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Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

Landry J, Sharov AA, Piao Y, Sharova LV, Xiao H, Southon E, Matta J, Tessarollo L, Zhang YE, Ko MS, Kuehn MR, Yamaguchi TP, Wu C - PLoS Genet. (2008)

Bottom Line: Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent.We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors.We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Cell Biology, National Cancer Institute, National Institutes of Heath, Bethesda, MD, USA. landrjos@mail.nih.gov

ABSTRACT
We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

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Bptf mutants manifest early embryonic growth defects.(A) Wild type (+/+), heterozygous (+/−), and homozygous (−/−)Bptf XG023 embryos at E5.5, E6.5, and E7.5 were removed from their decedua and genotyped using the PCR based method. Reduced growth rates are evident in the homozygous Bptf XG023 embryos as early as E5.5. (B) Hematoxylin and eosin (H&E) staining of E5.5, E6.5, and E7.5 mutant embryos show reduced growth of both embryonic and extra-embryonic tissues, the presence of visceral endoderm, and an absence of anterior–posterior asymmetry when compared to controls. Bptf mutant embryos were stained for phosphorylated histone H3, a marker of cell mitosis. Mutant embryos at E5.5, E6.5, and E7.5 have significantly reduced staining when compared to controls, suggesting that they have reduced cellular proliferation. Bptf-mutant embryos were stained for apoptotic cells using the TUNEL assay. Mutant embryos at E5.5, E6.5, and E7.5 do not have any positive staining by TUNEL when compared to controls, suggesting that they are not apoptotic. Scale Bars: E5.5 and E6.5 embryos = 50 µm, E7.5 embryos = 100 µm. Abbreviations: ve, visceral endoderm; ee, embryonic ectoderm; m, mesoderm; xe, extra-embryonic ectoderm; xve, extra-embryonic visceral endoderm.
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pgen-1000241-g001: Bptf mutants manifest early embryonic growth defects.(A) Wild type (+/+), heterozygous (+/−), and homozygous (−/−)Bptf XG023 embryos at E5.5, E6.5, and E7.5 were removed from their decedua and genotyped using the PCR based method. Reduced growth rates are evident in the homozygous Bptf XG023 embryos as early as E5.5. (B) Hematoxylin and eosin (H&E) staining of E5.5, E6.5, and E7.5 mutant embryos show reduced growth of both embryonic and extra-embryonic tissues, the presence of visceral endoderm, and an absence of anterior–posterior asymmetry when compared to controls. Bptf mutant embryos were stained for phosphorylated histone H3, a marker of cell mitosis. Mutant embryos at E5.5, E6.5, and E7.5 have significantly reduced staining when compared to controls, suggesting that they have reduced cellular proliferation. Bptf-mutant embryos were stained for apoptotic cells using the TUNEL assay. Mutant embryos at E5.5, E6.5, and E7.5 do not have any positive staining by TUNEL when compared to controls, suggesting that they are not apoptotic. Scale Bars: E5.5 and E6.5 embryos = 50 µm, E7.5 embryos = 100 µm. Abbreviations: ve, visceral endoderm; ee, embryonic ectoderm; m, mesoderm; xe, extra-embryonic ectoderm; xve, extra-embryonic visceral endoderm.

Mentions: To identify defects in the embryonic development of BptfXG023 and BptfΔExon2 homozygotes, we performed whole mount examinations of mutant embryos. Growth defects increasing in severity from E5.5 to E7.5 were observed (Figure 1A) (Figure S5A). Dissections conducted at E8.5 and E9.5 were uninformative, because a majority of the embryos had been completely reabsorbed (data not shown). We crossed BptfXG023 and BptfΔExon2 heterozygous mice to generate the trans-heterozygous BptfXG023/BptfΔExon2 embryos. The trans-heterozygote recapitulated the growth defects of the BptfXG023 and BptfΔExon2 homozygotes, indicating that the two mutations are functionally equivalent (Figure S5B). To determine whether the developmental defects originated before implantation, we harvested E3.5 blastocysts and performed blastocyst outgrowth assays. We observed normal E3.5 homozygous BptfXG023 and BptfΔExon2 blastocysts and normal outgrowths from the blastocysts after tissue culture for 5 days, suggesting that either Bptf is not essential for pre-implantation development or that the maternal Bptf protein or mRNA can mask pre-implantation phenotypes (Figure S6). The masking of pre-implantation phenotypes by maternal Bptf is possible because it is highly expressed in oocytes [27],[28].


Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

Landry J, Sharov AA, Piao Y, Sharova LV, Xiao H, Southon E, Matta J, Tessarollo L, Zhang YE, Ko MS, Kuehn MR, Yamaguchi TP, Wu C - PLoS Genet. (2008)

Bptf mutants manifest early embryonic growth defects.(A) Wild type (+/+), heterozygous (+/−), and homozygous (−/−)Bptf XG023 embryos at E5.5, E6.5, and E7.5 were removed from their decedua and genotyped using the PCR based method. Reduced growth rates are evident in the homozygous Bptf XG023 embryos as early as E5.5. (B) Hematoxylin and eosin (H&E) staining of E5.5, E6.5, and E7.5 mutant embryos show reduced growth of both embryonic and extra-embryonic tissues, the presence of visceral endoderm, and an absence of anterior–posterior asymmetry when compared to controls. Bptf mutant embryos were stained for phosphorylated histone H3, a marker of cell mitosis. Mutant embryos at E5.5, E6.5, and E7.5 have significantly reduced staining when compared to controls, suggesting that they have reduced cellular proliferation. Bptf-mutant embryos were stained for apoptotic cells using the TUNEL assay. Mutant embryos at E5.5, E6.5, and E7.5 do not have any positive staining by TUNEL when compared to controls, suggesting that they are not apoptotic. Scale Bars: E5.5 and E6.5 embryos = 50 µm, E7.5 embryos = 100 µm. Abbreviations: ve, visceral endoderm; ee, embryonic ectoderm; m, mesoderm; xe, extra-embryonic ectoderm; xve, extra-embryonic visceral endoderm.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1000241-g001: Bptf mutants manifest early embryonic growth defects.(A) Wild type (+/+), heterozygous (+/−), and homozygous (−/−)Bptf XG023 embryos at E5.5, E6.5, and E7.5 were removed from their decedua and genotyped using the PCR based method. Reduced growth rates are evident in the homozygous Bptf XG023 embryos as early as E5.5. (B) Hematoxylin and eosin (H&E) staining of E5.5, E6.5, and E7.5 mutant embryos show reduced growth of both embryonic and extra-embryonic tissues, the presence of visceral endoderm, and an absence of anterior–posterior asymmetry when compared to controls. Bptf mutant embryos were stained for phosphorylated histone H3, a marker of cell mitosis. Mutant embryos at E5.5, E6.5, and E7.5 have significantly reduced staining when compared to controls, suggesting that they have reduced cellular proliferation. Bptf-mutant embryos were stained for apoptotic cells using the TUNEL assay. Mutant embryos at E5.5, E6.5, and E7.5 do not have any positive staining by TUNEL when compared to controls, suggesting that they are not apoptotic. Scale Bars: E5.5 and E6.5 embryos = 50 µm, E7.5 embryos = 100 µm. Abbreviations: ve, visceral endoderm; ee, embryonic ectoderm; m, mesoderm; xe, extra-embryonic ectoderm; xve, extra-embryonic visceral endoderm.
Mentions: To identify defects in the embryonic development of BptfXG023 and BptfΔExon2 homozygotes, we performed whole mount examinations of mutant embryos. Growth defects increasing in severity from E5.5 to E7.5 were observed (Figure 1A) (Figure S5A). Dissections conducted at E8.5 and E9.5 were uninformative, because a majority of the embryos had been completely reabsorbed (data not shown). We crossed BptfXG023 and BptfΔExon2 heterozygous mice to generate the trans-heterozygous BptfXG023/BptfΔExon2 embryos. The trans-heterozygote recapitulated the growth defects of the BptfXG023 and BptfΔExon2 homozygotes, indicating that the two mutations are functionally equivalent (Figure S5B). To determine whether the developmental defects originated before implantation, we harvested E3.5 blastocysts and performed blastocyst outgrowth assays. We observed normal E3.5 homozygous BptfXG023 and BptfΔExon2 blastocysts and normal outgrowths from the blastocysts after tissue culture for 5 days, suggesting that either Bptf is not essential for pre-implantation development or that the maternal Bptf protein or mRNA can mask pre-implantation phenotypes (Figure S6). The masking of pre-implantation phenotypes by maternal Bptf is possible because it is highly expressed in oocytes [27],[28].

Bottom Line: Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent.We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors.We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Cell Biology, National Cancer Institute, National Institutes of Heath, Bethesda, MD, USA. landrjos@mail.nih.gov

ABSTRACT
We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

Show MeSH
Related in: MedlinePlus