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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

Apoptotic defects in PiT1 mutant fetal liver cells.(A–B) Cytokeratin 18 (CK18) staining (brown) reveals the presence of hepatoblasts in E12.5 mutant sections (black arrows). (C–D) Staining of Ter119 (brown) reveals great loss of erythroid cells in E12.5 mutant liver sections (black arrows). (E–F) Activated caspase 3 staining of E12.5 liver sections reveals excessive apoptotic activity in mutant liver sections (black arrow) that was not present in Ter119 positive cells (white arrows). Bar, 100 µm.
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pone-0009148-g006: Apoptotic defects in PiT1 mutant fetal liver cells.(A–B) Cytokeratin 18 (CK18) staining (brown) reveals the presence of hepatoblasts in E12.5 mutant sections (black arrows). (C–D) Staining of Ter119 (brown) reveals great loss of erythroid cells in E12.5 mutant liver sections (black arrows). (E–F) Activated caspase 3 staining of E12.5 liver sections reveals excessive apoptotic activity in mutant liver sections (black arrow) that was not present in Ter119 positive cells (white arrows). Bar, 100 µm.

Mentions: We next asked whether the reduction in liver cell density was caused by reduced proliferation or whether a loss of liver cells had occurred due to cell death. Histological sections revealed that most of the E12.5 mutant livers displayed abundant fragmented pyknotic nuclei, whereas only few were visible on control sections (Fig. 5A and B). TUNEL staining of liver sections confirmed the above observation (Fig. 5C to F). Of interest, the level of TUNEL staining in E11.5 mutant livers was comparable to that of wild-type controls (Fig. 5E and F). Immunodetection of activated caspase 3, revealed a significant number of apoptotic cells in E12.5 PiT1Δ5/Δ5 livers, while few apoptotic cells were found in control livers (Fig. 5G and H). The level of activated caspase 3 in E11.5 mutant livers was comparable to that of wild-type (Fig. 5I and J). We next examined the proliferation status of PiT1-deficient livers. Staining with the proliferation marker Ki67 was less intense in sections of E12.5 PiT1Δ5/Δ5 livers than in the wild-type (Fig. 5K and L). These results were confirmed by performing BrDU pulse-labeling of dividing cells in E11.5 pregnant females, and subsequent detection of BrDU-positive cells. Wild-type fetal livers showed heavy nuclear staining in the entire liver, whereas BrDU staining was reduced in PiT1- samples directly demonstrating a subnormal growth rate in this organ (Fig. 5M and N). Antibodies recognizing the fetal hepatocyte marker cytokeratin 18 (CK18) revealed label-positive cells in PiT1 mutant embryos at E12.5, suggesting that, at least at this stage, developing hepatocytes were present (Fig. 6A and B). However, it was notable that CK18 staining revealed disorganized tissue architecture and that marker-positive cells were often rounded up or pyknotic. Staining against the erythroid cell marker Ter119 revealed a significant reduction in marker-positive cells in mutant livers (Fig. 6C and D), correlating with the loss of erythroid lineage cells. Activated caspase 3 staining of sections from the same livers revealed that Ter119-positive cells were not labeled in mutant sections whereas apoptosis was evident in CK18-expressing cells (Fig. 6E and F). Thus, while developing hepatocytes emerge in PiT1 mutant livers at E12.5, loss of hepatocytes by apoptosis is a frequent occurrence whereas the scarcity of Ter119-labeled cells in mutant livers is unlikely to correlate with apoptotic activity.


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)

Apoptotic defects in PiT1 mutant fetal liver cells.(A–B) Cytokeratin 18 (CK18) staining (brown) reveals the presence of hepatoblasts in E12.5 mutant sections (black arrows). (C–D) Staining of Ter119 (brown) reveals great loss of erythroid cells in E12.5 mutant liver sections (black arrows). (E–F) Activated caspase 3 staining of E12.5 liver sections reveals excessive apoptotic activity in mutant liver sections (black arrow) that was not present in Ter119 positive cells (white arrows). Bar, 100 µm.
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Related In: Results  -  Collection

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pone-0009148-g006: Apoptotic defects in PiT1 mutant fetal liver cells.(A–B) Cytokeratin 18 (CK18) staining (brown) reveals the presence of hepatoblasts in E12.5 mutant sections (black arrows). (C–D) Staining of Ter119 (brown) reveals great loss of erythroid cells in E12.5 mutant liver sections (black arrows). (E–F) Activated caspase 3 staining of E12.5 liver sections reveals excessive apoptotic activity in mutant liver sections (black arrow) that was not present in Ter119 positive cells (white arrows). Bar, 100 µm.
Mentions: We next asked whether the reduction in liver cell density was caused by reduced proliferation or whether a loss of liver cells had occurred due to cell death. Histological sections revealed that most of the E12.5 mutant livers displayed abundant fragmented pyknotic nuclei, whereas only few were visible on control sections (Fig. 5A and B). TUNEL staining of liver sections confirmed the above observation (Fig. 5C to F). Of interest, the level of TUNEL staining in E11.5 mutant livers was comparable to that of wild-type controls (Fig. 5E and F). Immunodetection of activated caspase 3, revealed a significant number of apoptotic cells in E12.5 PiT1Δ5/Δ5 livers, while few apoptotic cells were found in control livers (Fig. 5G and H). The level of activated caspase 3 in E11.5 mutant livers was comparable to that of wild-type (Fig. 5I and J). We next examined the proliferation status of PiT1-deficient livers. Staining with the proliferation marker Ki67 was less intense in sections of E12.5 PiT1Δ5/Δ5 livers than in the wild-type (Fig. 5K and L). These results were confirmed by performing BrDU pulse-labeling of dividing cells in E11.5 pregnant females, and subsequent detection of BrDU-positive cells. Wild-type fetal livers showed heavy nuclear staining in the entire liver, whereas BrDU staining was reduced in PiT1- samples directly demonstrating a subnormal growth rate in this organ (Fig. 5M and N). Antibodies recognizing the fetal hepatocyte marker cytokeratin 18 (CK18) revealed label-positive cells in PiT1 mutant embryos at E12.5, suggesting that, at least at this stage, developing hepatocytes were present (Fig. 6A and B). However, it was notable that CK18 staining revealed disorganized tissue architecture and that marker-positive cells were often rounded up or pyknotic. Staining against the erythroid cell marker Ter119 revealed a significant reduction in marker-positive cells in mutant livers (Fig. 6C and D), correlating with the loss of erythroid lineage cells. Activated caspase 3 staining of sections from the same livers revealed that Ter119-positive cells were not labeled in mutant sections whereas apoptosis was evident in CK18-expressing cells (Fig. 6E and F). Thus, while developing hepatocytes emerge in PiT1 mutant livers at E12.5, loss of hepatocytes by apoptosis is a frequent occurrence whereas the scarcity of Ter119-labeled cells in mutant livers is unlikely to correlate with apoptotic activity.

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