Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Benchmarking bulk and single-cell variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries.

Single-cell sequencing methodologies such as scRNA-seq and scATAC-seq have become widespread and effective tools to interrogate tissue composition. Increasingly, variant callers are being applied to these methodologies to resolve the genetic heterogeneity of a sample, especially in the case of detecting the clonal architecture of a tumor. Typically, traditional bulk DNA variant callers are applied to the pooled reads of a single-cell library to detect candidate mutations. Recently, multiple studies have applied such callers on reads from individual cells, with some citing the ability to detect rare variants with higher sensitivity. Many studies apply these two approaches to the Chromium (10x Genomics) scRNA-seq and scATAC-seq methodologies. However, Chromium-based libraries may offer additional challenges to variant calling compared with existing single-cell methodologies, raising questions regarding the validity of variants obtained from such a workflow. To determine the merits and challenges of various variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries, we use sample libraries with matched bulk whole-genome sequencing to evaluate the performance of callers. We review caller performance, finding that bulk callers applied on pooled reads significantly outperform individual-cell approaches. We also evaluate variants unique to scRNA-seq and scATAC-seq methodologies, finding patterns of noise but also potential capture of RNA-editing events. Finally, we review the notion that variant calling at the single-cell level can detect rare somatic variants, providing empirical results that suggest resolving such variants is infeasible in single-cell Chromium libraries.

Development and function of smooth muscle cells is modulated by Hic1 in mouse testis.

In mammalian testis, contractile peritubular myoid cells (PMCs) regulate the transport of sperm and luminal fluid, while secreting growth factors and extracellular matrix proteins to support the spermatogonial stem cell niche. However, little is known about the role of testicular smooth muscle cells during postnatal testicular development. Here we report age-dependent expression of hypermethylated in cancer 1 (Hic1; also known as ZBTB29) in testicular smooth muscle cells, including PMCs and vascular smooth muscle cells, in the mouse. Postnatal deletion of Hic1 in smooth muscle cells led to their increased proliferation and resulted in dilatation of seminiferous tubules, with increased numbers of PMCs. These seminiferous tubules contained fewer Sertoli cells and more spermatogonia, and fibronectin was not detected in their basement membrane. The expression levels of genes encoding smooth muscle contractile proteins, Acta2 and Cnn1, were downregulated in the smooth muscle cells lacking Hic1, and the seminiferous tubules appeared to have reduced contractility. These data imply a role for Hic1 in determining the size of seminiferous tubules by regulating postnatal smooth muscle cell proliferation, subsequently affecting spermatogenesis in adulthood.

Conventional laboratory housing increases morbidity and mortality in research rodents: results of a meta-analysis.

BACKGROUND: Over 120 million mice and rats are used annually in research, conventionally housed in shoebox-sized cages that restrict natural behaviours (e.g. nesting and burrowing). This can reduce physical fitness, impair thermoregulation and reduce welfare (e.g. inducing abnormal stereotypic behaviours). In humans, chronic stress has biological costs, increasing disease risks and potentially shortening life. Using a pre-registered protocol ( https://atrium.lib.uoguelph.ca/xmlui/handle/10214/17955 ), this meta-analysis therefore tested the hypothesis that, compared to rodents in 'enriched' housing that better meets their needs, conventional housing increases stress-related morbidity and all-cause mortality. RESULTS: Comprehensive searches (via Ovid, CABI, Web of Science, Proquest and SCOPUS on May 24 2020) yielded 10,094 publications. Screening for inclusion criteria (published in English, using mice or rats and providing 'enrichments' in long-term housing) yielded 214 studies (within 165 articles, using 6495 animals: 59.1% mice; 68.2% male; 31.8% isolation-housed), and data on all-cause mortality plus five experimentally induced stress-sensitive diseases: anxiety, cancer, cardiovascular disease, depression and stroke. The Systematic Review Center for Laboratory animal Experimentation (SYRCLE) tool assessed individual studies' risks of bias. Random-effects meta-analyses supported the hypothesis: conventional housing significantly exacerbated disease severity with medium to large effect sizes: cancer (SMD = 0.71, 95% CI = 0.54-0.88); cardiovascular disease (SMD = 0.72, 95% CI = 0.35-1.09); stroke (SMD = 0.87, 95% CI = 0.59-1.15); signs of anxiety (SMD = 0.91, 95% CI = 0.56-1.25); signs of depression (SMD = 1.24, 95% CI = 0.98-1.49). It also increased mortality rates (hazard ratio = 1.48, 95% CI = 1.25-1.74; relative median survival = 0.91, 95% CI = 0.89-0.94). Meta-regressions indicated that such housing effects were ubiquitous across species and sexes, but could not identify the most impactful improvements to conventional housing. Data variability (assessed via coefficient of variation) was also not increased by 'enriched' housing. CONCLUSIONS: Conventional housing appears sufficiently distressing to compromise rodent health, raising ethical concerns. Results also add to previous work to show that research rodents are typically CRAMPED (cold, rotund, abnormal, male-biased, poorly surviving, enclosed and distressed), raising questions about the validity and generalisability of the data they generate. This research was funded by NSERC, Canada.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

The Transcription Factor RORα Preserves ILC3 Lineage Identity and Function during Chronic Intestinal Infection.

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that Rorasg/sg bone marrow chimeric mice (C57BL/6J) were protected from Salmonella-induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual Rorasg/sg ILC3s have decreased expression of key signature genes, including Rorc and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

Single-cell analysis of mesenchymal cells in permeable neural vasculature reveals novel diverse subpopulations of fibroblasts.

BACKGROUND: In the choroid plexus and pituitary gland, vasculature is known to have a permeable, fenestrated phenotype which allows for the free passage of molecules in contrast to the blood brain barrier observed in the rest of the CNS. The endothelium of these compartments, along with secretory, neural-lineage cells (choroid epithelium and pituitary endocrine cells) have been studied in detail, but less attention has been given to the perivascular mesenchymal cells of these compartments. METHODS: The Hic1CreERT2 Rosa26LSL-TdTomato mouse model was used in conjunction with a PdgfraH2B-EGFP mouse model to examine mesenchymal cells, which can be subdivided into Pdgfra+ fibroblasts and Pdgfra- pericytes within the choroid plexus (CP) and pituitary gland (PG), by histological, immunofluorescence staining and single-cell RNA-sequencing analyses. RESULTS: We found that both CP and PG possess substantial populations of distinct Hic1+ mesenchymal cells, including an abundance of Pdgfra+ fibroblasts. Within the pituitary, we identified distinct subpopulations of Hic1+ fibroblasts in the glandular anterior pituitary and the neurosecretory posterior pituitary. We also identified multiple distinct markers of CP, PG, and the meningeal mesenchymal compartment, including alkaline phosphatase, indole-n-methyltransferase and CD34. CONCLUSIONS: Novel, distinct subpopulations of mesenchymal cells can be found in permeable vascular interfaces, including the CP, PG, and meninges, and make distinct contributions to both organs through the production of structural proteins, enzymes, transporters, and trophic molecules.

Hic1 identifies a specialized mesenchymal progenitor population in the embryonic limb responsible for bone superstructure formation.

The musculoskeletal system relies on the integration of multiple components with diverse physical properties, such as striated muscle, tendon, and bone, that enable locomotion and structural stability. This relies on the emergence of specialized, but poorly characterized, interfaces between these various elements during embryonic development. Within the appendicular skeleton, we show that a subset of mesenchymal progenitors (MPs), identified by Hic1, do not contribute to the primary cartilaginous anlagen but represent the MP population, whose progeny directly contribute to the interfaces that connect bone to tendon (entheses), tendon to muscle (myotendinous junctions), and the associated superstructures. Furthermore, deletion of Hic1 leads to skeletal defects reflective of deficient muscle-bone coupling and, consequently, perturbation of ambulation. Collectively, these findings show that Hic1 identifies a unique MP population that contributes to a secondary wave of bone sculpting critical to skeletal morphogenesis.

Aberrant gene activation in synovial sarcoma relies on SSX specificity and increased PRC1.1 stability.

The SS18-SSX fusion drives oncogenic transformation in synovial sarcoma by bridging SS18, a member of the mSWI/SNF (BAF) complex, to Polycomb repressive complex 1 (PRC1) target genes. Here we show that the ability of SS18-SSX to occupy H2AK119ub1-rich regions is an intrinsic property of its SSX C terminus, which can be exploited by fusion to transcriptional regulators beyond SS18. Accordingly, SS18-SSX recruitment occurs in a manner that is independent of the core components and catalytic activity of BAF. Alternative SSX fusions are also recruited to H2AK119ub1-rich chromatin and reproduce the expression signatures of SS18-SSX by engaging with transcriptional activators. Variant Polycomb repressive complex 1.1 (PRC1.1) acts as the main depositor of H2AK119ub1 and is therefore required for SS18-SSX occupancy. Importantly, the SSX C terminus not only depends on H2AK119ub1 for localization, but also further increases it by promoting PRC1.1 complex stability. Consequently, high H2AK119ub1 levels are a feature of murine and human synovial sarcomas. These results uncover a critical role for SSX-C in mediating gene deregulation in synovial sarcoma by providing specificity to chromatin and further enabling oncofusion binding by enhancing PRC1.1 stability and H2AK119ub1 deposition.

Cellular taxonomy of Hic1+ mesenchymal progenitor derivatives in the limb: from embryo to adult.

Tissue development and regeneration rely on the cooperation of multiple mesenchymal progenitor (MP) subpopulations. We recently identified Hic1 as a marker of quiescent MPs in multiple adult tissues. Here, we describe the embryonic origin of appendicular Hic1+ MPs and demonstrate that they arise in the hypaxial somite, and migrate into the developing limb at embryonic day 11.5, well after limb bud initiation. Time-resolved single-cell-omics analyses coupled with lineage tracing reveal that Hic1+ cells generate a unique MP hierarchy, that includes both recently identified adult universal fibroblast populations (Dpt+, Pi16+ and Dpt+ Col15a1+) and more specialised mesenchymal derivatives such as, peri and endoneurial cells, pericytes, bone marrow stromal cells, myotenocytes, tenocytes, fascia-resident fibroblasts, with limited contributions to chondrocytes and osteocytes within the skeletal elements. MPs endure within these compartments, continue to express Hic1 and represent a critical reservoir to support post-natal growth and regeneration.

Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors.

Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.

Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors.

The cardiac stroma contains multipotent mesenchymal progenitors. However, lineage relationships within cardiac stromal cells are poorly defined. Here, we identified heart-resident PDGFRa+ SCA-1+ cells as cardiac fibro/adipogenic progenitors (cFAPs) and show that they respond to ischemic damage by generating fibrogenic cells. Pharmacological blockade of this differentiation step with an anti-fibrotic tyrosine kinase inhibitor decreases post-myocardial infarction (post-MI) remodeling and leads to improvement in cardiac function. In the undamaged heart, activation of cFAPs through lineage-specific deletion of the gene encoding the quiescence-associated factor HIC1 reveals additional pathogenic potential, causing fibrofatty infiltration within the myocardium and driving major pathological features pathognomonic in arrhythmogenic cardiomyopathy (AC). In this regard, cFAPs contribute to multiple pathogenic cell types within cardiac tissue and therapeutic strategies aimed at modifying their activity are expected to have tremendous benefit for the treatment of diverse cardiac diseases.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.

Hic1 Defines Quiescent Mesenchymal Progenitor Subpopulations with Distinct Functions and Fates in Skeletal Muscle Regeneration.

Many adult tissues contain resident stem cells, such as the Pax7+ satellite cells within skeletal muscle, that regenerate parenchymal elements following damage. Tissue-resident mesenchymal progenitors (MPs) also participate in regeneration, although their function and fate in this process are unclear. Here, we identify Hypermethylated in cancer 1 (Hic1) as a marker of MPs in skeletal muscle and further show that Hic1 deletion leads to MP hyperplasia. Single-cell RNA-seq and ATAC-seq analysis of Hic1+ MPs in skeletal muscle shows multiple subpopulations, which we further show have distinct functions and lineage potential. Hic1+ MPs orchestrate multiple aspects of skeletal muscle regeneration by providing stage-specific immunomodulation and trophic and mechanical support. During muscle regeneration, Hic1+ derivatives directly contribute to several mesenchymal compartments including Col22a1-expressing cells within the myotendinous junction. Collectively, these findings demonstrate that HIC1 regulates MP quiescence and identifies MP subpopulations with transient and enduring roles in muscle regeneration.

Methods and Strategies for Lineage Tracing of Mesenchymal Progenitor Cells.

Mesenchymal progenitors (MP) are found to varying extents in most tissues and organs. Their relationship to bone marrow-derived mesenchymal stem cells (MSCs) remains unclear, however, both populations appear to share a number of properties as defined by functional assays, clonogenic activity, and genetic and cell surface markers. MSCs were originally defined by their in vitro colony forming unit-fibroblast (CFU-F) activity and their ability to contribute to various mesenchymal lineages (i.e. cartilage, bone, and fat). MSCs also appear to exhibit some unique properties, in that expanded clones in the absence of bone-inducing factors generate bone spicules/organs in vivo. Subsequent analysis of these elements has demonstrated that the transplanted cells directly contribute to multiple mesenchymal lineages. Our ability to study MP and/or MSC behavior and lineage potential in vivo has been hampered by a lack of suitable Cre lines in which to effectively genetically mark and follow the fate and activity of these cells in development, growth, homeostasis and following injury or in disease. The emergence of several new genetic lines is enabling us to now address critical questions regarding MP/MSC location, behavior, function, and fate. The use of these lines and others in conjunction with suitable reporter lines will be described for MP/MSC cell fate analysis.

Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice.

Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl4)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl4-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis.