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

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.

Mapping Human Reproduction with Single-Cell Genomics.

The trillions of cells in the human body develop as a result of the fusion of two extremely specialized cells: an oocyte and a sperm. This process is essential for the continuation of our species, as it ensures that parental genetic information is mixed and passed on from generation to generation. In addition to producing oocytes, the female reproductive system must provide the environment for the appropriate development of the fetus until birth. New genomic and computational tools offer unique opportunities to study the tight spatiotemporal regulatory mechanisms that are required for the cycle of human reproduction. This review explores how single-cell technologies have been used to build cellular atlases of the human reproductive system across the life span and how these maps have proven useful to better understand reproductive pathologies and dissect the heterogeneity of in vitro model systems.

Plant-microbe interactions in the rhizosphere via a circular metabolic economy.

Chemical exchange often serves as the first step in plant-microbe interactions and exchanges of various signals, nutrients, and metabolites continue throughout the interaction. Here, we highlight the role of metabolite exchanges and metabolic crosstalk in the microbiome-root-shoot-environment nexus. Roots secret a diverse set of metabolites; this assortment of root exudates, including secondary metabolites such as benzoxazinoids, coumarins, flavonoids, indolic compounds, and terpenes, shapes the rhizosphere microbiome. In turn, the rhizosphere microbiome affects plant growth and defense. These inter-kingdom chemical interactions are based on a metabolic circular economy, a seemingly wasteless system in which rhizosphere members exchange (i.e. consume, reuse, and redesign) metabolites. This review also describes the recently discovered phenomenon "Systemically Induced Root Exudation of Metabolites" in which the rhizosphere microbiome governs plant metabolism by inducing systemic responses that shift the metabolic profiles of root exudates. Metabolic exchange in the rhizosphere is based on chemical gradients that form specific microhabitats for microbial colonization and we describe recently developed high-resolution methods to study chemical interactions in the rhizosphere. Finally, we propose an action plan to advance the metabolic circular economy in the rhizosphere for sustainable solutions to the cumulative degradation of soil health in agricultural lands.

Spatial gene expression maps of the intestinal lymphoid follicle and associated epithelium identify zonated expression programs.

The intestine is lined with isolated lymphoid follicles (ILFs) that facilitate sampling of luminal antigens to elicit immune responses. Technical challenges related to the scarcity and small sizes of ILFs and their follicle-associated epithelium (FAE) impeded the characterization of their spatial gene expression programs. Here, we combined RNA sequencing of laser capture microdissected tissues with single-molecule transcript imaging to obtain a spatial gene expression map of the ILF and its associated FAE in the mouse small intestine. We identified zonated expression programs in both follicles and FAEs, with a decrease in enterocyte antimicrobial and absorption programs and a partial induction of expression programs normally observed at the villus tip. We further identified Lepr+ subepithelial telocytes at the FAE top, which are distinct from villus tip Lgr5+ telocytes. Our analysis exposes the epithelial and mesenchymal cell states associated with ILFs.

Rhizosphere microbiome mediates systemic root metabolite exudation by root-to-root signaling.

Microbial communities associated with roots confer specific functions to their hosts, thereby modulating plant growth, health, and productivity. Yet, seminal questions remain largely unaddressed including whether and how the rhizosphere microbiome modulates root metabolism and exudation and, consequently, how plants fine tune this complex belowground web of interactions. Here we show that, through a process termed systemically induced root exudation of metabolites (SIREM), different microbial communities induce specific systemic changes in tomato root exudation. For instance, systemic exudation of acylsugars secondary metabolites is triggered by local colonization of bacteria affiliated with the genus Bacillus Moreover, both leaf and systemic root metabolomes and transcriptomes change according to the rhizosphere microbial community structure. Analysis of the systemic root metabolome points to glycosylated azelaic acid as a potential microbiome-induced signaling molecule that is subsequently exuded as free azelaic acid. Our results demonstrate that rhizosphere microbiome assembly drives the SIREM process at the molecular and chemical levels. It highlights a thus-far unexplored long-distance signaling phenomenon that may regulate soil conditioning.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

A single cell atlas of the human liver tumor microenvironment.

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.

Live imaging of root-bacteria interactions in a microfluidics setup.

Plant roots play a dominant role in shaping the rhizosphere, the environment in which interaction with diverse microorganisms occurs. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited because of methodological intricacy. Here, we describe a microfluidics-based approach enabling direct imaging of root-bacteria interactions in real time. The microfluidic device, which we termed tracking root interactions system (TRIS), consists of nine independent chambers that can be monitored in parallel. The principal assay reported here monitors behavior of fluorescently labeled Bacillus subtilis as it colonizes the root of Arabidopsis thaliana within the TRIS device. Our results show a distinct chemotactic behavior of B. subtilis toward a particular root segment, which we identify as the root elongation zone, followed by rapid colonization of that same segment over the first 6 h of root-bacteria interaction. Using dual inoculation experiments, we further show active exclusion of Escherichia coli cells from the root surface after B. subtilis colonization, suggesting a possible protection mechanism against root pathogens. Furthermore, we assembled a double-channel TRIS device that allows simultaneous tracking of two root systems in one chamber and performed real-time monitoring of bacterial preference between WT and mutant root genotypes. Thus, the TRIS microfluidics device provides unique insights into the microscale microbial ecology of the complex root microenvironment and is, therefore, likely to enhance the current rate of discoveries in this momentous field of research.

Indole Derivatives Maintain the Status Quo Between Beneficial Biofilms and Their Plant Hosts.

Biofilms formed by bacteria on plant roots play an important role in maintaining an optimal rhizosphere environment that supports plant growth and fitness. Bacillus subtilis is a potent plant growth promoter, forming biofilms that play a key role in protecting the host from fungal and bacterial infections. In this work, we demonstrate that the development of B. subtilis biofilms is antagonized by specific indole derivatives that accumulate during symbiotic interactions with plant hosts. Indole derivatives are more potent signals when the plant polysaccharide xylan serves as a carbon source, a mechanism to sustain beneficial biofilms at a biomass that can be supported by the plant. Moreover, B. subtilis biofilms formed by mutants resistant to indole derivatives become deleterious to the plants due to their capacity to consume and recycle plant polysaccharides. These results demonstrate how a dynamic metabolite-based dialogue can promote homeostasis between plant hosts and their beneficial biofilm communities.

MYB107 and MYB9 Homologs Regulate Suberin Deposition in Angiosperms.

Suberin, a polymer composed of both aliphatic and aromatic domains, is deposited as a rough matrix upon plant surface damage and during normal growth in the root endodermis, bark, specialized organs (e.g., potato [Solanum tuberosum] tubers), and seed coats. To identify genes associated with the developmental control of suberin deposition, we investigated the chemical composition and transcriptomes of suberized tomato (Solanum lycopersicum) and russet apple (Malus x domestica) fruit surfaces. Consequently, a gene expression signature for suberin polymer assembly was revealed that is highly conserved in angiosperms. Seed permeability assays of knockout mutants corresponding to signature genes revealed regulatory proteins (i.e., AtMYB9 and AtMYB107) required for suberin assembly in the Arabidopsis thaliana seed coat. Seeds of myb107 and myb9 Arabidopsis mutants displayed a significant reduction in suberin monomers and altered levels of other seed coat-associated metabolites. They also exhibited increased permeability, and lower germination capacities under osmotic and salt stress. AtMYB9 and AtMYB107 appear to synchronize the transcriptional induction of aliphatic and aromatic monomer biosynthesis and transport and suberin polymerization in the seed outer integument layer. Collectively, our findings establish a regulatory system controlling developmentally deposited suberin, which likely differs from the one of stress-induced polymer assembly recognized to date.

Small molecules below-ground: the role of specialized metabolites in the rhizosphere.

Soil communities are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in below-ground interactions. While plants are known to release an extremely high portion of the fixated carbon to the soil, less information is known about the composition and role of C-containing compounds in the rhizosphere, in particular those involved in chemical communication. Specialized metabolites (or secondary metabolites) produced by plants and their associated microbes have a critical role in various biological activities that modulate the behavior of neighboring organisms. Thus, elucidating the chemical composition and function of specialized metabolites in the rhizosphere is a key element in understanding interactions in this below-ground environment. Here, we review key classes of specialized metabolites that occur as mostly non-volatile compounds in root exudates or are emitted as volatile organic compounds (VOCs). The role of these metabolites in below-ground interactions and response to nutrient deficiency, as well as their tissue and cell type-specific biosynthesis and release are discussed in detail.

Cell isolation induces fate changes of bone marrow mesenchymal cells leading to loss or alternatively to acquisition of new differentiation potentials.

Mesenchymal stromal cell populations include a fraction, termed mesenchymal stem cells, exhibiting multipotency. Other cells within this population possess a lesser differentiation range. This was assumed to be due to a mesenchymal cellular cascade topped by a multipotent cell, which gives rise to progeny with diminishing differentiation potentials. Here, we show that mesenchymal cells, a priori exhibiting a limited differentiation potential, may gain new capacities and become multipotent following single-cell isolation. These fate changes were accompanied by upregulation of differentiation promoting genes, many of which also became H4K20me1 methylated. Early events in the process included TGFß and Wnt modulation, and downregulation of hypoxia signaling. Indeed, hypoxic conditions inhibited the observed cell changes. Overall, cell isolation from neighboring partners caused major molecular changes and particularly, a newly established epigenetic state, ultimately leading to the acquisition of new differentiation potentials and an altered cell fate.

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma.

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

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.

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.

Tracking Root Interactions System (TRIS) Experiment and Quality Control.

Soil organisms are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in shaping the rhizosphere. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited due to methodological intricacy. In this study, we developed a novel microfluidics-based device enabling direct imaging of root-bacteria interactions in real time.

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

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.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.