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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 is expressed throughout embryonic development.The LacZ reporter shown in Figure 1 was used here as a proxy for PTBP1 promoter activity in heterozygous embryonic stem cells (ESCs) and embryos at different stages of development. PTBP1 expression was high in ESCs. In embryos 6.5 days after fertilisation (E6.5), PTBP1 was expressed especially in the epiblast but also in the visceral endoderm surrounding the embryo. The expression pattern was similar at E7.5 where the activation of maternal PTBP1 was seen in the decidua surrounding the tip of the embryo. No LacZ staining was observed in the wild-type (wt) littermate (small inlet). The reporter was expressed in almost all cells at E8.5 and in most cells at E9, when only the areas at the roof of the future head, the primitive ventricle of the heart, and some structures in the tail appeared to express little or no PTBP1. In E12.5 embryos most developing tissues and organs expressed the reporter, most notably the cells lining the ventricles of the brain, the spinal cord, the pituitary and the heart. Similar organs were stained in E16.5 embryos and in addition the nasal cavities, the inner ear and the brown fat pad on the back of the embryo. PTBP1 was high in the ventral rib cartilage but much lower in the dorsal rib bones that already showed a central cavity. While at E12.5 there was no unspecific staining, at E16.5 the intestine and to a lesser degree the stomach displayed an X-gal signal not due to the PTBP1 reporter. The signal result from endogenous β-galactosidase. See Figure S3 for a whole-mount LacZ at E12 and E16.
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pone-0016992-g002: PTBP1 is expressed throughout embryonic development.The LacZ reporter shown in Figure 1 was used here as a proxy for PTBP1 promoter activity in heterozygous embryonic stem cells (ESCs) and embryos at different stages of development. PTBP1 expression was high in ESCs. In embryos 6.5 days after fertilisation (E6.5), PTBP1 was expressed especially in the epiblast but also in the visceral endoderm surrounding the embryo. The expression pattern was similar at E7.5 where the activation of maternal PTBP1 was seen in the decidua surrounding the tip of the embryo. No LacZ staining was observed in the wild-type (wt) littermate (small inlet). The reporter was expressed in almost all cells at E8.5 and in most cells at E9, when only the areas at the roof of the future head, the primitive ventricle of the heart, and some structures in the tail appeared to express little or no PTBP1. In E12.5 embryos most developing tissues and organs expressed the reporter, most notably the cells lining the ventricles of the brain, the spinal cord, the pituitary and the heart. Similar organs were stained in E16.5 embryos and in addition the nasal cavities, the inner ear and the brown fat pad on the back of the embryo. PTBP1 was high in the ventral rib cartilage but much lower in the dorsal rib bones that already showed a central cavity. While at E12.5 there was no unspecific staining, at E16.5 the intestine and to a lesser degree the stomach displayed an X-gal signal not due to the PTBP1 reporter. The signal result from endogenous β-galactosidase. See Figure S3 for a whole-mount LacZ at E12 and E16.

Mentions: In order to gauge the importance of PTBP1 in embryonic development, we performed X-gal staining of heterozygous embryonic stem cells (ESCs) and heterozygous whole embryos at equal time intervals between implantation at E4-4.5 and birth at E20-21 (Figure 2). PTBP1 displayed a striking expression pattern in E6.5 and E7.5 basically labelling the epiblast. In E6.5, we observed PTBP1 reporter in visceral endoderm but not in yolk sac mesoderm, chorionic ectoderm or the ectoplacental cone. Heterozygous but not wild-type decidua expresses PTBP1 in the area surrounding the epiblast. Since X-gal only penetrates 1–2 mm into tissue, we performed X-gal staining on cryosections in addition to whole mount preparations for E12.5 and E16.5. Accordingly, the LacZ signal in the skin was very strong in whole mount preparations (Figure S3), but barely detectable in the cryosections (Figure 2), which instead revealed strong PTBP1 expression in developing internal organs and tissues such the brain cortex and subventricular zone, where neuronal precursors are found, the trigeminal ganglion, the nasal cavity, the inner ear, the medulla oblongata, the spinal cord, the thyroid, the heart, the lung, the kidney, possibly the pituitary and several cartilage primordia but not more ossified structures (see especially the E16.5 rib sections in Figure 2). PTBP1 expression became more restricted with increasing embryonic age. While the E8.5 embryo was almost completely stained by X-gal, the signal became more differentiated in E12.5 and E16.5. We conclude that PTBP1 is strongly and broadly expressed during the entire gestational period and in the cultured embryonic stem cells used to generate this knockout mouse model.


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 is expressed throughout embryonic development.The LacZ reporter shown in Figure 1 was used here as a proxy for PTBP1 promoter activity in heterozygous embryonic stem cells (ESCs) and embryos at different stages of development. PTBP1 expression was high in ESCs. In embryos 6.5 days after fertilisation (E6.5), PTBP1 was expressed especially in the epiblast but also in the visceral endoderm surrounding the embryo. The expression pattern was similar at E7.5 where the activation of maternal PTBP1 was seen in the decidua surrounding the tip of the embryo. No LacZ staining was observed in the wild-type (wt) littermate (small inlet). The reporter was expressed in almost all cells at E8.5 and in most cells at E9, when only the areas at the roof of the future head, the primitive ventricle of the heart, and some structures in the tail appeared to express little or no PTBP1. In E12.5 embryos most developing tissues and organs expressed the reporter, most notably the cells lining the ventricles of the brain, the spinal cord, the pituitary and the heart. Similar organs were stained in E16.5 embryos and in addition the nasal cavities, the inner ear and the brown fat pad on the back of the embryo. PTBP1 was high in the ventral rib cartilage but much lower in the dorsal rib bones that already showed a central cavity. While at E12.5 there was no unspecific staining, at E16.5 the intestine and to a lesser degree the stomach displayed an X-gal signal not due to the PTBP1 reporter. The signal result from endogenous β-galactosidase. See Figure S3 for a whole-mount LacZ at E12 and E16.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016992-g002: PTBP1 is expressed throughout embryonic development.The LacZ reporter shown in Figure 1 was used here as a proxy for PTBP1 promoter activity in heterozygous embryonic stem cells (ESCs) and embryos at different stages of development. PTBP1 expression was high in ESCs. In embryos 6.5 days after fertilisation (E6.5), PTBP1 was expressed especially in the epiblast but also in the visceral endoderm surrounding the embryo. The expression pattern was similar at E7.5 where the activation of maternal PTBP1 was seen in the decidua surrounding the tip of the embryo. No LacZ staining was observed in the wild-type (wt) littermate (small inlet). The reporter was expressed in almost all cells at E8.5 and in most cells at E9, when only the areas at the roof of the future head, the primitive ventricle of the heart, and some structures in the tail appeared to express little or no PTBP1. In E12.5 embryos most developing tissues and organs expressed the reporter, most notably the cells lining the ventricles of the brain, the spinal cord, the pituitary and the heart. Similar organs were stained in E16.5 embryos and in addition the nasal cavities, the inner ear and the brown fat pad on the back of the embryo. PTBP1 was high in the ventral rib cartilage but much lower in the dorsal rib bones that already showed a central cavity. While at E12.5 there was no unspecific staining, at E16.5 the intestine and to a lesser degree the stomach displayed an X-gal signal not due to the PTBP1 reporter. The signal result from endogenous β-galactosidase. See Figure S3 for a whole-mount LacZ at E12 and E16.
Mentions: In order to gauge the importance of PTBP1 in embryonic development, we performed X-gal staining of heterozygous embryonic stem cells (ESCs) and heterozygous whole embryos at equal time intervals between implantation at E4-4.5 and birth at E20-21 (Figure 2). PTBP1 displayed a striking expression pattern in E6.5 and E7.5 basically labelling the epiblast. In E6.5, we observed PTBP1 reporter in visceral endoderm but not in yolk sac mesoderm, chorionic ectoderm or the ectoplacental cone. Heterozygous but not wild-type decidua expresses PTBP1 in the area surrounding the epiblast. Since X-gal only penetrates 1–2 mm into tissue, we performed X-gal staining on cryosections in addition to whole mount preparations for E12.5 and E16.5. Accordingly, the LacZ signal in the skin was very strong in whole mount preparations (Figure S3), but barely detectable in the cryosections (Figure 2), which instead revealed strong PTBP1 expression in developing internal organs and tissues such the brain cortex and subventricular zone, where neuronal precursors are found, the trigeminal ganglion, the nasal cavity, the inner ear, the medulla oblongata, the spinal cord, the thyroid, the heart, the lung, the kidney, possibly the pituitary and several cartilage primordia but not more ossified structures (see especially the E16.5 rib sections in Figure 2). PTBP1 expression became more restricted with increasing embryonic age. While the E8.5 embryo was almost completely stained by X-gal, the signal became more differentiated in E12.5 and E16.5. We conclude that PTBP1 is strongly and broadly expressed during the entire gestational period and in the cultured embryonic stem cells used to generate this knockout mouse model.

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