THE AMERICAN JOURNAL OF ANATOMY 187:287-302 (1990)

Reappraisal of Histogenesis in the Bursa1 Lymphoid Follicle of the Chicken MARIO LUPETTI, AMELIO DOLFI, FRANCESCO GIANNESSI, FRANCESCO BIANCHI, AND SERGIO MICHELUCCI Histology and General Embryology, Institute of Anatomy, Medical School, University of Pisa, Pisa, Italy

The development of the bursal ABSTRACT follicle and the appearance of the follicle-associated epithelial (FAE) cell and the reticuloepithelial (REp) cell were studied. The stages of development of the bursal follicle were observed by light and electron microscopy; an anticytokeratin monoclonal antibody was also used. At the beginning of follicle development, a mesenchymal cell cluster is observed in the tunica propria; the cluster becomes wedged in a niche of the surface epithelium, and gradually it is completely surrounded by the epithelium itself, which closes under the clump of mesenchymal cells. The epithelial cells lying upon the mesenchymal clump become necrotic, and a number of mesenchymal cells bulge out, forming the FAE cells. The epithelial cells that have closed under the mesenchymal nodule become stratified and form the REp cells; they become star-shaped because the medullarylymphoid cells grow between them. Finally, the cortex is formed, possibly as a result of the migration of medullary cells before they peripheralize. It is concluded that FAE cells are not specialized epithelial cells, as they do not react to an anticytokeratin monoclonal antibody; on the contrary, they are formed by mesenchymal stemcells that bulge into the lumen and change their character after moving into the epithelium. The REp cells appear in the follicular primordium shortly after the bursal follicle begins to develop; the pronounced reactivity of the REp cells to an anticytokeratin monoclonal antibody supports the hypothesis of their epithelial origin.

tudinal plicae grow and protrude into the bursal lumen. Ackerman and Knouff (1959) described the formation of the bursal lymphoid follicle in the chick embryo. They observed, in particular, that undifferentiated epithelial cells undergo cell division a t various sites in the basal and intermediate layers of the epithelium, beginning from the 12th to the 16th day of embryonic life. These cells become larger and increase in number. In these areas, localized enlargements are formed in the epithelial layer and protrude into the tunica propria. These epithelial buds give rise to a solid group of cells, which continues to grow. The capillary vessels form a framework around the growing bud near the basal membrane. The central cells of the bud still have a n undifferentiated pattern; certain morphological changes may be observed when they become lymphoblastic cells. Other mesenchyme-derived cells have not been observed to gain access to the bud. In this way, the follicle increases in volume, up to 100-300 pm; its medullary part is made up of lymphoid cells, together with epithelial cells, which become star-shaped. As the medulla is built up, the cells belonging to the bud and facing the bursal lumen undergo histological and histochemical changes. At first, they are flattened and cover the upper surface of the follicle. They then become columnar in shape and have less easily detectable cellular borders; they contain pale, oval nuclei with several nucleoli. These cells give rise to the epithelial tufts that subsequently will be called lymphoidfollicle-associated epithelial (FAE) cells (Bockman and Cooper, 1971,1973).The cortex then is formed from the mesenchymatic cells of the tunica propria and from the undifferentiated epithelial cells that migrated to the underlying tunica propria before the onset of bud formation. Using chromosomic labeling in parabiotic embryos, it was possible to show that the medullary lymphoid INTRODUCTION cells of the bursal follicle did not originate from the Since 1621, when Fabricius ab Acquapendente first epithelium, but from the hemopoietic cells that had described that particular organ connected with the clo- migrated into the bud (Moore and Owen, 1966, 1967). aca in birds which is now called the bursa of Fabricius The same conclusion was drawn from studies using (Fabricius, 1621), it has been the object of extensive quail-chick chimeras (Le Douarin and Houssaint, 1974; embryological, comparative, and experimental re- Dieterlen-Lievre, 1975). Murphy and Cho (1971) showed that mononucleated search by numerous authors. The bursal anlage rises from the entoderm as a solid cells with a basophilic cytoplasm exist in the blood vesepithelial mass. It quickly loses its connection with its site of origin, becomes empty, and subsequently is joined by a n ectodermic infolding of the proctodeum. This ectodermic infolding will form the final link between the bursa and the cloaca by establishing a direct Received February 27, 1989. Accepted August 26, 1989 connection with the bursal anlage itself (Pera, 1958). Address reprint requests to Prof. M. Lupetti, c/o Istituto Anatomico, Around the 10th day of embryonic life, 12 to 14 longi- Via Roma, 55 1-56126 Pisa, Italy. 0 1990 WILEY-LISS, INC.

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sels and in the tunica propria before the onset of bud formation. Shortly afterward, these cells migrate through the tunica propria and gather in subepithelial foci, which pass through the basal membrane and take up positions inside the epithelium. Taking this observation a s their starting point, Edwards et al. (1975) saw that if the development of lymphoid follicles is considered to begin not on the 12th day of embryonic life but on the 9th, i.e., from the time the hemopoietic stem cells start migrating, a gradual migration of leukocytes and lymphocytes is found beneath the basal membrane of the epithelium, where these cells often form groups. The stem cells migrate into the epithelium, where they grow in nodules surrounded by the epithelial cells; there they increase in volume and push the basal membrane toward the tunica propria, forming the so-called epithelial buds. No connection is observed between epithelial and mesenchymal cells inside the buds. Therefore, the lymphoepithelial nodule is formed and is made up of a group of lymphoid cells proliferating a t the center and surrounded by epithelial cells. Houssaint and coworkers (1976) again used the quail-chick chimera experimental model and were able to graft the isolated bursal epithelial component of the quail-chick onto the heterospecific somatopleura a t various stages of development. They concluded that the epithelial buds start their formation from the 12th day in the chicken and from the 11th day in the quail. The migration of cells then takes place; because these cells have heterospecific features, they belong, of course, to the host of the graft and are of the hemopoietic cell line. These same workers also pointed out that the degree of host-cell migration into the grafted buds depends on the stage of the sampling of the grafted epithelium. Le Douarin et al. (1980, 1984) used the same quailchick chimera experimental model to study the influence of testosterone in epithelial-mesenchymal interactions. They observed that this hormone causes a thickening in the epithelial component, which blocks the formation of the epithelial buds; when testosterone is used on the host, however, the grafted entoderm generates normal follicles. The site of origin of the bursal stem cells was established by Lassila et al. (1978), who demonstrated that they originate in the yolk-sac wall. Studies on bursal follicle ontogenetic development have also taken into account the evolutive stages of the FAE cells; it has been observed that the surface epithelium overlying the buds changes its characteristics on the 13th day of embryonic life. At first, the FAE area bulges out toward the bursal lumen, and then its cells change their shape and their staining affinity in relation to the surrounding epithelium. On the 19th day of embryonic life, the FAE area becomes capable of taking up colloidal carbon (Naukkarinen et al., 1978). This capability was also reported by Beezhold et al. (1983), who found it was already present on the 13th day of embryonic life. The same authors concluded that the development of FAE cells requires the presence of lymphoid cells, and they hypothesized that these last may have an inductive influence on the epithelium to cause differentiation into FAE cells. Regarding the histogenesis of FAE cells, we have

suggested elsewhere that, possibly, they are not epithelial cells, but rather are mesenchymatic cells that have differentiated along the hystiocytic line (Dolfi et al., 1981; Lupetti and Dolfi, 1982; Lupetti et al., 1983a,b). The recent use of monoclonal antibodies by various authors has made it possible to gain further knowledge concerning both the histogenesis of the bursal follicle in toto and the origin of FAE cells. For this purpose, many monoclonal antibodies have been raised against the cells belonging to the bursal follicle. Houssaint et al. (1986) described the reactivity of BEP-1 and BEP-2 monoclonal antibodies, which recognize the surface bursal epithelium and the medullary epithelium, respectively. Reactivity during the embryonic period has been studied. Positivity toward BEP-1 appears a t about the 8th day along the surface epithelium; and, after the 12th day, i t has spread to the epithelial layer, which borders the follicles, whereas the central area of the follicle does not react. BEP-2 positivity appears more or less a t the time of hatching, and i t spreads over all the follicle medulla. The FAE cells are not significantly stained by either of the two monoclonal antibodies (Houssaint et al., 1986; Houssaint and Hallet, 1986). On the contrary, when FAE cells bulge into the bursal lumen, they appear to be reactive toward a monoclonal antibody (L 17) specific for chicken leukocytes (Houssaint and Hallet, 1986). Boyd et al. (1987) showed the positivity of FAE cells toward two monoclonal antibodies, namely MU1 51 and MU1 73, which do not react with other bursal epithelial regions; MU1 60 stains the non-FAE regions alone. By using interspecific quail-chick chimeras and transmission electron microscopic (TEM) studies, Shunde et al. (1988) showed that FAE cells do not develop from the epithelial cells, but they differentiate from mesenchymal cells that have migrated into the bursal anlage. Moreover, these authors reported the existence of specific stem cells, which may be considered to be the precursors of the FAE cells. Lupetti et al. (1986) hypothesized a different histogenetic sequence for the bursal follicle. In their opinion, mesenchymal cells, beginning from the 8th or 9th day of embryonic life, migrate beneath the epithelium, which forms little niches; subsequently, cells collect in these epithelial niches and form small clumps. As it grows, a clump approaches the bursal lumen, flattening the surface epithelium and finally disrupting it so as to bulge into the lumen itself. The mesenchymal cells protruding into the bursal lumen change their morphology and staining affinity to assume the features of FAE cells. The beginning of this histogenetic sequence has also been described recently by Glick and Olah (1987). They support the hypothesis that dark-coloured mesenchyma1 cells collect first in subepithelial niches and then migrate inside the surface epithelium, causing the epithelial buds to rise; conceivably, they produce inductive substances, a s testosterone treatment in ovo, which prevents bud formation, causes d e g r a d a t i o n of these cells. Using a n antihuman cytokeratin monoclonal antibody, Dolfi et al. (1989) observed the absence of reactivity in the FAE cells, positivity in the proximal part of the surface epithelium of the bursal plicae, and a n

HISTOGENESIS OF RURSAI, LYMPHOID FOLLICLE

intense reaction in the reticuloepithelial (REp) cells of the bursal follicle in 30-day-old chicks. These cells may be involved in B-lymphocyte differentiation (Boyd et al., 1983a, b). The present work examines a number of problems regarding the histogenesis of the bursal follicle in general and, in particular, the origin of FAE and REp cells. TEM studies and the observation of semithin sections are compared with the results obtained by using frozen sections stained by the PAP method after treatment with a n antihuman cytokeratin monoclonal antibody. MATERIALS AND METHODS Animals

White Leghorn eggs were used in these experiments, and the eggs were incubated in our laboratory in the usual way. After hatching, the chicks received food and water ad libitum. Beginning from the 7th day of embryonic life, four embryos were collected a t intervals of 6 hr between each sample. In the early developmental stages, the caudal part of each embryo was removed; in later stages, the bursae themselves were removed. At each sampling time, two bursae were used for electron microscopy and the other two bursae were used to obtain cryostat sections; these sections were treated with a n anticytokeratin monoclonal antibody, and then the immunoperoxidase method was used. Five chicks were killed when they were 15 days old in order to carry out the same procedures on their bursae. Light and Electron Microscopy

The bursae of Fabricius were removed, trimmed into small cubes 1 mm thick, and fixed by immersion of the fragments in a solution composed of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.3, for 4 hr. After washing in phosphate buffer for 12 hr, postfixation was carried out for 2 h r in 1.3% OsO,. The preparations subsequently were embedded in Epon and sectioned with a n Ultratome Nova LKB ultramicrotome; the semithin sections were stained with toluidine blue for light microscopy; the ultrathin sections were stained with a n LKB ultrostainer and observed under a Siemens Elmiskop 101 electron microscope. Cytokeratin Detection With the Anticytokeratin Antiserum

The anticytokeratin antibody used was supplied by Dakopatts (Denmark); i t was a monoclonal mouse antihuman cytokeratin antibody (DAKO CK1). The cell clone that produces this monoclonal antibody is LP 34. The antiserum reacts with the intermediate filaments of human keratin. During the immunoblot test, it recognizes a fair number of polypeptides, including keratin 18 (of simple epithelia) and 6 (of keratinocytes), following the classification of Moll et al. (1982). The monoclonal antibody does not react significantly with keratin 1,8,or 19. (For further details of the reactivity of the antiserum toward human keratin in normal and pathological cells, see the specification sheet supplied by Dakopatts.) The antiserum is composed of a n IgG 1. As regards the reactivity od DAKO CK1 in chicken cells, i t was tested on various tissues of chicks, and the following levels of reactivity were observed: high in the epidermis, the kidney, and the thymus; moderately positive in the intestinal epithelium; and negative in

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the spleen and liver. The method used was a s follows: the antibody was adjusted to the optimal dilution, which proved to be 1:150 in phosphate-buffered saline (PBS). Fifteen-micrometer sections of chicken tissues were fixed in acetone for 10 min a t room temperature. Subsequently, one drop of the diluted antiserum was placed on each section, and the sections were left in a humid chamber for 24 h r a t 4°C. The sections then were incubated for 30 min a t room temperature with an antimouse immunoglobulin antiserum (Dakopatts) diluted 1:25. Lastly, the sections were incubated for 30 min a t room temperature with mouse peroxidase-antiperoxidase (PAP) diluted 1:50. Each step was followed by rinsing for 10 min in PBS. In order to show up the PAP complexes, the sections were incubated €or 10 min in a solution of diaminobenzidine tetrachloride and 0.001% hydrogen peroxide in 0.1M phosphate buffer, pH 7.2. Negative controls comprised sections of spleen and sections incubated without the monoclonal antibody. RESULTS Semithin Section Observation

Because all bursal follicles do not develop a t the same time, i t seems best to consider four stages of development: a first stage that precedes the formation of the bud, (the word bud is maintained to infer results by others, whereas mesenchymal clump, cluster, nodule, and mass are used in the description of our results); a second stage in which the bud is formed and begins to grow; a third stage, when all the medullary components of the bursal follicle are complete; and, lastly, the fourth stage during which the cortex is organized. Stage preceding bud formation

Around the 7th to 8th day of embryonic life, the bursal surface epithelium lies upon a mesenchymal tissue composed of a small number of cells. Some are clear, whereas others are deeply basophilic. The proximal layer of the epithelium does not show any outfolding or infolding (Figs. 1, 13A). Dark-colored cells can be observed in the tunica propria and in the epithelium, independently from the presence of nodules (Fig. 2). Stage of bud organization

Both the dark-colored and the clear mesenchymal undifferentiated cells migrate toward the basal surface of the epithelium, where a small niche forms in which the cells become located. The niche becomes deeper and deeper a s the number of mesenchymal cells increases (Fig. 31, forming a small clump of cells wedged in the epithelium (Figs. 4, 13B). The little cluster of mesenchymal cells becomes more and more packed and pushes against the surface epithelium (Figs. 5 , 13C). The epithelium itself progressively flattens and bulges toward the bursal lumen (Figs. 5 , 6 ) .This bulge allows the basal layer of the epithelium to proliferate under the mesenchymal cluster and to close completely under it. Thus the epithelium encloses the cluster of mesenchymal cells (Figs. 6, 13D). The upper part of the cluster now bulges less (Fig. 7), and the overlying epithelium becomes necrotic (as electron microscopy clearly shows in Fig. 32). Thus, a number of mesenchymal cells now face the bursal lumen (Fig. 8). These upper cells of the follicle become elongated (Fig. 9) and possess

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a lower degree of staining affinity. They now resemble FAE cells (Figs. 10-12). Stage of formation of the bursal follicle medulla

The epithelium containing the mesenchymal clump may be divided into two portions: the first lies over the mesenchymal mass itself and faces the lumen (as stated above, it becomes necrotic and leaves room for the FAE cells); the second surrounds the mesenchymal mass and faces the basal membrane (Figs. 8,9,13E-G). We shall call this second part of the epithelium, particularly the segment situated between the mesenchyma1 clump and the basal membrane, the subnodular epithelium (Figs. 9, 10, 13E-G). Two histogenetic events are observed, one involving the mesenchymal clump, the other involving the subnodular epithelium. A number of mesenchymal cells inside the clump differentiate into lymphoid cells. In the meantime, the subnodular epithelium becomes stratified (Figs. 10, 13E-G), and lymphocytes progressively migrate between the epithelial cells (Figs. ll, 12, 13H). In the outermost part of this follicle primordium, a continuous layer of flattened epithelial cells lies upon the basal membrane (Figs. 11, 12). It corresponds to the so-called corticomedullar bordering epithelium, whereas the subnodular cells correspond to the REp cells. In conclusion, where the development has ended, the basal cells of the subnodular epithelium form the corticomedullar border cells; the other layers of the epithelium itself, between which lymphocytes have migrated, form the REp cells. Stage of the Organization of the cortical components of the bursal follicle

Beginning from the last embryonic days, organized lymphoid cells are observed outside the corticomedullar border (Figs. 11, 12, 131).The follicular cortex then begins its growth, to be completed a few days after hatching. TEM Observations Stage preceding bud formation

The pattern does not differ from that observed by means of light microscopy (Fig. 14). Stage of bud organization

At the beginning, the epithelium shows small infoldings of the basal surface; mesenchymal cells coming from the underlying tunica propria collect in these small infoldings (Fig. 15). The basal membrane maintains its continuity beneath the epithelial layer (Fig. 18). As the mesenchymal cells get nearer the epithelium (Fig. 16), the basal membrane is interrupted a t the point of contact between the epithelial cells and the mesenchymal cells (Fig. 17). When cells of the mesenchymal clump begin to be wedged in the epithelium (Fig. 19), the basal membrane is interrupted a t the point where the mesenchymal cells gain access to the epithelial layer (Fig. 20). Subsequently, when the mesenchymal cells increase in number and close together to form the nodule which pushes the epithelium toward the bursal lumen (Fig. 21), the basal membrane that is situated between the epithelium overlying the nodule and the nodule itself is no longer detectable (Figs. 24-

291, whereas the basal membrane is still present beneath the epithelial cells that border the mesenchymal clump (Figs. 22,23,30). At this stage, no sign of a basal membrane can be found in that part of the nodule facing the tunica propria (Figs. 24-29). At a later stage, the surface epithelial cells are flattened (Fig. 31) and then become necrotic (Fig. 32). The mesenchymal cells lying under the necrotic epithelium then face the bursal lumen and become organized, giving rise to a n epithelioid-like layer (Fig. 331, which substitutes for the necrotic epithelium. The cytoplasmic electron density of these mesenchymal cells diminishes; therefore, clearer cells become mixed with cells containing a more electron-dense cytoplasm (Fig. 34). Finally, the FAE area is formed as a homogeneous cellular complex; indeed, all of the mesenchymal cells now have a pale cytoplasm and have acquired the features of FAE cells (Fig. 35).

Figs. 1-1 2. Bursa1 plicae surface epithelium in chicken embryos a t 7 (Fig. l), 8 (Figs. 2, 3), 9 (Figs. 4-61, 10 (Fig. 7), 11 (Figs. 8, 9), 14 (Figs. 10, ll),and 19 days (Fig. 12). Karnovsky, osmium tetroxide, Epon 812, toluidine blue. Fig. 1. The bursal plicae surface epithelium forms a continuous layer of uniform thickness. x 120. Fig. 2. Clear and dark-stained cells are to be seen in the epithelium and in the underlying mesenchyme. The epithelium shows a small niche in its proximal part to which a dark-stained mesenchymatic cell is about to gain access (arrow). x 160. Fig. 3. A small clump (arrow) of mesenchymal cells migrates into an infolding of the proximal epithelial surface. x 160. Fig. 4. A small mass of mesenchymal cells becomes wedged in the epithelium and compresses it. x 160. Fig. 5. A nodule of mesenchymal cells tends to push the overlying epithelium toward the bursal lumen, and the epithelium decreases in thickness. x 365. Fig. 6. After the small clump made up of mesenchymal cells has passed beyond the level of the basal surface of the epithelium, by bulging toward the bursal lumen, the epithelium itself tends to close again under it. x 320. Fig. 7. The cells of the surface layer of the epithelium become very flat. x350. Fig. 8. A number of roundish mesenchymal cells bulge into the bursal lumen. Under them, a layer of mesenchymal cells becomes organized like a barrier. x 365. Flg. 9. The surface mesenchymal cells in the FAE area which migrate into the epithelium become elongated and epithelioid in shape. SN, subnodular epithelium. x 365. Fig. 10. The FAE area cells become clearer; the subnodular epithelium (SN) stratifies. Arrows indicate the extent of the epithelial thickening. x250. Fig. 11. The FAE area is now well defined and is made up of clearer cells. The medullary lymphocytes proliferate and infiltrate the subnodular epithelium. x 200. Fig. 12. Lymphocytes are clearly seen also outside the corticomedullar epithelium border (arrowheads), and they form the cortex. x 200.

HISTOGENESIS OF’ KUHSAL LYMPHOID FOLI,ICI,E

Figs. 1-12.

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SURFACE

EPITHELIUM

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HISTOGENESIS OF BURSAL LYMPHOID FOLLICLE

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deeper cells of the epithelium are positive to the CK1 Stage of formation of the bursal follicle medulla monoclonal antibody. The surface epithelial cells show Mitosis is observed involving the epithelial cells borno clear positivity, which is consistent with their difdering the mesenchymal mass (Fig. 36). Epithelial mi- ferentiation into mucus-secreting cells (Fig. 47). The tosis is easily distinguished from mesenchymal mitosis medullar epithelial cells (REP) are strongly positive, because the dividing epithelial cells are closely conthey form a thick network inside the medulla, nected to one another by desmosomes (Fig. 37). Subse- and which is more positive below the FAE area (Fig. 47). quently, the subnodular epithelial cells give rise to a The FAE area is always negative, even during postemnumber of cell layers that are always closely connected to one another. This epithelial thickening continues bryonic life. around the mesenchymal mass (Fig. 38). The epitheDISCUSSION AND CONCLUSION lium lies upon a continuous basal membrane (Fig. 39), The following conclusions may be drawn: which formed again after the epithelial closure under 1. The epithelial buds are not a swelling of the epithe clump. When the mesenchymal cells are almost thelium towards the tunica propria, but are small completely differentiated into lymphoid cells, they begin to invade between the epithelial cells and disar- clumps of mesenchymal cells collected in epithelial range them (Fig. 40). They move between the cells be- niches. The mesenchymal clumps grow, giving rise to longing to the deepest layer of the thickened the bursal follicles. 2. FAE cells are not specialized epithelial cells, but epithelium (Fig. 40). During the growth of lymphoid cells, the subnodular epithelial cells change their mesenchymal cells of the clumps that bulge into the shape, become star-shaped, and develop numerous thin bursal lumen change their pattern and become wedged processes that are connected by desmosomes to one an- in the lining epithelium. 3. REp cells appear shortly after follicle development other and also to the cells of the basal layer (Fig. 41). In the meantime, the lymphoid cells continue to grow and has begun. Their epithelial origin is confirmed by their pass through the basal layer of the subnodular epithe- positivity to the anticytokeratin monoclonal antibody. lium; they then migrate into the connective tissue that Histogenesis of the Bursa1 Follicular Complex surrounds the bursal follicle medulla (Fig. 42). The The histogenetic sequence we hypothesize clearly basal layer, on the contrary, contains a number of flatdiffers from that described by Ackerman and Knouff tened cells set in a single continuous layer bordering (1959). In their opinion, the epithelial bud is a localized the bursal follicle medulla (Fig. 43). swelling of the epithelial layer that bulges into the underlying tunica propria. The bud then gives rise to Stage of the formation of the bursal follicle cortex lymphoid cells, and “ . . . these deeply basophilic cells During the final days of embryonic life, the lymphoid are indistinguishable from lymphoblasts seen in other cells become organized outside the basal layer of the lymphoid organs. Thus these blast cells appear to be subnodular epithelium, together with the cells of the formed intraepithelially and the possibility of their tunica propria; these cells gradually form the bursal arising from mesenchymal cells in the tunica propria follicle cortex (Fig. 43). and migrating into the follicle seems highly unlikely from our observations, since similar cells are rarely Anticytokeratin Monoclonal Antibody lmmunoperoxidase seen outside of the epithelial bud during this period of In the stage preceding bud formation, a weak posi- bursal development.” tivity to CK1 is found all over the layering epithelium The histogenetic process we propose might explain of the plicae (Fig. 44). At the beginning of the stage of Ackerman and Knouff‘s observation; if the buds are bud organization, the clumps of mesenchymal cells are composed of mesenchymal cells, lymphoid cells might not stained; and small, roundish, unstained areas in- be derived from them. terrupt the positivity of the epithelial layer. These negThe observations reported by Murphy and Cho ative areas correspond to the primitive cluster of mes- (1971) and Edwards et al. (1975) seem to support our enchymal cells (Fig. 45). Later the clusters show a pale, results. Indeed, those authors point out that bud fordiffuse positivity in the form of a network lying be- mation must go back to a n earlier embryonic stage, on tween the cells. This pattern is seen from the 14th to the 9th day of incubation. The earliest phases of bud 15th days of embryonic life. The covering epithelium organization consist of the migration of mesenchymal increases its positivity, whereas the FAE areas remain cells toward the subepithelial layer, where they collect negative (Fig. 46). in small groups to migrate one by one into the epitheAt the stage of formation of the bursal follicle me- lium. Here a number of small mesenchymal nodules dulla, when the follicle is completely formed, the are formed and completely surrounded by the epithelium, which is pushed into the underlying tunica propria (Edwards et al., 1975). The above observations by Edwards and coworkers received little credit, and it was widely accepted that the first event of bursal folFig. 13. Diagram representing the histogenesis of the bursal lymlicle histogenesis was the formation of the epithelial phoid follicle. A: The stage preceeding bud formation; B: The mesenchymal nodule is formed; C: The mesenchymal nodule wedges into the bud as suggested by Ackerman and Knouff (1959; Le epithelium; D: The epithelium encloses the mesenchymal nodule; E: Douarin and Houssaint, 1974; Houssaint et al., 1976; The uppermost cells of the mesenchymal nodule become the FAE Le Douarin et al., 1984). Semithin sections and eleccells; F, G: The subnodular (SN) epithelium stratifies. Lymphocytes tron microscopy (Lupetti et al., 1986) show that mesdifferentiate from the cells of the nodule; H: Lymphocytes migrate enchymal cells are wedged in small niches which bebetween the subnodular epithelial cells; I: Lymphocytes migrate out come wider and wider a s the mesenchymal clumps of the epithelial layer and the cortex begins to grow.

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grow. Moreover, no continuity between the epithelium and the presumed bud was observed in the present work, and the mesenchymal clump always appears as a small cluster enclosed in the epithelium in all of the semithin sections examined. TEM shows that the early stage of follicle development consists of the clumping of mesenchymal cells, which collect into small infoldings of the epithelium. When the mesenchymal clump becomes larger, i t pushes the epithelium toward the bursal lumen and the epithelium completely encloses it. At this stage, observation of the basal membrane seems to support our view on bursal follicle histogenesis. Indeed, a t the beginning, the basal membrane is maintained between the epithelium and the small clump of mesenchymal cells; then, when the latter approach the epithelium, the basal membrane is interrupted at the sites of contact, while remaining intact beneath the epithelial layer. When the mesenchymal clump is partially wedged in the epithelium, there is no basal membrane on the surface facing the tunica propria. Lastly, the basal membrane becomes continuous again when the clump is enclosed in the epithelium. If the bud were a n epithelial swelling, the basal membrane should be present in every stage of follicle development; and only small interruptions might be expected because of mesenchymal cell migration into the bud itself. This is not the case, and our observations clearly show there is no basal membrane beneath the clump before the epithelium again closes under it. Our interpretation would seem to gain added support when we use a n anticytokeratin monoclonal antibody. Indeed, positivity is shown in the plicae epithelium alone, except in the mesenchymal clump from the 10th to the 13th days of embryonic life. Recently, Glick and Olah (19871, like Murphy and Cho (1971) and Edwards et al. (1975) before them, observed that the first histogenetic event in follicle development is the appearance of niches in the basal epithelial layer as mesenchymal cells approach it. This observation appears to show a picture very similar to the early stages we describe. Lymphoid follicle histogenesis was studied by Le Douarin and Houssaint (1974) and Houssaint et al. (1976) using a quail-chick chimera. We think it worthwhile to point out that bursal follicle maturation continues in time and that not all the buds are formed a t the same moment. Le Douarin et al. (1980) described a gradual maturation that proceeds from the bottom of the bursa to the bursocloacal stalk. Thus, the exact beginning of bud formation cannot be determined; it has been dated at around the 9th to 12th day of embryonic life in chicks (Houssaint and Hallet, 1986) and around the 11th day (Houssaint et al., 1976) in quails. We prefer, therefore, not to report age alone but rather to define the developmental stage of the buds in the fragment grafted; thus, in our discussion of this chimeric experiment, we shall deal with a stage preceding bud formation, a second stage a t which buds have begun to form, and a third stage a t which the buds examined have completed being organized. Le Douarin and Houssaint (1974; Houssaint e t al., 1976) specified that lymphoid cells differentiate from hemopoietic stem cells which have migrated into the buds; this is demonstrated by grafting quail bursae onto chick em-

bryo chorion-allantoic membrane. By repeating the grafts a t various ages, differing results are obtained. Quail bursae at the 6th embryonic day, or when the buds have not yet formed, grafted onto 3-day-old chick embryos, produce a number of follicles with a mixed lymphoid population. When bursae of quails a t least 11 days old, i.e., when buds have already formed, are grafted onto 3-day-old chick embryos, a number of follicles are produced made up of quail lymphocytes alone. Summarizing, in the first case, if the buds have not yet formed, they will develop from chick stem cells and so the follicles will be made up of chick cells. In the second case, the buds examined will start as quail stem cells, but their formation will end as chick stem cells; so the lymphoid population will be mixed. Finally, in the third case, the buds of quail bursae will already be formed, and they will not allow chick stem cells to populate them. Now it is crucial to discover what blocks the migration of chicken mesenchymal cells into the quail bursa in this last experiment. After the small nodule of mesenchymal cells is formed, the epithelium proliferates and surrounds the nodule. It would be tempting to hypothesize that closure of the epithelium beneath the nodule might block the migration of new stem cells. Other authors thought it unlikely that new cells might migrate into the buds (Ackerman and Knouff, 1959) while a medullo-cortical migration of cells is taking place (Rizzoli et al., 1975; Ratcliffe et al., 1987). Moreover, a small number of stem cells per follicle is able to create a clonal proliferation with the synthesis of various classes of Ig (Pink et al., 1985). Houssaint et al. (1986) studied the reactivity of two monoclonal antibodies (BEP-1 and BEP-2) in the bursal epithelium during ontogenesis. The surface epithelium of bursal plicae of embryos in which buds have not yet formed is positive; after bud formation, Houssaint e t al. note that ". . . the epithelial layer limiting the follicles, a s well as the epithelium lining the lumen, was highly positive, while the core of the follicles did

Figs. 14-20, Surface epithelium in the bursal plicae in chicken embryos at 7 (Fig. 14) and 9 days (Figs. 15-20). Karnovsky, osmium, Epon 812, uranyl acetate and lead citrate. Fig. 14. The epithelium does not show any infolding in its proximal surface. x 2,650. Fig. 15. A small niche is seen, inside of which are three mesenchyma1 cells. x 2,900. Fig. 16. An epithelial infolding is shown, inside of which a small mesenchymal clump is located. x 2,450. Fig. 17. Higher-power view of Figure 16. The basal membrane (arrowhead) is not present a t the point where a mesenchymal cell touches the epithelium. X 10,440. Fig. 18. Higher-power view of Figure 15. The basal membrane (BM) is present between the epithelium and the underlying mesenchymal cell. x 17,700. Fig. 19. A clump of mesenchymal cells is wedged in the epithelium. x 2,650. Fig. 20. Higher-power view of Figure 19. The basal membrane (arrowheads) is interrupted by the mesenchymal cell of the clump wedging into the epithelium. x 8,430.

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Figs. 14-20,

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Figs. 21-30

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Figs. 31-35. Surface epithelium of bursal plicae in chicken embryos at 9 days (Figs. 31,32) and 14 days (Figs. 33-35). Karnovsky, osmium tetroxide, Epon 812, uranyl acetate, lead citrate. Fig. 31. The cells of the upper part of the layering epithelium are flattened by the mesenchymal cell nodule, which bulges toward the bursal lumen. x 2,950. Fig. 32. The epithelial cells of the upper layer show clear signs of necrosis (arrowhead). The underlying mesenchymal cells are round, and wide spaces are visible between them. X 4,950.

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Fig. 33. The epithelial layer can be seen at the left with mucosecreting features. On the right, cells can be seen bulging toward the bursal lumen (arrowhead); they possess different features from those of the epithelial cells and are epithelioid in appearance. They correspond to the mesenchymal cells facing the bursal lumen after necrosis of the epithelial cells. x 2,700. Fig. 34. The epithelioid mesenchymal cells gradually change; a number of them have greater electron density, and others are less electron dense. x 4,600. Fig. 35. The mesenchymal cells belonging to the FAE area now show less electron density than t,he underlying mesenchymal cells. x 4.200.

Fig. 21. A mesenchymal clump is partially enclosed in the epithelium of a chicken embryo a t 10 days. x 2,380. Figs. 22-30. Details of Figure 21. The letters (a-i) indicate com-

mon reference points. Figs. 22, 23, 30: The basal membrane (BM and arrowheads) is present beneath the epithelial cells which are located on the sides of the clumps. Figs. 24-29: Basal membrane is not present under the mesenchymal clump. x 6,950.

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not react . . .”. If follicle histogenesis is due to a gradual infiltration of stem cells into a n epithelial bud, a network-like positivity in the bud center is to be expected. The negativity of this central part of the nodule might suggest that, a t this stage, it is formed by a perhaps small, nonreactive mesenchymal clump, which is completely surrounded by epithelial cells.

found to be affected by treatment with carrageenan (Dolfi e t al., 1981) and silica (Lupetti et al., 1983a), which are macrophage-toxic substances. Indeed, silica selectively affects these cells (Allison et al., 1966). The experiment carried out by Eerola (1980) seems to give indirect support to the hypothesis that FAE cells are not epithelial in origin. Eerola put bursal fragments into culture and observed that the epithelial compoHistogenesis of the Follicle-Associated Epithelium nent is preserved both in the surrounding layer of the (FA€) Celis plicae and in the cortico-medullar border, whereas the The FAE cells are a separate cellular type, and they FAE cells, together with the lymphoid compartment, have been described morphologically by Ackerman and leave the bursal follicle and completely disappear. Knouff (19591,Ackerman (19621, and then by Bockman Monoclonal antibodies against various bursal cells and Cooper (1971, 1973). Although FAE cells have dif- have been used to investigate the origin of FAE cells. ferent morphological and functional characteristics, Boyd et al. (1987) observed that FAE cells are positive they seem to belong to the epithelial layer. The char- for MU1 51 and MU1 73 monoclonal antibodies, which acteristics of FAE cells have suggested that they have do not react with other bursal epithelial regions a t all, a role in antigen uptake and transfer. Like macro- whereas MU1 60 monoclonal antibody reacts only with phages (Bockman and Cooper, 1973; Schaffner et al., non-FAE regions. The phenotype of FAE cells is there1974; Rizzoli et al., 1975; Gilmore and Bridges, 1977), fore different from that of the surrounding epithelial they possess numerous vescicles and vacuoles cells. (Bockman and Cooper, 1973; Schaffner et al., 1974; Houssaint and Hallet (1986) studied the origin of Bockman and Stevens, 1977; Glick, 1977; Naukkari- FAE cells using monoclonal antibodies and a quailnen et al., 1978); they also show a n intense positive chick chimera. The monoclonal antibodies were BEP-1 reaction to nonspecific esterase (Schaffner et al., 1974; and BEP-2, which are specific for overlying epithelial Ruuskanen et al., 19771, which is a feature of macro- cells and for bursal follicle medullary epithelial cells, phages (Yang et al., 1979; Joshua et al., 1980). No respectively, and L 17, specific for leukocytes. The rebasal membrane separates them from the underlying sults show that FAE cells do not react significantly follicle medulla, while a basal membrane supports all with antiepithelium monoclonal antibodies, whereas a the epithelial layers (Rizzoli et al., 1975). The basal weak positivity is observed after treatment with anmembrane infolds and surrounds the corticomedullar tileukocyte L 17 monoclonal antibody in FAE cells proborder epithelium (Ackerman and Knouff, 1959; truding into the lumen. The interfollicular epithelium Hodges, 1974; Rizzoli et al., 1975). does not react with the L 17 monoclonal antibody, but The FAE cells show micropinocytotic activity and a number of scattered cells react strongly, whereas are involved in the uptake of various kinds of sub- medullary and cortical lymphocytes are highly posistances like India ink (Bockman and Cooper, 1973; Sor- tive. As regards the scattered cells in the epithelium vari et al., 1975), ferritin (Bockman and Cooper, 1973; which reacted to the L 17 monoclonal antibody, they Gilmore and Bridges, 1977), colloidal carbon (Schaffner might be leukocytes, which are often present in et al., 1974; Naukkarinen and Sorvari, 1980; Beezhold chicken intestinal epithelia (Toner, 1965; Dolfi et al., et al., 19831, latex particles (Schaffner et al., 19741, and 1988).These results would seem to show that FAE cells horseradish peroxidase (Bockman and Stevens, 1977). are not epithelial, but Houssaint and Hallet gave anBeezhold et al. (1983) hypothesized that FAE cells may other interpretation of their results. The quail-chick be epithelial cells that are functionally modified due to chimera makes i t possible to observe chicken cells mixed with quail cells in the FAE areas of quail bursae the inductive effect of the underlying lymphoid cells. We have attempted to find evidence for the hypoth- grafted at the age of 6 days into the somatopleura of esis that FAE cells are not epithelial cells, but rather 3-day-old chick embryos. They concluded that FAE armesenchymal cells differentiated toward the hystio- eas are regions of mixed epithelial and hemopoietic cytic line. In this connection, FAE cells have been cellularity, because the epithelial FAE component of

Figs. 36-43. Bursa Fabricii follicles in chicken embryos a t 14 (Figs. 36-40) and 19 days (Figs. 41-43). Karnovsky, osmium tetroxide, Epon 812, uranyl acetate, lead citrate.

Fig. 40. Lymphocytes at various stages of maturation spread throughout the subnodular epithelium and change its pattern. x 2,100.

Fig. 36. Mitosis in an epithelial cell bordering the mesenchymal cell mass. x 5,450.

Fig. 41. The subnodular epithelial cells change shape, become starshaped, and assume the same features as the REp cells. D, desmosomes. x 5,450.

Fig. 37. Higher-power view of Figure 22 showing desmosome connections between adjacent epithelial cells. x 16,350. Fig. 38. The subnodular (SN) epithelium bordering the mesenchyma1 nodule (N)becomes stratified. x 2,300. Fig. 39. A continuous basal membrane (BM) separates the subnodular epithelium from the underlying tunica propria. x 7,600.

Fig. 42. Mesenchymal cell migrating through the basal layer of the subnodular stratified epithelium. C, cortex; Ep, cortico-medullar bordering epithelium; arrowheads, basal membrane. x 2,100. Fig. 43. The cortex of the bursal follicle is formed externally to the basal layer of the epithelium. x 2,100.

HISTOGENESIS OF BUKSAL LYMPHOID FOLLICLE;

Figs. 36-43

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Figs. 44-47. Immunoperoxidase reaction with an anticytokeratin monoclonal antibody on the bursae of Fabricius of chicken. Cryostat, acetone, immuno-HRP.

Fig. 46. At 14 days, the reaction is spread over all of the layering epithelium on the medullary epithelial cells, whereas the FAE area (F) does not react. x 365.

Fig. 44. A t 7 days, reactivity toward the anticytokeratin monoclonal antibody is spread over the surface epithelium of the bursal plicae but is only of low intensity. x 125.

Fig. 47. At 19 days, the positive reaction of the layering epithelium decreases and is present only in the basal part of the epithelium. The REp cells show an intense positive reaction, mainly under the FAE area (F);the reaction of the FAE area continues to be negative. x 365.

Fig. 45. At 9 days, the reactivity again involves the epithelium. Two roundish negative areas (N) corresponding to the early anlage of the buds. x355.

the quail would mix with a mesenchymal component of the chick, which migrated into the bud after grafting. Houssaint and Hallet’s interpretation would be accurate if the buds protruded from the epithelium; but if they were mesenchymal, a s we suggest, they would be a mixture of host cells and grafted cells, and this would not necessarily imply that the quail cells must be epithelial in every case. Indeed, Shunde et al. (1988) used the same quailchick experiment to study the origin of FAE cells. They showed that, by grafting bursal fragments from 8-dayold chick embryos or 7-day-old quail embryos onto interspecific hosts, lymphoid follicles develop containing host cells both in the follicle and in the FAE area. If the graft is carried out with a bursal fragment removed

from a n 11-day-old chick embryo or a 10-day-old quail embryo, chimeric follicles are obtained; the FAE area, in contrast, is made up only of donor cells. They conclude that FAE cells grow from mesenchymal cells during a n established period of follicle histogenesis and reach their site in the FAE area. Thereafter it is no longer possible to obtain a chimerism in the FAE area itself. The presence, shown by TEM, of hemopoietic stem cells which can be divided into two morphologically distinct kinds has been reported (Shunde et al., 1988). The first line is the precursor of the lymphoid cells, and the second is the precursor of the FAE cells. Houssaint et al. (1986) used a n antiserum produced in rabbits against human cytokeratin, and they ob-

HISTOGENESIS OF BURSAL LYMPHOID E'OI,LICIJE

served a positive reaction in the layering epithelium of the lumen, in the reticular framework of the follicle medulla, and in the corticomedullar border cells. The cortex was reported to be negative; the degree of reactivity of the FAE cells was not mentioned. The anticytokeratin monoclonal antibody we used showed that the FAE area is negative both in the embryonic period and after hatching (Dolfi et al., 1989). The histogenetic sequence we propose would also explain the histogenesis of the FAE area. First a mesenchymal clump becomes wedged in the epithelium, and then the epithelium surrounds it; the epithelial cells separating the cluster of mesenchymal cells from the bursal lumen become necrotic (Lupetti et al., 1986); and the mesenchymal cells now face the lumen. The more external cells become part of the luminal lining itself; others become organized to make the epithelial surface whole again; after this, their staining affinity decreases, and they take on the features of FAE cells. Histogenesis of Reticuloepithelial (REp) Cells

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corticomedullar border epithelium represents a developmental stage of the subnodular epithelium itself, so much so that when development ends it remains in close contact with the REp cells (Frazier, 1974; Dolfi et al., 1989). The cortex will be formed outside the medulla, probably due to the migration of medullary lymphocytes which peripheralize (Cooper et al., 1972; Rizzoli et al., 1975; Ratcliffe et al., 1987). Summarizing, the most significant stages of bursal follicle development would seem to be 1)the formation of the mesenchymal clump with its subsequent gradual and complete enclosure inside the bursal epithelium; 2) the closing of the epithelial layer beneath the clump and the outward bulging of a number of mesenchymal cells which form the FAE cells; 3) the stratification of the corticomedullary border cells with the consequent formation of the REp cells; 4) migration of lymphoid cells between the REp cells; and 5) the growth of the cortex, probably due to the migration of medullary cells before they peripheralize (Ratcliffe et al., 1987). The present work may open new fields of investigation regarding FAE and REp cells; these might be considered to be two different cell types involved in the maturation of lymphoid cells. REp cells might have a primordial role comparable to that of thymic nurse cells in the case of T-lymphocytes (Kyewski, 1986); FAE cells might be involved later in the uptake of antigens from the external environment, selecting them and passing them onto the already mature B cells to create a local and systemic immune response.

REp cells have been described frequently (Frazier, 1974; Naukkarinen and Sorvari, 1982; Boyd et al., 1983a,b; Dolfi et al., 1989). They have also been considered responsible for the maturation of B-lymphocytes (Boyd et al., 1983a,b). An intense positivity of these cells to a n anticytokeratin monoclonal antibody has been observed in 30-day-old chicks (Dolfi et al., 1989). The present work shows that a positive reaction to CK1 appears on the 14th or 15th day of embryonic life; this fact does not exclude the possibility that the REp ACKNOWLEDGMENTS cells may be present earlier. By continuous proliferaThis work was carried out with the aid of a grant tion, the lymphocytes grow among the epithelial cells, from the Italian Ministry of Education. which progressively stratify after the epithelium closes beneath the primitive mesenchymal clump. Therefore LITERATURE CITED the REp cells represent a n advanced stage of the development of the subnodular epithelium after the lym- Ackerman, G.A. 1962 Electron microscopy of the bursa of Fabricius of the embryonic chick with particular reference to the lympho-epphocytes have migrated into it. An experiment cited ithelial nodules. J . Cell Biol., 13:127-146. above (Eerola, 1980) is worth mentioning again. That Ackerman, G.A., and R.A. Knouff 1959 Lymphocytopoiesis in the bursa of' Fabricius. Am. J. Anat., 104:163-178. author observed that, after the bursal follicles are depleted of lymphoid cells, the corticomedullar border ep- Allison, A.C., J.S. Harington, and M. Birbeck 1966 An examination of' the cytotoxic effect of silica on macrophages. J. Exp. Med. 124: ithelium becomes stratified. 141-153. Stratification might easily be due to the fact that the Beezhold, D.H., G. Sachs, and P.I. Van Alten 1983 The development of transport ability by embryonic follicle-associated epithelium. J. REp cells belong to a single epithelial layer with the Reticuloendothel. SOC., 34:143-152. corticomedullar border epithelium as its basal layer; D.E., and M.D. Cooper 1971 Fine structural analysis of indeed, REp cells are connected to the corticomedullar Bockman, pinocytosis in lymphoid follicle-associated epithelium in chick border layer by numerous desmosomes located between bursa and rabbit appendix. Fed. Proc., 3Ut511. the membrane of their processes and the apical surface Bockman, D.E., and M.D. Cooper 1973 Pynocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appenof the corticomedullar cells (Dolfi et al., 1989). In our dix and Peyer's patches. An electron microscopic study. Am. J. opinion, the data reported by Eerola (1980) seem to Anat., 136:455-478. indicate that, in these conditions, there is a return to Bockman, D.E., and W. Stevens 1977 Gut-associated lymphoepithelial tissue: Bidirectional transport of tracer by specialized epithelial the embryonic stage preceding the growth of lymphoid cells associated with lymphoid follicles. J. Reticuloendothel. Soc., cells between the REp cells, when the mesenchymal 21:245-254. clump is completely surrounded by the epithelium. Boyd, R.L., H.A. Ward, and H.K. Muller 1983a Bursal and thymic One further consideration may be deduced from our reticular epithelial cells in the chicken: preparation of in vitro monolayer cultures. J. Reticuloendothel. SOC., 34,371-382. results: because the REp cells become visible on about the 14th or 15th embryonic day, judging from the Boyd, R.L., H.A. Ward, and H.K. Muller 1983b Bursal and thymic reticular epithelial cells in the chicken: induction of B- and Tmethods we employed, it would seem logical to hypothlymphocyte differentiation by in vitro monolayer cultures. J. Reesize that their role in B-lymphocyte development, sugticuloendothel. SOC.,341383-393. gested by Boyd e t al. (1983a,b), is limited to influenc- Boyd, R.L., T.J. Wilson, K. Mitrangas, and H.A. Ward 1987 Characterization of chicken thymic and bursal stromal cells. In: Avian ing their maturation from IgM surface cells to IgG Immunology. W.T. Weber and D.L. Ewert, eds. Alan R. Liss, New cells. Indeed, IgM-producing cells are already present York, pp. 29-39. before that date (Marinkovich and Baluda, 1966; Cooper, M.D., A.R. Lawton, and P.W. Kincade 1972 A developmental approach to the biological basis for antibody diversity. In: ConLerner et al., 1971; Lydyard et al., 1976). Lastly, the

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