Bisphenol A (BPA) is an endocrine disruptor that displays estrogenic activity. Several reports suggest that BPA may have estrogen receptor‐independent effects. In zebrafish, 50 μM BPA exposure induces otic vesicle abnormalities, including otolith aggregation. The purpose of this study was to test if BPA action was mediated in vivo during zebrafish development by the orphan nuclear estrogen related receptor (ERR) γ. Combining pharmacological and functional approaches, we demonstrate that the zebrafish ERRγ mediates BPA‐induced malformations in otoliths. Using different bisphenol derivatives, we show that different compounds can induce a similar otolith phenotype than BPA and that the binding affinity of these derivatives to the zebrafish ERRγ correlates with their ability to induce otolith malformations. Morpholino knockdown of ERRγ function suppresses the BPA effect on otoliths whereas overexpression of ERRγ led to a BPA‐like otolith phenotype. Moreover, a subphenotypical dose of BPA (1 μM) combined with ERRγ overexpression led to a full‐dose (50 μM) BPA otolith phenotype. We therefore conclude that ERRγ mediates the otic vesicle phenotype generated by BPA. Our results suggest that the range of pathways perturbed by this compound and its potential harmful effect are larger than expected.—Tohmé, M., Prud'homme, S. M., Boulahtouf, A., Samarut, E., Brunet, F., Bernard, L., Bourguet, W., Gibert, Y., Balaguer, P., Laudet, V. Estrogen‐related receptor γ is an in vivo receptor of bisphenol A. FASEB J . 28, 3124–3133 (2014). www.fasebj.org

The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located ±10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Summary: Objective: In humans, mutations of the γ‐aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy‐causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms ofGABRA1loss of function.Methods: We generated agabra1^−/−zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay ofgabra1^−/−larval brains.Results: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic–clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation.Significance: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1‐deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

The CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals. Here we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels. With this technique, we were able to genotype adult zebrafish using genomic DNA extracted from fin-clips in less than 2 h. We were also able to obtain a reliable and early read-out of the effectiveness of guide RNAs only 4 h after the embryos were injected with the constructs for the CRISPR/Cas9 mutagenic system. Furthermore, through mutagenesis kinetic assay, we identified that the 2-cell stage is the earliest time point at which indels can be observed. By combining an inexpensive and rapid genomic DNA extraction method with an HRM-based assay, our approach allows for high-throughput genotyping of adult zebrafish and embryos, and is more sensitive than standard PCR approaches, permitting early identification of CRISPR-induced indels and with applications for other model organisms as well.