/ . Embryol. exp. Morph. Vol. 57, pp. 219-232, 1980
Printed in Great Britain © Company of Biologists Limited 1980
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Development of amphibian thymus
I. Morphological differentiation, multiplication, migration
and lysis of thymocytes in the urodele Pleurodeles waltlii
By M. HENRY 1 AND J. CHARLEMAGNE 1
From the Laboratoire d'lmmunologie Comparee,
Universite Pierre et Marie Curie, Paris
SUMMARY
Different stages of thymus morphogenesis and thymocyte differentiation have been studied
at theultrastructural level in the urodele amphibian Pleurodeles waltlii Michah. From stage 38
to 42 the undifferentiated lymphoid stem-cells colonize the epithelial thymic buds. From
stage 43 to 45 the lymphoid stem-cells differentiate into lymphoblasts and then transform
into typical lymphocytes. A clasmatosis phenomenon seems to be involved in this transformation. From stage 46 to 52, a phase of intense proliferation occurs and relations between
dense reticular epithelial cells and lymphocytes are described. At stage 53, numerous lymphocytes die in the thymic tissue and are phagocytosed by macrophages. At the same time,
lymphocytes undergo migrations through the intra- and peri-thymic blood vessels. These
lymphocytes should populate the peripheral lymphoid organs, according to the previous
finding that stage 52 was the last developmental step for an efficient abrogation of cellmediated immunity by thymectomy in Pleurodeles.
INTRODUCTION
The urodele thymus is known to be implicated both in cell-mediated and in
humoral immunity (Charlemagne & Houillon, 1968; Cohen, 1969, 1970;
Tournefier, 1973; Charlemagne, 1974, 1977a; Fache & Charlemagne 1975;
Charlemagne & Tournefier, 1977). In Pleurodeles waltlii, larval stage 52 of the
developmental table (Gallien & Durocher, 1957) is the latest stage when
thymectomy abrogates allograft rejection (Fache & Charlemagne, 1975). In
the Mexican axolotl, allograft rejection is also abrogated by early larval
thymectomy (Cohen, 1969, 1970). Furthermore in this species, early or adult
thymectomy significantly enhances the primary anti-horse red blood cell antibody response, suggesting the presence of a short-lived suppressor T-cell
population (Charlemagne, 1977 a, 1979). The presence of a non species-specific
serum factor, active in promoting some T-cell markers on mouse rosetteforming cells, was demonstrated in several urodele species (Dardenne, Tournefier,
Charlemagne & Bach, 1973). The thymic origin of this factor is assessed by its
1
Author's address: Universite Pierre et Marie Curie, Laboratoire d'Immunologie Comparee, 9 Quai St Bernard, 75005 Paris, France.
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M. HENRY AND J. CHARLEMAGNE
absence in thymectomized animals. It would be interesting to be able to correlate immunological function with thymus morphology in urodeles. Since early
stages of differentiation in Pieurodeles thymus have been partially studied by
electron microscopy (Charlemagne, 1977b), this paper examines later stages,
until metamorphosis. Lymphoid cell morphology has been analysed by electron
microscopy from the stem-cell stage in the recently colonized thymus to its final
fate, death in situ or migration towards extra-thymic sites. Epithelial cell differentiation occurring during thymus development will be described in a subsequent work. We finally try to suggest correlations between thymus structure
and function, focusing attention on stage 52, the latest stage at which larval
thymectomy is reported to abrogate allograft rejection.
MATERIAL AND METHODS
Animals
Pieurodeles were obtained from spontaneous layings in our laboratory colony.
Larvae and adults were bred in tap water (22 ± 2 °C) and fed with Artemia salina
larvae, Chironomus larvae, minced meat and commercial fish food pellets.
Electron microscopy
Six to ten larvae were fixed at each stage of the developmental table. Whole
heads (young larvae) or isolated thymuses (oldest larvae) were fixed for 3 h in
Karnovsky's solution at room temperature (Karnovsky, 1965) and washed for
12 h in sodium cacodylate buffer (0-2 M; pH 7-4). Specimens were post-fixed
with 2 % osmium tetroxide in sodium cacodylate buffer. After a brief washing
in the same buffer, the samples were dehydrated through a graded series of
acetone dilutions and embedded in Epon-Araldite (Mollenhauer, 1964). Semithin sections (1 jum) were cut on a Reichert OMU-2 ultramicrotome, mounted
on glass slides and stained with basic fuchsin. Ultra-thin sections were collected
on 200-mesh copper grids and double-stained with uranyl acetate and lead
FIGURES 1-4
Fig. 1. Thymus at stage 38. The central lymphoid stem-cell (S) characterized by a
large nucleus (n), a thin cytoplasm (c) and numerous microvilli (mv) is free in the
intercellular space. The surrounding epitehlial cells (E)contain numerouscytoplasmic
yolk granules (yg) and are united by desmosomes (arrows), x 4950.
Fig. 2. Thymus at stage 44. A lymphoblast (Lb) is shown in close contact (arrows)
with the surrounding epithelial cells (E). Notice the presence of a Golgi apparatus (g)
and a cytoplasmic bud (b). x 3300.
Fig. 3. Numerous cytoplasmic buds (b) filled with ribosomes are protruding (arrows)
in the intercellular space of the thymic tissue. Stage 44. x 3000.
Fig. 4. A row of small clear vesicles (small arrows) suggests the detachment of a
cytoplasmic bud (b) from the lymphoblast (Lb). Stage 44. x 9450.
Development of amphibian thymus. I
221
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Cf
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M. HENRY AND J. CHARLEMAGNE
citrate. Grids were then examined with a Philips 300 electron microscope at
80 kV.
RESULTS
The thymus in Pleurodeles is a paired organ located in connective tissue at
the posterior junction of the third pair of gills with the body wall. Morphological events occurring during lymphoid cell differentiation were sequentially
examined.
Stages 38-42: the epithelial bud and lymphoid stem-cells
The thymus, until stage 42, remains connected with the ventral gill epithelium
by a thin epithelial bridge. The migration of stem-cells from the extra-thymic
environment to the intra-thymic sites is over (Charlemagne, 19776). The thymic
bud is a network of large stellate epithelial cells, the meshes of which are filled
with lymphoid stem-cells (Fig. 1). Epithelial cells, sometimes observed in
mitosis, are joined together by desmosomes and their cytoplasm contains large
and dense granules. Lymphoid stem-cells, round or ovoid in shape, present
numerous long and slender cytoplasmic microvilli and have a high nucleocytoplasmic ratio. The large nucleus contains heterochromatin associated with
the nucleolus, distributed around the nuclear margin and clotted throughout its
structure. The cytoplasm is almost filled with ribosomes and polysomes. Few
mitochondria, occasional long and flat ergastoplasmic lamellae and a poorly
developed Golgi apparatus are also seen.
At this stage, either typical junctions or even simple contacts are never seen
between lymphoid and epithelial cells. Lymphocytic mitosis is still infrequent.
Stages 43-45. Lymphoblasts and the clasmatosis phenomenon
The thymic tissue becomes more compact (Fig. 2). The epithelial cells, with
their cytoplasmic yolk granules, closely surround the lymphoid cells and contacts are frequently observed between the two cell types. The lymphoid cells
are enlarged and differentiated into lymphoblasts. Their round nucleus is
located at a cellular pole and contains a large nucleolus; the extensive cytoplasm contains many ribosomes grouped in rosettes, a well-developed Golgi
apparatus and numerous polysomes (Fig. 3). The microvilli have almost
FIGURES 5-7
Fig. 5. A dense reticular epithelial cell (DRC) showing a dense stellate nucleus (n)
and dark cytoplasmic projections (cp) extending between lymphocytes (L.). Close
contacts between DRC and lymphocytes are numerous farrows), x 8400.
Fig. 6. Semi-thin section (1 /*m) showing lymphocytes (L), some of them in mitosis
(M), surrounded by dense reticular cells (DRC). x 2000.
Fig. 7. A typical lymphocyte mitosis (M) in contact with a dense reticular cell (DRC).
Notice the nuclear membrane remnants (arrows), x 9750.
Development of amphibian thymus. I
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M. HENRY AND J. CHARLEMAGNE
disappeared but large cytoplasmic buds can be seen. Rows of clear vesicles are
often located at the base of these buds (Fig. 4). Moreover, observation of serial
sections allows the identification of frequent cytoplasmic fragments of lymphoblasts in the intercellular space, suggesting a cell fragmentation phenomenon
(clasmatosis).
Stages 46-52. Intense lymphocyte proliferation
Thymic growth is considerable during these stages. Blood vessels, first observed
at the thymus periphery (stage 43), are finally scattered all over the organ.
The thymus still mainly comprises epithelial and lymphoid cells but the morphologies of these two cellular populations have somewhat changed: the
epithelial cells develop a dense reticular cell (DRC) aspect (Figs. 5-7). These
DRC are characterized by a very high nuclear and cytoplasmic density and long,
slender cytoplasmic processes. These processes contain many ribosomes and
extend a considerable distance between lymphocytes. Dense reticular cells and
lymphocytes show numerous close contacts (Fig. 5) and a great number of
surrounded lymphocytes are dividing. Between stages 46 and 52 the mitotic
rate of the lymphoid cells increases while that of the epithelial cells decreases.
Lymphocytes in division can easily be distinguished by the presence of nuclear
membrane remnants at all stages of division (Fig. 7).
Stages 53-56. Fully developed larvae and metamorphosis stages
The thymus is becoming mature. A differentiated epithelial cell type, which
was uncommon at stages 51 and 52, is now increasing in number and activity.
These hypertrophic epithelial cells (HEP) are characterized by a very extensive
cytoplasm showing intense secretory activity. The thymus is now composed of
numerous lymphocytes, DRC and lympho-epithelial nodules formed by gathered
HEP and lymphocytes. Some granulocytes and macrophages are scattered all
over the thymus.
FIGURES 8—11
Thymus at stage 55. In each electron micrograph the thymus (T) is located on the
left while a blood vessel (BV) is on the right. —>, Thymic basal lamina; Z», vascular
basal lamina; ->•, openings; E, epithelial cell; En, endothelial cell; L, lymphocyte;
lu, lumen; n, nucleus; pet; perivascular connective tissue; u, uropod.
Fig. 8. The limiting layer of the thymus composed of epithelial cells covered with a
basal lamina is disrupted in several points, just by a blood capillary. A passage is
thereby opened between the thymus and the perivascular connective tissue, x 6630.
Fig. 9. A lymphocyte lies in the perivascular connective tissue, x 5610.
Fig. 10. Lymphocyte migrating across the vascular wall. A part of the cell (hyaloplasm and nucleus) is located in the vascular lumen and the remaining in the
peripheral connective tissue (nucleus and uropode). x 6630.
Fig. 11. Two lymphocytes located in the vascular lumen of a peripheral bloodvessel,
x 5610.
Development of amphibian thymus. I
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M. HENRY AND J. CHARLEMAGNE
Two important physiological events occur at this period:
(1) lymphocyte migration through the vascular walls of thymic blood vessels:
(2) lysis of lymphocytes in the thymic tissue.
(1) Migration of lymphocytes. At stage 53, but mostly at stage 54, numerous
lymphocytes are observed migrating through vascular walls. Although this
migration occurs within the thymus as well as at the peripheral level, it is better
observed at the thymic periphery and will be described at this level. Four steps
are observed (Figs 8 to 11 and 16).
Stage (a) (Figs 8, 16). The external epithelial layer limiting the thymus is
constituted of epithelial cells united by desmosomes and covered by a basal
lamina. This layer is disrupted at several places in the proximity of a perithymic capillary. A way is so opened for subjacent lymphocytes to exit from
the thymus, i.e. into the perivascular connective tissue. The proximal vessel is
well preserved, its lumen is limited by endothelial cells covered by a thin basal
lamina.
Stage (b) (Figs 9, 16). The lymphocyte is now lying between the thymus and
a blood vessel in the peripheral connective tissue. The eccentric nucleus faces
to the blood vessels, its uropod contains mitochondria and numerous ribosomes.
Stage (c) (Figs 10, 16). The lymphocyte is just migrating through the capillary
wall. One part of the nucleus surrounded by a thin hyaloplasm ring is located
in the vascular lumen and the other part, including the uropod, remains in the
perivascular space.
Stage (d) (Figs 11 and 16). Lymphocytes are located in the blood capillary
lumen which is now closed again.
(2) Lysis of thymocytes (Figs 12, 15). During the last period of thymus development, intra-thymic lymphocytes degenerate and their phagocytosis in situ
by macrophages was often observed in semi-thin sections (Figs 12, 13). Isolated
dead lymphocytes (Figs 12, 14) show a clear cytoplasm containing altered mitochondria; the nucleus presents a very dense chromatin in a clear nucleoplasm.
More often, the nuclear structures are segregated: heterochromatin is located
at the periphery and nucleoplasm in the centre, including the nucleolus pars
granulosa and pars fibrosa. This aspect is also present in lymphocytes engulfed
FIGURES 12-15
Figs. 12, 13. Semi-thin sections of thymuses at stage 55. x 2000.
Fig. 12. Ths central dead lymphocyte (dL) showing typical signs of pycnosis can be
easily distinguished from healthy lymphocytes (L).
Fig. 13. Dead lymphocytes (dL) located in the cytoplasm of a macrophage (Ma).
Fig. 14 and 15: electron micrographs of dead lymphocytes (dL) in a stage-55 thymus.
x 7820.
Fig. 14. Notice the pycnotic nucleus fn) and the empty cytoplasm (c).
Fig. 15. A dead lymphocyte engulfed by a macrophage (Ma).
Development of amphibian thymus. I
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M. HENRY AND J. CHARLEMAGNE
Fig. 16. Schematic interpretative representation of lymphocyte migration from the
thymus to the blood circulation. In the thymus, the epithelial cells (E) united by
desmosomes (d) form a network, the meshes of which are filled with lymphocytes (L).
The thymus, limited by a basal lamina (tbl), is surrounded by connective tissue (pet).
In this tissue, close to the thymus, a blood vessel is located. The vascular wall is
formed by endothelial cells (En) united by desmosomes (d) and covered by a basal
lamina (ebl). Four steps illustrate the stages of lymphocyte migration. Stage A:
lymphocytes, inside the thymus, are in front of a passage (small arrow) opening
into the external connective tissue. Stage B: the direction of migration is given by the
lymphocyte morphology: nucleus (n) ahead and uropod (u) behind. Stage C: a
lymphocyte is just migrating across the vascular wall. Stage D: free lymphocyte in
the vascular lumen.
by macrophages (Figs 13, 15). Dead lymphocytes are sometimes observed inside
epithelial cells.
During metamorphose (stages 55a-56) no more epithelial and lymphoid
differentiation occurs. An increase in the number of phagocytic cells and some
new cellular types can nevertheless be observed, i.e. mast cells, myoid cells and
rare plasma cells.
DISCUSSION
This ultrastructural study of Pleurodeles waltlii thymus describes four essential
phenomena concerning the behaviour of lymphocytes during ontogeny: differentiation, multiplication, migration and lysis.
Development of amphibian thymus. I
229
The blast transformation of individual mesenchymal stem-cells in close contact
with epithelial cells occurs from stage 43 to stage 45. These lymphoblasts present
numerous cytoplasmic buds. A clasmatosis phenomenon allows these buds to
be lost and in this way, lymphoblasts become typical lymphocytes. Such cytoplasmic loss is strikingly similar to the separating-off of cytoplasmic processes
described by Murray, Murray & Pizzo (1965) in rat lymphocytes, by Kabalov &
Grachova (1970) as 'leptons' and Sugimoto, Yasuda & Egashira (1977) in bird
lymphocytes. This thymocyte differentiation pathway seems to be a general
feature in vertebrates. Moreover, we agree with these authors in postulating that
direct contact between epithelial and lymphoid cells might be involved in this
phenomenon.
From stage 46 to stage 52 an important phase of lymphocyte proliferation
correlates with the general increase of thymus size. Mitoses are frequently seen
in close contact with epithelial cells, the latter showing the typical aspect of the
dense reticular cells described by Izard & de Harven (1968) in AKR leukaemic
mice. Reticular cells surrounding dividing lymphocytes were also described in
the subcapsular cortex of mammal thymuses by Metcalf (1966), Mandel (1969)
and Rappay, Bukulya & Bacsy (1971). As the number of DRC increases considerably in leukaemic lymphoid tissues, Metcalf, Ishidate & Brumby (1967)
suggested that these cells might be essential for continuous lymphocyte proliferation. All these data reinforce our assumption that DRC are involved in thymic
lymphopoiesis.
At stage 53 two important phenomena occur with respect to the thymic
lymphocytes. Some of them die within the thymus, others are observed to
migrate through the vascular walls. Dead lymphocytes are most frequently
phagocytosed by macrophages; nevertheless lymphocyte remnants are sometimes found within epithelial cells. The lymphocytic nature of altered or dead
cells can be assumed by the visualization of numerous transitory stages between
almost intact lymphocytes to advanced alteration stages. Lymphocyte death
within the developing thymus was also described in the rat by Klug (1965).
Toro et al. (1968), Pfoch & Welsch (1970) and Rappay, Bukulya & Bacsy (1971).
Several steps of lymphocyte migration through vascular walls are described.
It is now demonstrated that thevertebrate lymphocyte migrates with its nucleus
ahead and its uropod behind, and this phenomenon was confirmed in Pleurodeles (Charlemagne, unpublished materials). Our electron micrographs strongly
suggest that thymocytes, in accordance with their morphological assymmetry,
leave the thymus via the blood circulation and probably colonize peripheral
lymphoid tissues, as in higher vertebrates (Weissman, 1967). So a good correlation exists between the morphological observation of thymocytes seeding from
the thymus (stage 53) and the quoted 'end stage' for a complete efficiency of
larval thymectomy (stage 52).
The urodele thymus does not show a clear-cut differentiation into typical
cortical and medullary zones as in other terrestrial vertebrates (Klug, 1967;
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M. HENRY AND J. CHARLEMAGNE
Hightower & Saint Pierre, 1971; Desvaux, 1974). Rather, the urodele thymus
possesses a homogeneous cortical-like tissue spotted by medulla-like areas.
Our findings suggest that Pleurodeles thymus is, at the cytological level, not so
different from the anuran (Kapa, Olah & Toro 1968; Curtis, Volpe & Cowden,
1972; Rimmer, 1977) and higher vertebrate (Abe & Ito, 1970; Chapman &
Allen, 1971; Kaiserling, Stein & Miiller-Hermelink 1974; Jones, Thomas &
Jones Williams, 1975) thymuses.
The authors wish to thank Dr J. D. Horton (University of Durham, England) for kindly
reviewing this manuscript. Pr. R. Couteaux and Mr J. Escaig for their hospitality in
the Laboratoire de Microscopie electronique appliquee a la Biologie, 105 Bd. Raspail, 75272
Paris. The technical assistance of G. Westrelin was greatly appreciated. This work was supported by the Delegation a la Recherche Scientifique et Technique (DGRST, 74 704 08 and
77 700 37).
SOMMAIRE
Les differentes etapes de la morphogenese du thymus ont ete etudiees par microscopie
electronique chez Famphibien urodele Pleurodeles waltlii Michah. (Salamandridae). Le
travail s'attache tout particulierement a l'etude des cellules lymphoides au cours de l'ontogenese du thymus:
(a) du stade 38 au stade 42 les cellules souches lymphoides indifferenciees colonisent
1'ebauche epitheliale thymique;
(b) du stade 43 au stade 45 les cellules souches lymphoides se differencient en lymphoblastes. Ces lymphoblastes presentent de nombreux bourgeons cytoplasmiques qui se detacheraient, probablement par un processus de clasmatose. Finalement des lymphocytes typiques
sont observes;
(c) du stade 46 au stade 52 les thymocytes se multiplient activement; ils sont alors constamment en contact etroit avec les cellules reticulaires epitheliales denses;
(d) a partir du stade 53 la lyse de nombreux lymphocytes est observee dans le tissus thymique,
les cellules mortes etant secondairement phagocytees par des macrophages. Par ailleurs, une
migration des lymphocytes a travers les vaisseaux sanguins est observee. Ces lymphocytes
pourraient done des le stade 53 peupler les organes lymphoides peripheriques. De telles
donnees morphologiques sont en accord avec les resultats de l'analyse physiologique qui
permet de situer au stade 52 le seuil d'efficacite de la thymectomie chez cet urodele.
REFERENCES
ABE, K. & ITO, T. (1970). Fine structure of small lymphocytes in the thymus of the mouse:
qualitative and quantitative analysis by electron microscopy. Z. Zellforsch. mikrosk. Anat.
110, 321-335.
CHAPMAN, W. L. & ALLEN, J. R. (1971). The fine structure of the thymus of the fetal and
neonatal monkey (Macaca mulatto). Z. Zellforsch. mikrosk. Anat. 114, 220-233.
CHARLEMAGNE, J. (1974). Larval thymectomy and transplantation immunity in the urodele
Pleurodeles waltlii Michah. (Salamandridae). Eur. J. Immunol. 4, 390-392.
CHARLEMAGNE, J. (1977a). Anti-horse red blood cells antibody synthesis in the Mexican
axolotl {Ambystoma mexicanum). Effect of thymectomy. In Developmental Immunobiology
(ed. J. B. Solomon and J. D. Horton), pp. 267-275. Amsterdam: Elsevier/North-Holland
Biomedial Press.
CHARLEMAGNE, J. (19776). Thymus development in Amphibians: colonization of thymic
endodermal rudiments by lymphoid stem-cells of mesenchymal origin in the urodele
Pleurodeles waltlii Michah. Annls Immunol. {Inst. Pasteur), 128 C, 897-904.
CHARLEMAGNE, J. (1979). Thymus independent anti-horse erythrocyte antibody response and
suppressor T-cells in the Mexican axolotl (Amphibia, Urodela, Ambystoma mexicanum).
Immunology, 36, 643-648.
Development of amphibian thymus. I
231
CHARLEMAGNE, J. &HOUILLON, CH. (1968). Effets delathymectomielarvairechezl'Amphibien
urodele, Pleurodeles waltlii Michah. Production a l'etat adulte d'une tolerance aux allogreffes cutanees. C. r. hebd. Seanc. Acad. Sci., Paris 267, 253-256.
CHARLEMAGNE, J. & TOURNEFIER, A. (1977). Humoral response to Salmonella typhimurium
antigens in normal and thymectomized urodele Pleurodeles waltlii Michah. Eur. J. Immunol.
7, 500-502.
COHEN, N. (1969). Immunogenetic and developmental aspects of tissue transplantation
immunity in urodele amphibians. In Biology of Amphibian Tumors (ed. Merle Mizell),
pp. 153-168. New York: Springer-Verlag.
COHEN, N. (1970). Tissue transplantation immunity and immunologic memory in Urodela
and Apoda. Transplant. Proc. 2, 275-281.
CURTIS, S. K., VOLPE, E. P. & COWDEN, R. R. (1972). Ultrastructure of the developing
thymus of the Leopard Frog. (Rana pipiens). Z. Zellforsch. mikrosk. Anat. 127, 323-346.
DARDENNE, M., TOURNEFIER, A., CHARLEMAGNE, J. and BACH, J.-F. (1973). Studies on thymic
products. VII. Presence of thymic hormone in urodele serum. Annls Immunol. (Inst.
Pasteur) 124 C, 465-469.
DESVAUX, M. (1974). Etude de la morphogenese du thymus chez Pleurodeles waltlii Michah.
Bull. Soc. zool. France 99, 259-265.
FACHE, B. & CHARLEMAGNE, J. (1975). Influence on allograft rejection of thymectomy at
different stages of larval development in urodele amphibian Pleurodeles waltlii Michah.
(Salamandridae). Eur. J. Immunol. 5, 155-157.
GALLIEN, L. & DUROCHER, M. (1957). Table chronologique du developement chez Pleurodeles waltlii Michah. Bull. Soc. zool. Fr. et Beig. 91, 97-114.
HIGHTOWER, J. A. & ST PJERRE, R. L. (1971). Hemopoietic tissue in the adult newt, Notophtalmus viridiscens. J. Morph. 135, 299-308.
IZARD, J. & DE HARVEN, E. (1968). Increased numbers of a characteristic type of reticular
cell in the thymus and lymph nodes of leukemic mice: an electron microscopic study.
Cancer Res. 28, 421-433.
JONES, D. L., THOMAS, K. & JONES WILLIAMS, W. (1975). A fine structure study of human
thymus, Beitr. path. Bd. 156, 387-400.
KABAKOV, Y. N. & GRACHOVA, A.I. (1970). Clasmatosis: ultrastructure of cytoplasmic
fragments from the spleen, lymph nodes and thymus of the rat. Protoplasma 70, 167-177.
KAISERLING, E., STEIN, H. & MULLER-HERMELINK, U. K. (1974). Interdigitating reticulum cells
in the human thymus. Cell Tiss. Res. 155, 47-55.
KAPA, E., OLAH, I. & T6R6, I. (1968). Electron microscopic investigation of the thymus of
adult frog {Rana esculentd). Acta biol. hung. 19, 203-313.
KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for
use in electron microscopy. /. Cell. Biol. 27, 137A-138A.
KLUG, H. (1965). Elektronenmikroskopische Untersuchungen zur Phagozytose strahlengeschadigter lymphozyten im Thymus von Ratten. Z. Zellforsch. mikrosk. Anat. 68, 43-56.
KLUG, H. (1967). Submikrokopische zytologie des Thymus von Ambystoma mexicanum.
Z. Zellforsch. mikrosk. Anat. 78, 388-401.
MANDEL, T. (1969). Epithelial cells and lymphopoiesis in the cortex of guinea-pig thymus.
Aust. J. exp. Biol. med. Sci. 47, 153-155.
METCALF, D. (1966). The thymus. Its role in immune responses, leukemia development and
carcinogenesis. In Recent Results in Cancer Research (ed. P. Rentchnick), pp. 1-144.
Springer-Verlag, Berlin, Heidelberg, New York.
METCALF, D., ISHIDATE, M. & BRUMBY, M. (1967). Proliferation and behavior of phagocytic
cells in mouse lymphoma tissue. /. natn. Cancer Inst. 38, 527-539.
MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy.
/. Stain Techno!. 39, 111-114.
MURRAY, R. G., MURRAY, A. & Pizzo, A. (1965). The fine structure of the thymocytes of
young rat. Anat. Rec. 151, 17-39.
PFOCH, M. & WELSCH, U.(1970). AltersbhangigeVeranderungen in histochemischen Reaktiosmuster des Thymus bei Wistar-Ratten Z. Zellforsch. mikrosk. Anat. 104, 138-148.
RAPPAY, GY., BUKULYA, B. & BACSY, E. (1971). Fine structure, distribution and function of
the rat thymic reticular cells in the perinatal life. Acta. biol. hung. 22, 187-196.
232
M. HENRY AND J. CHARLEMAGNE
J. J. (1977). Electron microscopic studies of the developing amphibian thymus.
Dev. Comp. Immunol. 1, 321-332.
SUGIMOTO, M., YASUDA, T. & EGASHIRA, Y. (1977). Development of embryonic chicken
thymus. I. Characteristic synchronous morphogenesis of lymphocytes accompanied by
the appearance of an embryonic thymus-specific antigen. Devi Biol. 56, 281-292.
RIMMER,
TORO, I., BACSY, E., OKROS, I. VADASZ, GY. & RAPPAY, GY. (1968). Postirradiation changes
in ultrastructure and enzyme cytochemistry of rat thymus. Ada. med. Acad. Sci. Hung. 25,
355-366.
TOURNEFIER, A. (1973). Developpement des organes lymphoides chez l'amphibien urodele
Triturns alpestris Laur., tolerance des allogreffes apres la thymectomie larvaire. /. Embryol.
exp. Morph. 29, 383-396.
WEISSMAN, I. L. (1967). Thymus cell migration. / . exp. Med. 126, 291-304.
{Received 10 January 1980)