Many different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of circadian neurons, we optimized a single cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression. Transcription factor regulation is prominent and likely contributes to the robust circadian oscillation of many transcripts, including those that encode cell-surface proteins previously shown to be important for cell recognition and synapse formation during development. The many other clock-regulated genes also constitute an important resource for future mechanistic and functional studies between clock neurons and/or for temporal signaling to circuits elsewhere in the fly brain.

Localized translation plays a crucial role in synaptic plasticity and memory consolidation. However, it has not been possible to follow the dynamics of memory-associated mRNAs in living neurons in response to neuronal activity in real time. We have generated a novel mouse model where the endogenous Arc/Arg3.1 gene is tagged in its 3' untranslated region with stem-loops that bind a bacteriophage PP7 coat protein (PCP), allowing visualization of individual mRNAs in real time. The physiological response of the tagged gene to neuronal activity is identical to endogenous Arc and reports the true dynamics of Arc mRNA from transcription to degradation. The transcription dynamics of Arc in cultured hippocampal neurons revealed two novel results: (i) A robust transcriptional burst with prolonged ON state occurs after stimulation, and (ii) transcription cycles continue even after initial stimulation is removed. The correlation of stimulation with Arc transcription and mRNA transport in individual neurons revealed that stimulus-induced Ca activity was necessary but not sufficient for triggering Arc transcription and that blocking neuronal activity did not affect the dendritic transport of newly synthesized Arc mRNAs. This mouse will provide an important reagent to investigate how individual neurons transduce activity into spatiotemporal regulation of gene expression at the synapse.

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3’ untranslated region of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

Near-infrared (NIR) fluorescent reporters open interesting perspectives for multiplexed imaging with higher contrast and depth using less toxic light. Here, we propose nirFAST, a small (14 kDa) chemogenetic NIR fluorescent reporter, displaying higher cellular brightness compared to top-performing NIR fluorescent proteins. nirFAST binds and stabilizes the fluorescent state of synthetic cell permeant fluorogenic chromophores (so-called fluorogens), otherwise dark when free. nirFAST displays tunable NIR, far-red or red emission through change of fluorogen. nirFAST allows imaging and spectral multiplexing in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its suitability for stimulated emission depletion nanoscopy enabled protein imaging with subdiffraction resolution in live cells. nirFAST enabled the design of a two-color cell cycle indicator for monitoring the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.

bioRxiv preprint: https://doi.org/10.1101/2024.04.05.588310

Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.

Inference-based decision-making, which underlies a broad range of behavioral tasks, is typically studied using a small number of handcrafted models. We instead enumerate a complete ensemble of strategies that could be used to effectively, but not necessarily optimally, solve a dynamic foraging task. Each strategy is expressed as a behavioral "program" that uses a limited number of internal states to specify actions conditioned on past observations. We show that the ensemble of strategies is enormous-comprising a quarter million programs with up to five internal states-but can nevertheless be understood in terms of algorithmic "mutations" that alter the structure of individual programs. We devise embedding algorithms that reveal how mutations away from a Bayesian-like strategy can diversify behavior while preserving performance, and we construct a compositional description to link low-dimensional changes in algorithmic structure with high-dimensional changes in behavior. Together, this work provides an alternative approach for understanding individual variability in behavior across animals and tasks.

Genetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.