Arch. histol. jap., Vol. 37, No. 4 (1975) p. 291-299 Department of Veterinary Anatomy (Prof.M. MISU), Obihiro Universityof Agriculture and Veterinary Medicine,Obihiro* and Department of Veterinary Anatomy (Prof.N. KUDO), Faculty of Veterinary Medicine, Hokkaido University,Sapporo,** Japan
Mesenchymal
Origin of Bursal Lymphoid in Japanese Quails
Cells
Junzo YAMADA, * Makoto SUGIMURA ** and Norio KUDO ** Received
November
19,
1974
Summary. The origin of bursal lymphoid cells in Japanese quails of a quantitative morphological method during a 6 to 12 day incubation obtained in this study are summarized as follows. At 6 to 7 days' In 8 day embryonic
incubation, no lymphoid bursa, the first appearance
was studied by means period. The findings
cells were observed in the bursal of lymphoid cells was found
primordium. in the tunica
mucosa. In the epithelium, the first appearance of lymphoid cells was observed at 9.5 days' incubation. At 9.5 to 11 days' incubation, lymphoid cells were frequently observed to pass through the epithelial basement membrane. It may be concluded that the lymphoid cells found in the epithelial formation of epithelial
region had migrated from buds began at about 11 days'
to penetration of lymphoid cells into the It may be concluded from the present mesenchymal cells in Japanese quails.
The bursa of Fabricius
the bursal incubation,
epithelium. results that
is a cloacal lymphoid
bursal
mesenchymal region. but were never found lymphoid
cells
derive
organ unique to the Ayes.
The prior from
Lym-
phocytopoiesis of the bursa of Fabricius has been examined by many investigators, but since controversy continues as to whether bursal lymphocytes originate from the mesenchymal, or from the epithelial, or whether they have a dual origin (JOLLY, 1915; ACKERMAN
and KNOUFF,
1959; MOORE
and
OWEN,
1965, 1966; ARAMAKI,
1968;
LEENE, DUYZINGSand VAN STEEG, 1973), the origin of bursal lymphocytes is still a matter for further investigation. Many papers exist on bursal lymphocytopoiesis in chickens, but little work has been done in this area using Japanese quails (KOBAYASHI, 1968; ARAMAKI, 1968). The purposes of this study are to decide the site and time of the first appearance of lymphoid cells in the quails' bursa, and to estimate the origin of bursal lymphoid cells by means of a quantitative morphological method. Materials
and Methods
The bursa of Fabricius in the embryos of 84 Japanese quails, at 6 to 12 days incubation (120 and 264hrs after incubation respectively) were examined as materials. The specimens for light microscopy were fixed with Bouin's fluid and embedded in paraffine.
Serial
sections
cut
4 to 6μ were
stained
with
hematoxyline-eosin,
periodic
acid-Schiff and toluidine blue (pH 7.4). The specimens for electron microscopy were fixed with either 1% OsO4 only, or with 2.5% glutaraldehyde and 1% OsO4 in Millonig's buffer or in 0.1M cacodylate buffer. The fixed tissues were dehydrated in a graded series of ethanol-aceton and 291
292
J. YAMADA, M. SUGIMURA and N. KUDO:
embedded bedded
in epoxy
in resins,
resins.
1μ sections
For light were
cut
microscopical
with
glass
knives
observation on
an
of specimens
ultramicrotome
em-
(Porter-
Blum MT-1) and stained with toluidine blue or basic fuchsin-methylene blue. For electron microscopic investigations, ultrathin sections were cut with glass knives and stained with uranyl acetate and lead citrate, and then examined under a JEM-7 type electron microscope. For the purposes of this study, the number of lymphoid cells were counted in the tunica mucosa (mesenchymal region) and the epithelium of the bursa. The bursa was photographed by a light microscope. The negative films were projected on a screen and the bursal profiles were traced on paper, and measured by use of a planimeter. Then, the number of the lymphoid cells was converted into number per unit area
(100,000μ2).
Results Since the purpose of this study is to decide the site and time of the first appearance of the lymphoid cells in the quails' bursa and to estimate the origin of the bursal lymphoid cells, all cells of the lymphocytopoietic series were, for convenience, given the general term "lymphoid cells." The lymphoid cells, containing a round nucleus with coarse chromatin and one to three prominent nucleoli and having strongly basophilic cytoplasm, were clearly distinguished from neighbouring fixed mesenchymal cells and bursal epithelial cells under a light microscope. In electron microscopy, the lymphoid cells showed one to three prominent nucleoli, distinct marginal heterochromatin distribution in the nucleus and numerous polyribosomes in the cytoplasm. In the materials which were fixed with the hypertonic fixatives, the lymphoid cells were distinguished easily from other cells, because they shrank and showed dark profiles. The bursal primordium of the quails' embryo on the 6th day of incubation, the earliest stage investigated, was recognized as an epithelial mass showing a slightly Table
±: standard
error,
():
range
1.
Distributions
of lymphoid
cells
Mesenchymal
Fig. 1.
Six day embryo.
Origin of Bursal
Lymphoid
cells blue
present stain.
Lymphoid
Cells
in the posterior
293
cardinal
veins.
Toluidine
×500
Fig. 2. Eight day embryo. Lymhpoid cells present around the posterior cardinal veins. Note one lymphoid cell in the marginal portion of the bursal region (arrow). Toluidine blue stain. ×250
rounded protuberance of the urodaeal caudal membrane. No lymphoid cells occurred in and around the bursal primordium, but the lymphoid cells were found in small numbers in the posterior cardinal veins (Fig. 1). At 7 days' incubation, the bursal primordium were enlarged in size. The lymphoid cells occurred in and around the posterior cardinal veins, but were not detected in the region of the bursal primordium. In 8 day embryos, the bursa represented an oval sac located between the cloaca and the vertebrates. At this stage, only a small number of lymphoid cells were observed in the bursal tunica mucosa, but never found in the bursal epithelium (Fig. 2). The number of the lymphoid cells per unit area is shown in Table 1. At this stage, the
number
of the
lymphoid
cells
in the
tunica
mucosa
was
3.5±0.7,
The
same
cells
were observed in and around the posterior cardinal veins (Fig. 2). At 9 days' incubation, the bursal lumen was enlarged in size. The bursal wall was composed of a loose mesenchymal connective tissue covered by a stratified or cuboidal epithelium. The longitudinal folds and muscular layer were not clear. A larger number of lymphoid cells
(8.5±4.0)
were
observed
in
the
tunica
mucosa
than
at 8 days'incubation,
but
none were found in the epithelium at this stage (Fig. 3 and Table 1). In 9.5 day embryos, the first appearance of the lymphoid cells in the epithelium was
observed
in 4 of 7 materials
and
their
mean
number
was
6.2±5.9
(Table
1).
The
lymphoid cells in the epithelium were found mostly near the basement membrane or passing through it and the same cells were recognized in the tunica mucosa (Fig. 4).
294
Fig.
J. YAMADA, M. SUGIMURA and N. KUDO:
3.
Nine day embryo.
Some lymphoid
cells in the bursal tunica mucosa.
stain.
Toluidine
blue
×500
Fig. 4. Nine and half day embryo. Note one lymphoid cell found in the bursal epithelium. Some lymphoid cells are in the subepithelial layer and appear to be passing through the basement membrane. Many similar cells are observed in the tunica mucosa. Basic fuchcin-methylene
blue
stain.
×500
Figs. 5 and 6. Ten day embryo. Lymphoid cells can be seen with a cytoplasmic extension passing through the basement membrane into epithelium. Toluidine blue stain (Fig. 5). Basic fuchcin-methylene
blue
stain
(Fig.
6).
×1200
Mesenchymal
Fig. 7. (Bm)
Origin of Bursal Lymphoid
Cells
295
Ten day embryo. Lymphoid cells (L) migrating into the epithelium.
is interrupted
by
a penetrating
lymphoid
cell.
E epithelial
cell,
M
Basement membrane
mesenchymal
cell.
×5,000
At 10 days' incubation, the lymphoid cells were observed in the epithelium and the tunica mucosa (Fig. 5-7), but no lymphoid cells were found in the epithelium in 2 of 11 materials. The mean number of the lymphoid cells in the tunica mucosa was larger than that in the epithelium (Table 1). The epithelial bud was not observed in this stage. The lymphoid cells which were constricted into hand-mirror and dumb-bell shapes, were frequently observed to pass through the basement membrane (Fig. 5-7). In 11 day embryos, the lymphoid cells rapidly increased in number in the epithelium. The mean number of the lymphoid cells in the epithelium was larger than that in the tunica mucosa (Table 1). The epithelial buds were first recognized as protrusions from the surface epithelium into the tunica mucosa in 5 of 11 materials (Fig. 8). They consisted of undifferentiated epithelial cells and of lymphoid cells. At 12 days' incubation (Fig. 9, 10), the epithelial buds were recognized in all materials, having increased in size and number over that on the 11th day of incubation. The lymphoid cells were rapidly increased in the epithelium with the epithelial buds formation. Between 10 and 12 days' incubation, the lymphoid cells in the tunica mucosa increased in the
slowly
epithelium
to 144.2±67.1
in
number
(and (Table
the 1).
from epithelial
14.3±6.5 buds)
to
57.6±20.3,
increased
rapidly
whereas
the
in number
lymphoid from
cells 11.1±8.6
296
J. YAMADA, M. SUGIMURA and N. KUDO:
Mesenchymal
Origin of Bursal
Lymphoid
Cells
297
Discussion
There is no doubt that the bursa of Fabricius of the Ayes is a lymphocytopoietic organ which mainly consists of epithelial reticular cells and of lymphoid cells. However there are considerable disagreements in respect to the origin of bursal lymphocytes. It is not yet determined whether they are derived from the epithelial cells or the mesenchymal cells. The classical and extensive observation of JOLLY (1915) on bursal development laid the foundation for the theory of mesenchymal origin of bursal lymphocytes. ORTEGA, KATTINE and SPURLOCK (1965), NAKAGAWA (1969)and LEENE, DUYZINGS and VANSTEED (1973) also considered that bursal lymphocytopoiesis initiated from the migration of mesenchymal lymphoid cells into the epithelial buds. MOOREand OWEN (1965, 1966) concluded by several unique methods (chromosome marker and parabiosis techniques), that the lymphocytic transformation in the epithelial buds took place by immigration of lymphocytic cells derived from undifferentiated mesenchymal cells in the tunica propria or from other unknown sites. Most other investigators(FORBES, 1877; STIEDA, 1880; WENCKBACH, 1893; RETTERER and LELIEVRE,1913a, b) believed that epithelial buds developed from the lining epithelium of the bursa and transformed into typical lymphoid follicles. Thus they believed in the transformation and differentiation of epithelial cells into lymphocytes. This concept was revived by ACKERMAN and KNOUFF (1959), ACKERMAN (1962), TAKAGI (1967), ICHIMURA (1967) and BOSCH (1968). JAFFE and FECHHEIMER (1960) and ARAMAKI (1968),On the other hand, concluded that bursal lymphocytes are dual in origin arising from undifferentiated epithelial cells and from mesenchymal cells. In order to solve this problem by means of a quantitative morphological method, it seemed important, firstly to decide the critical stage in which the lymphoid cells begin to appear in the bursal tunica mucosa or in the bursal epithelium, secondly to determine the time when the epithelial bud formation begins and thirdly, to estimate the number of the lymphoid cells in the epithelium and in the tunica mucosa per unit area at different developmental stages. Analyzing the results obtained the present authors could reach the clear conclusion that the bursal lymphoid cells are of mesenchymal rather than epithelial origin. In their study on the bursa of Fabricius in the chicken, ACKERMAN and KNOUFF (1959) reported: "These blast cells appear to be formed intraepithelially and the possibility of their arising from mesenchymal cells in the tunica propria and migrating Fig. 8.
Eleven day embryo. Epithelial buds are seen as protuberances of the epithelium. Lymphoid cells are in the epithelial region and tunica mucosa (especially in the subepithelial layer). They are readily identified by their basophilic cytoplasm. Toluidine blue stain.
Fig. 9.
Twelve
day embryo.
also
increased
×500 are
in
Lymphoid size.
cells are increased
Toluidine
blue
stain.
in number
in the epithelial
buds which
×500
Fig. 10. Twelve day embryo. Early epithelial bud formation. Lymphoid cells (L) of dark appearance are present. Note a lymphoid cell migrating into the epithelial bud, and cells of the same nature around the bud. These lymphoid cells are identified easily by a shrinkage artifact caused by the hypertonic fixative (2.5% glutaraldehyde and 1% OsO4 in 0.1M cacodylate) used in this specimen. E epithelial cell, M mesenchymal cell, Bm basement membrane. ×5,000.
298
J. YAMADA, M. SUGIMURA and N. KUDO:
into follicle seems highly unlikely from our observations since similar cells are rarely seen out side of the epithelial bud during this period of bursal development." The authors' findings are diametrically opposed to their findings. The lymphoid cells were first found in the tunica mucosa at 8 days' incubation. The first appearance of the lymphoid cells in the epithelium was 1.5 days after that in the tunica mucosa. The epithelial buds began to form at about 11 days' incubation, and were never observed prior to penetration of the lymphoid cells into the bursal epithelium. At 9.5 to 11 days' incubation, the lymphoid cells were frequently observed to pass through the basement membrane. These cells showed handmirror or dumb-bell cell body and nucleus shapes. From the findings described above, the direction of the cell movement is believed to be from the mesenchymal to the epithelial region. In the Japanese quails, lymphocytopoiesis of the bursa of Fabricius has been
studied by only two investigators (KOBAYASHI, 1968; ARAMAKI, 1968). ARAMAKI (1968) has, however, investigated only one embryonic stage (15 days' incubation), and KOBAYASHI's (1968) description is brief and lacks photographs. KOBAYASHI (1968) has indicated that the epithelial buds appeared at 13 days' incubation, and that large lymphocytes appeared at about 15 days' incubation. As his description isbasophilic insufficient, it is difficult to be discussed in comparison with the present findings. ACKERMANand KNOUFF(1963) demonstrated a relatively intense alkaline phosphatase activity in the subjacent compact mesenchyme during the phase of bud formation, which subsequently declines with the maturation of follicles. They concluded that this metabolic change may reflect an epithelial-mesenchymal inductive interaction involved in the epithelial bud formation. ICHIMURA (1967) objected to the inductive function of this enzyme, and the authors (unpublished data) never observed a specific distribution of this enzyme in Japanese quails. Penetration of the lymphoid cells into the bursal epithelium may rather represent an inducing factor involved in the epithelial bud formation. The present results support neither the concept of transformation of the bursal epithelialcellsinto the lymphoid cellsby earlierinvestigators(FORBES, 1877; STIEDA, 1880; WENCKBACH, 1893; RETTERER and LELIEVRE, 1913a,b; ACKERMAN and KNOUFF, 1959; ACKERMAN, 1962; TAKAGI, 1967; ICHIMURA, 1967; BOSCH, 1968),nor the dual origin of the lymphoid cells (JAFFE and FECHHEIMER, 1966; ARAMAKI, 1968). The present results indicate that the bursal lymphoid cells derive from the mesenchymal cells in Japanese quails. Further investigations are need to determine more precisely the site of origin of the lymphoid cells in the earliest stage of bursal development.
ウ ズ ラ の フ ァブ リキ ウス 嚢 にお け る リンパ 球 様 細 胞 の 間 葉 由来 に つ い て 山 田 純 三, 杉 村 孵卵 後6日 て,
誠, 工 藤 規 雄
目 (120時 間) か ら12日 目の ウ ズ ラ胚 (84例)
の フ ァブ リキ ウ ス嚢 に お い
リンパ 球 様 細 胞 の 由 来 を計 量 形 態 学 的 に 観 察 した.
6日 目お よ び7日
目の 嚢 原 基 に は リンパ球 様 細 胞 は 認 め られ ず,
8日 目の嚢 固 有 層 に
初 め て, 少 数 の リンパ 球 様 細 胞 が 認 め られ た. 上 皮 に は これ よ り1.5日 遅 れ て, 9.5日
Mesenchymal
Origin of Bursal Lymphoid
Cells
299
目に 初 め て 本 細 胞 が 認 め られ た. 上 皮 蕾 は11日 目頃 に 形 成 さ れ 始 め た.
この 上 皮 蕾 形 成
は リンパ 球 様 細 胞 の 上 皮 へ の 侵 入 に 先 行 して 認 め られ る こ とは な か った.
9.5日 目か ら
11日 目に か け て, 基 底 膜 を 通 過 し て 上 皮 お よ び上 皮 蕾 に侵 入 しつ つ あ る細 胞 が 多 く認 め られ た. 以 上 の 所 見 よ り, ウ ズ ラの フ ァブ リキ ウ ス嚢 に お け る リンパ 球 様 細 胞 の 由来 を上 皮 に 求 め る よ り 間 葉 に 求 め る方 が よ り妥 当 と考 えた.
References Ackerman, G. A.: Electron microscopy of the bursa of Fabricius of the embryonic chick with particular reference to the lymphoepithelial nodules. J. Cell Biol. 13: 127-146 (1962). Ackerman, G. A. and R. A. Knouff: Lymphocytopoiesis in the bursa of Fabricius. Amer. J. Anat. 104: 163-205 (1959). --: Testosterone
suppression
of mesenchymal
alkaline
phosphatase
activity
and
lympho-epithelial nodule formation in the bursa of Fabricius in the embryonic chick. Anat. Rec. 146: 23-27 (1963), Aramaki, T.: Morphologic study of lymphatic tissues (XIII). Light and electron microscope observations on the bursa of Fabricius of chick and coturnix embryos with special reference to lymphocytopoiesis. Keio J. Med. 17: 135-155 (1968). Bosch, J.: Morphogenetische, histochemische und experimentelle Untersuchungen uber die Bursa Fabricii. Zool. Jahrb. Abt. Anat. 85: 327-385 (1968). Forbes, W. A.: On the bursa of Fabricius in birds. Proc. Zool. Soc. London, 1877. (p. 304-318). Ichimura, M.: Embryonic development and histochemical study of the bursa of Fabricius in the embryo of the chickens (in Japanese). J. Keio Med. Soc. 44: 571-582 (1966). Jaffe, W. P. and N. S. Fechheimer: Cell transport and bursa of Fabricius. Nature 212: 92 (1966). Jolly, J.: La bourse de Fabricius et les organes lymphoepitheliaux. Arch. Anat. microsc. 16: 363547 (1915). Kobayashi, T.: Structure and function of bursa of Fabricius (in Japanese). In: Symp. Immunobiol. Tokyo, Maruzen, 1968. (Vol. 1, p. 24-28). Leene, W., M. J. M. Duyzings and C. van Steege: Lymphoid stem cell identification in the developing thymus and bursa of Fabricius of the chick. Z. Zellforsch. 136: 521-533 (1973). Moore, M. A. and J. J. T. Owen: Chromosome marker studies on the development of the haemopoietic system in the chick embryo. Nature 208: 956-990 (1965). --: Experimental
studies
on
the
development
of
the bursa
of Fabricius. Devel.
Biol. 14: 40-51 (1966). Nakagawa, Y.: The early stage of the epithelial bud-formation in the bursa of Fabricius in chick embryo (in Japanese with English summary). Tohoku Igaku Zasshi, 79: 139-146 (1969). Ortega, L. G., A. A. Kattine and B. O. Spurlock: Lympho-epithelial interaction in the developing bursa of Fabricius. Fed. Proc. 24: 160 (1965). Retterer, E. et A. Lelievre: Nouvelles recherches sur la bourse de Fabricius. C. r. Soc. Biol. 74: 182-185 (1913a). --: Homologies de la bourse de Fabricius. C. r. Soc. Biol. 74: 382-385 (1913b). Stieda, L.: Uber den Bau und die Entwicklung der Bursa Fabricii. Z. wiss. Zool. 34: 296-309 (1880). Wenckbach, K. F.: Die Follikel der Bursa Fabricii. Anat. Anz. 11: 159-160 (1893).
山田純 三 〒080北 海道帯広市稲田町 帯広畜産大学獣医学科 家畜解剖学教室
Junzo YAMADA Department of Veterinary Anatomy Obihiro University of Agriculture and Veterinary Medicine Obihiro, Hokkaido, 080 Japan
Mesenchymal
Origin of Bursal Lymphoid in Japanese Quails
Cells
Junzo YAMADA, * Makoto SUGIMURA ** and Norio KUDO ** Received
November
19,
1974
Summary. The origin of bursal lymphoid cells in Japanese quails of a quantitative morphological method during a 6 to 12 day incubation obtained in this study are summarized as follows. At 6 to 7 days' In 8 day embryonic
incubation, no lymphoid bursa, the first appearance
was studied by means period. The findings
cells were observed in the bursal of lymphoid cells was found
primordium. in the tunica
mucosa. In the epithelium, the first appearance of lymphoid cells was observed at 9.5 days' incubation. At 9.5 to 11 days' incubation, lymphoid cells were frequently observed to pass through the epithelial basement membrane. It may be concluded that the lymphoid cells found in the epithelial formation of epithelial
region had migrated from buds began at about 11 days'
to penetration of lymphoid cells into the It may be concluded from the present mesenchymal cells in Japanese quails.
The bursa of Fabricius
the bursal incubation,
epithelium. results that
is a cloacal lymphoid
bursal
mesenchymal region. but were never found lymphoid
cells
derive
organ unique to the Ayes.
The prior from
Lym-
phocytopoiesis of the bursa of Fabricius has been examined by many investigators, but since controversy continues as to whether bursal lymphocytes originate from the mesenchymal, or from the epithelial, or whether they have a dual origin (JOLLY, 1915; ACKERMAN
and KNOUFF,
1959; MOORE
and
OWEN,
1965, 1966; ARAMAKI,
1968;
LEENE, DUYZINGSand VAN STEEG, 1973), the origin of bursal lymphocytes is still a matter for further investigation. Many papers exist on bursal lymphocytopoiesis in chickens, but little work has been done in this area using Japanese quails (KOBAYASHI, 1968; ARAMAKI, 1968). The purposes of this study are to decide the site and time of the first appearance of lymphoid cells in the quails' bursa, and to estimate the origin of bursal lymphoid cells by means of a quantitative morphological method. Materials
and Methods
The bursa of Fabricius in the embryos of 84 Japanese quails, at 6 to 12 days incubation (120 and 264hrs after incubation respectively) were examined as materials. The specimens for light microscopy were fixed with Bouin's fluid and embedded in paraffine.
Serial
sections
cut
4 to 6μ were
stained
with
hematoxyline-eosin,
periodic
acid-Schiff and toluidine blue (pH 7.4). The specimens for electron microscopy were fixed with either 1% OsO4 only, or with 2.5% glutaraldehyde and 1% OsO4 in Millonig's buffer or in 0.1M cacodylate buffer. The fixed tissues were dehydrated in a graded series of ethanol-aceton and 291
292
J. YAMADA, M. SUGIMURA and N. KUDO:
embedded bedded
in epoxy
in resins,
resins.
1μ sections
For light were
cut
microscopical
with
glass
knives
observation on
an
of specimens
ultramicrotome
em-
(Porter-
Blum MT-1) and stained with toluidine blue or basic fuchsin-methylene blue. For electron microscopic investigations, ultrathin sections were cut with glass knives and stained with uranyl acetate and lead citrate, and then examined under a JEM-7 type electron microscope. For the purposes of this study, the number of lymphoid cells were counted in the tunica mucosa (mesenchymal region) and the epithelium of the bursa. The bursa was photographed by a light microscope. The negative films were projected on a screen and the bursal profiles were traced on paper, and measured by use of a planimeter. Then, the number of the lymphoid cells was converted into number per unit area
(100,000μ2).
Results Since the purpose of this study is to decide the site and time of the first appearance of the lymphoid cells in the quails' bursa and to estimate the origin of the bursal lymphoid cells, all cells of the lymphocytopoietic series were, for convenience, given the general term "lymphoid cells." The lymphoid cells, containing a round nucleus with coarse chromatin and one to three prominent nucleoli and having strongly basophilic cytoplasm, were clearly distinguished from neighbouring fixed mesenchymal cells and bursal epithelial cells under a light microscope. In electron microscopy, the lymphoid cells showed one to three prominent nucleoli, distinct marginal heterochromatin distribution in the nucleus and numerous polyribosomes in the cytoplasm. In the materials which were fixed with the hypertonic fixatives, the lymphoid cells were distinguished easily from other cells, because they shrank and showed dark profiles. The bursal primordium of the quails' embryo on the 6th day of incubation, the earliest stage investigated, was recognized as an epithelial mass showing a slightly Table
±: standard
error,
():
range
1.
Distributions
of lymphoid
cells
Mesenchymal
Fig. 1.
Six day embryo.
Origin of Bursal
Lymphoid
cells blue
present stain.
Lymphoid
Cells
in the posterior
293
cardinal
veins.
Toluidine
×500
Fig. 2. Eight day embryo. Lymhpoid cells present around the posterior cardinal veins. Note one lymphoid cell in the marginal portion of the bursal region (arrow). Toluidine blue stain. ×250
rounded protuberance of the urodaeal caudal membrane. No lymphoid cells occurred in and around the bursal primordium, but the lymphoid cells were found in small numbers in the posterior cardinal veins (Fig. 1). At 7 days' incubation, the bursal primordium were enlarged in size. The lymphoid cells occurred in and around the posterior cardinal veins, but were not detected in the region of the bursal primordium. In 8 day embryos, the bursa represented an oval sac located between the cloaca and the vertebrates. At this stage, only a small number of lymphoid cells were observed in the bursal tunica mucosa, but never found in the bursal epithelium (Fig. 2). The number of the lymphoid cells per unit area is shown in Table 1. At this stage, the
number
of the
lymphoid
cells
in the
tunica
mucosa
was
3.5±0.7,
The
same
cells
were observed in and around the posterior cardinal veins (Fig. 2). At 9 days' incubation, the bursal lumen was enlarged in size. The bursal wall was composed of a loose mesenchymal connective tissue covered by a stratified or cuboidal epithelium. The longitudinal folds and muscular layer were not clear. A larger number of lymphoid cells
(8.5±4.0)
were
observed
in
the
tunica
mucosa
than
at 8 days'incubation,
but
none were found in the epithelium at this stage (Fig. 3 and Table 1). In 9.5 day embryos, the first appearance of the lymphoid cells in the epithelium was
observed
in 4 of 7 materials
and
their
mean
number
was
6.2±5.9
(Table
1).
The
lymphoid cells in the epithelium were found mostly near the basement membrane or passing through it and the same cells were recognized in the tunica mucosa (Fig. 4).
294
Fig.
J. YAMADA, M. SUGIMURA and N. KUDO:
3.
Nine day embryo.
Some lymphoid
cells in the bursal tunica mucosa.
stain.
Toluidine
blue
×500
Fig. 4. Nine and half day embryo. Note one lymphoid cell found in the bursal epithelium. Some lymphoid cells are in the subepithelial layer and appear to be passing through the basement membrane. Many similar cells are observed in the tunica mucosa. Basic fuchcin-methylene
blue
stain.
×500
Figs. 5 and 6. Ten day embryo. Lymphoid cells can be seen with a cytoplasmic extension passing through the basement membrane into epithelium. Toluidine blue stain (Fig. 5). Basic fuchcin-methylene
blue
stain
(Fig.
6).
×1200
Mesenchymal
Fig. 7. (Bm)
Origin of Bursal Lymphoid
Cells
295
Ten day embryo. Lymphoid cells (L) migrating into the epithelium.
is interrupted
by
a penetrating
lymphoid
cell.
E epithelial
cell,
M
Basement membrane
mesenchymal
cell.
×5,000
At 10 days' incubation, the lymphoid cells were observed in the epithelium and the tunica mucosa (Fig. 5-7), but no lymphoid cells were found in the epithelium in 2 of 11 materials. The mean number of the lymphoid cells in the tunica mucosa was larger than that in the epithelium (Table 1). The epithelial bud was not observed in this stage. The lymphoid cells which were constricted into hand-mirror and dumb-bell shapes, were frequently observed to pass through the basement membrane (Fig. 5-7). In 11 day embryos, the lymphoid cells rapidly increased in number in the epithelium. The mean number of the lymphoid cells in the epithelium was larger than that in the tunica mucosa (Table 1). The epithelial buds were first recognized as protrusions from the surface epithelium into the tunica mucosa in 5 of 11 materials (Fig. 8). They consisted of undifferentiated epithelial cells and of lymphoid cells. At 12 days' incubation (Fig. 9, 10), the epithelial buds were recognized in all materials, having increased in size and number over that on the 11th day of incubation. The lymphoid cells were rapidly increased in the epithelium with the epithelial buds formation. Between 10 and 12 days' incubation, the lymphoid cells in the tunica mucosa increased in the
slowly
epithelium
to 144.2±67.1
in
number
(and (Table
the 1).
from epithelial
14.3±6.5 buds)
to
57.6±20.3,
increased
rapidly
whereas
the
in number
lymphoid from
cells 11.1±8.6
296
J. YAMADA, M. SUGIMURA and N. KUDO:
Mesenchymal
Origin of Bursal
Lymphoid
Cells
297
Discussion
There is no doubt that the bursa of Fabricius of the Ayes is a lymphocytopoietic organ which mainly consists of epithelial reticular cells and of lymphoid cells. However there are considerable disagreements in respect to the origin of bursal lymphocytes. It is not yet determined whether they are derived from the epithelial cells or the mesenchymal cells. The classical and extensive observation of JOLLY (1915) on bursal development laid the foundation for the theory of mesenchymal origin of bursal lymphocytes. ORTEGA, KATTINE and SPURLOCK (1965), NAKAGAWA (1969)and LEENE, DUYZINGS and VANSTEED (1973) also considered that bursal lymphocytopoiesis initiated from the migration of mesenchymal lymphoid cells into the epithelial buds. MOOREand OWEN (1965, 1966) concluded by several unique methods (chromosome marker and parabiosis techniques), that the lymphocytic transformation in the epithelial buds took place by immigration of lymphocytic cells derived from undifferentiated mesenchymal cells in the tunica propria or from other unknown sites. Most other investigators(FORBES, 1877; STIEDA, 1880; WENCKBACH, 1893; RETTERER and LELIEVRE,1913a, b) believed that epithelial buds developed from the lining epithelium of the bursa and transformed into typical lymphoid follicles. Thus they believed in the transformation and differentiation of epithelial cells into lymphocytes. This concept was revived by ACKERMAN and KNOUFF (1959), ACKERMAN (1962), TAKAGI (1967), ICHIMURA (1967) and BOSCH (1968). JAFFE and FECHHEIMER (1960) and ARAMAKI (1968),On the other hand, concluded that bursal lymphocytes are dual in origin arising from undifferentiated epithelial cells and from mesenchymal cells. In order to solve this problem by means of a quantitative morphological method, it seemed important, firstly to decide the critical stage in which the lymphoid cells begin to appear in the bursal tunica mucosa or in the bursal epithelium, secondly to determine the time when the epithelial bud formation begins and thirdly, to estimate the number of the lymphoid cells in the epithelium and in the tunica mucosa per unit area at different developmental stages. Analyzing the results obtained the present authors could reach the clear conclusion that the bursal lymphoid cells are of mesenchymal rather than epithelial origin. In their study on the bursa of Fabricius in the chicken, ACKERMAN and KNOUFF (1959) reported: "These blast cells appear to be formed intraepithelially and the possibility of their arising from mesenchymal cells in the tunica propria and migrating Fig. 8.
Eleven day embryo. Epithelial buds are seen as protuberances of the epithelium. Lymphoid cells are in the epithelial region and tunica mucosa (especially in the subepithelial layer). They are readily identified by their basophilic cytoplasm. Toluidine blue stain.
Fig. 9.
Twelve
day embryo.
also
increased
×500 are
in
Lymphoid size.
cells are increased
Toluidine
blue
stain.
in number
in the epithelial
buds which
×500
Fig. 10. Twelve day embryo. Early epithelial bud formation. Lymphoid cells (L) of dark appearance are present. Note a lymphoid cell migrating into the epithelial bud, and cells of the same nature around the bud. These lymphoid cells are identified easily by a shrinkage artifact caused by the hypertonic fixative (2.5% glutaraldehyde and 1% OsO4 in 0.1M cacodylate) used in this specimen. E epithelial cell, M mesenchymal cell, Bm basement membrane. ×5,000.
298
J. YAMADA, M. SUGIMURA and N. KUDO:
into follicle seems highly unlikely from our observations since similar cells are rarely seen out side of the epithelial bud during this period of bursal development." The authors' findings are diametrically opposed to their findings. The lymphoid cells were first found in the tunica mucosa at 8 days' incubation. The first appearance of the lymphoid cells in the epithelium was 1.5 days after that in the tunica mucosa. The epithelial buds began to form at about 11 days' incubation, and were never observed prior to penetration of the lymphoid cells into the bursal epithelium. At 9.5 to 11 days' incubation, the lymphoid cells were frequently observed to pass through the basement membrane. These cells showed handmirror or dumb-bell cell body and nucleus shapes. From the findings described above, the direction of the cell movement is believed to be from the mesenchymal to the epithelial region. In the Japanese quails, lymphocytopoiesis of the bursa of Fabricius has been
studied by only two investigators (KOBAYASHI, 1968; ARAMAKI, 1968). ARAMAKI (1968) has, however, investigated only one embryonic stage (15 days' incubation), and KOBAYASHI's (1968) description is brief and lacks photographs. KOBAYASHI (1968) has indicated that the epithelial buds appeared at 13 days' incubation, and that large lymphocytes appeared at about 15 days' incubation. As his description isbasophilic insufficient, it is difficult to be discussed in comparison with the present findings. ACKERMANand KNOUFF(1963) demonstrated a relatively intense alkaline phosphatase activity in the subjacent compact mesenchyme during the phase of bud formation, which subsequently declines with the maturation of follicles. They concluded that this metabolic change may reflect an epithelial-mesenchymal inductive interaction involved in the epithelial bud formation. ICHIMURA (1967) objected to the inductive function of this enzyme, and the authors (unpublished data) never observed a specific distribution of this enzyme in Japanese quails. Penetration of the lymphoid cells into the bursal epithelium may rather represent an inducing factor involved in the epithelial bud formation. The present results support neither the concept of transformation of the bursal epithelialcellsinto the lymphoid cellsby earlierinvestigators(FORBES, 1877; STIEDA, 1880; WENCKBACH, 1893; RETTERER and LELIEVRE, 1913a,b; ACKERMAN and KNOUFF, 1959; ACKERMAN, 1962; TAKAGI, 1967; ICHIMURA, 1967; BOSCH, 1968),nor the dual origin of the lymphoid cells (JAFFE and FECHHEIMER, 1966; ARAMAKI, 1968). The present results indicate that the bursal lymphoid cells derive from the mesenchymal cells in Japanese quails. Further investigations are need to determine more precisely the site of origin of the lymphoid cells in the earliest stage of bursal development.
ウ ズ ラ の フ ァブ リキ ウス 嚢 にお け る リンパ 球 様 細 胞 の 間 葉 由来 に つ い て 山 田 純 三, 杉 村 孵卵 後6日 て,
誠, 工 藤 規 雄
目 (120時 間) か ら12日 目の ウ ズ ラ胚 (84例)
の フ ァブ リキ ウ ス嚢 に お い
リンパ 球 様 細 胞 の 由 来 を計 量 形 態 学 的 に 観 察 した.
6日 目お よ び7日
目の 嚢 原 基 に は リンパ球 様 細 胞 は 認 め られ ず,
8日 目の嚢 固 有 層 に
初 め て, 少 数 の リンパ 球 様 細 胞 が 認 め られ た. 上 皮 に は これ よ り1.5日 遅 れ て, 9.5日
Mesenchymal
Origin of Bursal Lymphoid
Cells
299
目に 初 め て 本 細 胞 が 認 め られ た. 上 皮 蕾 は11日 目頃 に 形 成 さ れ 始 め た.
この 上 皮 蕾 形 成
は リンパ 球 様 細 胞 の 上 皮 へ の 侵 入 に 先 行 して 認 め られ る こ とは な か った.
9.5日 目か ら
11日 目に か け て, 基 底 膜 を 通 過 し て 上 皮 お よ び上 皮 蕾 に侵 入 しつ つ あ る細 胞 が 多 く認 め られ た. 以 上 の 所 見 よ り, ウ ズ ラの フ ァブ リキ ウ ス嚢 に お け る リンパ 球 様 細 胞 の 由来 を上 皮 に 求 め る よ り 間 葉 に 求 め る方 が よ り妥 当 と考 えた.
References Ackerman, G. A.: Electron microscopy of the bursa of Fabricius of the embryonic chick with particular reference to the lymphoepithelial nodules. J. Cell Biol. 13: 127-146 (1962). Ackerman, G. A. and R. A. Knouff: Lymphocytopoiesis in the bursa of Fabricius. Amer. J. Anat. 104: 163-205 (1959). --: Testosterone
suppression
of mesenchymal
alkaline
phosphatase
activity
and
lympho-epithelial nodule formation in the bursa of Fabricius in the embryonic chick. Anat. Rec. 146: 23-27 (1963), Aramaki, T.: Morphologic study of lymphatic tissues (XIII). Light and electron microscope observations on the bursa of Fabricius of chick and coturnix embryos with special reference to lymphocytopoiesis. Keio J. Med. 17: 135-155 (1968). Bosch, J.: Morphogenetische, histochemische und experimentelle Untersuchungen uber die Bursa Fabricii. Zool. Jahrb. Abt. Anat. 85: 327-385 (1968). Forbes, W. A.: On the bursa of Fabricius in birds. Proc. Zool. Soc. London, 1877. (p. 304-318). Ichimura, M.: Embryonic development and histochemical study of the bursa of Fabricius in the embryo of the chickens (in Japanese). J. Keio Med. Soc. 44: 571-582 (1966). Jaffe, W. P. and N. S. Fechheimer: Cell transport and bursa of Fabricius. Nature 212: 92 (1966). Jolly, J.: La bourse de Fabricius et les organes lymphoepitheliaux. Arch. Anat. microsc. 16: 363547 (1915). Kobayashi, T.: Structure and function of bursa of Fabricius (in Japanese). In: Symp. Immunobiol. Tokyo, Maruzen, 1968. (Vol. 1, p. 24-28). Leene, W., M. J. M. Duyzings and C. van Steege: Lymphoid stem cell identification in the developing thymus and bursa of Fabricius of the chick. Z. Zellforsch. 136: 521-533 (1973). Moore, M. A. and J. J. T. Owen: Chromosome marker studies on the development of the haemopoietic system in the chick embryo. Nature 208: 956-990 (1965). --: Experimental
studies
on
the
development
of
the bursa
of Fabricius. Devel.
Biol. 14: 40-51 (1966). Nakagawa, Y.: The early stage of the epithelial bud-formation in the bursa of Fabricius in chick embryo (in Japanese with English summary). Tohoku Igaku Zasshi, 79: 139-146 (1969). Ortega, L. G., A. A. Kattine and B. O. Spurlock: Lympho-epithelial interaction in the developing bursa of Fabricius. Fed. Proc. 24: 160 (1965). Retterer, E. et A. Lelievre: Nouvelles recherches sur la bourse de Fabricius. C. r. Soc. Biol. 74: 182-185 (1913a). --: Homologies de la bourse de Fabricius. C. r. Soc. Biol. 74: 382-385 (1913b). Stieda, L.: Uber den Bau und die Entwicklung der Bursa Fabricii. Z. wiss. Zool. 34: 296-309 (1880). Wenckbach, K. F.: Die Follikel der Bursa Fabricii. Anat. Anz. 11: 159-160 (1893).
山田純 三 〒080北 海道帯広市稲田町 帯広畜産大学獣医学科 家畜解剖学教室
Junzo YAMADA Department of Veterinary Anatomy Obihiro University of Agriculture and Veterinary Medicine Obihiro, Hokkaido, 080 Japan
