The miR-430 locus with extreme promoter density forms a transcription body during the minor wave of zygotic genome activation.

In anamniote embryos, the major wave of zygotic genome activation starts during the mid-blastula transition. However, some genes escape global genome repression, are activated substantially earlier, and contribute to the minor wave of genome activation. The mechanisms underlying the minor wave of genome activation are little understood. We explored the genomic organization and cis-regulatory mechanisms of a transcription body, in which the minor wave of genome activation is first detected in zebrafish. We identified the miR-430 cluster as having excessive copy number and the highest density of Pol-II-transcribed promoters in the genome, and this is required for forming the transcription body. However, this transcription body is not essential for, nor does it encompasse, minor wave transcription globally. Instead, distinct minor-wave-specific promoter architecture suggests that promoter-autonomous mechanisms regulate the minor wave of genome activation. The minor-wave-specific features also suggest distinct transcription initiation mechanisms between the minor and major waves of genome activation.

In vivo High-Content Screening in Zebrafish for Developmental Nephrotoxicity of Approved Drugs.

Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the Tg(wt1b:EGFP) zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for in vivo large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.

A cell cycle-coordinated Polymerase II transcription compartment encompasses gene expression before global genome activation.

Most metazoan embryos commence development with rapid, transcriptionally silent cell divisions, with genome activation delayed until the mid-blastula transition (MBT). However, a set of genes escapes global repression and gets activated before MBT. Here we describe the formation and the spatio-temporal dynamics of a pair of distinct transcription compartments, which encompasses the earliest gene expression in zebrafish. 4D imaging of pri-miR430 and zinc-finger-gene activities by a novel, native transcription imaging approach reveals transcriptional sharing of nuclear compartments, which are regulated by homologous chromosome organisation. These compartments carry the majority of nascent-RNAs and active Polymerase II, are chromatin-depleted and represent the main sites of detectable transcription before MBT. Transcription occurs during the S-phase of increasingly permissive cleavage cycles. It is proposed, that the transcription compartment is part of the regulatory architecture of embryonic nuclei and offers a transcriptionally competent environment to facilitate early escape from repression before global genome activation.

Translational co-regulation of a ligand and inhibitor by a conserved RNA element.

In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3'UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3'UTR, the lft1 and lft2 3'UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a 'translational regulon'.