Combined constitutive canonical and non-canonical HH signaling caused by depletion of Patched 2 causes an MPN phenotype and transforms chronic MPNs into acute leukemias
Ontology highlight
ABSTRACT: Murine BM niche cell comparison
INSTRUMENT(S): BD FACSAria III, Qiagen RNeasy micro Kit
Project description:Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application. RNA-Seq of 181 single-cell samples from 8 FACS sorted cell types: 1. endothelial cells (samples E11.0_EC_xxxx. CD31+ VE-cadherin+CD41-CD43-CD45-Ter119-); 2. T1 pre-HSCs (samples E11.0_T1_xxxx. CD31+CD45-CD41low c-Kit+CD201high); 3. T1 CD201- cells (samples E11.0_T1CD201neg_xxxx, CD31+CD45-CD41low c-Kit+CD201low/-) ; 4. T2 pre-HSCs (samples E11.0_T2_35xx. CD31+CD45+c-Kit+CD201high), 5. T2 CD41low (samples E11.0_T2_21xx, E11.0_T2_24xx and E11.0_T2_27xx. CD31+CD45+CD41low); 6. E12 HSCs (samples E12.5_FL_xxxx. Lin-Sca-1+Mac-1lowCD201+); 7. E14 HSCs (samples E14.5_FL_xxxx. CD45+CD150+CD48-CD201+); 8. Adult HSCs (samples Adult_HSC_xxxx. CD45+CD150+CD48-CD201+). ECs, T1 pre-HSCs, T1 CD201- cells, T2 pre-HSCs, T2 CD41low cells were sorted from E11 AGM region. Mature HSCs were sorted from E12 or E14 fetal liver and adult bonemarrow.
Project description:Hematopoietic stem cells (HSCs) in adult are specified early from the endothelium-derived precursors (e.g., hemogenic endothelium, and pre-HSCs) in mouse mid-gestation embryos, the detailed process, however, is still largely unknown due to their rareness, transience, and current inability to prospectively isolate them efficiently . Here we developed a potent set of surface markers that could capture the earliest emerging HSCs, the CD45- pre-HSCs with high accuracy and purity, as rigorously and functionally verified by single-cell-initiated serial transplantation assays. Then we applied single-cell RNA-Seq technique to analyze five populations related to HSC formation: the CD45- (type 1) and CD45+ (type 2) pre-HSCs as well as endothelial cells in the E11 AGM region; and later mature HSCs in the E12 and E14 fetal livers. Compared to other cell populations, both type 1 and type 2 pre-HSCs have their unique signatures of transcription machinery, transcription factor network, signaling pathway, cell cycle status, metabolism state, and lncRNA expression patterns. Our work paves the way for dissection of the complex molecular mechanisms regulating the step-wise formation of HSCs from endothelial cells, thus informing future efforts on engineering HSCs for clinical application. RNA-Seq of 35 10-cell pooled samples from 5 FACS sorted cell types, i.e., endothelial cells (ECs), T1 and T2 pre-HSCs from E11 AGM region, as well as mature HSCs from E12 and E14 fetal liver
Project description:The lymph node is home to resident macrophage populations that are essential for healthy immune function and homeostasis. They are involved in multiple processes including the initiation of the local response to pathogens, halting viral and bacterial spread, and clearance of apoptotic cells, but the macrophage niche and factors that create it are largely undefined. Here we analyse fibroblastic reticular cells (FRCs) as an essential component of the lymph node macrophage niche using single-cell RNA-sequencing. Our analysis revealed that most reticular cell subsets within lymph nodes expressed master macrophage regulator CSF1. We further show that signalling through CSF1R was sufficient to support macrophage development, while in the presence of LPS, FRCs underwent a mechanistic switch and maintained support through CSF1R-independent mechanisms. Our data reveal a critically important role for FRCs in the creation of the parenchymal macrophage niche within LNs.
Project description:Inflammation leads the hypothalamus-pituitary-adrenal (HPA) axis activation and increased production of glucocorticoids, which produced by the adrenal cortex, exert potent anti-inflammatory effects. The use of omics, including proteomics revealed metabolic changes in steroidogenic adrenocortical cells, including downregulation of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, decreased ATP production and induction of oxidative stress after lipopolysaccharide (LPS)-induced inflammation.
Project description:Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in non-classical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). While it is well-established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of lymph nodes and other classical secondary lymphoid organs, it has remained elusive whether and how FRCs in FALCs contribute to peritoneal immunity. Using FRC-specific gene targeting, we found that FALCs are underpinned by an elaborated FRC network and that initiation of peritoneal immunity was governed through FRC activation via MyD88-dependent innate immunological sensing. FRC-specific ablation of Myd88 expression blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation. Moreover, containment of Salmonella infection was compromised in conditionally Myd88-deficient mice indicating that FRCs in FALCs function as initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.
Project description:Purpose : Elucidate post-natal role of SCA1+ thymic mesenchymal cells (tMCs) and evaluate the functional overlap between thymic, bone and skin MCs. Method : By high speed cell sorting, we isolated primary MCs (Lin- SCA1+ cells) from mouse thymus, bone and skin. We extracted their respective total RNA and compared their transcriptome by high-throughput RNA-sequencing. Results : We found a total of 2036 differentially expressed genes (FC>5, p-adj<0.1 and RPKM>1) between the 3 MC populations. IPA analyses revealed that each MC population was enriched for genes associated to phagocyte chemotaxis. We also denoted 2850 genes with shared expression across MC populations. IPA analysis of those shared genes also revealed an enrichment for genes influencing phagocyte migration, chemotaxis and function. Finally, MC transcriptomes showed that all 3 MC populations were expressing genes associated with hematopoietic stem and progenitor cell (HSPC) support, strongly suggesting that MCs from thymic, bone and skin all possess the ability to support HSPCs. Conclusion : Overall, our study highlighted 3 potential novel roles for tMCs : 1) Promoting macrophage/monocyte chemotaxis, 2) Enhacing the apoptotic cell clearance process and 3) setting an inviting niche for hematopoietic progenitors. These novel biological roles for tMCs could have substantial effets on thymic biology. Finally, our RNA-seq data offer a valuable resource to the community that can be mined to explore multiple questions related mesenchymal cell biology. Transcriptome comparison between MC populations
Project description:We analyzed the total proteome of group 2 innate lymphoid cells (ILC2s) after different stimulation with interleukin-33 (IL-33), a cytokine playing a critical role in human asthma, and TL1A, a TNF-family cytokine also known to activate ILC2s. Upon combined stimulation with IL-33 plus TL1A, we show that lung ILC2s produce high amounts of IL-9 and acquire a transient ‘ILC9’ phenotype. This phenotype is characterized by simultaneous production of large amounts of type 2 cytokines (IL-5, IL-13 and IL-9), induction of the IL-2 receptor CD25 (Il2ra), and of the transcription factors IRF4, JunB and BATF, that form immune-specific complexes known to induce IL-9 expression.
Project description:We have used a chimeric VEGFR-2 in which the extracellular domain of mouse VEGFR-2 was replaced with the extracellular domain of human CSF-1 receptor. VEGFR-2 was immunoprecipitated with anti-VEGFR-2 antibody from PAE cells ectopically expressing VEGFR-2. The immunoprecipitated proteins were eluted and separated on SDS-PAGE, followed by in-gel chymotrypsin or trypsin digestion. The digested samples were analyzed by nano LC/MS/MS on a Thermo Fisher LTQ Orbitrap XL. The LC-MS/MS data were analyzed using Proteome Discoverer (Thermo Fisher Scientific; Version 1.3.0.339). MS/MS search was carried out using Sequest search algorithm against the sequence of target mouse protein from the UniProtKB database. Search parameters included chymotrypsin as the enzyme with four missed cleavage allowed; methylation at lysine and arginine, phosphorylation of serine, threonine, and tyrosine, alkylation at cysteine, and oxidation of methionine were set as dynamic modifications. Precursor and fragment mass tolerance were set to 5 ppm and 0.8 Da, respectively. The false discovery rate was calculated by enabling the peptide sequence analysis using a decoy database. High confidence peptide identifications were obtained by setting a target false discovery rate threshold of 1% at the peptide level.
Project description:Neutrophil homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor CXCR4. Herein, we show that the ELR chemokines CXCL1 and CXCL2 are constitutively expressed by bone marrow endothelial cells and osteoblasts, and CXCL2 expression is induced in endothelial cells during granulocyte colony-stimulating factor (G-CSF)-induced neutrophil mobilization. Neutrophils lacking CXCR2, the receptor for CXCL1 and CXCL2, are preferentially retained in the bone marrow, reproducing a myelokathexis phenotype. Transient disruption of CXCR4 failed to mobilize CXCR2 neutrophils. However, doubly deficient neutrophils (CXCR2-/- CXCR4-/-) displayed constitutive mobilization, showing that CXCR4 plays a dominant role. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow. We used gene expression microarrays to determine the changes in osteoblasts and bone marrow endothelial cells after G-CSF treatment. 3 untreated and G-CSF-treated osteoblast samples and 4 untreated and G-CSF-treated endothelial samples.