie, 1899.
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= Walker, George: Beitrag zur Kenntnlss der Anatomie und Physiologie der Prostata, etc. Ibid., 1899. American Joukxal of Anatomy. — Vor-. V.
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74 Tlif lUood Vessels of the Prostate Cilaiicl
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filled the arteries, capillaries, and veins ; the blue passed into the arteries and arterioles, displacing the gelatine and filling them, but was stopped at the capillaries because the nltramarine-blue granules were too large to enter them. In a specimen thus prepared the arteries appear blue, and the capillaries and veins red. This is shown in Figure 1, with colors reversed, in order to present the conventional appearance. As it was impossible to get a perfectly complete injection in one specimen, several of the best were selected and the gaps filled in, with the results as shown in Figure 1. One section, however, is remarkably beautiful and presents a picture very closely resembling that seen in this figure.
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In order to map out the complete network of arteries surrounding a separate lobule, I injected them with Prussian blue, then opened the urethra, and injected carmine gelatine into a prostatic duct through a very fine blunt hypodermic needle. A specimen made in this way is shown in Figure 2 where the ducts are represented in brown. The capillaries were studied in a specimen which had been completely injected with carmine gelatine. A very thin section of this was stained with iron hematoxylin, and is shown in Figure 3. The basement membrane is artificially tinted with yellow so as to make it visible.
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The technique of the injecting is rendered difficult by the fact that the situation of the gland in the pelvis is somewhat remote. In all, about
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75 dogs were used before a complete circulation cycle could be seen. Cinnabar, lampblack, and various other substances were tried, but they did not prove as good as the combination of carmine gelatine followed by ultramarine-blue gelatine.
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When the ordinary directions for preparing carmine gelatine were followed, it always proved difficult to get a perfectly transparent substance. The trouble is connected with the neutralization of the ammonia by the acetic acid. The gelatine should be rendered practically neutral, but if the reaction is carried the least bit too far, the solution becomes cloudy. Sometimes two drops of the acetic acid are sufficient to make turbid a whole litre of the prepared carmine. After a good many trials, the following method was adopted : Take 10 cc. of the ordinary laboratory ammonia and dilute with 40 cc. of distilled water, then determine by titration the exact amount of the laboratory acetic acid which will neutralize it. After this determination has been made, 10 grms. of pure carmine are rubbed up with 50 cc. of distilled water; then 25 cc. of the ordinarj^ ammonia are measured, and a few drops at a time are poured into the carmine mixture which is kept constantly rubbed up. This process is very closely watched, and the ammonia is gradually added until the carmine is completely dissolved, and the mixture becomes
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George Walker 75
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translucent and assumes .a dark red color. Tlie amount of ammonia used is determined by referring to the vessel in which the 25 cc. have been measured. The gelatine in whatever proportion it is required — according as a thin or thick solution is desired — is dissolved in the distilled water, and the carmine solution is added to it. We then calculate how mucli acetic acid will be required for the amount of ammonia w^hich has been used; this is measured and added, drop l)y drop, to the mixture which is constantly stirred. A sufficient quantity of water is then added to bring the amount up to a litre. I found that in this way I could always obtain a beautifully clear gelatine and was never annoyod by the failures and uncertainties belonging to tlie other method.
 +
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Akteries. .
 +
 +
The prostate gland derives its arterial supply from the internal iliac arteries by means of four branches; the superior vesical, the inferior vesical, a small branch from the inferior hemorrhoidal, and a small terminal branch from the internal pudic artery. These vessels will be found illustrated in my paper published in 1899. The superior vesical, a branch from the internal iliac, which is given off high up, divides before reaching the bladder, into two fair-sized branches ; the lower and smaller branch extends downward and supplies the vesical third of the prostate ; this branch is sometimes called the middle vesical artery. The inferior vesical, which is a large branch, is practically the main blood vessel of the prostate gland, and should be called the prostatic artery for, in the majority of instances, it does not send any branches to the bladder. The major part of the gland is supplied by this vessel; it courses along the vesicorectal fascia and meets the prostate at its lower border, where it usually divides into seven branches, four of these enveloping the anterior, and three the posterior surface. The posterior are about one-half the size of the anterior lu'anchcs. These vessels are situated in the capsule of the gland and envelop it as the fingers of one's hand would do in clasping a round object. From these trunks a numljer of smaller ones are given off, so that a very close arterial network is formed over the surface of the gland. The ])ranch from the inferior liemorrhoidal is not constant; in fact, it appears to be more often absent than present. When it is seen, it occurs as one or two small branches wbicli ]uQci llic prostate in its urethral half, and extend over the surface as line vessels which anastomose with the vesica] artery. The internal ])iulic branch is fairly constant. It extends along Ibc membranous urotlira and ])lunges directly into the prostatic substance usually without giving off any l)ranclies to the surface.
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7G The Blood Vessels of the Prostate Gland
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A slight anastomosis is occasionally seen. The vessels supplying the two sides of the gland are distinct. The only anastomosis across the median line is by way of the venous channels around the urethra.
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From the large superficial branches above described, smaller ones are given off at right angles, and pierce the gland in places corresponding to the divisions of the lobules (Art. Fig. 1). Here they penetrate the fibrous-tissue septa, and extend to the urethra, becoming smaller and smaller, however, as they approach it, so that in this region they are seen as very delicate terminal vessels. As they pass down, they give off branches which penetrate into the lobule and finally divide into myriads of capillaries which pass around the alveoli, and come in very close relationship with the secreting cells. From these cells they are separated simply by a delicate basement membrane composed of fine fibrils. From the superficial vessels branches are given off which enter the lobule directly, that is, they do not pass first into the fibrous-tissue septa {8up. Br. Fig. 1). On the anterior surface there are usually two branches which do not give off as many smaller ones as the rest, and consequently remain larger and extend over to the middle line, where they dip into the median fissure and supply the median side of the lobules (Med. Br. Fig. 1).
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The arrangement on the posterior surface corresponds to that seen on the anterior surface, in so far as the supply of the lobules is concerned. On the posterior surface toward the bladder one vessel penetrates the substance of the gland and runs directly to the caput gallinaceum (Art. Col. Sem. Fig. 1). Here it divides into a fine network and supplies the erectile tissue of the organ. Before this vessel reaches the eminence a small trunk is given off which extends to the ejaculatory duct (Ai-t. duct. ej. Fig. 1). The branch supplying the caput gallinaceum is usually derived from the pudic; sometimes it comes from the inferior vesical.
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The arterial supply in the connective tissue toward the urethra is much poorer than in the secreting portion. Here the vessels terminate in fine branches, relatively somewhat sparsely scattered. The arterial arrangement is shown on the red side of Figure 1.
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Capillaries.
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The capillaries form a very complete and elaborate network around the alveoli of the lobule. Here, as is seen in Figure 3, they surround an alveolus in a more or less circular manner, and upon these vessels the cells rest almost directly, being separated only by the very delicate connective-tissue basement membrane. From this outside capillary, a folding in is seen, which forms a definite loop (Cap. L. Fig. 3.) This at
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George Walker 77
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first sight might appear to end blindly, but a more careful study reveals the two branches, which sometimes appear winding around each otlier, and presenting enlarged club-shaped ends. The cells rest on these as they do on the circular portion. Under the low power, the epithelial cells appear to be in direct contact with the capillaries, and it is only by the aid of the oil immersion that a very delicate connective-tissue basement membrane is seen. This is shown artificially colored as B. M. Fig, 3. This membrane contains a few elastic fibers.
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Veins.
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On the surface of the gland are veins corresponding to the arteries which lie in the capsule. As a rule they merge into two main trunks corresponding to the vesical arteries; occasionally several small branches pass off into the middle hemorrhoidal vein.
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The superficial veins do not drain the blood from the whole gland, but only from the outer fourth, as is shown in Fig. 1. From the inner threefourths of the gland the blood passes towards the centre, and into the large venous sinuses which are a continuation of the corpora spongiosa. (Co. 8p. Fig. 1). These immediately surround the urethra. The large venous trunks which collect the blood from the gland do not lie on the same plane as the arteries, but are situated in the fibrous septa some little distance removed from them. These run, as do the arteries, on the outside of the lobule, and are interlobular, not intralobular. For the venous return from the caput gallinaceum there is no distinct vessel corresponding with the artery, but there are anastemoses with the spongy plexus.
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The venous plexus around the urethra is, as before stated, a continuation of the corpora spongiosa. The blood from this region passes away into the internal pudic vein. Occasionally two or three small veins drain the tissues from this region, pass out of the prostate and run along the membranous urethra and off into the vesicorectal fascia.
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There is an anastomosis of the veins in the prostate and bladder where these organs come together, and also on the outside through the superior vesical veins. There is, of course, an anastomosis of the urethral veins through the corpora spongiosa plexus.
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SUMMAEY.
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The prostate gland is supplied with blood by branches of the internal iliac arteries, viz., the superior vesicals, inferior vesicals, inferior hemorrlioidals, and internal pudics; the main blood supply comes from the inferior vesicals.
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78 The Jilood Vessels of I lie Tr. .slate 11 land
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Branelies of llieso envelop iho siirfaec! of tlie <ilnii(l and f^ivo ofT smallcMon(>s, wliieli iieiielratc between tlie lohnles in (he fihrons-tissuc septa.
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The caiiilhii-ies are separated iVom I he ciiilhclial cells only by a very lliiii haseiiieiil iiieiiibraiie.
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'I'here are siipei-llcial vcins correspond iiiii' with the arteries.
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I'\)i- the Older superficial fourth of the gland the return flow is towards (lie siirfaei'. 'I'he inner three-fourths are drained by veins wliieb enijjiy into the venous plexus immediately aronnd the nretlira.
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The lobule is formed primarily by the individual gland dnets as shown in Figure 2. The main arteries surround this lobnle wliicb they penetrate at many points. The veins leave the lobule mainly at its peripheral 5ind central (urethral) ends as shown in Fig. ].
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EXPLANATION OF PLATP^S I AND II.
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Fid. 1 is Irom a section of a prostate gland of a dog injected with carmine gelatine and ultramarine-blue gelatine. The arteries in the section were blue, the veins and capillaries red. The section was cut free hand, about r.O// in thickness, and cleared both in glycerine and in creosote. In the figure this artery is red and this vein blue. Ar^., Arteries; Art. Col. Ficm.. Artery of the colliculus seminal is; Art. duct, cj.. Artery of the ejeculatory duct; Col. Scni., Colliculus s(>niinalis; V. J'L, Venous plexus around the urethra.
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Fio. 2. Lobule of prostate from a gland which had been injected with uHramarine-golatine blue into the artery, and with carmine gelatine into the prostatic duct. Pr. duct., Opening of the prostatic duct into the urethra; Gl. Tis., Gland tissues distended with carmine gelatine; Art., Surrounding artery. In this figure the artery is represented in red and the ducts in brown.
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Fig. 3. Very thin section from Ihe i)rostate gland of a dog. Capillaries in red, injected with carmine gelatine. Section stained with iron h;rmatoxylin, with artificial yellow tinting of basement membrane. Oil immersion with one inch eye-piece amplification. Cap., Capillaries; B. M., Basement membrane; 07. Ep., Glandular epithelium; Cap. L., Capillary loop.
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BLOOD VESSELS OF PROSTATE GLAND
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GEORGE WALKER
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PLATE
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a) CO
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Q
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AMERICAN JOURNAL OF ANATOMY — VOL. V
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BLOOD VESSELS OF PROSTATE GLAND
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GEORGE WALKER
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PLATE II
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GI.Tis.
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Art
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PKDuct
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Fig. 2
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Cq1>.L,
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NCap.
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Krcj. 3
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AMERICAN JOURNAL OF AN ATOM Y--VO L. V
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THE EMBKYONIC DEVELOPMENT OP THE EETE-CORDS AND SEX-COEDS OF CHRYSEMYS.
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BY
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BENNET M. ALLEN,
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Instructor in Yertehrate Anatomy, University of Wisconsin.
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With 1 Double Plate akd 6 Text Figures.
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A glance at the diagrams on the next page will at once serve to show the great difference of opinion that has prevailed in regard to the origin of the sex-cords and rete-cords of the Sauropsida. In fact, it is hard to conceive of any possible manner of origin that has not been held to be correct by some well-known embryologist.
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The Chelonia have remained almost untouched in the study of this problem. Only one work has appeared upon the rete-cords (Von Moller, 98), while no work has been published upon the subject of the sex-cords.
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Von ]\Ioller studied two turtles, one a specimen of Emys lutaria of 2.5 cm. plastron length, and the other Clemmys leprosa of 4.9 cm. plastron length. He observed no connection between the testis and Wolffian body. This caused him to remark :
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" Dieser Befund wird hochst auffallig, wenn mann bedenkt dass die Beobachtungen an Amphibien zeigen dass die Verbindungen zwischen Hoden und Wolffschen Gauge schon dann angelegt und vollendet werden, wenn die iibrigen Organe sich noch in der Entwickelung befinden, und wenn das Junge in der Eischale, respective in Uterus eingeschlossen ist. Die zwei von mir untersuchten Schildkroten hatten dagegen schon seit Monaten die Eischale verloren, und doch war bei ihnen noch kein einzige Verbindung zwischen Hoden und Wolffschen Gauge vorhanden, obwohl Anlagen dieser Verbindungen sich bereits vorfanden."
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It is quite unfortunate that he considered these stages to be early enough for his purpose, since my work has shown the rete-cords to be formed at a relatively early stage of development in Chrysemys.
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Von Moller sums up his results as follows : " Ich finde also bei diesem Thiere zwischen Hoden und Wolffschen Korper noch keine Verbindungen, dagegen im Mesorchium und im oberflachlichen Bindegewebe der Umiere solide Zellenstrange, fiir welche ich genotigt bin einen UrAmeeican Journal op Anatomy. — Vol. V.
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80
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The Eete-Cords and Sex-Cords of Chrysemys
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Text Fig. A.
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GER
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Text Figs. A-D. Diagrams ilhistrating various views held by authors whose Avritings are reviewed in this article.
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^D.— Fundament of the adrenal body. GER.— Germinal epithelium.
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il/.— Mesentery. M. P.— Malp'ghian corpuscle. J?.— Rete-cord. S. C.-Sex-cord. U. r.— TJriniferous tubule. W. I).— Wolffian duct.
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spningsort anzunehmen, der wedcr in den Geweben der Urniere noch in denen dcs Hodens liegt denn ieh woder im Stande bin einen Ziisammenhang mit den gewundenen Kanalchen des Hodens nachznweisen, noch einem solchen mit deni Epithel Bowmanscher Kapseln oder sonst mit Theilen der Urniere. Ich nehme daher an, dass sie vom Peritoneum stammen." No further allusion need be made to this article.
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Turning to the other groups of the Sauropsida, we find a large mass of literature. To intelligently discuss this, we must use precise terms. The sexcords are those masses or cords of cells which eventually become the seminiferous tubules of the testis or the medullary cords of the ovary. The rete-cords are those structures which eventually give rise to the canals which unite the seminiferous tubules or medullary cords of the sex-glands with the ducts of the mesonephros.
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It is not necessary to enter into a lengthy review of the literature upon this subject. That has been thoroughly done by Born, 94, MihalkovicS;, 85, Janosik, 85, Coert, 98, Winiwarter, 00, and others. A few diagrams will suffice to show, in a sufficiently vivid manner, the wide differences between the many views upon this subject as expressed in the papers most worthy of note.
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The names associated with the different diagrams, Text Figures A-D. are those of the authors who have held views represented by the diagrams so indicated. After the name of each
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Bennet M. Allen 81
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author are placed the names of the forms which he studied in arriving at his conclusions.
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A. Tubules arise from the Wolffian duct and grow into the sex-gland fundament. Their distal portions form the sex-cords while their proximal portions form the rete-tubules.
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70, Waldeyer — Chick (Gallus).
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B. According to this view, evaginations grow out from the capsule of Bowman. Distal branches from these stems pass down into the sexgland fundament to form sex-cords, while the more proximal portions of the evaginations remain attached to the capsules of Bowman and serve as rete-tubules.
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Brann, 77, Platydactylus, Tropidonotus.
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Weldon, 85, Lacerta.
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Hoffmann, 8g and 92, Lacerta, Hjematopsis, Sterna, Gallinula.
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Semon. 87, Gallus.
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Peter, 04, Lacerta. Brann, 77, considers the rete-sex-cords to be, in the strictest sense, segmental in arrangement. He expressly denies that the cells that contribute to the formation of the adrenal body are derived from branches of the evaginations from the capsules of Bowman, as asserted by Weldon, 85 and Hoffmann. 89 and 92. These two last named authors asserted that each evagination divides into a dorsal and a ventral branch, the former suppljdng the cells of the cortical portion of the adrenal body, and the latter forming the sex-cords. Semon, 87, was not so clear upon the question. He merely stated that the anastomosing cords arising from the capsule of Bowman pass into the adrenal and sex-gland fundaments, — the more dorsal to the former, the more ventral to the latter.
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C. Large numbers of cells migrate from the germinal epithelium into the underlying stroma. From this unorganized blastema, the sex-cords are formed, suddenly crystallized as it were. The rete-cords are formed of evaginations from the capsule of Bowman.
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Schmiegelow, 82, Gallus. Mihalkovics, 85, Lacerta, Gallus. Laulanie, 86, Gallus.
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D. According to Janosik, the sex-cords arise as direct ingrowths from the germinal epithelium. Cords of cells grow from their distal ends to the capsules of Bowman, thus forming the rete-cords. Cords of cells grow in from the peritoneum between the sex-gland fundament and the mesentery to form the cortical portion of the adrenal body.
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Janosik. 90, Gallus. .
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6
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83 The Kete-Cords and Sex-Cords of Chrysemys
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The following table will show the great difference of opinion held by authors working upon the same identical species. The view held is indicated in the same manner as above.
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Lacerta agilis — Weldon (B) ; Hoffmann (B) ; Mihalkovics (C). Chick (Gallus)— Waldeyer (A); Semon (B) ; Mihalkovics (C) ; Laulanie (C) ; Schmiegelow (C) ; Janosik (D) ; Weldon (?),
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We cannot close an account of the literature upon the subject without refeiring to the work of Semon, 91, upon Ichthyophis, one of the Gymnophiona, and a paper by Semper, 75, upon the Sex-glands of the Elasmobranchs.
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Semon^ 91, considers the nephrotome to be the ventral portion of the mesoblastic somite. This view, by the way, is also held by Brauer, 02. Semon states that after the nephrotome breaks away from the myotome and sclerotome, it still remains attached to the peritoneum (unsegmented mesoderm) by means of two bridges of cells — a lateral and a medial. The major part of each nephrotome forms a Malpighian corpuscle of the mesonephros. The lateral of the two bridges connecting it with the peritoneum becomes its peritoneal funnel (nephrostome), while the medial bridge sends out a process which divides into a dorsal branch passing to the adrenal body, and irre|gular branches (sex-cords) non-segmental in character, that pass to the sex-glands, there to come in contact, in the case of the male, with the seminal vesicles, which, are derived from the germinal epithelium. He holds a theory that the pronephros extends in rudiment, at least, along the entire length of the mesonephros, and that this pronephric rudiment develops into the adrenal body. He considers the dorsal branches spoken of above, to be these vestiges of the pronephros.
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Semper, 75, gives the most interesting account of the rete in the male of Acanthias. According to him, each of the 34 primary Malpighian corpuscles of the kidney is connected with the body cavity by a peritoneal funnel. Seven of the most anterior of these funnels lose their union with the peritoneum and take on the form of vesicles. Three or four of them now fuse together to form the " central canal," which lies at the base of the testis and parallel with it. From this central canal there arise a number of irregular anastomosing canals which extend into the testis and come in contact with the true sex-structures (Vorkeimketten) that have arisen from the germinal epithelium. This net-Avork of rete-cords he calls the rete-vasculosum.
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In other forms there exists a somewhat modified condition of considerable interest. In comparing Acanthias and Mustelus, Semper said: " Trotzdem scheint ein grosser Untershied in Bezug auf die Entstehung
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Bonnet j\r. Allen 83
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des Centralcanals des Ilodens /wisclien ]\[usleliis ;ind Acantliias zu bestehen. Bei dieser Gattung wird or seiner ganzer Liingc nach gebiklet durcli die Verwachsimg der seitlicli vom Segmantalgang nach vorn sich wendenden Trichterblasen. Seitliche Aiisbuchtiingen der letztoren bildeii den basalem Theil der rete vascolosum. Bei Mnstehis dagegen ist es nur der vorderste iiber die Hodenfalte hinaiis vorgreifende Abschnitt des Centralcanals den mann entstanden ansebcn konnte, denn nur an diesen setzen sich 2 (oder 3) Segmentalgange an. Der ganze iibrige viel langere Theil des Centralcanals entsteht aiis den in das Stroma der Epithelfalte eingestiilpten Keimepithel Zellen."
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Balfour, 78, shows that in the forms which he studied, the anterior end of the sex-gland only, was directly united to the mesonephros by means of the rete-canals.
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The condition in the lizard Platydactylus is, according to Braun, 77, quite similar. He considers the union to be formed in adult life by two or three rete-cords joining the anterior ends of mesonephros and testis; although he states that they are connected along the entire length of the testis in early stages. Ploffmann, 89, finds the union of rete-cords to be complete and intact along the entire length of the testis in Lacerta at the end of the first year. He did not study older specimens. Semon, 87, claims that there is a degeneration of the rete-cords at both the anterior and posterior ends of the sex-gland of the chick; but Janosik, go, denies this.
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Material axd Technique.
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Our lakes in the vicinity of Madison abound in the little painted tortoise, Chrysemys marginata. The number of embryos to be gathered in the season is limited only by ones patience in the work of preserving them. I have prepared a large number of serial sections of the mesonephros and sex-gland, as well as of entire embryos, comprising an unbroken chain of stages from gastrulation to adult life.
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As a fixative, TQllyesnitzky's Bichromate-acetic fluid was almost exclusively used, as it gave most excellent results. Haidenhein's iron-ahnn hsematoxylin stain proved unsatisfactory for early stages of the embryos under 7 mm. length. For later stages than this it gave excellent results and was used almost exclusively. A counter-stain of Congo red was also employed. The sections were cut at a thickness of 7 fi.
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Measurements Avere made of the distance between the cervical bend and the tail bend (C-T). In the later stages the length of carapace was also given.
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To more clearly understand the origin of the sex-cords, it will be
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84
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The Eete-C'ords and 8ex-Cords of Chrysemys
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necessary to first understand certain features in the development of the niesonephros. Reference to these features will be made only in so far as they concern the subject of this paper. In an early stage of development (C-T. 3.5 mm.), a section through the posterior part of the sexgland fundament shows the mesoblastic somites to be attached to the lateral plates by the unmodified middle plate (Text Figure E). The cells of the latter are arranged in two rows, in such a manner as to leave a line of weakness between, which may be considered as a rudimentary lumen, connecting the body-cavity on the one hand with the cavity of the
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^'iEF
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Text Fig. E. Transverse section through the middle of the mesonephros fundament of an embryo of 3.5 mm. C-T. length.
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AO. — Aorta. NO. — Notochord.
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EC. — Ectoderm. SO. — Somatopleiire.
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MY. — Myotome. 8P. — Splanchnopleure.
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N. — Neural canal. WD.— Wolffian duct.
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NEP. — Nephrotome.
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mesoblastic somite on the other. In the region posterior to this, these relations become even more marked.
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More anteriorly, just behind the interesting region which forms a transition between the pronephros and mesonephros, the middle plate is found to be wholly broken away from the mesoblastic somites, and to be divided by transverse intervals into nephrotomes which occur in the number of three to four per somite.
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I found no evidence of a primary metamerism of these nephrotomes. So soon as the middle piece appeared to be broken up at all, the number of nephrotomes here recorded appeared. Special investigation along this
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Bennet M. Allen 85
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line, however, might show a primary metamerism, from which the above described condition was derived by further secondary splitting of the nephrogenous tissue. Each nephrotome becomes vesicular to within a sliort distance of the peritoneum thus forming the primary Malpighian corpuscles. The remaining portion of the nephrotome uniting it with the peritoneum becomes, in later stages, the peritoneal funnel or nephrostome, while the uriniferous tubule arises as an outgrowth from the distal end of the nephrotome. The mesonephric peritoneal funnels are vestigial structures from the time of their origin.
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In later stages {C-T. 6 mm.), two sharply defined regions of the mesonephros may be distinguished from one another. In the anterior part of the sex-gland, only the primary Malpighian corpuscles are formed. Each is well developed, the glomerular invagination having already taken place. The 11th to 21st Malpighian corpuscles are connected with the peritoneum by peritoneal funnels (Plate I, Fig. 5), some of which are much better developed than others, there being great variation among them. In the best developed among them, the end attached to the peritoneum flares open to form an actual funnel-like mouth, yet this opening is never continuous with that of the Malpighian corpuscles. The greater part of the peritoneal funnel is merely a cord of cells. In some cases even, it has lost its continuity with the capsule of Bowman. At this stage the first ten Malpighian corpuscles are without peritoneal funnels.
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Caudad of the 21st Malpighian corpuscle, each nephrotome shows two or three rudimentary vesicular enlargements. Each enlargement is destined to form a Malpighian corpuscle. The most ventral of these we shall consider as the primary Malpighian corpuscle. It is still rather broadly connected with the peritoneum. This place of union we shall consider as a rudimentary peritoneal funnel, although it has no flaring opening.
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In later stages, secondary and tertiary Malpighian corpuscles appear in the anterior region described above, thus making the total number per somite approximately equal to that in the posterior region. Eoughly speaking, from nine to twelve Malpighian corpuscles in all, appear in each somite.
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Eeference to Plate I, Fig. 1, will show certain of the points mentioned above. Furthermore, one can see an elongated mass of tissue that extends from each peritoneal funnel dorso-mediad and which lies just laterad of the V. renalis revehens (vena cava). This we shall term the funnel-cord. They appear in both the anterior and posterior regions of the mesonephros as described above and are co-extensive with the sex-gland
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86 'I'lic li'i'ti'-('<)i-(ls and Sex-Cords of Chrysemys
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I'liiidaiiiciit. in fact (licy ai'c found foi' a slioi't distance anterior to it Naturally each I'unnel-iord lies ()|)])ositc a ])i'iniarv Malpit^'liiaii eorpnscle, and likewise to the series of secondary, tertiary, etc., corpuscles formed in a vci'tical row above it. Each cord is made np of rather loosely arranged cells that bear a rather close resemblance to the mesenchyme cells. In fact the nuclei of these cells, the funnel cells, and the cells of the" peritoneum are not to be distinguished from one another. Cytoplasmic differences alone appear and these depend upon the density of the tissue. In some cases a slight evagination of the capsule of Bowman is found at the point where it joins the peritoneal funnel. This evagination may take various forms and in many cases is -wholly absent. Such an appearance may have led to the view held by some authors that these cords arise as outgrowths from the capsules of Bowman. This view would be still further justified if the peritoneal funnel were to break away from the peritoneum at a stage prior to that observed. There can be no question, however, but that the funnel-cords are outgrowths from the peritoneal funnels ; in fact their bases are the funnels themselves.
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The distal portions of the funnel-cords lie above the vena cava in the fundament of the adrenal body, contributing the greater part of the tissue that in later stages constitutes the cortical substance of that gland. Peritoneal ingrowths may also be seen extending dorso-laterad from the peritoneum at a point near the base of the mesentery to the adrenal fundament. These also contribute to the cortical tissue of the adrenal body. They are of less regular occurrence than the funnel cords, and in later stages lose their connection with the peritoneum, although they are easily distinguishable in the stage of 7 mm. G-T. length.
 +
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The sex-gland can be clearly distinguished in the embryo of 6.8 mm. C-T. length. It extends through six somites, although the last ^ of it remains in a rudimentary condition. Even in this stage it consists merely of thickened peritoneum containing scattered primitive sex-cells (Ureier).
 +
 +
The sex-gland develops from a portion of the germinal epithelium lying between the bases of the funnel-cords and the base of the mesentery. In the embryo of 6 mm. C-T. length, a few primitive sex-cells were already beginning to appear in this region. At this time, the V. renalis revehens (vena cava) lies close above the germinal epithelium which has not yet begun to thicken to form the sex-cords. In an embrvo of 6.8 mm. C-T. length the germinal epithelium has sent out masses of cells towards the Y. renalis revehens, and has at the same time bent outward in such a manner as to form in transverse section, the periphery of a semi-circle, the interior of which is occupied by the sex-cords. The tips of the sex
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Bennet M. Allen 87
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cords remain stationary and almost, if not quite, in contact with the wall of the V. renalis revehens, while their bases grow peripherally with the germinal epithelium. Mesenchyme cells between the sex-cords are few and far between.
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At some points, the tips of the sex-cords penetrate to one side or the other of the Y. renalis revehens, and penetrate to the adrenal fundament to which they contribute.
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Plate I, Fig. 3 shows a wax plate reconstruction of a large part of the sex-gland of the 7 mm. C-T. stage. In this stage the carapace has just formed. The prominent funnel-cords afford the most striking feature of the model. Their bases are attached to the peritoneum at the lateral boundary of the sex-gland. They extend in a dorso-medial direction. It will be noticed that each is connected with a primary Malpighian corpuscle. The other Malpighian corpuscles are not shown in the model.
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Mediad of the funnel-cords the peritoneum is greatly thickened, forming numerous irregular elevations and ridges between which are deep clefts and pits. These thickenings are the sex-cords. They are solid and their cells show no evidence of a radial arrangement to form a lumen. The peritoneum is far more cut up than would appear from the model. Many slight fissures separating adjacent sex-cords do not appear. In any case many of these rudimentary sex-cords are from the first, united with the funnel-cords while others anastomose freely with one another, so that all are either directly or indirectly connected with the latter.
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Primitive sex-cells are frequently met with in the germinal epithelium, as well as in the distal parts of the funnel-cords. Aside from the scattered primitive sex-cells, these tissues are composed of ordinary peritoneal cells. The cells of the germinal epithelium are so crowded as to make it stain very deeply. The sex-cords are less dense, their cells being distinct and having clear, sharp outlines, thus differing from those of the sex-cords of the pig and rabbit, in which a syncytium is formed among the pure peritoneal cells. The cells of all but the most proximal parts of the funnel-cord are elongated in the direction in which the cords extend. This elongation of the cells is so marked that they resemble the surrounding mesenchyme save for the fact that their cytoplasm is more dense thasi that of the latter. The cells are so closely associated that these funnel-cords stand out quite clearly from the surrounding mesenchyme.
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The proximal part of each funnel-cord is met by one, or sometimes two, evaginations from the capsule of Bowman of the adjoining primary Malpighian corpuscle. These evaginations are very clearly distinguish
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88 The Eete-Cords and Sex-Cords of Chrysemys
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able in this stage from the tissue of the funnel-cords but are in close contact with tliem.
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In earlier stages the funnel-cords are not even in contact with the capsules of Bowman, although they lie close to them. In these stages there are no evaginations from the capsules of Bowman, although a thickening of the cells of the medio-dorsal portions of them indicates the general region where these evaginations will take place. In the much earlier stages described above, 6 mm. C-T., the primary union of Malpighian corpuscle, peritoneal funnel and funnel-cord has already been described. The later union of Malpighian corpuscle and funnel-cord is a secondary one, and has nothing to do with the temporary primary union. The breaking away and reuniting of these elements seems to be a useless process which I confess I am at a loss to explain. I can merely describe it. It is, however, a most easily demonstrated fact.
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In later stages, the evaginations from the Malpighian corpuscles closely fuse with the funnel-cords, and are not to be distinguished from them. As development proceeds, the primary Malpighian corpuscles are often drawn some distance laterad of the sex-gland, at the same time pulling the funnel-cords laterad and causing them to stretch. In these cases each funnel-cord becomes sharply bent at the point where the evagination from the capsule of Bowman meets it; it is then continued in a dorsomedial direction to the adrenal body. As shown above, each primary Malpighian corpuscle is connected with the sex-cord by a cord of tissue, formed by an evagination from the capsule of Bowman plus the basal portion of a funnel-cord. These strands uniting the mesonephros with the sex-gland are the rete-cords and constitute the rete-testis or rete-ovarii, as the case may be. In these later stages the funnel-cords are more elongated and slender, but far more compact than in the early stages.
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Plate I, Fig. 4 shows the rete-cords and the relation that they bear to the sex-cords and primary Malpighian corpuscles. Here the base of the funnel-cord lies within the sex-gland and forms one of the sex-cords. This has been observed in many cases. In very many instances, however, the funnel-cords lie wholly outside the sex-gland, their bases being still attached at a greater or less distance from the sex-gland to the peritoneum covering the mesonephros.
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It will be noticed that the two rete-cords shown in this model are united to one another by a thickening of each in the direction of the long axis of the sex-gland. This represents a tendency to form a longitudinal canal uniting the rete-cords as in the Amphibia and to a certain extent in the Elasmobranchs, and in the lizard (Braun, 77). This
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Bennet M. Allen " 89
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longitudinal canal remains incomplete, however, although it may unite several rcte-eords in the manner shown.
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Young males taken at the time of hatching, show many of the retccords to have already acquired a lumen in places. The rete-cords of females at this age do not show a lumen, nor do they at any time, because they have already paused in development. They are, however, still recognizable. Up to this point no distinction of sex has been noted although well marked differences had begun to appear in the stage of 13 mm. C-T. length. Close study has yet to be made to determine the earliest evidences of sex differentiation.
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It is not our aim to follow the later development of the rete-cords or sex-cords. In its general features, the further development of the sexglands of the turtle shows many points of similarity to that in the mammals. The sex-cords degenerate in the female forming the medullary cords while the " cords of Pfiiiger " arise as a later thickening of the germinal epithelium. In the males the sex-cords lengthen, assuming a more regular form and arrangement. Their thorough anastomosis with one another allows the semen to be poured from several into a common rete-cord. The mesonephros degenerates leaving a number of the uriniferous tubules to function as vasa efferentia. In the adult male the retecords are found to be reduced in number, there being nine in the specimen studied while sixteen were counted on the right side of an embryo of C-T. 8 mm. length. No attempt was made to determine how or when this reduction was brought about. It is quite probable that some retecords are weak and become broken by shifting of the organs in the process of growth. In any case there is no systematic degeneration of the rete-cords in any particular region or regions along the sex-gland.
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Summary and Conclusions.
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The sex-cords are formed from irregular ingrowths of the germinal epithelium. It is not until relatively late in development that they take on the semblance of cords. They are made up of ordinary peritoneal cells, together with primitive sex-cells which are also found in the peritoneum at this stage.
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The rete-testis and rete-ovarii are formed by the union of funnel-cords with evaginations from the capsules of Bowman. The funnel-cords are derived from the peritoneal funnels of the Malpighian corpuscles. They occupy a region lying along the lateral edge of the sex-gland, and not only co-extensive with the latter, but extending a short distance anterior to it. The bases of the funnel-cords may, or may not, be included in the sex-gland to form a part of the seminiferous tubules of the testis or
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90 The Eete-Cords and Sex-Cords of Chrysemys
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iiRHluUary cords; of the ovary, as the case may he. The ])roxiinal portions of these fi)nnel-cords go to form a large part of the rete-testis-ovarii, while the more distal portions join the adi'onal fundament and contril)nte the major portion of the cortical suhstance of that organ.
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This leads me to briefly consider the adrenal body, although this was not within the original plan of the present work. Soulie, 02, finds that in Lacerta and the chick, the cortical substance arises wholly from cords of cells proliferated from the peritoneum mediad of the sex-gland and at the base of the mesentery. He states, however, that these cords become closely applied to the capsule of Bowman of the Malpighian corpuscle. It is difficult to understand how, arising from the base of the mesentery, they could reach the Malpighian corpuscle without growing dorsad along the medial side of the Y. renalis revehens to the adrenal body fundament, and thence laterad and ventrad to the Malpighian corpuscle. It is difficult to understand how they could take this course, without passing through and beyond the fundament of the adrenal body. There certainly are, in the turtle, cords of cells that arise as Soulie and others claim, near the base of the mesentery, and these contribute to the formation of the adrenal body; but certain sex-cords and the funnel cords contribute to it as well, and in even greater measure. Brauer, 02, also holds a view similar to that of Soulie as regards Hypogeophis one of the Gymnophiona. Poll, 03, reached similar results with the Elasmobranchs, Acanthias and Spinax. Be this as it may, I feel quite sure of my ground in the case of Chrysemys, and the work of Weldon, 85, and Hoffmann, 89, would lend color to this view, though they hold views in some points radically different from mine. . In this connection it may be well to state that several of Hoffmann's, 89, figures of the " Sexual Strange " would serve fairly well to represent the funnel-cords as I have seen them. They certainly do not prove his contention that the cords in question, sex-cords and adrenal-cords, arise from the capsule of Bowman ; although he has so interpreted them.
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Those who held view C, probably used insufficient material and lacked the intermediate stages between the period just before the formation of the sex-cords and those subsequent to their separation from the germinal epithelium.
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Janosik, 85, D, worked upon the chick. It is quite possible that future work may in large part substantiate his results for that form. My results agree with his as regards the origin of the sex-cords,- but differ from his upon the origin of the rete-tissue, although even here there may be a reconciliation between our views.
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In the literature upon the morphological significance of the uro-genital
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Bennct M. Allen
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91
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system we have some melancholy examples of the futility of making rash hypotheses unsupported by a sufficient array of facts. Still it is of interest to consider the possible interpretation that may be placed upon these structures when they are viewed from the standpoint of phylogeny.
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I am inclined to consider tlie funnel-cords as modified sex-cords. The fact that their distal extremities contribute to the formation of tbe adrenal bodies does not conflict with this interpretation, because that is also true of undoubted sex-cords. The funnel-cords arise just laterad of the true sex-cords and in a very similar manner. The fact that they arise from the peritoneal funnel would not be contrary to this view if the
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Text Fig. F. Diagram to show essential structures of the mammalian
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sex-gland.
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M. — Mesonephros. MP. — Malpighian corpuscles.
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R. — Rete-region. R. C. — Rete-cord.
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S. — Sex-gland region.
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8. C— Sex-cord.
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y. — Vestigial portion of genital ridge. W.D.— Wolffian duct.
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funnels could be shown to be mere recesses of the peritoneum, and similar to the latter in histological character. A more careful study of the origin of the sex-glands in the Amphibia is much to be desired as it might throw new light upon this question. It will be of interest to compare the results of this paper with those of a previous paper upon the same structures in the pig and rabbit. Allen, 04.
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The very schematic diagram of the testis of the pig (Text Figure F), shows the following points seen in a sagittal section passing through the genital ridge and the mesonephros. The genital ridge may be divided into three regions : (1) rete, (3) sex-gland, (3) rudimentary sex-gland ridge. The rete-cords arc liomodynamous with the sex-cords, being formed at the same time and in tlie same luannei" as the latter, and occupying
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92 The Eete-Cords and Sex-Cords of Chrysemys
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the anterior third of the genital ridge, whose middle portion is occupied by the sex-gland. As the rete-cords develop, they come in contact with slight evaginations from the Malpighian corpuscles in that part of the mesonephros which lies nearest the rete region. They then grow back to the anterior portion of the sex-gland and at a relatively late ]ierio(l of development advance along its entire length, giving off numerous branches (tubuli recti) which fuse with the tips of the seminiferous tubules. The rete-cords of the mammals are the peritoneal ingrowths of the anterior part of the genital ridge. Speaking in terms of phylogeny they are the sex-cords of the anterior part of the sex-gland. The analogous structures of the turtle, the funnel-cords, appear at intervals along the entire lateral margin of the sex-gland.
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It is quite probable that the mammalian sex-gland was derived from that of some reptilian group and that some now existing groups of reptiles may show sex-gland conditions from which those of the mammals were derived. ]S[o existing group is more likely to show mammalian affinities than that of the Chelonia.
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jSTothing exactly corresponding to the funnel-cord has ever been found in the embryonic development of the mammals. It is true that Aichel, 00, has found that the cortical portion of the adrenal body of the rabbit (Lepus) arises from funnel-like invaginations of the peritoneum near the base of the mesentery. He is very positive in his claim that these are the peritoneal funnels of the mesonephros. Nevertheless, he does not claim to have followed these funnels back to stages in which they were actually connected with the Malpighian corpuscles. The rete-tubules that may have directly united the sex-gland proper along its entire length with the adjacent Malpighian corpuscles of the mesonephros have disappeared without leaving a recognized vestige, in any of the mammals thus far studied. The rete-region of these mammals has been evolved from that part of the genital ridge which was primitively the anterior part of the sex-gland in the ancestors of the mammals.^
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It is scarcely possible to be more specific as regards the nature of the rete-region of the mammals. Two assumptions are possible: one, that
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^ It will be well to note that in Chrysemys, several funnel-cords occur in a well-marked region, anterior to the sex-gland, in which the sex-cords remain vestigial. Upon closer study of some sagittal sections of the sex-gland and mesonephros of Chrysemys I have been struck with the resemblance that this region bears to the rete-region of the pig and rabbit as seen in similar sections. In Chrysemys the funnel-cords of this anterior region together with those of the sex-gland region are joined to form the central canal. This shows some points of resemblance to the portions of the rete-cords lying parallel to the peritoneum anterior to mammalian sex-gland.
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Bennet M. Allen ' 93
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the sex-cords have disappeared leaving only the funnel-cords, the other, that the sex-cords which primitively existed in this region have taken on the character and function of funnel-cords. It is difficult to decide this question, I can merely say that the latter assumption seems the more probable one, because often two or more rete-cords can be seen in a single transverse section to arise from more than one point of the peritoneum covering the rete ridge. In fact the strongest and most numerous rete-cords arise from the portion of it that lies nearest the mes'entery. This question might be solved with certainty by a study of the conditions in the Monotremes or even in other less primitive groups of mammals.
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LITERATURE CITED.
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AiCHEL, Otto, oo. — Vergleichende Entwickelungsgeschichte unci Stammes geschichte der Nebennieren. Arch. f. mikr. Anat., Bd. LVI, 1900. Allex, B. M., 04. — The Embryonic Development of the Ovary and Testis of
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the Mammals. American Journal of Anat., Vol. Ill, 1904. Balfour, P. M., 78. — A Monograph of the development of the Elasmobranch
 +
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Fishes. Works of F. M. Balfour, 1878. Braux, M., 77. — Das Urogenitalsystem der einheimischen Reptilien. Arb.
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Zool.-zoot. Institut, Wiirzburg, Bd. IV, 1877. HoFFMAxx, C. K., 89. — Zur Entwickelungsgeschichte der Urogenitalorgane bei
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den Reptilien. Zeitschr. f. wiss. Zool., Bd. XLVIII, 1889.
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92. — Sur le developpement de I'appareil. uro-genital des oiseaux. Verb.
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d. Koninklyke Akademie v. Wetenschappen te Amsterdam. Sectie 2, Deel I, No. 4, 1892. Jaxosik, J., 85. — Histologisch embryologische Untersuchungen iiber das Urogenitalsystem. Sitz. Ber. Akad. Wien, 3 Abth., Bd. XCI, 1885.
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90. — Bemerkungen iiber die Entwickelung des Genital Systems. Sitz.
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Ber. Akad. Wien, 3. Abth., Bd. XCIX, 1890. Laulaxie. F., 86. — Sur le mode d'evolution et la valeur de I'epithelium germi natif dans le testicule embryonnaire du Poulet. C. R. Soc. de
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Biologie, T. 3, 1886. Mihalkovics, v., 85. — Untersuchungen iiber die Entwickelung des Harn- und
 +
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Geschlechtsapparates der Amnioten. Inter. Monatschr. f. Anat. Hist.,
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Bd. II, 1885. MoLLER, F. v., 99. — Ueber das Urogenitalsystem einiger Schildkrbten. Zeitschr.
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f. wiss. Zool., Bd. LXV, 1899.
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The most plausible theory is that the rete-region of the mammals has not been directly derived from a condition like that in Chrysemys; but that the genital ridges of both have been derived from a type in which the anlage of the sex-cords was co-extensive with that of the funnel-cords.
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To be more exact then, the rete-region of the mammals corresponds to the anterior end of the sex-gland of the turtle plus the modified region of funnelcords anterior to it.
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94 The Rete-Cords iind Sex-Cords of Chrysemys
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Peter, K., 04. — Normeiitafel ziir Eutwickelungsgeschichte der Zauiieidechse
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 +
(Lacerta muralis). Normentafeln z. Entw. gesch. d. Wirbelthiere,
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Heft IV, 1904. Poll, H., 03. — Die Anlage der Zwischenniere bei den Haifischen. Arch. f.
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mikr. Anat., Bd. LXII, 1903. SCHMIEGELOW, E., 82. — Studien iiber die Entwickelung des Hodens und Neben hodens. His u. Brawne Archiv f. Anat. u. Physiol., 1882. Semox, R., 87. — Die indifferente Anlage der Keimdriisen beim Hiihnchen und
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ihre Differenzierung zum Hoden. Jena Zeitschr. f. Naturwiss., Bd.
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XXI, 1887.
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90. — Ueber die morphologische Bedeutung der Urniere in ihrem Ver haltniss zur Vorniere und Nebenniere und iiber ihre Verbindung mit dem Genitalsystem. Anat. Anz., Bd. V, 1890.
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91. — Studien iiber dem Bauplan des Urogenitalsystems der Wirbel thiere. Jenaische Zeitschr. f. Naturwiss., Bd. XXVI. Semper, C, 75. — Das Urogenitalsystem der Plagiostomen. Arb. Zool.-zoot.
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Institut, Wiirzburg, Bd. II, 1875. SovLiE, 04. — Recherches sur le developpement des capsules Surrenales chez
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les vertebres Superieurs. Journ. de I'Anat. et de la Phys., T. 39. Waldeyer, W., 70. — Eierstock und Ei. Leipzig, 1870. Weldon, W. F. R., 85. — On the Suprarenal Bodies of the Vertebrata. Quart.
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Journ. of Micr. Sci., Vol. XXV, 1885.
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EXPLANATION OF PLATE.
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Ao. — Aorta. P. — Peritoneum.
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Art. — Arterial branch passing to P. F. — Peritoneal funnel.
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the Malpighian corpuscle. P. C. — Posterior cardinal vein
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FC. — Funnel-cord. 8C. — Sex-cord.
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M. — Mesentery. Y. R. R. — V. renalis revehens.
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M.C. — Malpighian corpuscle. W.D. — Wolffian duct.
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Fig. 1. Transverse section of the mesonephros and sex-gland fundament of an embryo of 6 mm. C-T. length. X 190.
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Fig. 2. Transverse section of the sex-gland fundament of an embryo of 7 mm. C-T. length (carapace 5 mm. long). X 190.
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Fig. 3. Wax plate reconstruction of the indifferent sex-gland of an embryo of 7 mm. C-T. length (carapace 5 mm. long). This includes as much of the sex-gland as lies within a little more than two somites. X 190.
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Fig. 4. Reconstruction of a small part of the sex-gland of an embryo of 13 mm. C-T. length (carapace 12 mm. long). X 190.
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Fig. 5. Drawing of a part of a section adjacent to that shown in Fig. 1. The proximal portion of the peritoneal funnel is here better shown than in Fig. 1.
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THE RETE-CORDS AND SEX-CORDS OF CHRYSEMYS
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BENNET M. ALLEN
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%tt 4 "% '
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^ »> J
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AMERICAN JOURNAL OF ANATOMY~VOL. V
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PLATE I
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F,g4
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THE DEVELOPMENT OF THE LYMPHATIC SYSTEM IN
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BABBITS.
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BY
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FREDERIC T. LEWIS, A.M., M. D.
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From the EmbryoJogical Laboratory, Harvard Medical School.
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With 8 Text Figures.^
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In following the transformations of the subcardinal veins in rabbits, the writer observed that a portion of those veins seemed to become detached from the venous system, and to be transformed into l3'mphatic vessels (02, p. 238). This supposition is not identical with the theory that the lymphatic system is a gland-like outgrowth of venous endothelium, always connected with the veins by means of the lymphatic ducts. It differs also from the older idea that lymphatic vessels are excavations in mesenchyma.
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In favor of this mesenchymal origin, the work of Sala, 00, is the most convincing. He observed in the chick that both the posterior lymph heart and the thoracic duct arose independently of the veins or of other lymphatics, and that their permanent openings into the veins were acquired subsequently. In the rabbit, as will be shown presently, there are many disconnected lymphatic spaces, but to their origin from mesenchyma there are four objections : 1st. The lymjjhatic spaces do not resemble mesenchyma even when it is cedematous, but on the contrary, are scarcely distinguishable from blood-vessels (Langer). 2d. After being formed, the lymphatics increase like blood-vessels, by means of blind endothelial sprouts, and not by connecting with intercellular spaces (Langer, Eanvier, MacCallum, Sabin). 3d. In early embryos, detached blood-vessels may be seen without proving that blood-vessels are mesenchymal spaces. These detached vessels are not far from the main trunks, from which they may have arisen by slender endothelial strands, yet often the connecting strands cannot be demonstrated. A similar supposition would account for detached lymphatic vessels. 4th. The endothelium of the embryonic lymphatics is sometimes seen to be continuous with that of the veins.
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^ This investigation, and the one which follows, were accomplished with the aid of a Bullard Fellowship, established in memory of John Ware. Ameeican Journal of Anatomy. — Vol. V.
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96 The Development of the Lymphatic System in Eabbits
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The second thcoiy, that of the giand-like origin of the lymphatic system, is supported by the remarkable injections of pig embryos, made by Prof. Sabin.' She considers that in mammals, this system buds from the venous endothelium at four points, forming four lymphatic ducts. The ducts are dilated to form four lymph hearts, which, though destitute of muscles, correspond with the four lymph hearts of amphibia. Starting from these hearts, lymphatic outgrowths invade the body, and those from the anterior pair unite with those from the posterior pair. Then the posterior hearts lose their original openings into the veins, but those of the anterior hearts persist as the outlets for the thoracic and right lymphatic ducts respectively. The lymph hearts themselves are said to become transformed into lymph nodes (05, p. 355).
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According to this idea, the lymphatic vessels are true lymphatics from their earliest inception. They differ from other branches of the veins by their very oblique angle of entrance, and by failing to anastomose with arteries or veins. Anastomoses with other lymphatics are abundant, due to absorption of contiguous walls (Ranvier, 97, p. 74).
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The supposition suggested by the study of the subcardinal veins is intermediate between those of Sabin and Sala. The endothelium of the lymphatics is considered to be a derivative of that which lines the veins, since the lymphatics are at first a part of the venous system; but by becoming detached from their origins these lymphatics form closed sacs in the mesenchyma. Later they acquire permanent openings into the veins, and many connections with other lymphatics.
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In studying the development of the lymphatic vessels, several methods have been employed. Sala used serial sections, generally of injected embryos, and made wax reconstructions of the posterior hearts. Sabin perfected the method of injection whicli had been employed by Ranvier for pigs of 100 mm., so that it was applicable to those of 20 mm. By this means she studied the large jugular hmpli sacs, or " anterior hearts," which, as Saxer discovered (p. 370), are the earliest lymphatic vessels to appear. On the basis of injections she was enabled to present the first connected account of the development of the mammalian Ij^mphatic system. This was illustrated by a series of conventional diagrams, in which the blood-vessels are shown without details. Thus the internal
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^ Ranvier described the interesting analogies, botli functional and embryological, between typical glands and the lymphatic system. Sabin does not adopt the idea that the whole lymphatic system represents a few large glands. She does, however, describe it as arising from four blind epithelial (endothelial) outpocketings which ramify in the connective tissue, and this origin may be designated, after Ranvier, as " gland-like."
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Frcdoi'ic T. Lewis
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97
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and t'xtonial juuular veins are merged in an '" anterior cardinal vein," the subcardinals are omitted, the renal and iliac anastomoses are made continuous with one another, and the sciatic and femoral veins are reversed.
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Fig. 1. Rabbit, 13 days, 9.5 mm.. Harvard Embryological Collection, Series 498, X 13 diams. 3, 4, and 5 indicate the position of the corresponding cervical nerves in this, as in the following figures. The veins shown are those of the left side: D. C. duct of Cuvier; Ex. M.. external mammary; In. J., internal jugular; Pr. VI., primitive ulnar.
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It was thought that more accurate figures might bo obtained bv tlie graphic reconstruction of uninjected embryos. The possibility of overlooking minute orifices guarded by valves, and the limitation of this method to small embryos are obvious disadvantages, but these are offset 7
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98
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Tlie Dovclojiiiu'iit of tlio Lyniitliatic Svf^tein in Kabbits
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l)v the avoidance of rnptnre of very thin-walled vessels and by the opportunity of seeing lymphatics too small for injection. The method has been employed ^Yith the following results.
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Fig. 2. Rabbit, 14 days, 10 mm., H. E. C, Series 155, X 13 diams. The lymphatic vessels are lieavily shaded, as in all the following figures. The veins are those of the left side: An. T., anterior tibial; C, caudal: c. b.. " connecting branch "; D. C. duct of Cuvier; Ex. J., external jugular; Ex. M., external mammary; In. J., internal jugular; P.O., posterior cardinal; Pr.Fi., primitive fibular; Pr. VI., primitive ulnar.
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In a rabbit of 13 days, 9.5 mm., no lymphatics could be found. The reconstruction, Fig. 1, shows the veins along which the first lymphatics are soon to appear. The internal jugular vein receives a great many small
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Frederic T. Lewis 99
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branches. One of these, nearly parallel with the dorsal border of the vein and wider than the others, opens into the vein at either end. It is in relation with the third cervical nerve. From its position and appearance it is believed that this branch of the vein becomes a lymphatic vessel.
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The second reconstruction is a 10 mm. embryo of 14 days. In this specimen a chain of lymphatic spaces has appeared along the internal jugular and the dorsal root of the primitive ulnar veins. The most anterior segment of the chain extends back to the third cervical nerve. It sends out short blind sprouts like a vein and contains many blood corpuscles. The partition between it and the jugular vein is very thin, and at one point there is a suggestion of communication between the two, as shown in the figure. No opening into the vein can be demonstrated, however. The second segment of the chain, proceeding posteriorly, extends to the fifth nerve. It equals the internal jugular vein in diameter, and is closely applied to its wall. Behind the third nerve it sends a blind diverticulum around the ventral end of the dorsal body muscles, into the deep subcutaneous tissue of the back. This diverticulum, not matched on the opposite side of the embryo, contains blood which apparently entered it from rough treatment in preserving the specimen. The third segment of the chain, between the fifth and sixth nerves, seems to connect with the root of the ulnar vein. This connection, however, lies in the plane of section, and a thin intervening wall may have been carried away in the process of cutting. A detached lymph space follows the dorsal root of the ulnar vein. A small and somewhat questionable one, not matched on the opposite side, rests against the superior vena cava, between the roots of the ulnar vein. The most significant structure found in this embryo is a space filled with blood, which opens into the external jugular vein near its junction with the internal jugular. This space lies quite near the third segment of the lymphatic chain. On the opposite side of this embryo, and in the following one, this blood-filled sac connecting with the vein appears to . be replaced by a lymphatic space, detached from the vein, but connecting with the chain.
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Fig. 3. from an embryo of 1-1 days, 11 mm., shows the fusion of all the lymphatics of the previous stage into one large sac which encircles the external jugular vein. On neither side could this sac be seen to communicate with the veins. No lymphatic vessels were found which did not connect with the jugular sacs. The dorsal subcutaneous extension, described in the preceding stage, occurred on both sides. In the posterior part of the embryo, no lymphatics were found. The reconstruction of the cardinal veins is that already figured in this journal. Vol. I, Plate 2, Fig. 5 (following p. 244).
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100 The Development of the Lymphatic System in Rabbits
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The cardinal veins of the 14.5 mm. rabbit, Fig. 4, were also shown in the earlier paper (Plate 3, Fig. 7). In the plate, the lower portions of the subcardinal veins are detached from the rest, and, though colored blue
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Fig. 3. Rabbit, 14 days, 11 mm., X 13 diams. The structures drawn are the same as in Fig. 2, except that in the trunlt of the embryo the following^ veins, belonging to the median plane and to the right side, have been added: Az., azygos; G., gastric; R. A., renal anastomosis of the subcardinal veins; Sc, subcardinal; /S. M., superior mesenteric; V., vitelline; V. G. I., vena cava inferior.
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Frederic T. Lewis 101
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■ like the veins, they are deseribed and figured as " spaces in the mesentery " suggesting the lymph hearts of the chick (p. 238). It is stated that these spaces " may be siibcardinal derivatives." Ee-examination of this embrj'O has yielded no more definite information. The spaces which are midoubtedly lymphatic, as shown by their later development, seem to replace veins of the preceding stage. In the same way the lymphatic vessels in the mesentery, accompanying the superior mesenteric ancVthe gastric veins may have arisen as the branches of those vessels seen in Fig. 3. They extend around the superior mesenteric artery, which the corresponding vein accompanies. The fused vitelline vein is destitute of small branches, and is not provided with lymphatics.
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The jugular lymph sac in Fig. -t has completely surrounded the third and fourth cervical nerves. It envelops two-thirds of the circumference of the internal jugular vein. On the right side of the embryo, in one section (No. -176), a miniite orifice connected the sac and the vein. It was not in the position of the adult opening between these structures, and was not matched on the opposite side. The deep subcutaneous outgrowth from the jugular sac has become greatly dilated in its distal portion. Near the beginning of the external mammary vein, a large lymph space is found wedged between two converging venous branches. This space is not connected with the veins. It may be a remnant of the lymphatic vessels which in the preceding stage accompanied the dorsal root of the ulnar vein. A few slender detached lymphatics follow the external mammary vein. Finally there are two lymphatics which appear to have arisen from branches of the azygos vein, one near the vagus nerve (Fig. 4, x) and the other along the aorta (Fig. 4, y). The former connects with a small vein, the latter ends blindly not far from one. Obviously when a connection with a vein is well preserved the structure in question would be considered a venous branch; and after becoming detached, were it not for its endothelial wall, it might be called a mesenchymal excavation. The study of this and the following specimens seems to show that the lymphatics along the aorta (thoracic ducts) are derived in part from the azygos veins; below, from the subcardinals; and above, from the jugular sacs.
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In order to determine whether the lymphatic system of the rablut differed materially from that of other mammals, reconstructions were made of a 21 mm. pig, and a 15 mm. cat. The former is of special interest as a basis of comparison between the present work and > that of Prof. Sabin. The lymphatics in the pig (Fig. 5) consist of a pair of jugular lymph sacs, a pair of subcardinal sacs which fuse with one another irregularly and are variously subdivided by thin septa, and
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Fic. 4. Rabbit. 14 days 18 hours, 14.5 mm., H. E^ C Sen^^l^^'^^,/,": diams. X designates a lymphatic vessel '-^^^^^P^!^^"^?^ f ^/^ Jf ceiSc; .,. a lymphati^c along ^^e aorta. The veans of th -^^,^%^,_ ,; Pr. Ul.. primitive ulnar. Those ot tne leg axe. ^-i". ^ •• "connecting branch"; Fe., femoral; Pr. Fu, primitive fibular.
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Frederic T. Lewis ' 103
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finally some irregular spaces behind the aorta, probably derived from the azygos veins. These spaces also fnse across the median line at several points.
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The jngnlar sac is shaped like a D of which the chief portion is vertical and closely applied to the internal jugular vein. Through the aperture in the D pass the third, fourth, and fifth cervical nerves, and from its dorsal arch several deeply subcutaneous sprouts pass off, corresponding with the single large sac of the rabbit. ISTo connection between the jugular sac and the veins could be detected. Except for this point, the reconstruction agrees with, and combines, the figure and diagram presented by Prof. Sabin in this journal. Vol. 3, p. 184, and Vol. -1, p. 359. It does not agree so well with the diagram on p. 380 of A^ol. I. In the latter the subcardinal l3anph spaces are not shown. The posterior portion of the body contains instead two " lymph hearts " arising from tlie posterior cardinal veins " below the Wolffian body "" but anterior to the femoral vein. In later stages, outgrowths from these hearts invade the skin of the back, and nltimately, as has already been noted. Prof. Sabin considers that the hearts become transformed into lymph nodes. From this description, it appears that the posterior lymph hearts are in the position of the ilio-lumbar veins. In the pig embryo represented in Fig. 5, however, no lymphatics were found in relation with the ilio-lumbar vessels.
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Considering its lymphatic development the pig of 21 mm. is less advanced than the rabbit of 14.5 mm., since there are no lymphatic vessels along the external mammary vein nor in the mesentery. The cat of 15 mm. is more advanced than either. In this embryo the D formed by the jugular sac is almost bisected diagonally. The second, third, and fourth nerves pass through its aperture, but the fifth penetrates the posterior section of the sac by a separate opening. There are two deep subcutaneous diverticula corresponding with the single one in the rabbit and several in the pig. In one section (266) a branch of the jugular sac may enter the innominate vein a little anterior to the subclavian, but it is not clear that an actual opening exists and none can be found on the opposite side.
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"Where the external mammary vein joins the brachial there is a large sac, and the question arises M'hether or not the detached lymphatics following the mammai-y vein are independent formations, or are outgrowths from that sac. The occurrence of the lymphatics especially near the places where the veins l)ranch suggests that they may have budded at such points. On the other hand, as in the rabbit, their order of appearance is from the proximal ])art of the vein distally. Similarly there are
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vf w r Series 59, X 10 diams. The veins are:
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Fig. 5. Pig, 20 mm.. H. E. C., series oy, ^ Cuvier; Ea-. J., exter A^., azygos; Br., brachial: Ce cephalic D. ^^^^^^ f ^^'^^ _ ^^stric; In. J.,
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nal' jugular: i... M -^--^^ ^.^^^rn'osifof ^b'ardinals- ..n.. sciatic;
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rS!tp^e^S?;ies?nte^riV;?^^viSlfne; V. C. I. vena cava inferior.
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Fig. 6. Cat. 15 mm., H. E. C, Series 436, X 13 diams. The lettering Is the same as in Fig. 5.
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10() The Development of the Lymphatic System in Eabbits
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obscure spaces which ap})ear to ho lymphatic, along the aorta, and in relation with the azygos veins. An occasional apparent connection with the vein suggests their venous origin in situ. The mesenteric and subcardinal plexuses have united with one another. They do not empty into the veins. The subcardinal sacs extend from the renal anastomosis almost to the sciatic vein, connecting with one another across the median line, as in the pig. No lymphatic vessels follow tlie ilio-lumbar veins into the posterior body wall.
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Eeturning to the rabbit embryos it will be seen that Fig. 7 from a 21 mm. rabbit differs from Fig. 4, the 14.5 mm. embryo, chiefly in regard to the thoracic duct. The duct is represented by a pair of vessels which connect with one another and pass on to the left jugular sac. Sometimes in the adult rabbit, as figured by Gage (02, p. 650), and occasionally in man, the thoracic duct bifurcates anteriorly and passes to the jugular sacs on either side. This did not occur in the 21 mm. embryo, which exhibited the relations figured by Sabin, Vol. I, p. 383.
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In Fig. 7 scattered lymphatics are shown along the external jugular vein and its branches. One much larger than the rest occurs wdiere the anterior and posterior facial veins unite. From its isolation it probably arose independently of the large jugular sac. Other and more isolated lymphatic centers are seen in the oldest rabbit studied, one of 20 days, 29 mm., Fig. 8, notably along the pudic and the sciatic veins. They arise near the junction of several venous branches, with which, however, they are not in communication.
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In the oldest embryo the lymphatic system has invaded the skin to such an extent that it is impracticable to represent more than a small part of it. In entering the skin the lymph vessels accompany the veins, those of the head following chiefly the external jugular vein. The jugular sac has become relatively less important, and persists as the lymphatic sheath of the internal jugular vein. The deep subcutaneous extension has becom^ covered by a thin layer of muscle, presumably the panni cuius, and does not appear to connect with the more superficial vessels of the skin. There are no lymphatics in the distal part of the arm, Init the subcutaneous vessels of the shoulder are attended by rich netw^orks. These veins are the external mammary, and another w^hich is ventral to the scapula and posterior to the shoulder joint, — a subscapular vein. The lymphatics along this large subscapular vein do not connect with the jugular sac. At the point L. N., indicated in the figure, a small but very distinct lymph node has developed in relation to these subscapular lymphatics. A corresponding node is found on the opposite side of the body.
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Fig. 7. Rabbit, 17 clays, 21 mm., H. E. C, Series 738, X 10 diams. The veirs not previously lettered in the rabbit figures are: 7?., ilio-lumbar; Ss., subscapular; R., radial; Sci., sciatic.
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i^Z u ^^^'Y- ^^ ^f^^' ^^ "'"'•• ^- ^- C- Series 170, X 6.9 diams. The first lymph nodes develop at L. lY., along the subscapular vein. ^^s. ; and at J. «., along the iho-lumbar vein, II. The veins of the arm are: Br., brachialte cephalic: J. Ce.. jugulo-cephalic: R.. radial. Those of the legs are- A« T anterior tibial; Sci., sciatic: Po. T.. posterior tibial: Fe.. femoVal; c. &., connecting branch between femoral and sciatic. P. marks the pudic vein
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Frederic T. Lewis 109
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The jugular sac on the leCt side, except for an extensive rupture, does not connect with the vein. On the right, a pore is found leading from the sac to the internal jugular vein near its union with the external, but this also may he artificial. Thus in all the series of rabbits no bilateral communication of the lymphatics and veins, in the position of the adult openings, could be found. The pores, sometimes detected in various positions, are not adequate to empty the large sacs, and may indeed be artifacts. Communication with the veins in these stages must be by osmosis, therefore, and the permanent outlets of the lymphatic system must develop later.
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The left jugular sac in Fig. 8 connects with the thoracic duct, which arises from a plexus of lymphatics surrounding the aorta. Ventral to the aorta these vessels receive the lymphatics from the mesentery. There are none in the leg. The body wall is supplied by those which follow the external mammary A'ein in its anastomosis with the superficial epigastric, and by vessels accompanying the ilio-lumbar vein. The ilio-luml)ar vein of Krause, whicli Hochstetter named the posterior transverse lumbar, supplies the subcutaneous tissue of the back, and seems to be inversely homologous with the much larger subscapular vein. At the position I. n., indicated in Fig. 8, a node is found among the lymphatics accompanying this vein. A similar node exists on the opposite side, and the pair was identified in a duplicate series of a 20-day rabbit. These superior inguinal nodes (Krause) develop almost simultaneously with the subscapular nodes already described. The early appearance of the inguinal nodes further identifies the lymphatics of the ilio-lumbar vein with the " posterior lymph heart " of Prof. Sabiu. It is my opinion that an identification of this structure with the amphibian or avian lymph heart is, at present, not justified. The posterior heart of the bird empties into the coccygeal veins (Sala), and that of the frog into the transveree iliac vein, a vessel connecting the femoral with the sciatic vein (Gaupp). The ilio-lumbar vein is more anterior than either. Its lymphatics do not differ in form, from those accompanying other veins, and they are presumably non-contractile. If the first lymph nodes can be utilized in making comparisons, then this " posterior heart " of the rabbit should be compared with the lymphatics of the subscapular vein, and not with the jugular sac. The jugular sac itself does not empty into the vertebral vein, like the anterior heart of the frog. It is non-contractile, so far as known. 1 f it shall l)e found that the anterior heart of the frog develops from the first ]ym})hatics which are formed in that animal, a comparison between the jugular sac and a lymph heart may be possible. At present it is not evident that mammals possess any lymph hearts.
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110 'I'lio Dovi'lopinoiit of tlic Lympliatic Systom in liabbits
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Summary.
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The lymphatic system of rabbits begins along the internal jugular vein as a detached sac formed by the coalescence of several venous outgrowths.
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Similar though smaller sacs arise from the subcardinal and mesenteric veins at a slightly later date.
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Subsequently lymphatic vessels develop along the courses of the azygos and cutaneous veins, apparently from independent venous outgrowths. All of these vessels unite with one another to form a continuous system, which acquires new and permanent openings into the veins near the subclavian termination.
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The first lymph nodes observed are two pairs, one beside the subscapular vessels, and the other beside the ilio-lumbar vessels.
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In order to facilitate comparison with Prof. Sabin's work, the following conclusions may be added :
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The lymphatic system does not arise from the venous system by four outgrowths, but by several. It is not always in communication with the veins. The outlets of the thoracic and right lymphatic ducts are not persistent primary openings. An identification of mammalian lymph hearts, comparable with those of the amphibia, should not be made, on the evidence now available. Judged by their relation to the early lymph nodes, the jugular sac is not comparable with the lymphatics along the iliolumbar vein. However, the study of rabbit embryos confirms the chief conclusion established by Prof. Sabin, that the lymphatic system is a derivative of the venous system.
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LITERATURE CITED.
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Gage, Simon H., 02. — A Reference Handbook of the Medical Sciences. Edited
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by Albert H. Buck. 2d ed., Vol. 5, pp. 624-659, New York. Gaupp, Ernst, gg. — Anatomic des Frosches. 2d ed.. Part 2, Braunschweig. HocHSTETTER, FERDINAND, g^. — Beitragc zur Entwicklungsgeschichte des
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Venensystems der Amnioten, III. Morph. Jahrb., Vol. 20, pp. 543-648. Krause, W., 68. — Die Anatomic des Kaninchens. Leipzig. Langeb, C, 68. — Ueber das Lymphgefasssystems des Frosches, III. Sitz.-Ber.
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d. Akad. d. Wiss., Wien, Vol. 58, pp. 198-210. Lewis, Frederic T., 02, — The development of the vena cava inferior. Amer.
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Journ. of Anat. Vol, 1, pp. 229-244. MacCallum, W. G., 02. — Die Beziehung der Lymphgefasse zum Bindegewebe.
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Arch. f. Anat. u. Phys., Anat. Abth., pp. 273-291. Ranvier, L., gj. — Morphologic et developpement des vaisseaux lymphatiques
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Chez les mammiferes. Arch. d'Anat. mic. Vol. I, pp. 69-81. Sabin, Florence R., 02. — On the origin of the lymphatic system .from the
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veins and the development of the lymph hearts and thoracic duct in
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the pig. Amer. Journ. of Anat., Vol. 1, pp. 367-389.
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Frederic T. Lewis 111
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Saiun, Florence R., 04. — Onthe development of the superficial lymphatics in
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the skin of the pig. Amer. Journ. of Anat., Vol. 3, pp. 183-195. 05. — The development of the lymphatic nodes in the pig, and
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theii- relation to the lymph hearts. Amer. Journ. of Anat., Vol. 4,
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pp. 355-389. Sala, LuKii, 00. — Sullo svillupo dei cuori linfatici e dei dotti toracici nell'
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embrione di polio. Ric. fatte nel hab. di Anat. norm. d. R. Univ.
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di Roma, Vol. 7, pp. 263-296. Saxer, Fr., 96. — Ueber die Entwickelung und den Bau der normalen Lymph driisen und die Entstehung der roten und weissen Blutkorperchen.
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Anat. Hefte, Abt. 1, Vol. 6, pp. 349-532.
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THE DEVELOPMENT OF THE VEINS IN THE LIMBS OF BABBIT EMBEYOS.
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BY
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FREDERIC T. LEWIS, A. M., M. D.
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From the Emhryological Laboratory, Harvard Medical School.
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With 1 Text Figure.
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In connection with the preceding study of the lymphatic system it was necessary to reconstruct the veins of the shoulder and hip in a series of rabbit embrj^os. The reconstructions were then extended to include the distal portions of these vessels, complete figures of which had never been published. Hochstetter, in 1891, had observed the veins in the limbs of living rabbit embryos, and had studied them in serial sections. His drawings, however, show only detached portions of the veins such as could be seen under most favorable conditions, in living embryos. Ten years later Grosser described but did not reconstruct, the developing veins in the extremities of bats. To these two investigators embryology is indebted for the present knowledge of the veins in mammalian limbs. It is proposed to review their work, while describing the reconstructions, considering first the veins of the anterior extremity, then those of the posterior extremity, and finally the homologies which exist between the two sets.
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A^EINS OF THE ANTERIOR EXTREMlTY.
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In the youngest rabbit figured, an embryo of 13 days. Fig. 1, p. 97, the small vessels along the radial or anterior border of the arm unite to form a vein which follows the periphery of the limb to its posterior or ulnar border, and then ascends behind the brachial plexus to terminate near the junction of the anterior and posterior cardinal veins. It receives a branch which at this stage is not well defined, ascending in the body wall. This is the Seitenrumpfvene of Hochstetter, and becomes the external mammary vein of the adult.
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According to Hochstetter, in rabbits of 12 and 121/2 days, the " border vein " mak€s a complete circuit of the limb, and its radial part either empties into the ulnar vein near its termination or connects with the cardinal vein directly. But this radial vein is said to be hard to follow
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American Jocrxal of Anatomy. — Vol. V. 8
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114 I)evc4opment of the A'oiiis in the Limbs of Eabbit Embrvos
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because *" attended by several venous twigs of nearly tht' same caliber, and only shortly before its termination is it recognizahU' as a distinct (starkeres) vessel " (p. 24). In a 13-day rabbit the radial vein had disappeared, " since it had been but imperfectly marked out." Similarly (irosser found a radial vein emptying into the anterior cardinal close to the ulnar vein, in the youngest bat which he studied (4% mm.). In the next stage (614 nim.) it had vanished (p. 136). An examination of rabbits of 12 and I2I/2 days, together with younger ones in the Harvard ■ Collection, shows that the first vein of the arm develops along its ulnar margin, extending distally around the border to the radial side. Small and variable vessels such as Hochstetter described as a radial vein may occur, as shown in Fig. 1, p. 97, Init they do not form a structure comjDarable with the primitive ulnar vein. The latter may be called the primary vein of the arm.
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The rabbit of 14 days. Fig. 2, p. 98, presents the condition described by Hochstetter in embrvos of 13 days. The primitive ulnar vein has acquired a new outlet ventral to the brachial plexus, so that, by the persistence of the original dorsal termination, most of the plexus and the brachial artery are surrounded by a loop of vein. In the following rabbit, Fig. 3, p. 100, the ventral outlet of the idnar vein is the chief one. This specimen shows a small vessel extending from the external jugular vein toward the radial border of the arm.
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The next embryo, Fig. 4, p. 102, is considerably more advanced. The dorsum of the hand, which was previously its external surface has rotated and become anterior; the arm is in pronation. The differentiation of the fingers is indicated by the sinuous terminal border of the hand, and by shallow interdigital depressions on its dorsum. Beneath these, interdigital veins have been formed, probably from branches of the primitive ulnar vein. A new vein has grown from the external jugular down the anterior or radial border of the arm, and has united with the independently formed interdigital veins. This is the cephalic vein of the adult. It is embryologically the second vein of the arm.
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Hochstetter states that the cephalic vein in rabbits develops toward the body from the back of the hand, connecting with the ulnar vein at the elbow, and later continuing up the arm to the external jugular vein. The preceding reconstructions of the rabbit agree better with Grosser's description of the bats. He failed to find a stage in which the cephalic vein emptied into the ulnar. In the earliest specimen in which the cephalic vein was found, it connected with the external jugular vein.
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The cephalic vein of the 17-day rabbit is the chief vein of the limb, and has developed a In-aneh whicli follows the radial artery, the deej) radial
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Frederic T. Lewis 115
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vein, Fig. T, }>. 107. At 20 days, Fig. 8, j). 108, the eeplialic vein has acquired its new and permanent orifice near the axiUary vein. The jugulo-cephalic vein niarl-cs its former outlet.
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Witli tlie differentiation of the digits, the primitive ulnar veiii becomes greatly reduced by the loss of its distal portion. This is shown in Fig. •1. At 17 days, Fig. 7, p. 107, the continuity of the primitive ulnar vein has been interrupted at tlie elbow, resulting in further reduction. The vein then extends from the elbow to the superior vena cava, following the brachial artery, from around which it receives small branches. In tlie 20-day embryo, Fig. 8, p. 108, the brachial vein (proximal part of the primitive ulnar) is continued down the forearm following the ulnar artery. If we may judge from the position of this vessel, there lias been a re-establishment of the course which was interrupted in the younger embryo. Hochstetter, however, states (p. 28) that in rabbits the forearm section of the primitive ulnar vein seems to disappear, although in man (p. 33) the corresponding vessel is preserved throughout, and forms the basilic vein of the forearm and arm, the axillary and subclavian veins.
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The question arises whether the primitive ulnar vein should be described as producing the deep ulnar, brachial, and axillary veins, naming it for the adjacent arteries, or as forming the basilic and axillary veins, considering the cutaneous vein of the corresponding region as its more direct derivative. This uncertainty calls attention to the fact that both the superficial and deep sets of veins have a common origin, and that before their separation the embryonic vein may properly be called either brachial or l^asilic. The ra])I)it of 20 days is characterized by the establishment of this brachial (or l:)asilic) vein.
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In the develo])ment of the veins of the arm three stages have been distinguished :
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1st. The stage of the primitive ulnar vein.
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2 d. " " " " cephalic vein.
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3d. " " " " brachial vein, the cephalic vein persisting.
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Veins of the Posterior Extremity.
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A rabbit of IOI/2 days (Harvard Collection, No. 199) has a very large umbilical vein which sends l^ranches into both Jimbs. Those in the leg form a net which connects with the posterior cardinal vein, still a minute vessel in the caudal end of the l)ody. From the network a vein is developed, which after following the periphery of the limb and passing along its posterior or fibular l)order, empties into the cardinal vein. This vessel may be called tlie ])rimitive fibular vein. The original connections of the
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116 Developmeut of tlie Veins in the Limbs of Eabbit Embryos
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net with the umbilical vein do not form ;i well defined vessel and soon disappear. Although Ilochstetter recognizes this, he refers to the connection with the umbilical vein as a tibial border vein. Grosser could not identify such a vessel in any of his three youngest bats (p. 149).
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The primitive fibular vein as shown in Fig. 2, p. 98, is a vessel readily comparable with the primitive ulnar vein. Both course along the posterior borders of their respective limbs, in which they are the first veins developed. They are undoubtedly homologous. In later development, however, they constantly diverge from one another. Even at 14 days the fibular vein has two small branches which are not matched by any belonging to the ulnar vein. One of these, coming from twigs on the outer and caudal surface of the leg, becomes the anterior tibial vein, An. T. The other which extends mediad toward what at this stage is the inguinal line, may be referred to as the " connecting branch," c. b. In the more advanced embrj^o. Fig. 3, p. 100, the same branches appear in similar relations. They have become much larger at 14 days 18 hours. Fig. 4, p. 102. Here the anterior tibial branch has extended diagonally down the limb to the dorsum of the foot. The connecting branch has sent its twigs into the abdominal wall and the adjoining tibial border of the limb. The primitive fibular vein is still the chief vein of the leg.
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In the older rabbit, Fig. 7, p. lOT, the difl'erentiation of the toes has broken up the distal portion of the primitive fibular vein, wdiich has disappeared almost to the point where it receives its anterior tibial branch. This branch now arises from the interdigital veins on the dorsum of the foot and its main trunk appears continuous with the proximal part of the primitive fibular vein. The anterior tibial and primitive fibular veins together, now constitute the sciatic vein, wliich is embryologically the second vein of the leg.
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The reconstructions to wliich we have referred agree with Hochstetter's description of the development of the sciatic vein except in one detail. They do not show that a part of the primitive fibular vein distal to the anterior tibial branch persists as the small saphenous vein.
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In the rabbit of 14 days 18 hours, a third vein of the leg has begun its development. This is the femoral vein which terminates in the posterior cardinal anterior to the sciatic vein. It advances toward the tibial l^order of the limb. At 17 days, Fig. 7, p. 107, it is seen approaching the external mammary and the connecting branch of the sciatic vein. In the embryo of 20 days, Fig. 8, p. 108, it has anastomosed with both and passes down the leg as the posterior tibial vein, Po. T.
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Just as it is questionable in the arm whether the parent vessel should
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Frederic T. Lewis 117
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be designated hriu-liial or basilic, so in tbe leg there is the cboice between femoral and large saphenous vein. Both of the latter spring from the vessel which we have called femoral. The close relation between the two is shown by Krause's description of the veins in the adult rabbit, where the posterior tibial is considered to be the distal continuation of the large saphenous vein. It seems probable also, that the anteror tibial vein, which is quite superficial at 20 days, though it accompanies the artery, should give rise to the small saphenous vein, with which it anastomoses in the adult. Hochstetter, as already noted, assigns a somewhat different origin to the small saphenous vein.
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The condition found in the rabbit at 20 days, is essentially that of the adult. The sciatic vein remains a large vessel. In man, assuming that the embryological history is similar to that of the rabbit, the proximal section of the sciatic vein dwindles after the formation of the femoral anastomosis near the knee. The sciatic vein is represented, therefore, merely by the collateral circulation of the thigh, as figured by Charpy (Poirier's Anatomic, Vol. 2, p. 1052), and by Spalteholz (Handatlas, Vol. 2, p. 469).
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The preceding observations seem to establish three stages in the venous development of the leg, comparable with those in the arm.
 +
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1st. The stage of the primitive fibular vein. 2d. " " " " sciatic vein.
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3d. " " " " femoral vein, the sciatic vein persisting (in man, very much reduced ) .
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Homologies between the Veins of the Anterior and Posterior
 +
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Extremities.
 +
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Bardeleben's view that the primary vein of the arm consisted of the vena cephalica antibrachii, vena mediana cubiti, and vena basilica brachii, and that this was homologous with the vena saphena magna of the leg was rightfully criticized and condemned by Hochstetter. Nevertheless it is referred to somewhat favorably by Charpy.
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Krause finds that the cephalic and sciatic veins are analogous (p. 210). Hochstetter denies this, and arrives at the following conclusions. Since the ulnar and fibular borders of the limbs are homologous, the primitive veins which follow them are also homologous. The small saphenous vein and the basilic vein of the forearm, being presumably persistent portions of the primitive veins, are therefore homologous. The cephalic and large saphenous veins are secondary formations, and any comparison between
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118 l)ov('l()|)nuMit of the Veins in the Limbs of Eabbit Embryos
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them is uncertain. The femoral and brachial veins " sliow no a<ii-eement either in position or in origin " (p. ;}-")).
 +
 +
These conclusions clearly depend upon the serial homology of the limbs. If we should accept the idea of inverse homology, advocated by Wilder, WynuTn, and others, according to whom the thumb is comparable with the little toe, and the radial border with the ulnar, then conclusions almost the reverse of Hochstetter's would be expected. A third basis for comparison
 +
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 +
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Stage 1.
 +
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Stage 2.
 +
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Stage
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Diagram 1. Anterior view of the arm and leg in their three stages of venous development. In Stage 1, a and A show the arm and leg. respectively, before rotation; b and B. after rotation. The primitive ulnar and fibular veins are in solid black. The secondary cephalic and sciatic veins are drawn as double lines, and the tertiary brachial and femoral veins have transverse shading. The black lines in contact with the secondary and tertiary vessels indicate the portions of those veins which are formed from the primitive vessels of Stage 1.
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is supplied by the familiar rotation theory. According to it, the limbs are at first serially homologous. The thumb and great toe, the ulnar and fibular borders correspond. The external surfaces of both limbs are to be extensor and the inner surfaces flexor. Later a rotation of approximately 90° occurs in both limbs, but in opposite directions. The extensor surface of the arm becomes posterior, and that of the leg becomes anterior. The knee and elbow are thus brought to bend in opposite directions. The foot is rotated with the leg and its extensor surface
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FredtM-ic- 'V. I^ewis 119
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(dorsum) is directed anteriorly. Tlie hand is not rotated with the arm, but ordinarily in the reverse direction, so that its extensor surface is directed ant(M-iorly like that of tlie foot. Since the arm and hand are rotated in opposite directions, a crossing of the bones of the forearm is produced. In man the liand may, in later development, be rotated with the arm so that its dorsum looks posteriorly and the bones of the forearm are not crossed. In this position the inverse symmetry of the arm and leg is complete.
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The embryonic rotation of the limits is not to be compared witli their voluntary rotation in the adult, for the former is a complex shifting of tissues involving modifications in the shapes of the bones. These changes in the human leg are clearly shown by Bardeen's reconstructions in Vol. 4 of this journal. (Compare Figs. 3, 5, 9, 12, and 13, following p. 303.) The external appearances during rotation may be observed in the rabbit embryos figured by Minot and Taylor for Keibel's Normentafeln. From these it will be seen that rotation does not occur with mathematical precision.
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Interpreted according to the rotation theory, the fundamental veins of the arm correspond with those of the leg. Their homologies are shown in the accompanying diagram which is based upon the reconstructions previously described. The diagram presents throughout anterior views of the left limbs, the veins being drawn as they would appear if the limbs were transparent. In Stage 1, at a and A respectively, the arm and leg are shown before rotation. Serial homology between the primitive ulnar and fibular veins is complete. Then rotation occurs, whereby the fibular vein is carried from the posterior to the outer border of the leg, as shown at B. The arm, on the contrary, turns so that the ulnar vein is carried from the posterior to the inner border, as in h. The forearm rotates in the opposite direction from the upper arm, so that the ulnar vein crosses from the inner side above to the outer side below. Were the forearm in supination, the ulnar border would be internal throughout. After rotation the ulnar border is no longer homologous with the fibular, Imt corresponds with the tibial border.
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In Stage 2, veins are established along the inversely homologous external borders of the liml)s, the radial and fibular respectively. As shown in the diagram, the cephalic vein must be a new formation throughout, but the course of the sciatic vein is already partially occupied by the primitive fibular vein. Consequently the sciatic may incorporate a portion of the fibular vein. Thus it appears that a real homolog)' exists between the cephalic and sciatic veins, although, as Hochstettcr pointed out, they differ in their relations to the ])riniitive veins of the limbs.
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1'20 l)i'vi'l(i|iiiuMit of tlir Veins in tlic Limbs of I^abbit Embryos
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In Stage 3. the brachial and femoi'al veins develop along the inversely homologous nlnnr and tibial borders. In this case the vein of the arm may incorporate the remains of the primitive nlnar vein, as was found to occur in rabbit embryos. The femoral vein on the contrary must be new throughout.
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Thus the primitive ulnar and fibular veins, which develop before rotation, are serially homologous. The veins arising after rotation may be considered inversely homologous, the cephalic with the sciatic, and the brachial with the femoral.
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LITERATURE CITED.
 +
 +
Babdeen, Charles R., 05. — Studies of the Development of the Human Skeleton. Amer. Journ. of Anat., Vol. 4, pp. 265-302. Charpy, a., 98. — Traite d'Anatomie Humaine. Edited by Paul Poirier. Vol.
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2. Paris. Grosser, Otto, 01. — Zur Anatomie und Entwickelungsgeschichte des Gefass systemes der Chiropteren. Anat. Hefte, Abt. 1, Vol. 17, pp. 203-424. Hochstetter, Ferdinand, gi. — Ueber die Entwicklung der Extremitatsvenen
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bei den Amnioten. Morph. Jahrb., Vol. 17, pp. 1-43. Kbause, W., 68. — Die Anatomie des Kaninchens. Leipzig. MiNOT, Charles S., and Taylor, Ewing, 05. — Normentafeln zur Entwicklungs geschichte der Wirbelthiere. Edited by Franz Keibel. Part 5. Jena. Spalteholz, Werner, 01. — Handatlas der Anatomie des Menschen. Vol. 2.
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Leipzig. Wn^DER, Burt G., 71. — Intermembral homologies. Proc. of the Boston Soc.
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of Nat. Hist, Vol. 14, pp. 154-242. Wyman, Jeffries, 67. — On symmetry and homology in limbs. Proc. of the
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Boston Soc. of Nat. Hist., Vol. 11, pp. 246-278.
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===Further Experiments on The Development Of Peripheral Nerves===
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{{Ref-Harrison1906}}
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By
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Ross Granville Harrison.
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From the Anatomical Laboratory of the Johns Hopkins University.
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With Five Figures.
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Two main questions have arisen in connection with the study of the development of the peripheral nerves. The one concerns the constitution of the nerve fiber, i. e., whether it is a process of a single cell or derived from a chain of cells. The other has to do with the manner in which the connection between center and periphery is established, whether there is a continuity ab initio (protoplasmic bridges) or whether the connection is secondarily brought about by outgrowth from the center towards the periphery.
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Prior to the year 1904 all attempts to solve these problems were based on observations made upon successive stages of normal embryos. When one compares the careful analyses of their observations, as given by various authors, one cannot but be convinced of the futility of trying by this method to satisfy everyone that any particular view is correct. The only hope of settling these problems definitely lies, therefore, in experimentation.
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The question of the constitution of the nerve fiber, whether a cell process or a cell chain, may here be considered first.
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If one examines a developing nerve, one sees that there are numerous spindle shaped cells (cells of Schwann, sheath cells) throughout its course, and that these are very closely attached to the young nerve fiber ; on the other hand, it is also found that the nerve is connected with ganglion cells. The disputed point with which we here have to deal concerns primarily the respective roles played by these two kinds of cells in the genesis of the fiber. Some time ago I described a series of experiments "" in which the spindle shaped sheath cells were eliminated by the removal of their source, at least their principal source, in an early embryonic stage, before nerves of any kind are visible. The experiment consisted in removing the ganglion crest. This was done by cutting off a thin strip from the dorsal side of the body of embryos {Rami esculenta) from 2.7 to 3 mm. in length (Fig. 1). Since this operation removes the source of the spinal ganglia also, the embryos develop without sensory nerves and ganglia, but the motor nerves do develop, and instead of being cellular in structure, as is the case in normal specimens (Figs. 2 and 3), they consist of naked fibers, which can be traced in a number of cases as far as the extreme ventral part of the musculature, i. e., as far as the nerves extend in the adult organism (Fig. 4).^
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^ Read before the Association of American Anatomists at the meeting held at Ann Arbor, Mich., December 29, 1905.
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' Harrison, Neue Versuche und Beobachtungen iiber die Entwiclvlung der peripheren Nerven aer Wirbeltiere. Sitzungsber. d. niederrheinischen Ges. f. Natur u. Heilkunde. Bonn, 1904.
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The first experiments were made upon Rana esculenta; they have since been confirmed upon the embryos of two American species, R. syJvatica and R. palustris. These experiments concerned only the spinal nerves.
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Fig. 1. Profile view of frog embryo (Rana esculenta, 2.7 mm. long) at the stage of operation; the line (ab) indicates the incision.
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Last season an attempt was made to corroborate the results in cranial nerves. For this purpose the cranial ganglia, the skin covering the side of the head^ and the dorsal part of the brain were excised from one side of the embryo before closure of the medullary folds. With one exception
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^ The experiment was based upon the assumption that the ganglion crest is the source of the sheath cells. The result of the experiment proves this to be true, as far as the early stages of development are concerned. In certain lower vertebrates, particularly in Elasmobranchs, it has been shown that large numbers of cells are given off from the ventral part of the medullary cord, wandering out along the motor roots of both the cranial and spinal nerves, and giving rise at least in part to the nerve sheaths. The literature bearing upon this subject has recently been considered by Neal (Mark Anniversary .Volume, New York, 1903). In the frog such cells are not given off until the yolk is nearly gone but after this period cells do wander out singly along the motor roots, and in these features the frog embryo resembles closely the salmon (Harrison, Archiv f. mikrosk. Anat., Bd. 57, 1901). The cells do not, however, begin to come off until the motor nerves are well developed and have reached the extreme end of their course. Thus it happens that in the experiment the nerves are developed without sheath cells.
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these experiments gave inconclusive results, as small ganglia were always found later, showing either that their normal rudiment had not been entirely removed, or that they had regenerated from some other source. In one experiment, however, in which the embryo was preserved four days after the operation, an examination of the serial sections revealed no ganglia except several sporadic cells on the n. facialis and n. vagus. These nerves consist of naked fibers, except that several sheath cells are present near their origin. A nerve in front of the facial, probably the oculo-motor, but perhaps the motor part of the trigeminus * is entirely without sheath cells and the naked fibers may be traced from the brain to a mass of mesoderm cells in the region of the eye. The results of this experiment, therefore, confirm the first series, showing that the cranial nerves may develop without the aid of the sheath cells.
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Fig. 2. Profile view of frog larva (Rana palustris, 12 mm. long) after complete resorption of yolk. The relation of the segmental nerves and the primary abdominal muscles are shown.
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But while the sheath cells are thus denionstrated not to be a necessary factor in the formation of the nerve, it may still be urged that they, as well as the ganglion cells, might normally form some of the fibers. During the past year an effort was made to solve this question by studying the behavior of the sheath cells in the absence of processes from the nerve centers. The source of the motor nuclei (ventral half of the medullary cord) was removed from embryos of the same nge as in the previous experiments, leaving the dorsal part of the cord together with the ganglion crest intact. The object was to ascertain whether the sheath
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Owing to the absence of most of the important landmarks on the injured
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side the exact determination of this nerve is doubtful.
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cells from the ganglion crest would be able in the absence of the motor ganglion cells to form the purely motor rami of the spinal nerves. Thereare difficulties in the way of making this experiment because it is first necessary to cut off the dorsal half of the cord, leaving it attached at oneend, then by a second cut to remove the ventral half entirely, and
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Fig. 3. Semidiagrammatic view of the nerves of the abdominal walls of the frog larva (normal specimen). Abel. M., abdominal muscle; HL., rudiment of hind leg; Mot. N., motor branch of segmental nerve running in inscriptiO' tendinea of the primary abdominal muscle; Mot. Nuc, motor nucleus (ventral horn cells) in spinal cord; Seg. N., segmental (spinal) nerve; Sen. N., sensory branch of spinal nerve running to integument outside of muscle; 8p. C, spinal cord; 8p. G., spinal ganglion.
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finally to heal the first strip, which is very thin, l)aek in place. Even if this is done successfully, a scar is loft, which lies in the path of the spinal nerves, and which no doubt serves as a hindrance to their development. Again it seems to be practically impossible to remove entirely the motor elements from all regions of the cord. After several days the larva?, although almost completely paralyzed, regain some power of movement, showing usually a slight tremor in some part of their axial musculature, when stimulated mechanically. Sections show that in some
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Fig. 4. Semidiagrammatic view of the nerves of the abdominal walls of a frog larva from which the ganglion crest had been removed as shown in Fig. 1. Only motor nerves are present and these consist of axis cylinders without sheath cells.
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segments very fine motor roots are present, and the ventral part of the remaining medullary cord contains in these regions a few large motor •cells. These motor fibers supply, as the movements indicate, merely that part of the musculature lying close to the spinal cord. With the two exceptions below noted, no motor fibers whatever were found in the
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12(5 Experiint'iits on tlu^ Dcvolopinciil dI' I'tM'iphci'iil Xcrves
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abdominal walls, which were used especially for study, because it is only there that one can distinguish clearly between motor and sensory ninu ( Fig. 3 ) . The results of ten ' experiments were as follows : In seven cases sensory nerves were found in the abdominal walls, but no motor (Fig. 5), althougli the sheath cells, as shown particularly in one case, were in very
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Fig. 5. Semidiagrammatic view of the nerves of the abdominal walls of a frog larva from which the ventral half of the spinal cord had been removed at the stage represented in Fig. 1. Absence of the purely motor rami, which normally run in the inscriptiones tendineas.
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close proximity to the point where the terminal motor rami normally arise; often, however, the sensory nerves were not so well developed as in
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^ Each side of each individual specimen is counted as a case, because, as far as the factors in the experiment are concerned, the two sides of the body are mutually independent.
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normal specimens, in two cases motor as well as sensory nerves were fonnd; once in one, and once in two seo^ments, tlioiiol-, in other segments only sensory nerves were present. Here the motor nnclei had been less completely removed than in the other cases. In one case neither sensoiT nor motor branches were found. The last named case is to be explained as due to imperfect union of the parts, as is also the fact that in other cases the sensory nerves were often scantily developed. The results of these experiments show, therefore, that durin'g the period in which the specimens were kept imde.r observation, the sheath cells are unable by themselves to form nerve fibers. The negative character of the result renders it necessary, however, to secure further cases befQre this conclusion can be regarded as established beyond all question. Should it be nrged that time enongh was not given the sheath cells to form the nerves, it may be pointed ont that in normal specimens the motor fibers develop at a much earlier stage and that if the sheath cells nonnally contribute to their formation they should unquestionably act in the period allotted. The purpose of the experiment was to determine tl^- normal behavior of the cells and not any possible regulative action on thir part, which might take place later.
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The above experiments deal only with the motor nerves, and it has not been found practicable to experiment systematically with the sensory nerves because in the latter the ganglion and the sheath cells have a common place of origin. In studying the normal development of the sensory nerves in the amphibian embryos, we find important evidence bearing upon the question. For instance, the nerves derived from the dorsal (giant) cells of Eohon-Beard are formed without sheath cells. These fibers consist, in fact, of naked axis cylinders, wdiich branch and form a delicate plexus of nerves under the skin of the frog larva, and are entirely devoid of cells (or nuclei) . Again in the Triton larva, even some of the nerves derived from the spinal ganglia of the tail are for a short time devoid of sheath cells; these, together with the nerves from the dorsal cells form a non-cellular plexus in the fin folds." In the frog larva the nerves derived from the spinal ganglia have sheath cells from the beginning. Comparison of these instances show that these cells are a variable element in the voung nerve fiber: it may therefore, be concluded that they play no necessary part in
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« Since this fact was disputed by O. Schultze (Archiv f. mikrosk. Anat., Bd. 66, 1905, p. 68), I have again examined the specimens in question and have nothing'to correct in my former statement. It may be added, however, that I did not intend to include the n. lateralis, which is independent of the cutaneous plexus. This nerve of course has sheath cells at this stage.
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its formation. In addition to these noi'inal eases there are several experiments at hand, whieli show that even in the froo; embryo the spinal ganglion cells are by themselves capable of forming long peripheral nerve fibers. The cases in question are those in which relatively small fragments of ganglia had been dislocated or transplanted. In one such case four ganglion cells, transplanted to the abdominal wall, were found giving rise to a long nerve, which ran free through the peritoneal cavity of the larva. This nerve consisted solely of bundles of fibrillae without cells and could be traced for a distance of nearly two millimeters.
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These results differ from those recently reported by 0. Schultze (Op. cit.) based also upon the study of the amphibian larva. It is not possible to discuss this work in detail here, but it may be pointed out that by confining his studies to relatively late stages, Schultze has missed the early and fundamental phases of development and thus is led to consider the purely secondary connections of the sheath cells with the nerve fibers as a primary genetic relation.
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We may now take up the second great question, viz., the origin of the connection between ganglion cell and end organ. According to the one view a protoplasmic process grows out from the ganglion cell, makes its way through tissues and ultimately reaches its end organ, gradually differentiating into a nerve fiber. According to the second view (Hensen's ' hypothesis) protoplasmic connections remain between cells after division; those that are used, i. e., that function as conducting paths, persist and differentiate into nerves, the remainder disappearing.
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According to Hensen's hypothesis the nerve paths are thus developed much earlier than they seem to be, and they are present for some time before they become visible. If we consider the embryo of a stage just before the nerves do become visible, then the two theories might be distinguished as follows : according to the one, the center (ganglion cells) is the all important factor in forming the nerve; according to the other the nerve is formed in situ in the peripheral path. This difference affords the basis for experimentation, though unfortunately the distinction is not so clearly cut as could be desired, for the first view does not deny the importance of the periphery in forming paths along which the developing nerve grows, nor does the second altogether disclaim the influence of the ganglion cell upon the differentiation of the primitive protoplasmic connections into nerve fibers.
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The first set of experiments consisted in the extirpation of the center.
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' Virchow's Archiv, Bd. XXXI, 1864. Die Entwickelungsmechanik der Nervenbahnen im Embryo der Saugetiere. Kiel und Leipzig, 1903.
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This was clone by removing the medullary cord of the trunk shortly after its closure. The result is always the total absence of peripheral nerves, ■except the cranial. In the second set of experiments the peripheral path was altered. The simplest way to accomplish this is to remove the spinal €ord before any nerves are visible. After this the wound heals readily and during the next week at least no regeneration takes place. Above the notochord in the trunk of the embryo there is thus left a small space which becomes filled with mesenchyme. Into this the longitudinal bundle fibers arising in the brain grow, and after a few days they may be followed as far as six or eight segments from the cut end of the medullary tube. In other words, fibers which normally develop in the walls of the latter, develop here within the mesenchyme, which is a tissue as unlike that forming the normal path as it could possibly be.
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The third mode of experimentation, which is not formally different from the preceding, consisted in the transplantation of parts of the central organ. In one series of experiments the spinal cord of the embryo was extirpated, and in each case a small piece of the cord was transplanted under the skin of the abdominal walls. The normal nerves of the body ■of course do not develop in such cases, but small nerve trunks do arise from the transplanted pieces and run for some distance in various directions, usually remaining in the abdominal walls. Sometimes portions of the ganglion crest were transplanted with the cord, resulting in the formation of small ganglia. In one of these instances, already referred to •above, the nerve fibers, which were sheathless, ran free throu,gh the peritoneal cavity. While the great length of this nerve is due largely no doubt to the shifting of its peripheral attachment, it is nevertheless quite im|)ossible that preformed bridges could have been present in its course.
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The foregoing results can be interpreted in but one way. The nerve •center (ganglion cells) is shown to be the one necessary factor in the formation of the peripheral nerve. When the former is removed from the body of the embryo the latter fails to develop. When it is transplanted to abnormal positions in the body of the embryo it then gives rise to nerves which may follow paths, where normally no nerves rim, and likewise when the tissues surrounding the center are changed entirely, nerves proceeding from tliat center may develop as normally. The nerve fil)cr is therefore a product of the ganglion cell. The histological findings indicate that it is an outflow of the substance of the ganglion cell and not 3. more activation by contact of indifferent extra ganglionic substance.
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While Lewis's ' experiments upon the olfactory and optic nerves affording nq^ortant atldilional evidence I'or tliis view, the conclusion of Brans," who was the lii'st to exjieriment upon this phase of the question of iiei've clevelo])ment, are diametrically opposed to it. Ilraus interprets his results in accordance with Hensen's hypothesis. AVhile one cannot but admire his ingenuity of experimentation and argument, his results are not, in my opinion, in any way inconsistent with the outgrowth theory. The growth of strange (facial or pelvic) nerves into a transplanted fore limb can be accounted for on the assumption, for wliich there is good evidence, that the configuration of the various organs and tissues plays an important part in determining the course taken by growing nerve fibers. The failure of the nerves of the host to grow into " aneurogenic " buds, while they do grow into " euneurogenic " transplantations, might be due to the absence of the attraction afforded in the latter by the cut ends of the nerves." The large size of the nerves in the transplanted limb as com])ared with the nerves connecting them with the center, may be due partially to the presence of the sheath cells transplanted with the bud, and partially to an abnormal number of dividing fibers. Braus does not exclude beyond doul)t the possibility of the latter. In any case the evidence for autogeneration of fibers could be regarded as crucial only if nerves having no nervous connection whatever with the center are developed in the transplanted part. This condition Braus has failed to demonstrate. While the facts necessitate our deciding against the validity of Hensen's view, as far as the question of primary continuity is concerned, it should be pointed out before closing that this view is in so far correct as in many instances the nervous connection between center and end organs is established when the two are very close together, and the long nerve paths originate in such cases by the moving apart of center and innervated organ after the establishment of the connection. The best example of this is seen in the lateral line. Here the ganglion is practically in contact with the rudiment of the sense organs when the first nerves are developed. The cell processes have merely to grow out for a distance less than the diameter of a cell in order to make connection. Yet by the wandering of the sensory epithelium from the head to the tip of the tail the lateral branch of the vagus is ultimately drawn out to this enormous length. The observations of Kerr" upon' the motor nerves of Lepidosiren are, in my opinion, capable of a similar interpretation and are a valid support of Hensen's view only in the above modified sense. In other ^^'ords, the nervous connection, though formed very early, is by no means primary.
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^ W. H. Lewis, Proceedings Ass. Am. Anatomists. Am. Joiirn. of Anat., Vol. V, No. 2, 1906.
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" H. Braus, Verhandl. d. Anatom. Gesell., Jena, 1904. Anatom. Anz.. Bd. XXVI, 1905.
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'"Forssman (Ziegler's Beitrage, Bd. 24, 189S, and Bd. 27, 1900), lias shown beyond question that a tropism of this kind does play an important part in the regeneration of peripheral nerves.
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The results of the foregoing may be summarized as follows : The axis cylinder of the nerve fiber is the outgrowth of a single ganglion cell, with ■which it remains in continuity throughout life. It grows gradually from the center towards the periphery establishing secondarily connection with its end organ. The other elements, the cells of Schwann, which are found upon the developing nerve have nothing to do with its genesis, though they may play an important part in the nutrition and protection of the fibers.
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"J. Graham Kerr, Trans. Roy. Soc. Edinburgh, Vol. XLI, 1904.
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===The Gastrulation And Embryo Formation in Amia Calva===
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By
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Albert C. Eycleshymer And James Meredith Wilson.
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From the Anatomical Laboratory of St. Louis University. With 4 Double Plates.
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In an earlier paper entitled " The Egg of Amia and Its Cleavage,"^ Whitman and Eycleshymer, 96, described the development from the time of fertilization np to and inchiding the late blastula. The present paper is a continnation of the earlier study, and deals with the changes taking place between the late blastula and the time when the tail of the embryo becomes free from the yolk ; that is, from the time of the late blastula until the time when most of the organs are laid down.
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The material was killed in Flemming's fluid, Perenyi's fluid, chromosmic acid, picro-acetic acid, picro-snlphuric acid and corrosive sul)limate-acetic acid. For surface views, chrom-osmic acid gives most perfect pictures; the osmic acid blackens the lines of cleavage so that they stand out in bold relief. Another excellent method for surface study is faint staining with Delafield's hsematoxylin w^hich may be employed after any of the above-named fixing solutions. The best serial sections have been obtained after fixation in picro-acetic acid. Owing to the crumbling of the yolk we have been compelled to use celloidin as an imbedding mass. Serial sections were made after the method described elsewhere by the senior author, 91. Staining in toto is best accomplished by using Czoker's alum-cochineal for from twenty-four to forty-eight hours. Staining in section with Mayer's haemalum and alcoholic carmine has proved very satisfactory.
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So far as the writers are aware, but two papers have been published dealing with the phases of development under consideration. Both of these appeared in 1896. The first was published by Sobotta and contains a fairly accurate, but incomplete, description of the gastrulation stages. The illustrations, however, are few and highly diagrammatic. The second was written by Bashford Dean and is more extended, but less accurate. Dean's descriptions unfortunately were based upon the erronc American Jocrxal of Anatomy. — Vol. V.
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134 Gastriilation and iMiihrvo Formation in Amia Calva
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oils assumption tiiat the egg' of Amia is meroblastic. In view of these facts, the present writers have thought that a renewed study of these phases of development might bo profitable.
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The ages given in the following descrijition of stages have been determined from material taken from a single nest. The eggs were taken from the nest and placed in dishes which were submerged in the lake, a constant temperature of about 16° C. being thus maintained. It is well known that no two spawnings progress at precisely the same rate. It is thus obvious that the ages designated are only in a general way indicative of the degree of development. We have, therefore, given measurements of the extent of the blastodisc and embryo in addition to the age.
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The description of the latest stage studied by Whitman and Bycleshymer reads as follows : " The calotte, which has now begun to extend over the yolk, consists of thickly crowded spherical cells which marginally pass abruptly into the large yolk segments, while in the central portion they gradually increase in size and lie loosely scattered. The outer layer of the calotte is distinctly differentiated in that the cells are elongated and more densely granular. The entire yolk is irregularly cleft, the cells forming the lower portion are roughly polygonal and grade oif into the larger yolk spheres which lie at the center." This stage of development indicates the beginning of our study.
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DETAILED DESCRIPTION OF STAGES.
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Egg Nine Hours After Fertilization. Blastodisc Covers About 100° of the Circumference of the Egg. — A profile view of an egg of this age is shown in Fig. 1. An examination of the surface of the blastodisc shows an area at the upper pole of the egg in which cell division is most rapid. In addition to this, there are frequently found other areas in which cell division is accelerated. Often one side of the blastodisc is distinctly in advance of the other. Again, the most careful search results in a failure to detect such areas. We, therefore, are unable to say what relation, if an}^, these areas bear to the future embryo.
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In all eggs of this stage the surface of the yolk is cleft by thirty to forty furrows which pass in meridional planes. Many of these grooves have not as yet reached the vegetative pole. Some never reach the pole, but pass obliquely into the longer ones. Through this process a number of long triangular segments are cut off at the upper margin of the yolk, as shown in the figure. At the lower pole, where fifteen to twenty grooves converge, the yolk is irregularly cleft. In general it may be said that the cleavage of the yolk as compared with the cleavage of the blastodisc is exceedinglv slow.
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Albert C. Eyclesbymcr and James Meredith Wilson 135
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A study of ineridioual sections of many eggs in this stage shows that the blastodisc takes on different forms. In most eggs it is distinctly crescentic, but in some it is lenticular. When it takes on the crescentic form, as shown in Fig. 21, there is often a very distinct segmentation cavity (s. c.) present. The roof of the cavity is here made up of five or six layers of cells. The cells of the blastodisc contain finer granules than those contained in the large yolk segments. At the margin of the blastodisc, the cells pass over into those of the yolk by such imperceptible gradations that no sharp line of demarcation can be seen. The outermost layer of the blastodisc may be designated as the superficial layer of the ectoblast (s. ec.) and as stated by Whitman and Eycleshymer it early appears quite unlike the deeper ectoblastic layers {d. ec), in that it possesses granules which stain more intensely than those in the other layers.
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The upper ends of the 3^olk masses (y. m .) are, in the egg shown, smooth and only at the margin are the cells being cut off. Other eggs, however, show that the large yolk masses at the center of the egg are actively contributing to the blastodisc. In the section shown (Fig. 21) the yolk nuclei lie near the upper margin of the large masses and this upper portion is probably to be considered as homologous with the periblast of bony fishes. Not more than one-third of the cleavage grooves observed on the surface of the yolk have reached the center of the Cigg, leaving the yolk masses incompletely cleft and thus forming a great syncytium.
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Egg Twelve Hours After Fertilization. Blastodisc Covers About 110°. — The surface view (Fig. 2) shows that the rapid multiplication of the cells in the margin of the blastodisc has now given rise to greater uniformity in the size of all the cells of the blastodisc. Beyond this feature, surface views show no points worthy of special mention. Meridional sections (Fig. 22) show that the cells forming the superficial ectol)last {s. ec.) are smaller and more elongated than in the preceding stage. The lower layers of cells of the blastodisc are scattered through the upper portion of the segmentation cavity. The entoblastic cells, which have been cut off from the large yolk masses, are distinguished by their coarser granules and are also scattered through the segmentation cavity, many of them being found in its upper portion. Through these changes the segmentation cavity is more or less obscured.
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Egg Twenty Hours After Fertilization. Blastodisc Covering About 120°. — An egg of this stage (Fig. 3) shows a well-defined blastodisc with a sharply delimited margin in which, under the magnification given, cell boundaries are no longer distinguishable. No features have been observed which enable us to recognize the embryonic anlage. The yolk shows little advance in cleavage beyond. that described in the preceding
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136 Castriiliition and Embryo Foniiatioii in Amia Calva
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stage. In this particular egg the grooves, instead of following meridional lines as usual, diverge more widely than those in the eggs shown in Figs. 1 and 2.
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Sections show, although none are figured, that the blastodisc in this stage is made up of eight to ten layers of cells which gradually pass over into the yolk derivatives. The outermost layer of the blastodisc has undergone still further modification in that its cells are more elongated,, closely apposed, and more deeply stained. The large yolk masses are actively budding off cells not only around the margin of the blastodisc but also in the central portion of the yolk. The yolk nuclei, which in the earlier cleavage stages, were confined to the upper portion of the yolk masses are now frequently found more deeply situated.
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Egg Forty Hours After Fertilization. Blastodisc Covers 130°.-^Although the surface of the egg, as shown in Fig 4, presents no features worthy of special comment, changes are going on in. its interior which merit consideration. If a meridional section of an egg in approximately the same stage (Fig. 23) be examined it wall be seen that the blastodisc proper is made up of from ten to twelve layers of cells so closely apposed that they make a compact stratum. In addition to these cells, there is a large number of loosely scattered cells which lie in the space which we have hitherto designated as the segmentation cavity. These loosely scattered cells gradually pass over on the one hand into the cells of the blastodisc proper and on the other into the large yolk masses. The cells of the ten or twelve layers forming the upper portion are smaller, more uniform, more closely compacted and contain very fine granules as shown in the figure; while the loosely scattered yolk cells and those being budded off from the large yolk masses are larger, more irregular in outline and contain coarser yolk granules. These two portions cannot be considered as sharply marking off ectoblast and entoblast, since one finds in the portion which is largely ectoblastic, large cells which are filled with coarse granules; and if granules be the criterion for the separation of layers these cells must be regarded as recent derivatives from the large yolk cells which have wandered up from the lower portion of the blastodisc. If this interpretation be correct, it is a fact of some importance, since the part hitherto considered as exchisivehj ectoblast contains a considerable number of cells derived from the large yolk masses.
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It has already been pointed out that the outermost layer of the ectoblast ( s. ec.) can be readily distinguished from the underlying layers. A glance at Fig. 23 shows that in the locality where this layer passes over into the large yolk cells there is a marked proliferation of its elements. A study of the remaining sections shows that as yet there is no invagination.
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Albert C. Eycleshynicr and James Meredith Wilson 137
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This thickening of the superficial layer marks the anlage of the forthcoming dorsal lip (d. 1.) of the blastopore. The margin of the blastodisc in the embryonic region has a more rounded contour than at the opposite margin. It is also thicker and possesses a greater number of cells with fine granules; the periblast, too, is more actively engaged in budding off derivatives in this region than at the opposite margin. These several factors enable us at this time to orient the forthcoming embryo.
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The so-called periblastic nuclei are no longer confined to the upper margin of the large yolk masses, but are often widely scattered. These yolk masses sometimes contain several nuclei and the same is true of the scattered entoblastic cells. In other words, nuclear division here goes on far in advance of cytoplasmic division.
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Egg Fifty Hours After Fertilization. Blastodisc Covers Adout 180° . — If the surface of the egg be carefully examined in a stage intermediate between Figs. 4; and 5, it will be found that just above the equator on the side of the blastodisc which is least transparent there is a slight indentation w^hich indicates the beginning of the blastopore. As development progresses this indentation becomes a groove which extends in a latitudinal plane until it reaches the condition shown in Fig. 5, where it extends around some 20° of the egg's equatorial circumference.
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The meridional section represented in Fig. 24 is from an egg intermediate between those represented in Figs. 4 and 5. At this time, the blastodisc has taken a more definite form owing to the greater compactness of its layers. In extent it covers very nearly one-half of the egg's surface. It is noteworthy that at the time the blastopore appears the blastodisc reaches its maximal thickness.
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In the particular egg described, the entoblastic cells are less densely aggregated than usual, with very large intercellular spaces, while the large yolk masses extend well up towards the lower layers of the blastodisc. In this respect we find considerable variation. In some eggs they are even less densely aggregated than shown in Fig. 24 so that a well-marked segmentation cavity {s. c.) is shown between the large yolk masses and the blastodisc.
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In the stage under consideration we have the first actual appearance of the invagination to form the archenteron. As to the factors which initiate this process, we are as much in the dark as ever. Without attempting to discuss the various theories, we may simply say that thus far there is an infolding of the external layer and that this infolding is not in the locality where the transition between large and small cells is most abrupt, but in a locality where the superficial cells are largest and of fairly uniform character. One would at first, glance think the inlo"
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138 Gastrulaiion and l^mbryo Formation in Aiiiia Calva
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vauination weiv wholly in the })art of the surface layer which belongs to the epiblast. A conipai'ison with other forms, liowever, such as the various Amphibia, leads one to hesitate in such an interpretation. The crucial factor is the determination of the limits of the ectoblast. If the ectoblast be considered as extending to the point where the smaller cells pass over into the large yolk masses, then the invagination is in the ectoblast. If it does not extend to this point, there are no features which will enable us to determine how far it does extend.
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A more highly magnified view of the blastoporic region is shown in Fig. 25. The section is taken from an egg of the same age as that shown in Fig. 24. In many eggs of this stage, there is a stratum or tongue of cells wdiich is somewhat peculiar. This stratum is directly continuous with the deep ectoblast at the dorsal lip of the blastopore. Anteriorly its cells are separated from the deep ectoblast by the segmentation cavity above, while below they pass over into the entoblastic cells. In this stratum which is from four to five layers thick, two kinds of cells are present. The more numerous are cells which conform in structural peculiarities to those of the deep ectoblast. The less numerous are cells which possess the structural features of the entoblast. This layer of cells Sobotta, 96, has described as mesoblast. Since this layer not only contains mesoblast but also entoblast, we have decided to designate the layer as mes-entoblast
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In Fig. 26 there is represented a meridional section of an egg somewliat older than that just described, but younger than that shown in surface view in Fig. 5. The archenteron or gastral cavity is more extended and its dorsal wall is formed of cells which are so much like those of the superficial ectoblast that one is inclined to regard invagination as still playing the more important role. The rounded cells at the end of the gastral cavity are further confirmation. In short it may be said that thus far there are no reasons for considering delamination as a factor of any importance in the formation of the gastral cavity.
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No particular changes are noticed in the character of the cells in the region w^here the head of the embryo is about to appear. The yolk derivatives are widely scattered in the segmentation cavity and many cells which, from the character of their granules, would be called yolk derivatives are still to be found scattered among the ectoblast cells. The large yolk masses are still actively budding off cells and this process has gone on so rapidly in this particular egg that these masses have become greatly reduced in size.
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Egg Fifty-three Hours After Fertilization. BJnstodisc Covers About 200° . The anlage of the cmbrvo can now he faintlv recognized in sur
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Albert C. Eycleshymer and James Meredith Wilson 139
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face view (Fig. 5). It first appears as a light area with ill-defined outlines extending over some '80° towards the upper pole, where it shades off imperceptibly into the remainder of the blastodisc. That portion which is invaginated to form the dorsal lip of the blastopore stands out more prominently from the yolk than elsewhere. The line of invagination now extends some 60° along the margin of the blastodisc and appears as a crescentic fissure.
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A meridional section of an egg in a stage closely corresponding to that described above is represented in Fig. 37. Although this egg is but slightly older than the one shown in Fi,g. 26, some interesting changes have occurred. In the preceding stage the deeper ectoblast was seven or eight layers of cells thick; now it is only two or three. The embryonic margin of the blastodisc has likewise undergone a reduction, while the opposite side of the blastodisc is reduced to one-half the number of layers present in the preceding stage. In addition to these changes, the stratum of cells which we have designated as mes-entoblast extends well up toward the upper pole of the egg and it is probably through the extension of this layer tliat the surface views show faintly the anlage of the embryo.
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In most eggs in this and subsequent stages, there are relatively few entoblastic cells as compared with the earlier stages. The space which in most of the earlier stages was filled with small cells is now filled with large yolk masses with a few smaller cells scattered among them. It is possible that many of the entoblastic cells have found their way into the rapidly extending blastodisc.
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Fig. 28 represents a section of the blastoporic end of the embryo under much higlier magnification. The section is taken from another egg in about the same stage of development as that shown in Fig. 27. It will be here noted that the gastral cavity is lined above by a single layer of cells which strikingly resemble those of the superficial ectoblast in size, granular contents and staining capacity. The same can be said of the cells forming the ventral wall. The cells of this wall rest in this section upon the large yolk masses whose margins are regular and clearly delimited. A peculiar feature which was noticed in the preceding stage, but is here more clearly shown, is the striking differentiation of the innermost layer of the deep ectoblast. These cells stain more deeply than the remaining cells of this stratum.
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Fig. 29 represents a meridional section of an egg in a stage somewhat later than that last described, but earlier than the stage shown in Fig. 6. The blastodisc has undergone continual thinning at the upper pole until at present it is but two or three layers of cells thick. At the blastoporic margin the blastodisc is thickened, while on the opposite side of the egg
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140 Gastrulation and Embryo Formation in Amia Calva
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a slighter thickening of the margin gives rise to a condition which reminds one of the germ-ring of the teleost.
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An equatorial section, taken just al)0ve the equator of an egg in the same stage, is shown in Fig. 30. The ectoblast in the embryonic region is much thicker than elsewhere, and from this region of greatest thickness it shades off gradually on either side, showing that at this time there are no well-defined lateral boundaries of the embryonic aniage. Just beneatli the median portion of the embryonic aniage there is a compact arrangement of the mes-entoblastic cells which represents the beginning of the notochord (ch).
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Egg Fifty-five Hours After Fertilization. Blastodisc Covers About 2U0° . Embryo Extends Over 110°-120°. — The embryo now presents a profile (Fig. 6) which may be spoken of as somewhat triangular. Its anterior portion fades out in the region of the upper pole of the egg. Its .posterior portion, however, is more sharply defined owing to its being deeply infolded at the blastoporic margin. In many embryos of this stage, there is present a median thickening in the blastoporic margin which may doubtless be considered as the homologue of the caudal knob of the teleosts. The lower portion of an egg in about the same stage is shown in Fig. 7. It wall be noticed that the margin of the blastodisc is not only deeply infolded along the base of the embryo, but also slightly infolded on the opposite side of the egg. A comparison of Figs. 6 and 7 with Fig. 5 shows that the surface cleavage of the yolk is very slow\
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A meridional section of an egg in this stage is shown in Fig. 31. The embryonic aniage here shows as a thickening of the ectoblast. The area of maximal thickness {li) near the upper pole of the egg represents the anla,ge of the head. In this region the superficial ectoblast shows no changes, the thickening being due to the proliferation of the deeper ectoblast which is now twelve to fourteen layers thick as compared with six to eight in the preceding stage. The deeper layers decrease in number throughout the anterior trunk region and again increase at the blastoporic margin. In front of the aniage of the head (/;), the deep ectoblast becomes thinner until, in the region of the equator, it is but a single layer thick; beyond this region it again thickens and at the lilastoporic margin is three or four layers deep.
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The segmentation cavity, which in the preceding stage extended over the greater portion of the upper hemisphere, is no longer present above the level of the equator. The la^-er of ectoblastic cells forming its roof is still sharply diffrentiated from the other layers of the ectoblast.
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The gastral cavity {g. c.) has extended cephalad to the level of the posterior third of the embryo. Behind the dorsal lip of the blastopore, it
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Albert C. Eycleshymer and James Meredith Wilson 1-il
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extends around on either side of and behind the large yolk plug where it is continuous with that part of the cavity which is everywhere lined externally by a sharply differentiated layer of hypoblast. At the blastoporic margin, this layer of hypoblastic cells changes in character from the small elongated cells with deeply staining granules to larger cuboidal cells and these in turn shade off into the smaller elongated cells of the superficial ectoblast. The floor of the anterior portion of the gastral cavity is made up of entoblastic cells which are heavily laden with large yolk granules. Toward the exterior these cells increase in size as they extend over the sides of the yolk plug until they finally become continuous with the great yolk masses (//. m.)
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The laj'ers of mes-entoblast (m. en.) not only extend much farther forward in the embryonic region but also become well differentiated in the extra-embryonic portion of the blastodisc. By tracing these layers in serial sections it is readily found that the anlage of the mesoblast is peristomal.
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Embryo Sixty Hours After Fertilization. Blastodisc Covers About 2Jt5°. Embryo Extends Over 130° . — The outline of the embryo as yet is indistinct in the anterior region, but fairly well defined posteriorly (Fig. 8). The entire margin of the blastodisc is deeply infolded around the projecting yolk plug. In the posterior portion of the embryo, there is a shallow groove present. A comparison with other embryos in this same stage shows that this groove is variable, being sometimes more and sometimes less pronounced. Sometimes it terminates posteriorly in a deep indentation in the margin of the blastopore much like the condition observed in Batrachus or Ameiurus; at other times there is a well-defined caudal knob.
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The sagittal section represented by Fig. 32 is frdm an Qgg in the same stage. The deep ectoblast in the head region is notably thickened, being now twelve to sixteen layers in dorso-ventral thickness. In the trunk region these layers are further reduced while at the l^lastopore they remain practically unchangd. Anterior to the region of maximal thickness the deep ectoblast gradually thins until, as in the preceding stage, it is but one or two layers thick in the equatorial region; finally at the ventral lip there are four or five layers.
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The mes-entoblast has extended farther toward the upper pole, but to just what extent it is impossible to say since the cells are here indistinguishable, on the one hand, from those of the deep ectoblast, and on the other, from those of the yolk. At the blastoporic margin where the cells of the mes-entoblast and the deep ectoblast unite, they form a sharp angle. In this angle there now appears a peculiar group of cells
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142 Gastriilation and Embryo Formation in Ainia Calva
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wliieli has been derived from the mes-entoderm. We were at hrst inclined to regard these cells as exclusively mesodermal bnt since they later lose their distinctive character the question cannot be definitely settled.
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A sagittal section of an embryo slightly later than the preceding is shown in Fig. 33. The principal changes are the further extension of the blastodisc and the corresponding reduction in the diameter of the yolk plug. The peculiar differentiation of the inner layer of the deep ectoblast is here prominent. The segmentation cavity is vanishing, the gastral cavity enlarging. The yolk is being rapidly segmented, especially at its periphery.
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Embryo Sixty-five Hours After Fertilization. Blastodisc Covers About 355° . Embryo Extends Over About lJfO°. — The surface view of an egg in this stage is represented by Fig. 9. The embryo is now much better defined. The anterior portion is somewhat broader than the trunk, whicli in turn becomes narrowed towards the blastoporic end. The blastopore is almost closed. In its closure one rarely finds the condition so frequently found in the amphibia where the lateral lips approximate so much faster than tlie dorsal and ventral that a slit-like blastopore arises.
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Embryo Seventy Hours After Fertilization. Embryo Extends Over About 154°. The next surface view (Fig. 10) represents an embryo about five hours older than that shown in Fig. 9. The features noted in addition to those described in the preceding stage are the further elongation of the embryo; the presence of a well-marked neural trench; the further closure of the blastopore. At this time there are no external evidences of optic vesicles, protovertebra?, or pronephric ducts.
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A sagittal section of the posterior portion of an embryo in this stage is shown in Fig. 34. At this time the blastopore is nearly closed. The external epiblast, as in the earlier stages, is a single layer of cells which still retain their peculiar coarse granules and deep staining capacity. These cells are in direct continuity wdth the single layer of cuboidal cells liniriig the blastopore. These cuboidal cells in turn pass over into the elongated layer of hypoblastic cells which form the dorsal wall of the gastral cavity. The floor of the gastral cavity is made up for the most part of a single layer of entodermal cells. The appendicular portion of the gut {a. g.) is lined by cells similar to those just described.
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A transverse section through the blastopore of an embryo in the same stage is shown in Fig. 35. The relation of the lavers is here more clearlv shown. It will be noted that the section shows especially w^ell the gi*eat lateral sheets of mesoblast (mes.)
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A median sagittal section of an embryo a few hours later is shown in Figs. 36 and 37. The anterior and middle portions are shown in Fig. 36,
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Albert C. Eycleshymer and James Mereditli Wilson 143
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the poslci'ior in Fig. M . The general contour of the embryo shows some ailvancc Ijeyoml the conditions shown in Figs. 34 and ST). The head region shows a considerable increase in the number of ectoblastic cells, in the trunk region but two or three layers are present, while posteriorly they again show a marked increase in number. Just beneath the superficial ectoblast {s. ec), there is now differentiated a second layer of elongated cells. These cells, however, possess granules which are similar in staining capacity to those of the deep ectoblast and have thus been considered as derivatives from the deeper layer.
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In the mass of cells which makes up the anlage of the future brain, there is now observed a slight cavity (hr. c.) which is the first appearance of a cavity in the central nervous system. In front of this mass of cells is a second thickening which has been designated as the pre-cerebral mass (p. ch).
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The notochord (cJi.) is now well differentiated, being readily distinguished from the surrounding tissues by the loosely scattered arrangement of its cells. It extends from the undifferentiated caudal mass of cells to the anlage of the future optic vesicles.
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The gut has increased through both forward and lateral extension. In its anterior portion its dorsal and ventral walls are closely apposed, yet they can be readily traced as distinct layers to a point somewhat beyond the anterior end of the brain. In its middle and posterior portions it is widely open. Just anterior to the line of large yolk cells (hi.) which represent the closed blastopore, there is a dorsal diverticulum of the gut which has been regarded by others, as well as ourselves, as the homologue of Kupffer's vesicle.
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Emhri/o Seventy-five Hows After Fertilization. Emiryo Covers About 150°. — In Figs. 11 and 12 are represented, the anterior and posterior portions of an embryo of this age. The anterior trunk region is narrower and two or three protovertebrge are now present. Lateral thickenings at the anterior end represent the beginnings of the optic vesicles. On either side of the anterior end, there is a darkened area wliicli represents the lateral extension of the mesoblast.
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An oblique section through the optic thickenings (op. t.) is shown in Fig. 38. The superficial ectoblast which is now double layered passes over these thickenings unmodified. Xo lumen is present in the central nervous system at this level, but in sections intermediate between those represented in Figs. 38 and 39 there is a slight fissure present. The mesoblast shows as two wide lateral bands (mr.s.) on either side of the neural rod or keel. The foregut is present, but the close approximation of its walls makes its lumen obscure.
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144 Gastrulalion and ]Miil)rv() Fonnalion in Amia Calva
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Another section of the same enibrvo thi'onuii the posterior portion of the Innd l)rain is represented in Fig. 'A\). On either side of the neural keel and in close proximity to its dorso-lateral margin, there are deeply staining groups of cells which are probably spinal ganglia; although it should be said that in some preparations they appear to be proliferations of the inner layer of the superficial ectoblast. The notochord (nc.) is well differentiated at this level. Beneath it the layers forming the walls of the gut are in contact so that the lumen is here obscured. On either side of the notochord, however, the layers separate and the laterally extending gut cavity (g. c.) is obvious.
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Another transverse section at the level of Kupffer's vesicle is represented in Fig. 40. In the median line there is a groove in the superficial epiblast (n. t.) which we have interpreted as a neural trench. It extends backward to the point where the scattered coarsely granular cells indicate the line of closure of the blastopore (cf. Fig. 37). The deeper epiblast has not yet taken on the form of a neural keel, but extends laterally to a considerable distance. The notochord is well differentiated and consists of cells Avhose character lends confirmation to the view that they are derived from the mesoderm rather than the gut hypoblast. At any rate we have not observed the coarsely granular cells of the hypoblast participating in its formation.
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The section represented by Fig. 41 is taken through the posterior portion of an embryo of about the same age. In this embryo a deeper neural trench (n. t.) is present than in the preceding. The posterior end of the notochord, as it passes over into the mass of undifferentiated cells is barely defined by the peculiar arrangement of its cells. Kupffer's vesicle is smaller than in the preceding embryo. In this structure there are wide variations in size as may be inferred by glancing at the different figures.
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Embryo Eighty Hours After Fertilization. Embryo Covers About 160°. — The surface views (Figs. 13 and 14) show that the embryo is considerably advanced beyond the stage represented in Figs. 11 and 12. The body of the embryo is narrower; the optic vesicles are more prominent; seven to nine protovertebrse are differentiated; the pronephric ducts are forming. In the anterior portion of the embryo there are three fairly well defined regions which represent the primary divisions of the brain. Anterior to the optic vesicles the nervous sA'stem is continued into a conical process, the homologue of the structure which Salensky found in Acipenser and to which he gave the name " Stirnforsatz." This precerebral portion of the head is the anlage of several structures to which we shall hereafter refer in greater detail. The mid-brain is marked off by a constriction posteriorly and behind this constriction is a marked eulargement which forms the basis of the anterior portion of the medulla. In this region, as Keibel has pointed out, the anlage of the otic vesicles will later appear. The darkened zone around the anterior end of the embryo represents the extent of the mesoblast. Posteriorly the lateral boundaries of the mesoblast are poorly defined so that in surface views it is impossible to indicate them.
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Embri/o Ninety Hours After Fertilization. Emhri/o Covers About 1S0°. — Figs. 15 and 16 represent the anterior and posterior portions' of an embryo in this stage. Many striking changes have occurred. The subdivisions of the brain are more clearly defined and are more prominent. The optic vesicles are better defined. The precerebral portion extends forward as a distinct process. The first visceral arch has formed and, just behind it, is the first visceral cleft. The protovertebras have increased to sixteen or more pairs. The pronephric ducts have extended both anteriorly and posteriorly. There are at this time, however, no external indications of the olfactory, auditory or adhesive organs.
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Fig. 42 represents a section passing close to the median sagittal plane of an embryo slightly younger than that shown in Fig. IT. The superficial ectoblast (5. ec.) consists of two layers of cells which are invaginated at a point lying between the anterior margin of the fore brain (/. h.) and the median portion of the adhesive organs.
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 +
The brain cavities are now well defined. There is, however, as yet no indication of the infundibular or epiphysial evaginations. The notochord extends nearly to the level of the middle portion of the brain, as shown in the figure. The gut cavity is well defined beneath the posterior portion of the brain ; it is greatly reduced in size anteriorly. After reachui,g the level of the epiblastic invagination described above, it again expands into a wide cavity (g. d.). The walls of the gut show little change until the head region is passed when the dorsal wall is greatly thickened to form the beginnings of adhesive organs {a. 0.).
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 +
A transverse section through the extreme anterior end of the brain is represented in Fig. 43. The superficial ectoblast shows no modification in this section. Just beneath this layer the deep epiblast extends over the surface, but in the median line it is lost in the mass of cells which are radially disposed and w^hich represent the anterior end of the fore brain. Below the fore brain is a wide layer of mesoblast (mes.) which extends upward on each side. On either side of the median line the fore gut is greatly expanded. The layer of columnar cells covering these expanded portions, even at this early stage, is different from that forming the dorsal wall of the gut in other portions of the body. On either side the hypoblast extends peripherally, its cells take on the cuboidal form,
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14(1 Gastrulation and Embryo Fornialion in Amia Calva
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and become continuous with tht' yolk. In llic li^uiirc *i-iven the dorsal hypoblast has been too deeply shaded so that it is bronglit out in too strong contrast with the layer of yolk-bearing cells which form the floor of the fore gnt. On either side of the gut the anterior extremities of the ccelomic cavities (c.) are present.
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The next section described is represented in Fig. 44. The section passes through the optic vesicles (o. v.) which at this time are hollow and in wide communication with the fore brain. On either side there are slight depressions of the superficial epi blast which are probably artifacts dne to killing reagents. The loosely scattered cells of the mesoblast {mes.) form two large masses which extend laterally from the region where the floor of the fore brain rests directly upon the gut hypoblast. Just beyond the lateral boundary of the fore gut the mass separates into two layers, an outer somatic which is closely united with the ectoblast and an inner splanchnic which lies close to the gut hypol)last. Between these two layers are the ccelomic cavities (c). The gut (/. g.) is here widely expanded and, on either side, are seen the hypoblastic cells as they pass over into the anlagen of the adhesive organs.
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A section through the region where the mid brain passes over into the medulla is show in Fig. 45. The pharyngeal portion of the gut is here widely open and slight evaginations indicate the first appearance of the visceral clefts. Just external to these are mesoblastic masses (v. a.) which are the beginning of the visceral arches. The mesoblast extends down around the brain until it comes in immediate contact with the notochord. The walls of the ccelomic cavities (c.) are separated widely and are lined by a single layer of cells.
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Passing still further back we have selected a section (Fig. 46) through the region of the auditory vesicles. The cavity of the medulla is here widely open. Its lateral walls are made up of elongated epithelial cells arranged in one or two layers. Its roof, however, is very thin, so that when viewed from the surface it is very transparent. On either side are the auditory vesicles (a. v.) which have formed from thickenings of the deeper ectoblast. Above these the two layers of the superficial ectoblast are continuous. The vesicles which were earlier solid now show very small lumina. Just external to the vesicle on the right side there is a diverticulum of the gut. the pharyngeal portion of the third cleft, while Just outside this, a thickening in the mesoblast is the third arch. The cells of the liypoblast forming the dorsal wall of the gut are flattened, l)ut in the region of the clefts they become cuboidal, which character they retain until they pass over into the yolk cells. The ventral wall of the gut is
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Albert C. Evcloshynier and James Mcroditli Wilson 147
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still formed by a loosely scattered layer of entodcrinal cells which lie above the large yolk masses.
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The last section of this embryo, which we have represented in Fig. 47, is taken at the level of the last protovertebra. In this section we see that the neural keel of the earlier stages has taken on a cylindrical outline and has acquired a large well defined lumen in which there are no traces of cell degeneration. Below the neural tube and in contact with it, is the large notochord, and between the notochord and the gut hypoblast, is the sub-notochordal rod (h. cli.) which, from the character of its cells, seems to have arisen from the hypoblast of the gut.
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On either side of the notochord, are the large masses of mesoblast which form the last protovertebrge. At this level, the mesoblast shows no line of division between its somatic and splanchnic portions. In that portion which must be considered as potentially somatic, there is a slight proliferation which gives rise to a more or less well defined rod {p. d.) which soon becomes the pronephric duct.
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Embryo About One Hundred and Five Hours After Fertilization. Embryo Surrounds 220° . — The embryo (Fig. 17) shows a marked advance beyond the condition represented in Figs. 15 and 16. The divisions of the brain are more distinct. The hind brain shows a decided thinning of its dorsal wall. In front of the anterior end of the fore brain there is a slight pocket followed by a projection or median knob. On either side of this knob, are the large adhesive organs which are now apparent in surface view. The large optic vesicles lie just behind, and now show the first beginnings of the lenses. On either side of the medulla the auditory vesicles are faintly shown. The pronephric ducts have extended both anteriorly and posteriorly. The anterior portion of an embryo about five hours older is shown in Fig. 18.
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In this stage but few changes are noted beyond those described. The lateral walls of the medulla are more widely separated and the roof has become thinner. The visceral arches and clefts are more pronounced. There is no trace as yet of the olfactory organs. As a result of the uplifting of the embryo through growth, the adhesive organs have assumed an oblique position.
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A sagittal section, passing slightly to one side of the median plane of an embryo in this stage, is represented in Fig. 48. The lumen of the brain is enlarged and its subdivisions more clearly marked. The dorsal wall of the fore brain now shows a slight evagination which is the beginning of the epiphysis. Just opposite in the floor is another evagination which is the beginning of the infundibulum. Anteriorly the cavity narrows down in conformity with its external contour. Just in front of
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148 Gastrulalion and Eml)ryo Formation in Aniia Calva
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the autorior end of the fore bi'ain, the continued invagination of the superficial epiblast gives rise to a deep pocket in which a lumen is sometimes plainly apparent, while at other times its walls are so closely apposed that no lumen is discernible. The fore gut is here well shown with its forward extension into the precerebral region where it ends in a dilated cavity. The walls of this cavity, except the ventral, are made up of elon,gated hypoblastic cells. Just beneath this median evagination there is a large chamber surrounded by a double wall. The lining wall is made up of elongated cells which strongly resemble those lining the body cavity. Outside this layer is a second wall made up of large yolkladen entoblastic cells. This chamber represents the beginning of the heart. The discussion of its formation, however, may best be deferred until we have studied the series of transverse sections of the next stage. Embryo One Hundred and Tiventy-five Hours After Fertilization. Embryo Covers 260° . — The last stage of the embryo included in the present study is represented in Figs, 19 and 20, the anterior portion being shown in Fig. 19, while the posterior is represented by Fig. 20. The embryo has increased greatly in length and its body is more prominent above the surface of the yolk, while the tail is just becoming free from the yolk. The increase in the length of the head has caused further shifting in the position of the adhesive organs which, instead of having their surfaces directed above, have come to occupy such an oblique position that their surfaces are almost directed forward. The so-called "button" (Eeighard) is likewise carried forward and is no longer visible when the embryo is viewed from the dorsal surface. Just behind the adhesive organs are the two nasal pits which are visible for the first time in surface views. In the eyes, the lenses are plainly shown in the surface views. The mid brain has extended backward, while the hind brain has pushed forward in such a manner that its anterior portion envelops the posterior portion of the mid brain. In this embryo, the roof of the hind brain has been removed and one can plainly see in its floor a number of neuromeres. These are variable in the different embryos of this age, ranging from six to eight. On either side of the hind brain, the auditory vesicle shows as a deep pit. Three visceral arches are now well defined, as are also the three visceral clefts which appear as darker portions between them. The coelomic cavity shows as a darker circle around the margin of the embryo, although its boundaries are not as clearly defined as in some of the earlier stages. (Fig. 17). The protovertebrse have extended on either side until they now reach from the extreme posterior end nearly up to the auditory pits. The pronephric ducts have extended both anteriorly and posteriorly. At tlieir anterior ends they curve outward, then inward, in the form of a shepherd's crook.
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Albert C. Eycleshymer and James Meredith Wilson 149
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A transverse section of an embryo in this stage is represented in Fig. 49. This section is taken through the region just anterior to the adhesive organs. On either side the coelomic cavities show plainly as they approach the median line. The layers of the splanchnopleure are thus brought in such close contact above that the gut (g.) is almost closed off. During the time these layers are approaching they become folded backward into the coelom on either side. In the figure, the left side is considerably in advance of the right. Through this folding there is formed a second closed cavity (ht.) which is the beginning of the heart. There is present at this time a lining layer, but its origin is uncertain.
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The section represented in Fig. 50 passes somewhat obliquely through another embryo in about the same stage of development. The anterior end of the fore brain (/. h.) appears as a solid mass of elongated cells. In connection with its ventral wall, the optic stalk passes obliquely outward and terminates in the optic vesicle. On the other side the section passes through the anterior portion of the adhesive organ which here shows its connection with the anterior end of the fore gut (g.). The fore gut is almost closed off ventrally through the approximation of the coelomic cavities. Between the end of the brain and the optic vesicles, there is a slight invagination (n.) of the deep ectoblast to form the beginning of the nasal pits.
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The section represented in Fig. 51 is from the same series as the preceding and three sections farther back. The section through some oversight is not magnified quite so highly. The chief point of interest, as compared with the precedin,g is the rapid separation of the coelomic cavities so that the gut is here widely open upon the yolk. It should also be noted that the cavities of the optic stalk, the fore brain and the adhesive organs are becoming apparent.
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A section of the same series is shown in Fig. 52 at the level of the auditory vesicles. The section shows the extension of the cavities of these vesicles (a. v.) In other respects the section shows nothing more than is shown in Fig. 46.
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The section shown in Fig. 53 is taken in a horizontal plane and shows practically all the structures which have been described in the series of transverse sections. The divisions of the brain are very clearly shown. Just in front of the anterior end of the fore brain is the invagination of the superficial ectoblast which we have previously described. On either side are the nasal pits (n.) with well defined lumina. Just anterior to these are the adhesive organs {a. o.) made up of the coarsely granular hypoblastic cells. Behind these are the large optic vesicles (o. v.) in
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\vliii.-li the lenses are present, but not shown. Ik'hind tlicse in turn are tliree gill clefts {v. c.) and between them the mesoblastic bases of the correspondinig arches. Close to the medulla are the auditory vesicles (a. v.). Around the periphery the lines of darker cells represent the hypoblastic walls of the gut (g.) The section is cut so thick that the mesoblast shows above, making it almost appear as if the gut were filled with these cells.
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SUMMARY AND GENERAL REMARKS.
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The Segmextatiox Cavity.
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Before considering the stages which properly belong to the present paper, we are obliged to say a few words concerning the segmentation cavity. Whitman and Eycleshymer, 96, pointed out that there are to be found in the egg, even in the earliest cleavage stages, certain irregular cavities which sooner or later become continuous with the cleavage grooves and in many cases unite to form a common cavity. These cavities appear in eggs collected in different years and in different seasons of the same year and fixed and imbedded in various ways. Since it is from these spaces that the segmentation cavity later takes its origin they have been subjected to renewed study. As segmentation progresses the cleavage grooves, in many cases at least, expand into broad spaces as they approach the center of the egg, in which locality they become continuous with the earlier spaces described above. Often these cavities unite and give rise to a more spacious one as figured by Whitman and Eycleshymer. That the cavities should be regarded as artifacts seems highly improbal)le. In the first place no cellular fragments are to be found in the spaces which would indicate imperfections in cutting. Again these cavities often shade ofi" by imperceptible degrees into veritable intercellular spaces which no one would consider as artifacts. As the later stages of the blastula approach, the cavities no longer show as large irregular spaces, but become more or less obliterated by bein,g filled with the rapidly proliferating cells of the blastodise and yolk. In view of these facts, we cannot agree with Sobotta, 97, that these cavities are artifacts.
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Periblast.
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The periblast in ganoids was first discussed by Dean, 95, 96, who pointed out the homology of the upper layer of yolk cells in Acipenser, Lepidosteus and Amia with the periblast of teleosts. Sumner, 00, later gives two figures showing the periblast in Amia. In one of these (Fig. 16) he represents a well defined, clear zone lying on the large yolk masses
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Albert C. Eyelosliyiner and James Mereditli Wilson 151
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and covered by a cellular layer wliich is considered as periblast. Tbe author states that the tig-ure is slightly schematized, l)ut to wliat ]iart of the figure he refers is uncertain. If to the periblast we have no criticism to offer. If tiie author, however, intended to represent the periblast as it is actually found in Amia we must emphasize the fact that our material shows nothing of the sort. There is no layer of cells between the peril)last and the gastral cavity. The floor of the gastral cavity is, to our minds, the homologue of the periblast in teleosts. We find in -the ganoids a complete series of gradations from the teleostean to the amphibian conditions. In Lepidosteus, as described and figured by the senior author, 03, we find the closest approach to the teleostean periblast. Amia comes next with its homologous layer in the floor of the gastral cavity; this floor is made up in part of detached cells and in part by the projecting ends of the large yolk masses. From this condition we can readily pass to the homologue of the periblast in Acipenser which is the layer of cells forming the floor of the gastral cavity. We are thus prepared to support the statement of the Zieglers, 92, that the floor of the gastral cavity in the amphibia is the homologue of the periblast in fishes.
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The Mesoderm.
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The origin of the mesoderm in Amia has been previously studied by both Dean and Sobotta. Their descriptions, however, are quite unlike.
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Dean, 96, in describing a stage in which the blastopore has nearly closed, says : " The mesoblast is found to arise peristomal ; on the dorsal side it arises from the undifferentiated tissue (of the tail mass), thence extending forward as a separate cell layer, and finally appears to be ])lended with the loosely associated cells of the entoblast; ventrally the mesoderm, although distinctly to be recognized, is not to be separated from the cellular elements of the entoderm. In its early growth it extends forward as a wide and flattened cell mass, thinning distally and becoming confluent with the inner germ layer. As in the teleosts, gastral mesoderm is absent, and the division of the middle layer into its somatic and splanchnic layers is not apparent until a comparatively late stage of development."
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Sobotta. 96, describes the origin of the mesoderm in a much earlier stage in tlic following words: " N'un tritt, uoch ehe es zur Urdarmbildung. also zur eigentlichen Cxastrulation kommt, eine Differenzirung der Furchungszellen zu Keimblattern auf, indem sich eine compacte mehrschichtige Zellage an der Oberflache des Eies durch einen feinen Spalt von den darunten gelegenen, mit grosseren Dotterkornern beladenen Zellen sondert (Fig. 3). Diese Erscheinung trennt bereits jetzt das Ek
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153 Gastriilation and Embryo Formation in Aniia Calva
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todorni von dor spiitor zu Mesoderm nnd Entoderm werdenden Zellage — Selir bald beginnt nun am Aequator dcs Eeies die Urdarm-Bilduug, nnd zwar zuerst an der Stelle der spateren Embryonalanlage. Es entsteht dadnrch die dorsale Urmnndlippe (Fig. 4). Letztere ist ziir Zeit, wo der Urdarm als feiner Spalt sichtbar wird, sofort dentlich dreibliittrig, niclit zweiblattrig wie Dean angiebt. Die verschiedene Gehalt der Zellen an Dotterkornern, resp. die verschiedene Grosse derselben in den Zellen, ermoglicht die Unterscheidnng drier Keimblatter sehr leicht." The writer then points out the differences in the sizes of the granules in the different layers, stating that those in the cells of the ectoderm are all fine, those in the cells of the mesoderm are considerably larger, while those in the cells of the entoderm are coarse. The writer further states that when the gastral cavity has extended beneath the dorsal lip of the blastopore, the dorsal and ventral mesoderm are united.
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It is evident from our studies that we agree in general with Sobotta in that we find the ectoderm early separated from the underlying layer of cells by the slit-like remains of the segmentation cavity. This underlying layer represents, according to Sobotta, the mesoderm. We do not agree, however, that in the early gastrula the size of granules or their staining capacity will enable one, as Sobotta claims, to distinguish mesoderm from entoderm. It is not until the time when the blastopore is nearly closed that a differentiation of cells is apparent. Even then we are not certain that these cells represent the mesoderm since the marked contrast in the staining capacity later disappears. To know precisely when and how the mesoderm arises and how it extends in Amia will involve better methods of staining than we now possess.
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The Archexterox. Kupffer's Vesicle and Adhesive Organs.
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As the archenteric cavity extends the innermost layer of mes-entoderm early is differentiated into a well defined layer which we have called hypoblast. This layer, together with the invaginated dorsal ectoblast, forms the dorsal wall of the archenteron. At the same time there is differentiated a superficial yolk layer which forms the ventral wall of the archenteron. The extent of this primitive gut, however, does not correspond to the extent of the embryo. There is formed both an appendicular (Salensky) or post-annal gut and a precephalic gut. Since the changes in the posterior portion precede those in the anterior they will be considered first.
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It should be remarked here that the closure of the blastopore is complete, no portion persisting to form the anus. Its line of closure is in
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Albert C. Eycleshymer iuid James Meredith Wilson 153
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dicated for some time by the large coarsely granular cells which lined it. The anus arises through an invagination just behind the line of closure of the blastopore and soon becomes continuous with the appendicular gut. The portion posterior to this or the post-anal gut proper soon shows retrogressive changes. The walls lose their distinctive hypoblastic character, the lumen becomes obliterated and the entire structure later disappears, playing no part in the formation of later embryo.
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Kupffers vesicle has been studied previously in Amia by Dean, g6, and Sumner, 'oo. Dean writes as follows : " The coelenteron, now a deep cavity beneath the dorsal lip, extends forward below the entire head; its hinder dilation immediately below the dorsal lip is to be interpreted as representing Kupffer's vesicle."' Eegarding Dean's interpretation Sumner says : " Dean maintained that this cavity simply represented the angle formed by the blastoderm's margin, as it was mechanically turned in upon itself during its circumcrescence of the yolk. This simple mechanical explanation I cannot accept for the teleosts because (among other reasons) the vesicle in some fishes is not formed until the blastoderm has nearly or quite finished its journey over the yolk and thus the supposed mechanical cause no longer exists." In considering the function of this structure in fishes Sumner further says : " It has for some time been my view that this vesicle contains a more fluid yolk, partly assimilated through tlie activity of the periblast and intended for the nourishment of the growing embryo. I have also expressed the view that Kupffer's vesicle represents an embryonic digestive organ (more properly an organ of absorption)."
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In considering this structure it is necessary to recall what has been said regarding the periblast in the teleosts, ganoids, and amphibia. While the roof of the vesicle in all these forms is the same, the floor in the teleosts is sometimes of periblast and again there is a cellular floor lying upon the periblast. These facts are at first difficult to interpret, yet if as H. Y. Wilson, 91, suggests the latter condition is to be regarded as secondary, the difficulties are in a measure overcome. If it be accepted that the floor of Kupfer's vesicle is periblast in the teleost and that the periblast of Lepidosteus is the homologue of that of the teleost Eycleshymer, 03, we are placed in position to say that the ventral wall of the vesicle in Amia, Acipenser and the amphibia is represented by nothing more or less than the gastral floor. The vesicle then represents the posterior portion of the primitive digestive tract. This being the case in Amia no one need hesitate to accept Sumner's view that the vesicle may have had a digestive function.
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The first description of tlie growth of the adhesive organs is given by 11
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154 Gastnilation and Minhvvo Formation in Amia Calva
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Dean, 96, who says: "The mode of oi'igiii of the sucking disc gives the most interesting evidence of how precociously embryonic and larval structures may be developed. As far as histological evidence goes there is certainly no difference between the enlarged thick-walled, cup-shaped organs which arise on the snout of the late embryo of Amia or of Lepidosteus, and the typical pit organs, or sense buds, which later occur on oth'er integumental regions. It is found, in fact, that a gradation in size exists which connects the huge sucking organs of the snout with the inconspicuous pit organs of the trunk.
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These organs were later studied in Eeighard's laboratory by Miss Phelps, 99, who found that " the organ is formed in a very early stage as a diverticulum of the fore gut. This diverticulum subsequently divides into two, each of which continues to communicate for a time with the cavity of tlie fore gut." The author further observed that the organs open to the exterior, but become cut off from the fore gut and degenerate leaving no trace behind.
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Our studies show that these organs arise from paired diverticula of the fore gut and not from a single diverticulum which later divides. While it is undoubtedly correct to state that the paired gait diverticula are derived from an unpaired condition, there is not the slightest evidence that the anlagen of these structures appear before the gut diverticula are well established. The beginnings are first visible as slight thickenings of the hypoblast, forming the antero-dorsal walls of these diverticula. As development progresses these thickened areas evaginate and the cells begin to elongate. Soon a longitudinal constriction forms which divides each of these structures, giving rise to four. Meantime the lumen of each is reduced, the walls of the gut become apposed and the organs are cut off from further communication with the gut. After losing their connection with the gut they continue to divide until eight or more are form.ed. They then come in contact with the ectoblast whose cells undergo cytolysis, leaving the hypoblastic cells of the organs projecting to the free surface. We have not followed the later changes in these organs. Miss Phelps states that after being functional for a time the organs are pushed beneath the surface of the thickened ectoblast, become filtrated with leucocytes and finally disappear.
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As to the meaning of these remarkable organs we are in the dark. Their function may be only to hold the larva in position for a certain period. Again they may serve to convey some sort of nutriment to the digestive tract. That they are modified sense buds, as Dean suggests, seems highly improbable. Their interpretation from a phylogenetic standpoint is certainly most difficult. About all that can be said is that
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All)crt C. E3'clesliymer and James Meredith Wilson 155
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structures siu'li as these rt;ive rise to some of the nu3st perplexing problems with which (Mnl)ryolo«2:y has to deal. •
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In the surface views (Figs. 17, 18, 19) there is a peculiar median knob wdiich appears soon after the adhesive discs are differentiated. This structure lies between and somewhat anterior to the discs. It lias been observed, as earlier stated, by Eeighard (see Keibel, 03). Sagittal sections through embryos of this stage ( Fig. 48) show that in addition to the lateral evaginations of the fore gut there is a less marked evagination of the median wall. The hypoblast in this region is thickened and becomes continuous on either side with that of the adhesive discs. The epiblastic pocket behind separates this structure from the anterior end of the fore brain and together with the surrounding mesoblast gives it considerable prominence in surface views. We conclude that the so-called "button" is nothing more than the strongly evaginated median portion of the adhesive organs.
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The Chorda axd Hypochokda.
 +
 +
Dean, 96, has described the formation of the chorda in Amia as follows : " The notochord arises as in the sturgeon or gar-pike : it separates directly (i. e., delaminates) from the entoderm." We have studied the origin of the notochord in many series of embryos, but are unable to add much to Dean's description. In the great majority of cases examined the sections show conditions similar to that observed in Fig. 40; often the mesoblast has not yet separated from the axial rod as shown in Fig. 41. In but one instance have we found anything which would lead 'us to regard the chorda as formed by an evagination of the dorsal wall of the archenteron, as is known to be the case in many amphibia. We therefore conclude that the chorda is derived by delamination from the layers of cells which we have called mes-entoderm. The controversies that have been w^aged over this structure and the failure to homologize it in the various groups, especially amphibia and fishes, hold out little promise that a definite solution of the problem in harmony with the germ layer theory is near at hand.
 +
 +
The hypochorda, so far as we know, has not been previously seen in Amia. It arises from the hypoblast forming the dorsal wall of the gut in a stage just prior to the appearance of the pronephric ducts. It extends the length of the notochord and presents in sagittal sections appearances which lead us to regard it as irregularly segmented. This peculiar structure has been observed in many vertebrates and numerous suggestions offered as to its significance. By some it has been considered
 +
 +
 +
 +
156 Gastnilntion and Eiiil)rvo Formation in Am in Calva
 +
 +
tis the anlapc of a ligament or a blood vessel. By others it is regarded ■as the remains of a blood vessel or of a blood-forming organ. Still others think it entirely disappears. We incline toward the last view, but our later stages are not complete enough to settle the question definitely.
 +
 +
The Heart,
 +
 +
In considering the origin of the heart, it is necessary to recall that toward the anterior end of the embryo the coelomic cavities on either side approach the middle line. This approximation proceeds anteriorly until the two halves of the coelomic cavities are brought closely together. Just before they meet each becomes folded back at the edge. Through this folding back of the splanchnopleure there are formed two grooves; the edges of these two grooves unite across the median line to form a single oval sac which is open both anteriorly and posteriorly. This sac is lined by entoderm and surrounded by the splanchnic mesoblast. While these changes have been going on there has appeared within the heart cavity thus formed a layer of cells which have the appearance of mesoblast cells, but apparently they are derived from the hypoblast. Whether they are to be considered as mesoblast, that at this relatively late period has differentiated from the hypoblast, or whether they are to be considered as hypoblast we are unable to say. Only by knowing the fate of these layers could one hazard an interpretation.
 +
 +
The Central Nervous System and Sense Organs.
 +
 +
The central nervous system, as observed by Dean, 96, is first formed as a solid rod or keel from the deeper ectoblast. Soon after the appearance of the optic vesicles a lumen is formed, but whether through cytolysis or delamination or both is uncertain. There are indications which lead us to regard cytolysis as the most probable.
 +
 +
Towards the caudal end of the embryo the superficial ectoblast folds downward into the neural keel forming the neural trench, which at the posterior end passes over into the blastopore. The question whether or not this is to be interpreted as a neurenteric canal depends upon the significance of the neural trench. If this trench is to be considered as homologous with the extreme lower part of the medullary grooves in Amniota, as Kupfl'er regards it in the trout, we should certainly consider its continuation over into the blastopore as a reminiscence of the neurenteric canal. However, both Wilson's and Kupffer's views are questioned by Minot and others, and since the interpretation rests upon funda
 +
 +
 +
Albert C. Eycleshymer and James Meredith Wilson 157
 +
 +
mentally different conceptions which are at present beyond proof or disproof, we may dismiss the question without further comment.
 +
 +
Concerning- the neuromeres whicli aic so well shown in Fig. 19, we can only say that at present we are unable to interpret these structures. They have not been found in the preceding stages and have not been followed in the succeeding stages. Why they should appear at this time and be wanting in the stage shown in Fig. 18, is at present unexplafinable. It is impossible to state whether they are secondary foldings due to the formation of protovertebnB or whether they are formed independently in the floor of the hind brain and are the first definite expression of segments. If the latter be true, as many embryologists hold, then we should find in the hind brain of Amia indications of seven or eight primitive segments.
 +
 +
Tlie melian pit, which first appears in Fig. 42, has been followed in both tlie earlier and later sta,ges. After a careful study much doubt lingers in our minds as to whether or not it takes any part in the formation of the hypophysis. Ivupffer maintains that in Petromyzon and Acipenser this structure forms the hypophysis. It seems to us possible that the invagination of the gut to form the media^i portion of the adhesive organs, as shown in the figure, would carry the epiblast outward in such a manner that it results in an increase in this invagination. In other words, the mechanical factors operating could cause just the appearance observed. We should hesitate to regard this structure in Amia as of great value in phylogenetic interpretation.
 +
 +
The previous observations on the development of the optic vesicles in Amia are embodied in the following sentence by Dean, g6: " The mode of development of the eye and of the nasal and auditory capsules differs but little from that typical in the lower vertel)rates generally." Our studies show that the eyes first appear as solid outgrowths which shortly after become hollow.
 +
 +
Concerning the early development of the auditory vesicle there is nothing beyond the sentence quoted above. According to our observations, the ear likewise begins as a solid thickening of the deep epibhist over which the superficial layers pass unmodified. This thickening continues until there is an oval mass lying on either side of the anterior portion of the medulla. AVhen the embryo reaches the stage shown in Fig. 18. a cavity is present.
 +
 +
The olfactory organs first appear as proliferations of the deep ectoblast in the stage represented in Fig. 19. In this mass an invagination soon appears forming well defined pits.
 +
 +
 +
 +
158 Castnilalion and Embryo Formation in Amia Calva
 +
 +
The Pronephric Duct.
 +
 +
The proneplij'ic duel is preeeded by a solid rod of cells which arises throii,g"h a proliferation of the cells of the somatopleuric portion of the mesoderm, but before the appearance of a well defined coclom. We do not agree with the observations of Felix and Biihler, 04, who state that it arises as an evagination of the somatopleure. The further study does not come within the scope of the present paper.
 +
 +
Systematic Position of Amia.
 +
 +
Dean concludes his second paper, 96, on Amia with the statement that " at the base of a gradational series stands Lepidosteus, near it and in some ways even below it is Acipenser; next is Amia; next, and very closely related, is Amiurus; and, finally, are the many remaining forms of teleosts."
 +
 +
Previous studies by the senior author on x\mia and Lepidosteus, the present study of Amia, together with the unpublished work on i\.meiurus by the junior author, all indicate that such an arrangement, based upon early developmental characters, is not only premature but incorrect. The only conclusion which can be reached at the present time is that the evidence from oecology, anatomy, histology, embryology, is so fragmentary that it affords no secure basis for assigning the various ganoids their respective places within the group, nor the group its position in the vertebrate phylum.
 +
 +
BIBLIOGRAPHY.
 +
 +
Deax, Bashford. — The Early Development of Amia. Quart. Jour. Micr. Sc,
 +
 +
Vol. XXXVIII, 1896, pp. 413-444. Deax, Bashford. — On the larval development of Amia calva. Zool. Jahrb.
 +
 +
Abt. f. Syst, Bd IX, 1896, pp. 639-672. Eycleshymer, Albert C. — Notes on Celloidin Technique. Am. Nat., XXVI,
 +
 +
1892, pp. 354-358. Eycleshymer, Albert C. — The Cleavage of the Egg of Lepidosteus osseus.
 +
 +
Anat. Anz., Bd. XVI, 1899, pp. 529-536. Eycleshymer, Albert C. — The Early Development of Lepidosteus osseus.
 +
 +
Univ. of Chicago Decennial Publications, Chicago, 1903. Felix and Buhler. — Die Entwickelung der Ham und Geschlechtsorgane.
 +
 +
Handbuch d. vergl. u. exper. Entwickelung d. Wirbeltiere. O. Hert wig ed. Jena, 1904, Lief. XVIII, p. 135. Kerb, J. Graham. — The Development of Lepidosiren paradoxa. Part II.
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 +
Quart. Jour. Micr. Sc, Vol. XLV, 1901, pp. 1-40. KuPFFER, C. — Studien zur vergl. Entwickelungsgeschichte des Kopfes der Kra nioten. Heft I. Die Entwickelung des Kopfes von Acipenser Sturios
 +
 +
an Medianschnitten untersucht. Miinchen, 1893, pp. 1-95.
 +
 +
 +
 +
Albert C. P]vclcshvmer and James Meredith Wilson
 +
 +
 +
 +
159
 +
 +
 +
 +
Phelps. Jessie. — The Development of the Adhesive Organ of Amia. Science. N. S.. Vol. IX, 1899, p. 366.
 +
 +
SuMXER. F. B. — Kupffer's Vesicle and its Relation to Gastrulation and Concrescence. Mem. New York Acad. Sci., Vol. II, 1900, Pt. 2, pp. 48-80.
 +
 +
SoBOTTA. J. — Die Gastrulation von Amia calva. Verhandl. anat. Ges., 1896, pp. 108-111.
 +
 +
SoBOTTA, J. — Die Furchung des Wirbeltiereies. Ergebnisse Anat. und Entwickelungsgeschichte, Bd. VI, 1897, pp. 493-593. ,
 +
 +
Whitman axi) Eyclesiiymer. — The Egg of Amia and its Cleavage. Jour. Morph., Vol. XII, 1896, pp. 309-354.
 +
 +
Ziegler. H. E., und Ziegler, F. — Beitrage zur Entwicklungsgeschichte von Torpedo. Arch, fiir mikr. Anat., Bd. XXXIX, 1892, pp. 56-102.
 +
 +
 +
 +
ABBREVIATIONS.
 +
 +
 +
 +
a. g., appendicular gut. o. v., auditory vesicle.
 +
 +
b. c, brain cavity. cTi., chorda.
 +
 +
d. I., dorsal lip of blastopore.
 +
 +
f. ft., fore-brain. g.. gut.
 +
 +
g. d., gut diverticulum. h.. anlage of head. hch.. hypochorda.
 +
 +
m. b., mid-brain.
 +
 +
vies., mesoblast.
 +
 +
n. c, neural canal.
 +
 +
op. t., optic thickenings.
 +
 +
p. cb., pre-cerebral mass.
 +
 +
s. c. segmentation cavity.
 +
 +
V. a., visceral arch.
 +
 +
V. 1.. ventral lip of blastopore.
 +
 +
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a. o.. adhesive organ.
 +
 +
bl., blastopore.
 +
 +
c coelom.
 +
 +
d. ec, deep ectoblast.
 +
 +
env., envelope.
 +
 +
f. g., fore-gut.
 +
 +
g. c, gastral cavity. g. hy., gut hypoblast. h. b.. hind-brain.
 +
 +
M., heart.
 +
 +
m. en., mes-entoblast.
 +
 +
n., nasal pit.
 +
 +
n. t., neural trench.
 +
 +
o. v., optic vesicle.
 +
 +
p. d., pronephric duct.
 +
 +
s. ec, superficial ectoblast.
 +
 +
V. c, visceral cleft.
 +
 +
y. m., yolk mass.
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EXPLANATION OF PLATES.
 +
 +
Plate I.
 +
 +
Fig. 1. Profile view of egg about nine hours after fertilization.
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 +
Fig. 2. Profile view of egg about twelve hours after fertilization.
 +
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Fig. 3. Profile view of egg about twenty hours after fertilization.
 +
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Fig. 4. Profile view of egg about forty hours after fertilization.
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Fig. 5. Profile view of egg about fifty-three hours after fertilization, anlage of embryo faintly outlined.
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Fig. 6. Profile view of egg about fifty-five hours after fertilization, outline of embryo discernible.
 +
 +
Fig. 7. Same egg viewed from the lower pole, showing infolded blastodisc.
 +
 +
Fig. 8. Embryo about sixty hours after fertilization, viewed from above, embryo well defined posteriorly, neural (?) trench visible.
 +
 +
Fig. 9 . Embryo about sixty-five hours after fertilization, viewed from above, embryo narrower, better defined outline, blastopore nearly closed.
 +
 +
 +
Fig. 10. Embryo alioiit seventy hours after fertilization, eml)ryo longer, neural (?) trench extended, blastopore closing.
 +
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Figs. 11, 12. Anterior and posterior portions of embryo about seventy-five hours after fertilization, viewed from above, optic thickenings visible, two or three protovertebrae differentiated.
 +
 +
Figs. 13, 14. Anterior and posterior portions of embryo, eighty hours after fertilization, viewed from above, divisions of brain and precerebral process apparent, seven to nine protovertebrae, beginnings of pronephric ducts, extent of mesoblast indicated by dark area around head.
 +
 +
Figs. 1.5, 16. Anterior and posterior portions of embi-yo about ninety hours after fertilization, viewed from above, showing pre-cerebral process, optic thickenings, first gill arch and cleft, about sixteen protovertebrae, dark shading around embryo indicates extent of coelom.
 +
 +
Fig. 17. Anterior portion of embryo about one hundred and five hours after fertilization, viewed from above, showing divisions of brain, optic vesicles, adhesive organs, median knob, two gill clefts, beginnings of auditory vesicles.
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 +
Fig. 18. Anterior portion of embryo about one hundred and ten hours after fertilization, showing slight advance beyond condition represented in Fig. 17.
 +
 +
Figs. 19, 20. Anterior and posterior portions of embryo about one hundred and twenty-five hours after fertilization, viewed from above, showing, in addition to structures previously described, the anlage of the heart, the beginnings of the olfactory organs, neuromeres, three gill arches and their corresponding clefts.
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Plate II.
 +
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Fig. 21. Meridional section of an egg about nine hours after fertilization. Showing early condition of blastodisc segmentation cavity and yolk.
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Fig. 22. Meridional section of egg about twelve hours after fertilization. Showing the yolk masses actively budding off entoblastic cells, and obscuring more or less the segmentation cavity.
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Fig. 23.. Meridional section of egg about forty hours after fertilization showing proliferation of superficial ectoblast at the point where invagination begins.
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Fig. 24. Meridional section of egg. about fifty hours after fertilization showing beginning of invagination and maximal thickness of blastodisc.
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Fig. 25. Portion of meridional section of egg of about same age as above through region of blastopore showing first formation of mes-entoblast.
 +
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Fig. 26. Meridional section of egg slightly older than preceding stage showing further extension of gastral cavity.
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 +
Fig. 27. Meridional section of an egg about fifty-three hours after fertilization, showing reduction in number of entoblastic cells, also thinning of blastodisc.
 +
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Fig. 28. Portion of meridional section of egg in same stage as above more highly magnified, showing region of blastopore.
 +
 +
Fig. 29. Meridional section of an egg in stage somewhat later than above showing further thickening of margin of blastodisc.
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Fig. 30. Horizontal section of egg just above level of equator, showing lateral extent of embryonic anlage, also beginning of notochord.
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Plate III.
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Fig. 31. Meridional section of egg about fifty-five hours after fertilization, showing thickening of ectoblast to form head of embryo.
 +
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Fig. 32. Sagittal section of an embryo some sixty hours after fertilization showing further differentiation of embryo.
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Fig. 33. Sagittal section of an embryo about sixty-three hours after fertilization, showing further growth of embryo, the obliteration of the segmentation cavity, the extension of the gastral cavity, the reduction of yolk plug.
 +
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Fig. 34. Sagittal section of posterior end of embryo about seventy hours after fertilization, showing the closure of the blastopore.
 +
 +
Fig. 35. Transverse section through the blastopore of an embryo in same stage as above.
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Figs. 36, 37. Sagittal section of embryo about seventy-two hours after fertilization. The anterior portion of the embryo is shown in Fig. 36, while the posterior portion is shown in Fig. 37.
 +
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Fig. 38. Oblique section of an embryo seventy-five hours after fertilization showing the thickenings which later form the optic vesicles.
 +
 +
Fig. 39. Transverse section of an embryo of same age passing through the posterior portion of the hind brain.
 +
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Fig. 40. Transverse section of same embryo at the level of Kupffer's vesicle.
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 +
Fig. 41. Transverse section of an embryo of same age, showing deep neural trench and posterior end of notochord.
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 +
Fig. 42. Sagittal section of head end of embryo about ninety-five hours after fertilization, showing divisions of brain, notochord, gut, adhesive organs, and the peculiar invagination of the ectoblast just anterior to the fore-brain.
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Plate IV.
 +
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Fig. 43. Transverse section through embryo of same age at the level of the adhesive organs.
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Fig. 44. Transverse sections of same embryo at level of optic vesicles.
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Fig. 45. Transverse section of same embryo at level of anterior margin of medulla..
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Fig. 46. Transverse section of same embryo through the region of the auditory vesicles, showing the first appearance of pronephric duct and hypochorda.
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Fig. 47. Transverse section of same embryo at the level of last protovertebra.
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Fig. 48. Sagittal section of embryo one hundred and ten hours after fertilization, showing the beginning of the epiphysis and infundibulum, also the median portion of the adhesive organs and the heart.
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 +
Fig. 49. Transverse section of an embryo one hundred and twenty-five hours after fertilization taken just in front of the region of adhesive organs showing heart, gut and coelom.
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Fig. 50.. Obliquely transverse section of an embryo of same stage passing through the anterior margin of the adhesive organs on the one side and the optic stalk and vesicle on the other, showing the approximation of the layers of the splachnopleure to form the heart.
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162 (iastruhiiioii and Einlir\(i l-'ormalion in Aniia Calva
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 +
Fig. 51. Obliquely transverse section of same embryo taken a few sections behind that shown in Fig. 50, showing same structures as above.
 +
 +
Fig. 52. Transverse section of embryo in same stage showing the extension of the cavity of the auditory vesicles also the gut and coelomic cavities.
 +
 +
Fig. 53. Horizontal section through the head region of embryo in same stage showing various structures.
 +
 +
 +
===A Contributiox To The Anatomy And Development Of The Venous System Of Didelphys Marsupialis (H)===
 +
 +
Part II, Development.
 +
 +
{{Ref-McClure1906}}
 +
 +
BY
 +
 +
Charles F. W. McClure.
 +
 +
Professor of Comparative Anatomy, Princeton University.
 +
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With 5 Double Platks and 27 Text Figures.
 +
 +
A number of pul^lications have appeared, especially the monographs of Selenka, 86-7 and 91, and Semon, 94, in which have been described the arrangement of the blood vessels in the extra-embryonic vascular area of marsupials. So far as known to the writer, however, the only actual contributions that have been made to the development of the intra-embryonic venous system of this group of mammals are one by Broom, 98, and a preliminary notice by the writer, 02.
 +
 +
The publication of the present paper (Part II) has been unavoidably delayed, owing to the writer's inability to obtain embryos sufficiently young to show the earliest stages in the development of the postcaval vein. These early stages have, unfortunately, not yet been obtained, and were it not for the circumstance that so little has been published upon the development of tlie veins of marsupials, an apology would be due for presenting what must necessarily be an incomplete account.
 +
 +
In writing this paper the writer has fully borne in mind the danger involved of drawing conclusions from an incomplete series, and, in the case of Didelphys, the danger is especially great on account of the variable character of its venous system. A complete account of the development of the veins of Didelphys, especially of the variations of the postcava, necessitates not only a complete series of embryos and pouch young, but a number of examples from each stage as well. Such an
 +
 +
' The publication of this paper In two parts, one dealing with the Anatomy (Part I) and the other, or present paper (Part II), with the Development of the venous system, was unavoidable and It is, therefore, to be hoped that the frequent references made in the following pages to Part I will not prove too great a source of confusion or inconvenience to the reader.
 +
 +
Part I of this paper was published in The American Journal of Anatomy, Vol. II, No. .3, 1903.
 +
 +
American JontxAL of ,\natomy. — ^'o^,. V.
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164 A'enoiis System oC Didelphys Marsnpialis (L)
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 +
abundance of material the writer has been \iiiable io obtain, and, therefore, appreciates thai many modifications, as well as additions, to his account may possibly be necessary before a comjilete history of the veins is at hand.
 +
 +
In the tdllowin^u- pages an attempt has been made, on the basis of the material at liand, to present an account of the development of the postcava from a time soon after its first appearance until the adult stage is reached; also, an account of the development of the azygos veins, as well as the transformations which the umbilical and omphalomesenteric veins undergo during the diiferent stages of development.
 +
 +
An attempt to breed opossums in captivity proved only partially successful, the failure being due, I am convinced, to the unsuitable conditions which necessarily prevail in my laboratory. ^lost of my Didelphys material was therefore obtained outside of the laboratory, and, for this reason, it is impossible to give the exact age in days and hours of any of the embryos or pouch young studied. It has been possible, however, by means of Selenka's, 86 and 87, figures and descriptions to approximate their ages in a few case^ and to establish the fact that the embryo of Dasyurus which Dr. J. P. Hill, of the University of Sydney, kindly sent me is, in point of structure, relatively younger than my youngest Didelphys embryo.
 +
 +
According to Selenka, the interval between copulation and birth in Didelphys virginiana is about thirteen days (twelve days and twenty hours), while that lietween copulation and the beginning of cleavage is five days. In the following list where ages are mentioned the age has been reckoned from the beginning of cleavage.
 +
 +
List of Material Studied.
 +
 +
1. One Dasyurus embryo measuring about G mm. in length (crownrump measurement)." From a comparison of the structure and external characters of this embryo with that of Selenka's, 86 (Fig. 3, Taf. XXVI), six days old Didelphys embryo, it is evident that the latter is slightly more advanced than the Dasyurus embryo, which, in point of structure, corresponds to a Didelphys embryo of about five and one-half days.
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 +
2. Eleven Didelphys embryos averaging 8 mm. in length. From a comparison of their measurements these embryos may be a few hours older than Selenka's, 86, (Fig. 3, Taf. XXYI) six days old embryo, although in their external characters the two appear to be identical.
 +
 +
■ All measurements were made in this manner. Embryos and pouch young were measured by the writer after fixation.
 +
 +
 +
 +
Charles F. W. McGlure . 1G5
 +
 +
3. Three embrvos of Didelphys averaging 11.5-12 mm. in length. These embryos were kindly presented to me by Dr. Bremer, of Harvard University, to whom my thanks are -due.
 +
 +
It is a curious fact that these embryos measure more than certain of the pouch young studied by the writer; a circumstance which shows, as suggested by Professor Minot, that opossums may vary considerably as to the degree of development attained before they enter the pouch.
 +
 +
•1. One pouch young of Didelphys measuring 10.5 mm. in length. Harvard Embryological Collection, No. 614.
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 +
5. One pouch young of Didelphys measuring 11.5 mm. in length. Harvard Embryological Collection, No. 617. This measurement corresponds to that of Selenka's, 87 (Fig. 2, Taf. XXIX), newly-born pouch young of eight days (eight days after beginning of cleavage), but is apparently not constant for young of this age, since the Harvard specimen, No. 614, which is undoubtedly the younger of the two, measures only 10.5 mm.
 +
 +
6. Five pouch young of Didelphys averaging about 14 mm. in length.
 +
 +
7. Eight pouch young of Didelphys averaging about 15 mm. in length. According to measurements these correspond approximately to Selenka's (87, Fig. 8, Taf. XXX) four days old pouch young (twelve days from the beginning of cleavage).
 +
 +
8. One pouch young of Didelphys measuring 17 mm. in length.
 +
 +
Preparation of Material.'
 +
 +
The wTiter has experienced no difficulties as regards the fixation of the embryonic material. Any one of the ordinary fixing agents, such as picro-sublimate, Perenyi's fluid, or a 10% solution of formaldehyde/ will produce good results. The fixation of the pouch young, however, is a very difficult matter. In these the epitrichium is so impervious to the penetration of fluids that the writer has been unable to find any fixing
 +
 +
' I take much pleasure in expressing my thanks and appreciations to the following gentlemen for the unusual courtesies they have extended to me in connection with the preparation of this paper: To Professor Charles S. Minot of Harvard University for the loan of the opossum material in the Harvard Embryological Collection; to Dr. Bremer of Harvard University for three opossum embryos and three pouch young; to Dr. J. P. Hill of the University of Sydney for an embryo of Dasyurus; to Professor Bashford Dean of Columbia University for a number of kangaroo pouch young; to Mr. Stephen S. Palmer of New York for funds necessary to cover the cosi of several of the figures and plates in this paper, and to Professor Macloskie and Mr. Silvester of Princeton University for many helpful suggestions.
 +
 +
A 10 per cent solution of the 40 per cent commercial formaldehyde.
 +
 +
 +
IGG X'cnous Syslom of I )iilcl|iliys Miiisupialis (Tj)
 +
 +
iiiit'ut that, with the usual after-treatment, will fix the tissues of the older pouch vouiiig ^vithout, at the same time, producing a considerable shrinkage. Although I have not yet had the opportunity of trying this method, I am inclined to believe that shrinkage can only be avoided by removing the epitricliiuui liefore tlie pouch young are placed in the fixing ageut.
 +
 +
Two methods of staining which proved to be most satisfactory for the study of blood vessels were a combination of Delafield's hematoxylin and picric acid and one of bleu de Lyon and safranin.
 +
 +
It is evident from the recent investigations of Lewis, 02, that the development of the postcaval vein in mammals cannot be adequately considered without taking into account the role played by the subcardinal veins, since he has shown that a portion of the right subcardinal in the rabbit enters into its formation. Lewis' description of the subcardinal veins and his conclusions regarding the origin of the postcava in the rabbit, are given in the following quotation from his paper (page 241) :
 +
 +
" Small vessels from the mesentery pass into the cardinals. They anastomose in front of the aorta with vessels of the other side. They form a longitudinal anastomosis parallel with the cardinal vein, with which it is connected b}^ numerous short veins, and from which it is separated by a line of mesonephric arteries. This longitudinal vessel connected with the cardinal vein at l^otli ends, and bilaterally symmetrical in its early stages is tlic subcardinal vein."'
 +
 +
" The cross connections between the subcardinal veins give place to a single large cross anastomosis caudad to the origin of the superior mesenteric artery. Above this anastomosis the right subcardinal connects with the liver and rapidly enlarges; the left subcardinal becomes very small— Hochstetter says that it forms the left suprarenal of tlie adult. Below the anastomosis the subcardinals cease to exist as veins; they may persist as lymph spaces."
 +
 +
" The vena cava inferior is a compound vessel composed of parts of the heart, the vena hepatica communis, the hepatic sinusoids, the upper part of the right subcardinal, and the lower part of the right cardinal vein."
 +
 +
Miller, 03, under the direction of the writer, has followed the development of the postcaval vein in birds and has likewise noted and described a system of veins in the embryo which corresponds exactly to that described by Lewis in the rabbit as the subcardinal system of veins. He also found in birds that a portion of the right subcardinal vein, as in the rabbit, enters into the formation of the adult postcava and that, in
 +
 +
 +
 +
Cliavles F. W. McClure ■ 167
 +
 +
addition to this, the subeardinal veins persist in the adult as the left suprarenal and genital veins.
 +
 +
The veins which Lewis and Miller have described under the name of " subcardinals " were, so far as known to the writer, first described in the embryos of birds and mammals by Hochstetter, 88 and 93, who regarded them as the homologues of the revehent veins of the Wolffian bodies in reptiles. In tracing their subsequent development, however, Hochstetter found that they disappeared for the most part, and were represented in the adult by only the left suprarenal and possibly the genital veins in the chick, and by the left suprarenal vein in the rabbit.
 +
 +
In addition to the mammals, Lewis, 04, has also recently described the subeardinal veins as met with in the selachians (Torpedo and Acanthias). .amphibians (Necturus) and reptiles (Lacerta) and, in the writer's opinion, has correctly interpreted the role which these veins play in the formation of the adult renal portal system. He states that in Torpedo and Acanthias, after fusing to form the genital sinus, the subcardinals make connections anteriorly with the cranial ends of the postcardinals and with the latter form the revehent veins of the adult renal portal ' system. In N'ecturus the subcardinals fuse to form an unpaired vessel, which, after making connections with the hepatic circulation, constitutes the greater portion of the postcava. In Lacerta the subcardinals also form a large part of the postcava, although the fusion between the two veins is less complete here than in Xecturus (Lewis).
 +
 +
Lewis, so far as known to the writer, was the first investigator to interpret the development of the venous system of the selachians and amphibians in the terms of the subeardinal veins, and, although I feel confident his interpretations are correct, at the same time a more thorough investigation of the amphibia is to be desired before any definite conclusion can be established. In reptiles, however, Hochstetter, and, more recently, my pupil Stromsten, 05, have conclusively shown that the veins which form a large portion of the postcava are the homologues of the so-called subeardinal veins of birds and mammals. To what extent the subeardinal veins may be developed in the embryos of vertebrates other than those mentioned above, it is impossible to state without further investigation. From our present knowledge of the subcardinals. however, it is evident that tliey possess so great a morphological significance in certain vertebrates, that any interpretation of the vertebrate venous system must necessarily be incomplete witliout, at least, taking into consideration the presence or absence of these veins.
 +
 +
There can be no doubt as to the morphological significance of the subcardinal veins; that their development is primarily eorrelaterl with the
 +
 +
 +
 +
168 Venous System of Didelpliys ]\rarsiipi;ilis (li)
 +
 +
presence of a renal |)()rtal system as is the ease in selachians, (amphihians), reptiles and the embryos of l)ir(ls. Their presence, therefore, in the embryos of mammals in which a renal portal system is usually wanting' is most suggestive, and indicative of a "ground-type" of venous SA'stem of which the suhcardinal veins form a constituent element. It is evident, therefore, from what we at present know of the suhcardinal veins that they can no longer be regarded as transitory structures of little importance, since they form an essential and important element of the embryonic venous system in a number of vertebrates, and are retained in the adult, to a greater or lesser degree, in accordance with the presence or absence there of a renal portal system.
 +
 +
Since the suhcardinal veins play such an important role in the development of the mammalian postcava (rabbit) it may be well, in order to better appreciate the conditions in the marsupials, to first give a comparative sketch of the transformations which these veins undergo in reptiles, birds and the rabbit. ,
 +
 +
The figures recently published by Miller and Lewis show more clearly than has hitherto been observed the striking parallelism that exists, up to a certain period, between the development of the suhcardinal system in reptiles, birds and the rabbit. In the latter stages, however, this parallelism ceases to exist, owing to the divergence from the common groundplan which occurs in birds and the rabbit in connection with a partial degeneration of the suhcardinal veins. For convenience of description, therefore, the transformations which the suhcardinal veins undergo will be considered as they occur under the following periods: I. Period of parallelism — (a) before postcava is formed, (b) after postcava is formed; II. Period of divergence.
 +
 +
I. Period of Parallelism.
 +
 +
(a) Before Postcava is Fanned. — According to Hochstetter, 92, upon whose investigations the following account of the development of the reptilian venous system is based (Lacerta), the veins which subsequently become the Vv. revehentes anteriores and posteriores of the mesonephroi at first convey blood to these organs. These veins are branches of the caudal vein in Lacerta, but in Tropidonotus arise independently of this vein at the caudal end of the mesonephroi. They consist at first of two bilaterally symmetrical vessels (Text Fig. 1) which lie on the ventromedial side of the mesonephroi (see Hochstetter, 92,
 +
 +
' See reference to Perameles imder Resume and General Considerations (page 223).
 +
 +
 +
 +
Charles F. W. .ALcClure
 +
 +
 +
 +
169
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 +
 +
 +
Fig. 5, Plate XV), anastomose with the postcardinals, give off branches to the mesonephroi and receive tributaries from the tissue ventral to the aorta.
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LACERTA
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LACERTA
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PRECARDINAL • SUBCLAVIAN
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PRECARDINAL - -'
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SUBCARDINAL.
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HEPATICO-SUBCARDINAL JUNCTION
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SUBCARDINAL
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CAUDAL
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RIGHT POSTCARDINAL
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SUBCARDINAL SCIATIC CAUDAL --
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..POSTCAVA
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LEFT , ANT. REVEHENT SUBCARDINAL
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VENOUS RING
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OMPHALOMESENTERIC ARTERY
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Fjg. 1.
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Fig. 2.
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LACERTA
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RIGHT ANT. REVEHENT.
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\Jr
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HEPATICO -SUBCARDINAL -L X JUNCTION ^- y\
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PARS SUBCARDINALIS CROSS ANASTOMOSIS -r-p' POST. REVEHENT-'
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PRECAVA POSTCAVA
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LEFT ANT. REVEHENT
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POST. REVEHENT
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POSTCARDINAL-.'
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Fig. ?,.
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Figs. 1. 2 and 3. Diagrams illustrating the development of the veins in Lacerta. After Hochstetter.
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At a certain ])oriod of development the sul)carilinal vi'ins of !)irds (chick, ninety hours) and the ral)bit (twelve days and twelve hotirs) have recently I)een shown ])y ^liller (03, Figs. 1 and 4) and Lewis (02, 12
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ITO A'oiious System of Didclpliys Marsupialis (L)
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Toxt Fig. 7 and Figs. 1 and 2, Plate 1), rospeetively, to attain a high degree of develo])nient and to consist, as in reptiles, of two l)ilaterally symmetrical vessels wliich hold the same relation to the mesonejihroi and postcard inal veins as the hubcardinals do in reptiles.
 +
 +
(I)) After Postcard is Formed. — In connection witli tlie development of the postcava in reptiles the groivad-plan of the venous system, as represented hy Text Fig. 1, undergoes considerable modification. The proximal or hepatic portion of the impaired postcava in Lacerta grows caiidad from the Y. hepatica revehens dextra and, at a point slightly craniad of the origin of the omphalomesenteric artery, anastomoses with both subcardinal veins at a point which, for convenience of description, may be designated as the hepatico-snbcardinal junction. The subcardinal veins also anastomose with each other caudad of this artery so that a complete venous ring, ventral to the aorta, is formed about the origin of the omphalomesenteric artery (Text Fig. 2). This condition is only temporary, however, since the anastomosis craniad of the omphalomesenteric artery between the subcardinal of the left side and the hepatic portion of the postcava is not long retained, with the result that the right side of the venous ring (a portion of the right subcardinal) enters into the formation of a portion of the unpaired postcava (pars subcardinalis. Text Fig. 3). Correlated with the above changes the caudal vein (Lacerta) gives up its connections with the subcardinals (Text Fig. 3) and joins the postcardinals so that the latter, after giving up their connections with the ducts of Cuvier, function as the advehent veins of the niesonephroi. The subcardinal veins, on the other hand, through their connection with the unpaired portion of the postcava, function as the anterior and posterior revehent veins of the mesonephroi. The posterior and left anterior revehent veins open into the cross anastomosis between the subcardinals behind the omphalomesenteric artery ; wliile the right anterior revehent vein opens into the unpaired portion of the postcava, somewhat craniad of the anastomosis at the hepatico-subcardinal junction (Text Fig. 3).
 +
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A ground-plan of the venous system similar to that last descrilied for reptiles (Text Fig 3) is also met with in the embryos of birds (chick, five days incubation) and the rabbit (thirteen days) as described and figured by Miller (03, Fig. 6) and Lewis (02, Figs. 3 and 4, Plate 1" and Figs. 5 and 6, Plate 2), respectively. In the case of both the birds (Text Fig. 4) and the rabbit (Text Fig. G) the subcardinal veins have anastomosed with each other caudad of the origin of the omphalomesenteric artery and the right subcardinal has been " ta])ped "' by the hepatic circulation at the hepatico-subcardinal junction. The subcardinal sys
 +
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Charles F. W. :\IcChire
 +
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171
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RIGHT ANT. REVEHENT
 +
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HEPATICO SUBCARDINAL JUNCTION""
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PARS
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SUBCARDINALIS
 +
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POSTCAVA
 +
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CROSS --• ANASTOMOSIS POST. REVEHENT
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 +
 +
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, POSTCAVA
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 +
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LEFT ANT. REVEHENT
 +
 +
 +
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-. POSTCARDINAL
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OMPHALOMESENTERIC ARTERY
 +
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EXT. ILIAC
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PRECAVA --'
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CROSS ANASTOMOSIS
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GENITAL -,
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INT. ILIAC POSTCARDINAL
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POST. VERTEBRAL
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LEFT
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SUPRARENAL
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EXT. ILIAC 'GREAT RENAL
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Fig. 4.
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Fig. 5.
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RABBIT
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RABBIT
 +
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RIGHT ANT. REVEHENTHEPATICO-SUBCARDINAL JUNCTION
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PARS SUBCARDINALIS CROSS ANASTOMOSIS
 +
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POST. REVEHENT POSTCARDINAL
 +
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/ POSTCAVA
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\ J
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>
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POSTCARDINAL
 +
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POSTCAVA V ^
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^
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/ ,'
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LEFT
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PARS V 1
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SUBCARDINALIS » 1
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 +
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' , -/'SUPRARENAL
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ANT. REVEHENT
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CROSS ANASTOMOSIS '^M
 +
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^
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^S^ LEFT
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OMPHALO
 +
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RIGHT ^^m
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f^'RENAL
 +
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MESENTERIC
 +
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 +
renal" 1
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,\
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ARTERY
 +
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SPERMATIC --/ 1
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POSTCAVA '
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POSTCARDINAL
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\
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/ SPERMATIC /
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■* EXT. ILIAC
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Fig. 6.
 +
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Fig. 7.
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 +
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Figs. 4 and 5. Diagrams illustrating the development of the veins in birds. After Hochstetter and Miller.
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Figs. 6 and 7. Diagrams illustrating the development of the veins in the rabbit. After Hochstetter and Lewis.
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172 Venous SystiMr. of I)i<lolj)liys Mai'supialis (T.)
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 +
tiMu is now represented in birds and the rabbit, as in reptiles, (1) by an anterior and posterior pair of revehent veins which hold the same relations to the unpaired postea\a and cross anastomosis between the subcardinals, as the anterior and posterior revehent veins in reptiles; and (2) by a portion of the unpaired postcava (pars subcardinalis) which consists, approximately, of that portion of the right subcardinal vein which is included between the hepatico-subcardinal junction and the cross anastomosis.
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 +
It is evident from Hochstetter's figures and description of the veins in Lacerta that the right side of the venous ring which is formed around the origin of the omphalomesenteric artery, and which enters into the formation of the unpaired postcava is derived from the right subcardinal vein. Such being the case, we then have in Lacerta a portion of the unpaired postcava which corresponds in its relations to the subcardinal portion of the postcava descril)ed above for birds and the rabbit, since the right side of the venons ring in Lacerta is composed of that portion of the right subcardinal, which is included between the hepatico-subcardinal junction and the original cross anastomosis between the two subcardinals.
 +
 +
Miller, 03, has shown that in chick embryos the subcardinal veins may occasionally, as in reptiles, form a venous ring around the origin of the omphalomesenteric artery and has kindly permitted the writer to pul)lish his reconstruction of the same (Text Fig. 8). Although Miller did not publish this figure in his paper, he descril)ed it as follows on page 291: "At about the stage from which Fig. 6 was taken (fifth day of incubation) the writer found a most interesting exception to the general plan of development of the subcardinal system in birds, which exception shows a striking combination of the conditions described by Hochstetter in reptiles and Echidna. Anterior to the origin of the A. omphalomesenterica and ventral to the aorta there is present a large anastomosis between the right and left sulK-ardinals, just caudal to the point where the postcava joins the right sul)cardinal. Such a renuirkable similarity to the conditions found in the earlier stages of reptilian development is certainly unusual. Tlie reference to Echidna mentioned in the above quotation does not refer to the formation of a venous ring al)out the artery, but to the secondary anastomosis between the subcardinals, as shown in the reconstruction.
 +
 +
There can be no doubt as to the sul)cardinal character of the right side of the venous ring in Miller's figure of the chick and, also, that it corresponds, in all essential details, to tlie right side of the ring in Lacerta.
 +
 +
At the stages of development represented by Text Figs. 3. 4 and G, it is, therefore, seen that the unpaired ])ostcava, as thus far developed, con
 +
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C'hrtTles F. W. :\lcriuro
 +
 +
 +
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173
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 +
sists in roptilos, birds and the rabl)it of two principal subdivisions which are i>-enetically independent of each otlier ; one of which is formed betwecMi tlie sinus venosns and the liepatico-sidicardinal junction and the otlu'r between tlie hitter and the cross anastomosis lietween the sul)cardinals. Tlie latter subdivision is formed in all three cases from a portion of the riulit subcardinal vein: while the former has a somewhat different
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t
 +
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SUECARDI^-.
 +
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~r'
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POSTCAVA
 +
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r
 +
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PATiCO-sueCAR0iN'V_
 +
JUNCTION
 +
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Bt
 +
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PARS SUBCAROINALIS,
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OyPMAi.CyESE\TEC-ARTERY
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Fig. 8.
 +
 +
Fig. 8. Reconstruction of the venous system of a chick embryo of five days incubation in which the subcardinals form a venous ring about the origin of the omphalomesenteric artery as in Lacerta. After Miller. Ventral view.
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 +
origin in reptiles, bii'ds and the rabbit wbicb need not be mentioned here. There is one feature at thi.s period of development, however, in which the venous system of the rabbit differs from that of reptiles and birds. In the rabbit (Text Fig. 6) tlie unpaired postcava is connected at its caudal end with each postcardinal vein by means of large anastomoses so that a continuous and uninterrupted channel is established between the
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174 Venous System of T)i(lel|)liys i\rarsupialis (L)
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hinder end of the hody and the postcava. By this means the blood may flow directly to the heart without passing through the mesonephric circulation, as is the case in reptiles (Text Fig. 3) and the embryos of birds (Text Fig. 4 and 8) in which a renal portal system is present (see Miller, 03, Fig. (!).
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II. Period of Divergence.
 +
 +
The final changes wliicli take place in connection with the development of the venous system, subsequent to those represented by Text Figs. 3, 4 and 6, and which lead up to the adult condition are in reptiles, birds and the rabbit somewhat divergent.
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 +
In reptiles the fundamental plan of the venous system as represented by Text Fig. 3, is, with slight modifications, retained in the adult. The posterior revehent veins which remain, for the most part, separate in Lacerta and snakes and fuse to form a single vein in turtles (Stromsten, 05) function as the revehent veins of the permanent kidneys. The advehent veins, on the other hand, are formed from the caudal divisions of the postcardinals which, after giving up their connections with the ducts of Cuvier, return blood from the hinder end of the body to the permanent kidneys.
 +
 +
In order to attain the adult condition in birds (Text Fig. 5), in which a renal portal system is absent," the Vv. renales magnas (the revehent veins of the permanent kidneys) grow caudad from the caudal end of the postcava (pars subcardinalis) and, at the level of the external iliac veins, anastomose with the postcardinals. A continuous channel is thereby established, on each side, between the hinder end of the body and the postcava through which the blood may flow without previously passing through the mesonephric circulation (see Miller, 03, Fig. 7).
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The postcardinals which lie craniad of their anastomosis with the great renal veins atrophy, while, those which lie caudad of the same fuse at their caudal ends and persist in the adult as the so-called internal iliac veins (Miller). Unless the unpaired portion of the postcava together with the internal iliac veins (postcardinals) may be regarded as representing a type of bifurcated or double postcava, it is evident that the postcardinal veins or any part of the same do not, in birds, as in the rabbit, enter into the formation of the adult postcava. since the latter
 +
 +
'Parker and Haswell, 97 (page 375), figure and describe tlie presence of a partial renal portal system in adult birds (pigeon). This system in the adult, however, differs fundamentally from that in the embryo in that the revehent veins (Vv. renales magnae) are independent formations and are not formed from the subcardinal veins.
 +
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 +
Charles F. W. :\Icriur.e 175
 +
 +
toriiiinatcs in birds at the caiidal end of the pars subcardinalis which, in addition to the portion which is develoi)ed lietween the heart and the hcpatico-suhcardinal jnnction, constitutes the postcava in the adnlt.
 +
 +
The suhcardinal system is represented in the adult bird by the following veins : The left anterior revehent vein forms the left suprarenal ; the right anterior revehent probably atrophies; the section of the right subcardinal included between the hepatico-subcardinal junction and the original anastomosis between the two subcardinals forms the pars subcardinalis of the postcava, and the two posterior revehent veiris enter into the formation of the genital veins.
 +
 +
In birds the azygos veins, as met with in the rabbit, are not developed ; their place being taken, for the most part, by the newly formed posterior vertebral veins (Text Fig. 5) which open, on each side (chick) into the precava in common with the internal jugular and the subclavian veins (see McClure, 03, page oSl).
 +
 +
In the rabbit the fundamental plan of the venous system, as represented by Text Fig. (i, undergoes a number of changes before the adult stage is reached.
 +
 +
The section of the right postcardinal vein which lies caudad of its anastomosis with the pars subcardinalis of the postcava, after forming a collateral channel on the medial side of the ureter (Text Fig. 6), constitutes in the adult that portion of the postcava which lies caudad of the renal veins (Text Fig. 7). The corresponding section of the left postcardinal atrophies with the exception of a small portion at its proximal end which usually persists as the left spermatic vein, and of a portion at its caudal end wliieli fuses with the postcardinal of the opposite side to form the common internal iliac vein. The postcava of the adult rabl)it is thus seen to be a compound vessel which is formed from four distinct sets of veins: The vena hepatica communis, the hepatic sinusoids and portions of the right suhcardinal and right postcardinal veins.
 +
 +
Correlated with the completion of the adult postcava in the rabbit a number of changes, also take place in connection with the remaining portions of the postcardinal and suhcardinal veins. The postcardinals which lie craniad of the level of the renal veins entirely disappear with the exception of the proximal end of the vein of the right side which persists as the common trunk of the newly formed azygos veins. Also, with the exception of a section of the right suhcardinal which enters into the formation of the adult postcava, a portion of the left suhcardinal which forms the left suprarenal, and possibly a portion of the right which forms the right suprarenal vein ( Hochstetter, 03), the suhcardinal veins are completely lost at the time the adult stage is. reached.
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176 A'eiions SysttMii of Didclpliys ^rarsii]ii;ilis (L)
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 +
TlIK l)r.\ KI.OI'.MKXT OF THE VeNOUS SySTEM IN MaRSITIALS.
 +
 +
All of my marsupial embryos, as stated al)OY0, are too advanced to definitely determine tlie earliest stages in the development of the postcaval vein, as well as the condition presented by the subcardinal veins at a
 +
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 +
 +
DUCT OF CUVIER
 +
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POSTCARDINAL
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RIGHT HEPAT'C
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HEPATICO-SUBCARDINALJUNCTION
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MESONEPHRIC ARTERY
 +
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MESONEPHRIC ARTERi POSTCAVA PARS SUBCARDINALIS
 +
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MESONEPHRIC ARTERY
 +
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RIGHT
 +
 +
POST. REVEHENT
 +
 +
VEIN
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SUBCARDINAL
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POSTCARDINAI.
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PRECARDINAL
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POSTCARDINAL
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LEFT
 +
 +
NT. REVEHENT VEIN SUBCARDINAL LEFT HEPATIC VEIN
 +
 +
-UMBILICAL VEIN
 +
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OMPHALOMESENTERIC VEIN
 +
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MESONEPHRIC ARTERY
 +
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kNT. REVEHENT VEIN SUBCARDINAL
 +
 +
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__^ OMPHALOMESENTERIC
 +
 +
■ "* ARTERY
 +
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. CROSS ANASTOMOSIS
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LEFT POST. REVEHENT VEIN SUBCARDINAL
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POSTCARDINAL
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UMBILICAL ARTERY
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 +
Fig. 9.
 +
 +
Fig. 9. Reconstruction of the venous system of a 6 mm. embryo of Dasyurus. Ventral view.
 +
 +
time before the postcava is formed. There can be no donbt, however, so far as the subcardinal system is concerned, that it plays the same role in the marsupials, as thus far examined, as in the rabbit (Text Fig. 6), in which it enters into the formation of a portion of the postcava, as well as
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Olmrlcs F. W. :\lcriuiv 177
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into that of tlic antcrioi- and posterior rcvchont veins. This is clearly shown to h(> tlu> case by the reconstructions of the venons system of the () nun. Dasyurus (Text Fi^". !•) and S mm. Didelpliys embryos (Text Fi^y. Ill) in which the <?round-]>hin is fundamentally the same as that described by Lewis. 02 (Plate I, Figs, li and 4), for a rabbit embryo of thirteen days, where the right and left subcardinals have anatomosed in the median line, caudad of the origin of the omphalomesenteric artery, and the right suln-ardinal has been "tapped"' liy the he|)atic circulati-<in. The point at which the right subcardinal vein makes connection with the hepatic circulation is designated by the writer in the following pages as the hepatico-suhcardlniil junction.
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On account of the bilateral symmetry of its subcardinal veins the (S mm. embryo of Dasyurus undoubtedly represents a stage of development which is relativelv earlier than that of the S mm. embryo of Didelpliys, and, for purposes of comparison with the latter, a reconstruction of its venous system has been added to the text (Text Fig. 9).
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The Yexous System of the 8 :\[m. P^mbryos of Didelphys.
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The Postcardinal Veins. — In the majority of the 8 mm. embryos of Didelphys (Text Fig. 10) the postcardinal veins can be traced as continnous vessels between the ducts of Cuvier, into which they open dorsal ly, and the caudal end of the body where they are formed, on each side, through the union of the internal and external iliac veins. Caudal to the ori,gin of the omphalomesenteric artery each postcardinal vein joins the root of the postcava by means of a single large anastomosis. The postcardinals which lie caudad of this anastomosis with the postcava are vessels of large size and constitute its principal, though not direct, caudal continuation ; the latter being formed l)y the right posterior revehent vein (subcardinal). The relation of the postcardinal veins to the mnbilical arteries is most complex and will be treated more fully in connection with another topic. It may bo mentioned here, however, that the umbilical artery of each side, instead of lying ventral to the postcardinal vein as in most mammalian embryos or dorsal to the same as in Echidna (Hochstetter) and the 6 mm. embryo of Dasyurus (Text Fig. 9), is encircled by a circumarterial venous ring.
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Craniad of the anastomosis with the postcava the postcardinals are much reduced in size and slightly caudad of their union with the ducts of Cuvier each postcardinal receives a tributary which can be traced caudad for only a short distance as a continuous vessel. These two tributaries (Fig. 31, Plate II and Text Fig. 10) which lie lateral or dorso
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178
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\'eiious Svsleni oi' Uidclphys ]\rai-supialis (L)
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lateral io the aorta and ventral to its segmental branches, appear to be formed thnmuli a lonigitudinal anastomosis between branches of the postcardinals and undoubtedly represent a portion of the future azygos system of veins.
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77/ (• I'ostrava. — The ])ostcava of the 8 mm. embryos of Didelphys
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PRECARDINAL
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DUCT OF CUVIER
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RIGHT HEPATIC VEINS
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HEPATICO^SUBCARDINAf JUNCTION
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POSTCAVA PARS SUBCARDINALIS
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RIGHT
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POST. REVEHENT
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VEIN
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CARDINAL COLLATERAL
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VFiNS
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DUCT OF CUVIER
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OMPHALOMESENTERIC ARTERY
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CROSS ANASTOMOSIS
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MESONEPHRIC ARTERY OST. REVEHENT VEIN UBCARDINAL
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POSTCARDINAL 1BILICAL ARTERY — -EXT. ILIAC VEIN
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VENOUS RING
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Fig. 10.
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Fig. 10. Reconstruction of the venous system of an 8 mm. embryo of Didelphys. Ventral view.
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(Text Figs. 10 and 11), which represents only a portion of this vein as met with in the adult, is a vessel of relatively greater size than that found in the Dasyurus embryo. It extends as an unpaired vessel between the sinus venosus and a point slightly caudad of the origin of the omphalomesenteric artery Avhere it anastomoses with the right and left postcardinal veins which form its principal caudal continuation, and where it also receives the left anterior and the two posterior revehent veins.
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Cluu-lcs F. W. ]\rcClure
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179
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Between tlie sinus venosus and tlie ]ioint wliere it receives the right anterior rcvehent vein (hepatico-subcardinal junction. Text. Fig. 11) the postcava, at its cranial end, occupies a position ventral to the right lung (Fig. 31, Plate II), and further caudad is embedded in the liver. Here it receives the following trilnitaries (Text Fig. 10) : (a) One or two
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POSTCAROlNAL
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LEFT
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ANT. REVEHEN
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VEIN
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SUBCARDIN-\L
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CROSS ANASTOi:OSiS
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DUCT OF CUVIER
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POSTCAROlNAL
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RIGHT NT. REVEHENT
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VEIN SUBCARDINAL
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HEPATICO-SUBCARDINAL JUNCTION
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POST. REVEHENT VEIN
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SUBCARDINAL
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POSTCAROINAl,
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Fig. 11.
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Fig. 11. Dorsal view of the venous system of an 8 mm. embryo of Didelphys. Semi-diagrammatic.
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hepatic veins from the right side of the liver {\. hepatica reveheus dextra) ; (b) a large hepatic vein from the left side of the liver (V. hepatica revehens sinistra) which opens into the postcava in common with the hepatic continuation of the umbilical veins; (c) the continuation of the omphalomesenteric vein which, after tunnelling the liver, opens into the postcava independently of the umbilical veins and finally, (d) a number of small hepatic veins which open at irregular intervals. Be
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ISO N'ciious System of l)i(lcl|)liys M;ii'sii|»i;ilis ( L)
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twciMi till' sinus Ncnosus and tlu> hepatico-subcardinal junction (Toxt Fig. 11) the |i()steava is without doubt formed, as in tlie rabbit, independently of the right snbcardinal Ncin. 'V\]o remaining portion of the unpaired posteava, that section of the vein whicli lies caudad of the liepatico-snbcardinal junction, is formed from the right snbcardinal vein witli tlie exception of a portion near the hepatico-subcardinal junction wliieli is partially embedded in the parenchyma of the liver and which is formed from the hepatic sinusoids in conjunction with the right subcardinal vein. The term hepatico-subcardinal junction refers to the most cranial of the anastomoses that may exist between the right subcardinal and the hepatic circulation (Text Fig. 11) since in some cases the section of the posteava which is formed from the hepatic sinusoids and which lies partially embedded in the liver does not fuse along its entire extent, but only at intervals, with the right snbcardinal vein. Fig. 34, Plate II, represents the section preceding and Fig. 35, Plate II, a section taken through the hepatico-subcardinal junction in which it is seen that ventrally the posteava is formed by hepatic sinusoids and dorsally by the right snbcardinal vein. Slightly caudad of the hepatico-subcardinal junction the posteava lies upon the dorsal surface of the liver (Fig. 36, Plate TI), where, as well as caudad of the liver itself (Figs. 37 and 38, Plate III), it occupies the same relative position with respect to the mesonephros and suprarenal body and, with the exception of those from the liver, receives the same class of tributaries as the anterior revehent vein of the left side (left snbcardinal). These tributaries are veins from the suprarenal body, the mesonephros, the genital anlage and from the tissue ventral to the aorta. Finally, the ]iars subcardinalis of the posteava does not, as in Dasyurus, anastomose at intervals along its course with the right postcardinal vein ; the absence of such connections being probably correlated with the degeneration of the postcardinal vein.
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The Anterior Reveheiit Veins. — The right anterior revehent vein (Text Fig. 11 and Fig. 34, Plate II) is derived from that portion of the ri,ght snbcardinal wdiich lies craniad of the hepatico-subcardinal junction. The left anterior vein (Figs. 10 and 11 and Figs. 34, 35 and 3(i, Plate II, and Fig. 37, Plate III) consists of that portion of the left snbcardinal which lies craniad of the anastomosis (Fig. 38, Plate III) between the two subcardinals. In the 8 mm. embryos of Didelphys this anastomosis between the two subcardinals (cross anastomosis) is, as a rule, more extensive and complete than in the 6 mm. embryo of Dasyurus so that the left anterior revehent vein usually has the appearance of opening into the posteava rather than being directly continuous caudad, as in the Dasyurus embryo (Text Fig. 9), with the left posterior revehent vein. In one
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Charles F. W. :\[eClure.
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181
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of till' S inni. enil)i-V()s, however, th(> fusion hetween the two suheardinals was not as complete as in some of the others so that the left anterior revehent vein could be traced directly craniad from the left posterior revehent vein (See Text Fig. 12). Both of the anterior revehent veins occupy the same relative position in the embryo with respect to the suprarenal bodies and the mesonephroi and, with the exception of the direct anastomoses with the postcardinals which are wanting, receive the same class of subcardinal tributaries as the corresponding veins in the (> mm. embryo of Dasyurus.
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The Posterior Eerelieitt Veins. — The right and left posterior revehent veins (suheardinals) which lie caudad of the cross anastomosis can. in
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RIGHT ANT. REVEHENT
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VEIN SUBCARDINAL
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HEPATICO-SUBCARDINALJUNCTION
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POST CAVA PARS SUBCARDINALia
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CROSS ANASTOMOS
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LEFT
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ANT. REVEHENT VEIN SUBCARDINAL
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POSTCARDINAL
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POSTCAROINAL
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rOSTCROINi
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V
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POST. REVEHENT
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VEINS
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SUBCARDINAL
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Fig. 12.
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Fig. 12. Reconstruction of the venous system of an 8 mm. embryo of Didelphys in tlie region of the original cross anastomosis between the suheardinals. Ventral view.
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most of the 8 mm. emlnwos, be traced as continuous vessels between the cross anastomosis, into which they open cranially, and the hinder end of the body where they sometimes aid in the formation of the venous rings which encircle the uml)i Ileal arteries. Each posterior revehent vein lies ventral to the mesonejihric arteries on the medial side of the mesonephros and receives tributaries from the latter as well as from the genital anlage and tis.>ue ventral to tlie aorta (Fig. 39, Plate III). Each vein also anastomoses at intervals along its course with the postcardinal vein of the same side as well as with a complicated system of vessels which, for the most i)art, lies doi'sal or dorsohilcral to it and which I shall describe under the name of the cttrdiiKil colhilenil system of veins ( \'v. cardinales collaterales).
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182 Voiious Systom of Didclpliys Marsupialis (Ti)
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C'onvlatcMl with tlic atropliy of the niosoucplwoi. tlu> cai'diiial collateral veins, or veins which are tlerived from them, assume the function of the postcardinals in returning' the blood from the hind limbs and pelvic region to tlu' root of the ))ostcava (|)ars suhcai'dinalis) ; and, after fusing ventral to the aorta, constitute the greater portion of the stem of the postcava whicli is developed caudad of the original cross anastomosis between the subcardinals. From a physiological standpoint the cardinal collateral veins of Didelphys may be said to correspond to that portion of the postcardinal in the cat and rabbit which is formed on the medial side of the permanent kidney and ureter, respectively.
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The Cardinal CoUatei-al Veins. — The cardinal collateral veins, as represented in the reconstructions (Text Figs. 10 and 13) and in section (Figs. 40 and 41, Plate III) constitute an extremely complicated system of vessels which, in the 8 mm. eml)ryo. are so irregular in character that it is difficult, at this stage, to assign to them any definite ground-plan arrangement which may be regarded as characteristic of these veins in general.
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In some of the 8 mm. embryos examined the cardinal collateral veins appear to be present, for the most part on one side (Text Fig. 10), while in others they approach a bilateral arrangement as represented by Text Fig. 13. They may anastomose in front with the postcardinals (Text Fig. 10, left side) or, as is usually the case, with the root of the postcava as in Text Fig. 13. They may also extend caudad, on each side, parallel to the postcardinals either as single vessels or as a network of vessels which spread out in the space ventral to the aorta as in Text Fig. 13. The cardinal collateral veins often anastomose with each other in the median line ventral to the aorta (Text Fig. 10) ; they may also form frequent anastomoses with the postcardinal and posterior revehent (subcardinal) veins and, on being traced caudad, appear, in some cases to be directly continuous with that portion of the circumarterial venous ring which encircles the umbilical artery ventrally (Text Fig. 13, left side). In a few cases the cardinal collateral veins could be traced for a short distance caudad of the circumarterial venous rings where they appeared to terminate in capillary vessels (Text Fig. 13).
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The question as to origin of the cardinal collateral veins is difficult of solution and with the material at hand impo'ssiljle to determine definitely. They do not, however, appear to be formed through a longitudinal anastomosis between the dorsal somatic branches of the postcardinals. but rather through a longitudinal anastomosis l)etween the cross connections which exist between the post and subcardinal veins.
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Having considered the postcardinal, cardinal collateral and posterior
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Charlrs F. W. :\Ieriun:>
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1S3
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iwelu'iit (siibeardinal) veins of the 8 mm. emhrvo of Didelphys, we arc now in a position to consider the circumarterial venous rings or loops which encircle the umbilical arteries.
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The Circumarterial Venous Rings. — It has been stated aljove, as well as in a preceding paper (MeClnre, 02), that in the 8 mm. embryos of Didelphys the umbilical arteries, instead of lying ventral to the postcardinal veins, as in most mammals, or dorsal to the same, as in Echidna
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RIGHT POSTCARDINA
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POST. REVERENT SUBCARDINAL
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POSTCAVA LEFT
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PARS SUBCARDINALIS ANT. REVEHENT SUBCARDINAL
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POSTCARDINAL
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NASTOMOSIS
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POSTCARDINAL VENOUS RING
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UMBILICAL ARTERV
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Fig. 13.
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Fig. 13. Partial reconstruction of the venous system of an 8 mm. embryo of Didelphys showing the cardinal collateral and posterior revehent veins and the venous rings which encircle the umbilical arteries. Ventral view.
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( Iloehstetter) and Dasyurus (Text Fig. 9), are encircled near their origin by complete circumarterial venous rings. These venous rings are situated slightly craniad of the point of junction of the external and internal iliac veins, and, so far as their general make-up is concerned, are extremely variable in character, not only in the different embryos, but even upon opposite sides of the same individual. Two main types of venous rings may be distinguished : — One in which the portion of the ring which
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184 Venous Syslciu of Diddphys ,Mai'sii])ialis (L)
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eiU'irc-K's the umhilifal artery ventrallv ])ossesses a caliber either subeiiual with or greater than that which encircles it clorsally, as in Text Fig. 10 and Fig. 42. Plate III; the other, in which the portion of the venous ring whieh encircles the umbilical artery ventrallv possesses a smaller caliber than that which encircles it dorsally as in Text Fig. 13 and Fig. 41, Plate III. The latter type of ring is by far the more comnu:)n of the two since, with one exception, it was characteristic of all the S mm. eml)rvos examined.
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As regards the veins which enter into the formation of these venous rings there is also considerable variation and, in some cases, it is quite impossible to determine definitely how these rings are formed. In all of the rings the portion which encircles the umbilical artery dorsally is formed by the postcardinal vein. The portion of the ring wdiich encircles the umbilical artery ventrallv, however, may be formed exclusively by the cardinal collateral vein as in Text Fig. 13 (left side), or by a vein which appears to be formed as the result of a fusion between the cardinal collateral and posterior revehent (subcardinal) veins. In addition to the above, in one embryo (Text Fig. 10) the circumarterial venous rings appear to be formed exclusively by the postcardinal veins, although it is impossilile to determine in this case to what extent the cardinal collateral veins may have also entered into their formation.
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The variable character of these circumarterial venous rings foreshadows the unusual variations recently described by the writer in Part I of this paper as regularly occurring in the adult, and the relationship which exists between the two will be considered in connection with another topic.
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The presence of circumarterial venous rings about the origin of the umbilical artery is not so uncommon as is generally supposed to be the case. The writer has recently observed these rings in the embryos of a lizard (Sceloporus undulatus). Hochstetter, 88, and Miller, 03, have observed them in the embryos of the chick and the English sparrow (Passer domesticus), respectively, and Lewis, (02, Figs. 7 and 8, Plate 2) has recently figured them as occurring in a rabbit emlu'yo of 1-1.5 mm. in length. In Sceloporus the portion of the ring which encircles the umbilical artery dorsally disappears before the adult condition is reached, while in birds, as well as in the rabl)it. it is the ventral portion of the ring that atrophies. In Sceloporus and l)irds the portion of the ring which is not formed from the postcardinal vein appears to be f(U-med through a longitudinal anastomosis of the somatic branches of the [jostcardinals, while in the rabl)it it appears from Lewis' figures as if it might be formed from the subcardinal vein. Whatever the case mav be, I am
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Charles F. W. McClure 185
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inclined to believe that some of the abnormalities which are met with in adult mammals, in which the internal iliac artery passes through a foramen in the common iliac vein, as described by Treadwell, 96, McClure, 00, (1) and Weysse, 03, may be accounted for on the ground that they represent instances in which these embryonic circumarterial venous rings have persisted in the adult.
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Up to this point we have considered more or less in detail the general plan of the venous system as met with in the 8 mm. embryo of Didelphys and, since the latter represents the youngest stage of Didelphys possessed by the writer, its venous system may be taken as the starting point from which may be traced the subsequent transformations that' lead up to the adult condition. From now on, therefore, and beginning with the 8 mm. embryo of Didelphys, we will trace in a connected manner through the different stages of embryos and pouch young possessed by the writer the transformations which the different portions of the venous system undergo before arriving at the adult stage.
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The Azygos Veins.
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In the adult of Didelphys there is, as a rule, but one azygos vein present and that is situated on the left side (Fig. 28, Plate I). At its cranial end it opens into the left precava about opposite the head of the third rib, while at its caudal end it invariably joins the postcava caudad of the renal veins and about opposite the second lumbar vertebra. Between its point of union with the postcava and about the middle of the tenth thoracic vertebra, the left azygos vein lies dorsal to the segmental branches of the aorta; between the tenth thoracic vertebra and its connection with the precava, however, it lies ventral to these branches (see McClure, 03, pp. 381-2 and Fig. 28, Plate I, at the end of this paper).
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The right azygos vein, when present in the adult, opens into the precava about opposite the head of the second rib. It is always a small and insignificant vessel, and its tributaries are confined to the first five intercostal spaces of the right side.
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In the 8 mm. oml)ryo of Didelphys, as stated above, each postcardinal receives a tributary slightly caudad of its junction with the duct of Cuvier (Text Figs. 10 and 11). Each tributary, which can be traced caudad for only a short distance, lies lateral or dorsolateral to the aorta (Fig. 31, Plate II) and vontral to the latter^s segmental branches. These two tributaries, as stated above, which appear to be formed through a longitudinal anastomosis between the somatic branches of the postcardinals, together with the proximal ends of the two postcardinals, undoubtedly constitute the anlages of the right and left nzygos veins. 13
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186 A'enoiis System of Didelphys Marsupialis (L)
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It is a curious fact that tlic azygos veins are more advanced in development in the 11.5-12 mm. embryos than in the youngest of the pouch young studied by the writer (10.5 mm.). This circumstance clearly proves that the opossums are not born, in all cases, at a corresponding period of development, but that some are born at a more advanced stage than others. In order, therefore, to give a connected account of the development of the azygos system it will be necessary to describe the conditions as met with in the 11.5-12 mm. embryos after those of the youngest pouch young have been considered.
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In the 10.5 mm. pouch young two azygos veins are present in the thoracic region which, as in the 8 mm. embryo, open dorsally into the ducts of Cuvier, the opening of the right vein being somewhat craniad of that of the left. These two veins, as stated above, are formed from the cranial ends of the two postcardinals as well as from veins which have united with the latter and which have probably been formed through a longitudinal anastomosis between the somatic branches of the postcardinals.
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The right azygos can be traced caudad from its connection Avith the duct of Cuvier for about 89 sections where it appears to terminate as a small capillary vessel which lies on the ventral surface of the vertebral column. The left azygos is, however, of much greater extent and can be traced caudad as a continuous vessel for about 156 sections where it then appears to terminate in the region slightly caudad of the point where the omphalomesenteric vein enters the liver. Each azygos vein, along its entire extent, lies dorsolateral to the aorta and ventral to the segmental arteries and, at intervals along its course, receives tributaries from the body walls contiguous to the vertebral column (Fig. 43, Plate III). Somewhat caudad of the apparent termination of the left azygos vein (37 sections) small capillary vessels are met with which lie in the tissue dorsal and dorsolateral to the aorta and dorsal to the segmental arteries, which become more prominent near the origin of the omphalomesenteric artery and especially so, further caudad, in the neighborhood of the permanent kidneys (Fig. 46, Plate lA-"). These vessels can be traced without difficulty caudad of the anastomosis between the pars subcardinalis and the postcardinal veins where they form frequent anastomoses with vessels which lie in the tissue ventral to the aorta (Figs. 47 and 48, Plate IV). These latter or ventral vessels with which they anastomose (Figs. 47 and 48, Plate IV) are, in my estimation, representatives of the cardinal collateral veins which have been described above in connection with the S mm. cnil)ryos. They can be traced caudad almost as far as the origin of the umbilical arteries, but whether they join the postcardinal veins at their caudal ends I am unable to determine definitelv.
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Charles F. W. McClure " 187
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The separation of the azygos SA'stem into two subdivisions (thoracic and lumbar) is a marked feature at this stage of its development. The separation may, however, be apparent rather than actual since a capillary anastomosis may exist between the two which cannot be determined in section. In later stages the two subdivisions do become connected so that the blood from the lumbar azygos tributaries is returned to the heart, for the most part, by the left thoracic azygos vein. In the 10.5 mm. pouch young, however, the large size of the azygos veins in the lumbar region precludes the possibility of any such route for all of the blood collected by them; and I am, therefore, inclined to believe that it is returned, for the most part, through capillaries directly to the postcava, which is the course pursued at a subsequent stage of development in Avhich large and frequent anastomoses are formed between this vessel and the lumbar azygos veins.
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In the 8 mm. embryo the azygos veins of the lumbar region have not as yet been formed and this region is drained by the dorsal somatic tributaries of the postcardinal veins. This circumstance leads one to infer that the lumbar azygos veins as met with in the 10.5 mm. pouch young may also be formed from branches of the postcardinals and in the same manner as a portion of the azygos veins in the thoracic region, although, on account of the lack of intermediate stages, it is impossible to determine this question.
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The azygos veins in the 11.5 mm. pouch young appear to present the same arrangement as in the preceding stage, although on account of the circumstance that the specimen was cut along the frontal plane it is difficult to determine the exact extent of the thoracic azygos veins, as well as whether a direct anastomosis exists between them and the azygos veins of the lumbar region. There can be little doubt, however, if such an anastomosis exists that it is still of minor importance as compared with that at a later stage, and that the thoracic and the lumbar azygos veins are, as in the 10.5 mm. pouch young, practically independent of each other.
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At the caudal end of the body the sections are cut almost at right angles to the long axis of the body so that in this region the lumbar azygos veins are not difficult to follow.
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In the region of the permanent kidneys and craniad of the point where the left anterior revehent vein joins the postcava (Fig. ■19, Plate IV) the lumbar azygos veins are extremely prominent and lie, for the most part, dorsal to the segmental branches of the aorta. Between its junction with the left anterior revehent vein and that with the two postcardinals, the postcava gradually approaches the aorta and in the region
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188 W'lioiis System of Didelph^'s Marsiipialis (L)
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dorsal to this section of the postcava the hiinbar azygos veins send trihiitaries into the tissue ventral to the aorta. Tliese tributaries, if they do not already anastomose by means of capillaries with the postcava are at least preparing to do so, since direct anastomoses between these two veins are of constant occurrence in this region in more advanced stages.
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Slightly caudad of the junction of the two postcardinals with the postcava the lumbar azygos system anastomoses with the left postcardinal vein( Fig. 50, Plate IV) and further caudad becomes continuous with small vessels (cardinal collaterals, Fig. 51, Plate IV) which lie, one on each side, ventrolateral to the aorta between the aorta and the ureter, and which frequently anastomose with each other ventral to the aorta (Fig. 52, Plate IV). As in th