Phenotypic analysis of transcriptional co-activator, brd2, gene knockdowns in zebrafish (Danio rerio) embryos

Bromodomain-containing 2 (BRD2) is a transcriptional co-activator involved in early developmental regulation through a potential role in both proliferation and apoptosis. Brd2 plays a role in Drosophila pattern formation and is expressed differentially in zebrafish and mouse embryonic development, yet no brd2 knockout has been performed. Two single nucleotide polymorphisms (SNPs) present in the human brd2 promoter region correlate with susceptibility to juvenile myoclonic epilepsy (JME), implying that normal brain development requires proper brd2 expression. In this study, morpholino (MO) antisense oligonucleotides were used to knockdown brd2 gene expression in zebrafish embryos, and gross changes in morphology and apoptosis were assessed. MO-injected zebrafish at Prim-5 stage of development exhibit an undefined cerebellum, usually accompanied by a tail kink, compared to wild-type and dye-injected controls. Quantification of brain morphometric measurements by analysis of variance reveals a significant difference in hindbrain areas between MO-injected and control embryos. In terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assays, these embryos also show increased apoptosis in post-anal tail regions compared to wild-type controls, possibly related to the observed tail kink phenotype. This study represents the first in vivo brd2 gene knockdown and phenotypic characterization in a vertebrate. We provide evidence for brd2 function during proper central nervous system development and cell death regulation, supporting the idea that brd2 mis-expression during CNS development may generate JME-associated abnormalities.