Journal: Nature immunology

Article Title: ThPOK is a critical multifaceted regulator of myeloid lineage development

doi: 10.1038/s41590-023-01549-3

Figure Lengend Snippet: a-b) UMAP plots of (a) wild- type (left) and (b) ThPOK−/− (right) C-GMP myeloid progenitors (Lin-, Kit+, Sca1-, CD34hi, CD16/32+/−, gated as in Extended data Fig.5b). Shown cell populations were defined based on reference alignment to a prior curated murine cKit+ CITE-Seq dataset (Extended data Fig.5a). Overlaid arrows indicate predicted RNA velocities derived from spliced versus unspliced scRNA-Seq reads. Areas with the greatest predicted observed trajectory differences are denoted by purple or red circles. c) Number of differentially expressed genes (DEGs) that are up- or downregulated in ThPOK−/− versus wild-type cells by cellHarmony analysis for indicated cell populations. d) cellHarmony organized heatmap of dynamically regulated DEGs in ThPOK−/− and wt for the most frequency detected cell populations (n=1,685 genes, fold > 1.1 and empirical Bayes moderated t-test p<0.05, FDR corrected). Yellow = upregulated gene, blue = downregulated gene. Genes noted in the text are called out to the right of the heatmap. e) Relative statistical enrichment (GO-Elite Z-score) of ThPOK−/− versus wild-type up-regulated genes against all prior defined hematopoietic differentiation markers17, indicates altered differentiation programs in ThPOK−/− mice, for lineage priming (top), lineage specification (middle) and neutrophil commitment (bottom). f) Gene Ontology enrichment analysis (GO-Elite) of down- (right) and up-regulated genes in ThPOK−/− versus wt MDP cells, with example terms highlighted.

Article Snippet: After projecting labels from the 10x Genomics cKit data onto the Fluidigm transcriptome of WT cells ( – ), we observed a progressive increase in alternative splicing in proNeu-1 through preNeu-1 stages.

Techniques: Derivative Assay

Journal: Nature immunology

Article Title: ThPOK is a critical multifaceted regulator of myeloid lineage development

doi: 10.1038/s41590-023-01549-3

Figure Lengend Snippet: a) FACS analysis of cKit, Sca1, CD16/32 and CD34 expression by gated total lineage negative (Lin-) or Lin- cKit+ Sca1- BM cells, as indicated. CMP, GMP, MEP and CD34lo gates are shown in right panels. b) Plots showing frequency of Lin-Sca-cKit+ cells in total BM and of CMP, GMP, MEP and CD34lo subsets in gated cKit+ Sca1- Lin- BM cells from ThPOK-deficient or WT mice (same gates as panel a) (n = 5 biologically independent animals per genotype). Error bars represent standard deviations (centre refers to mean). Significant differences between ThPOK−/− and WT mice were determined by two sided unpaired T test with Welch’s correction, and indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). c) FACS comparison of CD11b, CD115, Ly6c, and Ly6g expression by gated cGMP (conventional GMP) (grey histograms) and atypical c-Kit+ CD34lo CD16hi Lin- (green histograms) BM subsets, from ThPOK-deficient mice. d) FACS analysis of CD11b, Ly6g, Vcam1, CD115, Ly6c, CD16/32, cKit and CD34 expression by indicated gated BM subsets. Gates for preNeu1-3 (preN1-3), proNeu1/2 and other precursor populations are shown. e) Plots showing frequency (left panels) or cell number (right panels) of indicated gated precursor populations in ThPOK-deficient or WT mice (same mice as panel d). Data are presented as mean values +/− SEM. Significant differences between ThPOK−/− and WT mice were determined by two sided unpaired T test, and indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). n = 5 biologically independent animals.

Article Snippet: After projecting labels from the 10x Genomics cKit data onto the Fluidigm transcriptome of WT cells ( – ), we observed a progressive increase in alternative splicing in proNeu-1 through preNeu-1 stages.

Techniques: Expressing, Comparison

Journal: Nature immunology

Article Title: ThPOK is a critical multifaceted regulator of myeloid lineage development

doi: 10.1038/s41590-023-01549-3

Figure Lengend Snippet: a) Bar graphs showing absolute cell numbers for CMP, GMP, MEP and CD34lo subsets among gated cKit+ Sca1- Lin- BM cells from ThPOK-deficient or WT mice (same mice as Fig. 2 A, ​,B)B) (n = 5 independent animals per genotype). Error bars represent SEM. Significant differences between ThPOK−/− and WT mice were determined by two-sided unpaired T test with Welch’s correction, and indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). b) FACS analysis of CD135 and CD115 expression by gated CMPs from WT and ThPOK−/− mice (left panels). Bar graph at right shows % of indicated gated subsets (n = 5 independent animals per genotype). c) FACS analysis of CD11b, CD115, Ly6c, and Ly6g expression by gated cGMP (conventional GMP) (grey histograms) and c-Kit+ CD34lo CD16hi Lin- (green histograms) BM subsets from WT mice. d) FACS analysis of CD16/32 and CD34 expression by gated Lin- c-Kit+ Sca1- BM cells from ThPOPK+/− mice. CMP, GMP, MEP and CD34lo subsets are marked. e) CFU assay of FACS-sorted Lin- BM cells, from indicated WT or ThPOK-deficient mice. Error bars represent SEM. Significant differences between ThPOK−/− and WT mice were determined by two-sided multiple multiple unpaired T with Welch’s correction, and indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). Note that ThPOK−/− Lin- progenitors exhibited a substantial (** p < 0.01) increase in CFU-GM myeloid colony production even after secondary plating. f) FACS analysis of CD11b, Thy1, CD41 and Ter119 lineage marker expression by WT and ThPOK-deficient cells after secondary CFU assay (same experiment as in panel d).

Article Snippet: After projecting labels from the 10x Genomics cKit data onto the Fluidigm transcriptome of WT cells ( – ), we observed a progressive increase in alternative splicing in proNeu-1 through preNeu-1 stages.

Techniques: Expressing, Colony-forming Unit Assay, Marker

Journal: Nature immunology

Article Title: ThPOK is a critical multifaceted regulator of myeloid lineage development

doi: 10.1038/s41590-023-01549-3

Figure Lengend Snippet: a) UMAP plot of curated cKit+ progenitor scRNA-Seq cell populations that serves as the reference for cellHarmony alignment analyses in these studies (see Methods), b) Flow cytometry selection of c-Kit+ BM C-GMP progenitors (gated cells denoted in blue). c) Unsupervised clustering UMAP plot of ~26,000 combined C-GMP CITE-Seq captured mRNA profiles (WT and ThPOK−/−) following analysis with the software ICGS2. Indicated cell-population labels are those automatically assigned by ICGS2 (AltAnalyze BioMarker database). d) UMAP plot of all WT and ThPOK−/− cells from panel c aligned to the reference described in panel a. e) UMAP plot from panel d showing the expression levels of ThPOK mRNA in the wild-type CITE-Seq populations. f) Cell-population percentage of distinct BM progenitor populations detected by CITE-Seq from WT and ThPOK−/− mice. g–h) Heatmap of relative normalized (TotalVI) CITE-Seq antibody derived-tag (ADT) intensities for cellHarmony cKit+ aligned cell populations. Panels (g) and (h) displays cells from WT and ThPOK−/− BM progenitors, respectively. i–p) Gene Ontology enrichment analyses from the software GO-Elite, for each of the indicated cell population differential expression analysis comparisons (all ThPOK−/− vs. WT).

Article Snippet: After projecting labels from the 10x Genomics cKit data onto the Fluidigm transcriptome of WT cells ( – ), we observed a progressive increase in alternative splicing in proNeu-1 through preNeu-1 stages.

Techniques: Flow Cytometry, Selection, Software, Biomarker Discovery, Expressing, Derivative Assay, Quantitative Proteomics

Journal: Nature immunology

Article Title: ThPOK is a critical multifaceted regulator of myeloid lineage development

doi: 10.1038/s41590-023-01549-3

Figure Lengend Snippet: (a–b) UMAP plot of single-cell Fluidigm RNA-Seq analysis of prior profiled (a) wild-type hematopoietic progenitors and (b) Ly6c-ThPOK−/− GMPs aligned to cKit+ CITE-Seq. Consistent alignment of cell populations in the UMAP space indicates a lack of apparent batch effects. c) Gene Ontology enrichment analysis of ThPOK−/− dependent alternative splicing events in proNeu-1 cells, for splicing events with the opposite pattern of exon inclusion in proNeu-1 versus MultiLin (discordant = more immature splicing) or (d) with the same pattern (concordant = promoting neutrophil specification associated splicing). (e‒f) Validation of deregulated gene expression in ThPOK−/− versus WT progenitors for important transcripts as observed in CITE seq data: e) qRTPCR analyses of indicated flow-sorted progenitors (pooled from 4 mice/genotype) for monocyte/DC genes (Irf8, Zeb2, Runx1), granulocyte lineage genes (Cebpe, Pde4d, Cd63 and Nkg7) and RNA binding protein genes (Ddx3x and Srsf5). The experiment was repeated 3 times independently with similar results. f) Expression ratio of Hmga1 alternate transcript versus reference transcript in indicated progenitor populations (pooled from 4 mice/genotype). g) Representative confocal micrographs of EZH2 protein (top), DAPI (middle), and H3K27Me3 (bottom) staining in indicated subsets. Each experiment was reproduced twice and significant differences between ThPOK−/− and WT mice were determined by two-sided unpaired T test with Welch’s correction, and indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). h) Heat maps showing relative mRNA expression of indicated genes in WT progenitors (left panel), or in ThPOK−/− versus WT progenitor subsets (right panel) for genes that are relatively up-regulated in any ThPOK−/− progenitor population and that are known Ezh2 targets in WT immune cells by ChIP-seq (GSE181873). Note that in WT mice most of these genes show marked stage-specific regulation.

Article Snippet: After projecting labels from the 10x Genomics cKit data onto the Fluidigm transcriptome of WT cells ( – ), we observed a progressive increase in alternative splicing in proNeu-1 through preNeu-1 stages.

Techniques: RNA Sequencing, Alternative Splicing, Biomarker Discovery, Gene Expression, RNA Binding Assay, Expressing, Staining, ChIP-sequencing

Journal: Molecular cell

Article Title: BRCA1 mutational complementation induces synthetic viability

doi: 10.1016/j.molcel.2020.04.006

Figure Lengend Snippet: (A) The Brca1CC allele was generated using the indicated sgRNA sequence that targeted exon 13. DNA and amino acids deleted are shown (red). Below, electropherogram showing DNA sequences of Brca1+/+ and Brca1CC/CC mice, deleted base pairs (box) and deletion location (arrow) are indicated. (B) MEFs with the indicated genotypes were assessed for Brca1 protein expression by Western blotting. (C) MEFs with the indicated genotypes were assessed for RAD51 IRIF by immunofluorescence (IF). Nuclei with >5 foci were considered positive. Mean and S.E.M. are shown, n=3. Representative images, scale bar is 10 μm. (D) Summary of live pups born as well as live embryos at E16.5 generated from Brca1+/CC x Brca1+/CC intercross. (E) Representative photographs of E16.5 embryos, scale bar 0.5 cm; and 3-week old littermates, scale bar 2 cm. Hypopigmented fur area is indicated with an arrow. (F) Weights of individual mice at 3–4 weeks of age. (G) White blood cell (WBC), red blood cell (RBC) numbers from peripheral blood, and (H) mononuclear bone marrow (BM) cell numbers were measured in 3–4-week-old littermates with the indicated genotypes. Inset, representative H&E staining of BM, scale bar 100 μm. (I) Total number of long-term hematopoietic stem cells (LT-HSC) (Linlow; cKit+; Sca+; CD150+; CD48−; CD34−) analyzed by FACS in 3–4-week-old mice. See Fig. S1 for more information. (J) Representative H&E staining of testes and ovaries, scale bar 100 μm. (K) Representative images of T-ALL from a Brca1CC/CC mouse. H&E staining of thymic tumor and CD3+ staining of tumor cell infiltrates from the same mouse, scale bar 100 μm. Summary of Brca1CC/CC lifespans (days) and cause of death when known (inset). *p < 0.05 **p < 0.01, *** p < 0.001 (unpaired t-test).

Article Snippet: Cells were then resuspended in 0.5 ml of F-PBS, counted, and incubated on ice for 60 minutes with the following antibodies: CD3 (145–2C11 Biolegend), CD4 (RM4–5 eBiosience), CD8 (53–6.7 eBioscience), CD19 (6D5 Biolegend), B220 (RA3–6B2 Biolegend), Gr1 (RB6–8C5 Biolegend), Ter119 (Ter119 Biolegend), cKit (2B8 BD, Pharmingen), Sca-1 (D7, eBioscience), CD150 (TC15–12F12.2 Biolegend), CD34 ( RAM34 BD Pharmingen), FcgRII/III (93 Biolegend), CD48 (HM48–1 Biolegend).

Techniques: Generated, Sequencing, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Molecular cell

Article Title: BRCA1 mutational complementation induces synthetic viability

doi: 10.1016/j.molcel.2020.04.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then resuspended in 0.5 ml of F-PBS, counted, and incubated on ice for 60 minutes with the following antibodies: CD3 (145–2C11 Biolegend), CD4 (RM4–5 eBiosience), CD8 (53–6.7 eBioscience), CD19 (6D5 Biolegend), B220 (RA3–6B2 Biolegend), Gr1 (RB6–8C5 Biolegend), Ter119 (Ter119 Biolegend), cKit (2B8 BD, Pharmingen), Sca-1 (D7, eBioscience), CD150 (TC15–12F12.2 Biolegend), CD34 ( RAM34 BD Pharmingen), FcgRII/III (93 Biolegend), CD48 (HM48–1 Biolegend).

Techniques: Recombinant, Plasmid Preparation, Staining, Mutagenesis, Modification, Software

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a Three-dimensional (3D) wholemount immunostaining with αSMA, CD31 and NG2 of E10.5 (31–38 somite pairs (sp)) WT dorsal aorta; b NG2 and Runx1 expression on single plane wholemount WT E10.5 sections. NG2 + Runx1 + vSMCs (arrows), hemogenic endothelial cells (arrowheads) and intra-aortic hematopoietic clusters (IAHCs, stars) (Table ); c Representative example of flow cytometric analysis of NG2 + Runx1(GFP) + (green box) in E10.5 Runx1-IRES-GFP AGM and E10.5 WT control. d Percentages of NG2 + Runx1(GFP) + cells in E9 (21-25sp) body ( n = 6), E10/E10.5/E11 AGMs ( n = 8/7/7), N = 5, Kruskal-Wallis and Dunn’s post-hoc test. e Representative examples of wholemount 3D-images showing αSMA, CD31 and NG2 in E10.5 cKO dorsal aortae; f αSMA, Runx1 and CD31 immunofluorescence of E11 WT and cKO transversal frozen sections; n = WT/cKO: 2/2, N = 2. g cKit and CD31 wholemount 3D-images in E10.5 WT and cKO AGM; h Number of intra-aortic hematopoietic clusters (IAHCs) in E10.5 AGM; n = WT/KO: 5/4, N = 4. Number of colony forming unit-culture (CFU-C) in i E10.5 (31-38sp) AGM; n = WT/HET/KO: 14/10/5 embryos; N = 7 and j E11 (43–52sp) AGM; n = WT/HET/KO: 22/8/19 embryos; N = 11; one-way ANOVA and Tukey’s post-hoc test (Table ). k Percentages of donor cell chimerism 4-months post-transplantation of 6 E11 WT (NG2 +/+ ;Runx1 fl/+ or NG2 +/+ ;Runx1 fl/fl ) , 7 HET ( NG2-Cre;Runx1 fl/+ ) and 6 cKO AGMs ( NG2-Cre;Runx1 fl/fl ) into sub-lethally adult irradiated recipients (1xAGM cells transplanted/recipient; N = 4). Each dot represents one recipient. Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood (dashed line) ; one-tailed Z score test for two population proportions (Tables and ). For wholemount staining in a , b , e , g : WT/cKO ( N = 6/4): αSMA ( n = 9/7), CD31 ( n = 10/7), cKit ( n = 3/2), NG2 ( n = 3/1) and WT Runx1 ( n = 4) in 3 distinct combinations (Table ). D = dorsal, V = ventral. N = number of independent experiments; n = number of biological samples (embryos). All data are presented as mean values ± SEM. Source data for d , h , i , j and k are provided as a file.

Article Snippet: As all E11 AGM HSPCs have been reported to express cKit , E11 AGM cells were also stained with cKit (1:500, BD Horizon, 562609) for further enrichment.

Techniques: Immunostaining, Expressing, Control, Immunofluorescence, Transplantation Assay, Irradiation, One-tailed Test, Staining