Publications by authors named "Shalev Itzkovitz"

84 Publications

Interleukin-6 produced by enteric neurons regulates the number and phenotype of microbe-responsive regulatory T cells in the gut.

Immunity 2021 Mar;54(3):499-513.e5

Department of Immunology, Harvard Medical School, Boston MA 02115, USA. Electronic address:

The immune and enteric nervous (ENS) systems monitor the frontier with commensal and pathogenic microbes in the colon. We investigated whether FoxP3 regulatory T (Treg) cells functionally interact with the ENS. Indeed, microbe-responsive RORγ and Helios subsets localized in close apposition to nitrergic and peptidergic nerve fibers in the colon lamina propria (LP). Enteric neurons inhibited in vitro Treg (iTreg) differentiation in a cell-contact-independent manner. A screen of neuron-secreted factors revealed a role for interleukin-6 (IL-6) in modulating iTreg formation and their RORγ proportion. Colonization of germfree mice with commensals, especially RORγ Treg inducers, broadly diminished colon neuronal density. Closing the triangle, conditional ablation of IL-6 in neurons increased total Treg cells but decreased the RORγ subset, as did depletion of two ENS neurotransmitters. Our findings suggest a regulatory circuit wherein microbial signals condition neuronal density and activation, thus tuning Treg cell generation and immunological tolerance in the gut.
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http://dx.doi.org/10.1016/j.immuni.2021.02.002DOI Listing
March 2021

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

Elife 2021 Jan 15;10. Epub 2021 Jan 15.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

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 and in developing limb mesenchyme impaired their differentiation.
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http://dx.doi.org/10.7554/eLife.55361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810463PMC
January 2021

Space-time logic of liver gene expression at sub-lobular scale.

Nat Metab 2021 01 11;3(1):43-58. Epub 2021 Jan 11.

Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

The mammalian liver is a central hub for systemic metabolic homeostasis. Liver tissue is spatially structured, with hepatocytes operating in repeating lobules, and sub-lobule zones performing distinct functions. The liver is also subject to extensive temporal regulation, orchestrated by the interplay of the circadian clock, systemic signals and feeding rhythms. However, liver zonation has previously been analysed as a static phenomenon, and liver chronobiology has been analysed at tissue-level resolution. Here, we use single-cell RNA-seq to investigate the interplay between gene regulation in space and time. Using mixed-effect models of messenger RNA expression and smFISH validations, we find that many genes in the liver are both zonated and rhythmic, and most of them show multiplicative space-time effects. Such dually regulated genes cover not only key hepatic functions such as lipid, carbohydrate and amino acid metabolism, but also previously unassociated processes involving protein chaperones. Our data also suggest that rhythmic and localized expression of Wnt targets could be explained by rhythmically expressed Wnt ligands from non-parenchymal cells near the central vein. Core circadian clock genes are expressed in a non-zonated manner, indicating that the liver clock is robust to zonation. Together, our scRNA-seq analysis reveals how liver function is compartmentalized spatio-temporally at the sub-lobular scale.
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http://dx.doi.org/10.1038/s42255-020-00323-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116850PMC
January 2021

A single cell atlas of the human liver tumor microenvironment.

Mol Syst Biol 2020 12;16(12):e9682

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Malignant cell growth is fueled by interactions between tumor cells and the stromal cells composing the tumor microenvironment. The human liver is a major site of tumors and metastases, but molecular identities and intercellular interactions of different cell types have not been resolved in these pathologies. Here, we apply single cell RNA-sequencing and spatial analysis of malignant and adjacent non-malignant liver tissues from five patients with cholangiocarcinoma or liver metastases. We find that stromal cells exhibit recurring, patient-independent expression programs, and reconstruct a ligand-receptor map that highlights recurring tumor-stroma interactions. By combining transcriptomics of laser-capture microdissected regions, we reconstruct a zonation atlas of hepatocytes in the non-malignant sites and characterize the spatial distribution of each cell type across the tumor microenvironment. Our analysis provides a resource for understanding human liver malignancies and may expose potential points of interventions.
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http://dx.doi.org/10.15252/msb.20209682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746227PMC
December 2020

Liver zonation.

J Hepatol 2021 Feb 11;74(2):466-468. Epub 2020 Dec 11.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2020.09.003DOI Listing
February 2021

Genome-wide detection of DNA double-strand breaks by in-suspension BLISS.

Nat Protoc 2020 12 2;15(12):3894-3941. Epub 2020 Nov 2.

Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.
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http://dx.doi.org/10.1038/s41596-020-0397-2DOI Listing
December 2020

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

STAR Protoc 2020 Jun 3;1(1):100007. Epub 2020 Jun 3.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

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.
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http://dx.doi.org/10.1016/j.xpro.2019.100007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580104PMC
June 2020

Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Cell 2020 09 3;182(6):1441-1459.e21. Epub 2020 Sep 3.

Immunology Department, Weizmann Institute of Science, Rehovot 7610001, Israel; Division of Cancer-Microbiome Research, DKFZ, Heidelberg 69120, Germany. Electronic address:

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10 lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.
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http://dx.doi.org/10.1016/j.cell.2020.08.027DOI Listing
September 2020

Zonation of Pancreatic Acinar Cells in Diabetic Mice.

Cell Rep 2020 08;32(7):108043

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2020.108043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443616PMC
August 2020

Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Nat Commun 2020 07 15;11(1):3547. Epub 2020 Jul 15.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81 mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.
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http://dx.doi.org/10.1038/s41467-020-17402-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363928PMC
July 2020

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

Nat Commun 2020 04 22;11(1):1936. Epub 2020 Apr 22.

Department of Molecular Cell Biology, Weizmann Institute of Science, 7610001, Rehovot, Israel.

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.
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http://dx.doi.org/10.1038/s41467-020-15714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176679PMC
April 2020

Spatial sorting enables comprehensive characterization of liver zonation.

Nat Metab 2019 09 16;1(9):899-911. Epub 2019 Sep 16.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

The mammalian liver is composed of repeating hexagonal units termed lobules. Spatially resolved single-cell transcriptomics revealed that about half of hepatocyte genes are differentially expressed across the lobule, yet technical limitations impeded reconstructing similar global spatial maps of other hepatocyte features. Here, we show how zonated surface markers can be used to sort hepatocytes from defined lobule zones with high spatial resolution. We apply transcriptomics, miRNA array measurements and mass spectrometry proteomics to reconstruct spatial atlases of multiple zonated features. We demonstrate that protein zonation largely overlaps with mRNA zonation, with the periportal HNF4α as an exception. We identify zonation of miRNAs such as miR-122, and inverse zonation of miRNAs and their hepatocyte target genes, highlighting potential regulation of protein levels through zonated mRNA degradation. Among the targets we find the pericentral Wnt receptors Fzd7 and Fzd8 and the periportal Wnt inhibitors Tcf7l1 and Ctnnbip1. Our approach facilitates reconstructing spatial atlases of multiple cellular features in the liver and other structured tissues.
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http://dx.doi.org/10.1038/s42255-019-0109-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751089PMC
September 2019

Spatial heterogeneity in the mammalian liver.

Nat Rev Gastroenterol Hepatol 2019 07;16(7):395-410

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Hepatocytes operate in highly structured repeating anatomical units termed liver lobules. Blood flow along the lobule radial axis creates gradients of oxygen, nutrients and hormones, which, together with morphogenetic fields, give rise to a highly variable microenvironment. In line with this spatial variability, key liver functions are expressed non-uniformly across the lobules, a phenomenon termed zonation. Technologies based on single-cell transcriptomics have constructed a global spatial map of hepatocyte gene expression in mice revealing that ~50% of hepatocyte genes are expressed in a zonated manner. This broad spatial heterogeneity suggests that hepatocytes in different lobule zones might have not only different gene expression profiles but also distinct epigenetic features, regenerative capacities, susceptibilities to damage and other functional aspects. Here, we present genomic approaches for studying liver zonation, describe the principles of liver zonation and discuss the intrinsic and extrinsic factors that dictate zonation patterns. We also explore the challenges and solutions for obtaining zonation maps of liver non-parenchymal cells. These approaches facilitate global characterization of liver function with high spatial resolution along physiological and pathological timescales.
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http://dx.doi.org/10.1038/s41575-019-0134-xDOI Listing
July 2019

Single-Cell Analysis of Diverse Pathogen Responses Defines a Molecular Roadmap for Generating Antigen-Specific Immunity.

Cell Syst 2019 02 13;8(2):109-121.e6. Epub 2019 Feb 13.

Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

The immune system generates pathogen-tailored responses. The precise innate immune cell types and pathways that direct robust adaptive immune responses have not been fully characterized. By using fluorescent pathogens combined with massively parallel single-cell RNA-seq, we comprehensively characterized the initial 48 h of the innate immune response to diverse pathogens. We found that across all pathogens tested, most of the lymph node cell types and states showed little pathogen specificity. In contrast, the rare antigen-positive cells displayed pathogen-specific transcriptional programs as early as 24 h after immunization. In addition, mycobacteria activated a specific NK-driven IFNγ response. Depletion of NK cells and IFNγ showed that IFNγ initiated a monocyte-specific signaling cascade, leading to the production of major chemokines and cytokines that promote Th1 development. Our systems immunology approach sheds light on early events in innate immune responses and may help further development of safe and efficient vaccines.
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http://dx.doi.org/10.1016/j.cels.2019.01.001DOI Listing
February 2019

Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma.

Nat Med 2018 12 6;24(12):1867-1876. Epub 2018 Dec 6.

Department of Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients.
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http://dx.doi.org/10.1038/s41591-018-0269-2DOI Listing
December 2018

Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas.

Dev Cell 2019 01 29;48(1):115-125.e4. Epub 2018 Nov 29.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.devcel.2018.11.001DOI Listing
January 2019

Lung Single-Cell Signaling Interaction Map Reveals Basophil Role in Macrophage Imprinting.

Cell 2018 11 11;175(4):1031-1044.e18. Epub 2018 Oct 11.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease.
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http://dx.doi.org/10.1016/j.cell.2018.09.009DOI Listing
November 2018

Spatial Reconstruction of Single Enterocytes Uncovers Broad Zonation along the Intestinal Villus Axis.

Cell 2018 11 27;175(4):1156-1167.e15. Epub 2018 Sep 27.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterium-rich environment. It is unknown whether these tasks are spatially zonated along the villus axis. Here, we extracted a large panel of landmark genes characterized by transcriptomics of laser capture microdissected villus segments and utilized it for single-cell spatial reconstruction, uncovering broad zonation of enterocyte function along the villus. We found that enterocytes at villus bottoms express an anti-bacterial gene program in a microbiome-dependent manner. They next shift to sequential expression of carbohydrates, peptides, and fat absorption machineries in distinct villus compartments. Finally, they induce a Cd73 immune-modulatory program at the villus tips. Our approach can be used to uncover zonation patterns in other organs when prior knowledge of landmark genes is lacking.
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http://dx.doi.org/10.1016/j.cell.2018.08.063DOI Listing
November 2018

Single molecule approaches for studying gene regulation in metabolic tissues.

Diabetes Obes Metab 2018 09;20 Suppl 2:145-156

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

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.
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http://dx.doi.org/10.1111/dom.13390DOI Listing
September 2018

Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells.

Nat Biotechnol 2018 11 17;36(10):962-970. Epub 2018 Sep 17.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell's identity and its position in a tissue. We recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here we used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs' tissue coordinates. We applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, we reconstructed LEC zonation and extracted a landmark gene panel that we used to spatially map LEC scRNAseq data. Our approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.
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http://dx.doi.org/10.1038/nbt.4231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546596PMC
November 2018

Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by Autologous FMT.

Cell 2018 09;174(6):1406-1423.e16

Immunology Department, Weizmann Institute of Science, 7610001 Rehovot, Israel. Electronic address:

Probiotics are widely prescribed for prevention of antibiotics-associated dysbiosis and related adverse effects. However, probiotic impact on post-antibiotic reconstitution of the gut mucosal host-microbiome niche remains elusive. We invasively examined the effects of multi-strain probiotics or autologous fecal microbiome transplantation (aFMT) on post-antibiotic reconstitution of the murine and human mucosal microbiome niche. Contrary to homeostasis, antibiotic perturbation enhanced probiotics colonization in the human mucosa but only mildly improved colonization in mice. Compared to spontaneous post-antibiotic recovery, probiotics induced a markedly delayed and persistently incomplete indigenous stool/mucosal microbiome reconstitution and host transcriptome recovery toward homeostatic configuration, while aFMT induced a rapid and near-complete recovery within days of administration. In vitro, Lactobacillus-secreted soluble factors contributed to probiotics-induced microbiome inhibition. Collectively, potential post-antibiotic probiotic benefits may be offset by a compromised gut mucosal recovery, highlighting a need of developing aFMT or personalized probiotic approaches achieving mucosal protection without compromising microbiome recolonization in the antibiotics-perturbed host.
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http://dx.doi.org/10.1016/j.cell.2018.08.047DOI Listing
September 2018

Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features.

Cell 2018 09;174(6):1388-1405.e21

Immunology Department, Weizmann Institute of Science, Rehovot, 7610001, Israel. Electronic address:

Empiric probiotics are commonly consumed by healthy individuals as means of life quality improvement and disease prevention. However, evidence of probiotic gut mucosal colonization efficacy remains sparse and controversial. We metagenomically characterized the murine and human mucosal-associated gastrointestinal microbiome and found it to only partially correlate with stool microbiome. A sequential invasive multi-omics measurement at baseline and during consumption of an 11-strain probiotic combination or placebo demonstrated that probiotics remain viable upon gastrointestinal passage. In colonized, but not germ-free mice, probiotics encountered a marked mucosal colonization resistance. In contrast, humans featured person-, region- and strain-specific mucosal colonization patterns, hallmarked by predictive baseline host and microbiome features, but indistinguishable by probiotics presence in stool. Consequently, probiotics induced a transient, individualized impact on mucosal community structure and gut transcriptome. Collectively, empiric probiotics supplementation may be limited in universally and persistently impacting the gut mucosa, meriting development of new personalized probiotic approaches.
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http://dx.doi.org/10.1016/j.cell.2018.08.041DOI Listing
September 2018

Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration.

Cell Stem Cell 2018 09 9;23(3):436-443.e7. Epub 2018 Aug 9.

Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK. Electronic address:

The intestinal epithelium is largely maintained by self-renewing stem cells but with apparently committed progenitors also contributing, particularly following tissue damage. However, the mechanism of, and requirement for, progenitor plasticity in mediating pathological response remain unknown. Here we show that phosphorylation of the transcription factor Atoh1 is required for both the contribution of secretory progenitors to the stem cell pool and for a robust regenerative response. As confirmed by lineage tracing, Atoh1 cells (Atoh1 mice) give rise to multilineage intestinal clones both in the steady state and after tissue damage. In a phosphomutant Atoh1 line, preventing phosphorylation of ATOH1 protein acts to promote secretory differentiation and inhibit the contribution of progenitors to self-renewal. Following chemical colitis, Atoh1 cells of Atoh1 mice have reduced clonogenicity that affects overall regeneration. Progenitor plasticity maintains robust self-renewal in the intestinal epithelium, and the balance between stem and progenitor fate is directly coordinated by ATOH1 multisite phosphorylation.
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http://dx.doi.org/10.1016/j.stem.2018.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138952PMC
September 2018

Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells.

Nature 2018 07 18;559(7715):622-626. Epub 2018 Jul 18.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.
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http://dx.doi.org/10.1038/s41586-018-0346-1DOI Listing
July 2018

Design principles of the paradoxical feedback between pancreatic alpha and beta cells.

Sci Rep 2018 Jul 16;8(1):10694. Epub 2018 Jul 16.

Weizmann Institute of Science, Department of Molecular Cell Biology, Wolfson Building, 234 Herzl St., Rehovot, 76100, Israel.

Mammalian glucose homeostasis is controlled by the antagonistic hormones insulin and glucagon, secreted by pancreatic beta and alpha cells respectively. These two cell types are adjacently located in the islets of Langerhans and affect each others' secretions in a paradoxical manner: while insulin inhibits glucagon secretion from alpha cells, glucagon seems to stimulate insulin secretion from beta cells. Here we ask what are the design principles of this negative feedback loop. We systematically simulate the dynamics of all possible islet inter-cellular connectivity patterns and analyze different performance criteria. We find that the observed circuit dampens overshoots of blood glucose levels after reversion of glucose drops. This feature is related to the temporal delay in the rise of insulin concentrations in peripheral tissues, compared to the immediate hormone action on the liver. In addition, we find that the circuit facilitates coordinate secretion of both hormones in response to protein meals. Our study highlights the advantages of a paradoxical paracrine feedback loop in maintaining metabolic homeostasis.
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http://dx.doi.org/10.1038/s41598-018-29084-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048053PMC
July 2018

Pax6 regulation of in the mouse retinal pigmented epithelium controls its timely differentiation and choroid vasculature development.

Development 2018 08 9;145(15). Epub 2018 Aug 9.

Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Sagol School of Neurosciences, Tel Aviv University, Tel Aviv 69978, Israel

The synchronized differentiation of neuronal and vascular tissues is crucial for normal organ development and function, although there is limited information about the mechanisms regulating the coordinated development of these tissues. The choroid vasculature of the eye serves as the main blood supply to the metabolically active photoreceptors, and develops together with the retinal pigmented epithelium (RPE). Here, we describe a novel regulatory relationship between the RPE transcription factors Pax6 and Sox9 that controls the timing of RPE differentiation and the adjacent choroid maturation. We used a novel machine learning algorithm tool to analyze high resolution imaging of the choroid in and conditional mutant mice. Additional unbiased transcriptomic analyses in mutant mice and RPE cells generated from human embryonic stem cells, as well as chromatin immunoprecipitation and high-throughput analyses, revealed secreted factors that are regulated by Pax6 and Sox9. These factors might be involved in choroid development and in the pathogenesis of the common blinding disease: age-related macular degeneration (AMD).
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http://dx.doi.org/10.1242/dev.163691DOI Listing
August 2018

Author Correction: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Nature 2018 08;560(7718):E29

Department of Genetics and Center for Molecular Studies in Digestive and Liver Diseases, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Change history: In this Letter, the surname of author Efi E. Massasa was misspelled 'Massassa'. This error has been corrected online.
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http://dx.doi.org/10.1038/s41586-018-0286-9DOI Listing
August 2018

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.

Nature 2018 05 2;557(7704):242-246. Epub 2018 May 2.

Department of Genetics and Center for Molecular Studies in Digestive and Liver Diseases, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1 telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1 telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1 telocytes are an important source of niche signals to intestinal stem cells.
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http://dx.doi.org/10.1038/s41586-018-0084-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966331PMC
May 2018

Casein kinase 1-epsilon or 1-delta required for Wnt-mediated intestinal stem cell maintenance.

EMBO J 2017 10 28;36(20):3046-3061. Epub 2017 Sep 28.

The Lautenberg Center for Immunology, Institute of Medical Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel

The intestinal epithelium holds an immense regenerative capacity mobilized by intestinal stem cells (ISCs), much of it supported by Wnt pathway activation. Several unique regulatory mechanisms ensuring optimal levels of Wnt signaling have been recognized in ISCs. Here, we identify another Wnt signaling amplifier, CKIε, which is specifically upregulated in ISCs and is essential for ISC maintenance, especially in the absence of its close isoform CKIδ. Co-ablation of CKIδ/ε in the mouse gut epithelium results in rapid ISC elimination, with subsequent growth arrest, crypt-villous shrinking, and rapid mouse death. Unexpectedly, Wnt activation is preserved in all CKIδ/ε-deficient enterocyte populations, with the exception of Lgr5 ISCs, which exhibit Dvl2-dependent Wnt signaling attenuation. CKIδ/ε-depleted gut organoids cease proliferating and die rapidly, yet survive and resume self-renewal upon reconstitution of Dvl2 expression. Our study underscores a unique regulation mode of the Wnt pathway in ISCs, possibly providing new means of stem cell enrichment for regenerative medicine.
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http://dx.doi.org/10.15252/embj.201696253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641904PMC
October 2017

Global mRNA polarization regulates translation efficiency in the intestinal epithelium.

Science 2017 09 10;357(6357):1299-1303. Epub 2017 Aug 10.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Asymmetric messenger RNA (mRNA) localization facilitates efficient translation in cells such as neurons and fibroblasts. However, the extent and importance of mRNA polarization in epithelial tissues are unclear. Here, we used single-molecule transcript imaging and subcellular transcriptomics to uncover global apical-basal intracellular polarization of mRNA in the mouse intestinal epithelium. The localization of mRNAs did not generally overlap protein localization. Instead, ribosomes were more abundant on the apical sides, and apical transcripts were consequently more efficiently translated. Refeeding of fasted mice elicited a basal-to-apical shift in polarization of mRNAs encoding ribosomal proteins, which was associated with a specific boost in their translation. This led to increased protein production, required for efficient nutrient absorption. These findings reveal a posttranscriptional regulatory mechanism involving dynamic polarization of mRNA and polarized translation.
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http://dx.doi.org/10.1126/science.aan2399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955215PMC
September 2017