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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01081

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Disruption of ferroportin 1 regulation causes dynamic alterations in iron homeostasis and erythropoiesis in polycythaemia mice

Henry Mok¹, Jaroslav Jelinek^2,*, Sonia Pai¹, Bruce M. Cattanach³, Josef T. Prchal², Hagop Youssoufian^1, and Armin Schumacher^1,

¹ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA
³ MRC Mammalian Genetics Unit, Medical Research Council, Harwell, Oxfordshire OX11 0RD, UK

View larger version (24K): [in a new window]   Fig. 1. Pcm heterozygotes are characterized by external ruddiness and transient polycythemia mapping to proximal chromosome 1. (A) Ears and hindpaws show increased redness in 7-week-old heterozygous mice, particularly in vasculature (arrowheads). (B) Microcapillary hematocrits measured at several postnatal timepoints demonstrate transient polycythemia in Pcm/+ mice, and a transient anemia in Pcm/Pcm mice. Asterisk, P<0.001 for mutant classes when compared with wild-type mice per timepoint. (C) Pcm critical region. Phenotype co-segregates with microsatellite marker D1Mit236; n represents ratio of Pcm/+ mutant progeny (Hct > 55%) retaining heterozygosity for parental alleles (101/HeH) of markers. Physical map position (Mb) as per the Sanger Centre mouse genome assembly (NCBI Build 30).

View larger version (21K): [in a new window]   Fig. 2. Deletion in promoter region of Fpn1 leads to altered transcriptional initiation, but not transcript abundance, in Pcm mutant mice. (A) A 58 bp deletion in the promoter region of Fpn1 constitutes the Pcm mutation. Black arrowheads indicate wild-type transcription initiation site determined by 5'RACE, red arrowheads indicate Pcm/Pcm initiation sites. ATG, translational start; IRE, iron responsive element; boxed region indicates TATA box. (B) Mutant phenotype co-segregates with deletion in Fpn1, as demonstrated by PCR genotyping using primers indicated by overlined and underlined sequences in A. Wild-type band 230 bp; mutant band 172 bp. (C) 5'RACE PCR demonstrates altered Fpn1 transcript identity in Pcm mutant mice. Wild-type band 630 bp. (D) Real-time RT-PCR to quantify Fpn1 transcript abundance reveals no statistically significant difference among genotypes at 3 weeks of age. The horizontal line indicates the median.

View larger version (36K): [in a new window]   Fig. 3. Iron deficiency anemia at birth and iron accumulation in young adults in Pcm mutant mice. (A) Peripheral blood smear remarkable for hypochromic, microcytic red blood cells in Pcm/Pcm mice at P0. Wright-Giemsa stain. (B) Prussian Blue staining of P0 liver sections reveals decreased parenchymal liver iron in Pcm/Pcm mice. (C) Quantification of total iron at P0 demonstrates organismal iron deficiency in Pcm mutant pups. (D) Prussian Blue staining of sections of 12-week-old liver shows numerous localized iron deposits in Pcm/Pcm mice. (E) Hepatic iron content significantly elevated by 12 weeks of age in Pcm/Pcm mice. (F) Decreased serum iron levels in Pcm mutants compared with wild type. Asterisk, P<0.05; double asterisk, P<0.01; triple asterisk, P<0.001.

View larger version (28K): [in a new window]   Fig. 4. Polycythemia is secondary to Epo-driven, bone marrow-derived red cell production. (A) Real-time RT-PCR reveals elevated Epo transcript levels in mutant liver and kidney at 3 weeks of age. The horizontal line indicates the median. *P<0.05; **P<0.01; ***P<0.001. (B) In vitro, FACS-based determination of late-progenitor and precursor erythroid cell differentiation to Ter119-positivity. Pcm/Pcm mice at 3 weeks of age show increased bone marrow erythroid activity at day 2. *P<0.001. (C) Increased hemoglobinization and cellularity of Pcm/+ bone marrow, as demonstrated by O-D staining of sections at 7 weeks of age. (D) In vitro analysis reveals increased bone marrow erythroid activity in Pcm/+ mice at 7 weeks of age. *P<0.001. (E) Real-time RT-PCR using 12-week kidney samples shows no statistically significant differences in Epo transcript levels among genotypes. The horizontal line indicates the median.

View larger version (56K): [in a new window]   Fig. 5. Altered Fpn1 protein and Hamp mRNA expression in Pcm mutant mice. (A) Western blot analysis reveals graded increase in Fpn1 protein expression in P0 mutant liver, with concomitant graded decrease in ferritin expression. Approximate molecular masses: Fpn1, 68 kDa; ferritin (Ftn), 20 kDa; actin, 41 kDa. (B) Three-week-old liver shows similar graded increase in Fpn1 protein expression on a western blot. (C) Seven-week-old liver exhibits persistence of Fpn1 protein expression only in polycythemic Pcm heterozygous mice on a western blot. (D) Seven-week-old duodenum demonstrates pattern of Fpn1 protein expression similar to liver on a western blot. (E) Twelve-week-old liver reveals downregulation of Fpn1 protein expression in all genotypes on a western blot. (F) Seven-week-old liver RT-PCR shows no significant difference in Fpn1 mRNA expression, but lack of Hamp mRNA expression in polycythemic mutant mice. Band sizes: Fpn1, 338 bp; Hamp, 171 bp; ß-actin, 250 bp. (G) Twelve-week-old liver RT-PCR demonstrates upregulation of Hamp mRNA expression in all animals.