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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

PiT1 expression during liver growth.(A) In situ hybridization (ISH) of a wild-type E12.5 embryo shows heavy PiT1 signal in the developing liver (black arrow), whereas low signal is detected in the brain (white arrows) and throughout the embryos. (B) ISH with the PiT1 sense probe gives no signal. Bar, 1 mm. (C) Ratios of PiT1 and PiT2 mRNA expression levels between embryonic (E12.5) and post-natal (P15) wild-type livers, as determined by real-time RT-PCR. (D) PiT1/PiT2 expression ratios in embryonic (E12.5) and post-natal (P15) livers, as determined by real-time RT-PCR. (E) Quantification of the expression of PiT1 and PiT2 in normal and PiT1Δ5/Δ5 E12.5 livers by real-time RT-PCR. Note the 1.5-fold overexpression of PiT2 in PiT1- livers. (F) Gene induction after partial hepatectomy. Total cellular RNA collected in a time course after partial hepatectomy in wild-type mice was analyzed by real time RT-PCR for the expression of PiT1, PiT2 and PCNA as a marker of proliferation. Results are reported after normalization to the expression of Pinin.
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pone-0009148-g007: PiT1 expression during liver growth.(A) In situ hybridization (ISH) of a wild-type E12.5 embryo shows heavy PiT1 signal in the developing liver (black arrow), whereas low signal is detected in the brain (white arrows) and throughout the embryos. (B) ISH with the PiT1 sense probe gives no signal. Bar, 1 mm. (C) Ratios of PiT1 and PiT2 mRNA expression levels between embryonic (E12.5) and post-natal (P15) wild-type livers, as determined by real-time RT-PCR. (D) PiT1/PiT2 expression ratios in embryonic (E12.5) and post-natal (P15) livers, as determined by real-time RT-PCR. (E) Quantification of the expression of PiT1 and PiT2 in normal and PiT1Δ5/Δ5 E12.5 livers by real-time RT-PCR. Note the 1.5-fold overexpression of PiT2 in PiT1- livers. (F) Gene induction after partial hepatectomy. Total cellular RNA collected in a time course after partial hepatectomy in wild-type mice was analyzed by real time RT-PCR for the expression of PiT1, PiT2 and PCNA as a marker of proliferation. Results are reported after normalization to the expression of Pinin.

Mentions: Although widespread, PiT1 expression in adult mouse, rat and human is rather variable between tissues and is low in the adult liver [2], [7], [8]. Our in situ hybridization (ISH) experiments on E12.5 wild-type embryo sections demonstrated that PiT1 was expressed at low levels throughout the embryo, except in the liver. Faint PiT1 expression was detected in the developing brain as previously described [8], but this signal was much lower than the high level of expression found in the fetal liver (Fig. 7A and B). This result is in sharp contrast with the low level of expression of PiT1 in adult livers, but is consistent with a role of PiT1 during developmental liver growth. We confirmed these results by quantifying the expression of PiT1 and PiT2 in fetal E12.5 and post-natal P15 wild-type livers by real-time RT-PCR. The level of PiT1 in the fetal liver was 4.4-fold higher than that in the post-natal liver, whereas fetal and post-natal PiT2 expression were similar (Fig. 7C). Moreover, quantification of the relative expression of PiT1 and PiT2 in wild-type fetal and post-natal livers revealed that PiT1 expression was 3.4-fold higher in fetal livers and 1.7-fold lower in post natal livers than PiT2 (Fig. 7D), further illustrating the need for PiT1 during developmental liver growth. Of importance, while PiT1 was undetectable in PiT1Δ5/Δ5 E12.5 livers, the expression of PiT2 was 1.5-fold higher than in the wild-type livers (Fig. 7E), a value that is comparable to the increase found in the whole mutant embryo (Fig. 1F). To determine in which cell types PiT1 was most expressed, we performed immunostaining of PiT1 on wild-type and mutant sections using PiT1 antibodies from commercial or laboratory sources [9]. Unfortunately, we are unable to generate a specific signal using the available anti-mouse antibodies, and the expression of PiT1 in specific liver cell types during mouse development remains an open question. However, PiT1 has been localized in adult hepatocytes, but not in cholangiocytes, in rat livers [36]. Consistent with this finding, we further illustrated the role of PiT1 in liver growth by performing partial hepatectomy on normal adult mice. In this model, the remnant liver undergoes rapid division to re-establish the original weight of the organ. Our results show that, although PiT2 expression is unchanged, PiT1 is highly (3.5-fold) induced within 2 h following partial hepatectomy (Fig. 7F) indicating that PiT1 is likely to be essential during the early stage of compensatory liver growth. Although further experiments are necessary, this observation could be consistent with a higher expression of PiT1 in developing versus adult fetal livers and the low proliferation rate of PiT1Δ5/Δ5 livers.


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)

PiT1 expression during liver growth.(A) In situ hybridization (ISH) of a wild-type E12.5 embryo shows heavy PiT1 signal in the developing liver (black arrow), whereas low signal is detected in the brain (white arrows) and throughout the embryos. (B) ISH with the PiT1 sense probe gives no signal. Bar, 1 mm. (C) Ratios of PiT1 and PiT2 mRNA expression levels between embryonic (E12.5) and post-natal (P15) wild-type livers, as determined by real-time RT-PCR. (D) PiT1/PiT2 expression ratios in embryonic (E12.5) and post-natal (P15) livers, as determined by real-time RT-PCR. (E) Quantification of the expression of PiT1 and PiT2 in normal and PiT1Δ5/Δ5 E12.5 livers by real-time RT-PCR. Note the 1.5-fold overexpression of PiT2 in PiT1- livers. (F) Gene induction after partial hepatectomy. Total cellular RNA collected in a time course after partial hepatectomy in wild-type mice was analyzed by real time RT-PCR for the expression of PiT1, PiT2 and PCNA as a marker of proliferation. Results are reported after normalization to the expression of Pinin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2818845&req=5

pone-0009148-g007: PiT1 expression during liver growth.(A) In situ hybridization (ISH) of a wild-type E12.5 embryo shows heavy PiT1 signal in the developing liver (black arrow), whereas low signal is detected in the brain (white arrows) and throughout the embryos. (B) ISH with the PiT1 sense probe gives no signal. Bar, 1 mm. (C) Ratios of PiT1 and PiT2 mRNA expression levels between embryonic (E12.5) and post-natal (P15) wild-type livers, as determined by real-time RT-PCR. (D) PiT1/PiT2 expression ratios in embryonic (E12.5) and post-natal (P15) livers, as determined by real-time RT-PCR. (E) Quantification of the expression of PiT1 and PiT2 in normal and PiT1Δ5/Δ5 E12.5 livers by real-time RT-PCR. Note the 1.5-fold overexpression of PiT2 in PiT1- livers. (F) Gene induction after partial hepatectomy. Total cellular RNA collected in a time course after partial hepatectomy in wild-type mice was analyzed by real time RT-PCR for the expression of PiT1, PiT2 and PCNA as a marker of proliferation. Results are reported after normalization to the expression of Pinin.
Mentions: Although widespread, PiT1 expression in adult mouse, rat and human is rather variable between tissues and is low in the adult liver [2], [7], [8]. Our in situ hybridization (ISH) experiments on E12.5 wild-type embryo sections demonstrated that PiT1 was expressed at low levels throughout the embryo, except in the liver. Faint PiT1 expression was detected in the developing brain as previously described [8], but this signal was much lower than the high level of expression found in the fetal liver (Fig. 7A and B). This result is in sharp contrast with the low level of expression of PiT1 in adult livers, but is consistent with a role of PiT1 during developmental liver growth. We confirmed these results by quantifying the expression of PiT1 and PiT2 in fetal E12.5 and post-natal P15 wild-type livers by real-time RT-PCR. The level of PiT1 in the fetal liver was 4.4-fold higher than that in the post-natal liver, whereas fetal and post-natal PiT2 expression were similar (Fig. 7C). Moreover, quantification of the relative expression of PiT1 and PiT2 in wild-type fetal and post-natal livers revealed that PiT1 expression was 3.4-fold higher in fetal livers and 1.7-fold lower in post natal livers than PiT2 (Fig. 7D), further illustrating the need for PiT1 during developmental liver growth. Of importance, while PiT1 was undetectable in PiT1Δ5/Δ5 E12.5 livers, the expression of PiT2 was 1.5-fold higher than in the wild-type livers (Fig. 7E), a value that is comparable to the increase found in the whole mutant embryo (Fig. 1F). To determine in which cell types PiT1 was most expressed, we performed immunostaining of PiT1 on wild-type and mutant sections using PiT1 antibodies from commercial or laboratory sources [9]. Unfortunately, we are unable to generate a specific signal using the available anti-mouse antibodies, and the expression of PiT1 in specific liver cell types during mouse development remains an open question. However, PiT1 has been localized in adult hepatocytes, but not in cholangiocytes, in rat livers [36]. Consistent with this finding, we further illustrated the role of PiT1 in liver growth by performing partial hepatectomy on normal adult mice. In this model, the remnant liver undergoes rapid division to re-establish the original weight of the organ. Our results show that, although PiT2 expression is unchanged, PiT1 is highly (3.5-fold) induced within 2 h following partial hepatectomy (Fig. 7F) indicating that PiT1 is likely to be essential during the early stage of compensatory liver growth. Although further experiments are necessary, this observation could be consistent with a higher expression of PiT1 in developing versus adult fetal livers and the low proliferation rate of PiT1Δ5/Δ5 livers.

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