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The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development.

Beck L, Leroy C, Beck-Cormier S, Forand A, Salaün C, Paris N, Bernier A, Ureña-Torres P, Prié D, Ollero M, Coulombel L, Friedlander G - PLoS ONE (2010)

Bottom Line: In contrast, mutant fetal livers display decreased proliferation and massive apoptosis.Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic.This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U845, Centre de Recherche Croissance et Signalisation, Paris, France. laurent.beck@inserm.fr

ABSTRACT

Background: PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na(+)-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/principal findings: To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and alleles in heterozygous compounds results in late embryonic lethality (E14.5-E16.5) with phenotypic features intermediate between and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/significance: This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

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Related in: MedlinePlus

Elevated proportion of circulating primitive erythrocytes and identical number of hematopoietic progenitors per fetal liver in PiT1Δ5/Δ5 embryos.(A, B) May-Grünwald/Giemsa staining of blood smears from E12.5 embryos. At E12.5 almost all blood cells are nucleated; enlarged cells represent primitive erythroid precursors. (C) Mean cell and nucleus diameter of erythroid cells in PiT1+/+ and PiT1Δ5/Δ5 E12.5 embryos, measured with the NIS-Elements AR 3.00 software. (D) RT-PCR analysis of the expression of globin chains in E12.5 PiT1+/+ and PiT1Δ5/Δ5 livers. Results are expressed as ratios between fetal and adult globin expression, as indicated. (E–H) In vitro differentiation of E12.5 fetal-liver cells from wild-type and PiT1Δ5/Δ5 embryos. The number of CFU-E (E), BFU-E (F), CFU-GM (G), and CFU-GEMM (H) colonies per fetal liver (FL) or per 105 nucleated fetal-liver cells are indicated. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).
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pone-0009148-g004: Elevated proportion of circulating primitive erythrocytes and identical number of hematopoietic progenitors per fetal liver in PiT1Δ5/Δ5 embryos.(A, B) May-Grünwald/Giemsa staining of blood smears from E12.5 embryos. At E12.5 almost all blood cells are nucleated; enlarged cells represent primitive erythroid precursors. (C) Mean cell and nucleus diameter of erythroid cells in PiT1+/+ and PiT1Δ5/Δ5 E12.5 embryos, measured with the NIS-Elements AR 3.00 software. (D) RT-PCR analysis of the expression of globin chains in E12.5 PiT1+/+ and PiT1Δ5/Δ5 livers. Results are expressed as ratios between fetal and adult globin expression, as indicated. (E–H) In vitro differentiation of E12.5 fetal-liver cells from wild-type and PiT1Δ5/Δ5 embryos. The number of CFU-E (E), BFU-E (F), CFU-GM (G), and CFU-GEMM (H) colonies per fetal liver (FL) or per 105 nucleated fetal-liver cells are indicated. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).

Mentions: Peripheral blood smears showed that the majority of erythroid cells in PiT1Δ5/Δ5 mutants were larger than in the wild-type (Fig. 4A and B), which was confirmed by measuring the cell and nucleus mean diameters of erythroid cells in the PiT1+/+ and PiT1Δ5/Δ5 mice (Fig. 4C). Since primitive erythroid precursors are approximately twice as large and contain twice as much hemoglobin than their mature counterparts [33], this suggests that PiT1Δ5/Δ5 embryos have a higher proportion of immature erythroid cells. We confirmed this by quantifying the expression of hemoglobin genes (Fig. 4D). Hemoglobin molecules contain globin chains derived both from the α-globin (Hbb-a) and β-globin (Hbb-b) gene loci. Although definitive erythroid cells in the mouse express α1-globin, α2-globin, β1-globin, and β2-globin, primitive erythroid cells in addition express ζ-globin (Hba-x), βH1-globin (Hbb-bh1), and εy-globin (Hbb-y) [33], [34]. We found that the amount of adult and fetal forms of globin chains in the mutant represented 2 to 9% and 19 to 25% of the wild-type, respectively. Accordingly, the ratios between fetal and adult forms of hemoglobin was higher in E12.5 PiT1Δ5/Δ5 livers than in the wild-type (Fig. 4D), suggesting that the maturational globin switching may be impaired or delayed in PiT1-deficient embryos. Alternatively, a defect in the developmental niche provided by the fetal liver for the maturation and enucleation of primitive erythrocytes may explain this phenotype [35].


The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development.

Beck L, Leroy C, Beck-Cormier S, Forand A, Salaün C, Paris N, Bernier A, Ureña-Torres P, Prié D, Ollero M, Coulombel L, Friedlander G - PLoS ONE (2010)

Elevated proportion of circulating primitive erythrocytes and identical number of hematopoietic progenitors per fetal liver in PiT1Δ5/Δ5 embryos.(A, B) May-Grünwald/Giemsa staining of blood smears from E12.5 embryos. At E12.5 almost all blood cells are nucleated; enlarged cells represent primitive erythroid precursors. (C) Mean cell and nucleus diameter of erythroid cells in PiT1+/+ and PiT1Δ5/Δ5 E12.5 embryos, measured with the NIS-Elements AR 3.00 software. (D) RT-PCR analysis of the expression of globin chains in E12.5 PiT1+/+ and PiT1Δ5/Δ5 livers. Results are expressed as ratios between fetal and adult globin expression, as indicated. (E–H) In vitro differentiation of E12.5 fetal-liver cells from wild-type and PiT1Δ5/Δ5 embryos. The number of CFU-E (E), BFU-E (F), CFU-GM (G), and CFU-GEMM (H) colonies per fetal liver (FL) or per 105 nucleated fetal-liver cells are indicated. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818845&req=5

pone-0009148-g004: Elevated proportion of circulating primitive erythrocytes and identical number of hematopoietic progenitors per fetal liver in PiT1Δ5/Δ5 embryos.(A, B) May-Grünwald/Giemsa staining of blood smears from E12.5 embryos. At E12.5 almost all blood cells are nucleated; enlarged cells represent primitive erythroid precursors. (C) Mean cell and nucleus diameter of erythroid cells in PiT1+/+ and PiT1Δ5/Δ5 E12.5 embryos, measured with the NIS-Elements AR 3.00 software. (D) RT-PCR analysis of the expression of globin chains in E12.5 PiT1+/+ and PiT1Δ5/Δ5 livers. Results are expressed as ratios between fetal and adult globin expression, as indicated. (E–H) In vitro differentiation of E12.5 fetal-liver cells from wild-type and PiT1Δ5/Δ5 embryos. The number of CFU-E (E), BFU-E (F), CFU-GM (G), and CFU-GEMM (H) colonies per fetal liver (FL) or per 105 nucleated fetal-liver cells are indicated. * and # indicate significant differences as compared to wild-type controls with P<0.05 and P<0.001, respectively (Student's t test).
Mentions: Peripheral blood smears showed that the majority of erythroid cells in PiT1Δ5/Δ5 mutants were larger than in the wild-type (Fig. 4A and B), which was confirmed by measuring the cell and nucleus mean diameters of erythroid cells in the PiT1+/+ and PiT1Δ5/Δ5 mice (Fig. 4C). Since primitive erythroid precursors are approximately twice as large and contain twice as much hemoglobin than their mature counterparts [33], this suggests that PiT1Δ5/Δ5 embryos have a higher proportion of immature erythroid cells. We confirmed this by quantifying the expression of hemoglobin genes (Fig. 4D). Hemoglobin molecules contain globin chains derived both from the α-globin (Hbb-a) and β-globin (Hbb-b) gene loci. Although definitive erythroid cells in the mouse express α1-globin, α2-globin, β1-globin, and β2-globin, primitive erythroid cells in addition express ζ-globin (Hba-x), βH1-globin (Hbb-bh1), and εy-globin (Hbb-y) [33], [34]. We found that the amount of adult and fetal forms of globin chains in the mutant represented 2 to 9% and 19 to 25% of the wild-type, respectively. Accordingly, the ratios between fetal and adult forms of hemoglobin was higher in E12.5 PiT1Δ5/Δ5 livers than in the wild-type (Fig. 4D), suggesting that the maturational globin switching may be impaired or delayed in PiT1-deficient embryos. Alternatively, a defect in the developmental niche provided by the fetal liver for the maturation and enucleation of primitive erythrocytes may explain this phenotype [35].

Bottom Line: In contrast, mutant fetal livers display decreased proliferation and massive apoptosis.Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic.This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U845, Centre de Recherche Croissance et Signalisation, Paris, France. laurent.beck@inserm.fr

ABSTRACT

Background: PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na(+)-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/principal findings: To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and alleles in heterozygous compounds results in late embryonic lethality (E14.5-E16.5) with phenotypic features intermediate between and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/significance: This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.

Show MeSH
Related in: MedlinePlus