Development of the embryonic chicken thymus

DEVELOPMENTAL

BIOLOGY

56, 293-305

Development II. Differentiation MASANOBU * Department

of the Embryonic

of the Epithelial

SUGIMOTO,*

of Pathology

Received

(1977)

and

Cells Studied

TOMOYOSHI t Department Shinagawa-ku,

October

4, 1976;

Chicken

YASUDA,?

of Technology Tokyo

accepted

by Electron AND

of National 141, Japan

in revised

Thymus

form

Microscopy

YASUYUKI Institute

Decem,ber

EGASHIRA” of Health,

Kamiosaki,

6, 1976

The differentiation of thymic epithelial cells in embryonic chickens was studied ultrastructurally from the tenth day of incubation to hatching. Reticuloepithelial cells first displayed functionally differentiated features between the twelfth and thirteenth days of incubation, whereas medullary epithelial cells with cysts and/or numerous granules did not display such features until the fifteenth day of incubation or later. Between the twelfth and thirteenth days, reticuloepithelial cells came to possess characteristic electron-lucent vacuoles and dense bodies. Furthermore, some of the vacuoles, probably as a result of fusion, seemed to contain the electron-dense bodies. The density of the material of such inclusion vacuoles tended to decrease after its entrance into the vacuoles. These findings suggested that the material in the inclusion vacuoles may be released into cytoplasm and, thereafter, may be secreted to exterior of the cells. Since a series of distinct morphological changes of reticuloepithelial cells took place simultaneously with an abrupt increase of lymphoctes bearing the embryonic thymus-specific antigen (T antigen), it is likely that the material supposedly released from the inclusion vacuoles of reticuloenithelial cells could be resoonsible for differentiation of lymphoid cells into embryonic T-antigen-bearing lymphocytes. INTRODUCTION

It is generally accepted that intrathymic differentiation of lymphoid cells as well as differentiation of thymus-derived lymphocytes in the peripheral lymphoid tissues, is influenced by humoral factors supposedly elaborated by thymic epithelial cells (Goldstein et aZ., 1971; Teodorczyk and Potworowski, 1975; Potworowski et al., 1975). The epithelial cells are generally classified as reticuloepithelial cells, present either in the cortex or medulla, and epithelial cells, found in the medulla alone. Reticuloepithelial cells are considered to produce thymic humoral factors which induce intrathymic differentiation of precursor lymphoid cells into thymus-specific (T)-antigen-bearing lymphocytes (Komuro and Boyse, 1973a,b; Teodorczyk and Potworowski, 1975; Teodorczyk et al., 1975). However, the mechanism which can elaborate such a factor is not clear. Medullary

epithelial cells may also be a candidate to regulate the differentiation of lymphoid cells. One of the interesting approaches to elucidate further the role of such epithelial cells may be to investigate, in the developing embryonic thymus, the morphological differentiation of epithelial cells compared to the differentiation of lymphoid cells. If differentiation of lymphoid cells are under the control of epithelial cells, there may be a temporal relationship between the two kinds of cells during their morphological and functional changes. In the developing thymus of embryonic chickens, a large proportion of lymphoid cells were found almost synchronously to show characteristics typical of differentiated thymic lymphocytes between the twelfth to the thirteenth days of incubation (Sugimoto et al., 1977): round-shaped cells, distinct heterochromatin in their nu-

293 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

294

DEVELOPMENTAL BIOLOGY

clei, and an embryonic T antigen on their cell surface. Prior to hatching, thymic lymphocytes develop partial immunocompetence, enabling them to mount a graftversus-host reaction (Jancovic et al., 1975) and to react with mitogens (Sallstrom and Alm, 1974). Ultrastructurally, it is important to note the differentiation of epithelial cells by comparing such morphological and functional transformation of lymphoid cells. In the present experiment, ultrastructural changes of the developing embryonic chicken thymus from the tenth day of incubation to hatching were studied. MATERIALS

AND

METHODS

Embryonic and posthatch chickens. White Leghorn chicken embryos from the tenth to the twentieth days of incubation and post-hatch chickens were used. Light-microscopical and electron-microscopical observations. The thymus was fixed at 0°C for 1 hr simultaneously with 2.5% glutaraldehyde and 2.0% 0~0, in 20 nN phosphate buffer (pH 7.4) containing 4.5% sucrose (Kurozumi, 1970). The fixed tissue was embedded in Epon 812 (Luft, 1961) and 2-pm sections were made with an LKB-8800 ultramicrotome and stained with toluidine blue for light-microscopical observations. Fifty-nanometer sections were similarly made and double-stained with uranyl acetate and lead citrate (Venable and Coggeshall, 1965) for ultrastructural observations with a Hitachi HU-11B or HS-8 electron microscope. RESULTS

Light-Microscopical

Observations

From Days 10 to 12 of incubation. On the tenth day of incubation, about equal numbers of epithelial cells and lymphoid cells existed intermingled. From the tenth to the twelfth days, the number of lymphoid cells progressively exceeded that of epithelial cells. Epithelial cells had clear nuclei with two nucleoli which contained

VOLUME 56, 1977

light-stained cytoplasm in comparison with lymphoid cells. From the thirteenth to the fifteenth days of incubation. Between the twelfth to the thirteenth days a large number of typical lymphocytes possessing distinct heterochromatin in their nuclei increased abruptly and populated the gland in a more dense manner (Fig. la). During this stage, the primitive cortex and medulla became individually distinguishable. Simultaneously with the changes of lymphocytes, epithelial cells also showed significant changes. First, typical reticuloepithelial cells developed which were scattered chiefly among the lymphoid cells, primarily in the cortex, and formed typical reticular structures (Fig. la). These reticuloepithelial cells were also characterized by their light vacuoles and fine granules. Second, several epithelial cells gathered to form primitive medullary zones, some of which hypertrophied extensively (Fig. lb). Most of these epithelial cells did not form reticular structures and were never present in the cortex alone unlike reticuloepithelial cells. These epithelial cells will be referred to as medullary epithelial cells distinguishing them from reticuloepithelial cells. From the sixteenth day of incubation to hatching. On the sixteenth day or later, the lobules of the thymus were more closely packed and the cortex became thicker. Small lymphocytes were by far the most numerous cell type (Fig. Id). Reticuloepithelial cells in the cortex had more numerous vacuoles and fine granules (Fig. Id). In the medulla, groups of hypertrophied epithelial cells were seen occasionally, containing cystic structures and also having many granules in their cytoplasm (Fig. lc). A few distinct Hassall’s bodies were observed just before hatching. Electron-Microscopical

Observations

From the tenth to the twelfth days of incubation. Epithelial cells of the tenth to the eleventh days of incubation had simi-

SUGIMOTO,

YASUDA,

AND

EGASHIRA

Thymic

Epithelial-Cell

Differentiation

295

FIG. 1. Light microscopy of the thymus from embryonic chickens and chickens at hatching. (a) Cortical area of 13-day embryonic thymus. Reticuloepithelial cells are seen among typical lymphocytes with distinct heterochromatin in their nuclei. (b) Primitive medullary area of 13-day embryonic thymus. Hypertrophied epithelial cells form a medullary area in the boundary between lobules. (c) Medulla with cysts of 16-day embryonic thymus. One large and two small cysts are seen (arrows). The epithelial cells with cysts have apparently hypertrophied cytoplasm with numerous cytoplasmic granules. (d) Cortex of the thymus of a chicken at hatching. Among lymphocytes, there is a typical reticuloepithelial cell which has dense granules and light vacuoles in its cytoplasm. The thymus was fixed simultaneously with glutaraldehyde and 0~0, and embedded in Epon 812. Two-micrometer sections were stained with toluidine blue. x 1000.

lar morphological features. Most of them had reticular structures enveloping lymphoid cells with their elongated cytoplasmic processes. The Golgi apparatus contained small vesicles and a few multivesicular bodies (Fig. 2). The multvesicular bodies contained amorphous material. The cytoplasm was rich in rough-surfaced endoplasmic reticulum (r-ER) but the r-ER did not have a large number of ribosomes on its membrane. There were a few lysosome-like bodies as well as stellate bodies in these cells. On Day 12, some of the multivesicular bodies became more electron-dense. Portions of the r-ER be-

came large, and round vacuoles were also apparent (Fig. 3). From the thirteenth to the fifteenth days of incubation. Ultrastructural changes in epithelial cells observed between the twelfth and thirteenth days of incubation were drastic. At least two types of epithelial cells, reticuloepithelial cells and medullary epithelial cells, could be discriminated from each other. Reticuloepithelial cells extended their cytoplasmic processes to embrace lymphocytes and remained linked to each other via the desmosomes of the processes (Figs. 4 and 5). They were characterized by electron-lucent vacuoles

FIG. 2. Ultrastructure of reticuloepithelial cells of lo-day embryonic thymus. Three multivesicular bodies (MV) are seen near Golgi apparatus. Amorphous material is contained in these bodies. Small vesicles are conspicuous in Golgi area. Cytoplasm is rich in round r-ER with a few ribosomes on its membrane. A desmosome (DS) with tonotibrils is seen in the boundary of two epithelial cells. The thymus was fixed simultaneously with glutaraldehyde and 0~0, and embedded in Epon 812. Fifty-nanometer sections were stained with uranyl acetate and lead citrate. x 17,500. FIG. 3. A reticuloepithelial cell of 12-day embryonic thymus. Two electron-dense bodies (DB) with small vesicles and one electron-lucent vacuole (V) are seen near the Golgi apparatus. See also the legend to Fig. 2. x 15,000. 296

FIG. 4. A reticuloepithelial cell of 13.day ous. Several electron-dense bodies (DB) are include small dense vesicles and granules. FIG. 5. A reticuloepithelial cell of 13-day dense bodies are seen. In addition, several and small dense granules are seen. See aso

embryonic thymus. Electron-lucent vacuoles (V) are also seen. It is noted that some of the electron-lucent See also the legend to Fig. 2. x 15,000. embryonic thymus. Both electron-lucent vacuoles and inclusion vacuoles (IV) with electron-dense amorphous the legend to Fig. 2. x 15,000. 297

conspicuvacuoles electronmasses

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(1 pm in average diameter) and electrondense bodies (0.5 pm). The electron-lucent vacuoles seemed to be derived from a part of the r-ER found in cells of earlier stages, since similar vacuoles with a few ribosomes were occasionally found. These vacuoles were filled with some electron-lucent material. The dense bodies contained small vesicles and granules. The multivesicular bodies found in cells on the twelfth day of incubation or earlier seemed to be precursors of these bodies. On Day 13, most of these bodies contained large amounts of dense amorphous material and small granules unlike those of earlier stages. The contents are assumed to be derived from the Golgi area, since dense small vesicles and granules have been observed near the Golgi area. A notable phenomenon of these cells was that the electron-lucent vacules sometimes contained either (i) small dense vesicles and granules or (ii) amorphous masses quite similar to the contents of the dense bodies, or, alternately, (i) and (ii). Occasionally, such a feature was observed as indicating fusion of the lucent vacuoles with the dense bodies (Fig. 8). These inclusion vacuoles were virtually absent on Days 10 and 11, found only occasionally on Day 12, and found very frequently on Day 13 or later. The other type of epithelial cell was found in the medulla. These cells were linked to similar adjacent epithelial cells by means of desmosomes, but contained few, if any, cytoplasmic processes and no electron-lucent vacuoles, unlike the previously described reticuloepithelial cells. They were characterized by a large cytoplasm with many mitochondria, a welldeveloped Golgi apparatus, and dense bodies. Most of these dense bodies contained homogeneous dense materials different in character from those of reticuloepithelial cells, although some dense bodies had vesicular structures similar to the dense bodies of reticuloepithelial cells. Cysts with microvilli were formed in the cytoplasm of some of these cells. Primitive microvilli

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were seen on Day 13, but typical cysts with microvilli were not seen before the fifteenth day of incubation. Precursors of secretory cells possessing numerous granules which will be described later were also identified in this stage. From the sixteenth day of incubation to hatching. In this stage, the dense bodies in the reticuloepithelial cells of the cortex were numerous, and most of the electronlucent vacuoles contained dense material (Fig. 61, although there were also undifferentiated reticuloepithelial cells containing lucent vacuoles without dense material or, alternately, having only a few vacuoles and dense bodies. The dense bodies were roughly classified into two types, one with clearly visible small vesicles and granules and the other with more homogeneous contents. We believe that the former represents the immature form of the latter. The inclusion vacuoles varied in their appearance: (i) vacuoles containing amorphous mass of various densities (Fig. 8); (ii) those with small vesicles and granules (Fig. 4); (iii) those including both of the latter; (iv) large vacuoles (3-4 pm in diameter) with numerous amorphous masses (Fig. 7); (v) vacuoles almost filled with several amorphous masses (Fig. 8). Fusion seemingly occurred not only between the vacuoles and dense bodies, but also between vacuoles and vacuoles or between dense bodies and dense bodies (Figs. 6-8). As a consequence, differentiated reticuloepithelial cells contained various types of dense bodies and inclusion vacuoles. There was also a small number of half-dense multivesicular bodies very similar to those found in loto 11-day cells. A significant observation at this stage is that the amorphous masses in the inclusion vacuoles were of varied densities and that some masses apparently underwent a reduction in their density (Figs. 6 and 8). These findings led to the conclusion that at least a part of the electron-dense material of the inclusion vacuoles was released outside the vacuoles. On the other hand,

FIG. 6. A differentiated reticuloepithelial cell of the thymus from a chicken at hatching. The reticuloepithelial cell is surrounded by small lymphocytes, elongating its cytoplasmic processes among them. There are numerous dense bodies and inclusion vacuoles of various types. Electron-lucent vacuoles without dense material as seen in 13-day cells (Figs. 4 and 5) are absent. See also the legend to Fig. 2. x 9000. FIG. 7. A reticuloepithelial cell with a large inclusion vacuole of the thymus from a chicken at hatching. See also the legend to Fig. 2. x 12,000. 299

FIG. 8. Inclusion vacuoles of a reticuloepithelial cell of the thymus from a chicken at hatching. One inclusion vacuole (IV,) reveals a feature indicating that an electron-lucent vacuole has just fused with a dense body. The other inclusion vacuoles contain low-density-amorphous masses, small dense granules, and vesicles (IV,). There is also a vacuole which is almost entirely filled with dense material (IV,). It is noted that the density of the amorphous masses of the inclusion vacuoles (IV,, IV,, IV,) is lower than that of the dense bodies. One electron-lucent vacuole has an opening (IV,). See also the legend to Fig. 2. A lysosomelike body is also seen (LY). x 19,200.

SUGIMOTO,

YASUDA,

AND

EGASHIRA

there was no indication that the vacuoles or dense bodies released their contents directly outside the cells by exocytosis. Medullary epithelial cells with cysts presented functionally differentiated features on the fifteenth day of incubation or later. Although not all cells with cysts demonstrated features of activity, some displayed apparent synthesizing activity of dense amorphous material (Figs. 9 and 10). Golgi apparatus, r-ER, and smoothsurfaced ER (s-ER) were well developed in these cells. Some of these cells also contained many dense granules. Among them were granules of different sizes characterized by electron-lucent patches. There were phagosome-like bodies supposedly including lysosomes. Some s-ER contained electron-dense material (Fig. 10). There was membranous debris in the lumen of most cysts. In some cases, amorphous dense material was observed in the lumen of the cysts (Fig. 9). The tips of the microvilli of such cysts were very electron dense (Fig. 9, inset). These findings suggested to us that some electron-dense material in the cytoplasm was secreted through the tips of the microvilli into the lumen. Many mitochondria were gathered at the base of the microvilli, reflecting active energy metabolism there (Fig. 9). Adjoining cells interdigitated each other by their narrow cytoplasmic processes. There were also a small number of cells with numerous dense granules but no cyst. These cells probably secreted the contents of these granules, since release of their contents outside the cells by exocytosis was observed (Fig. 11). DISCUSSION

As shown in the present study on the developing embryonic chicken thymus, reticuloepithelial cells which are mainly present in the cortex displayed functionally differentiated features between the twelfth and thirteenth days of incubation, a period of time when a large proportion of lymphoid cells first acquired embryonic T

Thymic Epithelial-Cell

Differentiation

301

antigen on their cell surfaces and distinct heterochromatin in their nuclei (Sugimoto et al., 1977). On the other hand, medullary epithelial cells with cysts or secretory granules did not manifest such features until the fifteenth day or later. These tindings strongly support the concept that reticuloepithelial cells may be the responsible element for lymphoid-cell differentiation. The pattern of the dense bodies and electron-lucent vacuoles is to be noted in reticuloepithelial-cell differentiation during embryonic development of the thymus. Typical dense bodies and electron-lucent vacuoles appeared first between the twelfth and thirteenth days of incubation, although their precursors were seen before the twelfth day. An interesting observation of these organelles was that the vacuoles contained the contents of the fused dense bodies as well as the small vesicles and granules found in the Golgi area (Figs. 5-8). These inclusion vacuoles observed in the chicken thymus may correspond to the cytoplasmic inclusions of Type I in the mouse thymus described by Tamaoki and Esaki (1974). Furthermore, the contents of these inclusion vacuoles might reflect digested or released products, since they showed various degrees of density, including extremely low density features in the differentiated reticuloepithelial cells (Figs. 6 and 8). These inclusion vacuoles are probably important in the process of elaboration of thymic humoral factor to be discussed. There was no indication that the contents of the dense bodies, electronlucent vacuoles, or inclusion vacuoles were released directly outside the cell by exocytosis. Several investigators have reported that thymic humoral factors are capable of initiating the appearance of T antigen in lymphoid precursor cells. These factors manifest their effects in vitro within 1.52.5 hr by increasing the concentration of cyclic adenosine monophosphate and/or augmenting transcription and translation (Komuro and Boyse, 1973a,b; Teodorczyk

FIG. 9. Cyst of epithelial cells from the thymus of chicken at hatching. Amorphous dense present in the lumen of the cyst. Tips of microvilli are dense. Dence particles with electron-lucent (DP) are also present. x 20,000. Inset: dense tips of microvilli and amorphous dense material near 48,500. See also the legend to Fig. 2. FIG. 10. Cyst of epithelial cells from the thymus of a chicken at hatching. Cytoplasm is very tilled with dense material. Narrow cytoplasmic processes of ajoining cells are interdigitated with See also the legend to Fig. 2. x 26,500. 302

material is patches the tips. x rich in s-ER each other.

SUGIMOTO,

FIG. apparent

11. Secretory exocytosis

YASUDA,

AND EGASHIRA

Thymic

cells with numerous granules of 17-day of the content of a granule is seen (inset).

and Potworowski, 1975; Scheid et al ., 1975; Storrie et al., 1976). With respect to the cytoplasmic organelles elaborating such humoral factors,Mandi and Glant (1973) demonstrated with the use of immunofluorescence that fine as well as coarse granules in the epithelial cells of the reticular structure were stained with anti-thymoet al. (1975) resine IgG. Potworowski ported that antibody to one such factor, when administered into embryogenated eggs, inhibited differentiation of lymphoid cells into T-antigen-bearing lymphocytes and also led to the destruction of reticuloepithelial cells. They called these vacuoles “secretory vacuoles”. From these facts together with our present ultrastructural findings on the reticuloepithelial cells as discussed before, it seems likely that the inclusion vacuoles of the reticuloepithelial cells may, in fact, be an apparatus elaborating the thymic humoral factor which in turn stimulates the appearance of T-antigen-bearing lymphoid cells. A hypothetical mechanism for the

Epithelial-Cell

embryonic x 27,000.

Differentiation

chicken thymus. See also the legend

303

x 17,000. An to Fig. 2.

elaboration of the thymic factor from inclusion vacuoles might be as follows: (i) The content of the dense bodies represents the precursor of thymic humoral factor, whereas that of the electron-lucent vacuoles represents an activator, i.e., a specific proteolytic enzyme; and (ii) the activator may transform the precursor into its active form in the inclusion vacuoles in an analogous manner to the peptidase (kallikrein) which transforms bradykininogen into its active form, bradykinin (a review, Wilhelm, 1973). The appearance of embryonic T-antigen-bearing lymphoid cells simultaneous with the development of the inclusion vacuoles of reticuloepithelial cells further supported the concept that elaboration of the thymic humoral factor is dependent upon the inclusion vacuoles. As shown in the preceding paper (Sugimoto et al., 19771, however, lymphoid cells of the embryonic chicken thymus showed a series of dramatic changes other than appearance of embryonic T antigen between the tenth and thirteenth days of incuba-

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tion which can be summarized as follows: (a) cytoplasm became stained deeply with toluidine blue; (b) large cytoplasmic processes were formed, and these processes were separated from the parent cells; and (c) distinct heterochromatin was formed in their nuclei. It is unlikely that the thymic humoral factor of reticuloepithelial cells, as discussed, is also responsible for all these changes. Another factor, for instance, the direct contact between reticuloepithelial cells and lymphoid cells, may be responsible for such changes. Stutman et al. (1970a,b) demonstrated the presence, in mice of two kinds of cells which are affected by the thymus gland. One type was those cells contained in adult and newborn tissues that had already received some of the thymic influence necessary to become a post-thymic population which is sensitive to the humoral activity of the thymus. The other type was those cells contained in embryonic tissues that still required thymic influence, through traffic into the organ, to become sensitive to the humoral activity of the thymus. These two types of cells were termed “post-thymic” and “prethymic”, respectively. In connection with this fact, it is not clear at present whether the thymic factor supposedly elaborated from the inclusion vacuoles, as discussed here, renders its effect locally in the thymus or systemically; in other words, whether it affects prethymic or post-thymic cells. Two types of active cells were observed in the medulla. First, epithelial cells with cysts appeared to contain some dense materials within the cyst lumen. Since some of these cells had s-ER containing electrondense material, the material in the lumen might be synthesized by s-ER and secreted into there. Second, there were epithelial cells with numerous granules which could apparently release the material of the granules directly to the outside of the cells. From these findings, it is highly probably that both of these epithelial cells in the medulla also secrete active substances.

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Such secretory functions became obvious on Day 15 of incubation or later. The presence of both types of cells in the thymus of young chickens was reported by Isler (1976). The immunocompetence of thymic lymphocytes as manifested by the ability to mount graft-versus-host reactions (Jancovic et al., 1975) and to respond to mitogens (Sallstrom and Alm, 1974) appears just before hatching. Therefore, it is possible that lymphoid cells are first influenced by reticuloepithelial cells to differentiate into embryonic T-antigen-bearing lymphocytes; then, these cells transform further into immunocompetent lymphocytes under the influence of epithelial cells in the medulla. The function of such epithelial cells, however, must be elucidated further, particularly since immunodeficient nude mice have thymus rudiments including epithelial cells with cysts (Tamaoki and Esaki, 1974). We thank Dr. S. Nakazawa of the present Institute for his support of this work and our colleagues in our laboratory for their kind discussion and advice. We thank Dr. T. Tsuruhara for his valuable advice. We also thank Mr. K. Yaginuma, Mrs. K. Miyanomae, and Mrs. M. Kitamura for their technical assistance and Miss M. Kimura for preparing this manuscript. Finally, we thank Dr. M. I. Greene for revising this manuscript and Mrs. Sharon Smith of the Harvard Medical School for editing it. REFERENCES GOLDSTEIN, A. L., GUHA, A., HOWE, M. L., AND WHITE, A. (1971). Ontogenesis of cell-mediated immunity in murine thymocytes and spleen cells and its acceleration by thymosin, a thymic hormone. J. Zmmunol. 106, 773-780. ISLER, H. (1976). Fine structure of chicken thymic epithelial vesicles. J. Cell Ski. 20, 135-147. JANKOVIC, B. D., ISAKOVIC, K., LUKIC, M. L., VUJANOVIC, N. L., PETROVIC, S., AND MARKOVIC, B. M. (1975). Immunological capacity of the chicken embryo. I. Relationship between the maturation of lymphoid tissues and the occurrence of cell-mediated immunity in the developing chicken embryo. Immunology 29, 497-508. KOMURO, K., AND BOYSE, E. A. (1973a). Zn vitro demonstration of thymic hormone in the mouse by conversion of precursor cells into lymphocytes. Lancet 1, 740-743. KOMURO, K., AND BOYSE, E. A. (197313). Induction of

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AND EGASHIRA

T lymphocytes from precursor cells in vitro by a product of the thymus. J. Erp. Med. 138479-482. KUROZUMI, K. (1970). “Preparative methods for the samples for electron microscopy (in Japanese).” (Kant0 Branch of Japanese Society of Electron Microscopy, ed.), pp. 303-304. Seibundoshinkosha, Tokyo. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MANDI, B., AND GLANT, T. (1973). Thymosine-producing cells of the thymus. Nature New Biol. 246, 25. POTWOROWSKI, E. F., LEFEBVRE, D., LUSSIER, G., AND TEODORCZYK, J. A. (1975). Inhibition of T-cell differentiation by an antibody to a soluble thymic factor. Immunology 28, 1115-1121. SKLLSTRBM, J. F., AND ALM, G. V. (1974). Mitogenreactive lymphocytes in the embryonic chicken thymus in organ culture. Znt. Arch. Allergy 47, 388-399. SHEID, M. P., GOLDSTEIN, G., HAMMERLING, U., AND BOYSE, E. A. (1975). Lymphocyte differentiation from precursor cells in uitro. Ann. N. Y. Acad. Sci. 249, 531-540. STORRIE, B., GOLDSTEIN, G., BOYSE, E. A., AND HAMMERLING, U. (1976). Differentiation of thymocytes: Evidence that inducion of the surface phenotype requires transcription and translation. J. Zmmunol. 116, 1358-1362. STUTMAN, O., YUNIS, E. J., AND GOOD, R. A. (1970a). Studies on thymus function. I. Cooperative effect of thymic function and lymphohemopoietic cells in

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restoration of neonatally thymectomized mice. J. Ewp. Med. 132, 583-600. STUTMAN, O., YUNIS, E. J., AND GOOD, R. A. (1970b). Studies on thymus function. II. Cooperative effect of newborn and embryonic hemopoietic liver cells with thymus function. J. Exp. Med. 132, 601-619. SUGIMOTO, M., YASUDA, T., AND EGASHIRA, Y. (1977). Development of the embryonic chicken thymus. I. Characteristic synchronous morphogenesis of lymphocytes accompanied by the appearance of an embryonic thymus-specific antigen. Develop. Biol. 56, 281-292. TAMAOKI, N., AND ESAKI, K. (1974). Electron microscopic observation of the thymus and lymph nodes of the nude mouse. “Proceedings of the First Internatinal Workshop on Nude Mice” (J. Rygaard and C. 0. Povsen, eds.), pp. 43-50. Gustav Fischer Verlag, Stuttgart. TEODORCZYK, J. A., POTWOROWSKI, E. F., AND SVICULIS, A. (1975). Cellular localization and antigenie species specificity of thymic factors. Nature (London) 258, 617-619. TEODORCZYK, J. A., AND POTWOROWSKI, E. F. (1975). Induction of T-cell differentiation in the bursa of Fabricius by a soluble thymus factor. Immunology 28, 711-717. VENABLE, J. H., AND COGGESHALL, R. (1965). A simple lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408. WILHELM, D. L. (1973). Chemical mediators. In “The Inflammatory Processes” (B. W. Zwifach, L. Grant, and R. T. McCluskey, eds.), Chapter 8, pp. 251-301. Academic Press, New York.