Branching topology of the human embryo transcriptome revealed by entropy sort feature weighting.

Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after sub-setting to highly variable genes (HVGs). Here we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous entropy sort feature weighting (cESFW) outperforms HVG selection in distinguishing cell state specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from 8-cell to post-implantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression we provide markers for staging progression from morula to blastocyst.

A branching model of lineage differentiation underpinning the neurogenic potential of enteric glia.

Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.

Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus.

Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.

Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network.

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We identify heterogeneity within the FDC network. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as the antigen reservoir, where different antigens arriving from subsequent immunizations colocalize. Mechanistically, central LZ FDCs constitutively express subtly higher CR2 membrane densities than peripheral LZ FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

Systematic diet composition swap in a mouse genome-scale metabolic model reveals determinants of obesogenic diet metabolism in liver cancer.

Dietary nutrient availability and gene expression, together, influence tissue metabolic activity. Here, we explore whether altering dietary nutrient composition in the context of mouse liver cancer suffices to overcome chronic gene expression changes that arise from tumorigenesis and western-style diet (WD). We construct a mouse genome-scale metabolic model and estimate metabolic fluxes in liver tumors and non-tumoral tissue after computationally varying the composition of input diet. This approach, called Systematic Diet Composition Swap (SyDiCoS), revealed that, compared to a control diet, WD increases production of glycerol and succinate irrespective of specific tissue gene expression patterns. Conversely, differences in fatty acid utilization pathways between tumor and non-tumor liver are amplified with WD by both dietary carbohydrates and lipids together. Our data suggest that combined dietary component modifications may be required to normalize the distinctive metabolic patterns that underlie selective targeting of tumor metabolism.

Microbe capture by splenic macrophages triggers sepsis via T cell-death-dependent neutrophil lifespan shortening.

The mechanisms linking systemic infection to hyperinflammation and immune dysfunction in sepsis are poorly understood. Extracellular histones promote sepsis pathology, but their source and mechanism of action remain unclear. Here, we show that by controlling fungi and bacteria captured by splenic macrophages, neutrophil-derived myeloperoxidase attenuates sepsis by suppressing histone release. In systemic candidiasis, microbial capture via the phagocytic receptor SIGNR1 neutralizes myeloperoxidase by facilitating marginal zone infiltration and T cell death-dependent histone release. Histones and hyphae induce cytokines in adjacent CD169 macrophages including G-CSF that selectively depletes mature Ly6Ghigh neutrophils by shortening their lifespan in favour of immature Ly6Glow neutrophils with a defective oxidative burst. In sepsis patient plasma, these mediators shorten mature neutrophil lifespan and correlate with neutrophil mortality markers. Consequently, high G-CSF levels and neutrophil lifespan shortening activity are associated with sepsis patient mortality. Hence, by exploiting phagocytic receptors, pathogens degrade innate and adaptive immunity through the detrimental impact of downstream effectors on neutrophil lifespan.

Molecular profiling of enteric nervous system cell lineages.

The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.

Disrupted control of origin activation compromises genome integrity upon destabilization of Polε and dysfunction of the TRP53-CDKN1A/P21 axis.

The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.

A local human Vδ1 T cell population is associated with survival in nonsmall-cell lung cancer.

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8+ T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA-CD27- effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103+ tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.

UBAP2/UBAP2L regulate UV-induced ubiquitylation of RNA polymerase II and are the human orthologues of yeast Def1.

During transcription, RNA polymerase II (RNAPII) faces numerous obstacles, including DNA damage, which can lead to stalling or arrest. One mechanism to contend with this situation is ubiquitylation and degradation of the largest RNAPII subunit, RPB1 - the 'last resort' pathway. This conserved, multi-step pathway was first identified in yeast, and the functional human orthologues of all but one protein, RNAPII Degradation Factor 1 (Def1), have been discovered. Here we show that following UV-irradiation, human Ubiquitin-associated protein 2 (UBAP2) or its paralogue UBAP2-like (UBAP2L) are involved in the ubiquitylation and degradation of RNAPII through the recruitment of Elongin-Cul5 ubiquitin ligase. Together, our data indicate that UBAP2 and UBAP2L are the human orthologues of yeast Def1, and so identify the key missing proteins in the human last resort pathway.

KLF17 promotes human naïve pluripotency but is not required for its establishment.

Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions.

Regulation of intestinal immunity and tissue repair by enteric glia.

Tissue maintenance and repair depend on the integrated activity of multiple cell types1. Whereas the contributions of epithelial2,3, immune4,5 and stromal cells6,7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here we uncover important roles of enteric glial cells (EGCs) in intestinal homeostasis, immunity and tissue repair. We demonstrate that infection of mice with Heligmosomoides polygyrus leads to enteric gliosis and the upregulation of an interferon gamma (IFNγ) gene signature. IFNγ-dependent gene modules were also induced in EGCs from patients with inflammatory bowel disease8. Single-cell transcriptomics analysis of the tunica muscularis showed that glia-specific abrogation of IFNγ signalling leads to tissue-wide activation of pro-inflammatory transcriptional programs. Furthermore, disruption of the IFNγ-EGC signalling axis enhanced the inflammatory and granulomatous response of the tunica muscularis to helminths. Mechanistically, we show that the upregulation of Cxcl10 is an early immediate response of EGCs to IFNγ signalling and provide evidence that this chemokine and the downstream amplification of IFNγ signalling in the tunica muscularis are required for a measured inflammatory response to helminths and resolution of the granulomatous pathology. Our study demonstrates that IFNγ signalling in enteric glia is central to intestinal homeostasis and reveals critical roles of the IFNγ-EGC-CXCL10 axis in immune response and tissue repair after infectious challenge.

Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture.

Astrocytes have essential functions in brain homeostasis that are established late in differentiation, but the mechanisms underlying the functional maturation of astrocytes are not well understood. Here we identify extensive transcriptional changes that occur during murine astrocyte maturation in vivo that are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lack expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induces distinct sets of mature astrocyte-specific transcripts. Culturing astrocytes in a three-dimensional matrix containing FGF2 induces expression of Rorb, Dbx2 and Lhx2 and improves astrocyte maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

Molecular characterization of projection neuron subtypes in the mouse olfactory bulb.

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion, and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy.

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway.

The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of nls-YAP5SA or knockout of Lats1/2 do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include Ctgf, Cyr61 and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation in vitro, possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation in vivo, and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.

Enteric glia as a source of neural progenitors in adult zebrafish.

The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrates is a matter of intense debate. Here, we demonstrate that the non-neuronal ENS cell compartment of teleosts shares molecular and morphological characteristics with mammalian enteric glia but cannot be identified by the expression of canonical glial markers. However, unlike their mammalian counterparts, which are generally quiescent and do not undergo neuronal differentiation during homeostasis, we show that a relatively high proportion of zebrafish enteric glia proliferate under physiological conditions giving rise to progeny that differentiate into enteric neurons. We also provide evidence that, similar to brain neural stem cells, the activation and neuronal differentiation of enteric glia are regulated by Notch signalling. Our experiments reveal remarkable similarities between enteric glia and brain neural stem cells in teleosts and open new possibilities for use of mammalian enteric glia as a potential source of neurons to restore the activity of intestinal neural circuits compromised by injury or disease.

CARD14E138A signalling in keratinocytes induces TNF-dependent skin and systemic inflammation.

To investigate how the CARD14E138A psoriasis-associated mutation induces skin inflammation, a knock-in mouse strain was generated that allows tamoxifen-induced expression of the homologous Card14E138A mutation from the endogenous mouse Card14 locus. Heterozygous expression of CARD14E138A rapidly induced skin acanthosis, immune cell infiltration and expression of psoriasis-associated pro-inflammatory genes. Homozygous expression of CARD14E138A induced more extensive skin inflammation and a severe systemic disease involving infiltration of myeloid cells in multiple organs, temperature reduction, weight loss and organ failure. This severe phenotype resembled acute exacerbations of generalised pustular psoriasis (GPP), a rare form of psoriasis that can be caused by CARD14 mutations in patients. CARD14E138A-induced skin inflammation and systemic disease were independent of adaptive immune cells, ameliorated by blocking TNF and induced by CARD14E138A signalling only in keratinocytes. These results suggest that anti-inflammatory therapies specifically targeting keratinocytes, rather than systemic biologicals, might be effective for GPP treatment early in disease progression.