The ability to image and manipulate specific cell populations in Drosophila enables the investigation of how neural circuits develop and coordinate appropriate motor behaviors. Gal4 lines give genetic access to many types of neurons, but the expression patterns of these reagents are often complex. Here, we present the generation and expression patterns of LexA lines based on the vesicular neurotransmitter transporters and Hox transcription factors. Intersections between these LexA lines and existing Gal4 collections provide a strategy for rationally subdividing complex expression patterns based on neurotransmitter or segmental identity.

Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis, with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

After spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site.

Neurons are inherently plastic, adjusting their structure, connectivity and excitability in response to changes in activity. How neurons sense changes in their activity level and then transduce these to structural changes remains to be fully elucidated. Working with the Drosophila larval locomotor network, we show that neurons use reactive oxygen species (ROS), metabolic byproducts, to monitor their activity. ROS signals are both necessary and sufficient for activity-dependent structural adjustments of both pre- and postsynaptic terminals and for network output, as measured by larval crawling behavior. We find the highly conserved Parkinsons disease-linked protein DJ-1b acts as a redox sensor in neurons where it regulates pre- and postsynaptic structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. Neuronal ROS thus play an important physiological role as second messengers required for neuronal and network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.

Virtual reality (VR) holds great promise as a tool to study the neural circuitry underlying animal behaviors. Here, we discuss the advantages of VR and the experimental paradigms and technologies that enable closed loop behavioral experiments. We review recent results from VR research in genetic model organisms where the potential combination of rich behaviors, genetic tools and cutting edge neural recording techniques are leading to breakthroughs in our understanding of the neural basis of behavior. We also discuss several key issues to consider when performing VR experiments and provide an outlook for the future of this exciting experimental toolkit.

Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell’s ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA–including initiation, elongation, and termination–at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

Although mRNA translation is a fundamental biological process, it has never been imaged in real-time with single molecule precision in vivo. To achieve this, we developed Nascent Chain Tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify single mRNA protein synthesis dynamics. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 s. Polysomes contain ~1 ribosome every 200-900 nucleotides and are globular rather than elongated in shape. By developing multi-color probes, we show most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

Electrical recordings from a large array of electrodes give us access to neural population activity with single-cell, single-spike resolution. These recordings contain extracellular spikes which must be correctly detected and assigned to individual neurons. Despite numerous spike-sorting techniques developed in the past, a lack of high-quality ground-truth datasets hinders the validation of spike-sorting approaches. Furthermore, existing approaches requiring manual corrections are not scalable for hours of recordings exceeding 100 channels. To address these issues, we built a comprehensive spike-sorting pipeline that performs reliably under noise and probe drift by incorporating a channel-covariance feature and a clustering based on fast density-peak finding. We validated performance of our workflow using multiple ground-truth datasets that recently became available. Our software scales linearly and processes a 1000-channel recording in real-time using a single workstation. Accurate, real-time spike sorting from large recording arrays will enable more precise control of closed-loop feedback experiments and brain-computer interfaces.