A program of successive gene expression in mouse one-cell embryos.

At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer.

Human embryonic genome activation initiates at the one-cell stage.

In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer.

Murine neuronatin deficiency is associated with a hypervariable food intake and bimodal obesity.

Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.

Innate Lymphoid Cells Promote Recovery of Ventricular Function After Myocardial Infarction.

BACKGROUND: Innate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. OBJECTIVES: ILC2s also reside in the pericardium but their role in postischemic injury is unknown. METHODS: We examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). RESULTS: We found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell-deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. CONCLUSIONS: ILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI.

SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation.

Macrophages exhibit a spectrum of activation states ranging from classical to alternative activation1. Alternatively, activated macrophages are involved in diverse pathophysiological processes such as confining tissue parasites2, improving insulin sensitivity3 or promoting an immune-tolerant microenvironment that facilitates tumour growth and metastasis4. Recently, the metabolic regulation of macrophage function has come into focus as both the classical and alternative activation programmes require specific regulated metabolic reprogramming5. While most of the studies regarding immunometabolism have focussed on the catabolic pathways activated to provide energy, little is known about the anabolic pathways mediating macrophage alternative activation. In this study, we show that the anabolic transcription factor sterol regulatory element binding protein 1 (SREBP1) is activated in response to the canonical T helper 2 cell cytokine interleukin-4 to trigger the de novo lipogenesis (DNL) programme, as a necessary step for macrophage alternative activation. Mechanistically, DNL consumes NADPH, partitioning it away from cellular antioxidant defences and raising reactive oxygen species levels. Reactive oxygen species serves as a second messenger, signalling sufficient DNL, and promoting macrophage alternative activation. The pathophysiological relevance of this mechanism is validated by showing that SREBP1/DNL is essential for macrophage alternative activation in vivo in a helminth infection model.

Combination of Ginsenosides Rb2 and Rg3 Promotes Angiogenic Phenotype of Human Endothelial Cells via PI3K/Akt and MAPK/ERK Pathways.

Shexiang Baoxin Pill (SBP) is an oral formulation of Chinese materia medica for the treatment of angina pectoris. It displays pleiotropic roles in protecting the cardiovascular system. However, the mode of action of SBP in promoting angiogenesis, and in particular the synergy between its constituents is currently not fully understood. The combination of ginsenosides Rb2 and Rg3 were studied in human umbilical vein endothelial cells (HUVECs) for their proangiogenic effects. To understand the mode of action of the combination in more mechanistic detail, RNA-Seq analysis was conducted, and differentially expressed genes (DEGs), pathway analysis and Weighted Gene Correlation Network Analysis (WGCNA) were applied to further identify important genes that a play pivotal role in the combination treatment. The effects of pathway-specific inhibitors were observed to provide further support for the hypothesized mode of action of the combination. Ginsenosides Rb2 and Rg3 synergistically promoted HUVEC proliferation and tube formation under defined culture conditions. Also, the combination of Rb2/Rg3 rescued cells from homocysteine-induced damage. mRNA expression of CXCL8, CYR61, FGF16 and FGFRL1 was significantly elevated by the Rb2/Rg3 treatment, and representative signaling pathways induced by these genes were found. The increase of protein levels of phosphorylated-Akt and ERK42/44 by the Rb2/Rg3 combination supports the notion that it promotes endothelial cell proliferation via the PI3K/Akt and MAPK/ERK signaling pathways. The present study provides the hypothesis that SBP, via ginsenosides Rb2 and Rg3, involves the CXCR1/2 CXCL8 (IL8)-mediated PI3K/Akt and MAPK/ERK signaling pathways in achieving its proangiogenic effects.

Phenotypic characterization of Adig null mice suggests roles for adipogenin in the regulation of fat mass accrual and leptin secretion.

Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig-/-) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig-/- mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion.

Impaired Autophagy in CD11b+ Dendritic Cells Expands CD4+ Regulatory T Cells and Limits Atherosclerosis in Mice.

RATIONALE: Atherosclerosis is a chronic inflammatory disease. Recent studies have shown that dysfunctional autophagy in endothelial cells, smooth muscle cells, and macrophages, plays a detrimental role during atherogenesis, leading to the suggestion that autophagy-stimulating approaches may provide benefit. OBJECTIVE: Dendritic cells (DCs) are at the crossroad of innate and adaptive immune responses and profoundly modulate the development of atherosclerosis. Intriguingly, the role of autophagy in DC function during atherosclerosis and how the autophagy process would impact disease development has not been addressed. METHODS AND RESULTS: Here, we show that the autophagic flux in atherosclerosis-susceptible Ldlr-/- (low-density lipoprotein receptor-deficient) mice is substantially higher in splenic and aortic DCs compared with macrophages and is further activated under hypercholesterolemic conditions. RNA sequencing and functional studies on selective cell populations reveal that disruption of autophagy through deletion of Atg16l1 differentially affects the biology and functions of DC subsets in Ldlr-/- mice under high-fat diet. Atg16l1 deficient CD11b+ DCs develop a TGF (transforming growth factor)-ß-dependent tolerogenic phenotype and promote the expansion of regulatory T cells, whereas no such effects are seen with Atg16l1 deficient CD8α+ DCs. Atg16l1 deletion in DCs (all CD11c-expressing cells) expands aortic regulatory T cells in vivo, limits the accumulation of T helper cells type 1, and reduces the development of atherosclerosis in Ldlr-/- mice. In contrast, no such effects are seen when Atg16l1 is deleted selectively in conventional CD8α+ DCs and CD103+ DCs. Total T-cell or selective regulatory T-cell depletion abrogates the atheroprotective effect of Atg16l1 deficient DCs. CONCLUSIONS: In contrast to its proatherogenic role in macrophages, autophagy disruption in DCs induces a counter-regulatory response that maintains immune homeostasis in Ldlr-/- mice under high-fat diet and limits atherogenesis. Selective modulation of autophagy in DCs could constitute an interesting therapeutic target in atherosclerosis.

Comparison of Human and Murine Enteroendocrine Cells by Transcriptomic and Peptidomic Profiling.

Enteroendocrine cells (EECs) produce hormones such as glucagon-like peptide 1 and peptide YY that regulate food absorption, insulin secretion, and appetite. Based on the success of glucagon-like peptide 1-based therapies for type 2 diabetes and obesity, EECs are themselves the focus of drug discovery programs to enhance gut hormone secretion. The aim of this study was to identify the transcriptome and peptidome of human EECs and to provide a cross-species comparison between humans and mice. By RNA sequencing of human EECs purified by flow cytometry after cell fixation and staining, we present a first transcriptomic analysis of human EEC populations and demonstrate a strong correlation with murine counterparts. RNA sequencing was deep enough to enable identification of low-abundance transcripts such as G-protein-coupled receptors and ion channels, revealing expression in human EECs of G-protein-coupled receptors previously found to play roles in postprandial nutrient detection. With liquid chromatography-tandem mass spectrometry, we profiled the gradients of peptide hormones along the human and mouse gut, including their sequences and posttranslational modifications. The transcriptomic and peptidomic profiles of human and mouse EECs and cross-species comparison will be valuable tools for drug discovery programs and for understanding human metabolism and the endocrine impacts of bariatric surgery.