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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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PTBP1 expression in normal and mutant embryos.Comparison of immunofluorescence from PTBP1 and its paralogue PTBP2 in control and PTBP1 KO embryos at E7.5. The top row shows serial cryosections of a normally sized control embryo stained for PTBP1 (left) and PTBP2 (right). PTBP1 was expressed in most embryonic cells, notably the visceral endoderm and the epiblast, comparable to the LacZ reporter staining in Figure 2. A strong PTBP1 signal was also observed in decidual nuclei. PTBP2 in controls was mostly expressed in the epiblast and absent from most decidual nuclei. PTBP1  embryos (bottom) showed no PTBP1 or PTBP2 signal, displayed here merged with the DAPI signal to reveal the embryo (dashed line). Again most cells surrounding the embryo displayed a nuclear PTBP1 signal while only very few showed a weak nuclear PTBP2 signal (arrow).
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pone-0016992-g007: PTBP1 expression in normal and mutant embryos.Comparison of immunofluorescence from PTBP1 and its paralogue PTBP2 in control and PTBP1 KO embryos at E7.5. The top row shows serial cryosections of a normally sized control embryo stained for PTBP1 (left) and PTBP2 (right). PTBP1 was expressed in most embryonic cells, notably the visceral endoderm and the epiblast, comparable to the LacZ reporter staining in Figure 2. A strong PTBP1 signal was also observed in decidual nuclei. PTBP2 in controls was mostly expressed in the epiblast and absent from most decidual nuclei. PTBP1 embryos (bottom) showed no PTBP1 or PTBP2 signal, displayed here merged with the DAPI signal to reveal the embryo (dashed line). Again most cells surrounding the embryo displayed a nuclear PTBP1 signal while only very few showed a weak nuclear PTBP2 signal (arrow).

Mentions: PTBP1 suppresses the expression of PTBP2, which is 70% identical in amino acid sequence and fulfils similar functions. In adult tissues, downregulation of PTBP1 often causes upregulation of PTBP2. We were therefore asking ourselves whether a similar compensation could be at work in the PTBP1 knockout embryos. Control embryos expressed both paralogues, albeit with a different distribution. PTBP1, apart from surrounding decidual cells was highly expressed in the visceral endoderm and the epiblast. This is in agreement with the expression pattern revealed with the LacZ reporter assay (Figure 2). In E7.5 embryos, the strongest PTBP2 fluorescence was found in the epiblast only. However, mutants showed neither PTBP1 nor PTBP2 immunofluorescent signals at E7.5 (Figure 7). We also looked at whether the 3rd, lesser known paralogue of the PTBP group, Rod1, is upregulated in PTBP1 embryo. Using RNA extracts from E7.5 embryos, we performed RT PCR and did not detect any amplification in the knockout or the control embryos (Figure S5).


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 expression in normal and mutant embryos.Comparison of immunofluorescence from PTBP1 and its paralogue PTBP2 in control and PTBP1 KO embryos at E7.5. The top row shows serial cryosections of a normally sized control embryo stained for PTBP1 (left) and PTBP2 (right). PTBP1 was expressed in most embryonic cells, notably the visceral endoderm and the epiblast, comparable to the LacZ reporter staining in Figure 2. A strong PTBP1 signal was also observed in decidual nuclei. PTBP2 in controls was mostly expressed in the epiblast and absent from most decidual nuclei. PTBP1  embryos (bottom) showed no PTBP1 or PTBP2 signal, displayed here merged with the DAPI signal to reveal the embryo (dashed line). Again most cells surrounding the embryo displayed a nuclear PTBP1 signal while only very few showed a weak nuclear PTBP2 signal (arrow).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016992-g007: PTBP1 expression in normal and mutant embryos.Comparison of immunofluorescence from PTBP1 and its paralogue PTBP2 in control and PTBP1 KO embryos at E7.5. The top row shows serial cryosections of a normally sized control embryo stained for PTBP1 (left) and PTBP2 (right). PTBP1 was expressed in most embryonic cells, notably the visceral endoderm and the epiblast, comparable to the LacZ reporter staining in Figure 2. A strong PTBP1 signal was also observed in decidual nuclei. PTBP2 in controls was mostly expressed in the epiblast and absent from most decidual nuclei. PTBP1 embryos (bottom) showed no PTBP1 or PTBP2 signal, displayed here merged with the DAPI signal to reveal the embryo (dashed line). Again most cells surrounding the embryo displayed a nuclear PTBP1 signal while only very few showed a weak nuclear PTBP2 signal (arrow).
Mentions: PTBP1 suppresses the expression of PTBP2, which is 70% identical in amino acid sequence and fulfils similar functions. In adult tissues, downregulation of PTBP1 often causes upregulation of PTBP2. We were therefore asking ourselves whether a similar compensation could be at work in the PTBP1 knockout embryos. Control embryos expressed both paralogues, albeit with a different distribution. PTBP1, apart from surrounding decidual cells was highly expressed in the visceral endoderm and the epiblast. This is in agreement with the expression pattern revealed with the LacZ reporter assay (Figure 2). In E7.5 embryos, the strongest PTBP2 fluorescence was found in the epiblast only. However, mutants showed neither PTBP1 nor PTBP2 immunofluorescent signals at E7.5 (Figure 7). We also looked at whether the 3rd, lesser known paralogue of the PTBP group, Rod1, is upregulated in PTBP1 embryo. Using RNA extracts from E7.5 embryos, we performed RT PCR and did not detect any amplification in the knockout or the control embryos (Figure S5).

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