The Ontogeny of Chicken Bursal Stromal Cells Defined by Monoclonal Antibodies

Molecules expressed on lymphoid stromal cells influence the differentiation of lymphocytes. We have examined the expression of stromal markers, identified by monoclonal antibodies, in the chicken bursa of Fabricius during ontogenic development. These results are also consistent with the hypothesis that medullary secretory cells are of mesenchymal origin, whereas the basement membrane-associated and some medullary epithelium are derived from the endoderm. Our results have demonstrated the complexity of bursal stromal development with determinants expressed on the adult medullary stellate cells (e.g., MUI-57 and 62) and cortical macrophages (e.g., MUI-66 and 72) detected on the early em.bryonic tunica propria (e.g., MUI-57, 66 and 72) or surface epithelium (e.g., MUI-62 and 66). In addition, we provide preliminary evidence regarding potential functions of these molecules in stem cell colonization (MUI-52), early B-cell differentiation (e.g., MUI-72), late bursal B-cell development (MUI-69 and 71) and the follicle-associated epithelium transport mechanism (MUI-61 and 73).


INTRODUCTION
The avian bursa of Fabricius is an epithelial lymphoid organ continuous with the cloacal epithelium. Its importance in the development of humoral immunity in birds is well established (reviewed by Glick, 1983), although the precise nature of the distinct microenvironments modulating B-cell differentiation remains poorly defined. It is clear, however, that the bursal epithelium influences the maturation of B cells. Induction of B-lineage differentiation markers on stem cells has been demonstrated in vitro by their incubation with bursal epithelial cultures or soluble factors derived from the epithelial conditioned medium (Eerola et al., 1982;Boyd et al., 1983). The bursal extract "bursopoietin" also induces such markers (Brand et al., 1983). Bursal ontogeny has been studied by many groups, although the results have contributed more *Corresponding author. Department of Pathology and Immunology, Monash Medical School, Commercial Road, Prahran, 3181, Victoria, Australia. }Present address: Division of Rheumatology/Allergy-Clinical Immunology, School of Medicine,TB 192,Davis,California,95616. to the understanding of lymphoid development than to that of the controlling microenvironment. Thus bloodborne stem cells have been established as the progenitor cells that colonize the bursa from 8-14 days of embryogenesis, where they proliferate and differentiate into functional B lymphocytes (Moore and Owen, 1966; Le Douarin et al., 1975;Houssaint et al., 1976;Lydyard et al., 1976;Pink et al., 1985). Ontogenic analysis of the bursal stromal components could potentially reveal important cells and factors involved in this sequence. Olah et al. (1986) have identified a mesenchymal secretory cell that appears to be involved in follicle formation. In addition, molecules associated with bursal epithelium, identified by monoclonal antibodies (mAb) BEP-1 and BEP-2, have initially been detected in ontogeny at 8 days and at hatching, respectively (Houssaint et al., 1986a), which may reflect their roles in different stages of lymphoid development. The present study utilizes a panel of mAb rea:tive with the stromal component of the avian thymus and bursa Boyd, Mitrangas, et al., 1987) to examine the appearance of these microenvironmental elements during ontogeny.
From our ontogenic analysis of the chicken bursa of Fabricius with these reagents, we present data in this paper that support the proposals that medullary secretory cells are derived from the mesenchyme (Olah et al., 1986), whereas medullary and basement membrane-associated epithelium (BMAE) are from endodermal origin. In addition, we have identified molecules expressed on various bursal elements that potentially modulate B-cell differentiation.

RESULTS
The expression of the antigens recognized by the mAb panel was examined at days 10, 12, 15, and 18 reactive with the surface epithelium (S.Ep), basement membrane (BM), BMAE, medulla, follicleassociated epithelium (FAE), interfollicular epithelium, and macrophages (MO)  . The expression of these molecules within the developing bursa can be divided into two main groups: (1) those initially reactive with the S.Ep (Table 1) and (2) those initially detected on one or more of the other components (Table 2). Of the 28 mAb screened, 17 were initially reactive on the S.Ep, 15 of these on 10-day embryonic bursae (Table 1). These antibodies were subsequently reactive on the epithelial buds later in ontogeny (15-18 days gestation), but react with different components of the adult bursa. For example, the of gestation in addition to immediately after antigens detected by 58,59,70,and 77 hatching. The antibodies included those normally were present on the adult basement membrane and   In older birds, two antigens identified by MUI-66 and MUI-81 were present primarily on cortical MO and medullary epithelium cells, respectively, with expression having been lost on the S.Ep during ontogeny (Fig. 2). This early expression may indicate an ontogenic relationship with the S.Ep, although identification of MUI-66-positive cells in the early tunica propria may be evidence for an alternative origin. Similarly, the origin of the molecule identified by MUI-53 is unclear. MUI-53 identified an antigen shared between the S.Ep and the BM.
During ontogenic analysis, this epitope was initially detected at day 10 on the S.Ep, however, at day 12, isolated cells bearing this marker were identified in the tunica propria. Thus, this antigen may develop from the S.Ep, but could possibly have multiple origins within the bursa.
Many medullary antigens were detected initially at 10-12 days of incubation on isolated cells in the tunica propria, some clustered near the S.Ep (Table   2), and were observed within the developing follicles during gestation. The majority of antigens that appear to develop from the S.Ep were also found on the epithelial buds between day 15 of embryogenesis and hatching. These included molecules that are expressed on the adult S.Ep (MUI-80) (Fig. 1) (Fig. 3). MUI-51 and 60 were S.Ep antigens that were expressed on the interfollicular epithelium, but not on the FAE or the epithelial buds. In the 10-day embryonic bursae, the MUI-60 antigen was expressed on some, but not all of the S.Ep, whereas MUI-51 was expressed on the entire S.Ep. Regions of the S.Ep, therefore, may undergo some differentiation prior to the formation of epithelial buds.
Molecules on the FAE, identified by MUI-52 (Fig.  4), were initially expressed concomitant with bud generation and the development of the FAE transport mechanism (Beezhold et al., 1983). Curiously, MUI-52 was only expressed until hatching in the bursa, being detected only on isolated cortical environmental elements are also derived from complex interactions between the precursor cell types, rather than only via a sequential differentiation process. Thus, from these results, we propose that these stromal components are predominantly progeny of the cells of the S.Ep (endoderm) and tunica propria (mesenchyme) (Le Douarin et al., 1975), differentiating following interaction between these structures. Many of the components are not mutually exclusive with respect to their origin, with different antigens expressed on the medullary epithelium, medullary stellate cells, BMAE and cortical MfD initially being detected on either the tunica propria or S.Ep.  being reactive on interstitial Mf in the adult and MUI-65, an antibody that initially detects the S.Ep thymic epithelial cells in the normal adult chicken, in the 10-day embryo and stains the S.Ep and The antigen detected by mAb MUI-71, however, medullary epithelium in the adult. Similar to a was not expressed at adult levels on the FAE until number of other mAb, MUI-65 also detected isolated posthatching. This marker was initially detected at cells in the tunica propria (mesenchyme) early in low level on the10-day embryonic S.Ep and showed ontogeny. It is therefore possible that some bursal no alteration during gestation. Posthatching this antigens are not restricted to expression on only antigen appeared in the medulla and on the FAE, endodermal or mesenchymal cells. Conversely, three whereas its expression was lost on the interfollicular other mAb also stained the medullary epithelium epithelium, after being initially reactive on cells in the tunica MUI-69 identified a medullary molecule on non-propria. Whether these molecules are expressed on epithelial cells in the adult, which first appeared in the same multipotential cell or on different mesenthe tunica propria of the 15-day embryo (Fig. 4). chymal cell types is unclear. The medullary region Throughout ontogeny, cells expressing this marker may thus contain epithelial cell populations derived were not detected in all follicles, from both endoderm and mesoderm, perhaps some form of hybrid cells. From our results, however, it appears improbable that it contains epithelium DISCUSSION exclusively derived from the endoderm. It also seems likely that there is an interaction This analysis of the ontogenic development of the between the endodermal and mesodermal cells prior bursa of Fabricius suggests that the bursal micro-to the formation of epithelial buds. Olah et al. (1986) observed that differentiated "dark" mesenchymal early developing bursa (Ackerman and Knouff, cells migrated to and entered the S.Ep before the 1964). Potentially, these molecules may be among induction of the epithelial buds. Our experiments those identified by the mAb reactive with the early have demonstrated MUI-67-positive cells clustered epithelium. The precursors then localize in the under the S.Ep and cells expressing MUI-75 within tunica propria, where induction of their differentiathe S.Ep of young embryos. These antibodies also tion into B cells may be initiated (Boyd and Ward, stain the adult bursal medulla, supporting Olah's 1978). It is possible mAb 64,68,72,74,and proposal that this interactive mesenchymal cell is a 78 identify stromal elements involved in this process precursor for medullary secretory cells (Olah et al., as they recognize nonlymphoid cells in the tunica 1986). Subsequent to, and possibly as a result of, propria early in ontogeny. MUI-72 also stains MO in this interaction, the FAE and epithelial buds are the adult bursal cortex, the more immature region of formed.  established that the bursa (Boyd and Ward, 1984), whereas the the FAE consisted of modified epithelial cells and of identification of the other markers (MUI-57 and 75) haemopoietic cells. Our data indicate that the FAE is in the embryonic cortex may be evidence of having primarily derived from cells of epithelial (endofunctions related to early steps in B-lymphocyte difdermal) origin since thirteen antigens initially ferentiation. expressed on the S.Ep were present on cells of the Similarly, molecules that appear in the bursal epithelial buds and FAE during ontogeny. Many of medulla later in ontogeny (e.g., MUI-69 and 71) are these molecules were lost late in ontogeny, although more likely to be involved in the latter stages of six were still expressed in the adult chicken. The B-cell differentiation. MUI-69 was not expressed on loss of these markers suggests that alterations in the all follicles, which may relate to the observation that epithelium are occurring as the FAE differentiates cyclophosphamide-treated, lymphoid reconstituted into a functional and antigenically distinct region, bursae have follicles that are either clonally repopu-The transient expression of some molecules (e.g., lated or empty (Sorvari et al., 1974;Pink et al., the antigen detected by MUI-52) on the FAE from 1985). In addition, the molecules that are lost as the day 15 until hatching corresponds chronologically to bird matures may possess a function that is only the recruitment of lymphoid precursors and their expressed early; e.g., rearrangement of the chicken subsequent localization within the lymphoid follicles Ig genes has been shown to occur only during (Houssaint et al., 1976;Ratcliffe et al., 1987). The ontogenic development (reviewed by  antigen is also re-expressed in neonatally naud, 1987). cyclophosphamide-treated birds (Wilson and Boyd, The ontogenic expression of the bursal stromal 1990). The other modifications of the FAE possibly antigens in this study concurs with earlier proposals involve molecules that have a function in the FAE regarding the mesenchymal origin of bursal medultransport mechanism (e.g., MUI-61 and 73). This is lary secretory cells (Olah et al., 1986) and the the means by which material from the bursal lumen endodermal origin of the BMAE and medullary is pinocytosed by the cells of the FAE and trans-epithelium (Le Douarin et al., 1975; ported into the follicles (Bockman and Cooper, 1973;, although our data indicate that the medullary Beezhold et al., 1983), where it presumably epithelium probably results from an interaction stimulates a specific B-cell response (Van Alten and between endodermal and mesenchymal cells. We Meuwissen, 1972). The current investigation of these have also provided preliminary evidence identifying molecules could provide important evidence potentially important molecules involved in the regarding the control of B-cell differentiation by bursal recruitment of precursor cells and the these stromal components, mechanisms controlling B-lymphocyte differentia-Haernopoietic stem cells seed the developing tion. These molecules have also been examined in embryonic bursa between days 8 and 14 of gestation birds with humoral immunodeficiency induced by (Le Douarin et al., 1975), possibly via adherence to testosterone propionate or cyclophosphamide the endothelium of blood vessels located in close . This comparative analysis proximity to the bursal epithelium (Le Douarin, has provided further insight into the 1986). It has been postulated that a molecule potential function of these antigens. Further secreted by the epithelium is involved in the chemoexperimentation regarding the functions of these attraction of these precursors (Le Douarin, 1978) and markers and their characterization is currently in haemopoietic activity has also been observed in the progress. Australorp xWhite Leghorn F1 hybrid chicken embryos were obtained from Research Poultry Farm (Research, Victoria, Australia) and incubated in a humidified incubator (Multiplo) at 39C.

Tissue Sections
Chick embryos were killed (six per group) at 10, 12, 15, and 18 days of incubation and immediately posthatching. Bursae, spleens, and thymuses were removed, immersed in Tissue-Tek (Miles Scientific) and snap-frozen on a liquid nitrogen-isopentane slurry.

Monoclonal Antibodies
A panel of mAb reactive with the avian bursal and thymic stroma was used in this study. It was prepared and characterized as described elsewhere .

Indirect Immunofluorescence
Cryostat sections (4/m) were incubated with culture supernatant from the hybridoma or NS-1 myeloma cells (20 rain), washed three times with PBS (1 xl min, 2 x5 min), incubated with FITCconjugated sheep antimouse immunoglobulin (1/100, Silenus Labs, Melbourne, Australia) for 20 min and washed as before. Sections were mounted using veronal buffered glycerol or Permafluor (Lipshaw, Detroit, Michigan) and examined using either a Leitz Diavert fluorescence or a Zeiss epifluorescence microscope or a Zeiss MC63 camera and Kodak Ektachrome 1600 ASA film were used for photography. Double labeling with rabbit antihuman keratin (Dako, Santa Barbara, California) and sheep antirabbit-rhodamine (1/50, Silenus) was also performed to enable recognition of epithelial cells.