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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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Generation of a mouse containing a novel multipurpose PTBP1 allele.We generated a multipurpose PTBP1 allele and targeted it into mice. A) The wild-type locus (blue) was modified with the insertion of a stop/detection cassette (red) into embryonic stem cells. The cassette can be flipped out using an FLP recombinase construct or strain. In addition, exons 3 to 7 have been framed with loxP sites for conditional removal by a Cre recombinase. The diagram indicates the Southern strategy as well as the location of the primers for validation of the construct and genotyping. B) shows the results of the long-range PCR to verify the 5′ junction resulting from the homologous recombination of the targeting vector with the wild-type PTBP1 locus. C) Southern blot to verify the correct 3′ junction in the mutant allele.
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pone-0016992-g001: Generation of a mouse containing a novel multipurpose PTBP1 allele.We generated a multipurpose PTBP1 allele and targeted it into mice. A) The wild-type locus (blue) was modified with the insertion of a stop/detection cassette (red) into embryonic stem cells. The cassette can be flipped out using an FLP recombinase construct or strain. In addition, exons 3 to 7 have been framed with loxP sites for conditional removal by a Cre recombinase. The diagram indicates the Southern strategy as well as the location of the primers for validation of the construct and genotyping. B) shows the results of the long-range PCR to verify the 5′ junction resulting from the homologous recombination of the targeting vector with the wild-type PTBP1 locus. C) Southern blot to verify the correct 3′ junction in the mutant allele.

Mentions: Using Red/ET homologous recombination [36] we generated a novel PTBP1 allele (Figure 1, Figure S1), which in its original state causes a systemic knockout and produces bacterial β-galactosidase (LacZ) for detection of expression from the PTBP1 locus. We verified the efficiency of the transcriptional block using RT PCR (Figure S2). The stop/detection cassette can be removed with FLP recombinase leading to an allele indistinguishable from the wild-type except for the presence of loxP sites flanking exons 3 to 7. In the last stage of the multi-purpose allele, this group of exons can be removed by Cre recombination, leading to a premature stop codon and a mutation of PTBP1.


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)

Generation of a mouse containing a novel multipurpose PTBP1 allele.We generated a multipurpose PTBP1 allele and targeted it into mice. A) The wild-type locus (blue) was modified with the insertion of a stop/detection cassette (red) into embryonic stem cells. The cassette can be flipped out using an FLP recombinase construct or strain. In addition, exons 3 to 7 have been framed with loxP sites for conditional removal by a Cre recombinase. The diagram indicates the Southern strategy as well as the location of the primers for validation of the construct and genotyping. B) shows the results of the long-range PCR to verify the 5′ junction resulting from the homologous recombination of the targeting vector with the wild-type PTBP1 locus. C) Southern blot to verify the correct 3′ junction in the mutant allele.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016992-g001: Generation of a mouse containing a novel multipurpose PTBP1 allele.We generated a multipurpose PTBP1 allele and targeted it into mice. A) The wild-type locus (blue) was modified with the insertion of a stop/detection cassette (red) into embryonic stem cells. The cassette can be flipped out using an FLP recombinase construct or strain. In addition, exons 3 to 7 have been framed with loxP sites for conditional removal by a Cre recombinase. The diagram indicates the Southern strategy as well as the location of the primers for validation of the construct and genotyping. B) shows the results of the long-range PCR to verify the 5′ junction resulting from the homologous recombination of the targeting vector with the wild-type PTBP1 locus. C) Southern blot to verify the correct 3′ junction in the mutant allele.
Mentions: Using Red/ET homologous recombination [36] we generated a novel PTBP1 allele (Figure 1, Figure S1), which in its original state causes a systemic knockout and produces bacterial β-galactosidase (LacZ) for detection of expression from the PTBP1 locus. We verified the efficiency of the transcriptional block using RT PCR (Figure S2). The stop/detection cassette can be removed with FLP recombinase leading to an allele indistinguishable from the wild-type except for the presence of loxP sites flanking exons 3 to 7. In the last stage of the multi-purpose allele, this group of exons can be removed by Cre recombination, leading to a premature stop codon and a mutation of PTBP1.

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