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PTBP1 is required for embryonic development before gastrulation.

Suckale J, Wendling O, Masjkur J, Jäger M, Münster C, Anastassiadis K, Stewart AF, Solimena M - PLoS ONE (2011)

Bottom Line: Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation.We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst.However, further post-implantation development requires PTBP1 and stalls in homozygous animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

View Article: PubMed Central - PubMed

Affiliation: Molecular Diabetology, Paul Langerhans Institute Dresden, School of Medicine and University Clinic Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.

ABSTRACT
Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

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Cell division & apoptosis in mutant embryos.The figure shows control and Ptbp1  embryo sections at E7.5 immunolabelled for BrdU (top row) and processed by TUNEL (bottom row). The former is a cell division assay measuring the integration of the nucleotide analogue bromodeoxyuridine (BrdU). The latter is an apoptosis assay based on terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Control embryos (1st column) showed strong BrdU signal especially in the epiblast, the yolk sac area, and the ectoplacental cone while being negative for TUNEL. Heterozygous decidua were typically BrdU-labelled most strongly in the periphery, visible only in KO1 and cropped in control1 and KO2. Homozygous mutant embryos showed less (KO1) to no BrdU labelling (KO2) varying with embryo and section. Most  mutants analysed showed some degree of TUNEL (exemplified by KO3 and KO4). The longest diameter of the embryo is indicated below each panel and the relative sizes of the panels are indicated with dashed lines.
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pone-0016992-g008: Cell division & apoptosis in mutant embryos.The figure shows control and Ptbp1 embryo sections at E7.5 immunolabelled for BrdU (top row) and processed by TUNEL (bottom row). The former is a cell division assay measuring the integration of the nucleotide analogue bromodeoxyuridine (BrdU). The latter is an apoptosis assay based on terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Control embryos (1st column) showed strong BrdU signal especially in the epiblast, the yolk sac area, and the ectoplacental cone while being negative for TUNEL. Heterozygous decidua were typically BrdU-labelled most strongly in the periphery, visible only in KO1 and cropped in control1 and KO2. Homozygous mutant embryos showed less (KO1) to no BrdU labelling (KO2) varying with embryo and section. Most mutants analysed showed some degree of TUNEL (exemplified by KO3 and KO4). The longest diameter of the embryo is indicated below each panel and the relative sizes of the panels are indicated with dashed lines.

Mentions: To take a more detailed look at cell proliferation in embryos lacking Ptbp1 versus those containing either 1 or 2 normal alleles, we performed BrdU and TUNEL assays (Figure 8). As estimated by BrdU incorporation into newly synthesised DNA, cell division was high throughout the control E7.5 embryos analysed, with especially strong signal coming from the epiblast, the visceral endoderm, the area of the yolk sac, and the ectoplacental cone. Interestingly, the epiblast and the endoderm were also labelled strongly using Ptbp antibodies (Figure 7). There appears to be a correlation between the expression levels of Ptbps and cell division in embryos. Also, mutants clearly synthesise less DNA.


PTBP1 is required for embryonic development before gastrulation.

Suckale J, Wendling O, Masjkur J, Jäger M, Münster C, Anastassiadis K, Stewart AF, Solimena M - PLoS ONE (2011)

Cell division & apoptosis in mutant embryos.The figure shows control and Ptbp1  embryo sections at E7.5 immunolabelled for BrdU (top row) and processed by TUNEL (bottom row). The former is a cell division assay measuring the integration of the nucleotide analogue bromodeoxyuridine (BrdU). The latter is an apoptosis assay based on terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Control embryos (1st column) showed strong BrdU signal especially in the epiblast, the yolk sac area, and the ectoplacental cone while being negative for TUNEL. Heterozygous decidua were typically BrdU-labelled most strongly in the periphery, visible only in KO1 and cropped in control1 and KO2. Homozygous mutant embryos showed less (KO1) to no BrdU labelling (KO2) varying with embryo and section. Most  mutants analysed showed some degree of TUNEL (exemplified by KO3 and KO4). The longest diameter of the embryo is indicated below each panel and the relative sizes of the panels are indicated with dashed lines.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040740&req=5

pone-0016992-g008: Cell division & apoptosis in mutant embryos.The figure shows control and Ptbp1 embryo sections at E7.5 immunolabelled for BrdU (top row) and processed by TUNEL (bottom row). The former is a cell division assay measuring the integration of the nucleotide analogue bromodeoxyuridine (BrdU). The latter is an apoptosis assay based on terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Control embryos (1st column) showed strong BrdU signal especially in the epiblast, the yolk sac area, and the ectoplacental cone while being negative for TUNEL. Heterozygous decidua were typically BrdU-labelled most strongly in the periphery, visible only in KO1 and cropped in control1 and KO2. Homozygous mutant embryos showed less (KO1) to no BrdU labelling (KO2) varying with embryo and section. Most mutants analysed showed some degree of TUNEL (exemplified by KO3 and KO4). The longest diameter of the embryo is indicated below each panel and the relative sizes of the panels are indicated with dashed lines.
Mentions: To take a more detailed look at cell proliferation in embryos lacking Ptbp1 versus those containing either 1 or 2 normal alleles, we performed BrdU and TUNEL assays (Figure 8). As estimated by BrdU incorporation into newly synthesised DNA, cell division was high throughout the control E7.5 embryos analysed, with especially strong signal coming from the epiblast, the visceral endoderm, the area of the yolk sac, and the ectoplacental cone. Interestingly, the epiblast and the endoderm were also labelled strongly using Ptbp antibodies (Figure 7). There appears to be a correlation between the expression levels of Ptbps and cell division in embryos. Also, mutants clearly synthesise less DNA.

Bottom Line: Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation.We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst.However, further post-implantation development requires PTBP1 and stalls in homozygous animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

View Article: PubMed Central - PubMed

Affiliation: Molecular Diabetology, Paul Langerhans Institute Dresden, School of Medicine and University Clinic Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.

ABSTRACT
Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

Show MeSH
Related in: MedlinePlus