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Experimental studies on the development of the bursa of Fabricius
DEVELOPMENTAL
BIOLOGY
Experimental
14, 40-51 ( 1986)
Studies Bursa
on
the
Development
of the
of Fabricius
M. A. S. MOORE AND J. J. T. OWEN Anthropology
Laboratory, University Accepted
Department of Oxford, February
of Human
Anatomy,
England 10, 1966
INTRODUCTION
The bursa of Fabricius is a lymphoid organ of the chick which plays an important role in the development of the immune system. In particular, it is concerned with humoral antibody production (Glick et d., 1956; Mueller et al., 1962; Graetzer et al., 1963; Cooper et al., 1965). Studies on the origin and differentiation of its lymphoid elements are therefore of some considerable interest. The bursa first appears on the tenth day of incubaticn as a diverticulum of the cloaca. The subsequent development of lymphoid follicles in mesenchyme underlying the surface epithelium and their separation into cortical and medullary regions by a basement membrane has been fully described in a number of morphological and histochemical studies ( Ackerman and Knouff, 1959, 1963; Ackerman, 1962). The broad conclusions of these investigations have been that lymphocytes in the follicular medulla arise exclusively by transformation of undifferentiated epithelial cells, whereas lymphocytes in the cortical region are derived both by migration of lymphoid cells from the medulla and by transformation of cortical mesenchymal cells in situ. Recently a technique has been developed in this laboratory for the preparation of abundant, clear chromosome preparations from various hemopoietic organs of the chick embryo (Owen, 1965; Owen et al. 1965). Male (ZZ) and female (ZW) cells can be readily distinguished from each other as the Z chromosome is the only large mediocentric element present. Thus a technique is available whereby cellular migration can be investigated using the sex chromosomes as cell markers. 40
DEVELOPMENT
OF
THE
BURSA
OF
FABRICIUS
41
A high degree of cell chimerism has been demonstrated in a number of hemopoietic organs, including the bursa of Fabricius, in parabiosed chick embryos by means of this marker technique (Moore and Owen, 1965). The results indicated that, contrary to previous suggestions, the lymphocytic elements of the bursa are derived from bloodborne progenitor cells rather than by transformation of cells of the bursal primordium. The validity of this conclusion has been examined further in the present study by using the chromosome marker technique in combination with various experimental procedures including parabiosis of embryos of similar major histocompatibility type, twin of bursal rudiments to the chorioalembryo studies, transplantation lantois, and hormonal modification of bursal development. MATERIALS
AND
METHOD
Parabiosis. In a previous investigation (Moore and Owen, 1965), White Leghorn eggs were parabiosed using a modification of the technique of Hasek (1953). Th e use of a tissue bridge was found unnecessary for the production of a parabiotic union, and vascular anastomosis was readily obtained by direct apposition of the chorioallantoic membranes. In the present study, the same technique was used to parabiose 8-day embryos of identical B blood group type ( B, z ) . After a further 7 days’ incubation each egg was injected, by way of the allantois, with Colcemid (Ciba) solution. In those instances where the parabionts were of opposite sex, various hemopoietic tissues were prepared for chromosome analysis, and in the case of bone marrow the right and left lower limb bones were sampled separately. Twin. embryos. In the previous investigation cell chimerism was demonstrated in the marrow, spleen, and thymus, but not the bursa, of two pairs of twin embryos of 11 and 12 days’ incubation. The analysis of an additional pair of embryos of 13 days’ incubation is reported in this study. Chorioalhtoic grafting. Bursas were removed aseptically from outbred embryos of known sex of lo-14 days’ incubation and were grafted to the chorioallantoic membranes of lo- or I2-day hosts using the technirlue described by Hancox (1947). The smaller bursas were grafted whole, but the 12- and 1Cday bursas were split and placed epithelial surface down on the chorioallantois so as to facilitate vascularization. The hosts were injected with Colcemid and the grafts 3
42
MOORE
AND
OWEN
were prepared for chromosome analysis after a further 7-9 days’ incubation. In an additional experiment lo-, I2-, and I4-day embryonic bursas were grafted to lo-day hosts as before, but were sampled for histological examination within 24 hours of transplantation and at daily intervals thereafter. Testosterone treatment. In the first series of experiments 0.5-0.75 mg of testosterone propionate in a mixture of arachis oil and sodium oleate (3: 2) was injected, by way of the allantois, into embryos of 5 days’ incubation. At 10 days’ incubation, these embryos were engrafted with normal 12-day bursal transplants. In a second series of experiments, embryos which had been injected with 1 mg of testosterone propionate at 9 days’ incubation were used as donors of 12-day bursal grafts. These testosterone-treated bursas were transplanted to lo-day untreated hosts and to IO-day hosts injected with 0.75 mg testosterone after 5 days’ incubation. All the grafts were sampled for histological analysis, and some for chromosomal analysis 9 days after transplantation. Chromosomal analysis. Three hours before sampling, eggs were injected with 0.1 ml Colcemid solution (containing 0.05-0.1 mg Colcemid according to stage of incubation). Chromosomes were then prepared from the various tissues using a modified hypotonic citrateair drying technique (Owen, 1965). Staining was carried out in lacticacetic-orcein and the preparations were mounted in Euparal and viewed by phase-contrast microscopy. Histological methods. Biopsies of transplanted bursas, host bursas, and bursas removed from parabiosed embryos were fixed in Susa, embedded in polyester wax, and sectioned at 7 p. Sections were stained with Harris’s hematoxylin and Gomori’s trichrome. RESULTS
The procedure of embryonic parabiosis high operative mortality. In addition, the provides abundant metaphase preparations dose for the embryo, and occasionally one were killed. In all, only a small proportion been parabiosed provided surviving pairs could be used for chromosome analysis.
is accompanied by a very dose of Colcemid which is very close to the toxic or both embryos of a pair of the eggs which had of dissimiliar sex which
DEVELOPMEST
OF
Buns.4
I’nir
THE
TABLE
1
OF
I?AHRICIT.‘S
Ko.
6
10
2 Outbred
6
19
3 Out,hed 4 Outbred 5 Outbred
7 8 8
20 1’) 17
6 Outbred 7 Outbred 8 Inbred
8 8 8
15 15 15
11
19
PII2
9 Outbred
from eggs, from eggs, from qgs,
OF
double1. double2. double3.
12 11 -
13
F
43
FABRICIUS
Sumbcr of cells scored
1 Outbr?d
Ii:mhryos yolked Emhyos yolked Embryos yolked
BURSA
261
h-umber of cells opposite sex
Percentage opposite sex
h1
30
97 8
37 27
F RI F RI F 31 13’ F F n1 Y M
300 68 21 50 100 41 I0 50 50 50 SO 100
i8 34 1:: I9 29 Is 5 0 2 4 14 49
26 50 43 38 2’) 39 50 0 4 8 47 49
F nr 11’ hl F RI
*in 50 50 50 50 50
0 0 0 0 2 1
0 0 0 0 4 2
The accumulated results are presented in Table 1. In terms of the outbred eggs previously studied, they may be summarized by stating that in embryos parabiosed at 6-11 days’ incubation and sampled at 17-20 days’ incubation between 26 and 50% of the dividing cells scored in the bursa of Fabricius were derived from the opposite partner. The degree of chimerism in embryos sampled at 15 days’ incubation was considerably less, with the exception of one embryo on which, however, only a small count was possible. Examination of histological sections of bursas removed from 17- to 20-day embryos previously treated with Colcemid showed that the overwhelming majority of mitosis were confined to lymphoid follicles, and phase contrast examination of bursal cell suspensions left no doubt that the predominant cell types sampled for chromosome analysis belonged to the lymphocyte series. It has been shown that during the later stages of incubation there
FIG. 1. Section bursa. Magnification: FE. 2. Section
showing lymphoid x 150. of a bursal graft
follicular made 44
from
development a lo-day
in a normal donor
to a host
l7-day of the
DEVELOPMEST
OF
THE
BURSA
OF
FABRICIUS
45
are cells in the chick embryo which are capable of being sensitized to transplantation antigens (Solomon, 1963). Further, it has been proposed that small numbers of immunologically competent cells appear in the embryo at about 15 days’ incubation and that they are capable of producing a weak homograft reaction (Solomon and Tucker, 1963). Since proliferation of adult immunologically competent cells takes place in embryonic hosts during the graft versus host reaction (Owen et al., 1965)) it might be argued that cell chimerism in parabiosed embryos is due to migration and proliferation of competent embryonic cells which are reacting against the foreign histocompatibility antigens of the partner. However, no macroscopic nor microscopic evidence of an immune reaction between embryonic partners was found, and chimerism was present in parabiosed embryos of the same B blood group locus and therefore of the same major histocompatibility type (Craig and McDermid, 1963). It seems extremely unlikely, therefore, that an immune mechanism is responsible for the cell chimerism noted. The degree of chimerism in the bursas of the B grouped embryos is low, but this is not unexpected as they were sampled at 15 days’ incubation when lymphoid follicle development was not far advanced. Their spleens contained 26% of dividing male cells in the female partner and 21% of dividing female cells in the male partner (based on 100 cells scored in each case). The comparable figures for the bone marrow were 15% cells of male sex in the female and 17% same incubation. The graft was sampled 7 days later and shows comparable follicular development to a normal l7-day bursa. Magnification: x 150. FIG. 3. Section of a normal 19-day bursa. Magnification: x 150. FIG. 4. Bursal graft made from a lo-day donor to a host of the same incubation and sampled 9 days later. Well-formed Iymphoid follicles are present. Magnification: X 150. FIG. 5. High power view of a graft biopsy removed from a Colcemid-treated embryo. The transplant was made from a lo-day donor to a host of the same age and was removed 9 days later. Note the numerous mitotic figures situated within the lymphoid follicles. Magnification: x 600. FIG. 6. Section of a bursal graft made from a normal 12-day donor to a IOday testosterone-treated host and sampled 9 days later. The graft has failed to develop and no lymphoid follicles are present. Magnification: x 150. FIG. 7. Section of a graft as in Fig. 6, but in this case the bursa shows good development with well formed follicles. Magnification: x 150. FIG. 8. Section of a testosterone-treated graft made from a 12-day donor to a normal lo-day host. Note the presence of lymphoid follicles 9 days later. Magnification: X 150.
46
MOORE
AND
OWEiS
cells of female sex in the male embryo. It is interesting to note that identical percentages were obtained on 100 marrow cells sampled from the right femur and 100 marrow cells from the left femur. Cell chimerism was not found in either thymus gland. It has been noted that two pairs of twin embryos have been examined for cell chimerism previously. In the present investigation a 10~ level of chimerism has been found in the bursas of a pair of 13-day twin embryos (Table 1) . The second experimental approach, namely that of chorioallantoic transplantation, provided results of considerable interest to the analysis of bursal development. Histological examination of grafts removed at various intervals after transplantation showed that lo-day bursal rudiments usually became vascularized within 2448 hours of transplantation to a lo-day host and thereafter their follicular development paralleled that of the host bursa (Figs, 14). On the other hand, 12and 1Cday bursal transplants vascularized less rapidly, and most underwent partial necrosis within 2 days of transplantation, This was followed by a period of regeneration, and although they showed an overall retardation in development, they ultimately reached a stage of follicular formation equivalent to that of the host bursa. A few grafts failed to be incorporated into the chorioallantois and subsequently showed grossly retarded development. The results of the chromosome analysis of those bursal grafts which were of opposite sex to the host are presented in Table 2. It can be seen that lo-day bursal grafts made to hosts of equiva!ent age contained SO-83% host metaphases and 86-94% host metaphases when sampled at 7 and 9 days later. Histological examination of a small biopsy removed from each transplant showed that in all cases the morphological development of the graft was equivalent to that of the host bursa. Nearly all the metaphase figures were confined to lymphoid follicles (Fig. 5) and there is little doubt that most of the cells sampled for chromosome analysis were of the lymphoid series. Twelve-day bursal transplants engrafted on hosts of 10 days’ incubation contained 72-92% host metaphases 9 days later, while transplants of the same age made to 12-day embryos contained 76% and 88% host dividing cells after a further 7 days’ incubation. Thirteen-day bursal grafts made to lo-day hosts showed a more variable host component (48-92%) after 9 days on the chorioallantois and a 1Cday graft which showed good follicular development contained 64% dividing
DEVELOPMENT
Age sampling
at
(days) 17 17 17 17 10 I9 19 I!) I!) 19 10 19 19 19 19 l!)
OF
THE
BURSA
OF
47
FABRICIUS
Age of host (days) IO
10
10 10 10 10 10 10 10 10 1% I:! 10 10 10 10 10
10 10
a Counts were made on 50 metaphases * The host in the cavz of graft 16 wits
10 10 IO 12 12 12 12 12 13 13 13 14 12
17 20
18 1X G 14 8 28 12 24 12 12 8 32 36 50
in all cases. testosterone treated.
host cells. Biopsies taken from these bursal grafts showed that they had reached a stage of development equivalent to that found in a normal 19-day bursa. In the final series of experiments, 12-day bursal transplants made to testosterone-treated hosts were found to show a wide range of histological development when sampled 9 days later (Figs. 6 and 7). In all cases the host bursas had completely failed to develop, but whereas some of the transplants were extremely retarded in development, some had reached a stage of follicular formation similar to that found in 12-day bursas grafted to normal embryos. Chromosome analysis of a graft showing good follicular development indicated that 50% of the dividing cells were host in type (Table 2). Twelve-day bursas removed from testosterone-treated embryos and then transplanted to normal lo-day hosts showed good follicular development 9 days later (Fig. 8). In fact their development was comparable to that of normal 12-day grafts on normal lo-day hosts. By way of contrast, treated bursas transplanted to treated hosts completely failed to develop.
48
MOORE
AND
OWEN
DISCUSSION
The high levels of cell chimerism found in the bursa of Fabricius of most parabiosed embryos indicates that there is an afferent stream of cells entering and proliferating in the organ during embryogenesis. As most mitotic figures were noted in bursal follicles and cell suspensions prepared for chromosome analysis were predominantly lymphoid, it seems reasonable to conclude that most if not all cells in the developing follicles are derived from blood-borne progenitor cells. Strong evidence for the belief that this is a normal feature of bursal development is provided by the fact that chimerism was found in parabiosed embryos of the same major histocompatibility type. This conclusion is substantiated by the results of the transplantation experiments. Ten-day bursal grafts transplanted to hosts of the same age did not undergo necrosis but showed normal bursal development. The fact that the great majority of dividing cells scored in these transplanted bursas after 7-9 days further incubation were of host rather than donor type supports the contention that bursal development is dependent on a stem cell inflow. Examination of graft biopsies showed that most mitotic figures were situated in lymphoid follicles suggesting that it is the lymphoid cells which have a blood-borne origin. Previous work on the cellular population of organ transplants such as thymus (Miller, 1963) and spleen (Metcalf and Wakonis-Vaartaja, 1964) has demonstrated a host cell repopulation of the tissues after an initial period of necrosis. In contrast to the lo-day transplants, 12and 1Cday bursal grafts also underwent partial necrosis on transplantation and subsequently were largely populated by host cells. Although this result cannot be interpreted as indicating a stem cell inflow into the normal bursa, it does add weight to the evidence already presented for such a process. It is well known that the administration of testosterone to chick embryos at an early stage of incubation results in an inhibition of bursal development (Warner and Burnet, 1961; Rao et al., 1962). By varying the dosage and time of administration, it is possible either to inhibit bursal development completely or to produce degrees of developmental retardation. An attempt has been made in the present study to gain some insight into the effect of testosterone administration on the availability and proliferation of progenitor cells in the bursa. The results have shown that normal bursal grafts can develop
DEVELOPMENT
OF
THE
BURSA
OF
FABRICIUS
49
to varying degrees on the chorioallantoic membranes of hormonally bursectomized hosts. The finding of a 50% dividing host cell population in one of these bursas indicates that bursal progenitor cells are available in the hormone-treated embryo. It is also apparent that these progenitor cells must originate from a source other than the host bursa (which had completely failed to develop). It has also been noted that a testosterone-treated bursa can recover if grafted to a normal host and later show good follicular development whereas no development takes place in hormonally treated bursas placed on testosterone-treated hosts. The inhibitory effect of testosterone on bursal development seems therefore to be on the proliferation rather than the availability of stem cells. It is unlikely that the effect is one of direct mitotic inhibition as some of the hormonally treated bursas show good epithelial proliferation. An inhibition of alkaline phosphatase activity has been demonstrated in bursal mesenchyme cells of testosterone-treated embryos (Ackerman and Knouff, 1963). Th us the action of the hormone may be on mesenchymal cell functions with a secondary failure of follicular development. In this context, it is interesting to note that mesenchyme activity has been shown to be necessary for induction of lymphoid transformation in the thymus ( Auerbach, 1961). In conclusion, it may he stated that the majority of bursal lymphocytes are not derived from cells intrinsic to the bursal primordium, but are produced by the proliferation of blood-borne progenitor cells which enter the primordium during embryogenesis. It is realized that by designating these cells as progenitor or stem cells, nothing is added to a knowledge of their structure or site of origin and these must remain points for future investigation. Finally it seems likely that the bursal primordium provides a specific environment for the proliferation and maturation of these cells, and testosterone treatment inhibits development by interfering with this environment rather than by limiting the supply of progenitor cells. SUMMARY
An analysis of the origin of lymphoid cells in the developing bursa of Fabricius has been made using a chromosome marker technique in combination with various experimental procedures including parabiosis of embryos of similar major histocompatibility type, twin embryo studies, transplantation of bursal rudiments to the chorioallan-
50
MOORE
AND
OWEN
tois, and hormonal modification of bursal development. The results indicate that the majority of bursal lymphoeytes are derived from blood-borne progenitor cells which enter the bursal primordium during embryogenesis. Testosterone treatment does not limit the supply of these cells but probably alters the environment in which they proliferate and mature. The structure and site of origin of the progenitor cells is unknown, and these remain points for future investigation. The graphs.
authors wish to thank Miss S. P. Warner
for preparing
the photomicro-
REFERENCES
G. A. (1962). Electron embryonic chick with particular J. Cell Biol. 13, 127-146.
ACKERMAN,
microscopy of the bursa of Fabricius of the reference to the lympho-epithelial nodules.
ACKERMAN, G. A., and KNOUFF, R. A. ( 1959). Lymphocytopoiesis in the bursa of Fabricius. Am. J. Anut. 104, 163-206. ACKERMAN, G. A., and KNOUFF, R. A. ( 1963). Testosterone suppression of mesenchymal alkaline phosphatase activity and lympo-epithelial nodule formation in the bursa of Fabricius in the embryonic chick. Anat. Record 146, 23-27. AUERBACH, R. ( 1961). Experimental analysis of the origin of cell types in the development of the mouse thymus. Develop. Biol. 3, 336-354. COOPER, M. D., PETERSON, R. D. A., and GOOD, R. A. (1965). Delineation of the thymic and bursal lymphoid systems in the chicken. Nature 205, 143-146. CRAIG, J. V., and MCDERMID, E. M. (19’63). Prolonged skin homograft survival and erythrocyte (B-locus) antigens in young chicks. Tmn.spZuntation 1, 191200. GLICK, B., GANG, T. S., and JAAP, R. G. (1956). The bursa of Fabricius and antibody production. Poultry Sci. 35, 224-245. GRAETZER, M. A., WOLFE, H. R., ASPINALL, R. L., and MEYER, R. K. (1963). Effect of thymectomy and bursectomy on precipitin and natural hemagglutinin production in the chicken. J. Immunol. 90, 878-887. of transplanted embryo bone grafted to HANCOX, N. M. ( 1947). Th e survival chorioallantoic membrane, and subsequent osteogenesis. .I. Physiol. (London)
106, 279-285.
.
HASEK, M. ( 1953). Parabiosis in birds during embryonic development. Ceskodov. Biol. 2, 25. METCALF, D., and WAKONIS-VAAHTA JA, R. (1964). Host-cell repopulation of normal spleen grafts. Lancet i, 1012-1014. MILLER, J. F. A. P. ( 1963 ). Immunity and the thymus. Lancet i, 43-45. MOORE, M. A. S., and OWEN, J. J. T. ( 1965). Chromosome marker studies on the development of the haemopoietic system in the chick embryo. Nature 208, 956 and 989-990. MUELLER, A. P., WOLFE, H. R., MEYER, R. K., and ASPINALL, R. L. (1962). Further studies on the role of the bursa of Fabricius in antibody production. J. Immunol. 88, 354-360.
DEVELOPMEKT
OF
THE
BURSA
OF
FABRICIUS
51
OWEK, J. J. T. ( 1965). Knryotype studies on Gallus domesticus. Chromosoma 16, 601-608. OWEN, J. J. T., MOORE, M. A. S., and HARRISON, G. A, (1965). Chromosome marker studies in the graft-versus-host reaction in the chick embryo. Nature 207, 313-315. Rao, M. A., ASPINALL, R. L., and ~IEYER, R. K. (1962). Effect of dose and time of administration of 19-nortestosterone on the differentiation of lymphoid tissue in the bursa Fabricii of chick embryos. EndocrinoZog~ 70, 159-166. SOLOMON, J. B. (1963). Actively acquired transplantation immunity in the chick embryo. Nature 198, 1171-1173. SOLOMON, J. B., and TUCKER, D. F. ( 1963). attack by adult cells I mmunological in the developing chick embryo: Influence of the vascularity of the host spleen and of homograft rejection by the embryo on splenomegaly. J. Embryol. Exptl. Morphol. 11, 119’-134. WARNER, X. L., and BUHNET, F. M. ( 1961). The influence of testosterone treatment on the development of the bursa of Fabricius in the chick embryo. Australian J. Biol. Sci. Med. Sci. 14, 580-587.
BIOLOGY
Experimental
14, 40-51 ( 1986)
Studies Bursa
on
the
Development
of the
of Fabricius
M. A. S. MOORE AND J. J. T. OWEN Anthropology
Laboratory, University Accepted
Department of Oxford, February
of Human
Anatomy,
England 10, 1966
INTRODUCTION
The bursa of Fabricius is a lymphoid organ of the chick which plays an important role in the development of the immune system. In particular, it is concerned with humoral antibody production (Glick et d., 1956; Mueller et al., 1962; Graetzer et al., 1963; Cooper et al., 1965). Studies on the origin and differentiation of its lymphoid elements are therefore of some considerable interest. The bursa first appears on the tenth day of incubaticn as a diverticulum of the cloaca. The subsequent development of lymphoid follicles in mesenchyme underlying the surface epithelium and their separation into cortical and medullary regions by a basement membrane has been fully described in a number of morphological and histochemical studies ( Ackerman and Knouff, 1959, 1963; Ackerman, 1962). The broad conclusions of these investigations have been that lymphocytes in the follicular medulla arise exclusively by transformation of undifferentiated epithelial cells, whereas lymphocytes in the cortical region are derived both by migration of lymphoid cells from the medulla and by transformation of cortical mesenchymal cells in situ. Recently a technique has been developed in this laboratory for the preparation of abundant, clear chromosome preparations from various hemopoietic organs of the chick embryo (Owen, 1965; Owen et al. 1965). Male (ZZ) and female (ZW) cells can be readily distinguished from each other as the Z chromosome is the only large mediocentric element present. Thus a technique is available whereby cellular migration can be investigated using the sex chromosomes as cell markers. 40
DEVELOPMENT
OF
THE
BURSA
OF
FABRICIUS
41
A high degree of cell chimerism has been demonstrated in a number of hemopoietic organs, including the bursa of Fabricius, in parabiosed chick embryos by means of this marker technique (Moore and Owen, 1965). The results indicated that, contrary to previous suggestions, the lymphocytic elements of the bursa are derived from bloodborne progenitor cells rather than by transformation of cells of the bursal primordium. The validity of this conclusion has been examined further in the present study by using the chromosome marker technique in combination with various experimental procedures including parabiosis of embryos of similar major histocompatibility type, twin of bursal rudiments to the chorioalembryo studies, transplantation lantois, and hormonal modification of bursal development. MATERIALS
AND
METHOD
Parabiosis. In a previous investigation (Moore and Owen, 1965), White Leghorn eggs were parabiosed using a modification of the technique of Hasek (1953). Th e use of a tissue bridge was found unnecessary for the production of a parabiotic union, and vascular anastomosis was readily obtained by direct apposition of the chorioallantoic membranes. In the present study, the same technique was used to parabiose 8-day embryos of identical B blood group type ( B, z ) . After a further 7 days’ incubation each egg was injected, by way of the allantois, with Colcemid (Ciba) solution. In those instances where the parabionts were of opposite sex, various hemopoietic tissues were prepared for chromosome analysis, and in the case of bone marrow the right and left lower limb bones were sampled separately. Twin. embryos. In the previous investigation cell chimerism was demonstrated in the marrow, spleen, and thymus, but not the bursa, of two pairs of twin embryos of 11 and 12 days’ incubation. The analysis of an additional pair of embryos of 13 days’ incubation is reported in this study. Chorioalhtoic grafting. Bursas were removed aseptically from outbred embryos of known sex of lo-14 days’ incubation and were grafted to the chorioallantoic membranes of lo- or I2-day hosts using the technirlue described by Hancox (1947). The smaller bursas were grafted whole, but the 12- and 1Cday bursas were split and placed epithelial surface down on the chorioallantois so as to facilitate vascularization. The hosts were injected with Colcemid and the grafts 3
42
MOORE
AND
OWEN
were prepared for chromosome analysis after a further 7-9 days’ incubation. In an additional experiment lo-, I2-, and I4-day embryonic bursas were grafted to lo-day hosts as before, but were sampled for histological examination within 24 hours of transplantation and at daily intervals thereafter. Testosterone treatment. In the first series of experiments 0.5-0.75 mg of testosterone propionate in a mixture of arachis oil and sodium oleate (3: 2) was injected, by way of the allantois, into embryos of 5 days’ incubation. At 10 days’ incubation, these embryos were engrafted with normal 12-day bursal transplants. In a second series of experiments, embryos which had been injected with 1 mg of testosterone propionate at 9 days’ incubation were used as donors of 12-day bursal grafts. These testosterone-treated bursas were transplanted to lo-day untreated hosts and to IO-day hosts injected with 0.75 mg testosterone after 5 days’ incubation. All the grafts were sampled for histological analysis, and some for chromosomal analysis 9 days after transplantation. Chromosomal analysis. Three hours before sampling, eggs were injected with 0.1 ml Colcemid solution (containing 0.05-0.1 mg Colcemid according to stage of incubation). Chromosomes were then prepared from the various tissues using a modified hypotonic citrateair drying technique (Owen, 1965). Staining was carried out in lacticacetic-orcein and the preparations were mounted in Euparal and viewed by phase-contrast microscopy. Histological methods. Biopsies of transplanted bursas, host bursas, and bursas removed from parabiosed embryos were fixed in Susa, embedded in polyester wax, and sectioned at 7 p. Sections were stained with Harris’s hematoxylin and Gomori’s trichrome. RESULTS
The procedure of embryonic parabiosis high operative mortality. In addition, the provides abundant metaphase preparations dose for the embryo, and occasionally one were killed. In all, only a small proportion been parabiosed provided surviving pairs could be used for chromosome analysis.
is accompanied by a very dose of Colcemid which is very close to the toxic or both embryos of a pair of the eggs which had of dissimiliar sex which
DEVELOPMEST
OF
Buns.4
I’nir
THE
TABLE
1
OF
I?AHRICIT.‘S
Ko.
6
10
2 Outbred
6
19
3 Out,hed 4 Outbred 5 Outbred
7 8 8
20 1’) 17
6 Outbred 7 Outbred 8 Inbred
8 8 8
15 15 15
11
19
PII2
9 Outbred
from eggs, from eggs, from qgs,
OF
double1. double2. double3.
12 11 -
13
F
43
FABRICIUS
Sumbcr of cells scored
1 Outbr?d
Ii:mhryos yolked Emhyos yolked Embryos yolked
BURSA
261
h-umber of cells opposite sex
Percentage opposite sex
h1
30
97 8
37 27
F RI F RI F 31 13’ F F n1 Y M
300 68 21 50 100 41 I0 50 50 50 SO 100
i8 34 1:: I9 29 Is 5 0 2 4 14 49
26 50 43 38 2’) 39 50 0 4 8 47 49
F nr 11’ hl F RI
*in 50 50 50 50 50
0 0 0 0 2 1
0 0 0 0 4 2
The accumulated results are presented in Table 1. In terms of the outbred eggs previously studied, they may be summarized by stating that in embryos parabiosed at 6-11 days’ incubation and sampled at 17-20 days’ incubation between 26 and 50% of the dividing cells scored in the bursa of Fabricius were derived from the opposite partner. The degree of chimerism in embryos sampled at 15 days’ incubation was considerably less, with the exception of one embryo on which, however, only a small count was possible. Examination of histological sections of bursas removed from 17- to 20-day embryos previously treated with Colcemid showed that the overwhelming majority of mitosis were confined to lymphoid follicles, and phase contrast examination of bursal cell suspensions left no doubt that the predominant cell types sampled for chromosome analysis belonged to the lymphocyte series. It has been shown that during the later stages of incubation there
FIG. 1. Section bursa. Magnification: FE. 2. Section
showing lymphoid x 150. of a bursal graft
follicular made 44
from
development a lo-day
in a normal donor
to a host
l7-day of the
DEVELOPMEST
OF
THE
BURSA
OF
FABRICIUS
45
are cells in the chick embryo which are capable of being sensitized to transplantation antigens (Solomon, 1963). Further, it has been proposed that small numbers of immunologically competent cells appear in the embryo at about 15 days’ incubation and that they are capable of producing a weak homograft reaction (Solomon and Tucker, 1963). Since proliferation of adult immunologically competent cells takes place in embryonic hosts during the graft versus host reaction (Owen et al., 1965)) it might be argued that cell chimerism in parabiosed embryos is due to migration and proliferation of competent embryonic cells which are reacting against the foreign histocompatibility antigens of the partner. However, no macroscopic nor microscopic evidence of an immune reaction between embryonic partners was found, and chimerism was present in parabiosed embryos of the same B blood group locus and therefore of the same major histocompatibility type (Craig and McDermid, 1963). It seems extremely unlikely, therefore, that an immune mechanism is responsible for the cell chimerism noted. The degree of chimerism in the bursas of the B grouped embryos is low, but this is not unexpected as they were sampled at 15 days’ incubation when lymphoid follicle development was not far advanced. Their spleens contained 26% of dividing male cells in the female partner and 21% of dividing female cells in the male partner (based on 100 cells scored in each case). The comparable figures for the bone marrow were 15% cells of male sex in the female and 17% same incubation. The graft was sampled 7 days later and shows comparable follicular development to a normal l7-day bursa. Magnification: x 150. FIG. 3. Section of a normal 19-day bursa. Magnification: x 150. FIG. 4. Bursal graft made from a lo-day donor to a host of the same incubation and sampled 9 days later. Well-formed Iymphoid follicles are present. Magnification: X 150. FIG. 5. High power view of a graft biopsy removed from a Colcemid-treated embryo. The transplant was made from a lo-day donor to a host of the same age and was removed 9 days later. Note the numerous mitotic figures situated within the lymphoid follicles. Magnification: x 600. FIG. 6. Section of a bursal graft made from a normal 12-day donor to a IOday testosterone-treated host and sampled 9 days later. The graft has failed to develop and no lymphoid follicles are present. Magnification: x 150. FIG. 7. Section of a graft as in Fig. 6, but in this case the bursa shows good development with well formed follicles. Magnification: x 150. FIG. 8. Section of a testosterone-treated graft made from a 12-day donor to a normal lo-day host. Note the presence of lymphoid follicles 9 days later. Magnification: X 150.
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cells of female sex in the male embryo. It is interesting to note that identical percentages were obtained on 100 marrow cells sampled from the right femur and 100 marrow cells from the left femur. Cell chimerism was not found in either thymus gland. It has been noted that two pairs of twin embryos have been examined for cell chimerism previously. In the present investigation a 10~ level of chimerism has been found in the bursas of a pair of 13-day twin embryos (Table 1) . The second experimental approach, namely that of chorioallantoic transplantation, provided results of considerable interest to the analysis of bursal development. Histological examination of grafts removed at various intervals after transplantation showed that lo-day bursal rudiments usually became vascularized within 2448 hours of transplantation to a lo-day host and thereafter their follicular development paralleled that of the host bursa (Figs, 14). On the other hand, 12and 1Cday bursal transplants vascularized less rapidly, and most underwent partial necrosis within 2 days of transplantation, This was followed by a period of regeneration, and although they showed an overall retardation in development, they ultimately reached a stage of follicular formation equivalent to that of the host bursa. A few grafts failed to be incorporated into the chorioallantois and subsequently showed grossly retarded development. The results of the chromosome analysis of those bursal grafts which were of opposite sex to the host are presented in Table 2. It can be seen that lo-day bursal grafts made to hosts of equiva!ent age contained SO-83% host metaphases and 86-94% host metaphases when sampled at 7 and 9 days later. Histological examination of a small biopsy removed from each transplant showed that in all cases the morphological development of the graft was equivalent to that of the host bursa. Nearly all the metaphase figures were confined to lymphoid follicles (Fig. 5) and there is little doubt that most of the cells sampled for chromosome analysis were of the lymphoid series. Twelve-day bursal transplants engrafted on hosts of 10 days’ incubation contained 72-92% host metaphases 9 days later, while transplants of the same age made to 12-day embryos contained 76% and 88% host dividing cells after a further 7 days’ incubation. Thirteen-day bursal grafts made to lo-day hosts showed a more variable host component (48-92%) after 9 days on the chorioallantois and a 1Cday graft which showed good follicular development contained 64% dividing
DEVELOPMENT
Age sampling
at
(days) 17 17 17 17 10 I9 19 I!) I!) 19 10 19 19 19 19 l!)
OF
THE
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OF
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FABRICIUS
Age of host (days) IO
10
10 10 10 10 10 10 10 10 1% I:! 10 10 10 10 10
10 10
a Counts were made on 50 metaphases * The host in the cavz of graft 16 wits
10 10 IO 12 12 12 12 12 13 13 13 14 12
17 20
18 1X G 14 8 28 12 24 12 12 8 32 36 50
in all cases. testosterone treated.
host cells. Biopsies taken from these bursal grafts showed that they had reached a stage of development equivalent to that found in a normal 19-day bursa. In the final series of experiments, 12-day bursal transplants made to testosterone-treated hosts were found to show a wide range of histological development when sampled 9 days later (Figs. 6 and 7). In all cases the host bursas had completely failed to develop, but whereas some of the transplants were extremely retarded in development, some had reached a stage of follicular formation similar to that found in 12-day bursas grafted to normal embryos. Chromosome analysis of a graft showing good follicular development indicated that 50% of the dividing cells were host in type (Table 2). Twelve-day bursas removed from testosterone-treated embryos and then transplanted to normal lo-day hosts showed good follicular development 9 days later (Fig. 8). In fact their development was comparable to that of normal 12-day grafts on normal lo-day hosts. By way of contrast, treated bursas transplanted to treated hosts completely failed to develop.
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DISCUSSION
The high levels of cell chimerism found in the bursa of Fabricius of most parabiosed embryos indicates that there is an afferent stream of cells entering and proliferating in the organ during embryogenesis. As most mitotic figures were noted in bursal follicles and cell suspensions prepared for chromosome analysis were predominantly lymphoid, it seems reasonable to conclude that most if not all cells in the developing follicles are derived from blood-borne progenitor cells. Strong evidence for the belief that this is a normal feature of bursal development is provided by the fact that chimerism was found in parabiosed embryos of the same major histocompatibility type. This conclusion is substantiated by the results of the transplantation experiments. Ten-day bursal grafts transplanted to hosts of the same age did not undergo necrosis but showed normal bursal development. The fact that the great majority of dividing cells scored in these transplanted bursas after 7-9 days further incubation were of host rather than donor type supports the contention that bursal development is dependent on a stem cell inflow. Examination of graft biopsies showed that most mitotic figures were situated in lymphoid follicles suggesting that it is the lymphoid cells which have a blood-borne origin. Previous work on the cellular population of organ transplants such as thymus (Miller, 1963) and spleen (Metcalf and Wakonis-Vaartaja, 1964) has demonstrated a host cell repopulation of the tissues after an initial period of necrosis. In contrast to the lo-day transplants, 12and 1Cday bursal grafts also underwent partial necrosis on transplantation and subsequently were largely populated by host cells. Although this result cannot be interpreted as indicating a stem cell inflow into the normal bursa, it does add weight to the evidence already presented for such a process. It is well known that the administration of testosterone to chick embryos at an early stage of incubation results in an inhibition of bursal development (Warner and Burnet, 1961; Rao et al., 1962). By varying the dosage and time of administration, it is possible either to inhibit bursal development completely or to produce degrees of developmental retardation. An attempt has been made in the present study to gain some insight into the effect of testosterone administration on the availability and proliferation of progenitor cells in the bursa. The results have shown that normal bursal grafts can develop
DEVELOPMENT
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THE
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49
to varying degrees on the chorioallantoic membranes of hormonally bursectomized hosts. The finding of a 50% dividing host cell population in one of these bursas indicates that bursal progenitor cells are available in the hormone-treated embryo. It is also apparent that these progenitor cells must originate from a source other than the host bursa (which had completely failed to develop). It has also been noted that a testosterone-treated bursa can recover if grafted to a normal host and later show good follicular development whereas no development takes place in hormonally treated bursas placed on testosterone-treated hosts. The inhibitory effect of testosterone on bursal development seems therefore to be on the proliferation rather than the availability of stem cells. It is unlikely that the effect is one of direct mitotic inhibition as some of the hormonally treated bursas show good epithelial proliferation. An inhibition of alkaline phosphatase activity has been demonstrated in bursal mesenchyme cells of testosterone-treated embryos (Ackerman and Knouff, 1963). Th us the action of the hormone may be on mesenchymal cell functions with a secondary failure of follicular development. In this context, it is interesting to note that mesenchyme activity has been shown to be necessary for induction of lymphoid transformation in the thymus ( Auerbach, 1961). In conclusion, it may he stated that the majority of bursal lymphocytes are not derived from cells intrinsic to the bursal primordium, but are produced by the proliferation of blood-borne progenitor cells which enter the primordium during embryogenesis. It is realized that by designating these cells as progenitor or stem cells, nothing is added to a knowledge of their structure or site of origin and these must remain points for future investigation. Finally it seems likely that the bursal primordium provides a specific environment for the proliferation and maturation of these cells, and testosterone treatment inhibits development by interfering with this environment rather than by limiting the supply of progenitor cells. SUMMARY
An analysis of the origin of lymphoid cells in the developing bursa of Fabricius has been made using a chromosome marker technique in combination with various experimental procedures including parabiosis of embryos of similar major histocompatibility type, twin embryo studies, transplantation of bursal rudiments to the chorioallan-
50
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tois, and hormonal modification of bursal development. The results indicate that the majority of bursal lymphoeytes are derived from blood-borne progenitor cells which enter the bursal primordium during embryogenesis. Testosterone treatment does not limit the supply of these cells but probably alters the environment in which they proliferate and mature. The structure and site of origin of the progenitor cells is unknown, and these remain points for future investigation. The graphs.
authors wish to thank Miss S. P. Warner
for preparing
the photomicro-
REFERENCES
G. A. (1962). Electron embryonic chick with particular J. Cell Biol. 13, 127-146.
ACKERMAN,
microscopy of the bursa of Fabricius of the reference to the lympho-epithelial nodules.
ACKERMAN, G. A., and KNOUFF, R. A. ( 1959). Lymphocytopoiesis in the bursa of Fabricius. Am. J. Anut. 104, 163-206. ACKERMAN, G. A., and KNOUFF, R. A. ( 1963). Testosterone suppression of mesenchymal alkaline phosphatase activity and lympo-epithelial nodule formation in the bursa of Fabricius in the embryonic chick. Anat. Record 146, 23-27. AUERBACH, R. ( 1961). Experimental analysis of the origin of cell types in the development of the mouse thymus. Develop. Biol. 3, 336-354. COOPER, M. D., PETERSON, R. D. A., and GOOD, R. A. (1965). Delineation of the thymic and bursal lymphoid systems in the chicken. Nature 205, 143-146. CRAIG, J. V., and MCDERMID, E. M. (19’63). Prolonged skin homograft survival and erythrocyte (B-locus) antigens in young chicks. Tmn.spZuntation 1, 191200. GLICK, B., GANG, T. S., and JAAP, R. G. (1956). The bursa of Fabricius and antibody production. Poultry Sci. 35, 224-245. GRAETZER, M. A., WOLFE, H. R., ASPINALL, R. L., and MEYER, R. K. (1963). Effect of thymectomy and bursectomy on precipitin and natural hemagglutinin production in the chicken. J. Immunol. 90, 878-887. of transplanted embryo bone grafted to HANCOX, N. M. ( 1947). Th e survival chorioallantoic membrane, and subsequent osteogenesis. .I. Physiol. (London)
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HASEK, M. ( 1953). Parabiosis in birds during embryonic development. Ceskodov. Biol. 2, 25. METCALF, D., and WAKONIS-VAAHTA JA, R. (1964). Host-cell repopulation of normal spleen grafts. Lancet i, 1012-1014. MILLER, J. F. A. P. ( 1963 ). Immunity and the thymus. Lancet i, 43-45. MOORE, M. A. S., and OWEN, J. J. T. ( 1965). Chromosome marker studies on the development of the haemopoietic system in the chick embryo. Nature 208, 956 and 989-990. MUELLER, A. P., WOLFE, H. R., MEYER, R. K., and ASPINALL, R. L. (1962). Further studies on the role of the bursa of Fabricius in antibody production. J. Immunol. 88, 354-360.
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OWEK, J. J. T. ( 1965). Knryotype studies on Gallus domesticus. Chromosoma 16, 601-608. OWEN, J. J. T., MOORE, M. A. S., and HARRISON, G. A, (1965). Chromosome marker studies in the graft-versus-host reaction in the chick embryo. Nature 207, 313-315. Rao, M. A., ASPINALL, R. L., and ~IEYER, R. K. (1962). Effect of dose and time of administration of 19-nortestosterone on the differentiation of lymphoid tissue in the bursa Fabricii of chick embryos. EndocrinoZog~ 70, 159-166. SOLOMON, J. B. (1963). Actively acquired transplantation immunity in the chick embryo. Nature 198, 1171-1173. SOLOMON, J. B., and TUCKER, D. F. ( 1963). attack by adult cells I mmunological in the developing chick embryo: Influence of the vascularity of the host spleen and of homograft rejection by the embryo on splenomegaly. J. Embryol. Exptl. Morphol. 11, 119’-134. WARNER, X. L., and BUHNET, F. M. ( 1961). The influence of testosterone treatment on the development of the bursa of Fabricius in the chick embryo. Australian J. Biol. Sci. Med. Sci. 14, 580-587.