1 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA
2 Division of Hematology, Baylor College of Medicine and Michael DeBakey VAH
Medical Center, Houston, TX 77030, USA
3 Department of Immunology and Biotechnology, University of Pécs,
Pécs, Hungary
* Author for correspondence (e-mail: armins{at}bcm.tmc.edu)
Accepted 6 July 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Ferroportin 1, Spleen, Iron homeostasis, Polycythaemia mutant, Apoptosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have recently identified a 58-base pair microdeletion in the promoter
region of the Fpn1 locus in radiation-induced polycythaemia
(Pcm) mice (Mok et al.,
2004). This deletion, located four nucleotides upstream of the
TATA box, conferred aberrant transcription initiation at the Fpn1
locus, which resulted in the absence of the iron-responsive element (IRE) in
the 5' untranslated region (UTR) of the vast majority of hepatic
Fpn1 transcripts in Pcm homozygotes. Consistent with a
critical in vivo role for the IRE in translational regulation of the
Fpn1 mRNA in response to cellular iron levels, Pcm mutant
mice displayed significant elevations in Fpn1 protein levels in liver and
duodenum, as well as organismal iron overload during early postnatal
development. Strikingly, an erythropoietin (Epo)-dependent polycythemia was
evident at 7 weeks of age in Pcm heterozygotes. Abrogation of the
iron accumulation and polycythemia in adult Pcm mutant animals
implicated hepcidin (Hamp), the hormonal regulator of iron homeostasis, in a
superimposed regulatory mechanism on Fpn1 protein levels
(Mok et al., 2004
).
Unexpectedly, Pcm mutant mice demonstrated a profound iron deficiency
at birth, with a severe hypochromic, microcytic anemia in homozygotes. In the
present study, we have identified dysregulated expression of placental Fpn1
during late gestation as the basis for neonatal iron deficiency in
Pcm mutants. Furthermore, we have uncovered evidence for tissue
specificity of developmental Fpn1 dysregulation, which associates with
apoptotic cell death and semidominant defects in Pcm spleen
organogenesis.
Surprisingly little is known about the development of the spleen, which is
derived from the coelomic epithelium and mesenchyme of the dorsal mesogastrium
(Green, 1967). The spleen
subserves important organismal functions, which can be attributed to distinct
anatomical regions within the organ. It represents the largest single lymphoid
organ and performs a critical immunological role reflected in the highly
organized arrangement of lymphoid and accessory cells within the white pulp
(for a review, see Fu and Chaplin,
1999
). The lymphocytic composition of the white pulp is different
from the red pulp, which also hosts a sizeable plasma cell population
(Nolte et al., 2000
;
Garcia De Vinuesa et al.,
1999
). Whereas the white pulp is primarily engaged in mounting
adaptive immune responses, the red pulp, composed of a stromal reticular
network of endothelial sinusoids, macrophages, erythroid and accessory cells,
performs a digestive function, clearing damaged and senescent red blood cells
from the circulation. This composite function of the spleen is a direct
reflection of embryonic spleen development, and results from colonization of a
stromal infrastructure, consisting of intrinsic mesenchyme, by extrinsic
hematopoietic cells at approximately embryonic (E) day 15 of murine
embryogenesis (Sasaki and Matsumura,
1988
).
Aside from the relative dearth of well-characterized splenic markers,
insight into the mechanisms regulating spleen development has been hampered by
a limited number of mutant alleles. Over the past decades, several murine
models of aberrant spleen development have been characterized, including
dominant hemimelia (Searle,
1959; Green,
1967
), and null mutants for transcription factors Hox11
(Roberts et al., 1994
;
Dear et al., 1995
;
Koehler et al., 2000
;
Kanzler and Dear, 2001
),
Wt1 (Herzer et al.,
1999
), Bapx1 (Lettice
et al., 1999
; Tribioli and
Lufkin, 1999
; Akazawa et al.,
2000
), and capsulin (Lu et
al., 2000
). Following specification of the spleen primordium
around E11.5 (Dear et al.,
1995
), these mutants display progressive spleen regression,
resulting in its complete absence as early as E13.5, but no later than E15.5.
Therefore, based on the early regression of the spleen, these models are
ill-suited to address hematopoietic cell colonization and potential cellular
interactions between intrinsic and extrinsic cell populations required for
structural and functional competence of the spleen. Here, we report a novel
defect in late embryonic spleen development in the context of aberrant fetal
iron homeostasis in Pcm mutant mice, which manifests as a severe
disruption of the red pulp sinusoidal endothelium in the postnatal spleen.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western blot analysis
Protein lysates from embryonic spleen, placenta and liver were prepared and
quantified as described previously (Mok et
al., 2004). Five µg of unboiled protein lysates from liver and
placenta and 2.5 µg of unboiled spleen lysate were separated by
electrophoresis on 8% SDS polyacrylamide gels and transferred to PVDF membrane
(BioRad). Blocking was achieved by incubation in TRIS-buffered saline
containing 5% BSA and 0.1% Tween 20. Membranes were incubated overnight at
4°C using primary antibodies against mouse Fpn1 (kindly provided by D.
Haile) at 1:2000 and mouse actin (Santa Cruz Biotechnology) at 1:5000.
Following washing, membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies (1:5000) against rabbit (for Fpn1)
or goat (for actin), and signals were detected using luminol reagent (Santa
Cruz Biotechnology).
mRNA analyses
Semi-quantitative RT-PCR for Hamp expression on E16.5 liver mRNA,
and 5' rapid amplification of cDNA ends (5'RACE) from E16.5
placenta and liver as well as E15.5 spleen were performed as described
previously (Mok et al., 2004).
Fpn1 mRNA expression was quantified via real-time RT-PCR on total RNA
samples isolated from E16.5 placental and liver as well as E15.5 spleen.
Reactions and signal detection were performed on an ABI Prism 7000 Sequence
Detection System (Applied Biosystems). Commercially available Fpn1
primers and probe were employed (Mm00489837_m1, Applied Biosystems). An
18S rRNA assay was performed using primers
5'-TCGAGGCCCTGTAATTGGAA-3' (forward),
5'-CCCTCCAATGGATCCTCGTT-3' (reverse), and TaqMan MGB probe
5'-AGTCCACTTTAAATCCTT-3' labeled with VIC (Applied Biosystems).
Relative amounts of mRNA were expressed as a ratio to 18S rRNA
levels, and normalized to an arbitrary wild-type (WT) ratio of 1.
Histology and immunohistochemistry
For histology and immunohistochemistry for Fpn1, F4/80 and Wt1, spleen,
placenta and liver were fixed in Bouin's reagent (postnatal stages) or 4%
PFA/PBS (embryonic stages), dehydrated in a graded series of ethanol, embedded
in paraffin and sectioned at 5-10 µm. Prussian blue staining for iron was
performed using the Accustain iron staining kit, according to the manufacturer
(Sigma). For Fpn1 and F4/80 antigen retrieval, sections were treated with 3%
H2O2 in methanol, whereas for Wt1, sections were boiled
in 0.01 M citric acid, pH 6.0. Blocking was achieved with goat (Fpn1 and Wt1)
or rabbit serum (F4/80). Primary antibody incubations were performed overnight
at 4°C using antibodies against Fpn1 at 1:100, F4/80 (clone CI:A3-1,
Serotec) at 1:100, or Wt1 (C-19, Santa Cruz Biotechnology) at 1:1000 dilution.
Secondary antibodies were peroxidase-conjugated with the Vectastain Elite ABC
Kit (Vector Laboratories), followed by signal detection with Vector NovaRED
substrate (Vector Laboratories). Immunohistochemistry for B220, CD3, Ter119,
MAdCAM-1, IBL-7/1, IBL-9/2 and IBL-7/22 was performed on frozen sections of
12-week-old spleens. Cryostat sections were stained with rat hybridoma
supernatants containing IBL-7/1, IBL-9/2 and IBL-7/22 antibodies followed by
biotin-amplified alkaline phosphatase detection as described
(Balázs et al., 2001).
Briefly, the sections were fixed in chilled acetone for 10 minutes, dried, and
were encircled with water-repellent wax pen. Following rehydration with PBS
containing 10% BSA for 20 minutes and removal of excess buffer, the sections
were incubated with undiluted hybridoma supernatants against lymphocyte
subsets and endothelial markers for 45 minutes. The Ly-76 erythroid antigen
was detected by the monoclonal antibody Ter119 (BD Pharmingen) at 1 µg/ml
concentration in PBS. The reaction was developed with biotinylated mouse
anti-rat kappa chain reagent (clone MRK-1, BD Pharmingen) at 1 µg/ml, and
ExtraAvidine-alkaline phosphatase conjugate (Sigma-Aldrich) using NBT/BCIP in
the presence of 1 mg/ml levamisole. Sections were counterstained with
methylene green. Rat monoclonal antibodies used: anti-B220 (clone RA3-6B2);
anti-CD3 (clone KT-3); anti-MAdCAM-1 (clone MECA-367); IBL-7/1, IBL-7/22 and
IBL-9/2 monoclonal antibodies were produced in the Department of Immunology
and Biotechnology, University of Pécs, Pécs, Hungary
(Balázs et al.,
2001
).
Determination of iron levels in embryonic tissues
The non-heme iron content of individual E15.5 liver samples was determined
as described previously (Mok et al.,
2004). Because of the small amount of tissue, E15.5 spleens were
pooled by genotype, dried, and quantified similar to liver samples.
TUNEL analysis
Embryonic spleen and liver were dissected out from E15.5 and E16.5 embryos,
fixed in 4% PFA/PBS for 2 hours at 4°C, dehydrated in a graded series of
ethanol, embedded in paraffin and sectioned at 5 µm. Sections were
processed for detection of TUNEL-positive cells using the In Situ Cell Death
Detection Kit (Roche), and mounted with the Vectashield Hard Set mounting
medium, containing DAPI (Vector Laboratories). Images were acquired via
fluorescence microscopy for fluorescein (TUNEL) and DAPI signals using
separate channels, and merged using Adobe Photoshop version 6.0.
Phenylhydrazine treatment of mice
To induce hemolytic anemia in 3- and 7-week-old wild-type and Pcm
mutant mice, phenylhydrazine hydrochloride dissolved in sterile saline was
administered intraperitoneally at a dose of 60 mg/kg body weight twice at a
24-hour interval on day 0 and day 1. Mice were euthanized on day 4, and
spleens were dissected out and weighed. Baseline spleen values were obtained
by comparison of spleen weights from untreated wild-type and heterozygous
mutant mice at 3 and 7 weeks of age.
Statistical analyses
All data are reported as the mean±s.d. All comparisons were
performed versus wild-type cohorts, and analyzed for statistically significant
differences using the Student's unpaired t-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Interestingly, Hamp mRNA levels were significantly reduced in
E16.5 Pcm mutant liver by semi-quantitative RT-PCR
(Fig. 1G), indicating that
Hamp, the principal negative hormonal regulator of iron balance
(Park et al., 2001;
Pigeon et al., 2001
) (for a
review, see Ganz, 2003
), is
also responsive to iron deficiency during embryogenesis.
Increased Fpn1 protein expression in the spleen of Pcm mutant embryos
In striking contrast to placenta and liver, Fpn1 protein levels were higher
in embryonic spleen from Pcm mutant embryos
(Fig. 1C). Importantly,
immunohistochemistry detected high levels of Fpn1 expression throughout the
spleen of Pcm homozygous embryos, consistent with upregulation of
Fpn1 expression in stromal cells (Fig.
1E). Thus, the present study uncovered evidence for
tissue-specific regulation of embryonic Fpn1 expression by virtue of
differential protein levels in Pcm mutant placenta and liver compared
with spleen.
Decreased Fpn1 mRNA levels in placenta, liver and spleen
To determine whether transcript abundance caused the observed changes in
embryonic Fpn1 protein levels, we performed real-time RT-PCR analysis for
Fpn1 mRNA expression. Fpn1 mRNA levels were statistically
significantly reduced in both placenta and liver from Pcm mutants
(Fig. 2A,B), consistent with
the decreased Fpn1 protein expression (Fig.
1A,B). Surprisingly, Fpn1 mRNA levels were also
significantly reduced in Pcm homozygous mutant spleen
(Fig. 2C), despite increased
Fpn1 protein levels (Fig.
1C,E). This discordance between Fpn1 mRNA and protein
expression in the spleen indicated that the upregulation of Fpn1 resulted from
a post-transcriptional mechanism.
|
Semidominant defect in Pcm mutant spleens during late embryogenesis
To ascertain whether aberrant Fpn1 protein expression in E15.5 spleen
associated with an organ phenotype, we conducted a time-course analysis of
spleen development. At E15.5, the spleen appeared to be consistent across all
genotypes with regard to organ size and gross morphology
(Fig. 3A). However, beginning
at E16.5 (data not shown) and increasingly evident at E17.5, Pcm
mutant spleens were reduced in size relative to wild type, more severe in
homozygotes (Fig. 3B). It is
noteworthy that the spleen size in homozygotes effectively regressed, rather
than merely stalled, as evident from comparison of spleen size at E17.5 and
E15.5 (Fig. 3A,B). At birth, a
completely penetrant, semidominant defect in spleen size was observed in
Pcm mutant pups (Fig.
3C). Thus, unlike previous models of defective splenogenesis
(Searle, 1959;
Roberts et al., 1994
;
Herzer et al., 1999
;
Lettice et al., 1999
;
Lu et al., 2000
), we observed
grossly intact spleens in Pcm mutants at E15.5
(Fig. 3A), indicating that
Pcm represents a novel murine model for disrupted splenogenesis.
|
|
The tumor suppressor Wt1 plays an essential role in organ
development, including that of the kidney, gonad and mesothelial structures
(Kreidberg et al., 1993). In
addition, Wt1 regulates spleen development, as Wt1 null mice
exhibit complete regression of the spleen by E15.5
(Herzer et al., 1999
). Using
Wt1 as a marker for splenic stromal cells, analysis at E15.5 demonstrated a
generalized pattern of Wt1 expression encompassing the majority of cells
present at this stage in both wild-type and Pcm mutant spleens
(Fig. 4D). At birth, similar to
other putative stromal cell markers in the spleen, such as Hox11
(Dear et al., 1995
;
Kanzler and Dear, 2001
) and
Bapx1 (Akazawa et al.,
2000
), Wt1 expression levels appeared significantly reduced and
were primarily restricted to the capsule and subcapsular region in wild type
(data not shown). Because of the low levels of Wt1 expression and abnormal
spleen structure in postnatal day 0 (P0) Pcm homozygotes
(Fig. 3C), Wt1 analysis at this
stage was not informative (data not shown). Importantly, at E15.5, the pattern
of Wt1 expression (Fig. 4D)
correlated with the Fpn1 expression pattern in Pcm mutant spleens
(Fig. 1E).
Taken together, the regression of Pcm spleens could not be reconciled with primarily macrophage cell death during spleen development. Rather, increased Fpn1 protein expression in stromal cells, in the context of organismal iron deficiency, associates with apoptotic death of stromal cells, resulting in the semidominant defect in spleen size at birth.
Postnatal changes in Pcm mutant spleens
Homozygous Pcm mutants maintained a decreased spleen size, as
observed at 7 weeks of age (Fig.
3D). Conversely, heterozygous spleens gradually increased in size
from birth, reaching wild-type size at 7 weeks of age
(Fig. 3D). Histologically, at
P0, Pcm heterozygous spleens appeared comparable to wild type,
whereas homozygous spleens displayed discrete alterations in cellular
composition, such as a decreased prevalence of basophilic cells
(Fig. 3E). Postnatal
organization and development of the white pulp appeared to occur to a
significant extent in both heterozygous and homozygous mutant spleens, as
distinct white pulp follicles were clearly identifiable at 7 weeks
(Fig. 3F). In addition,
heterozygous spleens were notable for increased eosinophilic cells within the
red pulp as well as the marginal sinus surrounding the white pulp follicles.
Seven-week-old homozygous spleens exhibited an increased white pulp to red
pulp ratio relative to wild type (Fig.
3F). Given the increased rate of red blood cell production
characteristic of Pcm heterozygotes at 7 weeks of age
(Mok et al., 2004), the
histological changes observed in the red pulp were consistent with increased
erythropoiesis in Pcm heterozygous spleens.
Impaired red pulp function in Pcm heterozygous spleen
Given the regression in spleen size during late gestation
(Fig. 3C,D), Pcm
homozygotes are likely to become functionally asplenic. In contrast,
Pcm heterozygotes retained a significant amount of spleen stroma, and
exhibited a significant increase in relative spleen size from during postnatal
development (Fig. 3C,D).
Consistent with its reduced size at P0
(Fig. 3C), heterozygous mutant
spleens demonstrated a significantly lower weight at 3 weeks of age as
compared with wild type (Fig.
5A). However, by 7 weeks of age, and correlating with the gross
appearance (Fig. 3D), similar
spleen weights were measured in wild-type and heterozygous mutant mice
(Fig. 5A). This increase in
relative spleen size was transient in nature, as by 12 weeks of age,
heterozygous spleen weights were again statistically significantly reduced as
compared with wild type (Fig.
5A).
|
To quantify the functional erythropoietic capacity of Pcm
heterozygous spleens, we performed phenylhydrazine treatment of mice to induce
hemolytic anemia (Itano et al.,
1975). Strikingly, Pcm heterozygous spleens were notable
for a significantly reduced hyperplastic response at both 3 and 7 weeks of age
(Fig. 5B,C), indicating a
decreased functional capacity to foster erythroid progenitor and precursor
proliferation. No increase in spleen size was evident in
phenylhydrazine-treated Pcm homozygotes at 3 weeks of age, consistent
with functional asplenia (data not shown).
Additional evidence for the functional capacity of Pcm heterozygous spleens was gleaned from the analysis of iron distribution in the context of erythrophagocytosis. At P0, no iron staining was observed within the Pcm heterozygous spleen, reflecting the perinatal iron deficiency in Pcm mutants (Fig. 5D). However, by 12 weeks of age, Pcm heterozygotes displayed a significant, punctate pattern of iron accumulation confined to the red pulp region (Fig. 5E). Given the polycythemia at 7 weeks of age and hematocrit normalization at 12 weeks of age in Pcm heterozygotes, this staining pattern probably reflects erythrophagocytosis followed by iron scavenging within the reticuloendothelial macrophages of the red pulp.
Taken together, the Pcm heterozygous mutant spleens appear to retain red pulp function with regard to erythrophagocytosis, as well as extramedullary hematopoiesis in response to stimulation, albeit at a significantly reduced level relative to wild type.
Characterization of hematopoietic cell lineages in postnatal Pcm mutant spleens
Ter119 represents a marker for late erythroid lineage cells
(Kina et al., 2000). Within
the red pulp, Ter119-positive erythroid cells were observed at a similar
density and distribution in wild-type and heterozygous spleens at 12 weeks of
age (Fig. 6A). Homozygotes
displayed a moderately reduced density of Ter119-positive cells in red pulp
regions (Fig. 6A). The mature
white pulp follicle consists of a T-cell rich, periarteriolar lymphoid sheath
(PALS) region, which is surrounded by primary follicles comprised of B cells,
the predominant lymphoid cell population of the spleen
(Veerman and van Ewijk, 1975
)
(for a review, see Fu and Chaplin,
1999
). Pcm heterozygous spleens displayed a similar
appearance of B- and T-cell regions, as demonstrated by B220 and CD3
expression, respectively (Fig.
6B,C). Interestingly, Pcm homozygotes exhibited an
increased ratio of B to T cells. In addition, the central arteriole, normally
positioned within a central location of the T-cell PALS region, was located
more peripherally in heterozygotes, and appeared within the B-cell region in
Pcm homozygotes (Fig.
6B,C). Thus, although the overall follicular structure of the
white pulp appeared largely intact, lymphocyte composition and positioning of
the central arteriole were abnormal in Pcm mutant spleens, more
severe in homozygotes.
|
In summary, significant abnormalities in red pulp sinusoidal architecture were observed in Pcm mutant spleens, which are consistent with the reduced functional competence of the red pulp during Epo-stimulated erythropoiesis in Pcm heterozygous spleens.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In striking contrast to placenta and liver, Fpn1 protein levels were
increased in Pcm mutant spleen, despite significantly decreased
Fpn1 mRNA levels. Based on Fpn1 transcript levels and
sequence, it appears unlikely that the increased Fpn1 protein levels result
from a transcriptional mechanism. Furthermore, although decreased
Hamp expression is consistent with increased Fpn1 protein levels in
the spleen, decreased Fpn1 protein expression in the placenta and liver
renders a systemic regulatory effect unlikely. Thus, similar to postnatal
development (Mok et al.,
2004), an unknown post-transcriptional mechanism appears to govern
Fpn1 protein levels during late gestation, which warrants additional studies.
In this context, it would be of significant interest to characterize further
the basis for decreased Fpn1 protein expression in embryonic liver, which,
strikingly, transitions to increased hepatic Fpn1 protein levels at birth
(Mok et al., 2004
). Regardless
of the mechanisms of tissue-specific Fpn1 regulation, increased Fpn1-mediated
iron efflux in stromal cells should potentiate cellular iron deficiency in the
spleen resulting from decreased embryonic iron levels. This pathophysiology
correlates with dramatic consequences at the cellular and organ level, which
become manifest as a semidominant defect in spleen development during late
gestation (for a model, see Fig.
7).
Consistent with its pleiotropic role in cellular metabolism, iron has long
been known to modulate pathways that regulate proliferation and cell death
(for a review, see Le and Richardson,
2002). For example, iron chelation has been demonstrated to induce
apoptosis (Fukuchi et al.,
1994
; Haq et al.,
1995
) and promote cell-cycle arrest
(Lederman et al., 1984
).
Similarly, cellular hypoxia represents a potent stimulus for the apoptotic
cascade (for a review, see Brunelle and
Chandel, 2002
). The effects of iron chelation mimic cellular
hypoxia (Wang and Semenza,
1993
), presumably via iron-dependent regulators of the hypoxia
signaling cascade, such as a prolyl hydroxylase
(Ivan et al., 2001
;
Jaakkola et al., 2001
), which
regulates hypoxia-inducible factor-1
(HIF-1
) (for a review, see
Semenza, 2003
). Therefore, it
is conceivable that apoptotic cell death detected in Pcm mutant
spleens results from a synergistic effect of iron deficiency and anemia during
development. Recently, upregulation of the pleomorphic adenomas gene-like 2
(PLAGL2) protein, a putative zinc-finger transcription factor, has been
demonstrated in response to hypoxia and iron chelation
(Furukawa et al., 2001
). In
turn, PLAGL2 induces apoptosis and upregulation of a proapoptotic factor BNip3
(Mizutani et al., 2002
).
Interestingly, BNip3 is itself induced by hypoxia, a unique feature of
regulation among the Bcl-2 family of apoptotic factors
(Bruick, 2000
). Thus, given the
relative specificity of these factors in response to iron and hypoxia stimuli,
we are currently assessing their potential involvement in the stromal cell
apoptosis in Pcm mutant spleens. Furthermore, to the best of our
knowledge, defects in spleen development have not been reported in extant
mouse models of iron deficiency and anemia. A detailed analysis of these
mutants would corroborate whether iron deficiency and/or Fpn1 protein
upregulation induce apoptosis in spleen stromal cells.
The defects in spleen organogenesis in Pcm mutant mice are
distinct from existing genetic mouse models of aberrant spleen development.
Complete asplenia is observed in dominant hemimelia
(Searle, 1959;
Green, 1967
), Hox11
(Roberts et al., 1994
;
Dear et al., 1995
;
Koehler et al., 2000
;
Kanzler and Dear, 2001
),
Wt1 (Herzer et al.,
1999
), Bapx1 (Lettice
et al., 1999
; Tribioli and
Lufkin, 1999
; Akazawa et al.,
2000
) and capsulin mutant mice
(Lu et al., 2000
). In these
mutants, primary induction of the splenic primordium is followed by complete
involution of the organ before E15.5. Evidence for apoptotic cell death within
the spleen has been observed in Hox11
(Dear et al., 1995
),
Wt1 (Herzer et al.,
1999
) and capsulin mutant animals
(Lu et al., 2000
). Because
Pcm mutant spleens appear intact at E15.5, this implicates Fpn1 and
iron homeostasis in the disruption of a distinct, subsequent developmental
phase in spleen organogenesis. Attempts to determine genetic interaction of
Hox11 with Wt1 (Koehler
et al., 2000
), Bapx1
(Akazawa et al., 2000
) and
capsulin (Lu et al., 2000
)
provided evidence that Wt1 functions downstream of Hox11
(Koehler et al., 2000
).
Interestingly, Pcm mutant spleens at E15.5 exhibit similar Wt1
expression levels and patterns compared with wild type, suggesting that the
defects in Pcm mice are independent of Hox11 and
Wt1 function. As Pcm heterozygotes display a reduced
severity of splenic disruption and homozygotes retain residual spleen tissue,
Pcm mice represent a complementary resource to the existing mutants
that will enable a more comprehensive understanding of the mechanisms and
pathways of spleen organogenesis throughout development.
In the mouse, the red pulp constitutes a significant erythropoietic organ
during normal development as well as in response to hypoxia, phlebotomy, or
exogenous Epo administration (Brodsky et
al., 1966; Bozzini et al.,
1970
). Pcm heterozygotes demonstrate an Epo-dependent
polycythemia at 7 weeks of age (Mok et
al., 2004
). Here, we show that Pcm heterozygous spleens
demonstrate changes consistent with elevated Epo levels, including a transient
increase in spleen weight and red pulp hyperplasia, which temporally coincide
with the transient polycythemia. In contrast, similar to genetically asplenic
or splenectomized mice, the spleen rudiment in Pcm homozygotes should
not respond to factors stimulating erythropoiesis
(Bozzini et al., 1970
;
Lozzio, 1972
). Therefore,
despite elevated Epo levels, functional asplenia and severe perinatal iron
deficiency probably represent the limiting factors toward the diminished rate
of productive erythropoiesis during postnatal development in Pcm
homozygotes, resulting in the lower peak hematocrit as compared with
heterozygous mutants.
The stromal defects during organogenesis of Pcm mutant spleens correlate well with the severe red pulp abnormalities postnatally, such as aberrant sinusoidal endothelial cell populations. In turn, these defects lead to decreased functional competence of Pcm mutant spleens, as reflected by impaired splenic hyperplasia in response to phenylhydrazine treatment. Furthermore, discrete abnormalities of the white pulp, as well as the marginal zone, have also been detected. Thus, it is likely that interactions between the intrinsic, stromal cell population, and extrinsic, hematopoietic cell lineages mediate proper structural and functional organization of the spleen during postnatal development. Therefore, further characterization of the patterning and organization of the white pulp in Pcm mutant spleens should yield additional insight into the mechanisms of spleen organogenesis in mammals.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abboud, S. and Haile, D. J. (2000). A novel
mammalian iron-regulated protein involved in intracellular iron metabolism.
J. Biol. Chem. 275,19906
-19912.
Aisen, P., Enns, C. and Wessling-Resnick, M. (2001). Chemistry and biology of eukaryotic iron metabolism. Int. J. Biochem. Cell. Biol. 33,940 -959.[CrossRef][Medline]
Akazawa, H., Komuro, I., Sugitani, Y., Yazaki, Y., Nagai, R. and
Noda, T. (2000). Targeted disruption of the homeobox
transcription factor Bapx1 results in lethal skeletal dysplasia with asplenia
and gastroduodenal malformation. Genes Cells
5, 499-513.
Andrews, N. C. (2000). Iron metabolism: iron deficiency and iron overload. Annu. Rev. Genomics Hum. Genet. 1,75 -98.[CrossRef][Medline]
Balazs, M., Grama, L. and Balogh, P. (1999). Detection of phenotypic heterogeneity within the murine splenic vasculature using rat monoclonal antibodies IBL-7/1 and IBL-7/22. Hybridoma 18,177 -182.[Medline]
Balazs, M., Horvath, G., Grama, L. and Balogh, P. (2001). Phenotypic identification and development of distinct microvascular compartments in the postnatal mouse spleen. Cell. Immunol. 212,126 -137.[CrossRef][Medline]
Bozzini, C. E., Barrio Rendo, M. E., Devoto, F. C. and Epper, C.
E. (1970). Studies on medullary and extramedullary
erythropoiesis in the adult mouse. Am. J. Physiol.
219,724
-728.
Brodsky, I., Dennis, L. H., Kahn, S. B. and Brady, L. W. (1966). Normal mouse erythropoiesis. I. The role of the spleen in mouse erythropoiesis. Cancer Res. 26,198 -201.[Medline]
Bruick, R. K. (2000). Expression of the gene
encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc.
Natl. Acad. Sci. USA 97,9082
-9087.
Brunelle, J. K. and Chandel, N. S. (2002). Oxygen deprivation induced cell death: an update. Apoptosis 7,475 -482.[CrossRef][Medline]
Cattanach, B. M. (1995). A dominant polycythaemia. Mouse Genome 93,1027 -1028.
Crowe, C., Dandekar, P., Fox, M., Dhingra, K., Bennet, L. and Hanson, M. A. (1995). The effects of anaemia on heart, placenta and body weight, and blood pressure in fetal and neonatal rats. J. Physiol. 488,515 -519.[Abstract]
Dear, T. N., Colledge, W. H., Carlton, M. B., Lavenir, I.,
Larson, T., Smith, A. J., Warren, A. J., Evans, M. J., Sofroniew, M. V. and
Rabbitts, T. H. (1995). The Hox11 gene is essential for cell
survival during spleen development. Development
121,2909
-2915.
Donovan, A., Brownlie, A., Zhou, Y., Shepard, J., Pratt, S. J., Moynihan, J., Paw, B. H., Drejer, A., Barut, B., Zapata, A. et al. (2000). Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403,776 -781.[CrossRef][Medline]
Fu, Y. X. and Chaplin, D. D. (1999). Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17,399 -433.[CrossRef][Medline]
Fukuchi, K., Tomoyasu, S., Tsuruoka, N. and Gomi, K. (1994). Iron deprivation-induced apoptosis in HL-60 cells. FEBS Lett. 350,139 -142.[CrossRef][Medline]
Furukawa, T., Adachi, Y., Fujisawa, J., Kambe, T., Yamaguchi-Iwai, Y., Sasaki, R., Kuwahara, J., Ikehara, S., Tokunaga, R. and Taketani, S. (2001). Involvement of PLAGL2 in activation of iron deficient- and hypoxia-induced gene expression in mouse cell lines. Oncogene 20,4718 -4727.[CrossRef][Medline]
Gambling, L., Danzeisen, R., Gair, S., Lea, R. G., Charania, Z., Solanky, N., Joory, K. D., Srai, S. K. and McArdle, H. J. (2001). Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and in vitro. Biochem. J. 356,883 -889.[CrossRef][Medline]
Ganz, T. (2003). Hepcidin, a key regulator of
iron metabolism and mediator of anemia of inflammation.
Blood 102,783
-788.
Garcia De Vinuesa, C., Gulbranson-Judge, A., Khan, M., O'Leary, P., Cascalho, M., Wabl, M., Klaus, G. G., Owen, M. J. and MacLennan, I. C. (1999). Dendritic cells associated with plasmablast survival. Eur. J. Immunol. 29,3712 -3721.[CrossRef][Medline]
Green, M. C. (1967). A defect of the splanchnic mesoderm caused by the mutant gene dominant hemimelia in the mouse. Dev. Biol. 15,62 -89.[CrossRef][Medline]
Haq, R. U., Wereley, J. P. and Chitambar, C. R. (1995). Induction of apoptosis by iron deprivation in human leukemic CCRF-CEM cells. Exp. Hematol. 23,428 -432.[Medline]
Hentze, M. W., Muckenthaler, M. U. and Andrews, N. C. (2004). Balancing acts: molecular control of mammalian iron metabolism. Cell 117,285 -297.[CrossRef][Medline]
Herzer, U., Crocoll, A., Barton, D., Howells, N. and Englert, C. (1999). The Wilms tumor suppressor gene wt1 is required for development of the spleen. Curr. Biol. 9, 837-840.[CrossRef][Medline]
Itano, H. A., Hirota, K. and Hosokawa, K. (1975). Mechanism of induction of haemolytic anaemia by phenylhydrazine. Nature 256,665 -667.[Medline]
Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M.,
Salic, A., Asara, J. M., Lane, W. S. and Kaelin, W. G., Jr
(2001). HIFalpha targeted for VHL-mediated destruction by proline
hydroxylation: implications for O2 sensing. Science
292,464
-468.
Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert,
J., Gaskell, S. J., Kriegsheim, A. V., Hebestreit, H. F., Mukherji, M.,
Schofield, C. J. et al. (2001). Targeting of HIF-alpha to the
von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
Science 292,468
-472.
Kanzler, B. and Dear, T. N. (2001). Hox11 acts cell autonomously in spleen development and its absence results in altered cell fate of mesenchymal spleen precursors. Dev. Biol. 234,231 -243.[CrossRef][Medline]
Kina, T., Ikuta, K., Takayama, E., Wada, K., Majumdar, A. S., Weissman, I. L. and Katsura, Y. (2000). The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage. Br. J. Haematol. 109,280 -287.[CrossRef][Medline]
Koehler, K., Franz, T. and Dear, T. N. (2000). Hox11 is required to maintain normal Wt1 mRNA levels in the developing spleen. Dev. Dyn. 218,201 -206.[CrossRef][Medline]
Kraal, G., Schornagel, K., Streeter, P. R., Holzmann, B. and Butcher, E. C. (1995). Expression of the mucosal vascular addressin, MAdCAM-1, on sinus-lining cells in the spleen. Am. J. Pathol. 147,763 -771.[Abstract]
Kreidberg, J. A., Sariola, H., Loring, J. M., Maeda, M., Pelletier, J., Housman, D. and Jaenisch, R. (1993). WT-1 is required for early kidney development. Cell 74,679 -691.[Medline]
Le, N. T. and Richardson, D. R. (2002). The role of iron in cell cycle progression and the proliferation of neoplastic cells. Biochim. Biophys. Acta. 1603,31 -46.[CrossRef][Medline]
Lederman, H. M., Cohen, A., Lee, J. W., Freedman, M. H. and Gelfand, E. W. (1984). Deferoxamine: a reversible S-phase inhibitor of human lymphocyte proliferation. Blood 64,748 -753.[Abstract]
Lettice, L. A., Purdie, L. A., Carlson, G. J., Kilanowski, F.,
Dorin, J. and Hill, R. E. (1999). The mouse bagpipe gene
controls development of axial skeleton, skull, and spleen. Proc.
Natl. Acad. Sci. USA 96,9695
-9700.
Liu, X., Hill, P. and Haile, D. J. (2002). Role of the ferroportin iron-responsive element in iron and nitric oxide dependent gene regulation. Blood Cells Mol. Dis. 29,315 -326.[CrossRef][Medline]
Lozzio, B. B. (1972). Hematopoiesis in
congenitally asplenic mice. Am. J. Physiol.
222,290
-295.
Lu, J., Chang, P., Richardson, J. A., Gan, L., Weiler, H. and
Olson, E. N. (2000). The basic helix-loop-helix transcription
factor capsulin controls spleen organogenesis. Proc. Natl. Acad.
Sci. USA 97,9525
-9530.
McArdle, H. J., Danzeisen, R., Fosset, C. and Gambling, L. (2003). The role of the placenta in iron transfer from mother to fetus and the relationship between iron status and fetal outcome. Biometals 16,161 -167.[CrossRef][Medline]
McKie, A. T. and Barlow, D. J. (2004). The SLC40 basolateral iron transporter family (IREG1/ferroportin/MTP1). Pflügers Arch. 447,801 -806.[CrossRef][Medline]
McKie, A. T., Marciani, P., Rolfs, A., Brennan, K., Wehr, K., Barrow, D., Miret, S., Bomford, A., Peters, T. J., Farzaneh, F. et al. (2000). A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol. Cell. 5,299 -309.[Medline]
Mizutani, A., Furukawa, T., Adachi, Y., Ikehara, S. and
Taketani, S. (2002). A zinc-finger protein, PLAGL2, induces
the expression of a proapoptotic protein Nip3, leading to cellular apoptosis.
J. Biol. Chem. 277,15851
-15858.
Mok, H., Jelinek, J., Pai, S., Cattanach, B. M., Prchal, J. T.,
Youssoufian, H. and Schumacher, A. (2004). Disruption of
ferroportin 1 regulation causes dynamic alterations in iron homeostasis and
erythropoiesis in polycythaemia mice. Development
131,1859
-1868.
Morris, L., Graham, C. F. and Gordon, S. (1991). Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80. Development 112,517 -526.[Abstract]
Nicolas, G., Bennoun, M., Porteu, A., Mativet, S., Beaumont, C.,
Grandchamp, B., Sirito, M., Sawadogo, M., Kahn, A. and Vaulont, S.
(2002). Severe iron deficiency anemia in transgenic mice
expressing liver hepcidin. Proc. Natl. Acad. Sci. USA
99,4596
-4601.
Nolte, M. A., Hoen, E. N., van Stijn, A., Kraal, G. and Mebius, R. E. (2000). Isolation of the intact white pulp. Quantitative and qualitative analysis of the cellular composition of the splenic compartments. Eur. J. Immunol. 30,626 -634.[CrossRef][Medline]
Park, C. H., Valore, E. V., Waring, A. J. and Ganz, T.
(2001). Hepcidin, a urinary antimicrobial peptide synthesized in
the liver. J. Biol. Chem.
276,7806
-7810.
Pigeon, C., Ilyin, G., Courselaud, B., Leroyer, P., Turlin, B.,
Brissot, P. and Loreal, O. (2001). A new mouse liver-specific
gene, encoding a protein homologous to human antimicrobial peptide hepcidin,
is overexpressed during iron overload. J. Biol. Chem.
276,7811
-7819.
Roberts, C. W., Shutter, J. R. and Korsmeyer, S. J. (1994). Hox11 controls the genesis of the spleen. Nature 368,747 -749.[CrossRef][Medline]
Sasaki, K. and Matsumura, G. (1988). Spleen lymphocytes and haemopoiesis in the mouse embryo. J. Anat. 160,27 -37.[Medline]
Schmidt, E. E., MacDonald, I. C. and Groom, A. C. (1993). Comparative aspects of splenic microcirculatory pathways in mammals: the region bordering the white pulp. Scanning Microsc. 7,613 -628.[Medline]
Searle, A. G. (1959). Hereditary absence of spleen in the mouse. Nature 184,1419 -1420.
Semenza, G. L. (2003). Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721-732.[CrossRef][Medline]
Srai, S. K., Bomford, A. and McArdle, H. J. (2002). Iron transport across cell membranes: molecular understanding of duodenal and placental iron uptake. Best Pract. Res. Clin. Haematol. 15,243 -259.[CrossRef][Medline]
Tribioli, C. and Lufkin, T. (1999). The murine
Bapx1 homeobox gene plays a critical role in embryonic development of the
axial skeleton and spleen. Development
126,5699
-5711.
Veerman, A. J. and van Ewijk, W. (1975). White pulp compartments in the spleen of rats and mice. A light and electron microscopic study of lymphoid and non-lymphoid celltypes in T- and B-areas. Cell Tissue Res. 156,417 -441.[Medline]
Wang, G. L. and Semenza, G. L. (1993). Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82,3610 -3615.[Abstract]
Related articles in Development: