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A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs (1) and recruits general transcription factors to initiate transcription (2). The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle (3-6). Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition (7). This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression (8), and determined the positions of H3K4me3-marked promoter-associated nucleosomes (9). Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (11) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts., Mapping of TSSs using cap analysis of gene expression (CAGE) (8) identified two major promoter classes with respect to TSS precision (10,11): 'sharp' promoters with one predominant TSS, often associated [...]

In the telencephalon of adult zebrafish, the inhibitor of DNA binding 1 (id1) gene is expressed in radial glial cells (RGCs), behaving as neural stem cells (NSCs), during constitutive and regenerative neurogenesis. Id1 controls the balance between resting and proliferating states of RGCs by promoting quiescence. Here, we identified a phylogenetically conserved cis‐regulatory module (CRM) mediating the specific expression of id1 in RGCs. Systematic deletion mapping and mutation of conserved transcription factor binding sites in stable transgenic zebrafish lines reveal that this CRM operates via conserved smad1/5 and 4 binding motifs under both homeostatic and regenerative conditions. Transcriptome analysis of injured and uninjured telencephala as well as pharmacological inhibition experiments identify a crucial role of bone morphogenetic protein (BMP) signaling for the function of the CRM. Our data highlight that BMP signals control id1 expression and thus NSC proliferation during constitutive and induced neurogenesis. [ABSTRACT FROM AUTHOR]

Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a- and gata3-dependent precursors coexpressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses. [ABSTRACT FROM AUTHOR]

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals. S tem C ells 2015;33:892-903 [ABSTRACT FROM AUTHOR]

Zebrafish embryos offer a unique combination of high-throughput capabilities and the complexity of the vertebrate animal for a variety of phenotypic screening applications. However, there is a need for automation of imaging technologies to exploit the potential of the transparent embryo. Here we report a high-throughput pipeline for registering domain-specific reporter expression in zebrafish embryos with the aim of mapping the interactions between cis-regulatory modules and core promoters. Automated microscopy coupled with custom-built embryo detection and segmentation software allowed the spatial registration of reporter activity for 202 enhancer-promoter combinations, based on images of thousands of embryos. The diversity of promoter-enhancer interaction specificities underscores the importance of the core promoter sequence in cis-regulatory interactions and provides a promoter resource for transgenic reporter studies. The technology described here is also suitable for the spatial analysis of fluorescence readouts in genetic, pharmaceutical or toxicological screens. [ABSTRACT FROM AUTHOR]

Abstract: Transcription is the primary step in the retrieval of genetic information. A substantial proportion of the protein repertoire of each organism consists of transcriptional regulators (TRs). It is believed that the differential expression and combinatorial action of these TRs is essential for vertebrate development and body homeostasis. We mined the zebrafish genome exhaustively for genes encoding TRs and determined their expression in the zebrafish embryo by sequencing to saturation and in situ hybridisation. At the evolutionary conserved phylotypic stage, 75% of the 3302 TR genes encoded in the genome are already expressed. The number of expressed TR genes increases only marginally in subsequent stages and is maintained during adulthood suggesting important roles of the TR genes in body homeostasis. Fewer than half of the TR genes (45%, n=1711 genes) are expressed in a tissue-restricted manner in the embryo. Transcripts of 207 genes were detected in a single tissue in the 24h embryo, potentially acting as regulators of specific processes. Other TR genes were expressed in multiple tissues. However, with the exception of certain territories in the nervous system, we did not find significant synexpression suggesting that most tissue-restricted TRs act in a freely combinatorial fashion. Our data indicate that elaboration of body pattern and function from the phylotypic stage onward relies mostly on redeployment of TRs and post-transcriptional processes. [Copyright &y& Elsevier]

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression.

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals., (© 2014 AlphaMed Press.)