Journal: PLOS One

Article Title: Single-cell profiling unveils key regulators of skeletal stem cells in chicken and human embryonic limb development

doi: 10.1371/journal.pone.0346514

Figure Lengend Snippet: Expression of COL5A2, COL1A2, PRRX1, and TGFβ2 was detected in the periosteal region and the primary ossification centers. A. Merged and single-channel images of COL5A2 (red) and DAPI (blue) staining. B. Merged and single-channel images of COL1A2 (red) and DAPI (blue) staining. C. Merged and single-channel images of PRRX1 (red) and DAPI (blue) staining. D. Merged and single-channel images of TGFβ2 (red) and DAPI (blue) staining. All experiments were independently repeated at least three times. Scale bars in snapshot image, 200 μm; scale bars in magnified images, 50 μm.

Article Snippet: The following antibodies were used: anti-COL5A2 antibody ( GB111012 , 1:500, Servicebio, Wuhan, China), anti-COL1A2 antibody (AF7001, 1:200, Affinity Biosciences, Jiangsu, China), anti-PRRX1 antibody (DF4274, 1:200, Affinity Biosciences, Jiangsu, China), anti-TGFβ2 antibody (AF0260, 1:100, Affinity Biosciences, Jiangsu, China), and Cy3-conjugated goat anti-rabbit IgG (GB21303, 1:300, Servicebio, Wuhan, China).

Techniques: Expressing, Staining

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) clustering of 434,810 cells from eight public scRNA-seq datasets. ( B ) Dot plot showing relative Cdh5 and Runx1 co-expression across clusters; clusters 8 and 50 co-express both genes. ( C ) UMAP highlighting clusters 8 and 50; all other clusters shown in grey. ( D ) Violin plots of doublet scores across Leiden clusters. Clusters 50 and 8 show no evidence of doublet enrichment. ( E ) Datasets proportional contribution to clusters 50 and 8; each dataset is color-coded. ( F ) Dot plot showing expression of selected marker genes in clusters 50 and 8 (from the public sc RNA-seq datasets listed in ) and from clusters 0, 1, 13, 22, and 14 (from Polylox scRNA-seq; ). Results reflect mean expression and fraction of cells in group. ( G ) Cdh5, Runx1, and Col1a2 co-expression in the indicated clusters as a fraction of cells in the cluster. ( H, I ) t-SNE plot of ECs from 11 murine tissues ( G ) and Venn diagram ( H ) showing rare co-expression of Cdh5, Runx1, and Col1a2 in these tissues.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Expressing, Marker, RNA Sequencing

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Schematic representation of the Col1a2 tracking lines. ( B ) Representative confocal image of a BM section from a tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mouse, showing widespread distribution of ZsGreen + cells. Flow cytometric identification of RUNX1 + VE-Cadherin + CD45 − endothelial cells (ECs) in the BM of peanut oil-treated ( n = 6) and tamoxifen-treated ( n = 6) Col1a2-Cre ERT2 /ZsGreen adult mice; Cre − mice ( n = 5) ( C ); WT C57Bl/6 mice ( n = 6) and Fluorescence Minus One (FMO) control ( n = 5) ( D ); and Cdh5-Cre ERT2 (PAC)/ZsGreen mice treated with peanut oil ( n = 6) or tamoxifen ( n = 6); Cre − mice ( n = 5) ( E ). Left panels: quantification of cells identified by the indicated gates as a percentage of total BM ECs; each dot represents one mouse (1 femur + 1 tibia). Middle and right panels: representative gating strategies. ( F ) Representative confocal microscopy image of a BM section from a tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen adult mouse showing a ZsGreen + Endomucin + cell lining a vascular structure (white arrows). ( G ) Representative confocal image of a blood smear from a Col1a2-Cre ERT2 /ZsGreen mouse treated with tamoxifen showing the presence of a nucleate CD45 + cell tracked by ZsGreen/Col1a2 fluorescence (pointed by the arrow). ( H ) Representative confocal image of a blood smear from a Col1a2-Cre ERT2 /mTmG mouse treated with tamoxifen showing the presence of a nucleated CD45 + cell tracked by EGFP/Col1a2 fluorescence (pointed by the arrow). Dots represent individual mice. Data are shown as mean ± SD. ***p < 0.001 by Student’s t -test.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Fluorescence, Control, Confocal Microscopy

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Percent EGFP + CD45 + cells in bone marrow (BM) and blood of tamoxifen-treated ( n = 6) or oil-treated ( n = 5) Col1a2-Cre ERT2 /mTmG mice ( A ) and tamoxifen-treated ( n = 4) or oil-treated ( n = 3) Col1a2-Cre ERT2 /ZsGreen mice ( B ). Cre-control mice ( n = 5 in A, and n = 2 in B). ( C ) Transplant experiment: sorted VE-Cadherin + CD45 − ZsGreen + /Col1a2 + cells from tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mice are transplanted into 5-FU-conditioned WT recipients. Detection ( D ) and characterization ( E ) of ZsGreen + CD45 + cells in BM and blood of WT 5-FU-conditioned mice ( n = 5), 4 weeks post-transplant of VE-Cadherin + CD45⁻ZsGreen + /Col1a2 + cells. Control FU-conditioned WT mice ( n = 4) received no cell transplant ( D ). ( F ) Time course of ZsGreen + peripheral blood mononuclear cell (PBMC) detection in control (Cdh5-Cre + /ZsGreen + ) and Runx1 EC-KI (Cdh5-Cre + /ZsGreen + /Runx1-KI) mice ( n = 10 per group). Representative images ( G ) and quantification ( H ) of ZsGreen + cells from OP9 cell-supported cultures of BM cells from tamoxifen-treated Cdh5-Cre + /ZsGreen + ( n = 11) and Runx1 EC-KI mice ( n = 5). Representative flow cytometry plots ( I ) and quantification ( J ) of CD45 + ZsGreen + cells from OP9 cell-supported BM cell cultures ( n = 5/group). Dots represent individual mice. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001 by Student’s t -test.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Control, Flow Cytometry

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Representative FACS gating strategy used to isolate BM hematopoietic cells, ECs, stromal cells, and Col1a2-tracked ECs from tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mice. ( B ) Gene expression profiling of unsorted BM and sorted BM populations defined in ( A ). Results from qRT-PCR are normalized by Gapdh and unsorted BM. Dots reflect experimental triplicates. ( C ) Representative confocal image of a BM section from a transplant recipient showing a CD45 + CD11b + ZsGreen + cell (arrow), indicating hematopoietic derivation from transplanted ZsGreen + VE-Cadherin + CD45 − (Col1a2 + ) cells. ( D ) Representative image of a blood smear from a WT recipient mouse transplanted with ZsGreen (Col1a2) + BM ECs from Col1a2-Cre ERT2 /ZsGreen mice. ZsGreen tracked cells are pointed by the arrows. ( E ) Schematic diagram of the Cdh5-Cre ERT2 /ZsGreen/Runx1-Knock-in (Runx1 EC-KI ) mouse line used to trace ECs with Runx1 expression induced upon tamoxifen treatment.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Gene Expression, Quantitative RT-PCR, Knock-In, Expressing