Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 (“SNAG-mGluR2”) evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics.
Spatial transcriptomics extends single-cell RNA sequencing (scRNA-seq) by providing spatial context for cell type identification and analysis. Imaging-based spatial technologies such as multiplexed error-robust fluorescence in situ hybridization (MERFISH) can achieve single-cell resolution, directly mapping single-cell identities to spatial positions. MERFISH produces a different data type than scRNA-seq, and a technical comparison between the two modalities is necessary to ascertain how to best integrate them. We performed MERFISH on the mouse liver and kidney and compared the resulting bulk and single-cell RNA statistics with those from the Tabula Muris Senis cell atlas and from two Visium datasets. MERFISH quantitatively reproduced the bulk RNA-seq and scRNA-seq results with improvements in overall dropout rates and sensitivity. Finally, we found that MERFISH independently resolved distinct cell types and spatial structure in both the liver and kidney. Computational integration with the Tabula Muris Senis atlas did not enhance these results. We conclude that MERFISH provides a quantitatively comparable method for single-cell gene expression and can identify cell types without the need for computational integration with scRNA-seq atlases.
The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.
The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel computational technique to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters with high precision. These measurements, combined with theoretical modeling, reveal a significant variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.
Ulcerative colitis (UC) is an inflammatory intestinal disorder driven by mucosal immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin monoclonal antibody that is effective for treating UC. VDZ is thought to primarily inhibit lymphocyte trafficking to the intestine, but its effect on other cell subsets is less well characterized. To identify the inflammatory cells that contribute to colitis and respond to VDZ, we performed a single-cell transcriptomic and proteomic analysis of peripheral blood and colonic biopsies in healthy controls (HC) and patients with UC on either aminosalicylates or VDZ. We identified mononuclear phagocytes (MNPs) as a primary target of VDZ, with comparatively modest effects on lymphocytes. Spatial proteomics and transcriptomics demonstrated increased density and proximity of MNP and fibroblast subsets in UC biopsies when compared to HC, with inhibition by VDZ. VDZ non-responders were enriched for activated fibroblast and MNP signatures in a validation cohort.
A challenge in quantitative biology is to predict output patterns of gene expression from knowledge of input transcription factor patterns and from the arrangement of binding sites for these transcription factors on regulatory DNA. We tested whether widespread thermodynamic models could be used to infer parameters describing simple regulatory architectures that inform parameter-free predictions of more complex enhancers in the context of transcriptional repression by Runt in the early fruit fly embryo. By modulating the number and placement of Runt binding sites within an enhancer, and quantifying the resulting transcriptional activity using live imaging, we discovered that thermodynamic models call for higher-order cooperativity between multiple molecular players. This higher-order cooperativity capture the combinatorial complexity underlying eukaryotic transcriptional regulation and cannot be determined from simpler regulatory architectures, highlighting the challenges in reaching a predictive understanding of transcriptional regulation in eukaryotes and calling for approaches that quantitatively dissect their molecular nature.
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