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Fibroblasts, the principal cell type of connective tissue, secrete extracellular matrix components during tissue development, homeostasis, repair and disease. Despite this crucial role, the identification and distinction of fibroblasts from other cell types are challenging and laden with caveats. Rapid progress in single-cell transcriptomics now yields detailed molecular portraits of fibroblasts and other cell types in our bodies, which complement and enrich classical histological and immunological descriptions, improve cell class definitions and guide further studies on the functional heterogeneity of cell subtypes and states, origins and fates in physiological and pathological processes. In this review, we summarize and discuss recent advances in the understanding of fibroblast identification and heterogeneity and how they discriminate from other cell types. In this review, the authors look at how recent progress in single-cell transcriptomics complement and enrich the classical, largely morphological, portraits of fibroblasts. The detailed molecular information now available provides new insights into fibroblast identity, heterogeneity and function.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes. To define and distinguish fibroblasts from vascular mural cells have remained challenging. Here, using single-cell RNA sequencing and tissue imaging, the authors provide a molecular basis for cell type classification and reveal inter- and intra-organ diversity of these cell types.

Two aspects of the blood–brain barrier — the transport of lipids to the brain and the transport of molecules across cells lining blood vessels — have been shown to be regulated by the same protein, Mfsd2a. See Letters p.503 & p.507 Building the blood–brain barrier The blood–brain barrier serves a vital function in maintaining the necessary environment for brain function but is an inconvenient obstacle to brain-directed therapeutics. Two papers published in this issue of Nature report the involvement of Mfsd2a, a member of the major facilitator superfamily regarded previously as an orphan transporter, in two aspects of blood–brain barrier function. David Silver and colleagues identify Mfsd2a as the major transporter for uptake of the omega fatty acid docosahexaenoic acid (DHA) into the brain. Mfsd2a is exclusively expressed in the endothelium of the blood–brain barrier, and Mfsd2a -knockout mice have reduced levels brain DHA, neuronal loss and reduced brain size and function. Chenghua Gu and colleagues find a role for Mfsd2 as a regulator of blood–brain barrier development and function: the barrier becomes 'leaky' in Mfsd2a-deficient mice, possibly a result of increased transcellular vesicular transport.

Blood-brain barrier (BBB) dysfunction is associated with worse epilepsy outcomes however the underlying molecular mechanisms of BBB dysfunction remain to be elucidated. Tight junction proteins are important regulators of BBB integrity and in particular, the tight junction protein claudin-5 is the most enriched in brain endothelial cells and regulates size-selectivity at the BBB. Additionally, disruption of claudin-5 expression has been implicated in numerous disorders including schizophrenia, depression and traumatic brain injury, yet its role in epilepsy has not been fully deciphered. Here we report that claudin-5 protein levels are significantly diminished in surgically resected brain tissue from patients with treatment-resistant epilepsy. Concomitantly, dynamic contrast-enhanced MRI in these patients showed widespread BBB disruption. We show that targeted disruption of claudin-5 in the hippocampus or genetic heterozygosity of claudin-5 in mice exacerbates kainic acid-induced seizures and BBB disruption. Additionally, inducible knockdown of claudin-5 in mice leads to spontaneous recurrent seizures, severe neuroinflammation, and mortality. Finally, we identify that RepSox, a regulator of claudin-5 expression, can prevent seizure activity in experimental epilepsy. Altogether, we propose that BBB stabilizing drugs could represent a new generation of agents to prevent seizure activity in epilepsy patients. The mechanisms underlying epilepsy development are not well understood. Here the authors show that loss of a key component of the so called blood-brain barrier drives seizures in mice and is also lost in humans with treatment resistant epilepsy

The blood vasculature is an organ pervading all other organs (almost). During vascular development, cell–cell signaling by extracellular ligands and cell surface receptors ensure that new vessels sprout into non‐vascularized regions and simultaneously acquire organ‐specific specializations and adaptations that match the local physiological needs. The vessels thereby specialize in their permeability, molecular transport between blood and tissue, and ability to regulate blood flow on demand. Over the past decades, we have learnt about the generic cell–cell signaling mechanisms governing angiogenic sprouting, mural cell recruitment, and vascular remodeling, and we have obtained the first insights into signals that induce and maintain vascular organotypicity. However, intra‐organ vascular diversity and arterio‐venous hierarchies complicate the molecular characterization of the vasculature's cellular building blocks. Single‐cell RNA sequencing provides a way forward, as it allows elucidation at a genome‐wide and quantitative level of the transcriptional diversity occurring within the same cell types at different anatomical positions and levels of arterio‐venous hierarchy in the organs. In this Louis‐Jeantet Prize Winner: Commentary , I give a brief overview of vascular development and how recent advances in the field pave the way for more systematic efforts to explore vascular functions in health and disease. Graphical Abstract The 2018 Louis‐Jeantet Prize for Medicine winner Christer Betsholtz provides an interesting account of his work in vascular biology, in particular the characterisation of pericytes and their role in vascular development and permeability.

Single-cell RNA sequencing (scRNAseq) marks the birth of a new era in physiology and medicine. Within foreseeable future, we will know exactly what genes are expressed – and at what levels – in all the different cell types and subtypes that make up our bodies. We will also learn how a particular cell state, whether it occurs during development, tissue repair, or disease, reflects precise changes in gene expression. While profoundly impacting all areas of life science, scRNAseq may lead to a particular leap in vascular biology research. Blood vessels pervade and fulfill essential functions in all organs, but the functions differ. Innumerable organ-specific vascular adaptations and specializations are required. These, in turn, are dictated by differential gene expression by the two principal cellular building blocks of blood vessels: endothelial cells and mural cells. An organotypic vasculature is essential for functions as diverse as thinking, gas exchange, urine excretion, and xenobiotic detoxification in the brain, lung, kidney, and liver, respectively. In addition to the organotypicity, vascular cells also differ along the vascular arterio-venous axis, referred to as zonation, differences that are essential for the regulation of blood pressure and flow. Moreover, gene expression-based molecular changes dictate states of cellular activity, necessary for angiogenesis, vascular permeability, and immune cell trafficking, i.e. functions necessary for development, inflammation, and repair. These different levels of cellular heterogeneity create a nearly infinite phenotypic diversity among vascular cells. In this review, I summarize and exemplify what scRNAseq has brought to the picture in just a few years and point out where it will take us.

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature. Single-cell transcriptomic analysis of the murine blood–brain barrier provides molecular definitions of the main vascular cell types, classifies perivascular cell types and sheds light on the organization of the arteriovenous axis. Molecular map of brain vascular cells Good vascular health is essential to proper brain function. Yet brain vascular cells have not been systematically characterized at a molecular level. Christer Betsholtz and colleagues use single-cell transcriptomics to identify the molecular profiles of the main vascular cell types in the adult mouse brain. The molecular identity and phenotype of endothelial cells change gradually along the arteriovenous axis, whereas mural cells are precisely defined either as arterial or arteriole smooth muscle cells and pericytes, or as venous smooth muscle cells. The work also provides a comprehensive molecular definition of pericytes, showing that the pericytes of one organ are highly homogeneous but quite distinct from those of a different organ. Finally, the analyses uncover a novel perivascular cell type that shares some similarities with fibroblasts and that makes up the outer layer of all brain vessels except capillaries.

The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb -expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies. The molecular identity of renal glomerular cells is poorly characterized and rodent glomerulopathy models translate poorly to humans. Here, the authors show molecular signatures of glomerulus-associated cells using single cell RNA sequencing and highlight differences between mouse and human cells.

Proteolytical processing of the growth factor VEGFC through the concerted activity of CCBE1 and ADAMTS3 is required for lymphatic development to occur. How these factors act together in time and space, and which cell types produce these factors is not understood. Here we assess the function of Adamts3 and the related protease Adamts14 during zebrafish lymphangiogenesis and show both proteins to be able to process Vegfc. Only the simultaneous loss of both protein functions results in lymphatic defects identical to vegfc loss-of-function situations. Cell transplantation experiments demonstrate neuronal structures and/or fibroblasts to constitute cellular sources not only for both proteases but also for Ccbe1 and Vegfc. We further show that this locally restricted Vegfc maturation is needed to trigger normal lymphatic sprouting and directional migration. Our data provide a single-cell resolution model for establishing secretion and processing hubs for Vegfc during developmental lymphangiogenesis. How and where VEGF-C is processed in lymphangiogenesis is unclear. Here, the authors show that development of the zebrafish lymphatic system is locally restricted by Vegfc maturation causing lymphatic sprouting in certain regions, which is regulated by the metalloproteases ADAMTS3 and ADAMTS14.