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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 is required during the fourth day of development. (A) Representative composite spinning-disc images of anti-CHD4 (magenta) and SIN3A (green; used as a control) staining in wild-type and Chd4−/− 2-, 4-, 8- and 16-cell embryos. Images are representative of eight  2-cell embryos, 12  4-cell embryos, 11  8-cell embryos (including both Chd4−/− and Chd4Δ/Δ embryos) and four  16-cell embryos. Scale bars: 33 µm. (B) Composite confocal images of DAPI (blue) and CHD4 (magenta) staining in wild-type (WT) and Chd4−/− (KO) 3.5 dpc embryos. Scale bars: 50 µm. KO images are representative of >20 mutant blastocysts (including both Chd4−/− and Chd4Δ/Δ). (C) Phase contrast images of wild-type (WT) and Chd4−/− (KO) embryos flushed at different times on the fourth day post coitus (dpc). Scale bars: 50 µm. KO images are representative of 16 Chd4−/− embryos. (D) Average number of contractions per embryo genotype observed during live imaging (see supplementary material Movies 1-3). Each circle indicates the number of contractions for a single embryo of the indicated genotype. (E) Representative confocal 3D projection images of early 3.5 dpc embryos of the indicated genotypes stained for the indicated markers. KO images are  representative of six  embryos. Scale bars: 50 µm. (F) Two representative images per genotype of late 3.5 dpc embryos stained for E-cadherin (white) and DAPI (blue). Each image is a composite of four to six stacks, which allows visualisation of one entire cell layer across the distal end of the trophectoderm. Arrows indicate cells displaying mislocalised basal staining of E-cadherin. KO images are representative of eight  embryos. Scale bars: 50 µm.
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DEV125450F1: CHD4 is required during the fourth day of development. (A) Representative composite spinning-disc images of anti-CHD4 (magenta) and SIN3A (green; used as a control) staining in wild-type and Chd4−/− 2-, 4-, 8- and 16-cell embryos. Images are representative of eight 2-cell embryos, 12 4-cell embryos, 11 8-cell embryos (including both Chd4−/− and Chd4Δ/Δ embryos) and four 16-cell embryos. Scale bars: 33 µm. (B) Composite confocal images of DAPI (blue) and CHD4 (magenta) staining in wild-type (WT) and Chd4−/− (KO) 3.5 dpc embryos. Scale bars: 50 µm. KO images are representative of >20 mutant blastocysts (including both Chd4−/− and Chd4Δ/Δ). (C) Phase contrast images of wild-type (WT) and Chd4−/− (KO) embryos flushed at different times on the fourth day post coitus (dpc). Scale bars: 50 µm. KO images are representative of 16 Chd4−/− embryos. (D) Average number of contractions per embryo genotype observed during live imaging (see supplementary material Movies 1-3). Each circle indicates the number of contractions for a single embryo of the indicated genotype. (E) Representative confocal 3D projection images of early 3.5 dpc embryos of the indicated genotypes stained for the indicated markers. KO images are  representative of six embryos. Scale bars: 50 µm. (F) Two representative images per genotype of late 3.5 dpc embryos stained for E-cadherin (white) and DAPI (blue). Each image is a composite of four to six stacks, which allows visualisation of one entire cell layer across the distal end of the trophectoderm. Arrows indicate cells displaying mislocalised basal staining of E-cadherin. KO images are representative of eight embryos. Scale bars: 50 µm.

Mentions: CHD4 was present ubiquitously in cleavage-stage embryos (Fig. 1A). Nuclear CHD4 protein was detected at the 2-cell and 4-cell stages in both wild-type and Chd4−/− embryos, indicating that CHD4 protein either inherited from the oocyte or translated from maternally deposited mRNA was present in these early embryos (Fig. 1A). Null embryos at the 8-cell stage showed much reduced nuclear CHD4 staining, and staining was reduced to background levels in 16-cell mutant embryos. Similarly, ubiquitous nuclear CHD4 expression was detected in wild-type blastocysts, consistent with the X-gal staining seen in Chd4 heterozygote blastocysts, but no protein was detected in littermates produced from either allele (Fig. 1B; supplementary material Fig. S1B).Fig. 1.


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 is required during the fourth day of development. (A) Representative composite spinning-disc images of anti-CHD4 (magenta) and SIN3A (green; used as a control) staining in wild-type and Chd4−/− 2-, 4-, 8- and 16-cell embryos. Images are representative of eight  2-cell embryos, 12  4-cell embryos, 11  8-cell embryos (including both Chd4−/− and Chd4Δ/Δ embryos) and four  16-cell embryos. Scale bars: 33 µm. (B) Composite confocal images of DAPI (blue) and CHD4 (magenta) staining in wild-type (WT) and Chd4−/− (KO) 3.5 dpc embryos. Scale bars: 50 µm. KO images are representative of >20 mutant blastocysts (including both Chd4−/− and Chd4Δ/Δ). (C) Phase contrast images of wild-type (WT) and Chd4−/− (KO) embryos flushed at different times on the fourth day post coitus (dpc). Scale bars: 50 µm. KO images are representative of 16 Chd4−/− embryos. (D) Average number of contractions per embryo genotype observed during live imaging (see supplementary material Movies 1-3). Each circle indicates the number of contractions for a single embryo of the indicated genotype. (E) Representative confocal 3D projection images of early 3.5 dpc embryos of the indicated genotypes stained for the indicated markers. KO images are  representative of six  embryos. Scale bars: 50 µm. (F) Two representative images per genotype of late 3.5 dpc embryos stained for E-cadherin (white) and DAPI (blue). Each image is a composite of four to six stacks, which allows visualisation of one entire cell layer across the distal end of the trophectoderm. Arrows indicate cells displaying mislocalised basal staining of E-cadherin. KO images are representative of eight  embryos. Scale bars: 50 µm.
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DEV125450F1: CHD4 is required during the fourth day of development. (A) Representative composite spinning-disc images of anti-CHD4 (magenta) and SIN3A (green; used as a control) staining in wild-type and Chd4−/− 2-, 4-, 8- and 16-cell embryos. Images are representative of eight 2-cell embryos, 12 4-cell embryos, 11 8-cell embryos (including both Chd4−/− and Chd4Δ/Δ embryos) and four 16-cell embryos. Scale bars: 33 µm. (B) Composite confocal images of DAPI (blue) and CHD4 (magenta) staining in wild-type (WT) and Chd4−/− (KO) 3.5 dpc embryos. Scale bars: 50 µm. KO images are representative of >20 mutant blastocysts (including both Chd4−/− and Chd4Δ/Δ). (C) Phase contrast images of wild-type (WT) and Chd4−/− (KO) embryos flushed at different times on the fourth day post coitus (dpc). Scale bars: 50 µm. KO images are representative of 16 Chd4−/− embryos. (D) Average number of contractions per embryo genotype observed during live imaging (see supplementary material Movies 1-3). Each circle indicates the number of contractions for a single embryo of the indicated genotype. (E) Representative confocal 3D projection images of early 3.5 dpc embryos of the indicated genotypes stained for the indicated markers. KO images are  representative of six embryos. Scale bars: 50 µm. (F) Two representative images per genotype of late 3.5 dpc embryos stained for E-cadherin (white) and DAPI (blue). Each image is a composite of four to six stacks, which allows visualisation of one entire cell layer across the distal end of the trophectoderm. Arrows indicate cells displaying mislocalised basal staining of E-cadherin. KO images are representative of eight embryos. Scale bars: 50 µm.
Mentions: CHD4 was present ubiquitously in cleavage-stage embryos (Fig. 1A). Nuclear CHD4 protein was detected at the 2-cell and 4-cell stages in both wild-type and Chd4−/− embryos, indicating that CHD4 protein either inherited from the oocyte or translated from maternally deposited mRNA was present in these early embryos (Fig. 1A). Null embryos at the 8-cell stage showed much reduced nuclear CHD4 staining, and staining was reduced to background levels in 16-cell mutant embryos. Similarly, ubiquitous nuclear CHD4 expression was detected in wild-type blastocysts, consistent with the X-gal staining seen in Chd4 heterozygote blastocysts, but no protein was detected in littermates produced from either allele (Fig. 1B; supplementary material Fig. S1B).Fig. 1.

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