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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

Disruption of the PiT1 gene leads to defective liver development and anemia.(A–B) Comparison between wild-type and PiT1Δ5/Δ5 embryos at E10.5 (A) and E12.5 (B). Note the reduced size, pale appearance and small liver (black arrow) of the E12.5 mutant embryos. (C, D) Haematoxilin and eosin (H&E) staining of E12.5 sections from PiT1+/+ (C) and PiT1Δ5/Δ5 embryos (D). fl: fetal liver; mg: mid-gut loop; h: heart. Bar, 100 µm. (E) Morphological appearance of wild-type and mutant E12.5 fetal livers. Bar, 0.5 mm (F) Fetal liver nucleated cell counts in E12.5 wild-type and mutant embryos. (G) Quantification of PiT1, PiT2, albumin and a-fetoprotein mRNA expression by real-time RT-PCR in E12.5 PiT1+/+ and PiT1Δ5/Δ5 embryos. (H–K) H&E staining of E12.5 (H,I) and E11.5 (J,K) sagital sections of PiT1+/+ and PiT1Δ5/Δ5 livers illustrating the hypocellularity of the mutant livers. Bar, 100 µm. # indicate significant differences as compared to wild-type controls with P<0.001 (Student's t test).
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pone-0009148-g002: Disruption of the PiT1 gene leads to defective liver development and anemia.(A–B) Comparison between wild-type and PiT1Δ5/Δ5 embryos at E10.5 (A) and E12.5 (B). Note the reduced size, pale appearance and small liver (black arrow) of the E12.5 mutant embryos. (C, D) Haematoxilin and eosin (H&E) staining of E12.5 sections from PiT1+/+ (C) and PiT1Δ5/Δ5 embryos (D). fl: fetal liver; mg: mid-gut loop; h: heart. Bar, 100 µm. (E) Morphological appearance of wild-type and mutant E12.5 fetal livers. Bar, 0.5 mm (F) Fetal liver nucleated cell counts in E12.5 wild-type and mutant embryos. (G) Quantification of PiT1, PiT2, albumin and a-fetoprotein mRNA expression by real-time RT-PCR in E12.5 PiT1+/+ and PiT1Δ5/Δ5 embryos. (H–K) H&E staining of E12.5 (H,I) and E11.5 (J,K) sagital sections of PiT1+/+ and PiT1Δ5/Δ5 livers illustrating the hypocellularity of the mutant livers. Bar, 100 µm. # indicate significant differences as compared to wild-type controls with P<0.001 (Student's t test).

Mentions: To identify at which developmental stage PiT1- embryos die, genotyping of litters harvested from serial timed matings from E3.5 to birth was performed (Table 1). Results revealed that living PiT1Δ5/Δ5 embryos were detected up to E12.5, and that no PiT1Δ5/Δ5 embryos survived after this stage. Until E9.5 no macroscopic abnormalities were observed in PiT1Δ5/Δ5 embryos by gross anatomical and histological examinations. At E10.5, embryos were found to be slightly growth retarded, without morphological abnormalities (Fig. 2A). At E12.5, PiT1Δ5/Δ5 embryos could be easily recognized by their significant growth retardation and profound anemia (Fig. 2B). The most striking defect was observed in the developing liver, occupying only a small fraction of the abdominal areas (Fig. 2C and D). Morphological examination showed that E12.5 mutant livers, composed of four lobes, were extremely pale and severely reduced in size as compared to normal livers (Fig. 2E). Total liver nucleated cell counts represented only 2% of wild-type livers at this stage (Fig. 2F). Real-time RT-PCR analysis of RNA extracted from E12.5 PiT1+/+ and PiT1Δ5/Δ5 embryos demonstrated that the level of albumin and α-fetoprotein transcripts in mutant embryos was markedly reduced, further suggesting the decrease in hepatoblast number or function (Fig. 2G). Histological analysis of PiT1Δ5/Δ5 E12.5 livers further confirmed the reduced cellularity and increased empty space, indicating less dense packing of cells in the PiT1Δ5/Δ5 as compared to the wild-type livers (Fig. 2H and I). Hypocellularity and presence of large sinuses in the mutant livers were already visible at E11.5 (Fig. 2J and K).


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)

Disruption of the PiT1 gene leads to defective liver development and anemia.(A–B) Comparison between wild-type and PiT1Δ5/Δ5 embryos at E10.5 (A) and E12.5 (B). Note the reduced size, pale appearance and small liver (black arrow) of the E12.5 mutant embryos. (C, D) Haematoxilin and eosin (H&E) staining of E12.5 sections from PiT1+/+ (C) and PiT1Δ5/Δ5 embryos (D). fl: fetal liver; mg: mid-gut loop; h: heart. Bar, 100 µm. (E) Morphological appearance of wild-type and mutant E12.5 fetal livers. Bar, 0.5 mm (F) Fetal liver nucleated cell counts in E12.5 wild-type and mutant embryos. (G) Quantification of PiT1, PiT2, albumin and a-fetoprotein mRNA expression by real-time RT-PCR in E12.5 PiT1+/+ and PiT1Δ5/Δ5 embryos. (H–K) H&E staining of E12.5 (H,I) and E11.5 (J,K) sagital sections of PiT1+/+ and PiT1Δ5/Δ5 livers illustrating the hypocellularity of the mutant livers. Bar, 100 µm. # indicate significant differences as compared to wild-type controls with P<0.001 (Student's t test).
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Related In: Results  -  Collection

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pone-0009148-g002: Disruption of the PiT1 gene leads to defective liver development and anemia.(A–B) Comparison between wild-type and PiT1Δ5/Δ5 embryos at E10.5 (A) and E12.5 (B). Note the reduced size, pale appearance and small liver (black arrow) of the E12.5 mutant embryos. (C, D) Haematoxilin and eosin (H&E) staining of E12.5 sections from PiT1+/+ (C) and PiT1Δ5/Δ5 embryos (D). fl: fetal liver; mg: mid-gut loop; h: heart. Bar, 100 µm. (E) Morphological appearance of wild-type and mutant E12.5 fetal livers. Bar, 0.5 mm (F) Fetal liver nucleated cell counts in E12.5 wild-type and mutant embryos. (G) Quantification of PiT1, PiT2, albumin and a-fetoprotein mRNA expression by real-time RT-PCR in E12.5 PiT1+/+ and PiT1Δ5/Δ5 embryos. (H–K) H&E staining of E12.5 (H,I) and E11.5 (J,K) sagital sections of PiT1+/+ and PiT1Δ5/Δ5 livers illustrating the hypocellularity of the mutant livers. Bar, 100 µm. # indicate significant differences as compared to wild-type controls with P<0.001 (Student's t test).
Mentions: To identify at which developmental stage PiT1- embryos die, genotyping of litters harvested from serial timed matings from E3.5 to birth was performed (Table 1). Results revealed that living PiT1Δ5/Δ5 embryos were detected up to E12.5, and that no PiT1Δ5/Δ5 embryos survived after this stage. Until E9.5 no macroscopic abnormalities were observed in PiT1Δ5/Δ5 embryos by gross anatomical and histological examinations. At E10.5, embryos were found to be slightly growth retarded, without morphological abnormalities (Fig. 2A). At E12.5, PiT1Δ5/Δ5 embryos could be easily recognized by their significant growth retardation and profound anemia (Fig. 2B). The most striking defect was observed in the developing liver, occupying only a small fraction of the abdominal areas (Fig. 2C and D). Morphological examination showed that E12.5 mutant livers, composed of four lobes, were extremely pale and severely reduced in size as compared to normal livers (Fig. 2E). Total liver nucleated cell counts represented only 2% of wild-type livers at this stage (Fig. 2F). Real-time RT-PCR analysis of RNA extracted from E12.5 PiT1+/+ and PiT1Δ5/Δ5 embryos demonstrated that the level of albumin and α-fetoprotein transcripts in mutant embryos was markedly reduced, further suggesting the decrease in hepatoblast number or function (Fig. 2G). Histological analysis of PiT1Δ5/Δ5 E12.5 livers further confirmed the reduced cellularity and increased empty space, indicating less dense packing of cells in the PiT1Δ5/Δ5 as compared to the wild-type livers (Fig. 2H and I). Hypocellularity and presence of large sinuses in the mutant livers were already visible at E11.5 (Fig. 2J and K).

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