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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1586-1594
Structural and Functional Abnormalities in the Spleen of an
mFtz-F1 Gene-Disrupted Mouse
By
Ken-ichirou Morohashi,
Hisae Tsuboi-Asai,
Sumie Matsushita,
Masahiro Suda,
Manabu Nakashima,
Hironobu Sasano,
Yoshiaki Hataba,
Chun-Lin Li,
Junichi Fukata,
Junji Irie,
Takeshi Watanabe,
Hiroshi Nagura, and
En Li
From the Division of Cell Differentiation, Department of
Developmental Biology, National Institute for Basic Biology,
Myodaiji-cho, Okazaki; the Department of Molecular Biomechanics, School
of Life Science, The Graduate University for Advanced Studies,
Myodaiji-cho, Okazaki; the Department of Molecular Biology, Graduate
School of Medical Science, Kyushu University, Higashi-ku, Fukuoka; the
Clinical Laboratory, Kyushu University Hospital, Higashi-ku, Fukuoka;
the Department of Molecular Immunology, Medical Institute of
Bioregulation, Kyushu University, Higashi-ku, Fukuoka; the Department
of Pathology, Tohoku University School of Medicine, Sendai; the
Department of Molecular Cell Biology, Institute of DNA Medicine, Jikei
University School of Medicine, Minatoku, Tokyo; the First Department of
Internal Medicine, Kochi Medical School, Nankoku, Kochi; the Department
of Pathology, Nagasaki University School of Medicine, Nagasaki,
Japan; and the Cardiovascular Research Center, Massachusetts General
Hospital-East, Harvard Medical School, Charlestown, MA.
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ABSTRACT |
The spleen has two main functions. The first is to provide a proper
microenvironment to lymphoid and myeloid cells, whereas the second
involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of
the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirmed the
expression of Ad4BP/SF-1 in endothelial cells of the splenic venous
sinuses and pulp vein. In mFtz-F1 gene-disrupted (KO) mice, several structural abnormalities were detected in the spleen, including
underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses showed failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most
likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice included defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in
the vascular system were associated with a decrease in the proportion
of hematopoietic cells, although differentiation of these cells was not
affected significantly. A high number of abnormal red blood cells
containing Howell-Jolly bodies were noted in the KO mice, indicating
impaired clearance by the splenic vascular system. We also detected the presence of an mRNA-encoding cholesterol side-chain cleavage P450 in
the spleen, resembling the findings in steroidogenic tissues such as
the gonads and adrenal cortex. The mRNA transcript was not involved in
splenic structural defects as it was detected in the spleens of both
normal and KO mice, indicating that the regulatory mechanism of the
P450 gene in the spleen is different from that in steroidogenic
tissues. Our results indicate that a lack of the mFtz-F1 gene
in mice is associated with structural and functional abnormalities of
the splenic vascular system.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
Ad4BP/SF-1, A MEMBER of the nuclear
receptor family, was originally identified as a steroidogenic,
tissue-specific transcription factor implicated in the expression of
steroidogenic CYP genes encoding cytochrome
P450s.1,2 Expression of Ad4BP/SF-1 correlates with the
distribution of P450s in steroidogenic tissues such as gonads and
adrenal cortex.3-5 In addition, Ad4BP/SF-1 immunoreactive
cells are also present in nonsteroidogenic cells such as the pituitary
gonadotrophs and ventromedial hypothalamic neurons.6-8
These two cell types are involved in the establishment and
maintenance of reproductive function by displaying a coordinated function with the gonads.9
Recent studies of genes downstream of Ad4BP/SF-1 have shown that
several genes are under the control of Ad4BP/SF-1. These include the
3 -hydroxysteroid dehydrogenase gene,10 the steroidogenic acute regulatory protein gene,11,12 the prolactin receptor gene,13 the adrenocorticotropin receptor
gene,14 the oxytocin gene,15 the
Müllerian inhibitory substance gene,16 the
gonadotropin gene,17-20 and the Leydig insulin-like
gene.21 These genes are closely related to functions
specific for the corresponding tissues. In addition to the functions
identified in the studies mentioned, gene disruption studies of the
mammalian Ftz-F1 (mFtz-F1)-encoding Ad4BP/SF-1
indicated an additional role for the transcription factor by showing
that both gonads and the adrenal gland are absent in mFtz-F1
gene-disrupted mice.8,22,23 Therefore, it is assumed that
Ad4BP/SF-1 also plays an important role in the differentiation and
development of steroidogenic tissues, although the precise mechanism
remains to be clarified.
In addition to the above functionally or developmentally related
tissues, recent studies using reverse transcription-polymerase chain
reaction (RT-PCR) or in situ hybridization also detected the
mFtz-F1 gene transcript in the nonsteroidogenic
spleen.24,25 The spleen of adult animals consists of two
major parts, the red pulp and white pulp. The former contains a large
number of erythrocytes, whereas the latter contains tightly packed
lymphatic cells.26 In the white pulp, lymphopoiesis occurs
from immature lymphoid cells analogous to lymph nodes, whereas in the
red pulp, abnormal erythrocytes are thought to be eliminated when they
pass through the meshwork structure of the splenic vasculature. In
addition, the spleen transiently exhibits erythropoietic activity
similar to the liver during fetal life.
The spleen has a unique vascular system composed of venous sinuses as
well as a network of arteries and veins. Blood enters through splenic
arteries that branch into trabecular arteries. These run into the white
pulps making further branches as the central arteries. Some of fine
branches from the central arteries run out of the white pulps and
terminate in the surrounding marginal zone or in the red pulp. After
flowing into the splenic cord, blood is collected in the venous sinus
and thereafter runs directly into the pulp vein. Finally, the pulp
veins coalesce forming the trabecular veins. Because the lymphopoietic
and erythropoietic cells originate from their stem cells, which are not
intrinsic splenic cells, they are transferred through the vascular
system to settle into the spleen. Therefore, with respect to
hematopoiesis in the spleen, an inherent function of the tissue is to
provide a proper microenvironment for the settlement and
differentiation of different blood cells.
In the present study, we examined the expression of Ad4BP/SF-1 in the
spleen of normal and mFtz-F1 gene-disrupted mice. Our results
showed that the gene product, Ad4BP/SF-1, is implicated in
differentiation of splenic architecture and functions.
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MATERIALS AND METHODS |
Immunoblot analysis.
Spleens were isolated from the E16.5 mouse fetuses, newborn and adult
mice, and then lysed with 50 mmol/L Tris-HCl (pH 7.5) containing 2%
sodium dodecyl sulfate (SDS). This was followed by
sonication to disrupt viscous cellular DNA. Total cellular proteins (25 µg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of total tissue lysates prepared from the adrenal
glands of the adult mice were used as control. The procedure for
immunoblotting using a rabbit antiserum to bovine Ad4BP/SF-1 was
described previously.5,27 Electrochemiluminescence (ECL) Western blot reagents (Amersham, Arlington Height, IL)
were used for the detection.
Immunohistochemical analysis.
For immunohistochemical analysis, the spleens were fixed with 4%
paraformaldehyde-0.1 mol/L potassium phosphate (pH 7.0), dehydrated,
and embedded in paraffin. To remove blood cells, the spleens of the
adult animals were perfused, then fixed by using an arterial and venous
pressure-loading perfusion fixation method that was originally
developed for preparation of the specimen for scanning electron
microscopy, as described previously.28,29 The human spleen
was washed with phosphate-buffered saline (PBS) to remove red blood
cells before fixation. Sections were prepared for immunohistochemical
staining using the antiserum to Ad4BP/SF-1.5,30 For
detection of laminin, type-IV collagen, c-kit, and vimentin, frozen sections of newborn mice spleens were fixed with
acetone/methanol (1:1) at  20°C. A rat monoclonal antibody to mouse
laminin and a rabbit polyclonal antibody to mouse type-IV collagen were
purchased from Chemicon International Inc (Temecula, CA). An
anti-c-kit monoclonal antibody (ACK2) was kindly provided by
Dr Nishikawa (Kyoto University, Kyoto,
Japan).31 Mouse monoclonal antibody to bovine
vimentin was purchased from Boehringer Mannheim (Mannheim, Germany).
Antibody-antigen complexes were detected by the
streptavidin-biotin-peroxidase or -alkaline phosphatase method using
the Histofine kit (Nichirei Co, Tokyo, Japan).
Characterization of myeloid and lymphoid cells.
Single-cell suspensions were prepared from spleens of newborn mice. For
this purpose, splenocytes were stained with fluorescein dye-labeled
monoclonal antibodies for 30 minutes on ice in PBS containing 2% fetal
calf serum and 0.05% sodium azide. The cells were washed with PBS and
analyzed with FACScan cytometer using the Lysis II program (Becton
Dickinson, Mountain View, CA). The monoclonal antibodies
used for cell staining were phycoerythrin (PE)-conjugated anti-CD4
(Pharmingen, San Diego, CA), fluorescein isothiocyanate
(FITC)-conjugated anti-CD8 (Pharmingen), FITC-conjugated anti-CD3
(Pharmingen), PE-conjugated anti-B220 (GIBCO-BRL,
Gaithersburg, MD), and biotin-anti-Mac1 in conjunction with
streptavidin-Red670 (GIBCO-BRL).
The number of erythrocytes containing Howell-Jolly bodies was
determined by the method described by Higashikuni et al.32 In brief, blood samples obtained from newborn mice were placed on a
glass slide coated with acridine orange. Howell-Jolly bodies were
detected under a fluorescence microscope. At least 1,000 erythrocytes
were examined in each specimen.
RT-PCR.
Complementary DNAs (cDNAs) were obtained by reverse-transcription using
total RNAs (1 µg) prepared from the spleens of newborn and adult
mice. One-twentieth of the cDNAs was subjected to amplification of the
Ad4BP/SF-1, Cyp11A, and -actin transcripts using specific primers.
The primers for Ad4BP/SF-1 were 5'-GACCAGATGACACTGCTGC-3' and
5'-TCCTTGGCCTGCATGCTCA-3',4 those for Cyp11A were
5'-GCACACAACTTGAAGGTACAGGAG-3' and
5'-CAGCCAAAGCCCAAGTACCGGAAG-3',33 and those for -actin were 5'-GCTGTATTCCCCTCCATCGTG-3' and 5'-CGGTTGGCCTTAGGGTTCAGG-3'. The
expected lengths of the amplified products by the above sets of primers
are 400 bp for the Ad4BP/SF-1, 343 bp for the Cyp11A, and 265 bp for
-actin.34
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RESULTS |
Microscopic and macroscopic features of the spleen of an
mFtz-F1 gene-disrupted mouse.
The results of studies by Ninomiya et al24 and Ramayya et
al,25 in which the Ad4BP/SF-1 transcript was detected in
the spleen, prompted us to examine the tissue of the mFtz-F1
gene-disrupted (KO) mice. As described elsewhere,8,22,23
the KO mouse dies within 1 week after birth, probably because of very
low levels of circulating adrenocorticoids due to a developmental
defect of the adrenal gland.34 Accordingly, we examined
spleen tissues obtained from the KO fetuses and newborn mice.
Interestingly, the spleen of the KO mouse was abnormal macroscopically
and microscopically (Fig 1A). The size of
the spleen was significantly smaller than the normal spleen. Wheras the
spleen of the wild-type litter mates was uniformly red in color, the
spleen of the KO mouse was characterized by the presence of scattered
red spots that varied in size, distribution, and intensity from one
animal to another. Histological examination of the spleen of the
wild-type showed densely packed myeloid and lymphoid cells at various
stages of maturation (Fig 1B), together with features of erythropoiesis
throughout the whole tissue. In contrast, the spleen of the KO mouse
contained sparsely packed areas among densely packed areas. These areas
correspond to the white and red areas, respectively, shown
macroscopically in Fig 1A. The densely packed red area contained mainly
erythrocytes as indicated by arrowheads in Fig 1C. Furthermore, we also
detected several megakaryocytes in the spleen of the KO mouse (Fig 1D).
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| Fig 1.
Macroscopic and microscopic features of the spleen of
mFtz-F1 gene-disrupted mice. (A) Comparison of macroscopic
features of wild-type spleens (Wild) and mFtz-F1
gene-disrupted (KO) newborn mice. Arrowheads show red spots
characteristic of the KO spleen. Photomicrographs of splenic tissues of
the wild-type (B) and KO (C and D) mice stained with hematoxylin and
eosin. Note the erythrocyte-rich region (encircled by arrowheads in C),
probably corresponding to the red regions in (A). (D) Photomicrograph
showing megakaryocytes in the spleen of KO mouse (arrowheads). Scale
bars: 100 µm in B and C; 25 µm in D.
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Expression of Ad4BP/SF-1 in the adult spleen.
Expression of Ad4BP/SF-1 was detected in total tissue lysates of spleen
tissues of adult and fetal (E16.5) mice using immunoblot analysis (Fig
2A). As expected, the signal was not
detected in the spleen of the KO mouse. The significantly low level of
Ad4BP/SF-1 expression in the spleen of this mouse, relative to that in
the adrenal gland, was probably due to a limited expression in certain cell types or otherwise represented at a uniformly low level throughout the entire tissue. In the next step, we examined the distribution of
Ad4BP/SF-1 immunoreactive cells by immunohistochemistry. In the spleen
of adult mice, Ad4BP/SF-1-immunoreactive cells were observed in the
red pulp (indicated by "r" in Fig 2B) but not in the white pulp
(indicated by "w" in Fig 2B). Although some immunoreactive cells
showed an irregular distribution pattern, others were arranged in
distinct patterns (Fig 2B). Because the sinus structure in the red pulp
varies among animal species, we also investigated the spleen of the
adult rat. Interestingly, several immunoreactive cells in the rat were
arranged in a regular pattern probably along the sinuses (Fig 2C). Our
results were, however, inconclusive with regard to the exact cell type
that was immunoreactive for Ad4BP/SF-1 due to the large number of blood cells. Therefore, further investigation was performed using perfused spleens. Even after perfusion, immunoreactive cells remained at the
luminal side of the pulp vein (indicated by a black asterisk in Fig 2D)
and venous sinuses (indicated by red asterisks in Fig 2D). Examination
at a higher magnification clearly indicated that these cells were
attached to the basement membrane of the venous sinuses (arrowheads in
Fig 2E). Alignment of immunoreactive cells along the venous sinus was
also seen in the marginal sinus surrounding the white pulp (indicated
by arrowheads in Fig 2F). A similar staining pattern was noted in the
perfused spleens of adult rats (Fig 2G) and washed spleens of humans
(Fig 2H). As in the mouse, Ad4BP/SF-1 immunoreactive cells were found
to lay inside the sinus in both rats and humans. Furthermore, the pulp
vein of the rat spleen was found to contain immunoreactive cells in its
luminal side (data not shown).
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| Fig 2.
Expression of Ad4BP/SF-1 in the spleen. Expression of
Ad4BP/SF-1 was investigated using immunoblot (A) and
immunohistochemical analysis (B, C, D, E, F, G, and H) using the
antiserum to Ad4BP/SF-1. Total tissue lysates (25 µg) were prepared
from spleen tissues of adult (Adult), E16.5 fetus (E16.5), and wild
[B0 (Nor)] and KO [B0 (KO)] newborn mice. Adrenal glands (Adrenal)
of adult mice were used as the positive control for detection of
Ad4BP/SF-1. Molecular weight markers are indicated on the left. Arrows
indicate signals for Ad4BP/SF-1. Immunohistochemistry was performed
using mouse (B) and rat (C) splenic tissues, and perfused mouse (D, E,
and F) and rat (G) spleen tissues. A tissue sample of human spleen was
washed with PBS to remove erythrocytes and immunostained (H). Note that
almost all positive nuclei are localized in the red pulp (r) but not in
the white pulp (w) (B and C). Red and black asterisks in D, F, G, and H
indicate venous sinuses and pulp vein, respectively. Arrowheads in E
indicate the basement membrane of the venous sinus. Arrowheads in F
indicate Ad4BP/SF-1 immunoreactive nuclei surrounding the white pulp.
Ca, central artery. Scale bars: 50 µm in B, C, D, F, and H; 120 µm
in E and G.
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Expression of Ad4BP/SF-1 in the fetal spleen.
Immunohistochemical analysis was also performed using spleen tissues of
wild and KO newborn mice. Ad4BP/SF-1-immunoreactive cells were
detected in the wild mouse but not in the KO mouse (Fig
3A and B). As in the case of adult spleen,
a large number of erythrocytes packed the entire spleen of the newborn
control mice, making it difficult to determine the exact cell type that was immunoreactive to Ad4BP/SF-1. Therefore, we examined the spleen at
an early developmental stage before the onset of erythropoiesis. Expression of Ad4BP/SF-1 appeared in the spleen of wild-type mice at
day 14.5 of gestation (Fig 3C). Interestingly, some of the immunoreactive cells formed tubular structures in concert with other
immunoreactive cells (indicated by arrowheads in Fig 3C). Although it
is difficult to establish that these structures develop into the
splenic vascular system, some of the tubular structures were observed
to contain erythrocytes, suggesting that they are embryonic components
of the vascular system. The tubular structures, which were sectioned
longitudinally during tissue processing, were also occasionally seen in
the wild-type spleen (indicated by arrowheads in Fig 3D). In contrast,
the spleens of KO mice of the same littermate were less likely to
contain similar well-assembled tubular structures.
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| Fig 3.
Expression of Ad4BP/SF-1 in fetal and newborn
spleens and structural changes observed in the early stages of splenic
development. Immunohistochemical analysis was performed using splenic
tissues from wild-type (A) and KO (B) newborn mice and spleen of an E
14.5 mouse fetus. Arrowheads in (C) indicate tubular structure composed
of Ad4BP/SF-1 immunoreactive cells. Inset: higher magnification of the
tubular structure. Spleens of E16.5 fetuses of wild-type (D) and KO (E)
mice were histologically examined with hematoxylin and eosin.
Arrowheads in D indicate longitudinal sections of the tubular
structures. Scale bars: 100 µm.
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Alteration of expression profiles of marker proteins.
In the next step, we compared the expression and distribution of
extracellular matrix proteins in spleen tissues of newborn wild mice
with those in KO mice by using silver-stained sections. Fibrous
structures were similarly stained in the spleens of both strains of
mice without any obvious differences (data not shown). Similar staining
patterns were also detected with the antibody to type-IV collagen.
However, staining along certain structures, notably trabeculae, was
clearly observed in the wild-type (arrowheads in Fig
4A) but not in the KO mouse (Fig 4E). More
importantly, striking differences were detected between the two spleens
when tissue samples were stained for another extracellular matrix
protein, laminin (Fig 4B and F). In the spleen of the newborn wild-type mouse, laminin was often assembled around vessel-like structures. In
contrast, in spite of the persistent presence of laminin, such a unique
pattern was never seen in the spleens of newborn KO mice. Staining
patterns similar to that of laminin were also observed in
c-kit- (Fig 4C and G) and vimentin- (Fig 4D and H) positive cells. Immunoreactive cells for these antigens were also assembled around the vessel-like structures in the spleen of the wild-type mouse.
In contrast, the spleen of the newborn KO mouse was characterized by
the absence of such assembly and fewer c-kit- and
vimentin-positive cells.
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| Fig 4.
Altered distribution of marker proteins. Frozen sections
of spleens of newborn wild-type (A, B, C, and D) and KO mice (E, F, G,
and H) stained with antibodies to type-IV collagen (A and E), laminin
(B and F), c-kit (C and G), and vimentin (D and H). Arrowheads
in (A) indicate a fibrous structure. Scale bars: 100 µm.
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Splenic hypofunction in KO mice.
Splenic venous sinuses are involved in the clearance of abnormal
erythrocytes. Accordingly, we counted the number of erythrocytes that
contained Howell-Jolly bodies as a measure of splenic function, using
blood smears prepared from adult and newborn mice (wild and KO mice at
postnatal day 0). A few Howell-Jolly body-containing erythrocytes were
present in healthy adult mice as well as newborn mice of the wild-type
(Fig 5). In newborn KO mice, there was a 2.6-fold increase in the number of abnormal erythrocytes (Fig 5).
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| Fig 5.
Percentage of erythrocytes containing Howell-Jolly
bodies. Blood samples were prepared from wild (Nor) and KO newborn mice
at postnatal day 0. Erythrocytes containing Howell-Jolly bodies were
counted as described in Materials and Methods. Data represent the mean ± SEM of 10 wild-type and 6 KO mice.
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To investigate if the structural changes described above affected the
differentiation of blood cells, we compared the percentages of
different cellular components in the spleens of two newborn wild-type
mice to those in a similar number of KO mice. Hypocellularity of both
lymphocytes and erythrocytes was evident in the spleen of the KO mouse
(<20% of those in wild-type mice) compared with those of the
wild-type (Table 1). These
findings correlated well with the small size of the organ and irregular
distribution of erythrocytes in the KO mouse. In newborn KO mice, the
numbers of mature CD3+ T cells, B220+ B cells,
and Mac-1+ macrophages in the spleens were greatly
decreased, but the proportion of T and B cells did not change as
compared with that in the wild-type. The major cell population in the
spleen of newborn mice was
CD3 B220Mac1 mononuclear
cells in both strains. Furthermore, the presence of megakaryocytes was
confirmed by histological examination of spleens of both KO and
wild-type mice (Fig 1D).
Expression of steroidogenic Cyp genes in the spleen.
Because the regulation of steroidogenic Cyp gene is mediated by
Ad4BP/SF-1 in steroidogenic tissues, we investigated the expression of
Cyp genes in the spleen by RT-PCR. When total RNA prepared from
the spleen of adult mice was used, the mRNA transcripts of both the
mFtz-F1 gene and Cyp11A gene, which encodes P450SCC, were detected (Fig 6), whereas transcripts
of other steroidogenic Cyp genes, Cyp11B1, Cyp11B2,
Cyp11B3, and Cyp17, were undetectable (data not shown). We
also employed RT-PCR using cDNA of the wild-type and KO spleens. The
mFtz-F1 gene transcript was detected in the spleen of wild-type
but not KO mice. In contrast, the Cyp11A gene transcript was
detected in both strains, showing a similar observation with the Cyp11A
expression in the placenta.23
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| Fig 6.
Expression of Cyp11A in the spleen. Total RNAs were
prepared from the spleens of wild-type adult (Adult), and wild-type
(Nor) and mFtz-F1 gene-disrupted (KO) new born (B0) mice.
cDNAs reverse-transcribed from the RNAs were used for PCR with specific
primers for Ad4BP/SF-1 (A), Cyp11A (B), and -actin (C) as described
in Materials and Methods. Arrows indicate the expected length of PCR
products.
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DISCUSSION |
The spleen has a unique structure composed of extrinsic cells derived
mainly from the bone marrow and intrinsic cells. The latter cells constitute the framework of the spleen and include blood
vessels, venous sinuses, and trabeculae, whereas both myeloid and
lymphoid cells differentiate from bone marrow cells. The white pulp
consists of T and B lymphocytes that can potentially produce antibodies
in response to antigenic stimulation, similar to the lymph nodes.
The red pulp, on the other hand, is packed with erythrocytes and
leukocytes, both of which are in the process of transport from the
arteries to the veins. Blood cells released from the arterial terminals
into the splenic cord pass through slitlike structures in the sinus
wall and enter into the luminal side. Examination by electron
microscopy26,35-37 and scanning electron microscopy29 has shown that the major component of the
sinus wall consists of endothelial cells arranged side-by-side, forming a tubular structure. Based on these morphologic studies, a remarkable feature of the endothelial cell is the presence of nuclei that protrude
into the luminal side of the sinus.
Recent studies using RT-PCR and in situ hybridization24,25
have confirmed the expression of Ad4BP/SF-1 in the spleen. However, the
exact cell type that expresses Ad4BP/SF-1 could not be identified. A
major finding of the present study was the localization of splenic cells that express Ad4BP/SF-1. In the first step, routine
immunohistochemical analysis showed the presence of a large number of
blood cells that interfered with the identification of immunoreactive
cells. Consequently, we used perfused spleens for immunodetection. Our results showed that immunoreactivity was localized to endothelial lining cells of venous sinuses and pulp veins. In a series of preliminary experiments, we also investigated the expression of Ad4BP/SF-1 in other tissues that are functionally related to the spleen. To this effect, immunohistochemical staining of the thymus, lymph node, fetal liver, and bone marrow showed that none of these tissues contained immunoreactive cells for Ad4BP/SF-1 (data not shown).
Gross examination of the spleen showed a clear underdevelopment in the
mFtz-F1 gene-disrupted mice. Furthermore, histological examination showed the presence of fewer erythrocytes that appeared in
clusters irregularly distributed in the spleen, reflecting a state of
hypoerythropoiesis. Thus, in the KO mouse, erythropoiesis is likely to
occur in limited regions of the spleen, in sharp contrast to the
wild-type mice, where it occurs throughout the whole organ. These
abnormal features were detected as early as day 14.5 of gestation.
During normal fetal development, the expression of Ad4BP/SF-1 appeared
in cells that formed the tubular framework, which probably form the
splenic vascular system, including the venous sinuses and pulp vein. In
contrast, such tubular structures could not be detected in the spleen
of KO mice, probably leading to a poor organization of the vascular
system. This conclusion is supported by our finding of the
altered profile of the expression of type-IV collagen and more
strikingly by that of laminin. Because these proteins are components of
the extracellular matrix, our results suggest that disruption of the
mFtz-F1 gene not only results in an immature vascular system
but also altered architecture of the spleen in the KO mouse.
Interestingly, our results showed a similar distribution of
c-kit, a receptor tyrosine kinase, and vimentin, an
intermediate filament, in normal and KO mice. These proteins are known
to be expressed in the immature hematopoietic cells, and their
expression is dependent on the cell lineage.38,39 Our
results showed that in the wild-type spleen, immunoreactive cells for
these proteins were arranged around structures that probably
represented immature blood vessels. In contrast, such peculiar assembly
was not detected in the KO spleen. Hematopoietic cells originate from
the bone marrow and then settle in other hematopoietic tissues,
including the spleen. The presence of structural and/or
functional defects in the embryonic splenic vascular system is likely
to interfere with the splenic blood flow, thereby affecting the
extension and settlement of hematopoietic cells in the spleen. In this
regard, our results showed a clear overlap in the distribution of
laminin with that of c-kit and vimentin. Laminin is known as a
ligand molecular for members of the integrin family and is expressed on
the surface of several cell lineages, including the hematopoietic cells.40 Therefore, altered expression of laminin is
probably associated with inefficient distribution of the
c-kit- and vimentin-expressing cells in the KO spleen. It is
also possible that the altered microenvironment in the KO spleen does
not support a proper differentiation of hematopoietic cells. To
investigate this possibility, we compared the cellular components of
the spleen in KO mice with those of the wild-type by using several
peripheral cell markers. Our results showed that full maturation was
persistent in all hematopoietic lines tested and no obvious difference
was detected, indicating clearly that differentiation of the
hematopoietic cells is not impaired in KO mice.
Our results showed a higher proportion of erythrocytes containing
Howell-Jolly bodies in the mFtz-F1 gene-disrupted mice than in
control mice. A similar finding was also reported in the asplenic mouse41 and Hox11 gene-disrupted mouse, both of
which showed failure of splenic development.42 These
results are also in agreement with the appearance in the blood of
erythrocytes containing Howell-Jolly bodies following splenectomy in
humans.43
Ad4BP/SF-1 was originally identified as a specific steroidogenic
transcription factor expressed in the adrenal cortex and gonads. In
these tissues, the steroidogenic Cyp genes are downstream of
Ad4BP/SF-1. Although the spleen is not classified as a steroidogenic tissue, we examined the expression of Cyp genes in the present study. Interestingly, we detected the Cyp11A gene
transcript-encoding cholesterol side-chain cleavage P450 in the
spleen. However, the same mRNA was also present in KO mice, strongly
suggesting that the Cyp11A gene is not under the control of
Ad4BP/SF-1 in the spleen. A similar Ad4BP/SF-1-independent regulation
has been reported for the steroidogenic Cyp11A gene in the
placenta,23 the primitive gut and dermal
mesenchyme,44 and for the Cyp19 gene-encoding aromatase P450 in the hypothalamus.8 Although the mechanism remains to be clarified, the Cyp genes can be transcribed under a distinct mechanism present in steroidogenic tissues where Ad4BP/SF-1 is the crucial transcription factor for Cyp genes.
Ad4BP/SF-1 is thought to play a crucial role in the establishment of
hypothalamic-pituitary-gonadal and -adrenal axes, in addition to its
role in steroid hormone biosynthesis through the regulation of
steroidogenic P450 gene transcription. In the present study, we
identified a new function for Ad4BP/SF-1, ie, the construction of basic
splenic architecture based on its expression in endothelial cells
lining splenic venous sinuses and pulp vein. However, the exact
function of Ad4BP/SF-1 in splenic cells has not yet been explained.
Therefore, the mechanisms that lead to impaired splenogenesis in KO
mice could not be clearly identified. This is mainly due to a lack of
information on the exact gene regulated by Ad4BP/SF-1 in the spleen.
Identification of the downstream genes will help explain the mechanisms
involved in the development of splenic architecture, including the
vascular system.
| |
ACKNOWLEDGMENT |
The authors thank Drs N. Yanai (Tohoku University), K. Sasaki (Kawasaki
Medical School), and S. Sutou (Itoham Central Research Institute) for
their critical discussions. The antibody for c-kit was
generously provided by Dr Nishikawa (Kyoto University).
| |
FOOTNOTES |
Submitted July 29, 1998; accepted October 15, 1998.
Supported by a Grant-in-Aid for Scientific Research from Ministry of
Education, Science, Sports, and Culture of Japan, and grants from Naito
Foundation, Sumitomo Foundation, Suzuken Memorial Foundation, and
Uehara Memorial Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ken-ichirou Morohashi, Prof, PhD, Division
of Cell Differentiation, Department of Developmental Biology, National
Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan;
e-mail:moro{at}nibb.ac.jp.
| |
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Abstract
Introduction