Double bromodomain-containing gene Brd2 is essential for embryonic development in mouse - PubMed (original) (raw)

Double bromodomain-containing gene Brd2 is essential for embryonic development in mouse

Enyuan Shang et al. Dev Dyn. 2009 Apr.

Abstract

The BET subfamily of bromodomain-containing genes is characterized by the presence of two bromodomains and a unique ET domain at their carboxyl termini. Here, we show that the founding member of this subfamily, Brd2, is an essential gene by generating a mutant mouse line lacking Brd2 function. Homozygous Brd2 mutants are embryonic lethal, with most Brd2(-/-) embryos dying by embryonic day 11.5. Before death, the homozygous embryos were notably smaller and exhibited abnormalities in the neural tube where the gene is highly expressed. Brd2-deficient embryonic fibroblast cells were observed to proliferate more slowly than controls. Experiments to explore whether placental insufficiency could be a cause of the embryonic lethality showed that injecting diploid mutant embryonic stem cells into tetraploid wild-type blastocysts did not rescue the lethality; that is Brd2-deficient embryos could not be rescued by wild-type extraembryonic tissues. Furthermore, there were enhanced levels of cell death in Brd2-deficient embryos.

Copyright 2009 Wiley-Liss, Inc.

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Figures

Figure 1

Figure 1

Panel A: Schematic depiction of the inactivation of the Brd2 gene by a gene-trap approach. The gene trap vector was inserted into the first intron of the Brd2 gene, near the putative translation start codon ATG. The gene trap insertion is about 8 kb in size and is not depicted in proportion to the Brd2 gene. Arrows indicate the position of PCR primers used for verification of the insertion event and for genotyping progeny carrying the mutant or wild-type alleles. The primers are: EX1F (exon 1 forward), IN1R (intron 1 reverse), and GT1R (genetrap vector reverse). Panel B: Visualization of the PCR products used to genotype the progeny. The use of EX1F + GT1R gives the higher band (500 bp) representing the mutated allele; EX1F + IN1R produces the lower band (220 bp)--wildtype allele. Since the insertion is 8 kb, under our PCR condition (1 min extension time), this pair of primers would not yield a band corresponding to the mutant allele.

Figure 2

Figure 2

Panel A: Photographs of wild-type (Brd2+/+, left) and _Brd2_−/− (right) embryos at E9.5 of gestation. The mutant embryo is smaller and the developing neural tube is unfused (arrow). Panel B: A litter of 11 embryos at E9.5 of gestation were fixed in 4% paraformaldehyde and photographed together to portray the variations in phenotype among the embryos. The genotype and weight of the embryos are, from the top row and left to right: Top Row: +/+, 4.6 mg; +/+, 6.5 mg; +/−, 4.3 mg; +/−, 2.3 mg; Middle Row: +/−, 1.9 mg; +/+, 2.0 mg; −/−, 2.2 mg; +/−, 1.9 mg; and Bottom Row: −/−, 1.1 mg; −/−, 1.0 mg; −/−, 1.2 mg. The mean and SD for each genotype are: +/+, 5.1 ± 1.2 mg; +/−, 2.6 ± 1.3 mg; −/−, 1.4 ± 0.6 mg. Panel C: Sagittal sections of E11.5 embryos of wildtype and mutant embryos. Grossly normal appearing heart, lung, etc. are seen in the mutant, but the brain of the mutant is aberrant. FB, forebrain; MB, midbrain; HB, hindbrain. Panel D: Whole-mount in situ hybridization of wild-type and Brd2 mutant E9.5 embryos using digoxygenin-labeled Brd2 RNA probes (see Materials and Methods). Brd2 mRNA is detected in the wild-type developing neural system but is absent in the mutant.

Figure 3

Figure 3

Panel A: Growth parameters of mouse embryonic fibroblast (MEFs) derived from Brd2+/+, Brd2+/−, and _Brd2_−/− embryos of E9.5 (157-5 and 157-11), E10.5 (128-1 and 128-2), and E11.5 (127-2 and 127-7). The cells were plated at 104 cells/well at day 0 and the number of cells/well was monitored at daily intervals. At each time point, 3 wells of cells were counted and the number of cells is shown as average of the three samples. The culture media was changed at day 4. The _Brd2_−/− cells grew very poorly as compared to the wild-type cells, and the heterozygous Brd2+/− MEFs grew at an intermediate rate as compared to the Brd2+/+ and _Brd2_−/−cells. Panel B: Flow cytometry analysis of cell cycle of MEFs of E9.5 _Brd2_−/− (157-5) and Brd2+/+ (157-11) embryos, showing an elevated G1 peak (2N) in the mutant cells, indicating that the cell cycle of _Brd2_−/− MEFs is delayed at the G1 phase.

Figure 4

Figure 4

Lysotracker staining to detect apoptotic cells in E9.5 embryos. Embryos were dissected and processed for Lysotracker staining in the whole embryos (see materials and Methods). Photomicrographs of the entire embryos of Brd2+/+ versus _Brd2_−/− are shown. There are enhanced levels of positive lysotracker staining in the _Brd2_-deficient embryo.

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