Paper - The development of the thymus in the pig 2 (1915)

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Badertscher JA. The development of the thymus in the pig. II. Histogenesis. (1915) Amer. J Anat. 17(4): 437-493.

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This historic 1915 second paper by Badertscher describes the development of the thymus in the pig. See also: Badertscher JA. The development of the thymus in the pig. I. Morphogenesis. (1915) Amer. J Anat. 17(3): 317-337. Badertscher JA. The development of the thymus in the pig. II. Histogenesis. (1915) Amer. J Anat. 17(4): 437-493. Modern Notes: thymus | pig Endocrine Links: Introduction | BGD Lecture | Science Lecture | Lecture Movie | pineal | hypothalamus‎ | pituitary | thyroid | parathyroid | thymus | pancreas | adrenal | endocrine gonad‎ | endocrine placenta | other tissues | Stage 22 | endocrine abnormalities | Hormones | Category:Endocrine Historic Embryology - Endocrine   1903 Islets of Langerhans | 1903 Pig Adrenal | 1904 interstitial Cells | 1908 Pancreas Different Species | 1908 Pituitary | 1908 Pituitary histology | 1911 Rathke's pouch | 1912 Suprarenal Bodies | 1914 Suprarenal Organs | 1915 Pharynx | 1916 Thyroid | 1918 Rabbit Hypophysis | 1920 Adrenal | 1935 Mammalian Hypophysis | 1926 Human Hypophysis | 1927 Adrenal | 1927 Hypophyseal fossa | 1930 Adrenal | 1932 Pineal Gland and Cysts | 1935 Hypophysis | 1935 Pineal | 1937 Pineal | 1935 Parathyroid | 1940 Adrenal | 1941 Thyroid | 1950 Thyroid Parathyroid Thymus | 1957 Adrenal Pig Links: Introduction | Estrous Cycle | 1897 Pig Embryo Development Plates | 1951 Pig Embryology | Category:Pig   Historic Papers: 1894 Blastodermic Vesicle | 1903 12mm Pig | 1903 Pig Adrenal | 1905 Thymus | 1906 Testis | 1908 Pancreas | 1908 Pharyngeal Pouches | 1908 Intestinal Diverticula | 1910 Hypoglossal Ganglia | 1911 Prenatal Growth | 1911 Embryo 7.8 mm | 1916 Colon | 1916 Yolk Sac | 1918 Wolffian body | 1919 Corpus Luteum | 1919 Postnatal Thyroid | 1919 Placental Cord | 1921 Estrous and Implantation | 1922 Limb Arteries | 1924 Pig | 1937 Coronary Circulatory | 1938 Abnormal Brain PubMed Search: pig thymus development | thymus development

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The Development Of The Thymus In The Pig Ii. Histogenesis

J. A. Badertscher

From the Department of Histology and Embryology, Cornell University, Ithaca, N. Y.

Three Plates (Eight Figures)

Introduction

There is perhaps no organ in the body whose mode of development has given rise to so bitterly contested and so widely divergent views as has that of the thymus. This is particularly true of its histogenesis. The source and nature of the small round cells that make up the greatest mass of the organ in its fully developed condition; the origin and nature of its intralobular supporting structure; the origin and significance of its granular cells and of the Hassall's corpuscles; the extent to which red blood-cells and granular leucocytes are formed in it; have during the past thirty-five years attracted the attention of many investigators, and yet the only point upon which they unanimously agree is that the thymus is, in part at least, of epithelial origin. This disparity of views cannot be due to differences in the development of the thymus in different animal forms, for some workers who have extended their investigations over a comparatively wide range of species and classes of animals have found that the developmental processes involved are practically the same for the different types of animals investigated.

While the investigation presented in this paper deals with the histogenesis of the thymus as a whole, special consideration is given, (1) to the origin and nature of the small round cells, and (2) to the origin of free erythrocytes and eosinophile cells that are present in both the interlobular septa and the parenchyma of the thymus in later developmental stages. Though comparatively little attention has been paid to the development of the reticulum and the thymic bodies, they nevertheless have received a consideration sufficient to determine their origin.

Material and Methods

The material used for the histogenesis of the thymus was collected in great abundance at a packing house. Often the embryos still showed signs of life while they were being measured and prepared for the fixing fluid. The upper jaw, the cranium, and the posterior thoracic wall were removed from embryos from 10 to 20 mm. in length. The part containing the thymus was thus made comparativel}^ small and fixed well. Embryos from 20 to 55 mm. in length were treated in a similar manner and in addition the sides were trimmed and the cervical vertebrae removed in order to reduce the size of the piece. From embryos ranging from 60 to 165 mm. in length only portions of the thymus with some of the surrounding tissues were removed. From all these stages the entire superficial thjmius and thymus head, and parts of the mid-cervical and thoracic portions were procured. From embryos 180 to 280 mm. in length (full term) the superficial thymus and portions of the thymus head and midcervical segment were removed. The left thymus was usually selected in those stages from which only a portion of the organ was removed. The lengths in millimeters of the different developmental stages of which the thymus was prepared for a study of its histogenesis are as follows: 17, 20, 23, 25, 26, 27, 28, 30, 33, 35, 36, 37, 40, 42, 45, 50, 50, 55, 60, 65, 68, 78, 85, 100, 110, 115, 125, 135, 140, 165, 180, 190, 210, 230, 270, and 280. These figures represent the length of the embryos while in a fresh condition.

Helly's fluid (Zenker-formol) was aknost exclusively used for fixing the material. This fixer does not destroy the basophilic character of the cytoplasm of the lymphocytes and apparently produces no appreciable alteration in the hemoglobin of the red blood-cells found in the thymus. A few embryos of different developmental stages were fixed in Zenker's fluid, mainly to check up the results of the work done by investigators who emploj^ed this fixer for a histogenetic investigation of the thymus. It was found that Zenker's fluid is not at all suitable for work of this nature since, to a large extent, it destroys the basophilic character of the lymphocytes, the preservation of which is of inestimable value in tracing out the origin of the first lymphocytes found in the thymus. The tissue was embedded in paraffine and cut in sections 3 to 5 /x in thickness. Only such sect'ions as were desired were spread on slides.

These always included sections of the superficial thymus, the thymus head, and cervical and thoracic segments.' For the preservation of cells with basophilic granules the material was fixed in 95 per cent alcohol.

Basting's modification of Nocht's Romanowsky blood stain proved to be of the greatest value for this work and was almost exclusively used in the investigation of the histogenesis of the thymus. In properly differentiated sections the cytoplasm of the lymphocytes, which has a distinctly basophilic character, stains a light blue, while the cytoplasm of the epithelial cells of the thymus and that of the mesenchymal cells stains a light red color. The Ijonphocytes can thus be distinguished from the epithelial nuclei with comparative ease. Erythrocytes and the granules of eosinophile cells are stained intensely red, while the granules of the cells with basophilic granules (fixed in 95 per cent alcohol, are stained blue. In tissue fixed in Zenker's fluid (used by Bell) the basophilic character of the cytoplasm of the lymphocytes is lost, thus rendering it diflricult to distinguish a large or medium sized lymphocyte from some of the smaller epithelial nuclei of the thymus. Mallory's connective tissue stain was also used for staining the connective tissue fibers of the thymus.

1 For a discussion of the different regions of the thymus in the pig reference should be made to Part I of this investigation ('14).

Historical

Only a brief historical sketch will be given to outline, in a general way, the views regarding the histogenesis of the thjmius. For a comprehensive review of the literature on this subject reference should be made to Hammer's work of 1910.

The investigations that have been made of the histogenesis of the thymus of various classes and species of animals have led to the formation of two general theories, viz., the pseudomorphosis theory and the transformation theory, each of which has been more or less modified by the difi"erent investigators of this subject. As it is beyond the scope of this work to give a detailed discussion of each theor}^ and its modifications they will be discussed only in a general way. The pseudomorphosis theory will be first considered. In its original setting this theory held that the epithelial anlage of the thymus is gradually invaded by mesenchymal and adenoid tissue. This process displaces the epithelial cells and the only remnants of them in the fully developed thymus are the Hassall's corpuscles. This view was held by Maurer for the thymus of teleosts ('86) and for the thymus of Urodela and Anura ('88). He gives no detailed description of the development of the reticulum, or of the invasion of the epithelial anlage of the thymus by the lymphocytes.

Von Ebner held a somewhat modified view of the pseudomorphosis theory as set forth above. According to him, the reticulum and Hassall's corpuscles of the medulla are derived directly from the cells of the original epithelial anlage, while the entire cortex with its reticulum, lymphocytes, and blood vessels, and also the lymphocytes of the medulla, are of mesenchymal origin.

The latest modification of the pseudomorphosis theory which is now generally accepted by investigators belonging to this school, was set forth by Hammer in his investigations of the thymus of human embryos ('05). According to him, the reticulum of both the cortex and medulla and the Hassall's corpuscles are of epithelial origin. He then was in doubt as to the origin of the lymphocytes in the thymus, but called attention to the presence of 'wanderzellen' (lymphocytes) in the immediate vicinity of the organ before and some time after they were present in it. He also observed darkly stained cells in the thymus of early developmental stages which, with only moderately high magnification, could easily be mistaken for lymphocytes, but with very high magnification could readily be recognized as degenerating epithelial cells. He thus cast a doubt on the origin of lymphocytes from the epithelial cells of the thymus, as is still held by some investigators, and pointed out as probable an infiltration of the thymus with extrathymic lymphocytes which have migrated into it from the surrounding mesenchyme. This last view he was unable to prove conclusively on account of a lack of sufficient range of developmental stages. His investigations on numerous developmental stages of the Teleostean thymus ('08) also led him to conclude that the fixed elements of the thymus are of epithelial origin. He fully believed, however, that the lymphocytes first present in the thymus of the Teleost migrate into it from the mesenchyme and there, through repeated division, give rise to the numerous lymphocytes found in it in its fully developed condition. In his latest work ('11) on the development of the hmnan thymus he makes no mention of a migration of lymphocytes into the thymus from the surrounding mesenchyme.

Maximow in his work ('09 b) on the developing thymus of mammals (rabbit, guinea-pig, cat, rat and mouse) and on the thymus of the Axolotol ('12) also holds that the fixed elements of the thymus are of epithelial origin, while the lymphocytes first present in that organ have migrated into it from the mesenchyme and, through repeated division, form the numerous small lymphocytes of the thjmius in later developmental stages.

His views of the developing mammalian thymus are, therefore, similar to Hammer's views of the developing Teleostean thymus.

The chief exponents of the transformation theory are Prenant ('94), Maurer in his later work ('99), Bell ('06)', Stohr ('06), and Dustin ('11). The main point of this theory which they most strenuously defend is that the lymphocytes arise from transformed epithelial cells of the thymus. The epithelial cells proliferate rapidly. A part of the daughter cells transform into ' lymphocytes while the undifferentiated portions continue to proliferate and are the source of succeeding generations of epithelial cells and lymphocytes. All hold that the epithelial cells give rise to the reticulum and Hassall's corpuscles, excepting Dustin ('11) who claims that the reticulum is of mesenchjTnal origin. These investigators regard the small round cells of the thymus as real lymphocytes.

Stohr also derived the small round cells of the thj^mus from epithelial cells, but claimed that except for their sunilarity in structure to the small lymphocytes in the blood, they have nothing in common with them. They never enter the blood stream, they remain epithelial cells as long as they exist, and have the power to enlarge and change back to typical epithelial .cells. In the medulla, according to Stohr, are found real lymphocytes that have entered from the blood. He, however, fails to explain how they can be distinguished from the small round cells (epithelial cells) when they lie side by side. He also derives the reticuhun and Hassall's corpuscles from the epithelial cells of the thymus.

It is now generally accepted that the thymic bodies are derived from the epithelial cells of the thymus. Hammar and Bell have given a thorough description of their development.

From this brief historical sketch it can be seen that the nature of the developnient of the thymus is by no means a settled question. I wish to state at this point that the results of my investigation of the histogenesis of the thymus agree with these of Hammar ('08) and Maximow.

A brief historical sketch relative to the origin and development of the free erythrocytes and granular cells of the thymus will be given in connection with their consideration.

Histogenesis

To determine the origin of the different cellular elements that are found in the fully developed thymus it is necessary to begin with stages in which the thymus is purely epithelial, and to use a differentia stain by which one can definitely distinguish a l3rmphocyte from an epithelial cell. The latter fact was emphasized by Maxim ow ('09 b) who accomplished this differentiation by fixing the tissue in Helly's fluid and staining with eosin-azure. A similar differential staining was accomplished by me by fixing the tissue, as stated above, in Helly's fluid and staining with Hasting's Nocht's blood stain. The material prepared for the histogenesis of the thymus begins with an embryo 17 mm. in length. Of the many stages that were prepared and examined there are chosen for description only a few series of successively older stages, each of which is decidedly advanced in development over the previous stage and yet closely enough connected with it so that the developmental history will be continuous.

The histogenesis of the thymus may conveniently be divided into epochs, each of which is characterized by more or less distinct developmental features. They are: (1) a purely epithelial epoch which extends from its earliest development as an outpocketing from the third pharyngeal pouch and the formation of the cervical vesicle to the appearance of the first lymphocytes in the epithelial anlage of the organ; (2) The epoch of lymphocyte infiltration and lymphocyte proliferation, and the formation of the reticulum. This epoch begins with the appearance of the first lymphocytes in the thymus. The invasion of the thymus by lymphocytes from the surrounding mesenchyme continues probably up to stages 180 mm. in length while the proliferation of the lymphocytes in the thymus still continues in full term embryos and doubtless after birth. During this epoch the cortical and medullary portions of the lobules appear first in stages ranging from 65 to 75 mm. in length. The reticulum, which according to the nature of its development is formed gradually, is fully developed in embryos 180 mm. in length; (3) the epoch of the formation of red blood-cells and the development of granular leucocytes. Although an occasional red bloodcell is formed in the thymus shortly after the appearance of the first lymphocytes, this epoch properly begins in embryos about 55 mm. in length for it is at this developmental stage that erythrocytes are beginning to be formed in comparatively large numbers. Granular cells appear first in appreciably large numbers in embryos 125 mm. in length. The formation of both erythrocj^tes and granular cells in the thymus still continued in the full term embryo.

1. The purely epithelial epoch

A 23 mm. embryo is the first stage in which the histological structure of the thymus will be described. The thymus at this stage is a purely epithelial structure. It has the form of a greatly elongated mass of protoplasm and is a syncytium. No cell walls are present. The cytoplasm of the superficial thymus, the thymus head, and the mid-cervical and thoracic segments contain many vacuoles which vary in size anjovhere from the (apparent) size of a small pinhead to that of an epithelial nucleus when magnified 1300 diameters. The vacuoles in the intermediary and cervico-thoracic cords are comparatively few in number. Fine, branching, and rather deeply stained protoplasmic threads give the syncytium a distinctly reticular appearance. These protoplasmic threads, however, must not be confused with the reticulum in later developmental stages. The outer surface of the enlarged portions of the thymus is already quite irregular, being studded over with blunt epithelial buds which are the beginnings of lobules. No basement membrane is present.

The form and size of the epithelial nuclei vary considerably. While the majority are slightly ellipsoidal in shape, some are spherical and others slightly irregular in outline. They are quite regularly distributed through the different segments of the organ, lying farther apart in the more vacuolar regions than in the intermediary and cervico-thoracic cords where only a few vacuoles are present. Those lying near the surface are quite regularly arranged. The long axis of the oval ones is usually perpendicular to the surface. The more centrally located cells have no regular arrangement. A very distinct nuclear membrane is present. They possess a quite rich supply of chromatin which is distributed mostly in the form of fine threads, giving it a reticular structure. About one-half of the nuclei possess two nucleoli while the other half contains but one. Occasionally one can be found with three nucleoli. They are large and occupy no definite position in the nucleus. In the oval nuclei they may lie near the ends or near the center, while in the round nuclei they may occupy an eccentric position. The nuclei at this stage do not all stain with the same intensity. In the enlarged segments of the thymus some stain much more intensely than the majority and with only moderately high magnification could easily be mistaken for transforming stages leading to the development of lymphocytes, which, however, is not the case. A consideration of their real significance will be given in connection with a discussion of the origin of the lymphocytes in the thymus. Nuclear division at this stage goes on rapidly in the enlarged segments of the thymus. Even with a magnification of 1300 diameters often three nuclei in mitotic division can be brought into a microscopic field.

To determine whether or not cells migrate into the thymus it is necessary to make a study of the connective tissue, at least in the earlier stages, as painstaking as the study of the thymus itself. The mesenchymal cells are of the spindle or stellate type. Their protoplasmic processes often unite with those of neighboring cells. Many fine fibers are scattered in the meshes between the cells giving the appearance of a network. The mesenchyme so closely invests the thymus that in places the cytoplasmic processes of the mesenchymal cells appear to be fused with the cytoplasm of the epithelial cells. The nuclei are large and spherical or oval in shape, and contain about as much chromatin as the epithelial nuclei of the thymus.

Large lymph ocytes^ are found scattered here and there throughout the mesenchyme of the neck and upper thoracic regions which were the only regions examined. They are characterized by a wide rim of basophilic, nongranular cytoplasm and a large nucleus containing a generous amount of chromatin. Their shape varies; some are nearly spherical while others have an irregular outline with one or more projecting pseud opodia. When treated with Hasting's Nocht's blood stain the cytoplasm takes on a distinct bluish hue the deepness of which may vary in different lymphocytes found in a single section, thus indicating that some are more basophilic than others. The relation of faintly stained to the more deeply stained cells will be considered later on in this paper. The nucleus is sharply defined from the cytoplasm by a distinct nuclear membrane. The chromatin is in the form of irregular and deeply stained granules which vary much in size. Some of the granules adhere to the nuclear membrane, while others are scattered in the less deeply stained nucleoplasm. In most nuclei only one nucleolus is present but some contain two. The shape of the nucleus often conforms to the shape of the cell body. In round lymphocytes it usually is round while in the irregular shaped lymphocytes it may also be irregular in shape. It is impossible to mistake an irregularly shaped lymphocyte with its blue stained cytoplasm and its nucleus rich in chromatin for a spindle or stellate shaped mesenchjnnal cell with slightly reticulated and lightly red stained cytoplasm and a nucleus containing considerably less chromatin.

• The 'Wanderzellen' of Maximow and other investigators are regarded as being identical to the large lymphocytes.

While the lymphocytes are scattered singly throughout the entire mesenchyme, local accumulations are also found. These are most pronounced in the upper thoracic region near the large blood vessels and the thoracic segments of the thymus. Most profound growth activity is apparent in the mesenchyme surrounding the thoracic segments and it is here that transition stages from mesenchymal cells to lymphocytes frequently occur. Large lymphocytes are quite numerous. Even with a magnification of 1300 diameters five were found in a single microscopic field. Those with only a slightly basophilic cytoplasm are quite numerous.

Other cellular elements such as giant cells, the megaloblasts of Maximow, normoblasts and definitive erythrocytes also occur. These elements and the granular cells found in the thymus in later developmental stages are discussed further on in this paper.

The occurrence of lymphocytes in the mesenchyme before they are present in the thymus is of the greatest significance from a histogenetic view point, for Bell ('06) in describing the thymus of a 45 mm. pig embryo says: 'There are no lymphocytes in the connective tissue around the thymus or in the blood at this stage," and of a 70 mm. embryo he says: "There are a few lymphocytes outside the thymus in the interlobular tissue in this region; * * * * j have never seen lymphocytes outside the thymus where there were none inside it, but they appear outside shortly after they are formed here." The results of my observations are contradictory to those of Bell. In a 17 mm. embryo an occasional lymphocyte can be found in the mesenchyme while in the 23 mm. embryo an occasional lymphocyte was found in the blood. His failure to detect lymphocytes in the mesenchyme in stages less than 70 mm. in length was perhaps due to the use of Zenker's fixing fluid which, as stated above, destroys the basophilic character of the cytoplasm. Beard ('02), in his work on the smooth skate, positively asserts that the first lymphocytes in the body are found in the thymus.

Embryo of 26 mm. In this stage the lobes of the superficial thymus, the thjmius bead, and the thoracic segments have greatly enlarged. Lobules are beginning to grow out from them. The cervical segment, which enters its period of rapid development a little later than the thymus head and the thoracic segment, is now quite pronounced and is studded over with short, blunt epithelial buds. No lobules have yet started to grow out from the intermediary and cervico- thoracic cords. The surface of the thymus is quite definitely marked from the mesenchjTiie but no basement membrane is present. The vacuoles in the syncytium are somewhat more numerous than in the preceding stage. They vary greatly in size. Some are in contact with the epithelial nuclei while others have no connection with them. No consideration was given to the mode of their formation. They will again be considered in a later developmental stage.

Large lymphocytes, as in the previous stage, are found plentifully in the mesenchj^me surrounding the thoracic segment of the thymus. They are more numerous in the region of the larger blood vessels of the thorax than in those parts of the connective tissue containing only smaller vessels where, however, they can be found without much searching. Around the superficial and head thymus local accmnulations now occur. In general, they are more numerous than in the joreceding stage.

2. The epoch of lymphocyte infiltration and lymphocyte proliferation and the formation of the reticulum

SO mm. embryo. The thymus of this stage is decidedly in advance of the 26 mm. stage just described. The lobules of the superficial and head thymus and the cervical and thoracic segments have greatly enlarged while those of the intermediary and cervico-thoracic cords have started to develop. The mesenchyme occupies all the spaces between the lobules and is somewhat denser than that surrounding the thymus. Blood vessels are numerous in the connective tissue septa but none are present in the lobules. At this stage the lumen of the blood vessels is comparatively large, and their walls are thin, being made up of large endothelial cells only.

The structure of the epithelial nuclei of the thymus is the same as in the preceding stage. Mitoses (fig. 1, M.e.N., also fig. 2, 37 mm.) are quite frequent. The "large dark nuclei" and "small dark nuclei (lymphoblasts)," (figs. 1 and 2, D.e.N.), according to Bell's nomenclature, are present as in the 23 mm. embryo. These I regard as epithelial nuclei in the first stages of degeneration. A completely degenerated epithelial nucleus is also present (fig. l,D.e.X'). Its chromatin has massed into deeply stained clumps which lie in a clear space that has almost the same size and shape as a normal nucleus.

The cytoplasmic syncytium of the epithelial anlage has the same general structure as that in a 23 mm. embryo. The vacuoles (fig. 1, V) are, however, more numerous and the vacuolation of the cytoplasm has reached its greatest height in this stage. No basement membrane is present but the anlage is quite sharply defined from the surrounding mesenchyme which closely invests the thymus. As in earlier stages some of the protoplasmic processes of the mesenchymal cells are apparently fused with the outer surface of the cytoplasmic syncytium of the epithelium.

The point of greatest interest and importance in this developmental stage is the presence of lymphocytes (fig. 1, L.L.) in the thymus anlage. They are present in small numbers in the superficial thymus, thymus head, and in the thoracic segment. None were found in the mid-cervical segment which in this and younger stages is not as far advanced as the head and thoracic segments. Lymphocytes, however, occur in the thymus before the 30 mm. developmental stage. In a 25 mm. embryo a single lymphocyte was found in the thymus head. None were seen in the thymus of a 26 mm. embryo. In a 27 mm. embryo one lymphocyte was found in one of eleven sections prepared from the thoracic segment. In a 28 mm. embryo only a few could be demonstrated. In the stage being described they are present in small but appreciable numbers, hence, this stage was chosen for the discussion of the origin of the lymphocytes in the thymus. Their location in the lobules varies. Some are found in or near the center of the lobules while others lie near the periphery. All the lymphocytes that were found in the thymus in this and somewhat later stages are large lymphocytes. No small lymphocytes such as make up the bulk of the organ in late developmental stages, are present. The large lymphocytes are characterized by a generous amount of nongranular cytoplasm which is distinctly basophilic in its character. This basophilic character of the cytoplasm enables one to distinguish it unmistakably from the cytoplasm of the epithelial cells. On account of their power of undergoing amoeboid movement they take on various forms. Some are round with a layer of cytoplasm of uniform thickness around the nucleus. Some are irregularly oblong with the bulk of the cytoplasm massed at one or both poles of the nucleus. Others are very irregular in outline, sending out one or more pseudopods. The size of the large lymphocytes varies somewhat but all possess a large amount of cytoplasm. In the smaller of the large lymphocytes the nucleus is proportionally smaller than in the larger ones.

The nuclei of the lymphocytes are large, but on the whole a little smaller than the epithelial nuclei, i.e., the nuclei of the large lymphocytes are smaller than the large epithelial nuclei while the nuclei of the smaller lymphocytes are smaller than the small epithelial nuclei. A distinct nuclear membrane is present. The form of the nuclei may vary considerably. Some are spherical, some oval in outline, while others have a very irregular shape. The varied forms of nuclei are undoubtedly brought about through the motile activity of the lymphocytes. The chromatin is generally distributed in the form of larger or smaller irregular granules some of which are attached to the nuclear membrane. These are often united with each other by fine irregular chromatin threads. The nucleoli are large, and usually very irregular in outline, having an extremely jagged surface. On the whole the nucleus of a large lymphocyte contains a more generous supply of chromatin than does an epithelial nucleus. The amount of chromatin present and the manner of its distribution in the two kinds of nuclei is, however, not the main feature by which one can readily distinguish a large Ijrmphocyte from an epithelial nucleus. The basophilic character of the cytoplasm of the lymphocytes is the main distinguishing feature between them. At no stage in the histogenesis of the thymus is the value of a differential stain more appreciable than at the stage marking the appearance of the first Ij^mphocytes, for, by the use of it, a lymphocyte can be distinguished unmistakably from an epithelial cell or nucleus.

The most critical developmental stages in which the source of the lymphocytes in the thymus is to be determined, are those in which only a few lymphocytes are found. The evidence indicating a transformation of epithelial cells into lymphocytes, and the evidence indicating an infiltration of the epithelial thymus by Ijinphocytes from the mesenchyme surrounding it or from the blood, were carefully followed out. The transformation theory will be first considered.

In describing the structure of the thymus of a 23 mm. embryo it was stated that not all of the epithelial nuclei stain with the same intensity. In the enlarged portions of the thymus — superficial thjnnus, thymus head and mid-cervical and thoracic segments — are found normally shaped nuclei (spherical and ellipsoidal forms; fig. 1, D.e.N.), of both the larger and smaller types, the nucleoplasm of which stains much more intensely than it does in the great majority of epithelial nuclei present. The nucleoplasm, however, does not stain with the same degree of intensity in all of the darkly stained nuclei for, in some the deeply stained nucleoplasm almost completely masks the chromatin fibrils and granules while in others the chromatin is seen some what more clearly. Thus the transition forms, observed by Bell, occur between the more usual clear type of nuclei (which I regard as the normal nuclei) and the more deeply stained ones. In the stage (30 mm.) being described they are less numerous in the intermediary and cervico-thoracic cords than in the enlarged segments of the thymus. In a single section through the thoracic segment nineteen were found. In a 23 mm. embryo only an occasionally one was found in the intermediary and cervico-thoracic cords, while in a section through the superficial thymus ten were counted, thus indicating that they appear first in those portions of the organ that develop, most rapidly. In a 17 mm. embryo none of the intensely deeply stained nuclei were present although a few were found that were stained somewhat more deeply than the great majority of normal nuclei present.

A consideration of these darkly stained nuclei is of the greatest importance, for Prenant ('94), Bell ('06), and others apparently have taken these cells to be the forerunners of the first formed lymphocytes in the thymus. A close examination of them, therefore, is necessary in order to reveal their fate. Besides the normal shaped dark nuclei others are found that are very irregular in outline and greatly distorted. Varying degrees of deformed nuclei can be found between the more deeply stained normally shaped types and the greatly distorted ones. Others are again found that are, without doubt, undergoing degeneration. Figures 2 and 5 represent each a small portion of a section through the thymus head of a 37 and 36 mm. embryo respectively and were drawn specially to show the degenerating cells. ^ In some of the degenerated epithelial nuclei {D.e.N'.) represented in figure 5 the chromatin is massed together in one, two or three clumps that stain intensely deep blue or blue-black. These chromatin masses usually lie in clear spaces around some of which the distorted nuclear membrane apparently still persists. Occasionally a slightly elongated nucleus can be found in one end of which is a clump of chromatin lying in a nuclear vacuole while in the opposite end the nuclear threads and the nucleoplasm are stained as deeply as that in the nuclei described above. Again, here and there in the cytoplasm of epithelial cells may be found larger and smaller deeply stained particles which are evidently the debris of degenerated epithelial nuclei (fig. 5, D.d.e.N.). To the succession of microscopic pictures — deeply stained and normal shaped nuclei, deeply stained and distorted nuclei, and nuclei that have fallen to pieces — described above and drawn in figures 1, 2, and 5, only one interpretation, it seems to me, can be given, namely, that the deeply stained normal shaped epithelial nuclei are on their way to degeneration and do not transform into lymphocytes. Some of the epithelial cells show signs of degeneration in comparatively early stages as stated above. They are, however, most numerous and most pronounced in stages from about 30 mm. to about 45 mm. in, length. They are also found in later stages and will be referred to again.

' Judging from the greater number of lymphocytes present and the great number of completely degenerated epithelial cells, the thymus of the 36 mm. embryo is slightly farther advanced in its development than that of the 37 mm. stage.

With a proper fixer, a suitable stain, and a high magnification, such as was used for this work, the plump lymphocytes stand out in sharp contrast to the dark epithelial nuclei. Now, what structural features of the dark epithelial nuclei give them a semblance to small lymphocytes? As the amount of cytoplasm around the nucleus of a small lymphocyte is meager and often difficult to demonstrate, a comparison between the two must be confined largely to the structure of the nucleus. Prenant observed that the nucleoplasm of the small dark epithelial nuclei stained so intensely as to mask its internal structure. Bell makes no mention of the affinity of the nucleoplasm for basic stains. They apparently contain no greater amount of chromatin than do the normal epithelial nuclei. Their diffused dark color is due to the affinity of the nucleoplasm for a basic stain. This is not the case of the nuclei of the small lymphocyces. Their dark color is due to the deeply stained chromatin granules which, in proportion to the size of the cells, is much greater in amount than that found in the epithelial nuclei. The nucleoplasm of the small l5aTiphocytes, which is meager in amount, but with a high magnification easily demonstrable, is quite as clear as that ordinarily found in nuclei, for example in normal epithelial nuclei of the thymus anlage. This fact alone, namely, clear nucleoplasm and an abundance of chromatin in the nucleus of a small lymphocyte in contrast to the deeply stained nucleoplasm and a smaller amount of chromatin in the small dark epithelial cells, is sufficient to cause one immediately to doubt the identity of the two kinds of cells. Prenant writes of degenerated epithelial nuclei in the thymus of a 28 mm. and later stages of sheep embryos but apparently saw no connection between them and the darkly stained nuclei. Bell does not mention degenerating nuclei.

Most of the investigators who have made a detailed study of the histogenesis of the thymus and who adhere to the transformation theory of the formation of the lymphocytes lay a great de^l of stress on the vacuolation of the cytoplasmic syncytium as a factor in the histogenesis of the small lymphocytes. This is particularly true of Prenant and Bell. The latter calls the small dark epithelial nu(;lei, while still imbedded in the cytoplasm of the epithelial cells, lymplioblasts. During the process of vacuolation some of the lymphoblasts become free and lie in vacuoles. They then are called lymphocytes. My observation on the histogenesis of the thymus in the numerous pig embryos examined warrants no distinction in the nomenclature between the two. Referring again to figures 1, 2 and 5, it will be seen that some of the small dark epithelial nuclei lie entirely in the cytoplasm and some in contact with vacuoles. Occasionally one can be found that lies free in a vacuole (none of the latter happened to be present in the portions of the sections from which the figures were drawn) . The structure of the nucleus is the same whether they lie in the vacuoles or in the cytoplasm. This also is true of the large lymphocytes (L.L.). Some are entirely imbedded in the cytoplasm of the epithelial cells while others are in contact with or entirely in the vacuoles. The structure of all is the same and there is no reason why they should not all bear the same name. The significance of the vacuoles and their mode of formation in the thymus is unknown to me. They are already present in embryos of 17 mm. in length, and slightly increased in size and number in later stages (30 to 40 mm.).

In considering the genetic relationship of the small dark epithelial nuclei, the 'lymphoblasts' of Bell, to the large dark epithelial nuclei, Bell expresses a doubt by saying that the large dark nuclei probably divide by mitosis and form the lymphoblasts." I was unable to find any of the dark epithelial nuclei in mitosis although I earnestly searched for them. To me it appears that the small and large dark nuclei are derived respectively from small and large normal epithelial cells, their degree of darkness in stained preparations depending on the extent of degeneration. Also it cannot be that the small dark nuclei are formed through a contraction of the large ones for the structure of both is the same. Prenant, in the developing thymus of the sheep, described and figured direct cell division. I was unable to find amitotic cell division in the thjonus of the pig.

The true source of the lymphocytes first present in the thynms will now be considered. Reference has already been made to the presence of lymphocytes in the thymus anlage of a 30 mm. embryo. Some are represented in figure 1 (L.L.). In slightly later stages they have become more numerous as represented in figures 2 and 5 {L.L.). All the lymphocytes present in the thymus of these early stages are large lymphocytes. No small lymphocytes are present. No transition forms from the normal epithelial nuclei to the large plump lymphocytes with a generous amount of basophilic cytoplasm can be seen. When first present in the thymus they are there in a fully developed condition. It is, therefore, evident that their source must not be sought in the thymus anlage. It was stated above that large lymphocytes were present here and there in the mesenchyme of a 17 mm. embryo. In successively older stages their numbers gradually increase until in stages ranging in length from 25 to 30 mm. they can be found in all parts of the mesenchyme without much searching. In these later stages, however, they are most nimierous in the neighborhood of the thymus and the large blood vessels in the anterior portion of the thorax. Figure 1 represents a portion of a lobule of the thymus head and surrounding mesenchyme of a 30 mm. embryo. Three lymphocytes (L.L.) can be seen in the mesenchyme, two of which have a structure identical to those in the thymus. In one (lower corner to the right) the cytoplasm has a distinctly lighter hue, i.e., less basophilic, than the other two. Only very seldom can this latter type be found in the thymus anlage (fig. 2, L.L.; lower border to the left). These will be considered farther on in the paper. One of the lymphocytes (fig. 1) is in contact with the surface of the thymus. The microscopic picture, which is reproduced in the figure, is suggestive. Since lymphocytes are found in the mesenchyme in -the neighborhood of the thymus before they are found in it, and since there are no transition forms between epithelial cells and lymphocytes nor any blood vessels in the thymus anlage, only one conclusion can be drawn in regard to the source of the lymphocytes first present in the thymus, namely, that they have ixiigrated into the thymus from the surrounding mesenchyme. The lymphocyte bordering on the surface of the thymus was apparently about to enter it when the material was fixed. Many similar conditions exist, indicating the entrance of lymphocytes into the thymus at the time of the fixation of the material.

Usually there are no indications on the surface of the lobules to mark the place where lymphocytes have entered it. On account of the plasticity of the cytoplasmic syncytium we can assume that the gaps formed in it by the entrance of the lymphocytes immediately close up. Not infrequently, however, places can be found where the surface of a lobule is dented in and a lymphocyte located in the thymus near the depression (fig. 2, L.L., lower border to the left). Also occasionally a lymphocyte can be found in a lobule some distance away from the periphery with a trail (fig. 2, T.) leading to the surface of the lobule. This trail apparently marks the path that a very active lymphocyte took in its migration from its place of entrance to the position it now occupies. To similar microscopic pictures as represented above, Maximow has given a like interpretation. The first lymphocyte present in the thymus then must come from the mesenchyme and not from transformed epithelial nuclei.

Another type of cells which are comparatively few in number and found only in the earlier developmental stages deserves mention before passing on to a later developmental stage. These cells (fig. 1, X.) are characterized by rather deeply stained nuclei which resemble closely the degenerating epithelial nuclei discussed above. The cell wall, if present, is indistinct. Their cytoplasm can be distinguished from the cytoplasm of the epithelial cells only by its darker color. The majority of the cells are long and drawn out and usually lie near the surface of the thymus anlage with the long axis of the cell nearly perpendicular to the surface. These were most numerous in the 17 mm. stage and entirely absent from the 40 mm. and later developmental stages. This type of cells was also observed by Maximow ('09 b) who derived them from epithelial cells which for a time assume such form then revert to the usual type of epithelial cells. Their origin and significance are unknown to me.

Embryo ^2 mm. (fig. 3). The lobes of the superficial thymus, the thymus head, and of the mid-cervical and thoracic segments have greatly increased in size. A few lymphocytes are now present in the intermediary and cervico-thoracic cords. No blood vessels are present in the thymus. The walls of the blood vessels of the interlobular connective tissue septa are made up of endothelium only. The mesenchyme around the superficial and head thymus and the thoracic segment is much looser in its structure than in the corresponding regions in previous stages. Around the intermediary and cervico-thoracic cords and the mid-cervical segment it has a somewhat denser structure than around the above named regions of this stage.

As in previous stages completely degenerated epithelial nuclei (fig. 3, D.e.N'.) are present. Epithelial nuclei (D.e.N.) in the first stages of degeneration are also present but they are not as numerous as in the 36 mm. embryo. Mitoses of epithelial nuclei (M.e.N.) are quite numerous. The vacuoles are not as numerous as in stages ranging from 25 to 37 mm. in length. The most striking feature of this stage, however, is the large number of lymphocytes that are present in the thymus. They no longer all belong to the type of large lymphocytes but now and then a small lymphocyte {S.L.) is found. These are characterized by a rather small nucleus which is richly laden with chromatin and surrounded bj^ only a very thin layer of cytoplasm which is often difficult to demonstrate. Intermediate stages between the large and small lymphocytes make up a relatively large proportion of all present. Some in mitotic division can be found without much searching. Mitosis of epithelial nuclei and large lymphocytes can be distinguished from each other without much difficulty. The chromosomes of the lymphocytes are shorter, somewhat thicker, and more closely packed together than those of epithelial cells. The basophilic cytoplasm of the lymphocytes also is sharply outlined in contrast to the cytoplasm of epithelial syncytium. The absence of blood vessels in the thymus at this stage, the absence of transition forms between epithelial cells and lymphocytes, the unbroken' series of intermediate stages between the large and small lymphocytes, and the frequent mitoses found among them all, are evidences that undoubtedly point to the conclusion that the large lymphocytes through repeated division give rise to the small lymphocytes. This view of the origin of the small lymphocytes in the thymus is in accord with that of Hammer for teleosts ('08) and with that of Maximow for mammals ('09 b).

The mesenchyme of the interlobular septa (S.i.) in the head and thoracic segments contains a large number of large and intermediate sized lymphocytes. A few small lymphocytes are also present. The mesenchyme surrounding the above named regions also contains a relatively greater number of lymphocytes than it does in corresponding regions of the previous stage described.

An occasional nucleated red blood-cell can be found lying free in the thymus. At this stage they are scarcely more numerous than in the previous stage. None were present in that part of the section from which the figure was drawn. Eosinophile cells also can be found occasionally in any part of the mesenchyme. They were first found in embryos 35 mm. in length. None were seen in the thymus.

Embryo of 66 mm. (fig. 4)- The lobules are more numerous than in the previous stage along the entire extent of the organ. Those of the enlarged regions of the thymus are much more voluminous than those of the intermediary and cervico-thoracic cords. An almost interrupted layer of greatly attenuated mesenchymal cells closely invests the outer surface of the lobes and apparently forms a limiting membrane (fig. 3, L.M.) for the outer surface of the thymus. This membrane is present in slightly earlier stages. Blood vessels {Bl.V.) are numerous in the interlobular septa. No thick walled vessels are yet present. The walls of most of them are made up of endothelium only. A few small blood vessels of an essentially capillary nature can now be found in the center as well as in the periphery of the thymic lobules.

The thymus now contains many lymphocytes. The small lymphocytes (S.L.) are more numerous than in the previous stage. The medium-sized lymphocytes have also greatly increased in number while the number of large lymphocytes has remained about the same. Mitoses of both the lymphocytes (M.L.) and the epithelial nuclei (M.c.N.) are of frequent occurrence. Only an occasional deeply stained epithelial cell can be found. Completely degenerated epithelial nuclei can be seen scattered here and there throughout an entire section. The connective tissue of the interlobular septa now contains numerous lymphocytes of all sizes. The deep portions of some of the septa are so completely gorged with them that it is difficult to distinguish clearly where the septa end. An especially favorable place for lymphocytes to collect seems to be along the course of blood vessels of the septa. They can be found strung along in rows on one side of the vessels or the accumulation may extend entirely around it. The vacuoles of the syncytium are not as numerous as in the preceding stage. Most of them have become occupied with lymphocytes. In later stages they are altogether absent.

An almost uninterrupted zone of epithelial syncytium (7.pr.) extends around the periphery of the thymus. It is from one to three epithelial nuclei deep and, on account of the few lymphocytes which it contains, appears quite clear in contrast to the deeper portion of the syncytium in which are found many lymphocytes. It is not pronounced along the interlobular septa. Mitoses of the epithelial nuclei are more numerous in this zone than they are in the deeper portions of the lobules, hence, Prenant called it the zone of proliferation. According to him both lymphocytes and reticulum cells are formed from this zone. This, however, cannot be the case for the transition forms from epithelial nuclei to lymphocytes are not present. Considering the fact that the epithelial zone is most pronounced only on the convex peripheral surface of the lobules, and that it is present only during the period of rapid growth of the thymus, it can rightly be regarded as a zone of proliferation for epithelial cells but not for lymphocytes. It is mainly from this zone that the reticulum of the peripheral margin of the cortex is formed while the thjanus is rapidly growing in thickness. This interpretation of the significance of this zone is in accord with that of Maxim ow.

This develomental stage marks the appearance of the medulla. Longitudinal sections through the thymus head show that the epithelial syncytium of ahnost the entire central stem has undergone changes. The medulla of the lobes, in some of which at this stage it is but slightly developed, is continuous with that of the central stem. The deep portions of some of the interlobular septa are almost in contact with it while others are separated from it by a cortical layer of considerable thickness. The medulla is formed directly from the epithelial syncytium. In sections stained with Hasting's Nocht's stain it is easily distinguished from the cytoplasmic syncytium of the cortex by its brighter red color. The initiative changes marking its appearance are apparently chemical in their nature as pointed out by Bell, for the syncytium in some parts of the central stem and in the center of some of the lobules is stained a bright red even before any morphological changes have set in. The morphological changes of the epithelial structure occur very soon after, or almost simultaneously with, the chemical changes. The epithelial cells hypertrophy. The nuclei become large and relatively clear when compared with those of the cortex. The cytoplasm of the syncytium also increases in amount. Its anastomosing processes are no longer thin and attenuated as they now appear in the cortex but have become more or less massive bands. Although the cortex and medulla are quite sharply defined the cytoplasmic processes of the epithelial cells lying along the line of demarcation between these two structures are continuous with each other.

Soon after the medulla has started to develop some of the epithelial nuclei contained in it greatly increase in size, growing much larger than the majority of hypertrophied nuclei. These may be found singly or in groups of two or three and mark the beginning -of Hassall's corpuscles.

All the different types of lymphocytes (large, medium-sized, and small) found in the cortex are also found scattered in the meshes of the reticulum of the medulla where they are, however, much less numerous than in the former place. According to Maximow ('09 b) the disappearance of the lymphocytes from the medulla, when it is first formed, is due to their migration into the cortex and to degeneration. His interpretation does not seem to explain similar conditions existing in the thymus of the pig, for only very seldom can degenerated cells be found which may be degenerated epithelial nuclei, and as to whether they migrate into the cortex it is indeed difficult to establish in fixed material when no circumstantial evidence is present indicative of their migration. A more plausible interpretation seems to be that during the hypertrophy of the epithelial cells the medullary portion of the thymus greatly increases in volume through the enlarging of both the nuclei and anastomosing processes of the syncytium thus separating the lymphocytes farther apart. Just as many are present in the rapidly newly formed medulla as there were in the syncytium from which the medulla was formed only they are scattered over a larger area making them to appear less numerous. This interpretation is made plausible when the great rapidity of its initial development is considered, e.g., in the thymus of a 60 mm. embryo no traces of the medulla were present while in a 65 mm. embryo it has reached a stage of development as described above.

The reticulum of the cortex also is formed from the cytoplasmic syncytium of the epithelial cells. Its development, unHke that of the medulla, is gradual. The change from the rather coarse syncytial network of younger stages to fine and greatly attentuated threads making up the reticulum in the fully developed thymus is due to the lymphocytes constantly increasing in numbers in its meshes thus gradually separating the cell bodies of the reticulum farther apart. In all of the developmental stages studied mitosis of the epithelial nuclei could be found, being, however, more numerous in younger than in later developmental stages.

In this and slightly earlier stages (55 mm.) nucleated and nonnucleated red blood-cells lying free in the parenchyma of the thymus are of frequent occurrence. While some are scattered about singly they usually occur in groups. An occasional eosinophile cell can also be found. In the interlobular septa phagocytes can be found without much searching.

The thymus head and superficial thymus were so oriented on the microtome that sections of both of these regions were made by a single stroke of the knife. This made a comparison of their histological structure easy as they lay side by side on the sUde. No difference in structure could be distinguished between the two, thus indicating that the histogenetic processes of that portion of the thymus derived from the ectoderm keep pace with that portion derived from the entoderm.

Embryos 85, 100, 125, 165, and 180 mm. in length. In these developmental stages all the structures found in the fully developed thymus are laid down and will, therefore, be considered only briefly. The average size of the lobules and the thymus as a whole increases in the successively older stages. In the first four stages the thymic septa have still a very loose structure while in the 180 mm. embryo they are quite narrow and correspondingly denser. Many of the septa are broadly expanded where the larger interlobular blood vessels are harbored. The thymic septa of the 85 and 100 mm. stages are characterized by the large number of all types of lymphocytes (large, mediumsized, and small) which they contain. The presence of so many lymphocytes in the septa is a feature that is most marked in developmental stages from 65 to about 115 mm. in length. The septa of the 125 and 165 mm. stages contain many lymphocytes but on the whole they are less numerous than in the earher stages cited. In the 180 mm. embryo the lymphocytes are mostly confined to the deep expanded portions of the septa which are often gorged with them. The number of lymphocytes in the connective tissue immediately surrounding the thymus is relatively small when compared with the number present in the septa. Mitoses of all types of lymphocytes are of frequent occurrence.

A marked feature of the cortex in these stages is the large number of small lymphocytes which it contains. Excepting the 85 mm. stage they make up the largest proportion of all the lymphocytes present. Mitoses of all types of lymphocytes are of frequent occurrence while mitoses of epithelial nuclei can occasionally be found. The epithelial (reticulum) nuclei are, in general, smaller than those found in younger stages but their structure has remained unchanged. The reticulum composing the strands are greatly attenuated and only in very thin sections can it be satisfactorily demonstrated. Its meshes are filled with lymphocytes. Vacuoles are no longer present.

The clear epithelial zone around the periphery of the thymus is present in all the stages excepting the 180 mm. embryo. Around the thymus head of the 165 mm. embryo it is at its highest development. This is contradictory to the observations of Bell who states that this zone has disappeared in a 140 mm. embryo. Mitoses of epithelial cells in this zone are quite numerous and in no stage is it entirely free from lymphocytes. The limiting membrane could no longer be distinguished around the thymic lobules in the 180 mm, stage. It apparently has become blended with the thin capsule that invests the thymus of this and later stages.

In all these stages the medulla contains a relatively much larger number of lymphocytes than in the 65 mm. embryo, which makes it appear less conspicuous. This is especially the case in the 180 mm. embryo, but even in that stage in suitably stained preparations it is still quite sharply defined from the cortex. Mitoses of all types of lymphocytes occur here as in the cortex. In the 180 mm. embryo Hassall's corpuscles are more numerous than in the earlier stages, while some are still in the process of formation. The reticulum on the whole is much coarser than in the cortex and hence more easily demonstrable.

In the 180 mm. embryo deeply stained (degenerating) epithelial nuclei can be found only after prolonged searching while in the earlier stages they are of more frequent occurrence in both the cortex and medulla. Debris of degenerated cells, some of which in these stages is undoubtedly composed of nuclei extruded from normoblasts, also occurs.

A discussion of the red blood-cells and granular leucocytes will be considered later.

Embryo 270 mm. (full term). Since one of the main objects of the investigation of the developing thymus was to determine the origin and fate of the superficial thymus its histological structure will, therefore, be considered. The lobules are now closely packed together. The cortex has greatly increased in thickness over that of the 180 mm. embryo. The lymphocytes are no more closely packed together than in the previous stage, room having been made for the additional number by an increase in the volume of the organ. While the small lymphocytes are by far the most numerous, large ones are still plentiful in all parts of the cortex. Even with a magnification of 1300 diameters eleven were counted in a single microscopic field. All gradations between the large and small ones are present. Mitoses of all the different types occur.

The medulla is still quite sharply defined from the cortex. It contains less lymphocytes than the cortex. In some places where the medulla of the lobules joins with that of the central stem it comes in contact with the deep portion of the interlobular septa. It contains all the different types of lymphocytes that are present in the cortex and mitoses among them can be demonstrated without much difficulty. Hassall's corpuscles are more numerous than in the 180 mm. stage and an occasional one can still be found in the process of formation.

In the medulla the strands of the reticulum are often wavy and in general are much coarser than those of the cortex. Also •the epithelial nuclei are on the whole larger and clearer, and surrounded by a more generous amount of cytoplasm than those in the cortex. Mitoses of epithelial nuclei in both the cortex and medulla can only very seldom be found. In sections treated with Mallory's connective tissue stain fibrillae can be seen to come off from the interlobular septa and the capsule and extend a distance of from one to four cells deep into the cortex. In both the cortex and medulla the same condition prevails between the adventitia of the larger blood vessels (which are very few) and the reticulum. I was unable to determine whether the connective tissue fibers fuse with the reticulum. This intimate relation of the connective tissue of the septa and of the large blood vessels to the reticulum was observed by Mietens ('08) but denied by Maximow ('09 b). No connective tissue fibers aside from those mentioned above could be demonstrated in either the cortex or medulla. Bell, however, by using Jackson's modification of Mallory's stain states that he was able to demonstrate them thinly scattered through both the cortex and medulla in all late developmental stages.

The interlobular connective tissue septa are greatly reduced in thickness, being widest in those places which lodge the larger blood vessels and at the points of intersection of two or more septa. In some places prolongations of the septa dip down into the cortex of the lobules. These secondary septa approach very nearly the medulla but seldom enter it and are usually expanded at their deeper ends where they may lodge larger blood vessels. The structure of the wider portions of the septa is usually looser than the thinner parts. Small blood vessels of a capillary nature are found through the septa and can often be seen entering the cortex. Lymphocytes are present only in comparatively small numbers. In the more compact portions of the septa they may be entirely absent.

A discussion of the red blood-cells and the granular leucocytes in full term embryos will follow.

3. The epoch of the formation of the red hlood-cells and the development of granular leucocytes

Investigators disagree as to the extent of the formation of red blood-cells in the thymus. Many have observed red bloodcells lying free in the parenchyma of the thymus during both its growth and involution but to my knowledge no extended investigation through a wide range of developmental stages has yet been made of their origin. Afanassiew (77) apparently was the first to consider their origin. He held that during the development of the thymus a rearrangement of some of the blood vessels took place resulting in the formation of the concentric (Hassall's) corpuscles. During this process some of the blood vessels are ruptured thus permitting erythrocytes as well as leucocytes to escape into the parenchyma where they then may be found singly or in groups. In mammals the red blood-cells usually underwent degeneration. He regarded the thymus a hemolytic organ.

Watney ('82) also observed erythrocytes, 'hemoglobin masses,' and cells containing fragments of hemoglobin in their cytoplasm in the thymus of mammals, birds, reptiles, and fishes. He does not state whether the erythrocytes and hemoglobin masses are derived from cells in the parenchyma or whether they have passed into it from blood vessels but regards the thymus as a source of some of the "colored blood corpuscles."

Prymac ('02) holds that during involution of the teleostean thymus numerous red blood cells are formed from the small round cells. Erthrocytes also escape into the parenchyma from the blood vessels. All undergo degeneration. The products of degeneration of the greater number of red cells are granules which are taken up by indifferent thymus elements, while that of the smaller portion is in the form of pigment which accumulates in masses in the parenchyma.

Schaffer ('93) in the thymus of the rabbit and cat found red blood-cells in various stages of development, and transition forms between the leucocytes and nucleated red blood-cells. He believes that the thymus has a hematopoietic function.

Bell ('06) in the thymus of a 240 mm. pig found numerous erythrocytes, lying singly and in groups, in the cortex while free erythrocytes were rarely to be found in the medulla. He does not consider their origin.

Maximow ('09) was not able to recognize definite erythroblasts or transition forms in the parenchyma of the thymus but thinks that lymphocytes have been confused with them. In the interlobular septa, however, he found collections of erythroblasts and normoblasts, or briefly, all transition forms from large lymphocytes to erythrocytes. He does not believe that red blood-cells are formed in the thymus.

Other investigators have observed free erythrocytes in the thymus in various stages of its development. Some make no mention of their origin while others suggest a possible origin but do not trace out their cytomorphosis.

Granular cells have been observed in the thymus by many investigators but the views regarding their origin and nature, which will be only briefly summarized, are conflicting. During the latter part of fetal life and the remaining period of growth and involution, Watney ('82) found in the medulla of the thymus of birds, reptiles, and mammals many granular cells. He divided them into 'four classes which were connected with each other by intermediate forms. He found them especially numerous along the course of blood vessels to the outer tunic of which many were attached. He derived them all from connective tissue cells.

Schaffer ('91) apparently was the first investigator to observe eosinophile cells in the thymus. In various developmental stages of the human thymus he found large numbers of eosinophile cells in the connective tissue surrounding the thymus, in the interlobular septa, and along the course of some of the capillaries in the medulla. A few also were found in the cortex. The size of the granules vary and the nucleus he described as being round. His investigations of the eosinophile cells in the thymus were then too incomplete to say anything definitely regarding their origin but he believed that they were not identical with the Agranular cells' of Watney. In the medulla of the involuting thymus of the mouse he ('09) found many eosinophile cells and numerous free granules that stained intensely with eosin. These granules he regarded as products of degenerated epithelial cells. Within the lobules and in the interlobular septa of the involuting thymus many plasma cells were also present. These he derived from the small lymphocytes of the thymus. Many had the appearance of undergoing degeneration.

Goodall ('05) is of the opinion that the pseudo-eosinophile cells in the region of Hassall's corpuscles in the thymus of the guinea-pig, are derived from the blood.

Maximow ('09) claims that different types of granular cells were found in the thymus in different species of mammals. In the more advanced stages of rabbit embryos an appreciable number of pseudo-eosinophile myelocytes were found in the interlobular septa,- cortex and medulla. Only a few mast cells were found, most of which were in the septa. In guinea-pig embryos psuedo-eosinophile myelocytes were seldom found. In cat embryos 35 to 50 mm. in length special myelocytes and leucocytes were found in quite large numbers, while in embryos 120 to 130 mm. long numerous mast cells were found in the deeper portions of the cortex and in the medulla. In the septa they were less numerous. Only an occasional eosinophile cell was found in the interlobular septa of the thymus in the above named animals. A few were found in the parenchyma of the thymus in a rat embryo 19 mm. in length. The different types of cells named above are derived from lymphocytes and the granules in all of them are products of the cell in which they are contained.

In the description of the later developmental stages of the thymus mention was made of free nucleated and non-nucleated red blood-cells and eosinophile cells in both the parenchyma and interlobular septa of the thymus. Through the investigations of Maximow ('09 a), and others, who have traced the development of the blood from early developmental stages in which the cells of the blood islands were still undifferentiated to later stages in which all the different types of blood-cells were found in the circulating blood, the view of a common ancestor for all the different types of blood-cells has been quite generally accepted. This primitive or undifferentiated blood-cell is structurally very much like a large lymphocyte and by some regarded identical with it. Also, Maximow and others have shown that erythrocytes, and granular cells develop from mesenchymal cells of the intraembryonic mesenchyme.^

Since erythrocytes were already present in the blood streams of the youngest embryos collected for this work, the blood islands and other hematopoietic regions were not investigated. In the mesenchyme, however, the development of the free erythrocytes and granular cells was traced apparently from their source. A consideration, therefore, of the source of the above named cells found in the interlobular septa (mesenchyme) of the thymus will be made first, for a knowledge of their origin will aid in determining the origin of free erythrocytes and granular cells

For a detailed account of the development of free blood-cells in the mesenchyme, reference should be made to Maximow's work of 1906. With the methods of technic used for this work I was able to confirm most of his conclusions regarding the origin of mesenchymal blood-cells. Hence, I have adopted tentatively the nomenclature employed by him. A detailed account of my observations would unnecessarily lengthen this article. The descriptions and drawings, therefore, will be only sufficiently detailed to be within the limits of clearness and accuracJ^ The primitive blood-cells or 'WanderzcUen' have been termed 'large lymphocytes' throughout this work.

in the parenchyma of the thymus which, to anticipate, develop from the same type of undifferentiated cells as those in the mesenchyme.

In early stages the development of the free blood-cells can be demonstrated in most any portion of the mesenchyme in the neck and upper thoracic region of young embryos. There are, however, locahzed regions where this process is carried on even in later developmental stages. The interlobular septa of the thymus are particularly favorable places to study the development of blood-cells in well advanced embryos. The thymus of an embryo 125 mm. in length was selected for the cytomorphosis of the erythrocytes, eosinophile cells, and phagocytes, although somewhat later stages could have been used. The connective tissue of the septa is loosely arranged and contains many transitional forms leading from the connective tissue cells to the above named elements. A few stellate-shaped connective tissue cells are still found but the spindle shaped type is most numerous. In figure 7 is a series of diagrams representing a suggested 'cell lineage' between connective tissue cells and their derivatives, i.e., erythrocytes (blood-plastids of Minot), phagocytes, and granular cells. Diagram a represents a connective tissue cell. The cytoplasm is finely granular and is only very slightly basophilic. In some small vacuoles occur. No cell membrane could be demonstrated. The nucleus may be round or oval and has a distinct nuclear membrane. The chromatin is in the form of small irregularly shaped granules many of which are joined together by very fine chromatin threads. The nuclei vary in number from one to three. Diagram e represents a transformed mesenchymal cell. Its protoplasmic processes are retracted and now lie free in the septa. The cytoplasm has slightly but appreciably increased in amount and has become more basophilic and now is homogeneous. The nuclear changes are represented apparently by a slight massing of the chromatin, the granules becoming slightly coarser and less numerous. In some connective tissue cells the cytoplasm becomes more basophilic and the nuclear changes occur even before its processes have been retracted (diagram h). This type of connective tissue cells is interesting in that its transformation to the free cell can be easily followed. Now and then a transformed mesenchymal cell, d, can be found, the cytoplasm of which is quite pale, being no more or only slightly more basophilic than that in the ordinary connective tissue cells. Its cytoplasm, however, is homogeneous and its nuclear structure similar to that of ordinary large lymphocytes. This type of cells evidently has the power to wander about in the mesenchyme for occasionally (very seldom) can one be seen in the epithelial anlage of the thymus (fig 2, lower border to the left). These were observed by Maximow who claims that their cytoplasm soon turns basophilic after they are formed. Judging from their structure and the small number present his interpretation is correct and they must therefore be considered as belonging to the same type of cells as those in which the cytoplasm is more basophilic. In some transforming mesenchymal cells, c, the cytoplasm becomes basophilic and the chromatin increases in amount while the protoplasmic processes are being retracted, i.e., the cytoplasmic and nuclear changes take place simultaneously. This process results in the formation of a cell in which the cytoplasm is less basophilic and the nucleus contains less coarse granules than in a fully developed large lymphocyte. These cells (young lymphocytes) transform into the typical large lymphocytes as represented in diagram e. Since this type of cells is of more frequent occurrence than those represented in diagrams b and d it is assumed that this is the most usual manner by which a mesenchymal cell transforms into a lymphocyte. The type of cells under consideration and represented in diagrams c, d and e are the primitive mesamoeboids of Minot and the primary wandering cells of Maximow and others. With Maximow and others, I agree that they are essentially identical with the large Ijanphocytes which term was given them in the account of the histogenesis of the thymus in the early stages of its development.

The power of the lymphocytes to develop in different directions is clearly manifested in the interlobular septa of this stage in which many lymphocytes of all types are found. Judging from the mitoses that some are undergoing, the large lymphocytes through repeated divisions become smaller and form small lymphocytes, /. Whether or not the small lymphocytes have the power to grow and again form large lymphocytes, as claimed by some investigators, is difficult to demonstrate. The point of interest and importance is the development of erythrocytes and granular cells from the lymphocytes. In some of the transition stages between the large and small lymphocytes, or for convenience, the large and medium-sized lymphocytes, changes occur in both their nucleus and cytoplasm. The latter stains a faint brick-red indicating the presence of hemoglobin while the nucleus becomes granular. These are the megaloblasts of Maximow or erythroblasts, g. In some cells, h, the nucleus has the characteristic granular structure of the erythroblasts while the cytoplasm still retains its basophilic character, or is dimmed only slightly by a faint trace of hemoglobin. These are the younger forms of erythroblasts and aid in tracing the source of the older ones. They may be found lying singly but usually occur in groups. Mitoses of erythroblasts can be found without much searching. Diagram i represents a normoblast. The cells of this type are on the whole a little smaller than the erythroblasts from which they are derived. Through the extrusion of their nuclei they are transformed into erythrocytes, j. That the nuclei are extruded is indicated by deeply stained degenerating nuclei or fragments of them lying free among the cells in a group made up of a mixture of both erythrocytes and normoblasts. Thus the free erythrocytes of the interlobular septa, as stated by Maximow, are derived from the lymphocytes, their ultimate source being from transformed mesenchymal cells. Whether or not they enter the circulation will be considered presently. While they are found in the septa in quite early stages they are most numerous in this region in embryos ranging from 115 to 165 mm. in length, the greatest number being present at about the 125 mm. stage. The superficial and head thymus of a 270 mm. (full term) fetus contained a few, singly and in groups, in the deeper and looser portions of the septa.

With this brief review of the origin of the erythrocytes in the interlobular septa we are prepared to consider their source in the cortex and medulla of the thymus. In every stage from the 55 mm. to the full term embryo that was examined, red bloodcells were found singly and in groups in the thymus. In the developmental stages approaching maturity a larger number of the lobules contain groups of red cells than in younger stages. Some lobules contain two or three groups some of which are quite large. Also red cells lying singly in the thymus are more numerous in the later than in the earlier stages. The superficial thymus and the thymus head were found to contain a relatively larger number than the mid-cervical segment. Unfortunately, the thoracic segment of late developmental stages was not collected, so I was unable to make a comparison of their number with that of the other segments of the thymus. The superficial thymi and the thymus heads of two full term fetuses (270 and 280 mm in length) contained a comparatively larger number of red blood-cells than the corresponding segments of somewhat earlier stages. In the thymus of the 280 mm. fetus the red blood-cells were about equally distributed in the two segments while the red cells in the superficial thymus of the 270 mm. embryo were much more numerous than in the thymus head. In full term embryos groups of red blood-cells are found in both the cortex and medulla of the thymus. In the younger stages no groups of red blood-cells were found in the medulla although they may be found lying singly in that region.

An occasional nucleated red blood-cell can be found in the thymus of embryos 35 to 50 mm. in length. Erythrocytes in these stages are very seldom found. They do not come from the blood for blood capillaries have not yet penetrated the lobules at this stage. In an embryo 55 mm. in length, in which only a few capillaries are found in the lobules, they are much more numerous than in the preceding stages. Nucleated and nonnucleated red cells can be found singly among the lymphocytes which at this stage are already quite numerous but the striking feature is that they are present in groups (fig. 6). They vary somewhat in size as do those in the interlobular septa but the majority in the thymus have a smaller average diameter than those in the latter place. Their contour is often very irregular which is due to their lying closely together when found in groups or wedged in between lymphocytes and epithelial cells when they occur singly. The nuclei of some have the characteristic coarsely granular structure of erythroblasts (Erb.) and young normoblasts, while in others the nuclei are pyknotic. The reddish hemoglobin-containing cytoplasm of the nucleated red cells varies in its amount in different cells but is easily recognized even with moderately high magnification in the larger and medium sized cells. Some of the nucleated red cells have two nucleoli {A. Erb.) which stain intensely, are round, and of equal size. These apparently are undergoing amitotic division. Erythrocytes {Ere.) are quite numerously scattered among the erj^hroblasts. Aside from the irregular outline of some of the nucleated red cells found in the thymus of this stage they compare favorably in all other respects with the free erythroblasts and normoblasts found in the septa. Also they have the same origin, namely, from the lymphocytes.

The lymphocytes in the thymus in which the origin of the red blood-cells was just considered belong almost entirely to the large and medium-sized type. Only a very few small ones are present. It is, therefore, necessary to consider the origin of the numerous free erythrocytes in the thymus of late developmental stages in which the large majority of all the lymphocytes belong to the small type. The superficial thymus of a 270 mm. (full term) fetus was selected for this purpose because the red blood-cells in the thymus of this stage are more numerous than in any other examined. The great majority lie in groups which, in a section, appear as smaller or larger bright red irregular patches or as long tortuous streamers. The proportion of lymphocytes to the red cells varies in different groups. In some the lymphocytes are most numerous, in others they are about equal in number, while in still others the red cells greatly predominate. Also, in some groups the erythrocytes make up nearly the entire num.ber of red blood-cells, only a few nucleated red cells being present. In some groups many nucleated red cells are found among the erythrocytes, while other groups are composed almost entirely of nucleated red cells. Both the erythrocytes and nucleated red blood-cells vary in size, but the small ones greatly predominate over the medium sized and larger ones. They are usually irregular in outline. The structure of the nuclei vary as those in the 55 mm. stage. An interesting and most helpful feature in tracing out the origin of erythrocytes in late developmental stages is the presence of lymphocytes with granular nuclei, the structure of which is the same as that of the erythroblasts and normoblasts found in the interlobular septa, the only difference being their smaller size. Only a few small lymphocytes with this type of nucleus were found in the thymus of the 55 mm. stage and in the interlobular septa of the 125 mm. embryo. They stain intensely and when examined with lenses of low magnification appear as black dots in contrast to the other small lymphocytes among which they lie. Maximow ('09 b) in writing of the erythropoetic function of the thymus, which he denies, makes mention of this type of lymphocytes but on the ground that they contained no hemoglobin he does not consider them to be normoblasts. It is true that in many lymphocytes with this type of nucleus, sometimes entire groups, no traces ol hemoglobin can be detected in their cytoplasm even when highly magnified (X 2000). But, many small cells can also be found with similarly granular nuclei and with a distinct reddish tinge which indicates the presence of hemoglobin in their cytoplasm. These are small erythroblasts that are derived from small lymphocytes and the small cells referred to by Maximow, and so plentifully found in the thymus of this developmental stage, are transition forms between the ordinary small lymphocytes and the small erythroblasts. On account of the meagre amount of cytoplasm in these erythroblasts they appear much as if the nucleoplasm was stained slightly red. But that is not the case for the red stained cytoplasm, of those in the late normoblast stage in which the nucleus has become shrunken and pyknotic, stands out sharply, although it is small in amount. Transition forms are often scattered along the border of groups of red cells containing many erythrocytes, and in groups of nucleated red cells they are almost invariably found scattered among the erythroblasts and normoblasts. Typical large erythroblasts and normoblasts such as occur in the thymus of the 55 mm. stage are also present in the thymus of late developmental stages; but in these stages they make up only a small proportion of the erythroblasts. This iS' accounted for by the fact that the small nucleated red cells are derived from the small lymphocytes while the large ones are derived from large and medium-sized lymphocytes which are comparatively few in number in late developmental stages.

The debris of degenerated nuclei extruded from the normoblasts can often be found scattered among erythrocytes. It is not uncommon for this debris to collect in heaps which appear in sections as deeply stained dark structureless patches in a group of erythrocytes. Why the degenerated nuclei have a tendency to flow together can only be conjectured; also why the red bloodcells are mostly formed in groups instead of uniformly throughout a lobule is unknown to me.

The blood stream of a 55 mm. embryo contains nucleated red blood-cells and since capillaries are already found in the thymus in this developmental stage it might still be argued,' as is held by some, that the free erythrocytes in the thymus are derived from the blood. This, however, cannot be the case for in a full term fetus in which many erythrocytes and nucleated red blood-cells are found in the parenchyma of the thymus no nucleated red cells were found in the blood stream.

From the observations cited above I must conclude that the erythrocytes in the meshes of the reticulum of the thymus are derived from the lymphocytes. Furthermore, on this conclusion are hinged three important features two of which strongly reflect on the nature of the small round cells of the thymus, while one furnishes additional proof for the pseudomorphosis theory of the histogenesis of the thymus. They are: (1) Since both the lymphocytes of the mesenchyme and the small round cells of the thymus have the power to transform into erythrocytes they are potentially alike. The small thymic cells are, therefore, Ijonphocytes and not epithelial cells as held by Stohr ('06); (2) The lymphocytes can be regarded as undifferentiated blood-cells and under certain conditions are, in some organs, potentially like the primitive blood-cells, and (3) The like potentiality of the lymphocytes in both the thymus and the mesenchyme is additional evidence that the lymphocytes first present in the thymus have migrated into it from the mesenchyme.

Whether or not any of the erythrocytes formed in the thymus or in the mesenchyme surrounding it enter the circulation is difficult to determine in fixed material. Some undoubtedly undergo degeneration. In the mesenchyme of early stages and in the interlobular septa of later developmental stages some erythrocytes are present the cytoplasm of which is granular instead of homogeneous. In some cells the granules are small, round, and of a quite uniform size while in others the granules vary greatly in size. Some are apparently about to break up into a small number of irregularly shaped fragments. I am confident that these erythrocytes are degenerating forms and are not artifacts, for in the same microscopic field may be found numerous other cellular elements (connective tissue cells, lymphocytes, nucleated and non-nucleated red cells) all of which have the appearance of a good preservation. Also not infrequently erythrocytes can be found that have completely fallen to pieces, the debris of degeneration being in the form of varying sized globules and irregularly shaped fragments, or irregular groups or long drawn out rows of small deeply red stained (eosinophile) granules. The latter may be derived directly from the disintegration of granular erythrocytes or from the further disintegration of large fragments of them. Some of the red cells undergo degeneration while still in the normoblast stage. These are characterized by a pyknotic nucleus and more or less granular cytoplasm. Except for their small size and the type of nucleus they contain, some could easily be mistaken for small eosinophile cells.

Degeneration of some of the free erythrocytes in the lobules of the thymus takes place in a manner similar to that described above. Groups of free eosinophile granules can be found in the thymus of all developmental stages in which red blood-cells are formed in that organ. The free eosinophile granules are never very numerous but the thymus of late developmental stages in which the erythrocytes are comparatively numerous contains more than the thymus of early stages. Erythrocytes and normoblasts with granular (degenerating) cytoplasm are also present. The red blood-cells in the thymus usually have an irregular outline. This is not a sign of degeneration but is brought about by purely mechanical factors as stated above. In late developmental stages phagocytes ingested with erythrocytes and other degenerated products can occasionally be found in the lobules of the thymus. These, however, appear first and are more numerous in the interlobular septa. In some groups of red cells in the superficial thymus of the 270 mm. fetus some of the erythrocytes are apparently fused, forming as seen in section, irregularly and deeply red stained and quite homogeneous patches which contain only a few lymphocytes. Whether or not the fused erythrocytes undergo degeneration could not be determined with the material at hand. The thymus of post-natal pigs needs to be investigated to determine the fate of the numerous free erythrocytes present in that organ. It is evident, however, that not all, if any, enter the circulation.

In all the developmental stages examined eosinophile cells^ are, in general, more numerous in the connective tissue of the interlobular septa than in the lobules of the thymus. A consideration first of their origin in the former place will, therefore, aid in determining their origin in the lobules. Eosinophile cells are already present in the mesenchyme of quite young stages (20 to 25 mm.). In all these and in somewhat older stages (55 mm.) they are not found in localized areas but may be found in almost any part of the connective tissue. In the 55 mm. embryo they are somewhat more numerous than in the younger stages but can be found only after considerable searching. From embryos more than 55 mm. in length only the thymus was removed. In late developmental stages, therefore, only the eosinophile cells in the interlobular septa will be considered. The septa of the thymus in embryos from 65 to 85 mm. in length contain only a few eosinophile cells. In some sections none were found. In the 100 mm. stage they can be found without much searching while in the 110 mm. stage a single group was found in the sections prepared from the thymus head while those lying singly are more numerous than in the previous stage. In a 125 mm. embryo groups of eosinophile cells are of frequent occurrence and lie usually along the course of blood vessels but some are also present in the deep looser portions of the septa. They are also found lying singly in the septa. The greatest numbers occur in stages 165 and 180 mm. long. In the former stage they were more numerous in the superficial thymus than in the thymus head or cervical segment, and on the whole more numerous than in the latter stage in which their distribution was about equal in the different segments examined. In the full term fetus (270 mm.) they are much less numerous in the septa than in the 180 mm. stage. In the last three stages many eosinophile cells are found lying singly in the deep looser portions of the septa but the large majority are found in groups which almost without exception are found in the immediate vicinity of the larger blood vessels where the structure of the septa is comparatively loose. Some groups extend entirely around blood vessels (fig. 8, Eo.C.) while others lie only to one side of them. In some groups the eosinophile cells he closely together while in others they are more loosely arranged. Without exception a greater or less number of large and medium-sized lymphocytes are promiscuously scattered among the eosinophile cells.

^ The value of the distinction of eosinophile myelocytes (myeloid eosinophiles) and eosinophile leucocytes is not considered and cells containing eosinophile granules are therefore simply designated as eosinophile cells regardless of the shape of their nucleus.

The eosinophile cells vary in size from very large to mediumsized lymphocytes. The outline of the greater number is spherical but when they lie closely together or in close contact with other cellular elements they may have an irregular shape. The eosinophile granules are coarse, round, and of a nearly uniform size. Their number varies greatly in different cells. In some a few granules may be found in a group to one side of the nucleus, in others they are thinly and quite evenly scattered throughout the basophilic cytoplasm, while still others are completely gorged with them.

A striking peculiarity is that the large majority are mononuclear. Only very seldom can one of the polymorphonuclear type be found. The nuclei are round, slightly indented, or crescentic in outline and are usually eccentrically located in the cell. In those cells that are gorged with granules the nuclei are crowded to one edge of the cell and stand out conspicuously among the eosinophile granules. The structure of the nuclei is identical with that of the nuclei in the large lymphocytes which have been described.

The thymus of a 125 mm. embryo was chosen to consider the origin of the eosinophile cells. In this stage the interlobular septa, loose in structure, contain numerous lymphocytes, red blood-cells, and many eosinophile cells lying both singly and in groups. Also in a single group can be found eosinophile cells containing varying numbers of granules, as stated above. An interesting and instructive feature often to be observed is the presence of large lymphocytes containing only from one to three or four eosinophile granules which are of the same size and shape as those found in cells completely gorged with them. Often in very hmited areas — covered by very slightly moving the slide under high magnification — can be found large lymphocytes and* a series of eosinophile cells with gradually increasing numbers of granules (fig. 7, l.m.n.). Only one interpretation can be given to microscopic pictures of this kind, namely, that the eosinophile cells are derived from lymphocytes. This conclusion also accounts for the large numbers of eosinophile cells along the course of blood vessels in late developmental stages, for it is along the blood vessels — in the loose portions of the septa — that the lymphocytes are most numerous.

I believe that the groups of eosinophile cells in the septa are identical with the granular cells of Watney which he found in the interlobular septa of the thymus in various classes of animals, although none of the cells were attached to the tunica externa of the vessels, as was observed by him. The ultimate source of the eosinophile cells in the interlobular septa of the thymus of the pig is the same as that of the granular cells of Watney, the only difference is that he derived them directly from connective tissue cells while in the pig thymus they are derived from transformed connective tissue cells, the large lymphocytes.

Of course, in fixed material it is difficult to determine whether all the lymphocytes along the blood vessels are derived from the loose connective tissue in which the vessels lie or whether some come from the blood. Two features are in favor of the former view; (1) transition forms from connective tissue cells to lymphocytes are of frequent occurrence. The lymphocytes thus formed through division also increase in number; (2) diapedesis of the leucocytes is thought of as taking place only through thin walled blood vessels, but the lymphocytes and eosinophile cells are as numerous along the course of thick walled vessels as along those of a capillary nature. Another possible source of the lymphocytes in the septa is from the parenchyma of the thymus. However, in late stages that portion of the thymus contains mostly small lymphocytes and judging from the small number of small lymphocytes present in the septa very few have migrated into them from the parenchyma. Only a few eosinophile cells were found undergoing mitosis, so the number of this type of cells formed through their proliferation is almost neglible.

The source and nature of all the granules in eosinophile cells is difficult to determine. There is, however, no evidence indicating that the granules are debris of degenerated epithelial cells, as held by Schaffer ('09), but ample evidence that not all are products of the protoplasmic activities of the cells containing them, which view is held by Maximow for the origin of the granules of the myelocytes found in the thymus of various animals. Mention was made of free eosinophile granules (fig.. 8, Eo.G.) in the interlobular septa where free red bloodcells also occur. These can be traced directly to degenerated red blood-cells, but the free granules usually observed in the septa of any developmental stage do not seem to be numerous enough to account for all of the granules in the numerous eosinophile cells even though all should be ingested by lymphocytes. However, lymphocytes with only a few granules in their cytoplasm and lying among free eosinophile granules suggests that some eosinophile cells are simply lymphocytes ingested with debris of degenerated erythrocytes. This view of the origin of the granules in eosinophile cells is held by Weidenreich ('08, '08, mammals), and by Badertscher ('13, amphibia) in a somewhat modified form in that some of the granules are also formed from the debris of degenerated muscle tissue. Also circumstantial evidence indicating the formation of eosinophile granules from erythrocytes is not wanting and may be enumerated as follows: (1) The free red blood-cells appear in the interlobular septa in advance of eosinophile cells; (2) The red blood-cells appear in large numbers in earlier developmental stages than do large numbers of eosinophile cells, e.g., in the septa of the thymus of a 125 mm. embryo the red blood-cells are more numerous than in any other developmental stage while the largest number of eosinophile cells occur in the septa of the thymus of a 165 mm. fetus; (3) As the free red blood-cells in the septa of late stages begin to decrease in number the eosinophile cells decrease in number in correspondingly later stages, e.g., the red blood-cells in the thymic septa of 165 and 180 mm. fetuses are not as numerous as in the 125 mm. embryo but the eosinophile cells in the 270 mm. embryo are less numerous than in the 165 and 180 mm. fetuses. These facts can be stated in a general way by saying that the height and decrease of erythrocyte formation in the septa are followed respectively by the height and decrease of eosinophile cell formation in somewhat later stages. If the granules in eosinophile cells are products of degenerated erythrocytes this apparent relationship existing between these two types of cells can be accounted for only on the assumption that the majority of free red cells in the septa undergo dissolution and the products of degeneration taken up by the lymphocytes, possibly in soluble form, and in them transformed into granules.

Cells of a peculiar type (fig. 7, k). are quite frequently found among lymphocytes and eosinophile cells in the thymic septa. They are derived from large lymiDhocytes and are characterized by a part of or the entire superficial layer of the basophilic cytoplasm staining a deep red similar to the erythrocytes or the granules in eosinophile cells. Their nuclei have the characteristic structure of those in the lymphocytes or eosinophile cells. They cannot, therefore, be erythroblasts which have granular nuclei but must be classed with the eosinophile cells. The cells of this type are never very numerous and the youngest stage in which they, were found was in the body mesenchyme of a 25 mm. embryo. They occur most frequently in the thymic septa of quite late developmental stages.

The origin of the eosinophile cells in the lobules of the thymus can now be discussed briefly. Their structure is the same as of those in the interlobular septa. They belong to the mononuclear type. They were first found in the lobules of the thymus of a 42 mm. embryo. In this stage they are very rare and can be found only after prolonged searching. Their number increases in successively advanced developmental stages. In the 125 mm. embryo they are readily found m both the cortex and medulla. In the 180 mm. embryo a group of them was found in the medulla of the mid-cervical segment while those lying singly are more numerous than in younger stages. In the full term fetus they are present in appreciably greater numbers than in the previous stage, groups of them being found in both the cortex and medulla and many can be found lying singly. Since the red blood-cells were considered particularly in the superficial thymus of a 270 mm. (full term) fetus the eosinophile cells also in that region will be emphasized. Some groups of eosinophile cells are found in the immediate vicinity of blood vessels but as many are found that are not associated with the vessels. The groups occur most frequently along the border of or near the vicinity of groups of erythrocytes but some groups are isolated and as far as position is cfoncerned their origin does not seem to bear any relation to erythrocytes. Here as in the interlobular septa the origin of some is, undoubtedly, from the large lymphocytes that have mgested eosinophile granules (debris of degenerated erythrocytes) which as was stated above can be found lying free in the meshes of the reticulum among the lymphocytes. The free eosinophile granules do not seem to be numerous enough, as in the case of the septa, to account for all granules found in eosinophile cells. However, an apparent general relationship exists between the latter type of cells and the erythrocytes which indicates that at least some of the granules of eosinophile leucocytes are derived from degenerated erythrocytes. The features indicating this relationship may be expressed as follows: (1) As in the thymic septa, the red bloodcells are present in advance of the eosinophile cells; (2) The eosinophile cells increase in numbers in successively advanced developmental stages as do also the red blood-cells; (3) They are most numerous in the thymus of a full term fetus in which developmental stage the red blood cells are also most numerous; (4) In the thymus of a 270 mm. embryo the eosinophile cells are more numerous in the superficial thymus than in the thymus head, the difference in the numbers corresponding favorably to the difference in the numbers of red blood-cells which are much more numerous in the former than in the latter segment. Here also it must be said that if all the granules of the eosinophile leucocytes in the lobules are derived from degenerated erythrocytes it must also be assumed that their degenerated products are taken up in soluble form by the lymphocytes in which it is transformed into granules.

Phagocytes (fig. 7, o. and p.) are found in the interlobular septa of the thymus in a wide range of developmental stages. They are most numerous in those stages in which the septa have a loose structure and contain many lymphocytes. They possess a large amount of cytoplasm which in some cells is vacuolar. Some are gorged with ingested material which consists mainly of lymphocytes (apparently) in various stages of degeneration. Occasionally one can be found in which an entire erythrocyte or a part of one makes up a part of the ingested material. They arise directly from connective tissue cells some of which contain ingested particles even before their protoplasmic processes have been withdrawn. The phagocytes vary greatly in size. Some are from two to three times as large as the largest lymphocytes. Only a few were found in the lobules of the thymus of late developmental stages.

Cysts were found in the thymus in embr3^os 55, 65, 110, 125, 165 and 180 mm. in length. They vary in size and shape and all are lined with simple cuboidal or low columnar epithelium which is ciliated only in patches. The cilia are long and slender. No consideration was given to their origin.

Conclusions

1. The lymphocytes first present in the thymus are all large lymphocytes and have migrated into it from the mesenchyme.
2. The numerous small round cells of the thymus are formed by the repeated division of the large lymphocytes which thus become small, and also by their own proliferation.
3. Judging from the source and structure of the small round cells they are small lymphocytes and are identical with the small lymphocytes of the blood. The thymus, therefore, may well be considered as a source of some of the small lymphocytes found in the circulating blood.
4. The reticulum of the thymus is of epithelial origin and is formed passively by its meshes becoming filled with lymphocytes which separate the nodal nuclei farther apart and thus greatly attenuate the protoplasmic processes of the syncytium.
5. The Hassall's corpuscles are of epithelial origin.
6. The free red blood-cells and eosinophile cells found in both interlobular septa and the thymic lobules are derived from lymphocytes in situ.
7. Whether or not any of the erythrocytes formed in the thymus enter the circulating blood is difficult to determine in fixed material. Some of the free erythrocytes undoubtedly undergo degeneration and the products of disintegration of those existing in the form of eosinophile granules are taken up by the lymphocytes which thus become transformed into eosinophile leucocytes.
8. It was impossible to trace the origin of all the eosinophile granules in the eosinophile cells directly to degenerated red blood-cells. However, the fact, that the height and decrease of the formation of red blood-cells in the septa is followed by the height and decrease of the formation of eosinophile cells, is circmnstantial evidence that a relationship exists between the disappearance of the free erythrocytes and the formation of free eosinophile cells.
9. The histogenesis of the thymus may be divided into epochs each of which is characterized by more or less distinct developmental features. They are:
   1. The purely epithelial epoch which extends from its origin as an outpocketing from the third pharyngeal pouch and the formation of the cervical vesicle to the appearance of the first lymphocytes in the thymus.
   2. The epoch of lymphocyte infiltration and lymphocj^te proliferation and the formation of the reticulum. The infiltration of the thymus by extra thymic lymphocytes from the mesenchyme surrounding it begins in embryos from 25 to 30 mm. in length and probably continues up to stages 180 mm. in length, while their proliferation in the thymus undoubtedly continues after birth. The reticulum, which according to the nature of its development is formed gradually, differentiates into the cortex and the medulla in developmental stages 65 to 75 mm. in length, and is fully formed in embryos 180 mm. in length.
   3. The epoch of the formation of red blood-cells and the development of granular cells. An occasional red blood-cell is found in the thymic lobules shortly after lymphocytes are found in them. They are, however, first present in appreciably large numbers in stages of about 55 mm. in length and are most numerous in the thymus of full term embryos. In the interlobular septa of the thymus the greatest number occurs in stages of about 125 mm. in length while only a few are found in embryos of 180 mm. in length to full term.

Eosinophile cells were first found in the thymic lobules of a 42 mm. embryo but occur first in appreciably large numbers in embryos of about 180 mm. in length and are most numerous in the parenchyma of the thymus of full term embryos. In the interlobular septa they are seldom found in embryos from 65 to 85 mm. in length. They occur first in appreciably large numbers in the septa of embryos of about 125 mm. in length and are most numerous in embryos 165 to 185 mm. long but are still present in the septa in full term embryos.

I wish to thank Prof. B. F. Kingsbury for the aid and encouragement given me on this work, and Prof. S. H. Gage for many suggestions.

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1915 The development of the thymus in the pig. Part I: Morphogenesis. Am. Jour. Anat., vol. 17.

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Zool. Jahrb. Abt. f. Anat. u. Ontogen., Bd. 17.

Bell, E. T. 1906 The development of the thymus. Amer. Jour. Anat., vol. 5.

DusTiN, A. P. 1911 Le thymus de I'AxoIotl. Arch, de Biolog., vol. 26.

VON Ebner, V. 1902 A. Koelliker's Handbuch der Gewebelehre des Men schen, Bd. 3, 6 Aufl, p. 328.

GooDALL, A. 1905 The post natal changes in the thymus of guinea-pigs and the effect of castration on the thjaiius structure. Jour. Physiol., vol. 32.

Hammar, J. A. 1905 Zur Histogenese und Involution der Th^Tnusdruse. Anat. Anz., Bd. 27.

1908 Zur Kenntnis der Teleostierthymus. Arch. f. mikr. Anat., Bd. 73.

1910 Fiinfzig Jahre Thymusforschung. Kritische tlbersicht der normalen Morphologic. Ergeb. d. Anat. u. Entwickl., Bd. 19.

1911 Zur groberen Morphologic und Morphogenie der Menschenthymus. Anat. Hefte, vol. 43.

Maurer, F. 1886 Schilddrlise und Thymus der Teleostier. Morph. Jahrb., Bd. 11.

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Maximow, a. 1906 iJber die ZcUfonnen des lockeren Bindegewebes. Arch. f. mikr. Anat., Bd. 67.

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Plates

References On Plates

A. Ere, amitosis of erythroblasts

hl.v., blood vessel

D.d.e.N., debris of degenerated epithelial nuclei

D.e.N., degenerating epithelial nucelus

D.e.N'., completely degenerated epithelial nucleus

E.N., epithelial nucleus

Eo.C, eosinophile cells

Eo.G., free eosinophile granules

Erb., erythroblast

Ere, erythrocyte

L.L., large lymphocyte

L M., limiting membrane

M.C., mesenchymal ceil

M.e.N., mitosis of epithelial nuclei

Me.L., medium sized lymphocyte

M.L., mitosis of lymphocyte

Nmb., normoblast

Pc, phagocyte

S.i., interlobular septa

S.L., small lymphocyte

T., trail

v., vacuole

.\., cell of unknown origin and significance

Z.pr., zone of rapid i>rolifpration of epithelial cells

PLATE 1

EXPLANATION OP FIGURES

1 Camera lucida drawing of a portion of a lobule of a section of the right thymus head in a 30 mm. embryo. The infiltration of the thymus by extrathymic lymphocytes from the surrounding mesenchyme has just begun. In order to reduce the size of the drawing the very large lymphocyte represented in the outer border of the mesenchyme was drawn a little nearer the thymus than it really is. L5 mm. Zeiss App. objective, X 12 Comp. ocular; reduced one-half.

2 Camera lucida drawing of a portion of a lobule of a section of the left thoracic segment of the thymus in a 37 mm. embryo. The thymus in this developmental stage is slightly more advanced in development than in the 30 mm. embryo. This drawing was made to show particularly the large number of epithelial nuclei that are in the first stages of degeneration and the trail that was apparently made by an active lymphocyte that migrated into the thymus from the mesenchyme surrounding it. 1.5 mm. Zeiss App. objective, X 12 Comp. ocular; reduced one-half.

PLATE 2

EXPLANATION OF FIGURES

3 Camera lucida drawing of portions of two lobules of the left thymus head in an embryo 42 mm. in length. In the thjmius of this stage many lymphocytes are present most of which are large and medium-sized. Only a few small lymphocytes are present. Mitoses of both epithelial nuclei and lymphocytes occur. The lymphocytes in the interlobular septa are quite numerous. 1.5 mm. App. objective, X 4 Comp. ocular; reduced one-fourth.

4 Camera lucida drawing of portions of two lobules of the left thymus head in a 65 mm. embryo. The thymus of this stage contains numerous lymphocytes most of which are small ones. Mitoses of lymphocytes are comparatively numerous. Many lymphocytes are found in the interlobular septum. 1.5 mm. App. objective, X 4 Comp. ocular; reduced one-foiu-th.

PLATE 3

KXPLANATION OF FIGURES

5 Camera lucida drawing of a portion of a thymic lobule in a 36 nun. enibrjo to show specially epithelial nuclei in various stages of degeneration. 1.5 mm. Zeiss App. objective, X 8 Comp. ocular; reduced one-half.

6 Camera lucida drawing of a portion ot a lobule of the thymus in a 55 mm. embryo to show especially free nucleated and non-nucleated red blood-cells. In the portion drawn one erythroblast is in mitotic division while several are in amitotic division. 1.5 mm. Zeiss App. objective X 8 Comp. ocular; reduced one-half.

7 Diagrams showing the different types of cells that are derived from mesenchymal cells. The direction of the arrows shows the relation of the different types of cells to each other; a, mesenchymal cell; b, c and d, transforming mesenchymal cells; e, large lymphocyte;/, small Ijmtiphocyte; g and h, erythroblasts; i, normoblast; j, erythrocyte; k, lymphocyte capped with a layer of hemoglobin; I, m and n show the formation of eosinophile leucocytes, and o and j), phagocytes; o, b, k and p are camera lucida drawings while the remainder are free-hand drawings from actual specimens. All were drawn from specimens in the interlobular septa of a 125 mm. embryo excepting o and p, which were drawn from specimens in an interlobular septum of a 110 mm. embryo. 1.5 mm. App. objective, X 8 Comp. ocular; reduced one-half.

8 Camera lucida drawing of a portion of an interlobular septum of the thymus in a 165 mm. embryo showing specially eosinophile leucocytes and a few free eosinophile granules.

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