RESEARCH REPORT 3019
Development 137, 3019-3023 (2010) doi:10.1242/dev.055194
© 2010. Published by The Company of Biologists Ltd
Experimental evidence for the ectodermal origin of the
epithelial anlage of the chicken bursa of Fabricius
Nándor Nagy and Imre Oláh*
SUMMARY
The bursa of Fabricius (BF) is a central lymphoid organ of birds responsible for B-cell maturation within bursal follicles of
epithelial origin. Despite the fundamental importance of the BF to the birth of B lymphocytes in the immune system, the
embryological origin of the epithelial component of the BF remains unknown. The BF arises in the tail bud, caudal to the cloaca
and in close association with the cloacal membrane, where the anal invagination (anal sinus) of ectoderm and the caudal
endodermal wall of the cloaca are juxtaposed. Serial semi-thin sections of the tail bud show that the anal sinus gradually
transforms into the bursal duct and proctodeum, which joins the distal part of the cloaca during late embryogenesis. These
anatomical findings raise the possibility that the ectoderm may contribute to the epithelial anlage of the BF. The expression of
sonic hedgehog and its receptor in the embryonic gut, but not in the BF, further supports an ectodermal origin for the bursal
rudiment. Using chick-quail chimeras, quail tail bud ectoderm was homotopically transplanted into ectoderm-ablated chick,
resulting in quail-derived bursal follicle formation. Chimeric bursal anlagen were generated in vitro by recombining chick bursal
mesenchyme with quail ectoderm or endoderm and grafting the recombination into the chick coelomic cavity. After
hematopoietic cell colonization, bursal follicles formed only in grafts containing BF mesenchyme and tail bud ectoderm. These
results strongly support the central role of the ectoderm in the development of the bursal epithelium and hence in the
maturation of B lymphocytes.
INTRODUCTION
The secondary lymphoid organs develop from the mesoderm, but
they communicate with the environment via ectoderm and
endoderm. The primary lymphoid organs, such as the thymus and
bursa of Fabricius (BF) of birds, develop in genetically determined
locations, where the ectoderm and endoderm are juxtaposed at the
branchial pouches and cloacal membrane, respectively. The
functional framework for both primary lymphoid organs is the
lympho-epithelial (LE) tissue, in which the stellate-shaped
epithelial cells form a 3D meshwork. This structure creates a
micro-environment for T- and B-cell maturation (Glick, 1985;
Glick and Oláh, 1993; Cooper et al., 2006). The epithelial anlage
of the thymic primordium is of endodermal origin (Jolly, 1915;
Hammond, 1954; Le Douarin and Jotereau, 1975; Gordon et al.,
2004) and completely separated from the body surface. By contrast,
the BF is connected via the bursal duct to the third portion of the
cloaca: the proctodeum (Boyden, 1922; Romanoff, 1960; Oláh et
al., 1986).
In mammals, the cloaca partitions into a ventral urogenital sinus
and a dorsal anal canal. In birds, the cranio-caudal differentiation
of the cloaca results in cranial coprodeum (continuation of the
hindgut endoderm) and caudal urodeum, where the ureters enter.
The third part of the cloaca is the proctodeum, which receives the
bursal duct and develops from the ectodermal anal invagination.
Department of Human Morphology and Developmental Biology, Faculty of
Medicine, Semmelweis University, Tüzoltó str. 58, 1094 Budapest, Hungary.
*Author for correspondence ([email protected])
Accepted 27 June 2010
The proctodeum joins the urodeum in late embryogenesis: at E17.
These anatomical connections raise the possibility of an ectodermal
origin for the bursal epithelial primordium (Retterer, 1885; Minot,
1900; Oláh et al., 1986), but experimental evidence for this has
been lacking.
In adult birds, the BF, a chestnut-sized organ between the cloaca
and the sacrum, is the site of B-lymphocyte differentiation (Glick,
1985; Glick, 1991). Bursal differentiation can be staged as follows:
(1) appearance of the pre-programmed epithelial anlage in the tail
bud mesenchyme around E4.5-E5; (2) follicle formation, when
hematopoietic cells enter the bursal mesenchyme and subsequently
the surface epithelium, establishing LE tissue between E11 and
E14; (3) follicle-associated epithelium differentiation at E15; and
(4) development of cortical region after hatching. This study
focuses on the first two stages of bursal development. These are
crucial stages because the functional activity of the BF, namely Bcell differentiation, requires formation of bursal follicles and LE
tissue. The bursal epithelial primordium is generally believed to be
a cloacal diverticulum of endodermal origin (Wenkebach, 1888;
Jolly, 1915; Boyden, 1922; Pera, 1958; Miller and Briglin, 1996).
The vacuolization of the cloacal plate endoderm has been
postulated to give rise to the epithelial rudiment (Boyden, 1922;
Miller and Briglin, 1996). However, the pluripotent mesenchymal
cell mass (rest of Hensen’s node) of the tail bud is confluent with
the overlying ectodermal and underlying endodermal cells
(Schoenwolf, 1979; Schoenwolf, 1981), which makes the origin of
the bursal epithelial primordium uncertain.
The purpose of this study was to determine the origin of the
bursal epithelial anlage. Using chick-quail chimeras and tissue
recombination, we provide experimental evidence for the
ectodermal origin of the bursal epithelial primordium. This finding
contributes to our understanding of the micro-environment that
influences B lymphocyte differentiation.
DEVELOPMENT
KEY WORDS: Bursa of Fabricius, Ectoderm, Chicken, Chimera, Tail bud
3020 RESEARCH REPORT
Development 137 (18)
MATERIALS AND METHODS
Animals
Fertilized White Leghorn chicken and quail (Coturnix coturnix japonica)
eggs were obtained from commercial breeders and maintained at 37°C in
a humidified incubator. Embryos were staged according to Hamburger and
Hamilton (HH) tables (Hamburger and Hamilton, 1992) or the number of
embryonic days (E). The design and condition of the animal experiments
were approved by the Animal Ethical Committee of Semmelweis
University, Budapest, Hungary.
Histological procedures
The tissue samples were fixed in 4% buffered glutaraldehyde and
embedded in Polybed/Araldite 6500 mixture (Polyscience, Warrington,
PA). The 1 m semi-thin sections were stained by Toluidine Blue.
Immunocytochemistry
Avian embryos and the coelomic grafts were fixed in 4%
paraformaldehyde, embedded in gelatin and 10 m frozen sections placed
onto poly-L-lysine coated slides (Sigma-Aldrich, Hungary). The primary
antibodies are listed in Table 1.
Sections were incubated with primary antibodies for 45 minutes,
followed by biotinylated goat anti-mouse IgG (Vector Labs, Burlingame,
CA) and avidin-biotinylated peroxidase complex (Vectastain Elite ABC kit,
Vector Labs). Endogenous peroxidase activity was quenched with 3%
hydrogen peroxide (Sigma) for 10 minutes. The binding sites of the
primary antibodies were visualized by 4-chloro-1-naphthol (Sigma).
For double-immunofluorescence, sections were incubated with primary
and fluorescent-labeled secondary antibodies: anti-mouse IgG Alexa Fluor
594, anti-mouse IgG2a Alexa Fluor 488; anti-mouse IgG1 Alexa Fluor 594
(Molecular Probes); and anti-mouse IgG Pacific Blue (Jackson
ImmunoResearch). Images were compiled using Adobe Photoshop.
In situ hybridization
In situ hybridization was performed for chick sonic hedgehog (Shh) and
Ptc1 on 12 m frozen sections using digoxigenin-labeled riboprobes
[plasmids provided by Cliff Tabin (Riddle et al., 1993; Roberts et al.,
1998)]. Riboprobe synthesis and in situ hybridization were performed as
previously described (Nagy and Goldstein, 2006).
Ablation of tail bud ectoderm
The tail bud ectoderm of 15-16 HH stage (E2.5) chick embryos was
labeled with 2% Nile Blue Sulfate in PBS and the epithelium removed with
a tungsten needle. Control embryos received only Nile Blue Sulphate
solution. Embryos were collected at 8 days after surgery. Bursal epithelial
primordium was labeled with anti-cytokeratin staining and hematopoietic
cell colonization was marked with CD45 monoclonal antibody.
Chick-quail tail bud chimera
To clarify the contribution of ectoderm to BF, quail tail bud ectoderm was
grafted in ovo homotopically to chick embryo after tail bud ectoderm
ablation. The operation was performed on HH stages 14-15 embryos,
caudal to the 24-25th somites. The quail (donor) embryo was isolated from
the yolk and the tail bud ectoderm with the associated mesoderm removed
with tungsten needle. Microsurgery of the chick embryo (host) was
performed in ovo. After windowing the eggshell and the soft shell
membrane, a slit was made on the vitelline membrane covering the
prospective tail bud region. The tail bud ectoderm, neural tube and the
associated somatopleural mesoderm were discarded and the quail tail bud
ectoderm, with the neural tube and mesoderm, was transferred to the chick
embryo (see Fig. S1 in the supplementary material). After careful
orientation of the graft, the chimeric embryos were incubated for a further
12 days.
In vitro recombination of chicken bursal mesenchyme with quail
ectoderm and endoderm
Chick BF is colonized by hematopoietic cells at E9-E10. We therefore
selected E8 bursal mesenchyme for tissue recombination. Tail bud
ectoderm and endoderm from 15 HH stage, trunk ectoderm from 10 HH
stage, hindgut endoderm from 27 HH stage and E8 bursa epithelium of
quail embryos were recombined with E8 chick bursal mesenchyme. For
separation of epithelium from mesenchyme, tissues were incubated in
DMEM containing 0.03% collagenase (Sigma) at 37°C for 15 minutes.
After enzymatic digestion, tissues were extensively washed with DMEM
containing 10% fetal calf serum (Gibco), 5% chicken serum (Sigma),
glutamine, non-essential amino acids, 100 U/ml penicillin G and 0.1 mg/ml
streptomycin mixture to inactivate the collagenase activity. The in vitro
tissue recombinants were embedded into a three-dimensional collagen gel
matrix (BD Bioscience) described previously (Nagy and Goldstein, 2006).
After overnight incubation, the recombinant chimeric epithelialmesenchymal anlage was removed from the collagen gel and
immunocytochemically checked for chicken and quail cells by 8F3 and
QCPN mAb, respectively. The recombinant tissue was implanted into E3
(HH19 stage) chick coelomic cavity (Nagy et al., 2004). During this
incubation, chick hematopoietic cells colonized the implanted ‘artificially’
produced bursal epithelial-mesenchymal primordium. After 14 days of
incubation, the grafts were removed and checked for bursal follicle and LE
formation. These studies included a total of 69 chimeric experiments
comprising three separate series.
Table 1. Primary antibodies
Antibody specificity
Species specificity
Antibody
Source
Quail cells
Chicken cells
Hematopoietic and
endothelial cells
Hematopoietic cells
Unknown nuclear antigen
Unknown antigen
-Macroglobulin
Quail
Chick
Quail
Clone QCPN (mouse IgG1)
Clone 8F3 (mouse IgG1)
Clone QH1 (mouse IgG1)
DSHB
DSHB
DSHB
CD45
Chick
Clone HISC7 (mouse IgG2a)
Dendritic cells
Unknown antigen
Chick
Clone 74.3 (mouse IgG1)
Antigen presenting
cells
B lymphocytes
B lymphocytes
B lymphocytes
B lymphocytes
Epithelial cells
Epithelial cells
Endoderm
MHC II
Chick
Clone TAP1 (mouse)
Cedi Diagnostics,
The Netherlands
Cedi Diagnostics,
The Netherlands
DSHB
IgM
Bu-1 antigen
Bu-1a alloantigen
Bu-1b alloantigen
E-cadherin
Pan-cytokeratin
Sonic hedgehog
Chick
Chick/quail
Quail
Chick
Chick/quail
Chick/quail
Chick/quail
Clone M1 (mouse IgG2b)
Clone BoA1 (mouse IgG1)
Clone L22 (mouse IgG1)
Clone 11G2 (mouse IgG1)
Clone 36 (mouse IgG2a)
Clone Lu5 (mouse IgG1)
Clone 5E1 (mouse IgG1)
Southern Biotech
Igyarto et al., 2008
Santa Cruz Biotechnology
Southern Biotech
BD Biosciences
BMA, Biomedicals AG
DSHB
DSHB, Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained by University of Iowa, Dept of Biological Sciences, Iowa
City, IA 52242, USA).
DEVELOPMENT
Cells/structures identified
Development of the bursa of Fabricius
RESEARCH REPORT 3021
Fig. 1. Formation of the bursal duct and proctodeum. Sagittal
sections of the tail region from E5 (A,C,D) and E8 (B) chicken embryos.
(A)The anal sinus (As) is separated from the cloaca (Cl) by the cloacal
membrane (cm). (B)The bursal duct (Bd) is established between the
bursal lumen and cloacal membrane and caudal to the cloacal
membrane, and the proctodeum (Pr) is formed between the dorsal
(dap) and ventral anal lips (vap). (C-F)Shh immunocytochemistry (C,D)
and in situ hybridization of Shh (E) and Ptc1 genes (F). (C)Sagittal
section of the E5 tail bud. The boxed area is magnified in D. The strong
Shh immunoreactivity in the vacuolized endoderm is clearly demarcated
from the ectoderm (broken line). (E)Shh gene expression in the hindgut
and cloacal membrane but not in the bursa and bursal duct. (F)Ptc1
expression in the subepithelial mesenchyme of the distal hindgut but
not in the bursa. hg, hindgut; nt, neural tube.
RESULTS AND DISCUSSION
Formation of the proctodeum, bursal duct and
epithelial anlage of the BF
The epithelial rudiment of the BF emerges in the tail bud
mesenchyme at around E4-E5 days. In the E5 chicken embryo, the
thickened cloacal membrane (anal plate) is located between the
anal sinus and cloaca (Fig. 1A). By E8, the anal sinus transforms
into cranial and caudal portions from which the bursal duct and the
proctodeum, respectively, develop (Fig. 1B). At this stage, the
bursal epithelial anlage is a vesicle-like structure and the duct is
still separated from the cloaca by the cloacal membrane (Fig. 1B).
During the next developmental stage, the immigrant CD45+
hematopoietic cells colonize the epithelial-mesenchymal anlage of
the BF, which results in LE follicle formation, and subsequently Blymphocyte maturation (Nagy et al., 2004).
Sonic hedgehog (Shh) is expressed universally by the whole
digestive endoderm, including the cloaca, but not the ectoderm
(Roberts et al., 1995; Narita et al., 1998; Liu et al., 2007).
Therefore, we examined the tail bud region and cloacal membrane
for endoderm specific Shh protein and for expression of Shh and
its Ptc1 receptor transcripts. At E5, the hindgut endoderm and the
caudal wall of the cloaca are immunoreactive for Shh, unlike the
ectoderm-derived anal sinus (Fig. 1C). Shh transcript is expressed
in the hindgut and endodermal part of the cloacal epithelium (Fig.
1D), but not in bursal duct and epithelium (Fig. 1E). Ptc1 is present
in the mesenchyme of the cloaca, but not in the bursal mesenchyme
(Fig. 1F). These expression patterns support the hypothesis that the
bursal primordium may arise from the ectoderm, not the endoderm.
The vacuolization of the endodermal part of the cloacal plate was
the strongest evidence for the endodermal origin of the BF.
However, unlike the bursal duct and epithelium, these vacuoles
express Shh (Fig. 1D). In addition, the vacuolization does not
explain the origin of the bursal duct.
In order to study the contribution of tail ectoderm to the bursal
epithelium, first we removed microsurgically the tail bud ectoderm
of 16 HH chicken embryo. By E10, the anal invagination of control
birds is clearly transformed to a proximal bursal duct and distal
proctodeum (Fig. 2A) and the hematopoietic cells accumulate in
the most proximal side of the bursal cavity (Fig. 2B). Eight days
after microsurgery in ectoderm-ablated embryos, there is no sign
of bursal duct and BF development (Fig. 2C), although CD45+
hematopoietic cells immigrated into the tail bud, but they remained
homogeneously scattered throughout the mesenchyme (Fig. 2D).
The ablation of tail bud ectoderm abolished the bursal duct and
follicle formation. In ectoderm-ablated birds, the hematopoietic
cells are evenly scattered over the tail bud mesenchyme, whereas
in the control birds the hematopoietic cells are clustered at the most
cranial part of the bursal primordium, indicating some
chemoattractant effect of the bursal epithelial anlage.
DEVELOPMENT
Fig. 2. Tail bud ectoderm ablation. (A)In control bird, the bursal
lumen (Bl) and bursal duct (Bd) as well as the proctodeum (Pr) are well
developed. (C)Cloacal lumen (cl) is formed, but no sign for bursal
anlage in the ablated embryo. (B)In control embryos, CD45+
hematopoietic cells are scattered over the mesenchyme but they are
accumulated at the most proximal part of the bursa (outlined). (D)In
the ablated embryos, the CD45+ cells enter the tail bud mesenchyme,
but they do not accumulate at a specific site.
Fig. 3. Chick-quail tail bud chimera. (A)Inset shows the tail bud
chimeric embryo at day 14. The presence of pigmented and
nonpigmented feathers covers the quail and chick derivatives,
respectively. Sagittal section (white line) made from the chimeric tail
bud is shows the QCPN+ quail cells (red) forming the surface
epithelium, the bursal epithelial anlage and the mesenchyme under the
skin. (B)Cytokeratin staining confirms the epithelium (blue). (C)Chicken
CD45+ hematopoietic cells (green) are scattered over the mesenchyme
and enter the epithelium (blue). (D)Another combination of
immunostaining confirms that the quail epithelial bud (red, QCPN) is
colonized by chicken hematopoietic cells (green, CD45). (E-I)The
developing follicle contains phenotypically functional, Bu1b+/IgMproducing B lymphocytes (E,F); (G) bursal secretory dendritic cells (74.3);
and (H) MHC class II+ cells. However, granulocytes (Grl-1) appear only
in the mesenchyme (I). Epithelial bud is outlined.
Development 137 (18)
Chicken-quail tail bud chimera and in vitro tissue
recombination of chicken bursal mesenchyme
with quail epithelium
In recombination experiments, the chick tail bud ectoderm was
replaced by age-matched quail ectoderm, including the
mesenchyme and neural tube (see Fig. S1 in the supplementary
material). The chimeric embryos contain quail ectoderm, chick
endoderm and hematopoietic cells, as well as mixed (quail and
chick) mesoderm.
QCPN and cytokeratin immunostainings of the tail bud region
indicate that the skin epithelium and the anal invagination with the
primordium of the BF and part of the dorsal mesenchyme are
derived from the transplanted quail ectoderm (Fig. 3A,B). Double
staining of cytokeratin and CD45 indicates that chick
hematopoietic cells enter the quail epithelium and initiate bud
formation (Fig. 3C). These buds represent true tissue chimerism:
the chick CD45+ cells colonize the quail-derived epithelium (Fig.
3C,D). The LE tissue of buds was further analyzed for
differentiation of hematopoietic cells by using markers for B
lymphocyte (Bu-1), immunoglobulin (IgM), chicken dendritic cell
(74.3), MHC class II and granulocyte (Grl-1) (Fig. 3E,F,G,H,I).
These chick-specific markers confirmed the formation of functional
bursal follicles in these chimeras.
The next series of chimeric experiments were carried out to obtain
information about the signaling role of the endoderm. In a pilot
experiment, bursal follicle formation was tested by combining E8
chicken bursal mesenchyme with E8 quail bursal epithelium (see
Fig. S2A,B,C in the supplementary material). The recombinant tissue
(‘artificial’ bursal epithelial-mesenchymal anlage) was implanted into
E3 chicken coelom for hematopoietic cell colonization and follicle
formation. After 14 days of incubation, QCPN and 8F3 mAb
stainings clearly indicate that the epithelium and mesenchyme
including the hematopoietic cells of the BF are quail and chicken
derived, respectively (see Fig. S2D,E,F in the supplementary
material). These experiments provided evidence that chimeras are
able to form follicles and LE tissue. Furthermore, the cells of the
follicles express chick-specific antigens, such as Bu-1b, 74.3 and
MHC class II (see Fig. S2G,H,I in the supplementary material).
Fig. 4. Tissue recombination of chicken bursal
mesenchyme with different quail epithelium. In vitro
recombination of chicken bursal mesenchyme with
(A) quail tail bud endoderm, (B) quail tail bud ectoderm,
(C) quail trunk ectoderm and (D) hindgut endoderm. The
recombinant tissue was implanted in the chick embryo
coelomic cavity for hematopoietic cell colonization. QCPN
shows that bursal epithelium is of quail origin, and the
8F3 and CD45 staining prove that the mesenchyme and
hematopoietic cells are chick derived. Bu-1
immunostaining indicates the B lymphocytes. (B) Chicken
mesenchyme is capable of follicle formation only with
quail tail bud ectoderm.
DEVELOPMENT
3022 RESEARCH REPORT
In the next series of experiments, chick bursal mesenchyme was
combined with either quail ectoderm or endoderm to determine
which epithelial layer supports follicle formation (Fig. 4). In every
tissue recombination, the ‘artificial’ bursal anlagen were capable
of receiving CD45+ hematopoietic cells. Bu-1-positive B
lymphocytes were present in all varieties of tissue recombinations,
indicating that early B cell differentiation can take place without
bursal micro-environment. However, follicle formation happened
only if the chick bursal mesenchyme was recombined with quail
tail bud ectoderm (Fig. 4B). In these chimeric experiments, the
transplanted ectoderm was removed well before the formation of
the anal plate; thus, endodermal instruction to ectodermal follicle
formation is unlikely. Our findings obtained from the chimeric
recombination experiments are reminiscent of those of Gordon et
al. (Gordon et al., 2004) in stating that the thymic epithelial anlage
does not require ectodermal signaling as the third branchial pouch
can form functionally active thymic tissue without ectoderm.
Similarly, tail bud ectoderm can form bursal follicles without
endodermal contribution.
In birds, antigen-specific IgG production requires bursal LE
tissue micro-environment. In the majority of mammals, B-cell
maturation takes place in the bone marrow (Abdou and Abdou,
1972) or in the Peyer’s patches of ruminants (Reynaud et al., 1991),
which are so-called bursa equivalent organs. Bone marrow
histologically is not comparable with the LE tissue of BF. It seems
likely that the B-lymphocyte maturation is not connected to a
histologically determined structure, unlike T cells, which
exclusively develop in the thymic LE tissue. If the bursa-equivalent
organs of mammals are histologically not comparable with the LE
tissue of BF, but the function of the immune system of birds and
mammals is the same, it may be assumed that a ‘bursal function
equivalent stromal cell’ exists, which establishes the microenvironment for B cell differentiation. In jawed vertebrates, the
dichotomous nature of lymphocytes (T and B cells are responsible
for cellular and humoral immunity of adaptive immune response,
respectively) is well established. While T cells develop exclusively
in the endodermal-derived thymus, B lymphopoiesis occurs in
different anatomical locations of vertebrate classes, i.e. pronephros,
liver, spleen and gut-associated lymphoid tissue. In birds, the BF is
a unique evolutionary invention for B-cell maturation. Thus, T- and
B-lymphocyte maturation is orchestrated by endodermal and
ectodermal-derived micro-environments, respectively.
Acknowledgements
We thank Allan M. Goldstein for helpful discussion and editing the
manuscript; and Jutka Fügedi and Zsuzsa Vidra for technical assistance. This
work was supported by OTKA grant number 69061.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.055194/-/DC1
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DEVELOPMENT
Development of the bursa of Fabricius