Angela DiBenedetto

Zebrafish brd2a and brd2b are paralogous members of the bromodomain-ET (BET) family of transcriptional coregulators that show structural and expression divergence

BackgroundBrd2 belongs to the bromodomain-extraterminal domain (BET) family of transcriptional co-regulators, and functions as a pivotal histone-directed recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. Brd2 facilitates expression of genes promoting proliferation and is implicated in apoptosis and in egg maturation and meiotic competence in mammals; it is also a susceptibility gene for juvenile myoclonic epilepsy (JME) in humans. The brd2 ortholog in Drosophila is a maternal effect, embryonic lethal gene that regulates several homeotic loci, including Ultrabithorax. Despite its importance, there are few systematic studies of Brd2 developmental expression in any organism. To help elucidate both conserved and novel gene functions, we cloned and characterized expression of brd2 cDNAs in zebrafish, a vertebrate system useful for genetic analysis of development and disease, and for study of the evolution of gene families and functional diversity in chordates.ResultsWe identify cDNAs representing two paralogous brd2 loci in zebrafish, brd2a on chromosome 19 and brd2b on chromosome 16. By sequence similarity, syntenic and phylogenetic analyses, we present evidence for structural divergence of brd2 after gene duplication in fishes. brd2 paralogs show potential for modular domain combinations, and exhibit distinct RNA expression patterns throughout development. RNA in situ hybridizations in oocytes and embryos implicate brd2a and brd2b as maternal effect genes involved in egg polarity and egg to embryo transition, and as zygotic genes important for development of the vertebrate nervous system and for morphogenesis and differentiation of the digestive tract. Patterns of brd2 developmental expression in zebrafish are consistent with its proposed role in Homeobox gene regulation.ConclusionExpression profiles of zebrafish brd2 paralogs support a role in vertebrate developmental patterning and morphogenesis. Our study uncovers both maternal and zygotic contributions of brd2, the analysis of which may provide insight into the earliest events in vertebrate development, and the etiology of some forms of epilepsy, for which zebrafish is an important model. Knockdowns of brd2 paralogs in zebrafish may now test proposed function and interaction with homeotic loci in vertebrates, and help reveal the extent to which functional novelty or partitioning has occurred after gene duplication.

Chilling causes perivitelline granule formation in activated zebrafish oocytes

Chilling sensitivity in oocytes of the zebrafish represents a potential obstacle to their successful cryopreservation. Here, we report the first cryomicroscopic observations of the response of zebrafish oocytes to chilling conditions. In activated stage V oocytes that had been exposed to hypothermic temperatures, we observed a latent effect of chilling, manifesting as a granular precipitate that appeared in the perivitelline fluid upon return to 28.5 °C. The granules were visible in unstained oocytes under transmitted light microscopy, and the resulting perivitelline turbidity increased in a dose-dependent manner with decreasing chilling temperature (p < 0.001), as well as with increasing time of hypothermic exposure (p < 0.0001). The change in appearance of the perivitelline space in oocytes that had been chilled and rewarmed became statistically significant after a 7-min exposure to 10 °C and after only 30 s at 1 °C (p < 0.05). Thus, even moderate chilling exposures can lead to detectable changes in activated zebrafish oocytes.

Knockdown of epigenetic transcriptional co-regulator Brd2a disrupts apoptosis and proper formation of hindbrain and midbrain-hindbrain boundary (MHB) region in zebrafish

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.

High-Speed Imaging of Intra-Embryonic Phase Transformation Events During Rapid Freezing of Zebrafish Embryos

The zebrafish (Danio rerio) represents an increasingly popular vertebrate animal model valuable for genetic and developmental biology research, due to its rapid rate of reproduction and the ability to directly observe the growing embryos, which are optically clear and develop ex vivo. However, the need to maintain live stock of each genetic strain (the number of which is growing exponentially) is risky and prohibitively costly. Although long-term banking of frozen embryos would solve this problem, to date, no adequate method for cryopreservation of zebrafish embryos has been found (Hagedorn et al., 2004).© 2011 ASME