Cell Tiss. Res. 179, 87
Cell and Tissue Research
96 (1977)
(() by Springer-Verlag 1977
Electron Microscopic Study on the Early Histogenesis of Thymus in the Toad, Xenopus laevis * Saburo Nagata Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan
Summary. Sequential electron microscopic observations of thymic histogenesis in the toad, Xenopus laevis, reveal that the thymus arises as epithelial buddings of the visceral pouches at Nieuwkoop-Faber stage 40, and acquires its basic histological features at stages 48-49. In the rudiments and the surrounding mesenchyme at stages 43-45, there are non-epithelial cells with pseudopodia, abundant ribosomes, and marginated heterochromatin. These cells, possible precursor cells of thymic lymphocytes, are frequently observed to attach and pass through the basal lamina which coats the thymic rudiment. The proliferation and differentiation of large lymphocytes are evident at stage 47. During stages 48-49 the small lymphocytes, lymphoid cortex and epithelial medulla including the thymic cysts, differentiate, and vascularization occurs. The results provide an ultrastructural basis for recent experimental evidence that the thymus exerts its essential function at stages 47-48. The possibility of non-epithelial derivation of thymic lymphocytes is discussed. Key words: Thymus -
Histogenesis -
Electron microscopy -
Xenopus.
Introduction Recent immunobiological studies have established that, in the Amphibia, the thymus plays a similar role in the development of immunity as in homoiotherms (for reviews, see Du Pasquier, 1973; Cooper, 1976). Among amphibians, the correlation between maturation of immune responsiveness and lymphoid histogenesis has been best documented in the toad, Xenopus laevis (Horton, 1969; Kidder et al., 1973; Jurd et al., 1975). In addition, larval thymectomy at the Send of[print requests to." Saburo Nagata, Zoological Institute, Faculty of Science, Hokkaido
University, Sapporo 060, Japan * The author wishes to express his thanks to Asst. Prof. Ch. Katagiri for his helpful advice during the course of this study
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early stages of its histogenesis ( H o r t o n a n d M a n n i n g , 1972; T u r n e r a n d M a n ning, 1974; T o c h i n a i a n d Katagiri, 1975) has made it possible to define the exact stage(s) when the t h y m u s initiates its essential function. A l t h o u g h extensive light microscopic studies on the histogenesis of the Xenopus t h y m u s (Sterba, 1950; M a n n i n g a n d H o r t o n , 1969) are available, these o b s e r v a t i o n s do n o t elucidate the events which occur at the cellular level d u r i n g the critical stages of the f u n c t i o n a l d e v e l o p m e n t of this organ. Curtis et al. (1972) attempted to correlate the ultrastructure of larval t h y m u s with the d e v e l o p m e n t of allograft rejection capacity in Rana pipiens, b u t they did n o t e x a m i n e the earlier stages. The ultrastructure of well-differentiated t h y m u s has been investigated in larval a n d a d u l t Xenopus (Nagata, 1976), a n d in adults of other a n u r a n species (Klug, 1967; K a p a et al., 1968). However, n o i n f o r m a t i o n is available on the ultrastrucrural changes which occur in the t h y m u s d u r i n g the early stages of differentiation, a n d the present study attempts to alleviate this deficiency. Particular emphasis has been given to the first a p p e a r a n c e of l y m p h o i d cells a n d their derivation.
Materials and Methods The material used was the South African clawed toad, Xenopus laevis Daudin. Fertilized eggs were obtained by induced mating, following injection of chorionic gonadotropin into mature males and females. Embryos and larvae were reared in aerated aquaria at 23~ C, and from 4th day post-fertilization onward the larvae were fed boiled alfalfa. Developmental stages of larvae were determined according to the Normal Table of Nieuwkoop and Faber (1956). Larvae ranging from stage 40 to 50 (2.5-15 days of age) were used for sequential electron microscopic observations of thymic histogenesis. For larvae of stages 40~7, the head was cut transversely, without anesthetization, at the level immediately posterior to the ear vesicles, and the isolated heads fixed in ice-chilled5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). For larvae older than stage 48, the thymuses with surrounding connective tissue and epidermis were dissected and fixed in the same fixative. The material was postfixed with ice-chilled 1% OsO4, dehydrated in a graded series of acetone, and embedded in Epon 812 (Luft, 1961). Ultrathin sections were cut with glass knives on a Porter-Blum MT-1 ultramicrotome, stained with uranyl acetate and lead citrate (Reynolds, 1963), and observed with a Hitachi HS-7 electron microscope. The "thick" sections adjacent to ultrathin sections were routinely stained with toluidine blue for light microscopy. To localize acid phosphatase activity on ultrathin sections, the glutaraldehyde-fixed specimens were incubated for 2 h at 37~ C in a substrate medium (Gomori, 1952). The control run consisted of incubation in the medium without fl-sodium glycerophosphate. After incubation, specimens were thoroughly rinsed, postfixed with OsO4, dehydrated and embedded by the procedure described above. Sections were observed after staining with uranyl acetate and lead citrate. For ultrastructural localization of carbohydrates, the periodic acid-chromic acid-silver methenamine (PA-CrA-Ag) method was used according to Rambourg and Leblond (1967; for details, see also Nagata, 1976). Incubation in silver methenamine solution was always for 30 min.
Results The t h y m u s p r i m o r d i u m arises at stage 40 (2.5 days of age) as b u d d i n g s of the epithelium from the second visceral pouches stage, a pair of the buds consists of a b o u t 20 cells each of large yolk platelets a n d lipid droplets in the cytoplasm. These are a p p a r e n t l y epithelial in that they are tightly j o i n e d to each
a pair of dorsal (Fig. 1). A t this which c o n t a i n s c o m p o n e n t cells other by desmo-
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Fig. !. Transverse section of pharynx in stage 40 larva, showing thymic bud (7). E, ear vesicle. N, notochord. Epon-toluidine blue. x 160
Fig. 2. Part of thymus rudiment at stage 43, showing phagolysosome (P) in epithelial cell. Specific activity of acid phosphatase is found as lead precipitates, z 10,000
somes, and the basal lamina coating them is continuous with that lining the pharyngeal epithelium. In the thymic bud at stage 43 (3 days of age), the epithelial cells display highly branched processes and occasionally possess the membrane-limited, myelinated bodies which show acid phosphatase activity (phagolysosome; Fig. 2). Also at this stage the basophilic, free cells first appear in the thymic bud and the surrounding mesenchyme. These cells differ from epithelial cells and common mesenchymal cells by the presence of a higher density of ribosomes
Fig. 3. Part of thymus rudiment at stage 43, showing epithelial cell and a possible lymphoid precursor cell (L). Note fibrous basal lamina (arrow) coating epithelial cell at thymic surface, x 11,400 Fig. 4. Degenerating cells (*) found in the basal portion of the stage 43 thymic bud, showing that they are engulfed by other epithelial cell process. D, desmosome. Y, yolk platelet, x 7600
Fig. 5. Mesenchymal lymphoid precursor cell (L) in contact with the surface of the stage 45 thymus rudiment. 1I, yolk platelet, x 5700 Fig. 6. Enlargement of the rectangular area shown in Figure 5. Note the lack of basal lamina at the area indicated by arrows, where lymphoid precursor cell and thymic epithelial cell are in close apposition, x 28,500 Fig. 7. Lymphoid precursor cell (L) crossing epithelial cells. Arrows indicate basal lamina, x 9500
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Fig. 8. Thymus in stage 47 larva. Stronglybasophilic, large lymphocytesare readily distinguishable from epithelial cells. Arrow indicates a dividing large lymphocyte. M, mesenchymalcells. PE, pharyngeal epithelium. Epon-toluidineblue. x 800 (monoribosomes) in a relatively thin cytoplasmic rim, a marginated heterochromatin and a prominent nucleolus (Fig. 3). The mitochondria with dense matrix and well developed cristae also contrast with those of epithelial cells. Another feature in the thymic bud of this stage is the frequent occurrence of very deeplystained, degenerating cells which are engulfed by thin cytoplasmic processes of epithelial cells (Fig. 4). Such cell death may contribute to the "pinching off"' of the bud from the pharyngeal epithelium. At stage 45 (4 days of age), when the thymic rudiment has detached from the pharyngeal epithelium, the number of basophilic, ribosome-rich cells in the rudiment and the surrounding mesenchyme has increased from that observed in earlier stages. Those cells found in the mesenchyme are polarized, with the nucleus displaced toward the side facing the thymic surface (Fig. 5). Frequently these cells attach to the thymic basal lamina by means of pseudopodial processes. Figures 6 and 7 illustrate the cells making a direct contact with a thymic epithelial cell (Fig. 6), and tearing or passing through the basal lamina (Fig. 7). These observations may represent the migration of the lymphoid precursor cells from the extrathymic connective tissue space to the thymus. The epithelial cells still possess yolk platelets and lipid droplets, as well as phagolysosomes (Figs. 5 and 7). By stage 47 (6 days of age), the population of basophilic cells relative to epithelial cells has greatly increased (Fig. 8). In the former, there is a decrease in the number of mitochondria, resulting in a predominance of free ribosomes throughout the cytoplasm. These features make the cells resemble large lymphocytes, Epithelial cells, which now lack yolk platelets and lipid droplets, form a reticular meshwork in the area between the large lymphocytes. Basophilic cells are now rarely observed in the surrounding mesenchyme.
Fig. 9. Part of thymic cortex in stage 49, showing two types of small lymphocytes with different populations of cytoplasmic ribosomes, x 3800 Fig. 10. Part of thymic cortex in stage 49~ showing possible degenerating tymphocytes (*) engulfed in the cytoplasm of an epithelial cell x 9500 Figs. II and 12. Parts of intercellular cysts in thymic medulla of stage 49 larva. Note a number of granules and their possible secretory products (extracellular amorphous materials). Periodic acid-chromic acid-silver methenamine method (Fig. 12) specifically stains the granules and cystic contents (arrows). C, cilia, x 8075 and x 5700, respectively
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At stage 48 (8 days of age), the thymus is fully encapsulated by the mesenchymal cells, which begin the process of encapsulation in the preceding stage (cf., Fig, 8). In stage 49 (12 days of age) the thymus exhibits all the major histological features which persist in later stages of development. These include the differentiation of essentially lymphoid cortex, epithelial medulla, and small lymphocytes, initial appearance of intra- and intercellular thymic cysts, as well as vascularization. The occurrence of myoid cells and melanocytes is also evident. Small lymphocytes (Fig. 9) predominate in the cortex, and are found also in the medulla of this stage. Although its physiological significance is not clear, two types of small lymphocytes occur in the stage 49-50 thymus: the smaller cells have a cytoplasmic electron density which is similar to that of large lymphocytes, whereas the larger and less electron-dense cells are indistinguishable from the typical small lymphocytes found in more advanced larval and adult stages (Fig. 9). In the cortex there frequently occur the degenerating small lymphocytes, with pycnotic nucleus and dark cytoplasm (Fig. 10). They are apparently engulfed by the epithelial cells. In the medulla, the intercellular thymic cyst (Fig. 11) is composed of cells with numerous cytoplasmic projections including cilia. The intracellular secretory granules and the contents of the cystic lumen are positive to the P A - C r A - A g reaction (Fig. 12), although the number and size of granules and the amount of their secretory materials in the lumen is smaller than in later stages.
Discussion
The general chronological processes of thymic histogenesis observed here agree with the previous light microscopic observations by Manning and Horton (1969). However, the present electron microscopic study elucidates the events that occur during the early stages of thymic morphogenesis, particularly with regard to the derivation and timing of the first appearance of lymphoid cells. The basophilic, free cells observed here are morphologically identical to the lymphoid precursor or lymphoid stem cells found in the embryo of higher vertebrates (cf., Moore and Owen, 1967; Leene et al., 1973). These cells are likely to be a lymphoid precursor cell which will proliferate actively and transform into a lymphocyte in the succeeding stages of thymic histogenesis. It should be stressed that the possible lymphoid precursor cells appear both in the thymic rudiment and the surrounding mesenchyme as early as stage 43 at which stage the rudiment has not detached from the pharyngeal epithelium. These cells probably correspond to the "am6boide Wanderzellen" described by Sterba (1950), who interpreted them to be precursors of thymocytes. Although the morphological evidence obtained in this study is not conclusive, the observations of stage 45 which are presented in Figures 5-7 support the proposal that some, if not all, thymic lymphocytes are derived from cells which migrate into the epithelial rudiment from the surrounding mesenchyme. Two theories have been advanced to explain the origin of thymic lymphocytes in homoiotherms; one view suggests that they arise by direct transformation
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of epithelial cells (Sanel, 1967; Tachibana et al., 1974), whereas the other proposes that they are derived from stem cells migrating into the anlage from outside (Ackerman and Hostetler, 1970; Leene et al., 1973). Recent experimental approaches employing chromosome markers or species specific chromatin patterns support the latter view, and indicate that the blood-borne stem cells that originate from the embryonic yolk sac may give rise to thymic lymphocytes (Moore and Owen, 1967; Owen and Ritter, 1969; Le Douarin and Jotereau, 1973). Such a source is not universal among amphibians, as experiments employing the technique of chimera formation in combination with the polyploid marker in Rana pipiens (Volpe and Turpen, 1975; Turpen and Cohen, 1976) indicate that in this species the thymic lymphocytes arise from cells which are not distributed in the blood islands. The first appearance of small lymphocytes in the stage 49 thymus confirms the report of Manning and Horton (1969). The present observation also provides the ultrastructural evidence that the epithelial cells showing secretory activity have differentiated in the same stage. The significance of these secretory materials is not clear in amphibians, although their role in relation to the endocrine function of the thymus has been debated (cf., Curtis et al., 1972). Whatever their exact role may be, it is clear that the major ultrastructural features found in the thymus of more advanced stages (Nagata, 1976) have been established at stage 49. The establishment of basic histological features of thymus at stage 49 provides morphological support for the experimental evidence that the response to allografts, as expressed by lymphocytic invasion, is first detected at this stage (Horton, 1969). Investigations involving early larval thymectomy followed by assay for the allograft rejection capacity (Horton and Manning, 1972; Tochinai and Katagiri, 1975) have provided unequivocal evidence that the thymus exerts its essential function for the establishment of immunity at stages 4748. Thus it is apparent that either the seeding of thymus-derived lymphocytes or thymic elaboration of humoral influence occurs in those stages which do not possess small lymphocytes in this organ. Of particular interest in this regard is the observation that the cell-surface immunoglobulins, a possible indicator of the functional differentiation of the thymus, are first detected on the thymic cells of these critical stages (Du Pasquier et al., 1972). Definition of the exact role of these surface immunoglobulins will be extremely important in understanding the thymic function in the development of immunity. To this end, the localization of these molecules on the developing thymic cells at the ultrastructural level is currently under study. References Ackerman, G.A., Hostetler, J.R.: Morphological studies of the embryonic rabbit thymus: the in situ epithelial versus the extrathymic derivation of the initial population of lymphocytes in the embryonic thymus. Anat. Rec. 166, 2 7 4 6 (1970) Cooper, E.L.: Immunity mechanism. In: Physiology of Amphibia, Vol. III. (B. Lofts, Ed.) New York-San Francisco-London : Academic Press 1976 Curtis, S.K., Volpe, E.P., Cowden, R.R.: Ultrastructure of the developing thymus of the leopard frog (Rana pipiens). Z. Zellforsch. 127, 323-346 (1972)
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Du Pasquier, L. : Ontogeny of the immune response in cold-blooded vertebrates. Curr. Top. Microbiol. lmmunol. 61, 37 88 (1973) Du Pasquier, L., Weiss, N., Loor, F.: Direct evidence for immunoglobulins on the surface of thymus lymphocytes of amphibian larvae. Europ. J. Immunol. 2, 366 370 (1972) Gomori, G.: Microscopic histochemistry. Principles and practice. Chicago: Chicago Univ. Press 1952 Horton, J.D.: Ontogeny of the immune response to skin allografts in relation to lymphoid organ development in the amphibian Xenopus laevis Daudin. J. exp. Zool. 170, 449~66 (1969) Horton, J.D., Manning, M.J.: Response to skin allografts in Xenopus laevis following thymectomy at early stages of lymphoid organ maturation. Transplantation 14, 141-154 (1972) Jurd, R.D., Luther-Davies, S.M., Stevenson, G.T.: Humoral antibodies to soluble antigens in larvae of Xenopus laevis. Comp. Biochem. Physiol. 50, 65 70 (1975) Kapa, E., Olfih, I., T6r6, I. : Electron-microscopic investigation of the thymus of adult frog (Rana esculenta). Acta Biol. Acad. Sci. hung. 19, 203-213 (1968) Kidder, G.M., Ruben, L.M., Stevens, J.M. : Cytodynamics and ontogeny of the immune response of Xenopus laevis against sheep erythrocytes. J. Embryol. exp. Morph. 29, 73 85 (1973) Klug, H.: Submikroskopische Zytologie des Thymus von Ambystoma mexicanum. Z. Zellforsch. 78, 388~01 (1967) Le Douarin, N.M., Jotereau, F.V. : Origin and renewal of lymphocytes in avian embryo thymuses studied in interspecific combinations. Nature (Lond.) New Biol. 246, 25 28 (1973) Leene, W., Dyzings, M.J.M., van Steeg, C.: Lymphoid stem cell identification in the thymus and bursa of Fabricius of the chick. Z. Zellforsch. 136, 521 533 (1973) Luft, J.H. : Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409~14 (1961) Manning, M.J., Horton, J.D.: Histogenesis of lymphoid organs in larvae of the South African clawed toad, Xenopus laevis (Daudin). J. Embryol. exp. Morph. 22, 265 277 (1969) Moore, M.A.S., Owen, J.J.T.: Experimental studies on the development of the thymus. J. exp. Med. 126, 715-726 (1967) Nagata, S. : An electron microscopic study on the thymus of larval and metamorphosed toads, Xenopus laevis Daudin. J. Fac. Sci. Hokkaido Univ., Ser. VI, Zool, 20, 263 27l (1976) Nieuwkoop, P.D., Faber, J. : Normal table of Xenopus laevis (Daudin). Amsterdam : North Holland Publ. 1956 Owen, J.J.T., Ritter, M.A. : Tissue interaction in the development of thymus lymphocytes. J. exp. Med. 129, 431-442 (1969) Rambourg, A., Leblond, C.D. : Electron microscopic observations on the carbohydrate-rich cell coat at the surface of cells in the rat. J. Cell Biol. 32, 27-53 (1967) Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208 211 (1963) Sanel, F.T.: Ultrastructure of differentiating cells during thymus histogenesis. A light and electron microscopic studies of epithelial and lymphoid cell differentiation during thymus histogenesis in C57 black mice. Z. Zellforsch. 83, 8-29 (1967) Sterba, G.: Ober die morphologischen und histogenetischen Thymusprobleme bei Xenopus laevis Daudin nebst einigen Bemerkungen tiber die Morphologie der Kaulquappen. Abh. s/ichs. Akad. Wiss. 44, 1-54 (1950) Tachibana, F., Imai, Y., Kojima, M.: Development and regeneration of the thymus: the epithelial origin of the lymphocytes in the thymus of the mouse and chick. J. reticuloendothel. Soc. 15, 475~96 (1974) Tochinai, S., Katagiri, Ch. : Complete abrogation of immune response to skin allografts and rabbit erythrocytes in the early thymectomized Xenopus. Develop. Growth Differ. 17, 383-394 (1975) Turner, R.J., Manning, M.J.: Thymic dependence of amphibian antibody responses. Europ. J. Immunol. 4, 343-346 (1974) Turpen, J.B., Cohen, N. : Localization of thymocyte stem cell precursors in the pharyngeal endoderm of early amphibian embryo. Cell Immunol. 24, 109 115 (1976) Volpe, E.P., Turpen, J.B.: Thymus: central role in the immune system of the frog. Science 190, 1101 1103 (1975)
Accepted November 14, 1976