High Throughput FISH Screening Identifies Small Molecules That Modulate Oncogenic lncRNA MALAT1 via GSK3B and hnRNPs.

Traditionally, small molecule-based drug discovery has mainly focused on proteins as the drug target. Opening RNA as an additional target space for small molecules offers the possibility to therapeutically modulate disease-driving non-coding RNA targets as well as mRNA of otherwise undruggable protein targets. MALAT1 is a highly conserved long-noncoding RNA whose overexpression correlates with poor overall patient survival in some cancers. We report here a fluorescence in-situ hybridization-based high-content imaging screen to identify small molecules that modulate the oncogenic lncRNA MALAT1 in a cellular setting. From a library of FDA approved drugs and known bioactive molecules, we identified two compounds, including Niclosamide, an FDA-approved drug, that lead to a rapid decrease of MALAT1 nuclear levels with good potency. Mode-of-action studies suggest a novel cellular regulatory pathway that impacts MALAT1 lncRNA nuclear levels by GSK3B activation and the involvement of the RNA modulating family of heterogenous nuclear ribonucleoproteins (hnRNPs). This study is the basis for the identification of novel targets that lead to a reduction of the oncogenic lncRNA MALAT1 in a cancer setting.

Physically interacting beta-delta pairs in the regenerating pancreas revealed by single-cell sequencing.

OBJECTIVES: Until recently, communication between neighboring cells in islets of Langerhans was overlooked by genomic technologies, which require rigorous tissue dissociation into single cells. METHODS: We utilize sorting of physically interacting cells (PICs) with single-cell RNA-sequencing to systematically map cellular interactions in the endocrine pancreas after pancreatectomy. RESULTS: The pancreas cellular landscape features pancreatectomy associated heterogeneity of beta-cells, including an interaction-specific program between paired beta and delta-cells. CONCLUSIONS: Our analysis suggests that the particular cluster of beta-cells that pairs with delta-cells benefits from stress protection, implying that the interaction between beta- and delta-cells might safeguard against pancreatectomy associated challenges. The work encourages testing the potential relevance of physically-interacting beta-delta-cells also in diabetes mellitus.

Insulin is expressed by enteroendocrine cells during human fetal development.

Generation of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here we reconstruct a single-cell atlas of the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and expose its molecular blueprint.

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors.

The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.

Protocol for Single-Molecule Fluorescence In Situ Hybridization for Intact Pancreatic Tissue.

We describe an optimized smFISH protocol for the intact pancreas. The protocol is adapted from Lyubimova et al. (2013), a generic tissue smFISH protocol that works for most tissues but not the pancreas. The main changes implemented include increasing the period of mRNA denaturation from 5 min to at least 3 h and increasing formamide concentrations from 10% to 30%. These modifications yield sensitive single mRNA visualization that is comparable to those achieved in other tissues using the standard protocol. For complete details on the use and execution of this protocol, please refer to Farack et al., 2018, Farack et al., 2019.

Zonation of Pancreatic Acinar Cells in Diabetic Mice.

The islets of Langerhans are dynamic structures that can change in size, number of cells, and molecular function in response to physiological and pathological stress. Molecular cues originating from the surrounding "peri-islet" acinar cells that could facilitate this plasticity have not been explored. Here, we combine single-molecule transcript imaging in the intact pancreas and transcriptomics to identify spatial heterogeneity of acinar cell gene expression. We find that peri-islet acinar cells exhibit a distinct molecular signature in db/db diabetic mice that includes upregulation of trypsin family genes and elevated mTOR activity. This zonated expression program seems to be induced by CCK that is secreted from islet cells. Elevated peri-islet trypsin secretion could facilitate the islet expansion observed in this model via modulation of the islet capsule matrix components. Our study highlights a molecular axis of communication between the pancreatic exocrine and endocrine compartments that may be relevant to islet expansion.

Lgr5+ telocytes are a signaling source at the intestinal villus tip.

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.

Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas.

Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. Here, we developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of "extreme" beta cells with elevated mRNA levels of insulin and other secretory genes. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER-enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in db/db diabetic mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales.

Single molecule approaches for studying gene regulation in metabolic tissues.

Gene expression in metabolic tissues can be regulated at multiple levels, ranging from the control of promoter accessibilities, transcription rates, mRNA degradation rates and mRNA localization. Modulating these processes can differentially affect important performance criteria of cells. These include precision, cellular economy, rapid response and maintenance of DNA integrity. In this review we will describe how distinct strategies of gene regulation impact the trade-offs between the cells' performance criteria. We will highlight tools based on single molecule visualization of transcripts that can be used to measure promoter states, transcription rates and mRNA degradation rates in intact tissues. These approaches revealed surprising recurrent patterns in mammalian tissues, that include transcriptional bursting, nuclear retention of mRNA, and coordination of mRNA lifetimes to facilitate rapid adaptation to changing metabolic inputs. The ability to characterize gene expression at the single molecule level can uncover the design principles of gene regulation in metabolic tissues such as the liver and the pancreas.

Prolonged Elimination of Negative Feedback Control Mechanisms Along the Insulin Signaling Pathway Impairs ß-Cell Function In Vivo.

Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-I signaling. We therefore examined the effects of mutation of five "inhibitory" Ser phosphorylation sites on IRS2 function in transgenic mice that overexpress, selectively in pancreatic ß-cells, either wild-type (WT) or a mutated IRS2 protein (IRS25A). Islets size, number, and mRNA levels of catalase and superoxide dismutase were increased, whereas those of nitric oxide synthase were decreased, in 7- to 10-week-old IRS25A-ß mice compared with IRS2WT-ß mice. However, glucose homeostasis and insulin secretion in IRS25A-ß mice were impaired when compared with IRS2WT-ß mice or to nontransgenic mice. This was associated with reduced mRNA levels of Glut2 and islet ß-cell transcription factors such as Nkx6.1 and MafA Similarly, components mediating the unfolded protein response were decreased in islets of IRS25A-ß mice in accordance with their decreased insulin secretion. The beneficial effects of IRS25A on ß-cell proliferation and ß-cell transcription factors were evident only in 5- to 8-day-old mice. These findings suggest that elimination of inhibitory Ser phosphorylation sites of IRS2 exerts short-term beneficial effects in vivo; however, their sustained elimination leads to impaired ß-cell function.

Synchrotron-based Phase Contrast-enhanced Micro-Computed Tomography Reveals Delaminations and Material Tearing in Water-expandable Root Fillings Ex Vivo.

INTRODUCTION: This study evaluated the integrity of calcium silicate sealer-based fillings made with hygro-expandable cones (HEC) that are commercially known as CPoint or Smartpoint. METHODS: Fourteen human canines were prepared according to a standardized, conventional endodontic treatment protocol and filled with the HEC/calcium silicate sealer. Three-dimensional imaging was performed with laboratory micro-computed tomography (µCT) at its highest resolution and was compared with synchrotron phase contrast-enhanced µCT (PCE-CT) scans of the treatment extending 1-7 mm from the apex. Conventional destructive optical microscopy validated observations by comparison with virtual slices in the tomographic data. RESULTS: Conventional laboratory µCT at 10-µm resolution did not reveal the existing voids and defects within the root canal fillings. PCE-CT revealed elongated interfacial delamination localized mainly at the HEC-sealer interface forming extended through-and-through gaps along the root canal filling. CONCLUSIONS: Endodontic studies that use conventional laboratory µCT may underestimate thin defects and delamination within root canal fillings made with HEC because of limited resolution and contrast of laboratory-based broad-spectrum low intensity x-ray sources. These limitations favor use of high-brilliance, monochromatic synchrotron-based PCE-CT to reveal the important micrometer details within large (millimeter sized) samples. PCE-CT revealed the existence of a range of significant structural defects in recently placed HEC fillings, confirmed by optical microscopy after physical sectioning. Substantial delamination spanning 20%-40% of the circumferential interface as well as other structural defects were identified within root canal fillings made of HEC and calcium silicate sealer.