Biology subjects

Blau, J.

Publications and source records attributed to Blau, J..

7 recordsLinked to original sources

bioRxiv2026

Rhythmic expression of a cAMP phosphodiesterase switches neuronal excitability states in the mammalian suprachiasmatic nucleus

How the transcriptome of a neuron determines its electrophysiological properties and how this change in different contexts are major questions in neurobiology. The intracellular molecular clocks in the circadian pacemaker neurons in the mammalian suprachiasmatic nucleus (SCN) drive 24hr rhythms in clock gene expression via a transcriptional-translational feedback loop1,2. These molecular clocks also control 24hr rhythms in the firing of SCN neurons3-5 - a subset of which enter a silent depolarized state around midday6. We used single-cell RNA sequencing and identified that the Avp / Nms and Vip / Nms are the SCN neurons that undergo daytime silencing. We also found high level and rhythmic expression of the Pde10a phosphodiesterase in these Nms SCN neurons. Acutely inhibiting Pde10a activity or elevating intracellular cAMP - to bypass high Pde10a levels - specifically shifts these Nms+ SCN neurons into a depolarized silent state hours ahead of schedule. A similar mechanism may occur in other neurons that switch to silent depolarized states.

neuroscience↗

bioRxiv2024

Circadian clock neurons use activity-regulated gene expression for structural plasticity

Drosophila s-LNv circadian pacemaker neurons show dramatic structural plasticity, with their projections expanded at dawn and then retracted by dusk. This predictable plasticity makes s-LNvs ideal to study molecular mechanisms of plasticity. Although s-LNv plasticity is controlled by their molecular clock, changing s-LNv excitability also regulates plasticity. Here, we tested the idea that s-LNvs use activity-regulated genes to control plasticity. We found that inducing expression of either of the activity-regulated transcription factors Hr38 or Sr (orthologs of mammalian Nr4a1 and Egr1) is sufficient to rapidly expand s-LNv projections. Conversely, transiently knocking down expression of either Hr38 or sr blocks expansion of s-LNv projections at dawn. We show that Hr38 rapidly induces transcription of sif, which encodes a Rac1 GEF required for s-LNv plasticity rhythms. We conclude that the s-LNv molecular clock controls s-LNv excitability, which couples to an activity-regulated gene expression program to control s-LNv plasticity.

neuroscience↗

bioRxiv2024

Tango-seq: overlaying transcriptomics on connectomics to identify neurons downstream of Drosophila clock neurons

Knowing how neural circuits change with neuronal plasticity and differ between individuals is important to fully understand behavior. Connectomes are typically assembled using electron microscopy, but this is low throughput and impractical for analyzing plasticity or mutations. Here, we modified the trans-Tango genetic circuit-tracing technique to identify neurons synaptically downstream of Drosophila s-LNv clock neurons, which show 24hr plasticity rhythms. s-LNv target neurons were labeled specifically in adult flies using a nuclear reporter gene, which facilitated their purification and then single cell sequencing. We call this Tango-seq, and it allows transcriptomic data - and thus cell identity - to be overlayed on top of anatomical data. We found that s-LNvs preferentially make synaptic connections with a subset of the CNMa+ DN1p clock neurons, and that these are likely plastic connections. We also identified synaptic connections between s-LNvs and mushroom body Kenyon cells. Tango-seq should be a useful addition to the connectomics toolkit.

neuroscience↗

bioRxiv2023

A rapid and dynamic role for FMRP in the plasticity of adult neurons

Fragile X syndrome (FXS) is a neuro-developmental disorder caused by silencing Fmr1, which encodes the RNA-binding protein FMRP. Although Fmr1 is expressed in adult neurons, it has been challenging to separate acute from chronic effects of loss of Fmr1 in models of FXS. We have used the precision of Drosophila genetics to test if Fmr1 acutely affects adult neuronal plasticity in vivo, focusing on the s-LNv circadian pacemaker neurons that show 24 hour rhythms in structural plasticity. We found that over-expressing Fmr1 for only 4 hours blocks the activity-dependent expansion of s-LNv projections without altering the circadian clock or activity- regulated gene expression. Conversely, acutely reducing Fmr1 expression prevented s-LNv projections from retracting. One FMRP target that we identified in s-LNvs is sif, which encodes a Rac1 GEF. Our data indicate that FMRP normally reduces sif mRNA translation at dusk to reduce Rac1 activity. Overall, our data reveal a previously unappreciated rapid and direct role for FMRP in acutely regulating neuronal plasticity in adult neurons, and underscore the importance of RNA-binding proteins in this process.

neuroscience↗

bioRxiv2019

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

AbstractMacrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation and maintenance of homeostasis. Understanding the genes involved in all aspects of macrophage biology is essential to gaining new insights into immune system dysregulation during diseases that range from autoinflammatory to cancer. Here we utilize high throughput clustered regularly interspaced short palindromic repeats (CRISPR) screening to generate a resource that identifies genes required for macrophage viability and function. First, we employ a pooled based CRISPR/Cas nuclease active screening approach to identify essential genes required for macrophage viability by targeting genes within coding exons. In addition, we also target 3UTRs to gain insights into new cis-regulatory regions that control expression of these essential genes. Second, using our recently generated NF-{kappa}B reporter macrophage line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen to identify regulators of inflammation. We identify a number of novel positive and negative regulators of the NF-{kappa}B pathway as well as unraveling complexities of the TNF signaling cascade showing it can function in an autocrine manner to negatively regulate the pathway. Utilizing a single complex library design we are capable of interrogating various aspects of macrophage biology, generating a resource for future studies.\n\nAuthor SummaryExcess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are important mediators of this inflammatory response. Defining the genes involved in their viability and effector function is needed to completely understand these two important aspects of macrophage biology. Here we screened over 21,000 genes and generated a resource guide of genes required for macrophage viability as well as novel positive and negative regulators of NF-{kappa}B signaling.

immunology↗

bioRxiv2019

Transcription of a plasticity gene is activated by neuronal hyperpolarization

Long term synaptic plasticity requires transcriptional changes in response to neuronal activity. While neuronal activity rapidly increases transcription of activity-regulated genes, it is less clear how neurons respond to neuronal inactivity. The plasticity gene Pura encodes a Rho1 GEF whose rhythmic expression drives 24 hour rhythms in the retraction of the projections of Drosophila LNv circadian pacemaker neurons. We found that Pura transcription in LNvs is repressed by neuronal activity and induced by neuronal inactivity - the opposite of canonical activity-regulated genes. We identified the Pax6 transcription factor Toy as the relevant activator of Pura transcription, and found that toy transcription itself is induced by neuronal inactivity and by reducing intracellular calcium. Thus we propose that neurons contain complementary gene expression programs that are activated by either increased or decreased neuronal activity.

neuroscience↗

bioRxiv2017

Decoding directional genetic dependencies through orthogonal CRISPR/Cas screens

Genetic interaction studies are a powerful approach to identify functional interactions between genes. This approach can reveal networks of regulatory hubs and connect uncharacterised genes to well-studied pathways. However, this approach has previously been limited to simple gene inactivation studies. Here, we present an orthogonal CRISPR/Cas-mediated genetic interaction approach that allows the systematic activation of one gene while simultaneously knocking out a second gene in the same cell. We have developed this concept into a quantitative and scalable combinatorial screening platform that allows the parallel interrogation of hundreds of thousands of genetic interactions. We demonstrate that the established platform works robustly to uncover genetic interactions in human cancer cells and to interpret the direction of the flow of genetic information.

molecular biology↗