Expression profiling by high throughput sequencing Other
Summary
Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-Seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-Seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ propDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs, and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s
Overall design
For transposon integration site cloning, bone marrow cells and splenocytes were isolated from the Pdzk1ip1CreERR26FlipJump mice at weeks 20 post tamoxifen induction. HSC, DC subsets (pDC, cDC1 and cDC2) and Immature B were sorted from isolated bone marrow cells and splenocytes. Splenocytes from Cx3cr1CreERYFP R26FlipJump mice were obtained on days 8 post tamoxifen induction. GFP+ YFPlo/- DC subsets (pDC, cDC1 and cDC2) were sorted. GFP+ DC subsets (pDC, cDC1 and cDC2) from Flt3L cultured Cx3cr1CreERYFPR26FlipJump mouse BM cells were sorted. For CITE-seq experiments, HSC-derived Flt3+ progenitors were isolated from the Pdzk1ip1CreERR26FlipJump mice at weeks 3, 4 or 5 following tamoxifen induction. Cx3cr1+ progenitors were isolated from Cx3cr1CreERYFP R26FlipJump at days 2 and 5 post tamoxifen induction. For bulk RNA-seq experiments, the pDC with GFP or without GFP was sorted from Cx3cr1CreERYFPR26FlipJump mouse BM cells at 8 days post tamoxifen induction.
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