Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that most GAF is chromatin bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for more than 2 min, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF's DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF's neighbors such as Heat Shock Factor. Evaluation of GAF kinetics together with its endogenous abundance indicates that, despite on-off dynamics, GAF constitutively and fully occupies major chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.

In this work, we find that CD8 T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49CD8 regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 T cells efficiently eliminated pathogenic gliadin-specific CD4 T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIRCD8 T cells, but not CD4 regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49CD8 T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8 T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

Histone-lysine N-methyltransferase 2 (KMT2) methyltransferases play critical roles in gene regulation, cell differentiation, animal development, and human diseases. KMT2 biological roles are often attributed to their methyltransferase activities on lysine 4 of histone H3 (H3K4). However, recent data indicate that KMT2 proteins also possess non-enzymatic functions. In this review, we discuss the current understanding of KMT2 family, with a focus on their enzymatic activity-dependent and -independent functions. Six mammalian KMT2 proteins of three subgroups, KMT2A/B (MLL1/2), KMT2C/D (MLL3/4), and KMT2F/G (SETD1A/B or SET1A/B), have shared and distinct protein domains, catalytic substrates, genomic localizations, and associated complex subunits. Recent studies have revealed the central role of KMT2C/D in enhancer regulation, differentiation, and development and have highlighted KMT2C/D enzymatic activity-dependent and independent roles in mouse embryonic development and cell differentiation. Catalytic dependent and independent roles for KMT2A/B and KMT2F/G in gene regulation, differentiation, and development are less understood. Finally, we provide our perspectives and lay out future research directions that may help advance the investigation on enzymatic activity-dependent and -independent biological roles and working mechanisms of KMT2 methyltransferases.

Rats have the ability to learn and perform sophisticated behavioral tasks, making them very useful for investigating neural circuit functions. In contrast to the extensive mouse genetic toolkit, the paucity of recombinase-expressing rat models has limited the ability to monitor and manipulate molecularly-defined neural populations in this species. Here we report the generation and validation of two knock-in rat strains expressing either Cre or Flp recombinase under the control of Parvalbumin (Pvalb), a gene expressed in the critical “fast-spiking” subset of inhibitory interneurons (FSIs). These strains were generated with CRISPR-Cas9 gene editing and show highly specific and penetrant labeling of Pvalb-expressing neurons, as demonstrated by in situ hybridization and immunohistochemistry. We validated these models in both prefrontal cortex and striatum using both ex vivo and in vivo approaches, including whole-cell recording, optogenetics, extracellular physiology and photometry. Our results demonstrate the utility of these new transgenic models for a wide range of neuroscience experiments.

Crustaceans possess remarkably diverse appendages, both between segments of a single individual as well as between species. Previous studies in a wide range of crustaceans have demonstrated a correlation between the anterior expression boundary of the homeotic (Hox) gene Ultrabithorax (Ubx) and the location and number of specialized thoracic feeding appendages, called maxillipeds. Given that Hox genes regulate regional identity in organisms as diverse as mice and flies, these observations in crustaceans led to the hypothesis that Ubx expression regulates the number of maxillipeds and that evolutionary changes in Ubx expression have generated various aspects of crustacean appendage diversity. Specifically, evolutionary changes in the expression boundary of Ubx have resulted in crustacean species with either 0, 1, 2, or 3 pairs of thoracic maxillipeds. Here we test this hypothesis by altering the expression of Ubx in Parhyale hawaiensis, a crustacean that normally possesses a single pair of maxillipeds. By reducing Ubx expression, we can generate Parhyale with additional maxillipeds in a pattern reminiscent of that seen in other crustacean species, and these morphological alterations are maintained as the animals molt and mature. These results provide critical evidence supporting the proposition that changes in Ubx expression have played a role in generating crustacean appendage diversity and lend general insights into the mechanisms of morphological evolution.

Juvenile hormone (JH) given at pupariation inhibits bristle formation and causes pupal cuticle formation in the abdomen of Drosophila melanogaster due to its prolongation of expression of the transcription factor Broad (BR). In a microarray analysis of JH-induced gene expression in abdominal integument, we found that Krüppel homolog 1 (Kr-h1) was up-regulated during most of adult development. Quantitative real-time PCR analyses showed that Kr-h1 up-regulation began at 10h after puparium formation (APF), and Kr-h1 up-regulation occurred in imaginal epidermal cells, persisting larval muscles, and larval oenocytes. Ectopic expression of Kr-h1 in abdominal epidermis using T155-Gal4 to drive UAS-Kr-h1 resulted in missing or short bristles in the dorsal midline. This phenotype was similar to that seen after a low dose of JH or after misexpression of br between 21 and 30 h APF. Ectopic expression of Kr-h1 prolonged the expression of BR protein in the pleura and the dorsal tergite. No Kr-h1 was seen after misexpression of br. Thus, Kr-h1 mediates some of the JH signaling in the adult abdominal epidermis and is upstream of br in this pathway. We also show for the first time that the JH-mediated maintenance of br expression in this epidermis is patterned and that JH delays the fusion of the imaginal cells and the disappearance of Dpp in the dorsal midline.

Dopamine (DA) receptor-mediated signal transduction and gene expression play a central role in many brain disorders from schizophrenia to Parkinson’s disease to addiction. While trying to evaluate the role of L-type Ca2+ channels in dopamine D1 receptor-mediated phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB), we found that activation of dopamine D1 receptors alters the properties of L-type Ca2+ channel inhibitors and turns them into facilitators of Ca2+ influx. In D1 receptor-stimulated neurons, L-type Ca2+ channel blockers promote cytosolic Ca2+ accumulation. This leads to the activation of a molecular signal transduction pathway and CREB phosphorylation. In the absence of dopamine receptor stimulation, L-type Ca2+ channel blockers inhibit CREB phosphorylation. The effect of dopamine on L-type Ca2+ channel blockers is dependent on protein kinase A (PKA), suggesting that protein phosphorylation plays a role in this phenomenon. Because of the adverse effect of activated dopamine receptors on L-type Ca2+ channel blocker action, the role of L-type Ca2+ channels in the dopamine D1 receptor signal transduction pathway cannot be assessed with pharmacological tools. However, with antisense technology, we demonstrate that L-type Ca2+ channels contribute to D1 receptor-mediated CREB phosphorylation. We conclude that the D1 receptor signal transduction pathway depends on L-type Ca2+ channels to mediate CREB phosphorylation.

Label-free vibrational imaging of biological samples has attracted significant interest due to its integration of structural and chemical information. Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging provide label-free chemical identification by targeting the characteristic resonances of biological compounds that are present in the mid-infrared fingerprint region (3 µm - 12 µm). High contrast imaging of subcellular features and chemical identification of protein secondary structures in unlabeled and labeled fibroblast cells embedded in a collagen-rich extracellular matrix is demonstrated by combining contrast from absorption signatures (amplitude signals) with sensitive detection of different heat properties (lock-in phase signals). We present that the detectability of nano-sized cell membranes is enhanced to well below the optical diffraction limit since the membranes are found to act as thermal barriers. VIPPS offers a novel combination of chemical imaging and thermal diffusion characterization that paves the way towards label-free imaging of cell models and tissues as well as the study of intracellular heat dynamics.

Hyperspectral stimulated Raman scattering microscopy is deployed to measure single-membrane vibrational spectrum as a function of membrane potential. Using erythrocyte ghost as a model, quantitative correlation between transmembrane potential and Raman spectral profile was found. Specifically, the ratio between the area under Raman band at ∼2930 cm−1 and that at ∼2850 cm−1 increased by ∼2.6 times when the potential across the erythrocyte ghost membrane varied from +10 mV to −10 mV. Our results show the feasibility of employing stimulated Raman scattering microscopy to probe the membrane potential without labeling.