Regional specialization and fate specification of mesenchymal stromal cells in skeletal development [bulk RNA-seq]
Ontology highlight
ABSTRACT: To gain insight into the molecular signatures and heterogenity of bone mesenchymal stromal cells population between metaphysis (mpMSC) and diaphysis (dpMSC). mpMSCs and dpMSCs were sorted from three weeks old Hey1-GFP metaphysis and Pdgfra-EGFP BM using a flow cytometer for bulk-RNA sequencing analysis.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived femoral diaphysis and metaphysis transcriptome profiling (RNA-seq) to determine pathways and networks dependent on Dlx3 during bone development and homeostasis. Methods: mRNA profiles of diaphysis and metaphysis isolated from the femur of 5-week-old wild-type (WT) and Dlx3Oc-cKO (OC-cre;Dlx3f/-) conditional knockout mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level by ANOVA (ANOVA) and TopHat. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq data were generated with Illumina's HiSeq 2000 system. Raw sequencing data were processed with CASAVA 1.8.2 to generate fastq files. Reads of 50 bases were mapped to the mouse transcriptome and genome mm9 using TopHat 1.3.2. Gene expression values (RPKM) were calculated with Partek Genomics Suite 6.6, which was also used for the ANOVA analysis to determine significantly differentially expressed genes. Conclusions: Our study represents the first detailed analysis of Dlx3Oc-cKO diaphysis and metaphysis from femurs, with biologic triplicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Diaphysis and metaphysis mRNA profiles of metaphysis and diaphysis from femurs of 5-wk-old (WT) and Dlx3Oc-cKO male mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived femoral diaphysis and metaphysis transcriptome profiling (RNA-seq) to determine pathways and networks dependent on Dlx3 during bone development and homeostasis. Methods: mRNA profiles of diaphysis and metaphysis isolated from the femur of 5-week-old wild-type (WT) and Dlx3Oc-cKO (OC-cre;Dlx3f/-) conditional knockout mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level by ANOVA (ANOVA) and TopHat. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq data were generated with Illumina's HiSeq 2000 system. Raw sequencing data were processed with CASAVA 1.8.2 to generate fastq files. Reads of 50 bases were mapped to the mouse transcriptome and genome mm9 using TopHat 1.3.2. Gene expression values (RPKM) were calculated with Partek Genomics Suite 6.6, which was also used for the ANOVA analysis to determine significantly differentially expressed genes. Conclusions: Our study represents the first detailed analysis of Dlx3Oc-cKO diaphysis and metaphysis from femurs, with biologic triplicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
Project description:Bone stroma contributes to the regulation of osteogenesis and hematopoiesis but also to fracture healing and disease processes. Mesenchymal stromal cells from bone (BMSCs) represent a heterogenous mixture of different subpopulations with distinct molecular and functional properties. The lineage relationship between BMSC subsets and their regulation by intrinsic and extrinsic factors are not well understood. Here, we show with mouse genetics, ex vivo cell differentiation assays, and transcriptional profiling that BMSCs from metaphysis (mpMSCs) and diaphysis (dpMSCs) are fundamentally distinct. Fate-tracking experiments and single-cell RNA sequencing indicate that bone-forming osteoblast lineage cells and dpMSCs, including leptin receptor-positive (LepR+) reticular cells in bone marrow, emerge from mpMSCs in the postnatal metaphysis. Finally, we show that BMSC fate is controlled by platelet-derived growth factor receptor β (PDGFRβ) signaling and the transcription factor Jun-B. The sum of our findings improves our understanding of BMSC development, lineage relationships, and differentiation.
Project description:To identify cellular heterogeneity and molecular signatures of bone marrow stromal (BMSCs) cells, we performed scRNA-seq of non-hematopoietic stromal cells from 3-week-old mice. In addition, to gain further insight into MSC differentiation in vivo, Pdgfrb-CreERT2 Rosa26-mTmG (Pdgfrb-Cre R26-mT/mG) double transgenic mice were treated with tamoxifen at P1-3 and sacrificed at P21 for the isolation of GFP+ cells from long bone followed by scRNA-seq analysis.
Project description:During immune-mediated severe aplastic anemia (SAA) monocytes (CD11b+ Ly6C+ Ly6G-) signficantly increase by both frequency and number within the bone marrow. We isolated BM monocytes from F1 hybrid mice (C57BL/6;Balb/c) induced with SAA via the splenocyte-transfer model. We utilized single-cell RNA sequencing (scRNA-seq) to analyze the heterogenity of BM monocytes during SAA.
Project description:Extensive neovascularization is a hallmark of glioblastoma (GBM). In addition to supplying oxygen and nutrients, vascular endothelial cells provide trophic support to GBM cells via paracrine signaling. Here we report that Endocan (ESM1), an endothelial-secreted proteoglycan, confers enhanced proliferative, migratory, and angiogenic properties to GBM cells and regulates their spatial identity. Mechanistically, Endocan exerts at least part of its functions via direct binding and activation of the PDGFRA receptor. Subsequent downstream signaling enhances chromatin accessibility of the Myc promoter and upregulates Myc expression inducing stable phenotypic changes in GBM cells. Furthermore, Endocan confers a radioprotection phenotype in GBM cells, both in vitro and in vivo. Inhibition of Endocan-PDGFRA signaling with ponatinib increases survival in the Esm1 wild-type but not in the Esm1 knock-out mouse GBM model. Our findings identify Endocan and its downstream signaling axis as a potential target to subdue GBM recurrence and highlight the importance of vascular-tumor interactions for GBM development.
Project description:Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate β-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather β-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.