Project description:Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-sequencing (scRNA-seq) technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, the novel identified fibroblast populations remain ill-defined, both in respect to the molecular cues driving their differentiation, and their subsequent role in fibrosis. Here we identify the metalloprotease ADAMTS12 in an unbiased manner as a fibroblast-specific gene that is strongly upregulated during fibrogenesis in mice and humans. In vivo knockout studies in mice confirmed that Adamts12 is critical during fibrogenesis in heart and kidney. Leveraging spatial transcriptomics, CRISPR-Cas9 gene editing and expression of catalytically active or inactive ADAMTS12 we demonstrate that the protease domain of Adamts12 controls fibrogenesis by licensing expansion and migration of a distinct fibroblast subset defined by Mt1 gene expression and strong JAK-STAT signaling.
Project description:Mouse embryonic fibroblast (MEFs) cell lines and liver samples from Dnmt2 and Nsun2 single mutant, double mutant and wildtype mice were used to identify potential mRNA substrates of both proteins.
Project description:Fibroblast-like synoviocytes (FLS) were isolated from the hind paws of Col6a1-cre Bmal1-flox PER2::LUC mice for single cell sequencing to determine the effect of Bmal1 knockout on FLS cell function. FLS cells were isolated from joint digests by selection for Podoplanin expression, then fixed and barcoded for single cell transcriptomic analysis using a split-pool ligation-based transcriptomic sequencing (SPLiT-seq) approach.
Project description:To better understand the role of the COX-2-dependent lung fibroblast program in modulating the lung immune microenvironment, we generated fibroblast-targeted Ptgs2 conditional knockout (cKO) mice by crossing Pdgfra-Cre mice with Ptgs2flox/flox mice. Then we determined how Ptgs2 deficiency in CD140a+ fibroblasts alters the lung immune microenvironment by performing single-cell RNA sequencing on lung CD45+ immune cells from WT and Ptgs2-cKO mice.
Project description:We conducted fibroblast-specific transcriptome analysis by next generation sequencing in order to investigate qualitative change and activation signatures of lung fibroblasts in bleomycin-induced pulmonary fibrosis. Lung fibroblasts were identified by using reporter mice of collagen-α2(I), in which collagen I-producing fibroblasts were labeled with EGFP. Lungs were dissociated with protease sollution, and single cell suspension were stained with lineage markers (Ter119, CD45, CD31, EpCAM). Lineage- GFP+ cells were sorted out and mRNA was collected. Using serial analysis of gene expression (SAGE) method, we identified 2,973,937 SAGE tags (1,080,798 tags from saline-treated GFP+ fibroblasts and 1,893,139 tags from bleomycin-treated GFP+ fibroblasts). We found that genes related to extracellular matrix construction were highly up-regulated in fibroblasts from belomycin-treated lungs. Moreover, an analysis of mRNA profiles revealed biological functions such as proliferation, invasion, adhesion, and migration were promoted in fibroblasts from bleomycin-treated lung, which recapitulated the role of fibroblasts in the fibrogenesis. These fibroblast-specific gene expression profiles will be important notions in future fibrosis studies. mRNA profiles of Lung fibroblasts from 3 mice at day 14 after saline or bleomycin treatment.
Project description:Mouse embryonic fibroblast (MEFs) cell lines and liver samples from Dnmt2 and Nsun2 single mutant, double mutant and wildtype mice were used to identify potential mRNA substrates of both proteins. MEFs were derived from day 13.5 embryos and immortalized by transfection with a plasmid expressing the SV40 large-T antigen. Total RNA of MEFs cell line at passage 9 and total RNA extracted from 2 months old mouse liver tissues of Dnmt2, Nsun2 single mutant, double mutant and wildtype mice were arrayed on Illumina MouseRef8 v2 chips.
Project description:<p>Renal fibrosis, a hallmark of chronic kidney diseases, is driven by the activation of renal fibroblasts. Recent studies have highlighted the role of glycolysis in this process. Nevertheless, one critical glycolytic activator, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), remains unexplored in renal fibrosis. Upon reanalyzing the single-cell sequencing data from Dr. Humphreys' lab, we noticed an upregulation of glycolysis, gluconeogenesis, and TGFβ signaling pathway in myofibroblasts from fibrotic kidneys after unilateral ureter obstruction (UUO) or kidney ischemia/reperfusion. Furthermore, our experiments showed significant induction of PFKFB3 in mouse kidneys following UUO or kidney ischemia/reperfusion. To delve deeper into the role of PFKFB3, we generated mice with Pfkfb3 deficiency specifically in myofibroblasts (Pfkfb3f/fPostnMCM). Following UUO or kidney ischemia/reperfusion, a substantial decrease of fibrosis in injured kidneys of Pfkfb3f/fPostnMCM mice was identified compared to their wild-type littermates. Additionally, in cultured renal fibroblast NRK-49F cells, PFKFB3 was elevated upon exposure to TGFβ1, accompanied by the increase of α-SMA and fibronectin. Notably, this upregulation was significantly diminished with PFKFB3 knockdown, correlated with a glycolysis suppression. Mechanistically, the glycolytic metabolite lactate promoted the fibrotic activation of NRK-49F. In conclusion, our study demonstrates the critical role of PFKFB3 in driving fibroblast activation and subsequent renal fibrosis.</p>