RESUMO
The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.
Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/metabolismo , Análise de Célula Única/métodos , Animais , Linhagem da Célula/genética , Separação Celular/métodos , Células Cultivadas , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Transplante HeterólogoRESUMO
Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.
Assuntos
Ratos/classificação , Ratos/genética , Animais , Modelos Animais de Doenças , Genoma , Fenótipo , Filogenia , Polimorfismo de Nucleotídeo Único , Ratos EndogâmicosRESUMO
OBJECTIVES: To define the host mechanisms contributing to the pathological interferon (IFN) type 1 signature in Juvenile dermatomyositis (JDM). METHODS: RNA-sequencing was performed on CD4+, CD8+, CD14+ and CD19+ cells sorted from pretreatment and on-treatment JDM (pretreatment n=10, on-treatment n=11) and age/sex-matched child healthy-control (CHC n=4) peripheral blood mononuclear cell (PBMC). Mitochondrial morphology and superoxide were assessed by fluorescence microscopy, cellular metabolism by 13C glucose uptake assays, and oxidised mitochondrial DNA (oxmtDNA) content by dot-blot. Healthy-control PBMC and JDM pretreatment PBMC were cultured with IFN-α, oxmtDNA, cGAS-inhibitor, TLR-9 antagonist and/or n-acetyl cysteine (NAC). IFN-stimulated gene (ISGs) expression was measured by qPCR. Total numbers of patient and controls for functional experiments, JDM n=82, total CHC n=35. RESULTS: Dysregulated mitochondrial-associated gene expression correlated with increased ISG expression in JDM CD14+ monocytes. Altered mitochondrial-associated gene expression was paralleled by altered mitochondrial biology, including 'megamitochondria', cellular metabolism and a decrease in gene expression of superoxide dismutase (SOD)1. This was associated with enhanced production of oxidised mitochondrial (oxmt)DNA. OxmtDNA induced ISG expression in healthy PBMC, which was blocked by targeting oxidative stress and intracellular nucleic acid sensing pathways. Complementary experiments showed that, under in vitro experimental conditions, targeting these pathways via the antioxidant drug NAC, TLR9 antagonist and to a lesser extent cGAS-inhibitor, suppressed ISG expression in pretreatment JDM PBMC. CONCLUSIONS: These results describe a novel pathway where altered mitochondrial biology in JDM CD14+ monocytes lead to oxmtDNA production and stimulates ISG expression. Targeting this pathway has therapeutical potential in JDM and other IFN type 1-driven autoimmune diseases.
Assuntos
Dermatomiosite , Interferon Tipo I , Criança , Humanos , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , DNA Mitocondrial , Interferon Tipo I/metabolismo , NucleotidiltransferasesRESUMO
The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.
Assuntos
Síndrome de Down , Reação Leucemoide , Animais , Diferenciação Celular , Fator de Transcrição GATA1/genética , Humanos , Recém-Nascido , Megacariócitos , CamundongosRESUMO
Schwann cell c-Jun is implicated in adaptive and maladaptive functions in peripheral nerves. In injured nerves, this transcription factor promotes the repair Schwann cell phenotype and regeneration and promotes Schwann-cell-mediated neurotrophic support in models of peripheral neuropathies. However, c-Jun is associated with tumor formation in some systems, potentially suppresses myelin genes, and has been implicated in demyelinating neuropathies. To clarify these issues and to determine how c-Jun levels determine its function, we have generated c-Jun OE/+ and c-Jun OE/OE mice with graded expression of c-Jun in Schwann cells and examined these lines during development, in adulthood, and after injury using RNA sequencing analysis, quantitative electron microscopic morphometry, Western blotting, and functional tests. Schwann cells are remarkably tolerant of elevated c-Jun because the nerves of c-Jun OE/+ mice, in which c-Jun is elevated â¼6-fold, are normal with the exception of modestly reduced myelin thickness. The stronger elevation of c-Jun in c-Jun OE/OE mice is, however, sufficient to induce significant hypomyelination pathology, implicating c-Jun as a potential player in demyelinating neuropathies. The tumor suppressor P19ARF is strongly activated in the nerves of these mice and, even in aged c-Jun OE/OE mice, there is no evidence of tumors. This is consistent with the fact that tumors do not form in injured nerves, although they contain proliferating Schwann cells with strikingly elevated c-Jun. Furthermore, in crushed nerves of c-Jun OE/+ mice, where c-Jun levels are overexpressed sufficiently to accelerate axonal regeneration, myelination and function are restored after injury.SIGNIFICANCE STATEMENT In injured and diseased nerves, the transcription factor c-Jun in Schwann cells is elevated and variously implicated in controlling beneficial or adverse functions, including trophic Schwann cell support for neurons, promotion of regeneration, tumorigenesis, and suppression of myelination. To analyze the functions of c-Jun, we have used transgenic mice with graded elevation of Schwann cell c-Jun. We show that high c-Jun elevation is a potential pathogenic mechanism because it inhibits myelination. Conversely, we did not find a link between c-Jun elevation and tumorigenesis. Modest c-Jun elevation, which is beneficial for regeneration, is well tolerated during Schwann cell development and in the adult and is compatible with restoration of myelination and nerve function after injury.
Assuntos
Dosagem de Genes , Bainha de Mielina/fisiologia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Células de Schwann/metabolismo , Animais , Axônios/patologia , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Bainha de Mielina/ultraestrutura , Compressão Nervosa , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/biossíntese , Recuperação de Função Fisiológica , Nervo Isquiático/lesões , Nervo Isquiático/patologiaRESUMO
FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. We have employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. We generated mathematical models to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. We tested Sprouty genes as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. We propose a model in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Mesencéfalo/embriologia , Células-Tronco Neurais/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Movimento Celular , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/crescimento & desenvolvimento , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS). Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10%) of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.
Assuntos
Cromatina/genética , Desoxirribonuclease I/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Desoxirribonuclease I/metabolismo , Camundongos , FenótipoRESUMO
Arterial and venous endothelial cells exhibit distinct molecular characteristics at early developmental stages. These lineage-specific molecular programs are instructive to the development of distinct vascular architectures and physiological conditions of arteries and veins, but their roles in angiogenesis remain unexplored. Here, we show that the caudal vein plexus in zebrafish forms by endothelial cell sprouting, migration and anastomosis, providing a venous-specific angiogenesis model. Using this model, we have identified a novel compound, aplexone, which effectively suppresses venous, but not arterial, angiogenesis. Multiple lines of evidence indicate that aplexone differentially regulates arteriovenous angiogenesis by targeting the HMG-CoA reductase (HMGCR) pathway. Treatment with aplexone affects the transcription of enzymes in the HMGCR pathway and reduces cellular cholesterol levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for protein prenylation in zebrafish and human endothelial cells.
Assuntos
Artérias/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Sulfonamidas/farmacologia , Veias/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Animais , Animais Geneticamente Modificados , Artérias/fisiologia , Células Cultivadas , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Embrião não Mamífero , Humanos , Terapia de Alvo Molecular , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Especificidade por Substrato/efeitos dos fármacos , Veias/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologiaRESUMO
Variations in serum amino acid levels are linked to a multitude of complex disorders. We report the largest genome-wide association study (GWAS) on nine serum amino acids in the UK Biobank participants (117 944, European descent). We identified 34 genomic loci for circulatory levels of alanine, 48 loci for glutamine, 44 loci for glycine, 16 loci for histidine, 11 loci for isoleucine, 19 loci for leucine, 9 loci for phenylalanine, 32 loci for tyrosine and 20 loci for valine. Our gene-based analysis mapped 46-293 genes associated with serum amino acids, including MIP, GLS2, SLC gene family, GCKR, LMO1, CPS1 and COBLL1.The gene-property analysis across 30 tissues highlighted enriched expression of the identified genes in liver tissues for all studied amino acids, except for isoleucine and valine, in muscle tissues for serum alanine and glycine, in adrenal gland tissues for serum isoleucine and leucine, and in pancreatic tissues for serum phenylalanine. Mendelian randomization (MR) phenome-wide association study analysis and subsequent two-sample MR analysis provided evidence that every standard deviation increase in valine is associated with 35% higher risk of type 2 diabetes and elevated levels of serum alanine and branched-chain amino acids with higher levels of total cholesterol, triglyceride and low-density lipoprotein, and lower levels of high-density lipoprotein. In contrast to reports by observational studies, MR analysis did not support a causal association between studied amino acids and coronary artery disease, Alzheimer's disease, breast cancer or prostate cancer. In conclusion, we explored the genetic architecture of serum amino acids and provided evidence supporting a causal role of amino acids in cardiometabolic health.
RESUMO
The two types of craniopharyngioma, adamantinomatous (ACP) and papillary (PCP), are clinically relevant tumours in children and adults. Although the biology of primary craniopharyngioma is starting to be unravelled, little is known about the biology of recurrence. To fill this gap in knowledge, we have analysed through methylation array, RNA sequencing and pERK1/2 immunohistochemistry a cohort of paired primary and recurrent samples (32 samples from 14 cases of ACP and 4 cases of PCP). We show the presence of copy number alterations and clonal evolution across recurrence in 6 cases of ACP, and analysis of additional whole genome sequencing data from the Children's Brain Tumour Network confirms chromosomal arm copy number changes in at least 7/67 ACP cases. The activation of the MAPK/ERK pathway, a feature previously shown in primary ACP, is observed in all but one recurrent cases of ACP. The only ACP without MAPK activation is an aggressive case of recurrent malignant human craniopharyngioma harbouring a CTNNB1 mutation and loss of TP53. Providing support for a functional role of this TP53 mutation, we show that Trp53 loss in a murine model of ACP results in aggressive tumours and reduced mouse survival. Finally, we characterise the tumour immune infiltrate showing differences in the cellular composition and spatial distribution between ACP and PCP. Together, these analyses have revealed novel insights into recurrent craniopharyngioma and provided preclinical evidence supporting the evaluation of MAPK pathway inhibitors and immunomodulatory approaches in clinical trials in against recurrent ACP.
Assuntos
Evolução Clonal , Craniofaringioma , Sistema de Sinalização das MAP Quinases , Recidiva Local de Neoplasia , Neoplasias Hipofisárias , Proteína Supressora de Tumor p53 , Animais , Feminino , Humanos , Masculino , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Evolução Clonal/genética , Craniofaringioma/genética , Craniofaringioma/patologia , Craniofaringioma/metabolismo , Progressão da Doença , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Morphoea can have a significant disease burden. Aetiopathogenesis remains poorly understood, with very limited existing genetic studies. Linear morphoea (LM) may follow Blascho's lines of epidermal development, providing potential pathogenic clues. OBJECTIVE: The first objective of this study was to identify the presence of primary somatic epidermal mosaicism in LM. The second objective was tTo explore differential gene expression in morphoea epidermis and dermis to identify potential pathogenic molecular pathways and tissue layer cross-talk. METHODOLOGY: Skin biopsies from paired affected and contralateral unaffected skin were taken from 16 patients with LM. Epidermis and dermis were isolated using a 2-step chemical-physical separation protocol. Whole Genome Sequencing (WGS; n = 4 epidermal) and RNA-seq (n = 5-epidermal, n = 5-dermal) with gene expression analysis via GSEA-MSigDBv6.3 and PANTHER-v14.1 pathway analyses, were performed. RTqPCR and immunohistochemistry were used to replicate key results. RESULTS: Sixteen participants (93.8% female, mean age 27.7 yrs disease-onset) were included. Epidermal WGS identified no single affected gene or SNV. However, many potential disease-relevant pathogenic variants were present, including ADAMTSL1 and ADAMTS16. A highly proliferative, inflammatory and profibrotic epidermis was seen, with significantly-overexpressed TNFα-via-NFkB, TGFß, IL6/JAKSTAT and IFN-signaling, apoptosis, p53 and KRAS-responses. Upregulated IFI27 and downregulated LAMA4 potentially represent initiating epidermal 'damage' signals and enhanced epidermal-dermal communication. Morphoea dermis exhibited significant profibrotic, B-cell and IFN-signatures, and upregulated morphogenic patterning pathways such as Wnt. CONCLUSION: This study supports the absence of somatic epidermal mosaicism in LM, and identifies potential disease-driving epidermal mechanisms, epidermal-dermal interactions and disease-specific dermal differential-gene-expression in morphoea. We propose a potential molecular narrative for morphoea aetiopathogenesis which could help guide future targeted studies and therapies.
Assuntos
Esclerodermia Localizada , Humanos , Feminino , Adulto , Masculino , Pele/patologia , Epiderme/patologia , RNA-Seq , BiópsiaRESUMO
Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.
Assuntos
Síndrome de Bardet-Biedl , Humanos , Camundongos , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Camundongos Knockout , Proteínas/metabolismo , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
After nerve injury, myelin and Remak Schwann cells reprogram to repair cells specialized for regeneration. Normally providing strong regenerative support, these cells fail in aging animals, and during chronic denervation that results from slow axon growth. This impairs axonal regeneration and causes significant clinical problems. In mice, we find that repair cells express reduced c-Jun protein as regenerative support provided by these cells declines during aging and chronic denervation. In both cases, genetically restoring Schwann cell c-Jun levels restores regeneration to control levels. We identify potential gene candidates mediating this effect and implicate Shh in the control of Schwann cell c-Jun levels. This establishes that a common mechanism, reduced c-Jun in Schwann cells, regulates success and failure of nerve repair both during aging and chronic denervation. This provides a molecular framework for addressing important clinical problems, suggesting molecular pathways that can be targeted to promote repair in the PNS.
Assuntos
Envelhecimento , Regeneração Nervosa , Proteínas Proto-Oncogênicas c-jun/genética , Células de Schwann/metabolismo , Animais , Feminino , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet (smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.
Assuntos
Proliferação de Células , Extremidades/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Epiderme/crescimento & desenvolvimento , Epiderme/fisiologia , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica , Mesoderma/crescimento & desenvolvimento , Mesoderma/fisiologia , Miocárdio/metabolismo , Osteogênese/fisiologia , Regeneração , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
To define renal molecular mechanisms that are affected by permanent hyperglycaemia and might promote phenotypes relevant to diabetic nephropathy, we carried out linkage analysis of genome-wide gene transcription in the kidneys of F2 offspring from the Goto-Kakizaki (GK) rat model of type 2 diabetes and normoglycaemic Brown Norway (BN) rats. We mapped 2526 statistically significant expression quantitative trait loci (eQTLs) in the cross. More than 40% of eQTLs mapped in the close vicinity of the linked transcripts, underlying possible cis-regulatory mechanisms of gene expression. We identified eQTL hotspots on chromosomes 5 and 9 regulating the expression of 80-165 genes, sex or cross direction effects, and enriched metabolic and immunological processes by segregating GK alleles. Comparative analysis with adipose tissue eQTLs in the same cross showed that 496 eQTLs, in addition to the top enriched biological pathways, are conserved in the two tissues. Extensive similarities in eQTLs mapped in the GK rat and in the spontaneously hypertensive rat (SHR) suggest a common aetiology of disease phenotypes common to the two strains, including insulin resistance, which is a prominent pathophysiological feature in both GK rats and SHRs. Our data shed light on shared and tissue-specific molecular mechanisms that might underlie aetiological aspects of insulin resistance in the context of spontaneously occurring hyperglycaemia and hypertension.
Assuntos
Tecido Adiposo/metabolismo , Modelos Animais de Doenças , Resistência à Insulina/genética , Rim/metabolismo , Transcriptoma , Animais , Mapeamento Cromossômico , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Locos de Características Quantitativas , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos SHRRESUMO
Non-alcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. To disentangle etiological relationships between these conditions and identify genetically-determined metabolites involved in NAFLD processes, we mapped 1H nuclear magnetic resonance (NMR) metabolomic and disease-related phenotypes in a mouse F2 cross derived from strains showing resistance (BALB/c) and increased susceptibility (129S6) to these diseases. Quantitative trait locus (QTL) analysis based on single nucleotide polymorphism (SNP) genotypes identified diet responsive QTLs in F2 mice fed control or high fat diet (HFD). In HFD fed F2 mice we mapped on chromosome 18 a QTL regulating liver micro- and macrovesicular steatosis and inflammation, independently from glucose intolerance and adiposity, which was linked to chromosome 4. Linkage analysis of liver metabolomic profiling data identified a QTL for octopamine, which co-localised with the QTL for liver histopathology in the cross. Functional relationship between these two QTLs was validated in vivo in mice chronically treated with octopamine, which exhibited reduction in liver histopathology and metabolic benefits, underlining its role as a mechanistic biomarker of fatty liver with potential therapeutic applications.
Assuntos
Cromossomos de Mamíferos/genética , Metabolômica/métodos , Hepatopatia Gordurosa não Alcoólica/genética , Octopamina/administração & dosagem , Polimorfismo de Nucleotídeo Único , Animais , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos Endogâmicos BALB C , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Octopamina/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Locos de Características Quantitativas , Biologia de Sistemas , Resultado do TratamentoRESUMO
During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors. At the same time-restricted stage, absence of SCL results in expansion of cardiac/paraxial cell populations and increased cardiac/paraxial gene expression, suggesting active suppression of alternative fates. Indeed, SCL normally activates expression of co-repressor ETO2 and Polycomb-PRC1 subunits (RYBP, PCGF5) and maintains levels of Polycomb-associated histone marks (H2AK119ub/H3K27me3). Genome-wide analyses reveal ETO2 and RYBP co-occupy most SCL target genes, including cardiac/paraxial loci. Reduction of Eto2 or Rybp expression mimics Scl-null cardiac phenotype. Therefore, SCL-mediated transcriptional repression prevents mis-specification of blood-fated cells, establishing active repression as central to fate determination processes.
Assuntos
Linhagem da Célula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Separação Celular/métodos , Embrião de Mamíferos , Citometria de Fluxo/métodos , Código das Histonas/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Células-Tronco Embrionárias Murinas , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Fatores de Transcrição/genéticaRESUMO
Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of the CD40-CD40L pathway (known to induce B cell IL-10 and IL-6) revealed similar expression of IL-10 and IL-6 in B cells cultured with CD40L from both JDM patients and controls. In conclusion, JDM patients with active disease have a significantly expanded immature transitional B cell population which correlated with the type I IFN signature. Activation through TLR7 and IFNα may drive the expansion of immature transitional B cells in JDM and skew the cells toward a pro-inflammatory phenotype.
RESUMO
The zebrafish (Danio rerio) embryo toxicity test (DarT) is under consideration as an alternative to the acute fish toxicity test. Microscopically visible developmental disorders or death are the endpoints used to report on toxicity in DarT. These endpoints are easily observed. They, however, rarely reveal mechanisms leading to a toxic effect and are relatively insensitive compared to chronic toxic effects. We hypothesized that, by using gene expression profiles as an additional endpoint, it may be possible to increase the sensitivity and predictive value of DarT. Therefore, as a proof of principle, we exposed zebrafish embryos to the reference compound 3,4-dichloroaniline (3,4-DCA) and analyzed gene expression patterns with a 14k oligonucleotide array. Important stress response genes not included in the microarray were additionally quantified by reverse transcriptase polymerase chain reaction. Six genes involved in biotransformation (cyp1a, ahr2), stress response (nfe212, maft, hmox1) and cell cycle control (fzr1) were significantly regulated. With the exception of fzr1, these genes proved to be differentially expressed in post hatch life stages as well. The identified genes point toward an aryl hydrocarbon receptor-mediated response. Differential gene expression in embryos exposed for 48 h was observed at 3,4-DCA concentrations as low as 0.78 microM, which is more than 10-fold below the concentrations that elicited visible toxic effects. Upon exposure for 5 days, differential expression was detected at concentrations as low as 0.22 microM of 3,4-DCA, which was close to the lowest observed effect concentration (0.11 microM) in the 30-day early life stage test. This study therefore indicates that gene expression analysis in DarT is able to reveal mechanistic information and may also be exploited for the development of replacement methods for chronic fish tests.
Assuntos
Determinação de Ponto Final/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Testes de Toxicidade/métodos , Peixe-Zebra/metabolismo , Compostos de Anilina/toxicidade , Animais , Primers do DNA , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a zebrafish ncx1 mutant, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase murf1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down murf1 activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the murf1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced murf1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated murf1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.