EMBO/SourceData huggingface dataset:
figf3(a) Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control.(b) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm.(c) Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2.(d) Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial morphologies. Data are means ± s.e.m. (c, n = 3; d, n = 4). Asterisk indicates P 0.05, double asterisks indicate P 0.01 and dagger indicates P > 0.05 (t-test).
figf4(a) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by treatment with hIFN-γ. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. (b) LC3 puncta per cell were quantified from cells treated as in a.(c) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by starvation. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm.(d) U937 cells were transfected with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified.(e) U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification.(f) U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data.
figf5(b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm.(c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative, as indicated in the inset.(d) Quantification of GFP+ MTR+ cells from an experiment performed as in b.(e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting.(f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads.(g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.
figf6(a) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b-d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only).(e)HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified.(f) HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified.(g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP-DRP1 (A) or GFP-IRGMd (B-G) and labelled with MTR (A-D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B-D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E-G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm.(h) HeLa cells, co-transfected with GFP-IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.(i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP-IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified.(j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP-IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.(k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.
figf7(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP-IRGMd that were rounded 48 h post-transfection.(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.(c) HeLa cells transfected with vectors encoding GFP-IRGMd versus GFP-IRGMdS47N and YFP-IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified.(d) HeLa cells were transfected with plamsids encoding GFP-IRGMd, GFP-IRGMDS47N or YFP-IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified.(e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top).(f) HeLa cells were transfected with plasmids encoding GFP-IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP-IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm.(g-i) Cells were transfected with plasmids encoding GFP or GFP-IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).
figf8(a, b) HeLa cells were transfected with plasmids encoding GFP, CFP-IRGMa, GFP-IRGMc or GFP-IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified.(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.
Figure 1Number of EMPs per embryo equivalent (e.e.) of wild type and Sl/Sl E9.5 and E11.5 YS, determined by flow cytometry (panels in B,D). E9.5 Kit+ CD41+ CD16/32+ EMP numbers are the mean±SD from 4 wild type and 3 Sl/Sl biological replicates, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of embryos analyzed: 7 +/+ (14-23sp), 5 Sl/Sl (17-25sp). E11.5 Kit+ CD41+ EMP numbers are the mean±SD of 5 +/+ and 6 Sl/Sl YS analyzed individually over 2 independent experiments. Total live cells per YS: 1.7±0.3x105 (wt), 1.7±0.2x105 (Sl/Sl) at E9.5; 4.2±1.4x105 (wt), 4.0±2.2x105 (Sl/Sl) at E11.5.Example of flow cytometry analysis of E9.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.Number of CFU-C in wild type and Sl/Sl E9.5 and E11.5 YS. E9.5 data (mean±SD) are from 3 wild type and 2 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of analyzed embryos: 5 +/+ (17-23sp), 3 Sl/Sl (17-25sp) over 2 independent experiments. For E11.5, data are from 4 wild type and 6 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YSs of the same genotype. Total number of embryos analyzed: 6 +/+, 7 Sl/Sl over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.Example of flow cytometry analysis of E11.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.Confocal WM-IF analysis of CD31+ pHH3+ Kit+ proliferating EMPs (arrowheads) in E9.5 wild type and Sl/Sl YS. A single 2.5μm-thick Z section is shown. For each YS, CD31+ Kit+ pHH3+ cells were counted in 3 to 5 fields using a 25x objective and the percentage of pHH3+ proliferating EMPs was calculated. Bar graphs represent the mean±SD from 3 wild type (21-24sp) and 3 Sl/Sl (22-23sp) YS. Scale bars: 50μm.Cell cycle analysis of wild type and Sl/Sl E11.5 YS EMPs. The percentage of EMPs (Ter119- Kit+ CD41+) in G0/G1 or S/G2/M was determined by flow cytometry on the basis of BrdU and 7-AAD incorporation. a: apoptotic (percentage not shown). Embryos were analyzed individually. N=6 (+/+), N=5 (Sl/Sl) over 3 independent experiments. E11.5 embryos were staged according to tail somite counts as described in [76] and were: 12-17 (+/+); 12-17 (Sl/Sl). Bar graphs show mean±SD, and FACS plots representative results.
Figure 2Flow cytometry analysis of EMPs (Ter119- Kit+ CD41+ CD16/32+) and early erythroid progenitors (Early E: Ter119- Kit+ CD41- CD71+ CD44+) in wild type and Sl/Sl E11.5 FL. EMP and early E data are the mean (±SD) of 6 wild type and 4 Sl/Sl biological replicates, with a total of 10 wild type and 6 Sl/Sl embryos analyzed in pools of 1-3 FL per genotype. Bar graphs on the right show corresponding absolute cell numbers (nr) per FL. Total FL cell counts are the mean (±SD) of 12 wild type and 13 Sl/Sl embryos. Tail somite range: 10-16 (wild type), 12-15 (Sl/Sl). Note that although EMP frequency in Sl/Sl FL is increased, their absolute number is severely decreased.CFU-C numbers in wild type and Sl/Sl E11.5 FL. Data are the mean (±SD) of 10 wild type and 10 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of cell from 1-2 FLs. A total of 12 wild type and 11 Sl/Sl FL were plated over 6 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.Total cell numbers (nr) of E12.5 wild type (n=7) and Sl/Sl (n=8) FL. Right: CFU-E numbers per wild type and Sl/Sl E12.5 FL. Data are the mean (±SD) of 3 biological replicates analyzed over 2 independent experiments. Each biological replicate consisted of 1-2 FL samples pooled according to genotype and was plated in duplicate into a methylcellulose based medium containing EPO; colonies were scored after 2 days. Total number of FLs analyzed: 4 wild type and 6 Sl/Sl.Quantification of erythroid lineage cells in E12.5 wild type and Sl/Sl FL. Absolute cell counts (top) and percentages (bottom) of proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE) and orthochromatic erythroblasts (OrthoE) were determined by imaging flow cytometry analysis. The number (nr) of cells per FL was based on total cellularity and their relative proportions. Data are the mean (±SD) of 3 biological replicates for each genotype, with replicates consisting of single or 2 pooled FL samples. A total of 4 wild type, 5 Sl/+ and 6 Sl/Sl FLs were analyzed.Representative examples of maturing erythroblast series in wild type and Sl/Sl E12.5 FL, as identified by imaging flow cytometry. Scale bars: 10μm.
Figure 3Number of EMPs in peripheral blood (PB) of wild type and Sl/Sl midgestation embryos, calculated from the percentage of PB EMPs (Ter119- Kit+ CD41+ CD16/32+) determined by flow cytometry (Figure EV1E,F) and the total cell count (4.2±1.6x105 (wt), 3.1±1.1x105 (Sl/Sl) at E9.75; 2.6±0.8x106 (wt), 2.3±1.4x106 (Sl/Sl) at E11.5). For E9.75-E10, PB from 2 wild type (28-30sp) and 5 Sl/Sl (27-31) individual embryos was analyzed. For E11.5, PB from 1-3 embryos was pooled and 6 wild type and 4 Sl/Sl biological replicates were analyzed, with 10 wild type and 6 Sl/Sl embryos analyzed in total. Graphs show the mean (±SD) of PB EMPs per embryo equivalent (e.e.).Representative confocal whole-mount immunofluorescence images of wild type and Sl/Sl FL at the onset of hematopoietic colonization. Kit+ EMPs are shown in white, CD31+ vasculature in green. Maximum intensity 3D projections from 350μm-thick Z stacks are shown. Scale bars: 100μm. Number of embryos analyzed: 3 wild type and 3 Sl/Sl for E9.75 (27-29sp), 7 wild type and 8 (Sl/Sl) for E10.5 (34-36sp).Confocal whole-mount immunofluorescence analysis of proliferating hematopoietic progenitors in wild type and Sl/Sl E10.5 (34-36sp) FL. Very few CD31+ pHH3+ Kit+ proliferating hematopoietic cells (arrowheads) were detected in the Sl/Sl FL. A single 2.5μm-thick slice is shown. Scale bars: 20μm. A total of 7 wild type and 8 Sl/Sl embryos were analyzed.Percentage of cycling Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid (Early E) cells in the E11.5 FL, determined by flow cytometric analysis of BrdU and 7-AAD incorporation. Data are the mean (±SD) of 6 wild type and 5 Sl/Sl FLs analyzed individually over 3 independent experiments. Tail somite range: 12-17 (+/+); 12-17 (Sl/Sl).Percentage of early apoptotic EMPs and Early E in wild type and Sl/Sl E11.5 FL, determined by flow cytometric analysis of AnnexinV staining among 7-AAD- Ter119- CD41+ Kit+ CD16/32+ EMPs and 7-AAD- Ter119- Kit+ CD41- CD71+ CD44+ Early E. Data are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments, with each replicate consisting of 1-3 FLs of identical genotypes. A total number of 13 wild type and 9 Sl/Sl embryos were analyzed. Tail somite range: 11-17 for both genotypes.
Figure 4Percentages of macrophage populations in wild type and Sl/Sl E14.5 brain, skin (dissected from the back), lungs and limb buds, as determined by flow cytometry. Cells were gated as Ter119- CD45+ and further gated into F4/80hi CD11blo or F4/80lo CD11bhi populations. Representative dot plots of skin analysis are shown in Appendix Figure S1A. Data are the mean (±SD) of 4 biological replicates for either genotype, with each replicate consisting of individual or pooled samples (from up to 3 embryos). A total of 8 wild type and 5 Sl/Sl embryos were analyzed over 2 independent experiments.Percentage and absolute numbers of tissue macrophages per E14.5 FL. Data are mean (±SD). Number of embryos analyzed: as in (A).Representative flow cytometry analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. Cells were gated as shown, with percentages in individual gates as indicated. Data are the mean (±SD) of 4 wild type (32-35 sp) or 3 Sl/Sl (34-35 sp) biological replicates, analyzed over 2 independent experiments. Embryos were analyzed individually. Quantification of flow cytometry analysis in (C), showing percentage of F4/80+CD11b+ macrophages within live cells. E11.5 data are the mean (±SD) of 4 wild type (10-13 tail somites) or 3 Sl/Sl (10-13 tail somites) biological replicates, analyzed over 3 independent experiments. Embryos were analyzed individually.Confocal whole-mount immunofluorescence analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. 3D panels show maximum intensity projections from 400μm-thick Z stacks. Slice panels show single 2.5μm-thick Z slices of clusters in the dorsal aorta (da). A total of 4 wild type (31-34 sp) and 3 Sl/Sl (31-33 sp) embryos were analyzed. Scale bars: 50μm (3D), 10μm (slice). Quantification of whole-mount immunofluorescence in (E), showing absolute numbers (mean ± SD) of F4/80+ macrophages within hematopoietic clusters in the dorsal aorta.
Figure 6Confocal whole-mount immunofluorescence (WM-IF) analysis of Kitl-tdTomato expression in the E8.5 (6-7sp) conceptus (maximum intensity 3D projection from a 500μm-thick Z-stack). Kitl-tdTomato expression (red) is seen in the YS, allantois (a; from which the umbilical artery derives), and para-aortic splanchnopleura (PAS) of Kitl-tdTomato::23GFP transgenic concepti (the embryo proper, from which the head has been removed, is outlined with a purple dashed line). The 23GFP transgene (green) labels the YS blood islands (bi; yellow dashed line); this GFP reporter is transcribed from a heterologous hsp68 promoter under the spatiotemporal control of the Runx1 hematopoietic +23 enhancer, resulting in GFP expression in all primitive erythroid(-fated) cells, hemogenic endothelium, and emerging HSPCs[2,74]. Endothelial cells (CD31+) are in white. Arrowheads indicate the paired aortae (pa). Purple boxed area is magnified in (B); yellow boxed area is magnified in (C). Scale bar: 100μm. N=2 embryos analyzed. Single 2.5μm-thick Z slice from the region highlighted in the purple box in (A). Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells in one of the paired aortae (pa). Scale bar: 25μm. Single 2.5μm-thick Z slice from the region highlighted in the yellow box in (A), showing Kitl-tdTomato expression in the E8.5 YS BI. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells; the arrow indicates a Kitl-tdTomato+ perivascular cell. Scale bar: 25μm. Single 2.5μm-thick Z slice of 10.5 (35sp) YS (WM-IF) showing Kitl-tdTomato (red), Kit (white) and CD31 (green) expression. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells. N=3 embryos analyzed. Scale bar: 50μm.Confocal WM-IF analysis of Kitl-tdTomato expression (red) in a E10.5 (36sp) AGM region. Top panels (3D view) show a maximum intensity 3D projection from a 400μm-thick Z stack. Bottom panels (slice view) show a single 2.5μm-thick Z slice of the dorsal aorta, from the boxed region in the top panels. CD31 (vasculature; green); Kit (hematopoietic progenitors; white). Scale bars: 100μm (top); 25μm (bottom). fl: fetal liver; va: vitelline artery plexus; da: dorsal aorta; fp: floor plate; nt: neural tube. N=6 embryos analyzed.Confocal immunofluorescence analysis of cryosections through the AGM region of E10.5 (34-36sp) Kitl-tdTomato transgenic embryos. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells (top), Kitl-tdTomato+ PDGFR-β+ (middle) or Kitl-tdTomato+ α-SMA+ (bottom) perivascular smooth muscle cells. Scale bars: 25μm or 50μm (PDGFR-β panel). N>6 embryos analyzed.Confocal immunofluorescence analysis of Kitl-tdTomato expression in the E10.5 FL. A cryosection through a E10.5 (35-36sp) Kitl-tdTomato transgenic FL is shown. Arrowheads indicate Kitl-tdTomato+ CD31+ Kit- endothelial cells. Asterisks indicate Kitl-tdTomato+ CD31- Kit- mesenchymal cells or hepatoblasts. Note proximity of Kitl-tdTomato+ cells and Kit+ hematopoietic cells. Scale bars: 50μm (left panel), 25μm (magnified inset). N>5 embryos analyzed.
Figure 7Left: number of Ter119- Kit+ CD41+ CD16/32+ EMPs in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 YS, determined by flow cytometry. Data are the mean (±SD) of 8 control and 7 Tie2Cre::Kitl∆/∆ YS analyzed individually over 4 independent experiments. Tail somite range: 13-16. Middle: number of pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-) and pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+) in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 AGM+VU, determined by flow cytometry. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ AGM+VU analyzed individually over 3 independent experiments. Tail somite range: 14-15 (Kitlf/f, f/+); 14-16 (Tie2Cre::Kitl∆/∆). Right: number of Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid cells in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 FL, determined by flow cytometry. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ FL analyzed individually over 3 independent experiments. Tail somite range: 13-16.CFU-C numbers in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 YS. Data are the mean (±SD) of 6 control and 3 Tie2Cre::Kitl∆/∆ YS plated in duplicate in 2 independent experiments. Tail somite range: 13-16.Analysis of long-term multi-lineage HSC potential in E11.5 Tie2Cre::Kitl∆/∆ and control Tie2Cre::Kitl∆/+ or Kitlf/f or f/+AGM+VU. Irradiated CD45.1 syngeneic mice were transplanted with 1 e.e. of AGM+VU cells. PB chimerism is represented as the percentage of donor CD45.2+ cells among total CD45+ cells, 16 weeks after transplant. A total of 29 recipients were transplanted with Tie2Cre::Kitl∆/+ or Kitlf/f or f/+ cells and 8 with Tie2Cre::Kitl∆/∆ cells, over 7 independent experiments. Kitlf/f or f/+ is represented with a circle, Tie2Cre::Kitl∆/+ a triangle and Tie2Cre::Kitl∆/∆ with a square. Tail somite range: 12-17 (control); 12-17 (Tie2Cre::Kitl∆/∆).CFU-C numbers in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 Fl. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ FL plated in duplicate in 3 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid. Tail somite range: 13-16.Number of phenotypic LSK, HPC and HSC per FL in E12.5 Tie2Cre::Kitl∆/∆and control (Kitlf/f or f/+) FL, determined by flow cytometry and total FL cell counts. Cells were gated as 7-AAD- Lin- (F4/80- CD3e- Nk1.1- Ter119- Gr-1- B220- CD19-). LSK: Lin- Sca1+ Kit+; HPC: Lin- Sca1+ Kit+ CD48+ CD150- and HSC: Lin- Sca1+ Kit+ CD48- CD150+. Gating strategy is shown in Figure EV4E. Data are the mean (±SD) of 12 control and 6 Tie2Cre::Kitl∆/∆ FLs analyzed individually over 3 independent experiments. Total cellularity of E12.5 Kitlf/f or f/+ (n=14) and Tie2Cre::Kitl∆/∆ (n=11) FL. Error bars represent SD.