Integrative spatial analysis reveals a multi-layered organization of glioblastoma.

Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.

Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs.

In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.

Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Genome-wide mapping of IBD segments in an Ashkenazi PD cohort identifies associated haplotypes.

The recent series of large genome-wide association studies in European and Japanese cohorts established that Parkinson disease (PD) has a substantial genetic component. To further investigate the genetic landscape of PD, we performed a genome-wide scan in the largest to date Ashkenazi Jewish cohort of 1130 Parkinson patients and 2611 pooled controls. Motivated by the reduced disease allele heterogeneity and a high degree of identical-by-descent (IBD) haplotype sharing in this founder population, we conducted a haplotype association study based on mapping of shared IBD segments. We observed significant haplotype association signals at three previously implicated Parkinson loci: LRRK2 (OR = 12.05, P = 1.23 × 10(-56)), MAPT (OR = 0.62, P = 1.78 × 10(-11)) and GBA (multiple distinct haplotypes, OR > 8.28, P = 1.13 × 10(-11) and OR = 2.50, P = 1.22 × 10(-9)). In addition, we identified a novel association signal on chr2q14.3 coming from a rare haplotype (OR = 22.58, P = 1.21 × 10(-10)) and replicated it in a secondary cohort of 306 Ashkenazi PD cases and 2583 controls. Our results highlight the power of our haplotype association method, particularly useful in studies of founder populations, and reaffirm the benefits of studying complex diseases in Ashkenazi Jewish cohorts.

Context-specific microRNA analysis: identification of functional microRNAs and their mRNA targets.

MicroRNAs (miRs) function primarily as post-transcriptional negative regulators of gene expression through binding to their mRNA targets. Reliable prediction of a miR's targets is a considerable bioinformatic challenge of great importance for inferring the miR's function. Sequence-based prediction algorithms have high false-positive rates, are not in agreement, and are not biological context specific. Here we introduce CoSMic (Context-Specific MicroRNA analysis), an algorithm that combines sequence-based prediction with miR and mRNA expression data. CoSMic differs from existing methods--it identifies miRs that play active roles in the specific biological system of interest and predicts with less false positives their functional targets. We applied CoSMic to search for miRs that regulate the migratory response of human mammary cells to epidermal growth factor (EGF) stimulation. Several such miRs, whose putative targets were significantly enriched by migration processes were identified. We tested three of these miRs experimentally, and showed that they indeed affected the migratory phenotype; we also tested three negative controls. In comparison to other algorithms CoSMic indeed filters out false positives and allows improved identification of context-specific targets. CoSMic can greatly facilitate miR research in general and, in particular, advance our understanding of individual miRs' function in a specific context.

Tuft cells and fibroblasts promote thymus regeneration through ILC2-mediated type 2 immune response.

The thymus is a primary lymphoid organ that is essential for the establishment of adaptive immunity through generation of immunocompetent T cells. In response to various stress signals, the thymus undergoes acute but reversible involution. However, the mechanisms governing its recovery are incompletely understood. Here, we used a dexamethasone-induced acute thymic involution mouse model to investigate how thymic hematopoietic cells (excluding T cells) contribute to thymic regeneration. scRNA-seq analysis revealed marked transcriptional and cellular changes in various thymic populations and highlighted thymus-resident innate lymphoid cells type 2 (ILC2) as a key cell type involved in the response to damage. We identified that ILC2 are activated by the alarmins IL-25 and IL-33 produced in response to tissue damage by thymic tuft cells and fibroblasts, respectively. Moreover, using mouse models deficient in either tuft cells and/or IL-33, we found that these alarmins are required for effective thymus regeneration after dexamethasone-induced damage. We also demonstrate that upon their damage-dependent activation, thymic ILC2 produce several effector molecules linked to tissue regeneration, such as amphiregulin and IL-13, which in turn promote thymic epithelial cell differentiation. Collectively, our study elucidates a previously undescribed role for thymic tuft cells and fibroblasts in thymus regeneration through activation of the type 2 immune response.

The transcriptional and regulatory identity of erythropoietin producing cells.

Erythropoietin (Epo) is the master regulator of erythropoiesis and oxygen homeostasis. Despite its physiological importance, the molecular and genomic contexts of the cells responsible for renal Epo production remain unclear, limiting more-effective therapies for anemia. Here, we performed single-cell RNA and transposase-accessible chromatin (ATAC) sequencing of an Epo reporter mouse to molecularly identify Epo-producing cells under hypoxic conditions. Our data indicate that a distinct population of kidney stroma, which we term Norn cells, is the major source of endocrine Epo production in mice. We use these datasets to identify the markers, signaling pathways and transcriptional circuits characteristic of Norn cells. Using single-cell RNA sequencing and RNA in situ hybridization in human kidney tissues, we further provide evidence that this cell population is conserved in humans. These preliminary findings open new avenues to functionally dissect EPO gene regulation in health and disease and may serve as groundwork to improve erythropoiesis-stimulating therapies.

Tnfα, Cox2 and AdipoQ adipokine gene expression levels are modulated in murine adipose tissues by both nicotine and nACh receptors containing the ß2 subunit.

Studies have provided evidences for the effects of nicotine on adipose tissues, as well as in inflammatory response. We hypothesized that nicotine affects adipokine gene expression in adipose tissues via specific neuronal nicotinic acetylcholine receptors (nAChRs). First, we described the expression of multiple nAChR subunit genes in mouse white and brown adipose tissues (WAT and BAT), and detected differential expression in WAT and BAT (α2>α5>ß2 and α2>ß2>ß4, respectively). Additionally, when nicotine was administered to wild-type mice, it significantly affected the expression of adipokine genes, such as Tnfα, AdipoQ, Haptoglobin and Mcp1 in WAT. Next, we demonstrated that in mice deficient for the ß2 nAChR subunit (ß2-/- mice), the expression levels of Cox2 and Ngfß genes in WAT, and Leptin, Cox2, AdipoQ and Haptoglobin in BAT, were significantly altered. Furthermore, interactions between mouse ß2 subunit and nicotine treatment affected the expression levels of the adipokine genes Tnfα, Cox2 and AdipoQ in WAT and of AdipoQ in BAT. Finally, analysis of a cellular model of cultured adipocytes demonstrated that application of nicotine after silencing of the ß2 nAChR subunit significantly elevated the expression level of Cox2 gene. Together, our data suggest a molecular link between the ß2 nACh receptor subunit and the expression levels of specific adipokines, which is also affected by nicotine.

Exploring differential exon usage via short- and long-read RNA sequencing strategies.

Alternative splicing produces various mRNAs, and thereby various protein products, from one gene, impacting a wide range of cellular activities. However, accurate reconstruction and quantification of full-length transcripts using short-reads is limited, due to their length. Long-reads sequencing technologies may provide a solution by sequencing full-length transcripts. We explored the use of both Illumina short-reads and two long Oxford Nanopore Technology (cDNA and Direct RNA) RNA-Seq reads for detecting global differential splicing during mouse embryonic stem cell differentiation, applying several bioinformatics strategies: gene-based, isoform-based and exon-based. We detected the strongest similarity among the sequencing platforms at the gene level compared to exon-based and isoform-based. Furthermore, the exon-based strategy discovered many differential exon usage (DEU) events, mostly in a platform-dependent manner and in non-differentially expressed genes. Thus, the platforms complemented each other in the ability to detect DEUs (i.e. long-reads exhibited an advantage in detecting DEUs at the UTRs, and short-reads detected more DEUs). Exons within 20 genes, detected in one or more platforms, were here validated by PCR, including key differentiation genes, such as Mdb3 and Aplp1. We provide an important analysis resource for discovering transcriptome changes during stem cell differentiation and insights for analysing such data.

FUS-P525L Juvenile Amyotrophic Lateral Sclerosis and Intellectual Disability: Evidence for Association and Oligogenic Inheritance.

Background and Objectives: Amyotrophic lateral sclerosis (ALS) is characterized by upper and lower motor neuron degeneration, with juvenile ALS (jALS) defined as disease with age at onset (AAO) before 25 years. We aimed to identify the genetic basis of 2 unrelated patients with jALS with very rapid deterioration and early age intellectual disability (ID) and to assess association of genetic findings with both phenotypes in a large cohort of patients with ALS and controls, and in the literature. Methods: Exome sequencing was performed in 2 unrelated probands and their parents. Trio analyses included de novo, rare homozygosity, and compound heterozygosity analyses. A TaqMan genotyping assay was used to genotype ALS cohorts. A systematic literature review was conducted and additional information from authors obtained to assess prevalence of fused in sarcoma (FUS)-ALS associated with ID. Results: A de novo mutation FUS-P525L was identified in both patients. Additional variations were identified in other genes related to intellectual disabilities. Among 8 additional unrelated juvenile patients, one carried the same FUS mutation and had a similar medical history of mild ID and fulminant ALS, whereas the others did not carry any FUS coding mutations and had no reported learning or intellectual disabilities (p = 0.0083). In addition, 486 patients with ALS with AAO ≥25 years were negative for this mutation. An extensive literature review showed that among all patients with FUS-related ALS with full phenotype reports, 10.3% exhibited additional learning/intellectual disabilities. Discussion: FUS-P525L mutation was identified in 3 among 10 patients with jALS (30%) in our clinical cohort, all with a very aggressive disease course and ID. Together with literature reports, these results support a novel association between mutations in FUS and early life ID. Additional variations identified in genes related to ID and brain development in our patients (GPT2, DNAH10, and SCUBE2) may suggest a complex oligogenic inheritance for this phenotype. We propose that this mutation should be screened in patients with ALS with very early AAO, aggressive disease course, and sporadic occurrence, especially when ALS is accompanied by ID.

DestVI identifies continuums of cell types in spatial transcriptomics data.

Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

A novel mutation in TARDBP segregates with amyotrophic lateral sclerosis in a large family with early onset and fast progression.

Objective: To identify the genetic background of ALS segregating in a large Bedouin family in Israel. Methods: Exome sequencing was carried out on three siblings in a family segregating ALS, two affected and one without neurological symptoms. Filtering for causative variants and for modifiers was carried out. Eight variants were confirmed by Sanger sequencing and genotyped on nine available members of the family (three affected and six unaffected). Results: We report the identification of a novel mutation in TARDBP, p.Ala321Asp, segregating in the family. The patients are affected with early onset (average age 34.5, 21-43 years old) and fast progressive disease. The mutation is in exon 6, in the glycin-rich domain, and is predicted to be deleterious. Additional rare, potentially deleterious variants were observed in the three patients, only one of them, PLEKHG5-Phe538Leu, which is located 4.5 Mb upstream to the TARDBP, was also fully segregating in the family. Conclusion: We identified a novel mutation in TARDBP which segregates with the disease in a large family. Additional rare variants were identified, and the combination of next-generation-sequencing together with linkage analysis was optimal to identify causality and modification, emphasizing the importance of combining the two analyses. Burden of deleterious variants may be associated with early age at onset.

Rare homozygosity in amyotrophic lateral sclerosis suggests the contribution of recessive variants to disease genetics.

OBJECTIVE: to determine the occurrence of homozygous rare, in-silico damaging variants in a genetically relatively homogenous group of amyotrophic lateral sclerosis (ALS) patients. METHODS: Whole-exome-sequencing of 43 ALS patients of North-Africa Jewish origin was performed. Data were filtered to identify very rare homozygous recessive in-silico damaging variants, in genes annotated to ALS-associated cellular pathways. RESULTS: We identified a rare missense homozygous variant, p.Arg663Cys in MFN2, predicted to be damaging, in a patient with an early age at disease onset (36 years) and fast progression. An additional ALS patient carried the mutation and together established its association to ALS (p = .01). Additional homozygous variants were identified, including the risk allele p.Arg261His in NEK1, as well as variants in genes known to be associated with other neurodegenerative diseases, such as HTT (Huntington's disease), ATM (Ataxia-Telangiectasia), and ZFYVE26 (SPG15), and variants in genes previously reported as upregulated (LZTS3) or downregulated (ARMC4, CFAP54, and MTHFSD) in ALS patients. Altogether, 13 patients (30%) carried at least one homozygous rare in-silico damaging variant, of them 10 carried either another rare homozygous variant and/or a variant in a known ALS gene, which is categorized as pathogenic, likely-pathogenic or variant of uncertain significance. CONCLUSIONS: Our results suggest the contribution of recessive alleles to ALS and the possibility of burden of mutations, emphasizing the complexity of ALS genetics.

Differential brain transcriptome of beta4 nAChR subunit-deficient mice: is it the effect of the null mutation or the background strain?

Studies using mice with beta4 nicotinic acetylcholine receptor (nAChR) subunit deficiency (beta4-/- mice) helped reveal the roles of this subunit in bradycardiac response to vagal stimulation, nicotine-induced seizure activity and anxiety. To identify genes that might be related to beta4-containing nAChRs activity, we compared the mRNA expression profiles of brains from beta4-/- and wild-type mice using Affymetrix U74Av2 microarray. Seventy-seven genes significantly differentiated between these two experimental groups. Of them, the two most downregulated were spastic paraplegia 21 (human) homolog (Spg21) and 6-pyruvoyl-tetrahydropterin synthase (Pts) genes. Since the targeted mutagenesis of the beta4 nAChR subunit was done by using two mouse strains, 129SvEv and C57BL/6J, it is possible that the genes closely linked to the mutated beta4 gene represent the 129SvEv allele and not the control C57BL/6J-driven allele. We examined this possibility by using public database and quantitative RT-PCR. The expression levels of Spg21 and Pts genes that, like the beta4 gene, are localized on mouse chromosome 9, as well as the expression levels of other genes located on this chromosome, were dependent on the mouse background strain. The 67 differentially expressed genes that are not located on chromosome 9 were further analyzed for overrepresented functional annotations and transcription regulatory elements compared with the entire microarray. Genes encoding for proteins involved in tyrosine phosphatase activity, calcium ion binding, cell growth and/or maintenance, and chromosome organization were overrepresented. Our data enhance the understanding of the molecular interactions involved in the beta4 nAChR subunit function. They also emphasize the need for careful interpretation of expression microarray studies done on genetically manipulated animals.

Expression changes in mouse brains following nicotine-induced seizures: the modulation of transcription factor networks.

Nicotine, acting through the neuronal nicotinic acetylcholine receptors (nAChRs), can induce seizures in mice. We aimed to study brain transcriptional response to seizure and to identify genes whose expression is altered after nicotine-induced seizures. Whole brains of untreated mice were compared with brains 1 h after seizure activity, using Affymetrix U74Av2 microarrays. Experimental groups included wild-type mice and both nicotine-induced seizure-sensitive and -resistant nAChR mutant mice. Each genotype group received different nicotine doses to generate seizures. This approach allowed the identification of significantly changed genes whose expression was dependent on seizure activity, nicotine administration, or both but not on the type of nAChR subunit mutation or the amount of nicotine injected. Significant expression changes were detected in 62 genes (P < 0.05, false discovery rate correction). Among them, gene ontology functional annotation analysis determined that the most significantly overrepresented categories were of genes encoding MAP kinase phosphatases, regulators of transcription and nucleosome assembly proteins. In silico bioinformatic analysis of the promoter regions of the 62 changed genes detected significant enrichments of 16 transcription regulatory elements (TREs), creating a network of transcriptional regulatory responses to seizures. The TREs for activating transcription factor and serum response factor were most significantly enriched, supporting their association with seizure activity. Our data suggest that nicotine-induced seizure in mice is a useful model to study seizure activity and its global brain transcriptional response. The differentially expressed genes detected here can help us to understand the molecular mechanisms underlying seizures in animal models and may also serve as candidate genes to study epilepsy in humans.

Increased severity of experimental colitis in alpha 5 nicotinic acetylcholine receptor subunit-deficient mice.

Substantial evidence suggests a negative association between cigarette smoking and the incidence and severity of ulcerative colitis, a common human inflammatory bowel disease. Nicotine has been implicated in this association. The detection of nicotinic acetylcholine receptors in colonic epithelium, the primary tissue affected in ulcerative colitis, suggests a role for these receptors in the beneficial effect of nicotine on colonic inflammation. Using an animal model, we demonstrate for the first time that alpha5 nicotinic acetylcholine receptor knockout mice have significantly more severe experimental colitis than wild-type controls and that nicotine significantly ameliorates its course when compared with wild-type controls. These findings suggest that alpha5-containing nicotinic acetylcholine receptors participate in the modulation of colitis in mice, but other nicotinic acetylcholine receptor subunits also mediate the antiinflammatory effects of nicotine.

Lower core body temperature and attenuated nicotine-induced hypothermic response in mice lacking the beta4 neuronal nicotinic acetylcholine receptor subunit.

Diverse physiological and pathological effects of nicotine, including the alteration of body temperature, are presumably mediated by neuronal nicotinic acetylcholine receptors (nAChR). Previous studies have suggested the involvement of distinct nAChR subunits in nicotine-induced thermoregulation. We studied genetically manipulated knockout mice lacking the alpha7, alpha5 or beta4 subunit genes, in order to assess the effects of subunit deficiency on temperature regulation. Using a telemetry system, core body temperature was monitored continuously prior to and following nicotine administration in mutant mice and in wild-type littermates. Mice lacking in the beta4 nAChR subunit gene had significantly lower baseline core body temperature than all other mouse strains studied. beta4 null mice also demonstrated a reduced nicotine-induced hypothermic response and impaired desensitization following repeat nicotine exposure. These findings suggest the involvement of the beta4 nAChR subunit in both core body temperature homeostasis and nicotine-elicited thermo-alterations in mice.

MicroRNAs and Growth Factors: An Alliance Propelling Tumor Progression.

Tumor progression requires cancer cell proliferation, migration, invasion, and attraction of blood and lymph vessels. These processes are tightly regulated by growth factors and their intracellular signaling pathways, which culminate in transcriptional programs. Hence, oncogenic mutations often capture growth factor signaling, and drugs able to intercept the underlying biochemical routes might retard cancer spread. Along with messenger RNAs, microRNAs play regulatory roles in growth factor signaling and in tumor progression. Because growth factors regulate abundance of certain microRNAs and the latter modulate the abundance of proteins necessary for growth factor signaling, the two classes of molecules form a dense web of interactions, which are dominated by a few recurring modules. We review specific examples of the alliance formed by growth factors and microRNAs and refer primarily to the epidermal growth factor (EGF) pathway. Clinical applications of the crosstalk between microRNAs and growth factors are described, including relevance to cancer therapy and to emergence of resistance to specific drugs.