The chromatin-targeting protein Brd2 is required for neural tube closure and embryogenesis

Abstract

Chromatin modifications are essential for directing transcription during embryonic development. Bromodomain-containing protein 2 (Brd2; also called RING3 and Fsrg1) is one of four BET (bromodomain and extra-terminal domain) family members known to selectively bind acetylated histones H3 and H4. Brd2 associates with multiple subunits of the transcriptional apparatus including the mediator, TFIID and Swi/Snf multiprotein complexes. While molecular interactions of Brd2 are known, the functions of Brd2 in mammalian embryogenesis remain unknown. In developing a mouse model deficient in Brd2, we find that Brd2 is required for the completion of embryogenesis and proper neural tube closure during development. Embryos lacking Brd2 expression survive up to embryonic day 13.5, soon after mid-gestation, and display fully penetrant neurulation defects that largely result in exencephaly of the developing hindbrain. In this study, we find that highest expression of Brd2 is detected in the developing neural tube, correlating with the neural tube defects found in Brd2-null embryos. Additionally, embryos lacking Brd2 expression display altered gene expression programs, including the mis-expression of multiple genes known to guide neuronal development. Together these results implicate essential roles for Brd2 as a critical integrator of chromatin structure and transcription during mammalian embryogenesis and neurogenesis.

Figure 1

Identification of Brd2-null embryos. (A) Schematic of genomic Brd2 loci with insertions of two independent gene trap constructs between the first and second coding exons. (B) Brd2 genotypic distribution of viable mice at four weeks of age born to Brd2 heterozygous intercrosses. Total number of mice and sex-distribution of Brd2 genotypes are shown with wild type (Brd2^+/+), Brd2 heterozygote (Brd2^+/−) and null homozygous Brd2 mutants (Brd2^−/−). (C) Quantitative RT-PCR analysis of mRNA derived from whole E9.5 embryos across the three Brd2 genotypes. Relative levels of Brd2 mRNA and mRNA from the LacZ transgene are graphed from a wild type embryo (+/+), a Brd2 heterozygous embryo (+/−) and a homozygous Brd2 mutant embryo (−/−) derived from the same litter. Relative mRNA expression levels were determined using ΔCt values and were normalized to 18S rRNA levels to correct for minor variations in starting RNA concentrations. The absence of signal in the Brd2^−/− embryo indicates that this embryo is devoid of wild type Brd2 transcript and represents a Brd2-null embryo. LacZ expression was only detected in the Brd2 heterozygous embryo (+/−) and the homozygous Brd2 mutant embryo (−/−) and Med26 expression was assayed as a control. Numbers at the top of each column are relative expression values for each gene set.

Figure 2

Growth retardation and hindbrain exencephaly of Brd2-null embryos. Whole-mount analysis of matched wild type and Brd2-null embryos from E9.0–E13.5. Each embryo is represented by a side-by-side comparison of lateral and dorsal images. The genotypes of the embryos are as follows: (A, B, E, F, I, J, M, N) wild-type (+/+) embryos and (C, D, G, H, K, L, O, P) Brd2-null embryos. Note the marked decrease in overall size and the open neural tube (white arrows) at the level of the mesencephalon (G, H) and rhombencephalon (K, L, O, P) of the Brd2-null embryos. The four images at each time point are shown at identical magnification.

Figure 3

Hindbrain exencephaly of Brd2^−/− embryos at E10.5 and E11.5. Whole-mount analysis of littermate Brd2^+/+ and Brd2^−/− embryos demonstrate the open hindbrain region and thickening of the edges of the open neural tube. Note the wavy, thickened neural tube in the Brd2^−/− E10.5 embryo. The four images at each time point are shown at identical magnification to illustrate relative size differences.

Figure 4

The caudal neural tubes of Brd2^−/− embryos display incomplete closure and frequent malformations. Whole-mount analysis of E13 Brd2^+/+ and Brd2^−/− littermates from two separate heterozygous crosses illustrate the closure defects frequently found in Brd2-null embryos. Note the wavy, open neural tubes of the null embryos (white arrows) compared to wild-type littermates.

Figure 5

Embryonic Brd2 expression in the developing neural tube. A matched wild type (+/+, A, B) embryo and LacZ-containing Brd2 heterozygous (+/LacZ, C, D) embryo from the same E13.0 litter is shown after β-galactosidase staining. Lateral (A, C) and dorsal (B, D) views are presented to highlight the expression of this Brd2 reporter gene in the entire developing neural tube.

Figure 6

Proliferation and apoptosis in the Brd2-null embryos. Sections from a matched wild type control (+/+, A, C) embryo and Brd2-null (−/−, B, D) embryo from the same E11.5 litter are shown after anti-phosphorylated histone H3 (A, B) and TUNEL staining (C, D). Phosphorylated histone H3 staining appears pink, TUNEL staining appears light green, and DAPI is shown in blue.

Figure 7

Decreased transcript levels of neuronal mRNAs in Brd2^−/− embryos. (A) Quantitative RT-PCR for mediator component Med26 and mesodermal marker Brachyury (controls) show little relative difference between Brd2^+/+ and Brd2^−/− embryos at E9.5, while mRNA levels of genes known to be involved in neuronal development NeuroD1, NeuroD4, Olig3 and SlitRK6 show a marked (3–4 fold) decrease in transcript level. (B) Expression of neuronal mRNAs in Brd2^+/+ and Brd2^−/− embryos with 37–39 somites. Relative mRNA expression levels were determined using ΔCt values and were normalized to 18S rRNA levels to correct for minor variations in starting RNA concentrations.