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Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification.

O'Shaughnessy-Kirwan A, Signolet J, Costello I, Gharbi S, Hendrich B - Development (2015)

Bottom Line: Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE).We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed.This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.

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Chd4−/− embryos are viable before implantation. (A) Left: Confocal 3D projections of 3.5 dpc embryos of the indicated genotypes stained for phosphorylated histone H3 (p-H3) and Ki67. Scale bars: 50 µm. Right: Quantification of the total number of p-H3-positive cells per embryo reveals no difference in wild type and heterozygous (WT/Het) versus knockout (KO) embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. (B) Confocal 3D projections of embryos of the indicated genotypes stained for γ-H2A.X (magenta) and DAPI (blue). KO images are representative of five  embryos. Scale bars: 50 µm. (C) Confocal images (left) of blastocysts of the indicated genotypes stained for cleaved caspase 3 (Casp; green), OCT4 (magenta) and DAPI (blue) scored for total cleaved caspase 3-positive cells per blastocyst (right). Arrowheads (left) indicate examples of positive cleaved caspase 3 staining. Scale bars: 50 µm. Numbers of embryos used to generate the box plots are indicated below each genotype. (D) Left: Confocal images of wild-type and Chd4−/− blastocysts assayed for TUNEL-positive cells (green). Scale bars: 50 µm. Right: Using confocal images, the total number of TUNEL-positive cells/embryo was calculated for each genotype. Numbers of embryos used to generate the box plots are indicated below each genotype. (E) Quantification of the average number of cells per embryo in early blastocysts of indicated genotypes. The numbers of embryos used to generate each box plot are indicated below the genotypes. (F) Schematic representation of aggregation experiments. Embryos from gene-trap litters (i.e. wild type, heterozygous or knockout) were aggregated to wild-type (WT) embryos. Heterozygous and knockout embryos contain the lacZ gene present in the gene trap allele, and hence express β-GAL (blue). (G) Confocal images of aggregation chimaeras, with genotypes of the test embryos, as inferred from anti-β-GAL (green) and anti-CHD4 (magenta) staining, indicated on the right. Scale bars: 50 µm. Images are representative of 11 different aggregations involving  embryos. (H) Plot of average number of cells from either heterozygous (HET) or Chd4−/− (KO) cells in chimaeric embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. All P-values were calculated using a two-tailed Mann–Whitney U-test.
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DEV125450F2: Chd4−/− embryos are viable before implantation. (A) Left: Confocal 3D projections of 3.5 dpc embryos of the indicated genotypes stained for phosphorylated histone H3 (p-H3) and Ki67. Scale bars: 50 µm. Right: Quantification of the total number of p-H3-positive cells per embryo reveals no difference in wild type and heterozygous (WT/Het) versus knockout (KO) embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. (B) Confocal 3D projections of embryos of the indicated genotypes stained for γ-H2A.X (magenta) and DAPI (blue). KO images are representative of five embryos. Scale bars: 50 µm. (C) Confocal images (left) of blastocysts of the indicated genotypes stained for cleaved caspase 3 (Casp; green), OCT4 (magenta) and DAPI (blue) scored for total cleaved caspase 3-positive cells per blastocyst (right). Arrowheads (left) indicate examples of positive cleaved caspase 3 staining. Scale bars: 50 µm. Numbers of embryos used to generate the box plots are indicated below each genotype. (D) Left: Confocal images of wild-type and Chd4−/− blastocysts assayed for TUNEL-positive cells (green). Scale bars: 50 µm. Right: Using confocal images, the total number of TUNEL-positive cells/embryo was calculated for each genotype. Numbers of embryos used to generate the box plots are indicated below each genotype. (E) Quantification of the average number of cells per embryo in early blastocysts of indicated genotypes. The numbers of embryos used to generate each box plot are indicated below the genotypes. (F) Schematic representation of aggregation experiments. Embryos from gene-trap litters (i.e. wild type, heterozygous or knockout) were aggregated to wild-type (WT) embryos. Heterozygous and knockout embryos contain the lacZ gene present in the gene trap allele, and hence express β-GAL (blue). (G) Confocal images of aggregation chimaeras, with genotypes of the test embryos, as inferred from anti-β-GAL (green) and anti-CHD4 (magenta) staining, indicated on the right. Scale bars: 50 µm. Images are representative of 11 different aggregations involving embryos. (H) Plot of average number of cells from either heterozygous (HET) or Chd4−/− (KO) cells in chimaeric embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. All P-values were calculated using a two-tailed Mann–Whitney U-test.

Mentions: The fact that morphologically normal Chd4−/− blastocysts were recovered early on the fourth day indicates that the cells were able to undergo at least two rounds of division in the absence of CHD4 protein, and therefore that CHD4 is not required for cell viability per se in cleavage stage embryos. This is in contrast to the situation in somatic cell lines, in which CHD4 has been shown to be important for cell cycle progression. Cells in Chd4−/− blastocysts stain positively for Ki67 (MKI67 − Mouse Genome Informatics) and show normal levels of phosphorylated histone H3 (Fig. 2A), a marker of mitotic cells, indicating that CHD4 is not required for cell cycle progression in blastocysts. Additionally, absence of CHD4 in blastocysts was not associated with notably increased levels of the histone variant γ-H2A.X, a marker of DNA damage (Fig. 2B), as was reported after CHD4 knockdown in somatic cells (Pegoraro et al., 2009). Nevertheless, Chd4−/− blastocysts show an increased proportion of apoptotic cells as measured by TUNEL or caspase staining (Fig. 2C,D) and, on average, contain a slightly reduced number of cells per embryo compared with wild-type or heterozygous littermates (Fig. 2E).Fig. 2.


Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification.

O'Shaughnessy-Kirwan A, Signolet J, Costello I, Gharbi S, Hendrich B - Development (2015)

Chd4−/− embryos are viable before implantation. (A) Left: Confocal 3D projections of 3.5 dpc embryos of the indicated genotypes stained for phosphorylated histone H3 (p-H3) and Ki67. Scale bars: 50 µm. Right: Quantification of the total number of p-H3-positive cells per embryo reveals no difference in wild type and heterozygous (WT/Het) versus knockout (KO) embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. (B) Confocal 3D projections of embryos of the indicated genotypes stained for γ-H2A.X (magenta) and DAPI (blue). KO images are representative of five  embryos. Scale bars: 50 µm. (C) Confocal images (left) of blastocysts of the indicated genotypes stained for cleaved caspase 3 (Casp; green), OCT4 (magenta) and DAPI (blue) scored for total cleaved caspase 3-positive cells per blastocyst (right). Arrowheads (left) indicate examples of positive cleaved caspase 3 staining. Scale bars: 50 µm. Numbers of embryos used to generate the box plots are indicated below each genotype. (D) Left: Confocal images of wild-type and Chd4−/− blastocysts assayed for TUNEL-positive cells (green). Scale bars: 50 µm. Right: Using confocal images, the total number of TUNEL-positive cells/embryo was calculated for each genotype. Numbers of embryos used to generate the box plots are indicated below each genotype. (E) Quantification of the average number of cells per embryo in early blastocysts of indicated genotypes. The numbers of embryos used to generate each box plot are indicated below the genotypes. (F) Schematic representation of aggregation experiments. Embryos from gene-trap litters (i.e. wild type, heterozygous or knockout) were aggregated to wild-type (WT) embryos. Heterozygous and knockout embryos contain the lacZ gene present in the gene trap allele, and hence express β-GAL (blue). (G) Confocal images of aggregation chimaeras, with genotypes of the test embryos, as inferred from anti-β-GAL (green) and anti-CHD4 (magenta) staining, indicated on the right. Scale bars: 50 µm. Images are representative of 11 different aggregations involving  embryos. (H) Plot of average number of cells from either heterozygous (HET) or Chd4−/− (KO) cells in chimaeric embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. All P-values were calculated using a two-tailed Mann–Whitney U-test.
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DEV125450F2: Chd4−/− embryos are viable before implantation. (A) Left: Confocal 3D projections of 3.5 dpc embryos of the indicated genotypes stained for phosphorylated histone H3 (p-H3) and Ki67. Scale bars: 50 µm. Right: Quantification of the total number of p-H3-positive cells per embryo reveals no difference in wild type and heterozygous (WT/Het) versus knockout (KO) embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. (B) Confocal 3D projections of embryos of the indicated genotypes stained for γ-H2A.X (magenta) and DAPI (blue). KO images are representative of five embryos. Scale bars: 50 µm. (C) Confocal images (left) of blastocysts of the indicated genotypes stained for cleaved caspase 3 (Casp; green), OCT4 (magenta) and DAPI (blue) scored for total cleaved caspase 3-positive cells per blastocyst (right). Arrowheads (left) indicate examples of positive cleaved caspase 3 staining. Scale bars: 50 µm. Numbers of embryos used to generate the box plots are indicated below each genotype. (D) Left: Confocal images of wild-type and Chd4−/− blastocysts assayed for TUNEL-positive cells (green). Scale bars: 50 µm. Right: Using confocal images, the total number of TUNEL-positive cells/embryo was calculated for each genotype. Numbers of embryos used to generate the box plots are indicated below each genotype. (E) Quantification of the average number of cells per embryo in early blastocysts of indicated genotypes. The numbers of embryos used to generate each box plot are indicated below the genotypes. (F) Schematic representation of aggregation experiments. Embryos from gene-trap litters (i.e. wild type, heterozygous or knockout) were aggregated to wild-type (WT) embryos. Heterozygous and knockout embryos contain the lacZ gene present in the gene trap allele, and hence express β-GAL (blue). (G) Confocal images of aggregation chimaeras, with genotypes of the test embryos, as inferred from anti-β-GAL (green) and anti-CHD4 (magenta) staining, indicated on the right. Scale bars: 50 µm. Images are representative of 11 different aggregations involving embryos. (H) Plot of average number of cells from either heterozygous (HET) or Chd4−/− (KO) cells in chimaeric embryos. The numbers of embryos used to generate each box plot are indicated below the genotypes. All P-values were calculated using a two-tailed Mann–Whitney U-test.
Mentions: The fact that morphologically normal Chd4−/− blastocysts were recovered early on the fourth day indicates that the cells were able to undergo at least two rounds of division in the absence of CHD4 protein, and therefore that CHD4 is not required for cell viability per se in cleavage stage embryos. This is in contrast to the situation in somatic cell lines, in which CHD4 has been shown to be important for cell cycle progression. Cells in Chd4−/− blastocysts stain positively for Ki67 (MKI67 − Mouse Genome Informatics) and show normal levels of phosphorylated histone H3 (Fig. 2A), a marker of mitotic cells, indicating that CHD4 is not required for cell cycle progression in blastocysts. Additionally, absence of CHD4 in blastocysts was not associated with notably increased levels of the histone variant γ-H2A.X, a marker of DNA damage (Fig. 2B), as was reported after CHD4 knockdown in somatic cells (Pegoraro et al., 2009). Nevertheless, Chd4−/− blastocysts show an increased proportion of apoptotic cells as measured by TUNEL or caspase staining (Fig. 2C,D) and, on average, contain a slightly reduced number of cells per embryo compared with wild-type or heterozygous littermates (Fig. 2E).Fig. 2.

Bottom Line: Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE).We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed.This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.

Show MeSH
Related in: MedlinePlus