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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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Related in: MedlinePlus

PTBP1 knockout embryos implant but are severely growth-retarded.The small category of embryos from a heterozygous intercross were identified by PCR and immunofluorescence as PTBP1 knockout embryos. A) PCR products for the stop cassette and PTBP1 intron 2 were separated on agarose gels and scored blindly. 5/5 small embryos were genotyped as homozygous. 15/17 large control embryos were genotyped as heterozygous or wild-type with 2 false negatives due to the less efficient intron PCR. B) Serial paraffin sections of E7.5 embryos were stained with hematoxylin and eosin (left column) or labelled with DAPI (middle column) and the PTBP1 antibody (right column). The top row shows a large embryo (in an oblique section) with strong nuclear PTBP1 staining of the embryo proper (dashed line) and the surrounding tissue. Both small embryos were characterised by a lack of the nuclear PTBP1 signal while showing nuclear PTBP1 in surrounding, most likely maternal cells. Interpretative diagrams and a quantification of the embryo section area are shown on the right.
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pone-0016992-g004: PTBP1 knockout embryos implant but are severely growth-retarded.The small category of embryos from a heterozygous intercross were identified by PCR and immunofluorescence as PTBP1 knockout embryos. A) PCR products for the stop cassette and PTBP1 intron 2 were separated on agarose gels and scored blindly. 5/5 small embryos were genotyped as homozygous. 15/17 large control embryos were genotyped as heterozygous or wild-type with 2 false negatives due to the less efficient intron PCR. B) Serial paraffin sections of E7.5 embryos were stained with hematoxylin and eosin (left column) or labelled with DAPI (middle column) and the PTBP1 antibody (right column). The top row shows a large embryo (in an oblique section) with strong nuclear PTBP1 staining of the embryo proper (dashed line) and the surrounding tissue. Both small embryos were characterised by a lack of the nuclear PTBP1 signal while showing nuclear PTBP1 in surrounding, most likely maternal cells. Interpretative diagrams and a quantification of the embryo section area are shown on the right.

Mentions: In order to ascertain the nature of the small implantations, we isolated embryos at E7.5, carefully avoiding contamination from the maternal decidua which would have biased the resulting genotype towards heterozygous. We analysed 5 small and 17 large embryos by PCR. All 5 small embryos were genotyped as homozygous for the PTBP1 knockout allele, 15 large embryos were either wild-type or heterozygous, with 2 large embryos being misclassified as homozygous probably due to problems in the intron PCR (Figure 4 and Table 1).


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)

PTBP1 knockout embryos implant but are severely growth-retarded.The small category of embryos from a heterozygous intercross were identified by PCR and immunofluorescence as PTBP1 knockout embryos. A) PCR products for the stop cassette and PTBP1 intron 2 were separated on agarose gels and scored blindly. 5/5 small embryos were genotyped as homozygous. 15/17 large control embryos were genotyped as heterozygous or wild-type with 2 false negatives due to the less efficient intron PCR. B) Serial paraffin sections of E7.5 embryos were stained with hematoxylin and eosin (left column) or labelled with DAPI (middle column) and the PTBP1 antibody (right column). The top row shows a large embryo (in an oblique section) with strong nuclear PTBP1 staining of the embryo proper (dashed line) and the surrounding tissue. Both small embryos were characterised by a lack of the nuclear PTBP1 signal while showing nuclear PTBP1 in surrounding, most likely maternal cells. Interpretative diagrams and a quantification of the embryo section area are shown on the right.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016992-g004: PTBP1 knockout embryos implant but are severely growth-retarded.The small category of embryos from a heterozygous intercross were identified by PCR and immunofluorescence as PTBP1 knockout embryos. A) PCR products for the stop cassette and PTBP1 intron 2 were separated on agarose gels and scored blindly. 5/5 small embryos were genotyped as homozygous. 15/17 large control embryos were genotyped as heterozygous or wild-type with 2 false negatives due to the less efficient intron PCR. B) Serial paraffin sections of E7.5 embryos were stained with hematoxylin and eosin (left column) or labelled with DAPI (middle column) and the PTBP1 antibody (right column). The top row shows a large embryo (in an oblique section) with strong nuclear PTBP1 staining of the embryo proper (dashed line) and the surrounding tissue. Both small embryos were characterised by a lack of the nuclear PTBP1 signal while showing nuclear PTBP1 in surrounding, most likely maternal cells. Interpretative diagrams and a quantification of the embryo section area are shown on the right.
Mentions: In order to ascertain the nature of the small implantations, we isolated embryos at E7.5, carefully avoiding contamination from the maternal decidua which would have biased the resulting genotype towards heterozygous. We analysed 5 small and 17 large embryos by PCR. All 5 small embryos were genotyped as homozygous for the PTBP1 knockout allele, 15 large embryos were either wild-type or heterozygous, with 2 large embryos being misclassified as homozygous probably due to problems in the intron PCR (Figure 4 and Table 1).

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