Cell type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues – the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons – where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

A fundamental goal in neuroscience is to uncover common principles by which different modalities of information are processed. In the mammalian brain, thalamus acts as the essential hub for forebrain circuits handling inputs from sensory, motor, limbic, and cognitive pathways. Whether thalamus imposes common transformations on each of these modalities is unknown. Molecular characterization offers a principled approach to revealing the organization of thalamus. Using near-comprehensive and projection-specific transcriptomic sequencing, we found that almost all thalamic nuclei fit into one of three profiles. These profiles lie on a single axis of genetic variance which is aligned with the mediolateral spatial axis of thalamus. Genes defining this axis of variance include receptors and ion channels, providing a systematic diversification of input/output transformations across the topography of thalamus. Single cell transcriptional profiling revealed graded heterogeneity within individual thalamic nuclei, demonstrating that a spectrum of cell types and potentially diverse input/output transforms exist within a given thalamic nucleus. Together, our data argue for an archetypal organization of pathways serving diverse input modalities, and provides a comprehensive organizational scheme for thalamus.

Faithful transcription of human mitochondrial DNA has been reproduced in vitro, using a fraction of mitochondrial proteins capable of accurate initiation at both the heavy- and light-strand promoters. Here we report the initial dissection of this system into two nonfunctional components which, upon mixing, reconstitute promoter-specific transcriptional capacity in vitro. One of these components copurifies with the major nonspecific RNA polymerase activity, suggesting its identity. The other component lacks significant polymerase activity, but contains a protein or proteins required for accurate initiation at the two individual promoters by isolated mitochondrial RNA polymerase. This factor facilitates specific transcription, but has little or no effect on nonspecific transcription of a synthetic copolymer (poly(dA-dT)), indicating a positive role in proper promoter recognition. The transcription factor markedly stimulates light-strand transcription, but only moderately enhances transcription initiation at the heavy-strand promoter, suggesting different or additional factor requirements for heavy-strand transcription. These requirements may reflect the functional differences between heavy- and light-strand transcription in vivo and, in particular, the role of the light-strand promoter in priming of heavy-strand DNA replication.

Intracellular Ca(2+) is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca(2+) (TRIC) in Drosophila that uses a binary expression system to report Ca(2+)-dependent interactions between calmodulin and its target peptide. We found that in vitro assays predicted in vivo properties of TRIC and that TRIC signals in sensory systems depend on neuronal activity. TRIC was able to quantitatively monitor neuronal responses that changed slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient-activated cells enhanced stress resistance, providing a proof of principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC's modular design should enable optimization and adaptation to other organisms.

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 provide additional colors for highly multiplexed imaging of cells and organisms, and enable imaging with less toxic light and higher contrast and depth. Here, we present the engineering of nirFAST, a small tunable chemogenetic NIR fluorescent reporter that is brighter than top-performing NIR fluorescent proteins in cultured mammalian cells. nirFAST is a small genetically encoded protein of 14 kDa that binds and stabilizes the fluorescent state of synthetic, highly cell-permeant, fluorogenic chromophores (so-called fluorogens) that are otherwise dark when free. Engineered to emit NIR light, nirFAST can also emit far-red or red lights through change of chromophore. nirFAST allows the imaging of proteins in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its near infrared fluorescence provides an additional color for high spectral multiplexing. We showed that nirFAST is well-suited for stimulated emission depletion (STED) nanoscopy, allowing the efficient imaging of proteins with subdiffraction resolution in live cells. nirFAST enabled the design of a chemogenetic green-NIR fluorescent ubiquitination-based cell cycle indicator (FUCCI) for the monitoring of the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a fluorogenic chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.

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.