Mechanisms bywhich macrophages deploy antibacterial zinc toxicity are poorly understood. To gain insight into this antimicrobial pathway, we developed bacterial reporter-paired single-cell RNA sequencing of human monocyte-derived macrophages (HMDM) infected with an Escherichia coli zinc-stress reporter strain. We identified HMDM subpopulations harboring zinc-stressed E. coli and corresponding mammalian genes predicted to be associated with either zinc toxicity or survival of zinc-stressed bacteria. Consistent with the latter, SLC30A4 that encodes zinc exporter ZNT4 was enriched in one subpopulation of HMDM containing zinc-stressed E. coli and its overexpression in human macrophages increased intracellular E. coli survival. At a population level, SLC30A4 expression was rapidly downregulated in human macrophages responding to E. coli and its ectopic expression in macrophages attenuated zinc starvation of intracellular E. coli. This is consistent with a model in which macrophages switch off SLC30A4 to engage zinc starvation, while also deploying zinc toxicity against bacteria adapting to a low-zinc environment. Consistent with this, intramacrophage E. coli rapidly upregulated znuA messenger RNA (mRNA) that is induced during zinc limitation, with zntA mRNA that is induced during zinc stress peaking later. Moreover, E. coli cultured under conditions of zinc limitation displayed greatly enhanced zinc sensitivity. Susceptibility of zinc-sensitive E. coli to killing by macrophages was also attenuated when zinc uptake by E. coli was inactivated, confirming the coordinated actions of zinc starvation and zinc toxicity in macrophage antibacterial responses. Strategies that enhance zinc starvation of intracellular bacteria could be exploited in the design of host-directed therapeutics that amplify macrophage-mediated antibacterial zinc toxicity.

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here, we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency, encompassing an eight-day time course with modulation of WNT, BMP, and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas, evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together, this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974–2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.

AbstractNeonatal meningitis is a devastating disease associated with high mortality and neurological sequelae.Escherichia coliis the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence factor was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75 and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggests that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Context Recent advances in molecular technology have allowed us to examine the cattle genome with an accuracy never before possible. Genetic variations, both small and large, as well as the transcriptional landscape of the bovine genome, have both been explored in many studies. However, the topological configuration of the genome has not been extensively investigated, largely due to the cost of the assays required. Such assays can both identify topologically associated domains and be used for genome scaffolding. Aims This study aimed to implement a chromatin conformation capture together with long-read nanopore sequencing (Pore-C) pipeline for scaffolding a draft assembly and identifying topologically associating domains (TADs) of a Bos indicus Brahman cow. Methods Genomic DNA from a liver sample was first cross-linked to proteins, preserving the spatial proximity of loci. Restriction digestion and proximity ligation were then used to join cross-linked fragments, followed by nucleic isolation. The Pore-C DNA extracts were then prepped and sequenced on a PromethION device. Two genome assemblies were used to analyse the data, namely, one generated from sequencing of the same Brahman cow, and the other is the ARS-UCD1.2 Bos taurus assembly. The Pore-C snakemake pipeline was used to map, assign bins and scaffold the draft and current annotated bovine assemblies. The contact matrices were then used to identify TADs. Key results The study scaffolded a chromosome-level Bos indicus assembly representing 30 chromosomes. The scaffolded assembly showed a total of 215 contigs (2.6 Gbp) with N50 of 44.8 Mb. The maximum contig length was 156.8 Mb. The GC content of the scaffold assembly is 41 ± 0.02%. Over 50% of mapped chimeric reads identified for both assemblies had three or more contacts. This is the first experimental study to identify TADs in bovine species. In total, 3036 and 3094 TADs across 30 chromosomes were identified for input Brahman and ARS-UCD1.2 assemblies respectively. Conclusions The Pore-C pipeline presented herein will be a valuable approach to scaffold draft assemblies for agricultural species and understand the chromatin structure at different scales. Implications The Pore-C approach will open a new era of 3D genome-organisation studies across agriculture species.

Unlike bulk and single-cell/single-nuclei RNA sequencing methods, spatial transcriptome sequencing (ST-seq) resolves transcriptome expression within the spatial context of intact tissue. This is achieved by integrating histology with RNA sequencing. These methodologies are completed sequentially on the same tissue section placed on a glass slide with printed oligo-dT spots, termed ST-spots. Transcriptomes within the tissue section are captured by the underlying ST-spots and receive a spatial barcode in the process. The sequenced ST-spot transcriptomes are subsequently aligned with the hematoxylin and eosin (H&E) image, giving morphological context to the gene expression signatures within intact tissue. We have successfully employed ST-seq to characterize mouse and human kidney tissue. Here, we describe in detail the application of Visium Spatial Tissue Optimization (TO) and Visium Spatial Gene Expression (GEx) protocols for ST-seq in fresh frozen kidney tissue.

Identifying tissue-specific molecular signatures of active regulatory elements is critical to understanding gene regulatory mechanisms. In this study, transcription start sites (TSS) and enhancers were identified using Cap analysis of gene expression (CAGE) across endometrial stromal cell (ESC) samples obtained from women with (n = 4) and without endometriosis (n = 4). ESC TSSs and enhancers were compared to those reported in other tissue and cell types in FANTOM5 and were integrated with RNA-seq and ATAC-seq data from the same samples for regulatory activity and network analyses. CAGE tag count differences between women with and without endometriosis were statistically tested and tags within close proximity to genetic variants associated with endometriosis risk were identified. Over 90% of tag clusters mapping to promoters were observed in cells and tissues in FANTOM5. However, some potential cell-type-specific promoters and enhancers were also observed. Regions of open chromatin identified using ATAC-seq provided further evidence of the active transcriptional regions identified by CAGE. Despite the small sample number, there was evidence of differences associated with endometriosis at 210 consensus clusters, including IGFBP5, CALD1 and OXTR. ESC TSSs were also located within loci associated with endometriosis risk from genome-wide association studies. This study provides novel evidence of transcriptional differences in endometrial stromal cells associated with endometriosis and provides a valuable cell-type specific resource of active TSSs and enhancers in endometrial stromal cells.

The human immune system displays substantial variation between individuals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression. We subsequently show how eQTLs have dynamic allelic effects in B cells that are transitioning from naïve to memory states and demonstrate how commonly segregating alleles lead to interindividual variation in immune function. Finally, using a Mendelian randomization approach, we identify the causal route by which 305 risk loci contribute to autoimmune disease at the cellular level. This work brings together genetic epidemiology with scRNA-seq to uncover drivers of interindividual variation in the immune system.

This study develops a versatile cell multiplexing and data analysis platform to gain knowledge gain into mechanisms of cell differentiation. We engineer a cell barcoding system in human cells enabling multiplexed single-cell RNA sequencing for high throughput perturbation of customisable and diverse experimental conditions. This is coupled with a new computational analysis pipeline that overcomes the limitations of conventional algorithms by using an unsupervised, genome-wide, orthogonal biological reference point to reveal the cell diversity and regulatory networks in the input scRNA-seq data set. We implement this pipeline by engineering transcribed barcodes into induced pluripotent stem cells and multiplex 62 independent experimental conditions comprising eight differentiation time points and nine developmental signalling perturbations in duplicates. We identify and deconstruct the temporal, signalling, and gene regulatory imperatives of iPSC differentiation into cell types of ectoderm, mesoderm, and endoderm lineages. This study provides a cellular and computational pipeline to study cell differentiation applicable to studies in developmental biology, drug discovery, and disease modelling.### Competing Interest StatementThe authors have declared no competing interest.

SUMMARY This study develops a versatile cell multiplexing and data analysis platform to accelerate knowledge gain into mechanisms of cell differentiation. We engineer a cell barcoding system in human cells enabling multiplexed single-cell RNA sequencing for high throughput perturbation of customisable and versatile experimental conditions. This is coupled with a new computational analysis pipeline that overcomes the limitations of existing algorithms by using an unsupervised, genome-wide, orthogonal biological reference point to reveal the cell diversity and regulatory networks of any input scRNA-seq data set. We implement this pipeline by engineering transcribed barcodes into induced pluripotent stem cells and multiplex 62 independent experimental conditions comprising eight differentiation time points and nine developmental signalling perturbations with duplicates. We identify and deconstruct the temporal, signalling, and gene regulatory basis of multilineage iPSC differentiation into ectoderm, mesoderm, and endoderm cell types. This study provides a cellular and computational pipeline to study cell differentiation applicable to diverse fields of developmental biology, drug discovery, and disease modelling.

Available transcriptomes of the mammalian kidney provide limited information on the spatial interplay between different functional nephron structures due to the required dissociation of tissue with traditional transcriptome-based methodologies. A deeper understanding of the complexity of functional nephron structures requires a non-dissociative transcriptomics approach, such as spatial transcriptomics sequencing (ST-seq). We hypothesize that the application of ST-seq in normal mammalian kidneys will give transcriptomic insights within and across species of physiology at the functional structure level and cellular communication at the cell level. Here, we applied ST-seq in six mice and four human kidneys that were histologically absent of any overt pathology. We defined the location of specific nephron structures in the captured ST-seq datasets using three lines of evidence: pathologist's annotation, marker gene expression, and integration with public single-cell and/or single-nucleus RNA-sequencing datasets. We compared the mouse and human cortical kidney regions. In the human ST-seq datasets, we further investigated the cellular communication within glomeruli and regions of proximal tubules-peritubular capillaries by screening for co-expression of ligand-receptor gene pairs. Gene expression signatures of distinct nephron structures and microvascular regions were spatially resolved within the mouse and human ST-seq datasets. We identified 7,370 differentially expressed genes (p adj < 0.05) distinguishing species, suggesting changes in energy production and metabolism in mouse cortical regions relative to human kidneys. Hundreds of potential ligand-receptor interactions were identified within glomeruli and regions of proximal tubules-peritubular capillaries, including known and novel interactions relevant to kidney physiology. Our application of ST-seq to normal human and murine kidneys confirms current knowledge and localization of transcripts within the kidney. Furthermore, the generated ST-seq datasets provide a valuable resource for the kidney community that can be used to inform future research into this complex organ.

SUMMARYThis study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

Conventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3-/- mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3-/- mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, andmaturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.

Here, we outline detailed protocols to isolate and profile murine splenic dendritic cells (DCs) through advanced flow cytometry of the myeloid compartment and single-cell transcriptomic profiling with integrated cell surface protein expression through CITE-seq. This protocol provides a general transferrable road map for different tissues and species.For complete details on the use and execution of this protocol, please refer to Lukowski et al. (2021).

We present Nanopore-DamID, a method to simultaneously detect cytosine methylation and DNA-protein interactions from single molecules, via selective sequencing of adenine-labelled DNA. Assaying LaminB1 and CTCF binding with Nanopore-DamID, we identify escape from LAD-associated repression of hypomethylated promoters amidst generalised hypermethylation of LaminB1-associated regulatory elements. We detect novel CTCF binding sites in highly repetitive regions, and allele-specific CTCF binding to imprinted genes and the active X chromosome. Nanopore-DamID highlights the importance of DNA methylation to transcription factor activity.

With the exception of a few master transcription factors, regulators of neutrophil maturation are poorly annotated in the intermediate phenotypes between the granulocyte-macrophage progenitor (GMP) and the mature neutrophil phenotype. Additional challenges in identifying gene expression regulators in differentiation pathways relate to challenges wherein starting cell populations are heterogeneous in lineage potential and development, are spread across various states of quiescence, as well as sample quality and input limitations. These factors contribute to data variability make it difficult to draw simple regulatory inferences. In response we have applied a multi-omics approach using primary blood progenitor cells primed for homogeneous proliferation and granulocyte differentiation states which combines whole transcriptome resequencing (Ampliseq RNA) supported by droplet digital PCR (ddPCR) validation and mass spectrometry-based proteomics in a hypothesis-generation study of neutrophil differentiation pathways. Primary CD34+ cells isolated from human cord blood were first precultured in non-lineage driving medium to achieve an active, proliferating phenotype from which a neutrophil primed progenitor was isolated and cultured in neutrophil lineage supportive medium. Samples were then taken at 24-hour intervals over 9 days and analysed by Ampliseq RNA and mass spectrometry. The Ampliseq dataset depth, breadth and quality allowed for several unexplored transcriptional regulators and ncRNAs to be identified using a combinatorial approach of hierarchical clustering, enriched transcription factor binding motifs, and network mapping. Network mapping in particular increased comprehension of neutrophil differentiation regulatory relationships by implicating ARNT, NHLH1, PLAG1, and 6 non-coding RNAs associated with PU.1 regulation as cell-engineering targets with the potential to increase total neutrophil culture output. Overall, this study develops and demonstrates an effective new hypothesis generation methodology for transcriptome profiling during differentiation, thereby enabling identification of novel gene targets for editing interventions.

The libraries generated by high-throughput single cell RNA-sequencing platforms such as the Chromium from 10x Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use this data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3’ libraries consisting of over 70,000 cells. Our results demonstrate a highly comparable performance between the NovaSeq 6000 and MGISEQ-2000 in sequencing quality, and cell, UMI, and gene detection. However, compared with the NextSeq 500, the MGISEQ-2000 platform performs consistently better, identifying more cells, genes, and UMIs at equalised read depth. We were able to call an additional 1,065,659 SNPs from sequence data generated by the BGI platform, enabling an additional 14% of cells to be assigned to the correct donor from a multiplexed library. However, both the NextSeq 500 and MGISEQ-2000 detected similar frequencies of gRNAs from a pooled CRISPR single cell screen. Our study provides a benchmark for high capacity sequencing platforms applied to high-throughput single cell RNA-seq libraries.

Acute gastrointestinal Graft-versus-Host-Disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem-cell transplantation (alloSCT). It is mediated by alloreactive donor CD4 + T cells that differentiate into pathogenic subsets expressing IFNγ, IL-17A or GM-CSF, and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between T-helper states during priming in mesenteric lymph nodes (mLN) and effector function in the GI tract remain undefined at genome-scale. We used scRNA-seq and computational modelling to create an atlas of putative differentiation pathways during GVHD. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLN. Importantly, we identified an unexpected second trajectory, categorised by little proliferation or cytokine expression, reduced glycolysis, and high TCF1 expression. TCF1 hi cells upregulated α4β7 prior to gut migration and failed to express cytokines therein. Nevertheless, they demonstrated recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced TCF1. Thus, scRNA-seq revealed divergence of allo-reactive CD4 + T cells into quiescent and effector states during gut GVHD, reflecting putative heterogenous priming in vivo . These findings, the first at a single-cell level during GVHD over time, can now be used to interrogate T cell differentiation in patients undergoing alloSCT.