Dynamic changes in the intermediate filaments of the epithelial cells during development of the chicken's bursa of Fabricius.

IMMUNOLOGY Dynamic Changes in the Intermediate Filaments of the Epithelial Cells During Development of the Chicken's Bursa of Fabricius1 IMRE OLAH2 and BRUCE GLICK3 Poultry Science Department, Clemson University, Clemson, South Carolina 29634r-0379

1992 Poultry Science 71:1857-1872

basement membrane and appear among the surface epithelial cells. Their migration Follicles in the bursa of Fabricius de- into the epithelium correlates with velop subsequent to the interaction of epithelial cell proliferation and consemesenchymal and epithelial cells (Figure quently bud formation, which is the 1). The secretory dendritic cell (SDC) anlage of the future follicular medulla. precursors differentiate from the bursal The current authors have described this mesenchyme between 9 and 11 days of developmental process (Olah et al., 1986; incubation (Olah et al., 1986; Glick and Glick and Olah, 1987) and recently reexaOlah, 1987). They are grouped under the mined it with an anti-vimentin monosurface epithelium; later they cross the clonal antibody (mAb) (Olah et al, 1992). It was observed that the vimentin-negative SDC precursors entered the vimentin-positive surface epithelium, where they exReceived for publication February 10, 1992. pressed vimentin-intermediate filaments Accepted for publication June 15, 1992. S u p p o r t e d , in part, by USDA Grant 88 (VIF) (Olah et al, 1992). This observation 34116-3652 and the Agricultural Animal Biotechnol- turned the authors' attention to vimentin ogy Program "Functional Enhancement of the Immune System During Embryonic Development". This and cytokeratin, which are intermediate is technical contribution Number 3202 of the South filaments of mesenchymal and epithelial Carolina Agricultural Experiment Station. cells. 2 On leave from Second Department of Anatomy, The bursal surface epithelium consists Semmelweis University Medical School, Budapest, of two types of cell; one is associated with Hungary. 3 To whom correspondence should be addressed. the follicle, the follicular-associated INTRODUCTION

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ABSTRACT The development of bursal follicles and the differentiation of the follicle-associated epithelium and interfollicular epithelium were studied immunohistochemically using monoclonal anti-vimentin and anti-cytokeratin antibodies. In 10-day-old embryos the entodermal and cloacal epithelia coexpressed vimentin- and cytokeratin-intermediate filaments. Both undifferentiated and differentiated bursal surface epithelium simultaneously expressed vimentin- and cytokeratin-intermediate filaments during the entire period of embryogenesis. Vimentin expression in reticuloepithelial cells was related to bursal cell differentiation but was not linked to immune function. Sequential loss of vimentin from interfollicular epithelium, follicle-associated epithelium, and reticuloepithelial cells may reflect sequential acquisition of maturity in these three compartments. The presence of cytokeratin-intermediate filaments suggested that follicle-associated epithelium was not of mesenchymal origin. Testosterone treatment did not influence the vimentin and cytokeratin filament expression in the epithelial cell. (Key words: bursa of Fabricius, cytokeratin, embryo, secretory dendritic cell, vimentin)

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FIGURE 1. The major differentiation steps of the follicle and surface epithelium are: A) Secretory dendritic cell (SDC) precursors (dark cells) differentiated from the bursal mesenchyme; B) The SDC precursors were grouped in a shallow ditch at the surface epithelium; C) The SDC precursors entered the epithelium through the basement membrane; D) The proliferating epithelial cells formed a bud that was the anlage of the follicular medulla; E) Medulla was completed by the seeding of the B cell precursors, dark dots; and F) Cortex (mesenchymal origin) formed around the bud that became the medulla of the follicle. BM = basal membrane; IFE = interfollicular epithelium; FAE = follicle-associated epithelium.

INTERMEDIATE FILAMENTS IN CELLS OF THE BURSA OF FABRIOUS

MATERIALS AND METHODS Anti-vimentin mAb (Clone 3B4] and anticytokeratin mAb (Clone Lu5)^ and biotinylated anti-mouse IgG and avidinbiotin-peroxidase complex (ABC)5 were purchased. A New Hampshire breed of chicken selected for small bursa of Fabricius size was used in these experiments (Glick and Dreesen, 1967). Four embryos and three chickens per age were sampled at 10,13,14,15,16,17,18, and 19 days of incubation, at hatch, and 2, 3, 8, 14, 43, and 70 days after hatching. The bursae were frozen in liquid nitrogen and 10- to 12-um thick cryostat sections were immediately fixed in acetone for 1 to 2 min. After rehydration in PBS, endogenous peroxidase was developed with diaminobenzidine, which gave a brown color reaction. After washing in PBS the sections were incubated with primary antibodies or nonimmune serum, which served as a control. The unbound

4 Boehringer-Mannheim GmbH, Indianapolis, IN 46250-0414. Sector Laboratories, Burlingame, CA 94010.

primary antibodies were washed out with PBS and the sections covered with biotinylated secondary antibody. The sections were washed and ABC complex was used to label the biotinylated antibody binding sites. 4-Chloro-napthol produced a blue-grey deposit when used as substrate for visualization of exogenous peroxidase. All incubations were made in a moisture chamber at room temperature for 30 min. Testosterone propionate (TP) treatment inhibited bursal follicle formation (Glick and Olah, 1984). In order to determine whether TP treatment influenced VIF, 5-day-old embryonated eggs were dipped in 2% TP in absolute ethanol for 5 s. Embryos were sampled at 13 and 16 days of incubation and stained with antivimentin and anti-cytokeratin mAb as described above. RESULTS Vimentin Expression in the Bursal Epithelial Compartment During Embryonic Development The anlage of the bursa of Fabricius developed from the dorsocaudal part of the cloaca. In 10-day-old embryos both the entoderm, cloacal epithelium and the surface epithelium of the developing bursal folds expressed vimentin (Figures 2 and 3). In the cloacal and bursal duct epithelium, VIF appeared spotted or filamentous and accumulated in the middle portion of the cylindrical epithelial cells. However, in the surface epithelium of the bursal folds, VIF appeared to cover the whole epithelial cell (Figure 4). The first sign of bud formation that can be detected by anti-vimentin mAb appeared in 14-day-old embryos (Figure Id). In the surface epithelium of the folds, faintly outlined circular-shaped areas with increased vimentin positivity appeared (Figure 5). Soon after this stage a few vimentin-positive SDC were recognizable in the circular area. Growth of the vimentinpositive circular structure into the mesenchyme was accompanied by an apparent increase in vimentin-positive SDC precursors (Figure 6). The vimentin-positive SDC


epithelium (FAE), and occupies about 10% of the bursal surface. The remaining 90% of the surface is covered by mucusproducing interfollicular epithelium (IFE). There is general consensus that the IFE differentiates from the epithelium of the embryonic bursal anlage. Unlike the IFE, the FAE was capable of pinocytosis (Bockman and Cooper, 1973) and was esterase positive (Dolfi et ah, 1981; Lupetti et ah, 1983a,b), like macrophages. This raises the possibility that the FAE developed from mesenchymal cells inserted in the surface epithelium during embryonic development (Dolfi et at, 1981; Lupetti et al, 1983a,b, 1990). In the present immunohistochemical study, anti-vimentin and anti-cytokeratin mAb were used to address the question of possible mesenchymal origin or differentiation of the FAE from the entoderm. Data will be presented to support the transient coexpression of VTF and cytokeratinintermediate filaments (CIF) during lymphoepithelial tissue formation.

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FIGURE 2. Ten-day-old embryo, stained for vimentin. The endoterm (E) was vacuolized and the surface epithelial cells expressed axially oriented intermediate filaments. In the bursal duct (Bd) and the cloacal (C) epithelium, the vimentin localized in the apical portion of the cylindrical epithelial cells. FIGURE 3. Ten-day-old embryo, stained for vimentin. The strongly vimentin-positive bursal folds were covered by weakly positive cylindrical epithelium. FIGURE 4. Ten-day-old embryo, stained for vimentin. The surface epithelium expressed vimentinintermediate filaments throughout the cells.

INTERMEDIATE FILAMENTS IN CELLS OF THE BURSA OF FABRICIUS

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FIGURE 5. Fourteen-day-old embryo, stained for vimentin. The vimentin-positive circular-shaped areas (bracket) of the surface epithelium indicated the developing buds. FIGURE 6. Fifteen-day-old embryo, stained for vimentin. The vimentin-positive cells, secretory dendritic cells (SDC) accumulated in the center of the bud, which was surrounded by a pale halo. The other bud had not yet revealed vimentin-positive cells (bracket). FIGURE 7. Seventeen-day-old embryo, stained for vimentin. Both the surface epithelium and the reticular epithelial compartment of the buds expressed vimentin. Vimentin-positive cells appeared in the bud. FIGURE 8. Nineteen-day-old embryo, stained for vimentin. Every epithelial component of the bursa expressed vimentin: interfoUicular, epithelium (IFE), follicle-associated epithelium (FAE), and reticular epithelial cell (REC). FIGURE 9. Nineteen-day-old embryo, stained for vimentin. The cloacal epithelium ceased to express vimentin.

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FIGURE 10. At hatch, stained for vimentin. The vimentin was still expressed in every epithelial compartment. FIGURE 11. Sixteen-day-old embryo, stained for vimentin. Bird treated with testosterone propionate. No sign of bud formation. The epithelium expressed vimentin-intermediate filaments (VIF), which accumulated close to the apical part of the cell. This VIF pattern was very similar to that in the cloacal and bursal duct epithelium at Day 10 of incubation (Figure 2). Scattered vimentin-positive cells occurred in the epithelium. FIGURE 12. Two-day-old chick, stained for vimentin. The follicle-associated epithelium (FAE) and the reticular epithelial cells of the bud were stained whereas the interfoUicular epithelium revealed vimentin only in weak traces. FIGURE 13. Three-day-old chick, stained for vimentin. InterfoUicular epithelium was vimentin-negative whereas the follicle-associated epithelium and reticular epithelial cell of the bud continued to produce vimentinintermediate filaments.

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FIGURE 25. Eight-day-old chick, stained for cytokeratin. The Hassall-body-like structure (arrow) was connected with the epithelium underlying the follicle-associated epithelium (FAE). The epithelial layers separated the cytokeratin-negative FAE from the lymphoepithelial tissue of the follicular medulla. FIGURE 26. Forty-three-day-old chick, stained for cytokeratin. There was a dense reticular epithelial network under the follicle-associated epithelium. At the corticomedullary border the epithelial arches were completed. FIGURE 27. Seventy-day-old chick, stained for cytokeratin. Higher magnification of the epithelial arches. The seemingly "empty" bricks of the epithelial arches contained vimentin- and cytokeratin-negative lymphoid cells. FIGURE 28. Forty-three-day-old chick. The follicle-associated epithelium (FAE) was weakly positive for cytokeratin. The strongly cytokeratin-positive layer under the FAE was in connection with the reticular epithelial cells of the medulla.


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TABLE 1. The presence of vimentin (V) and cytokeratin (C) in the bursa of Fabricius after hatching1 REC

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The entodermal and cloacal epithelia in 10-day-old embryos express vimentin (Clone 3B4) simultaneously with cytokeratin (Clone Lu5). This observation confirms previous embryological findings that in early embryos the entoderm was capable of producing both types of intermediate filaments (Lane et al, 1983; Page, 1989). In early embryos, vimentin expression may be related with a reduced cell-to-cell contact (Lane et al, 1983) and cell migration (Page, 1989). In 10-day-old embryos, the rapid growth and canaliculization of entoderm and subsequent reshaping of the epithelial cells might reflect the vimentin expression in these cells. In the superficial layer of entoderm the transient vimentin filaments are axially oriented, which may reflect the acquisition of the columnar shape of the intestinal epithelium. A similar observation has been made in Sertoli cells (Paranko et al, 1986). Both changes in the epithelium require cell plasticity and motility, which breaks up the tight epithelial cell connections. Similar changes in epithelia were accompanied by reduced cytokeratin and increased vimentin expression in tumor growth (Summerhayes et al, 1981; Connell and Rheinwald, 1983; Venetianer et al, 1983; Mork et al, 1990), in tissue cultures (Connell and Rheinwald, 1983; Ramaekers et al, 1983; Schmid et al, 1983; Ben-Ze'ev, 1984), and during embryogenesis (Franke et al, 1982, 1983; Lane et al, 1983; Page, 1989).

than the IFE. This period of rapid bursa growth appeared to be accompanied by a reduction in the reticular epithelial network and direct B cell contact with epithelial membranes (Figure 23). Two major changes in cytokeratin staining occurred at 8 days of age; the FAE became cytokeratin-negative and a portion of the reticular epithelium immediately beneath the FAE stained heavily with cytokeratin (Figures 24 and 25). Between 8 and 43 days, posthatch, epithelial arches became prominent along the corticomedullary border (Figure 26). The cytokeratin-positive epithelial arches Reticular Epithelial Cells enclosed cytokeratin- and vimentin- of the Follicular Medulla negative cells, resulting in an empty "brick In 14-day-old embryos, the site of bud layered" pattern along the corticomedullary border (Figure 27). With increasing age formation is indicated by an increase in (43 and 70 days), weakly stained vimentin expression in a restricted, circular cytokeratin-positive FAE appeared occa- area of the surface epithelium. At this time sionally (Figure 28). the SDC precursors were present in the epithelium (Olah et al, 1986; Glick and Olah, 1987) and the separation of the tightly DISCUSSION associated epithelial cells had begun. DurThe bursa of Fabricius develops from ing rapid growth of the bud there is a an epithelial anlage of entodermal origin. continuous increase in the number of SDC Changes in the intermediate filaments of and, later, B cells intercalate among the the epithelial compartment occur during epithelial cells. These events contribute to the development of the follicles and dur- the change in the shape of the cuboidal ing differentiation of the surface epithelial cells. The cells become stellate and form a three-dimensional reticulum epithelium into FAE and IFE.


REC = reticular epithelial cells (medullary component); FAE = follicle-associated epithelium; and IFE = interfollicular epithelium. 2 A11 bursal surface epithelium of embryos was positive for V and C. 3 Weakly positive cells. 4 An occasional bursal follicle exhibited positive IFE. 5 An occasional bursal follicle exhibited positive FAE.

Entodermal and Cloacal Epithelia


distance between them. The presence of vimentin-positive cells in these buds suggests that bud formation may potentially occur beyond 14 days of embryonic life. Differentiation of the Surface Epithelium

It is generally accepted that the IFE differentiates from embryonic surface epithelium of the bursa. However, during the last decade the possibility emerged that the FAE was formed from mesenchymal cells that immigrated into the surface epithelium between 11 and 14 days of embryogenesis (Dolfi et al, 1981; Lupetti et al, 1983a,b, 1990). Both optical and transmission microscopic studies indicated that the development of buds was preceded by the migration of mesenchymal cells into the undifferentiated epithelium (Olah et al, 1986; Glick and Olah, 1987; Lupetti et al, 1990). The FAE differentiated in association with the follicles (Naukkarinen et al, 1978; Boyd et al, 1987). At Day 13 of embryogenesis, the epithelium bulges into the lumen where mesenchymal cells have previously appeared. At this time there is no other detectable cytological differentiation observed either by optical or electron microscopy (Naukkarinen et al, 1978). At Day 15, it is difficult to distinguish FAE cells from reticuloepithelial cells, but transmission microscopy clearly indicates that the FAE is already vesiculated whereas the IFE is beginning to produce mucin-containing secretory granules. The FAE shows a flat, elongated shape and sometimes forms more than one layer. At Day 19 the shape of the cells gradually changes from flat to cylindrical. At hatching, follicles with FAE are in transition between a flat and a cylindrical shape. The current timetable for FAE differentiation confirms previous light and electron microscopic studies (Edwards et al, 1975; Naukkarinen et al, 1978) but contradicts the findings of Lupetti et al. (1990), who observed well-developed FAE as early as 11 days of embryogenesis. Vimentin

Vimentin (Clone 3B4) and cytokeratin (Clone Lu5) are coexpressed during the entire period of embryogenesis by both the


with an increase in VIF and reduction in CIF. These data confirm a published report that intermediate filaments were involved in special functions related to differentiation and did not serve in a "housekeeping" function (Franke, 1987). Generally, cells produce one type of intermediate filament (Lazarides, 1982), but the simultaneous expression of two types of intermediate filaments is also a well-known phenomenon (Tapscott et al, 1981; Osborn and Weber, 1982; Connell and Rheinwald, 1983; Dinges et al, 1991). One of the intermediate filaments, VIF, was expressed in all three germ layers (Klymkowsky et al, 1989). In tissue culture, epithelial cells frequently expressed vimentin when the polarized, oriented cell structure was lost (Schmid et al, 1983; Ben-Ze'ev, 1984; Mork et al, 1990). In situ, the transient presence of vimentin was most frequently related with cell transformation (Summerhayes et al, 1981; Ramaekers et al, 1983; Lilienboum et al, 1986; Viale et al, 1988; Sommers, 1989) differentiation (Dellagi et al, 1983; Ngai, et al, 1984; Franke et al, 1987; Steinert and Roop, 1988; Klymkowsky et al, 1989; Tsuru et al, 1990), which resulted in reshaping of cells. The current authors agree with those who suggested that vimentin and cytokeratin expression in the epithelia were related to, or regulated by, reduced cell-to-cell contact (Cremer et al, 1981; Summerhayes et al, 1981; Lane et al, 1983; Ben-Ze'ev, 1984) but did not confirm that the lack of desmosomes was related to vimentin production (Ramaekers et al, 1980; Franke et al, 1983; Docherty and Edwards, 1984; Paranko et al, 1986; Erickson et al, 1987). The epithelial cells are connected to one another by desmosomes during the lymphoepithelial tissue formation. Vimentin expression by the reticular epithelial cells seems not to change between 16 and 19 days of incubation. At 19 days of embryonic development, vimentin expression declines and by 8 days posthatch some follicles are vimentin-negative. The differences in vimentin expression from follicle to follicle might relate to the "age" of the follicle. Thus, follicles that lack vimentin at Day 8 may have differentiated earlier than those possessing vimentin. Cytokeratin staining at Day 16 of embryogenesis reveals buds of different size with a highly variable

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Cytokeratin Before 19 days of embryonic life the developing FAE and the IFE are cytokeratin-positive whereas the other cells of the bud are cytokeratin-negative. After 19 days of embryogeneis the expression of cytokeratin declines in the FAE cells. The axial orientation of CIF becomes more prominent by Day 3 posthatch when the FAE is both histologically and functionally fully developed. Cytokeratin expression ceases by Day 8 in the FAE, but some follicles reexpress cytokeratin by Day 43. Reexpression of cytokeratin also occurred in rapidly growing mesothelial cells (Connell and Rheinwald, 1983). The present authors have used antibody to cytokeratin (Clone Lu5) pan Component 19, a major cytoskeletal component of simple epithelia. This might explain why the current results contradicted that of Lupetti et al. (1990), whose fundamental evidence for the mesenchymal origin of the FAE was the lack of immunostaining by anti-cytokeratin Component 1. However, the antibody against Component 1 identified larger molecular weight polypeptides in keratinizing epithelia, mainly in epider-

mis (Moll et al, 1982; Copper et al, 1985). In addition, failure to identify CIF with mAb does not rule out the presence of cytokeratin polypeptides, because folding or masking by other molecules, or nonfilamentous pools of cytokeratin may prevent detection by immunostaining (Lehtonen et al, 1983). Biochemical analysis is needed to determine the actual presence of cytokeratin in the FAE. The conversion of mesenchymal cells into epithelial cells takes place during the formation of germ layers and kidney development. These observations were extrapolated by Dolfi et al (1981) and Lupetti et al (1983a,b; 1990) to FAE. Fundamental changes during the mesenchymal-epithelial transformation were the mesenchymal cell aggregation and subsequently the gradual emergency of a highly polarized cytological structure (Klein et al, 1988; Ekblom, 1989). None of these fundamental processes appear applicable for FAE formation from mesenchymal cells. The appropriate aggregation of mesenchymal cells is prevented by the presence of reticuloepithelial cells and B cells. The basement membrane, which guides the polarization of mesenchymal cells, is absent beneath the FAE. Previous histological studies indicated a gradual transformation of undifferentiated epithelial cells into FAE (Bockman and Cooper, 1973; Edwards et al, 1975; Naukkarinen et al, 1978). Boyd et al (1987), using mAb raised against bursal epithelial cells, clearly showed continuity between IFE and FAE. The grafted quail bursal epithelium contained chick mesenchymal cells, suggesting that the epithelium was of donor origin (Houssaint and Hallet, 1986). The authors' recent immunohistochemical studies on the cytoskeletal structure of the FAE also indicates that the FAE-like IFE differentiated from undifferentiated embryonic surface epithelium. Effect of Testosterone Propionate on Cytoskeletal Protein Expression LeDouarin et al (1980) suggested that the TP treatment inhibited the capability of the bursal epithelial anlage to receive hemopoietic stem cells from the mesenchyme, and thus resulted in bursectomy. Recently, Wilson and Boyd (1990) published that in


undifferentiated surface epithelium and later by FAE and IFE. The loss of vimentin begins around Day 19 of embryonic life, beginning with the IFE and progressing to the FAE and reticuloepifhelial cells (Table 1). The highly proliferating epithelial cells expressed VIF but not CIF (Connell and Rheinwald, 1983; Mork et al, 1990). Unlike this transient expression of vimentin, some epithelial derivatives of mesoderm such as synovial tissue (Kasper et al, 1988) and mesothelial cells (Czernobilsky, 1985) permanently produced VIF. The disappearance of VIF may signal the completion of follicular development in the bursal follicles. The loss of vimentin expression by the IFE late in embryogenesis paralleled the period of IFE development (Edwards et al, 1975). Also, the disappearance of vimentin from the FAE after hatching paralleled the acquisition of endocytic activity by the FAE (Naukkarinen et al, 1978). The retention of vimentin in reticuloepithelial cells may reflect the rapid growth of the bursa during the posthatch period.


ACKNOWLEDGMENTS

The authors thank Gloria Freeman for her secretarial assistance and Suzanne Olah for her laboratory assistance. REFERENCES Ben-Ze'ev, A., 1984. Differential control of cytokeratins and vimentin synthesis by cell-cell contact and cell spreading in cultured epithelial cells. J. Cell Biol. 99:1424-1433. Bockman, D. E., and M. D. Cooper, 1973. Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix, and Peyer's patches. An electron microscopic study. Am. J. Anat. 136:455-478. Boyd, R. L., T. J. Wilson, K. Mitranges, and H. A. Ward, 1987. Characterization of chicken thymic and bursal stromal cells. Pages 29-39 in: Avian Immunology. W. T. Weber and D. L. Ewert, ed. Alan R. Liss, New York, NY. Connell, N. D., and J. G. Rheinwald, 1983. Regulation of the cytoskeleton in mesothelial cells: Reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell 34:245-253. Cooper, D., A. Schermer, and T. T. Sun, 1985. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations. Lab. Invest. 52:243-256. Cremer, M., I. Treiss, T. Cremer, D. Hager, and W. W. Franke, 1981. Characterization of cells of

amniotic fluids by immunological identification of intermediate sized filaments: Presence of cells of different tissue origin. Hum. Genet. 59: 373^389. Czernobilsky, B., 1985. Coexpression of cytokeratin and vimentin filaments in mesothelial, granulosa and rete ovarii cells of the human ovary. Eur. J. Cell Biol. 37:175-190. Dellagi, K., W. Vainchenker, G. Vinci, D. Pauliu, and J. C. Brouet, 1983. Alteration of vimentin intermediate filament expression during differentiation of human hemopoietic cells. Eur. Mol. Biol. Organ. J. 2:1509-1514. Dinges, H. P., K. Zatlowkal, C. Schmid, S. Mair, and G. Wirnsbeyer, 1991. Coexpression of cytokeratin and vimentin filaments in rat testis and epididymis. Virchows Arch. Pathol. Anat. 418: 119-127. Docherty, R. J., and J. G. Edwards, 1984. Chick embryonic pigmented retina is one of the group of epitheloid tissues that lack cytokeratins and desmosomes and have intermediate filaments composed of vimentin. J. Cell Sci. 71:61-74. Dolfi, A., M. Lupetti, and F. Giannessi, 1981. Toxic effect of carrageenan on lymphoid-follicle associated epithelial cells of the bursa of Fabricius of chickens. Cell Tissue Res. 221:67-75. Edwards, J. L., R. C. Murphy, and Y. Cho, 1975. On the development of the lymphoid follicles of the bursa of Fabricius. Anat. Rec. 181:735-754. Ekblom, P., 1989. Developmentally regulated conversion of mesenchyme to epithelium. Fed. Am. Soc. Exp. Biol. J. 3:2141-2159. Erickson, C. A., R. P. Tucker, and B. F. Edwards, 1987. Changes in the distribution of intermediate filament types in Japanese quail embryos during morphogenesis. Differentiation 34:88-97. Franke, W. W., 1987. Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell 48:3-4. Franke, W. W., C. Grund, B. W. Jackson, and K. Dlmensee, 1983. Formation of cytoskeletal elements during mouse embryogenesis. IV. Ultrastructure of primary mesenchymal cells and their cell-cell interactions. Differentiation 25:121-141. Franke, W. W., C. Grund, C. Kuhn, B. W. Jackson, and K. Qlmensee, 1982. Formation of cytoskeletal elements during embryogenesis IE. Primary mesenchymal cells and the first appearance of vimentin filaments. Differentiation 23:43-59. Franke, W. W., M. Hergt, and C. Grund, 1987. Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules. Cell 49:131-141. Glick, B., and L. J. Dreesen, 1967. The influence of selecting for large and small bursa size on adrenal, spleen, and thymus weight. Poultry Sci. 46:396-402. Glick, B., and I. Olah, 1984. Methods in bursectomy. Pages 3-9 in: Methods in Enzymology. Vol. 180. D. Sabato, J. Langone, H. Van Vunakis, ed. Academic Press, Orlando, FL. Glick, B., and I. Olah, 1987. Contribution of a specialized dendritic cell, secretory cell, to the microenvironment of the bursa of Fabricius. Pages 53-66 in: Avian Immunology. W. T. Weber and D. L. Ewert, ed. Alan R. Liss, Inc., New York, NY.


the early stage of follicle formation the FAE was TP-sensitive. The current observation that bursal epithelial cells were uniquely positive for vimentin suggests that VIF may be the target of TP. However, in the TPtreated birds, vimentin and cytokeratin staining are similar to that of controls at Day 13 and Day 16 of embryogenesis. Because vimentin expression is associated with a developmental stage of a cell and TP did not modify vimentin and cytokeratin expression in the bursal epithelium, one might assume either that the cytoskeletal system of the bursal epithelial cell is not influenced by TP treatment or the cytoskeleton filaments are not involved in the capability of the epithelial cells to receive emigrating cells. The presence of a few vimentin-positive cells in the bursal epithelium of TP-treated birds suggests that TP treatment inhibited mesenchymal cell transformation into SDC (Olah et al, 1986; Glick and Olah, 1987) rather than preventing epithelial cells from receiving SDC precursors. In the absence of SDC, bud formation and subsequent FAE differentiation are prevented.

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Toxic effect of NiCl2 on development of the bursa of Fabricius in broiler chickens.

Nurse cells of the bursa of Fabricius: do they exist?

The effect of infectious laryngotracheitis virus on the bursa of Fabricius of young chickens.

Effect of extracts of the bursa of Fabricius on IgG antibody production in hormonally bursectomized chickens.

Endogenous oncornaviral antigen in the bursa of Fabricius of 15B X 7(2) chickens.

The bursa of Fabricius and immunoglobulin synthesis.

On the development of the lymphoid follicles of the bursa of Fabricius.

Origin of IgM(+)IgG(+) lymphocytes in the bursa of Fabricius.

Follicle-associated epithelium and medullary epithelial tissue of the bursa of fabricius are two different compartments.

The ontogeny and cloning of B cells in the bursa of Fabricius.

Forsythiaside attenuates lipopolysaccharide-induced inflammatory responses in the bursa of Fabricius of chickens by downregulating the NF-κB signaling pathway.

Antiserum specific for reticulin of the bursa of fabricius.

New insight into the origin of IgG-bearing cells in the bursa of Fabricius.

Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells.

Developmental changes in cell proliferation and apoptosis in the normal duck bursa of Fabricius.

Analyzing dynamic properties of intermediate filaments.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

Intermediate filaments in rat ovarian surface epithelial cells: changes with neoplastic progression in culture.

Effect of bursa Fabricius extracts on antibody production in bursectomized or bursal cell autografted chickens.

Intermediate Filaments at the Junction of Mechanotransduction, Migration, and Development.

Historical perspective: the bursa of Fabricius and its influence on B-cell development, past and present.

Lymphocyte-differentiating hormone of bursa of fabricius.

Bursopentin (BP5) from chicken bursa of fabricius attenuates the immune function of dendritic cells.