First published online 1 September 2004
doi: 10.1242/dev.01342
Development 131, 4871-4881 (2004)
Published by The Company of Biologists 2004
Dysregulation of ferroportin 1 interferes with spleen organogenesis in polycythaemia mice
Henry Mok1,
Miriam Mendoza1,
Josef T. Prchal2,
Péter Balogh3 and
Armin Schumacher1,*
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
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Fig. 1. Decreased Fpn1 protein expression in Pcm mutant placenta and
embryonic liver is consistent with fetal iron deficiency, and contrasts with
increased Fpn1 expression in embryonic spleen. At E16.5, placenta (A) and
liver (B) show a graded decrease in Fpn1 protein expression on western blot.
Approximate molecular mass: Fpn1, 68 kDa; actin, 41 kDa. (C) Western blot
analysis reveals increased Fpn1 protein expression in E15.5 spleen. (D)
Immunohistochemistry demonstrates a significant reduction of labyrinth cell
expression of Fpn1 in E16.5 placenta from Pcm homozygotes. Original
magnification 400x. (E) Immunohistochemistry reveals a widespread
increase in Fpn1 expression in E15.5 Pcm mutant spleens. Original
magnification 400x. (F) Analysis of non-heme iron demonstrates
significant reduction of iron content in E15.5 Pcm livers, more
severe in homozygotes. *, P<0.0001. (G)
Semiquantitative RT-PCR of E16.5 liver indicates lower Hamp mRNA
expression in Pcm mutants. Band sizes: Hamp, 171 bp;
ß-actin, 250 bp.
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Fig. 2. Embryonic Fpn1 mRNA expression is decreased in Pcm mutant
mice. (A) Real-time RT-PCR analysis of E15.5 spleen shows reduction of
Fpn1 mRNA in Pcm homozygous mutants. **,
P<0.01. Decreased mRNA levels in placenta (B) and liver (C) in
E16.5 Pcm mutants. *, P<0.05; **,
P<0.01; ***, P<0.001. 5'RACE PCR of
E15.5 spleen (D) and E16.5 placenta mRNA (E) demonstrates aberrant transcripts
in Pcm homozygous mutants. Wild-type band, 630 bp.
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Fig. 3. Pcm mutant mice show a semidominant defect in spleen development
during late embryogenesis. (A) Spleen development and size appear consistent
across all genotypes at E15.5. Original magnification 40x. (B) By E17.5,
significant reduction in spleen size is observed in Pcm mutants, more
severe in homozygotes. Compare with A and note that spleen size in homozygotes
regresses. Original magnification 40x. At P0 (C) and 7 weeks (D),
spleens (arrowheads) of homozygous mutants maintain consistent decrease in
size, whereas heterozygotes undergo a significant increase in size from P0 to
7 weeks. Original magnification (C) 16x and (D) 6x. H&E
staining of spleen sections at P0 (E) and 7 weeks (F) reveals red and white
pulp compartmentalization of wild-type and mutant spleens; arrowhead indicates
white pulp follicle. Original magnification (E) 200x and (F)
50x.
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Fig. 4. Increased apoptosis detected in Pcm mutant spleens. TUNEL analysis
at E15.5 (A) and E16.5 (B) indicates a significant increase in TUNEL-positive
foci in Pcm mutant spleens, higher in homozygotes. Images represent
merged composites of TUNEL (fluorescein) and DAPI-counterstained sections,
original magnification 200x. (C) Immunohistochemistry for F4/80 at E15.5
reveals similar distribution of positive cells within spleens across all
genotypes. Original magnification, 400x. (D) Immunohistochemistry for
Wt1 at E15.5 demonstrates widespread stromal cell staining within the spleen.
Original magnification, 400x.
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Fig. 5. Pcm heterozygous mutant spleens exhibit functional competence for
red pulp hyperplasia and red blood cell clearance. (A) Spleen weights
demonstrate a transient increase in Pcm heterozygotes at 7 weeks of
age. *, P<0.01. Phenylhydrazine treatment of 3-week (B)
and 7-week-old mice (C) demonstrates reduced splenic hyperplasia in
Pcm heterozygotes. **, P<0.0001. (D) Prussian
blue staining of P0 spleen reveals no iron accumulation in wild-type or
Pcm heterozygous mutants. Original magnification, 200x. (E) At
12 weeks of age, Pcm heterozygotes display significant red pulp iron
accumulation. Original magnification, 100x.
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Fig. 6. Intact white pulp follicles but aberrant red pulp sinusoidal architecture
in Pcm mutant spleens. Immunohistochemistry of hematopoietic lineage
markers Ter119 (A), B220 (B), and CD3 (C) within the spleen at 12 weeks of
age. Arrowheads denote central arteriole. (D) MAdCAM-1 immunohistochemistry
indicates the presence of marginal sinuses in Pcm mutant spleens at
12 weeks of age. Immunohistochemistry of IBL-7/1 (E), IBL-9/2 (F), and
IBL-7/22 (G) demonstrates abnormal arrangement of the microvasculature
endothelial components of the red pulp in Pcm mutant spleens at 12
weeks of age. Original magnification, 200x.
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Fig. 7. Model for Fpn1-mediated embryonic iron deficiency and spleen stromal
defects in Pcm mice. Decreased Fpn1 mRNA and protein expression in
placental syncytiotrophoblast cells leads to decreased maternal-to-fetal iron
transport and embryonic iron deficiency. Although the spleen exhibits
decreased Fpn1 mRNA, increased Fpn1 protein is observed, which should
mediate cellular iron efflux from stromal cells. Under the influence of iron
deficiency and/or hypoxia, spleen stromal cell death translates to decreased
organ size and defects of the red pulp vascular endothelium. The broken line
indicates the uncertain relationship between decreased Fpn1 expression in the
fetal liver and the functional consequences, for instance with regard to fetal
erythropoiesis.
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© The Company of Biologists Ltd 2004
