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

Abstract

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.

Zebrafish Paralogs brd2a and brd2b Are Needed for Proper Circulatory, Excretory and Central Nervous System Formation and Act as Genetic Antagonists during Development

“…In fact, quantitative population data obtained through anti-Pax2a immunofluorescence confocal imaging show, on average, 65.4% of spinal interneurons are unpaired in brd2bMO morphant embryos, compared to only 3.1% unpaired interneurons in controls (Figure 6M,N; p < 0.0001, chi square contingency, Fisher's exact tests). This displays a similar trend to what we saw previously in brd2aMO morphants, which show unpaired interneurons 83% of the time, with 34.5% showing an unrecognizable pattern, compared to uninjected or control-injected embryos, which show, on average, mispairing only 3% of the time [27]. Thus, while patterning genes in the hindbrain and MHB region are misexpressed only in brd2a morphants, HOX-regulated patterning of spinal interneurons is disrupted by deficiencies in either paralog.…”

“…Since Brd2b knockdown results in morphological defects of the ventral trunk, including a malformed and clogged pronephric duct and disorganized PBI, we looked in this region to assess cell death. As in Brd2a knockdowns [27], we consistently see excess cell death overall in the post-anal tail, PBI, and dorsal spinal cord of brd2bMO morphants (Figure 4B,D vs. Figure 4A,C; trunk insets, bottom arrows).…”

“…Eventually, complete lack of circulation leads to degeneration of the trunk (Figure 3I,J) and death typically by 5-7 dpf. This contrasts with brd2aMO morphants, which can survive up to two weeks or more with continued circulation [27]. Similarly to what we observed in Brd2a knockdown studies [27], brd2bMO morphant defects show variable expressivity and, to a lesser degree, incomplete penetrance; both the severity and penetrance of defects increase with dose for brd2bMO1 (p < 0.0001, chi square contingency and Cochran-Mantel-Haenszel tests).…”

“…Significantly, brd2b produces a tissue-and stage-specific transcript variant and possibly several protein isoforms, and is required uniquely for the proper formation of the pronephros and cloaca and for ongoing blood circulation in developing embryos. Since we knew that a deficiency of Brd2a leads to similarly reduced and defective brains as seen here, and also to dramatic increases in neural cell death [27], we examined levels of apoptosis in Brd2b-deficient morphants as well. A minimum of 30 embryos per treatment group were subjected to whole-mount fluorescent TUNEL assay at prim 5 followed by laser-scanning confocal microscopy to produce optical sections and maximum projection images.…”

“…In each of these species, Brd2 produces both maternal and zygotic gene products necessary for survival and normal embryogenesis, suggesting an ancient, conserved role in germline formation, egg-to-embryo transition, and/or early embryo developmental events [ 23 , 24 , 25 ]. In addition, induced deficiencies of Brd2 during development demonstrate its necessity for the proper patterning, differentiation and morphogenesis of body segments in invertebrates [ 4 , 23 , 26 ], and of segmented tissues, most prominently the central nervous system (CNS), in vertebrates [ 27 ]. In Drosophila, ortholog Fsh1 acts as an upstream regulator of various segment-determining homeobox genes and functions in concert with Trithorax group (Trx- G ) proteins, which form the major activating epigenetic axis in metazoan development [ 4 , 23 , 28 ].…”