Cell identity is governed by graded transcription factor (TF) activity, yet current single-cell tools do not resolve how TF dosage shapes both gene expression and chromatin accessibility. We present DoseH-seq, a dosage-resolved single-nucleus RNA+ATAC multiome assay built on standard 10x Genomics workflows. DoseH-seq integrates sample hashing with quantitative tracking of continuous lentiviral overexpression and knockdown across multiplexed conditions and time points. We validate DoseH-seq in both single and multiperturb designs, including dual overexpression and overexpression/knockdown experiments, across different cell types and conditions. We apply DoseH-seq to resolve the dose and context-dependent roles of NFIX, a somatic TF enriched at regulatory elements more active in youthful cells, in fibroblasts, pluripotent stem cells (PSCs) and during reprogramming. In fibroblasts, increased NFIX opens regulatory elements whose motifs compete with myofibroblast identity TFs, consistent with counteracting mesenchymal drift and a restricted developmental reversion. During reprogramming, high NFIX overexpression activates AP-1 and stabilizes the somatic state. Conversely, transitory moderate-level NFIX overexpression, when Yamanaka factors are limiting, synergistically opens chromatin transiently to dismantle the somatic network, with potential analogous roles in oncogenic identity remodelling. During NFIX-induced PSC differentiation, transient reprogramming elements bearing NFI and degenerate pluripotency TF motifs (including OCT4) are re-engaged, consistent with developmental roles, mechanistically linking reprogramming with differentiation. Our data reveal graded dosage effects in somatic and pluripotency TF interactions, highlighting DoseH-seq as a generalisable perturbation-multiomics platform for resolving gene-dosage interactions governing cell identity and cell-state transitions.

Single-cell technology has allowed researchers to probe tissue complexity and dynamics at unprecedented depth in health and disease. However, the generation of high-dimensionality single-cell atlases and virtual three-dimensional tissues requires integrated reference maps that harmonize disparate experimental designs, analytical pipelines, and taxonomies. Here, we present a comprehensive single-cell transcriptome integration map of cardiac fibrosis, which underpins pathophysiology in most cardiovascular diseases. Our findings reveal similarity between cardiac fibroblast (CF) identities and dynamics in ischemic versus pressure overload models of cardiomyopathy. We also describe timelines for commitment of activated CFs to proliferation and myofibrogenesis, profibrotic and antifibrotic polarization of myofibroblasts and matrifibrocytes, and CF conservation across mouse and human healthy and diseased hearts. These insights have the potential to inform knowledge-based therapies.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

Reversal of ischemia is mediated by neo-angiogenesis requiring endothelial cell (EC) and pericyte interactions to form stable microvascular networks. We describe an unrecognized role for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in potentiating neo-angiogenesis and vessel stabilization. We show that the endothelium is a major source of TRAIL in the healthy circulation compromised in peripheral artery disease (PAD). EC deletion of TRAIL in vivo or in vitro inhibited neo-angiogenesis, pericyte recruitment, and vessel stabilization, resulting in reduced lower-limb blood perfusion with ischemia. Activation of the TRAIL receptor (TRAIL-R) restored blood perfusion and stable blood vessel networks in mice. Proof-of-concept studies showed that Conatumumab, an agonistic TRAIL-R2 antibody, promoted vascular sprouts from explanted patient arteries. Single-cell RNA sequencing revealed heparin-binding EGF-like growth factor in mediating EC-pericyte communications dependent on TRAIL. These studies highlight unique TRAIL-dependent mechanisms mediating neo-angiogenesis and vessel stabilization and the potential of repurposing TRAIL-R2 agonists to stimulate stable and functional microvessel networks to treat ischemia in PAD.

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1–derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Despite the high prevalence of heart failure in the western world, there are few effective treatments. Fibulin-3 is a protein involved in extracellular matrix (ECM) structural integrity, however its role in the heart is unknown. We have demonstrated, using single cell RNA-seq, that fibulin-3 was highly expressed in quiescent murine cardiac fibroblasts, with expression highest prior to injury and late post-infarct (from ~ day-28 to week-8). In humans, fibulin-3 was upregulated in left ventricular tissue and plasma of heart failure patients. Fibulin-3 knockout ( Efemp1 −/− ) and wildtype mice were subjected to experimental myocardial infarction. Fibulin-3 deletion resulted in significantly higher rate of cardiac rupture days 3–6 post-infarct, indicating a weak and poorly formed scar, with severe ventricular remodelling in surviving mice at day-28 post-infarct. Fibulin-3 knockout mice demonstrated less collagen deposition at day-3 post-infarct, with abnormal collagen fibre-alignment. RNA-seq on day-3 infarct tissue revealed upregulation of ECM degradation and inflammatory genes, but downregulation of ECM assembly/structure/organisation genes in fibulin-3 knockout mice. GSEA pathway analysis showed enrichment of inflammatory pathways and a depletion of ECM organisation pathways. Fibulin-3 originates from cardiac fibroblasts, is upregulated in human heart failure, and is necessary for correct ECM organisation/structural integrity of fibrotic tissue to prevent cardiac rupture post-infarct.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Endothelial cell (EC)-pericyte crosstalk is essential for generating stable capillary networks. Capillary function and development is disrupted in CVD, and processes mediating this are poorly understood. TNF-related apoptosis-inducing ligand (TRAIL) stimulates blood vessel development in pre-clinical models, while circulating levels are suppressed in CVD patients. The contribution of EC-specific TRAIL to angiogenesis in ischemia is unknown. To address this, an EC-specific TRAIL knockout ( Trail EC-/- ) was generated. Compared to Trail EC+/+ , Trail EC-/- mice had ~60% reduction in plasma TRAIL, revealing the endothelium as a significant source of TRAIL in the healthy circulation. Angiogenesis was quantified in the Matrigel plug, aortic sprouting and hindlimb ischemia (HLI) models. EC/pericyte content in plugs were ~50-60% less in Trail EC-/- than Trail EC+/+ mice, with a ~50% reduction in mRNA expression of angiogenesis/pericyte markers. Trail EC-/- aortic segments had reduced microvascular sprouts in hypoxia, and ECs lacking TRAIL had an impaired ability to form tubules and recruit pericytes. CD31 + SMA + microvessel numbers (measure of EC-pericyte interaction) were significantly reduced in Trail EC-/- ischemic limbs, associating with decreased expression of pericyte markers and ~50% reduction in blood perfusion. Similar findings were observed in Trail-receptor -/- ( Trail-R -/- ) mice. In contrast, administration of an agonistic anti-mouse TRAIL-R mAb (MD5-1), restored blood perfusion and increased EC-pericyte content in ischaemic Trail EC-/- limbs. MD5-1 also stimulated aortic sprouts. scRNA-seq data identified 5 EC clusters, 2 of which were markedly altered in Trail EC+/+ but not in Trail EC-/- ischemic limbs and multiple EC-pericyte interactions dependent on TRAIL were identified. In humans, TRAIL induces apoptosis through TRAIL-R1 and TRAIL-R2. Importantly, TRAIL and TRAIL-R2 mRNA and cell surface expression was augmented, and TRAIL physically interacted with TRAIL-R2 (but not TRAIL-R1) under hypoxic conditions. These studies provide a novel pathway mediating angiogenesis and vessel stabilisation. TRAIL-R agonists could be used as a potential new therapy to stimulate stable capillary networks in peripheral artery disease.

Irreversible fibrosis is a hallmark of myocardial infarction (MI) and heart failure. Extracellular matrix protein-1 (ECM-1) is up-regulated in these hearts, localized to fibrotic, inflammatory, and perivascular areas. ECM-1 originates predominantly from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts, and from M1 and M2 macrophages and myofibroblasts after MI. ECM-1 stimulates fibroblast-to-myofibroblast transition, up-regulates key fibrotic and inflammatory pathways, and inhibits cardiac fibroblast migration. ECM-1 binds HuCFb cell surface receptor LRP1, and LRP1 inhibition blocks ECM-1 from stimulating fibroblast-to-myofibroblast transition, confirming a novel ECM-1-LRP1 fibrotic signaling axis. ECM-1 may represent a novel mechanism facilitating inflammation-fibrosis crosstalk.

Endothelial cell (EC) and pericyte interactions are essential for the generation of stable microvascular networks in ischaemia, but these processes are disrupted in peripheral artery disease (PAD). We uncovered ECs are a major contributor of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in the circulation that is compromised in PAD, and associates with reduced stable microvessels in ischaemic amputated tissues. Angiogenesis was assessed by Matrigel plug, wound healing, hind-limb ischaemia (HLI), aortic sprouting and in cells. EC-specific Trail-/- mice (TrailEC-/-) revealed ∼50-60% less EC/pericyte content in plugs and in wounds and reduced aortic microvascular sprouting in hypoxia. In vitro, TRAIL-deleted ECs formed fewer tubules, recruited less pericytes and prevented pericyte stabilisation. TrailEC-/- mice had impaired formation of stable microvessels in HLI, which associated with reduced blood perfusion to the limbs; findings also observed inTRAIL-receptor knockout (Trail-R-/-) mice. Remarkably, activating the TRAIL-R using MD5-1, an anti-mouse agonistic TRAIL-R mAb, in vivo restored blood perfusion and increased stable microvessels in ischaemic limbs and ex vivo stimulated aortic sprouts in TrailEC-/- mice. MD5-1 also stimulated angiogenesis in vitro. Single cell RNA-sequencing of ischaemic tissues identified multiple EC-pericyte interactions disrupted with TRAIL deletion in the endothelium. These studies identify EC-derived TRAIL as a new player in mediating angiogenesis and vessel stabilisation necessary for effective microvascular function. TRAIL-receptor agonists already used in clinical trials for cancer could be repurposed to stimulate stable and functional microvessel networks in PAD.

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication. One sentence summary Lack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

We report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was ∼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues. CF proliferation was associated with higher reactive oxygen species (ROS) as occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ). The mitochondrial-targeted antioxidant MitoTEMPO rescued Hif-1a mutant phenotypes. Thus, HIF-1α in CFs provides a critical braking mechanism against excessive post-ischemic CF activation and proliferation through regulation of mitochondrial ROS. CFs are potential cellular targets for designer antioxidant therapies in cardiovascular disease.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVβ3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVβ3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch.

High-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for studying gene expression in single cells. Most current scRNA-seq bioinformatics tools focus on analysing overall expression levels, largely ignoring alternative mRNA isoform expression. We present a computational pipeline, Sierra, that readily detects differential transcript usage from data generated by commonly used polyA-captured scRNA-seq technology. We validate Sierra by comparing cardiac scRNA-seq cell types to bulk RNA-seq of matched populations, finding significant overlap in differential transcripts. Sierra detects differential transcript usage across human peripheral blood mononuclear cells and the Tabula Muris, and 3 'UTR shortening in cardiac fibroblasts. Sierra is available at https://github.com/VCCRI/Sierra .

Aging is a major risk factor for cardiovascular disease. Although the impact of aging has been extensively studied, little is known regarding the aging processes in cells of the heart. Here we analyzed the transcriptomes of hearts of 12-week-old and 18-month-old mice by single-nucleus RNA-sequencing. Among all cell types, aged fibroblasts showed most significant differential gene expression, increased RNA dynamics, and network entropy. Aged fibroblasts exhibited significantly changed expression patterns of inflammatory, extracellular matrix organization angiogenesis, and osteogenic genes. Functional analyses indicated deterioration of paracrine signatures between fibroblasts and endothelial cells in old hearts. Aged heart-derived fibroblasts had impaired endothelial cell angiogenesis and autophagy and augmented proinflammatory response. In particular, expression of Serpine1 and Serpine2 were significantly increased and secreted by old fibroblasts to exert antiangiogenic effects on endothelial cells, an effect that could be significantly prevented by using neutralizing antibodies. Moreover, we found an enlarged subpopulation of aged fibroblasts expressing osteoblast genes in the epicardial layer associated with increased calcification. Taken together this study provides system-wide insights and identifies molecular changes of aging cardiac fibroblasts, which may contribute to declined heart function.

Edits have been made to Development (2019) 146, dev178673 (doi:10.1242/dev.178673) to clarify that statements made comparing this and previous studies relate to the published analyses of data within those studies and not the detection of genes within the datasets themselves.The changes made are shown below and both the online full-text and PDF versions have been updated.In the Introduction, the following statement was changed:OriginalHowever, existing datasets have apparently not provided the transcriptional depth to identify the signalling pathways that operate within the human fetal kidney, and fail to detect several known ligand and receptor expression patterns in mouse.CorrectedThe analyses performed on existing datasets have not comprehensively identified the signalling pathway components known to be operating within the mouse fetal kidney.In the Discussion, the following statements were changed:OriginalFor example, although >20,000 cells were profiled at P1 (Adam et al., 2017), several known signalling molecules with functionally validated roles in the nephrogenic niche such as Gdnf, Fgf20, Fgf9, Bmp7, Wnt4 and Fgf8 were not detected in that analysis, precluding further insight into signalling interactions.CorrectedAlthough >20,000 cells were profiled at P1 (Adam et al., 2017), several known signalling molecules with functionally validated roles in the nephrogenic niche were not highlighted in that analysis.OriginalThe improved resolution of gene expression in our study may be due to sequencing depth (∼3000 genes detected per cell), biological replication and differential expression analysis with the edgeR method, which has recently been shown to be a top performer in a comparison of 36 differential expression analysis methods for scRNA-seq data (Soneson and Robinson, 2018).CorrectedThe improved analysis of signalling interactions provided in this study may be due to sequencing depth (∼3000 genes detected per cell), biological replication and differential expression analysis with the edgeR method, which has recently been shown to be a top performer in a comparison of 36 differential expression analysis methods for scRNA-seq data (Soneson and Robinson, 2018).In addition, a sentence (underlined) was moved as shown below:OriginalLineage tracing was performed to investigate the possibility of these cells representing a transitional state. Using a constitutively active Six2-Cre (Six2TGC), Six2-derived cells were observed in the cortical and medullary stroma in all samples (Fig. 5F). However, as this Six2-Cre is active from E11.5 or earlier, labelled stromal cells may reflect the early plasticity between stromal and nephron lineages rather than continued transdifferentiation. Using an inducible Six2-Cre (Six2GCE, induced from E12.5) to assess nephron progenitor contributions to stroma after the establishment of the proposed lineage boundary did result in rare Six2-derived cells in the nephrogenic zone that did not express SIX2 protein, but labelled cells were observed at a frequency lower than expected based on NP7 cluster size, and most labelled cells were unusually small, suggesting they may be undergoing apoptosis (Fig. 5G). Evidence of lineage transition was also observed deeper in the kidney.CorrectedLineage tracing was performed to investigate the possibility of these cells representing a transitional state. Using a constitutively active Six2-Cre (Six2TGC), Six2-derived cells were observed in the cortical and medullary stroma in all samples (Fig. 5F). Evidence of lineage transition was also observed deeper in the kidney. However, as this Six2-Cre is active from E11.5 or earlier, labelled stromal cells may reflect the early plasticity between stromal and nephron lineages rather than continued transdifferentiation. Using an inducible Six2-Cre (Six2GCE, induced from E12.5) to assess nephron progenitor contributions to stroma after the establishment of the proposed lineage boundary did result in rare Six2-derived cells in the nephrogenic zone that did not express SIX2 protein, but labelled cells were observed at a frequency lower than expected based on NP7 cluster size, and most labelled cells were unusually small, suggesting they may be undergoing apoptosis (Fig. 5G).The authors apologise for any lack of clarity in the original version of this paper. We believe the text now accurately reflects the contributions made by previous studies and the present work.