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2529 Janelia Publications
Showing 1041-1050 of 2529 resultsMetazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, we studied the conserved Hairy/Enhancer of Split family repressor Hairy, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. Our findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. We speculate that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits.
Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, and Tail-up (Islet) to guide the stereotyped differentiation, connectivity, and function of a subset of neurons in Drosophila. The role of Hb9 in directing neuronal differentiation is well documented, but the lineage of Hb9(+) neurons is only partly characterized, its regulation is poorly understood, and most of the downstream genes through which it acts remain at large. Here, we complete the lineage tracing of all embryonic Hb9(+) neurons (to eight neuronal lineages) and provide evidence that hb9, lim3, and tail-up are coordinately regulated by a common set of upstream factors. Through the parallel use of micro-array gene expression profiling and the Dam-ID method, we searched for Hb9-regulated genes, uncovering transcription factors as the most over-represented class of genes regulated by Hb9 (and Nkx6) in the CNS. By a nearly ten-to-one ratio, Hb9 represses rather than activates transcription factors, highlighting transcriptional repression of other transcription factors as a core mechanism by which Hb9 governs neuronal determination. From the small set of genes activated by Hb9, we characterized the expression and function of two - fd59a/foxd, which encodes a transcription factor, and Nitric oxide synthase. Under standard lab conditions, both genes are dispensable for Drosophila development, but Nos appears to inhibit hyper-active behavior and fd59a appears to act in octopaminergic neurons to control egg-laying behavior. Together our data clarify the mechanisms through which Hb9 governs neuronal specification and differentiation and provide an initial characterization of the expression and function of Nos and fd59a in the Drosophila CNS.
Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.
Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
View Publication PageThe identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.
Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe a ratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca(2+) oscillations in cultured astrocytes, (2) ex vivo functional mapping of cholinergic receptors triggering ER Ca(2+) release in acute hippocampal slices from transgenic mice, and (3) in vivo sarcoplasmic reticulum Ca(2+) dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca(2+) dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca(2+) signaling in animal models of health and disease.
When a behavior repeatedly fails to achieve its goal, animals often give up and become passive, which can be strategic for preserving energy or regrouping between attempts. It is unknown how the brain identifies behavioral failures and mediates this behavioral-state switch. In larval zebrafish swimming in virtual reality, visual feedback can be withheld so that swim attempts fail to trigger expected visual flow. After tens of seconds of such motor futility, animals became passive for similar durations. Whole-brain calcium imaging revealed noradrenergic neurons that responded specifically to failed swim attempts and radial astrocytes whose calcium levels accumulated with increasing numbers of failed attempts. Using cell ablation and optogenetic or chemogenetic activation, we found that noradrenergic neurons progressively activated brainstem radial astrocytes, which then suppressed swimming. Thus, radial astrocytes perform a computation critical for behavior: they accumulate evidence that current actions are ineffective and consequently drive changes in behavioral states.
We found that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila. Glial myoglianin upregulated neuronal expression of an ecdysone nuclear receptor that triggered neurite remodeling following the late-larval ecdysone peak. Thus glia orchestrate developmental neural remodeling not only by engulfment of unwanted neurites but also by enabling neuron remodeling.
Cell and tissue specific gene expression is a defining feature of embryonic development in multi-cellular organisms. However, the range of gene expression patterns, the extent of the correlation of expression with function, and the classes of genes whose spatial expression are tightly regulated have been unclear due to the lack of an unbiased, genome-wide survey of gene expression patterns.
Neuroscience research is becoming increasingly more collaborative and interdisciplinary with partnerships between industry and academia and insights from fields beyond neuroscience. In the age of institutional initiatives and multi-investigator collaborations, scientists from around the world shared their perspectives on the effectiveness of large-scale collaborations versus single-lab, hypothesis-driven science.