Figure 5.
Svep1 regulates the cellularity and fate of clusters. (A-B) Number of cluster cells per E10.5 (A) and E12.5 (B) Svep1+/+ and Svep1−/− aortas (at E10.5: n = 5 and 9 Svep1+/+ and Svep1−/− embryos, respectively; at E12.5: n = 7 and 5 Svep1+/+ and Svep1−/− embryos, respectively; n = 3 independent experiments). Bars represent means ± SEM. (C-D) Enlarged examples of clusters after maximal projection of E10.5 (C) and E12.5 (D) Svep1+/+ (top panels) and Svep1−/− (bottom panels) aortas stained with anti-CD31 (endothelial and hematopoietic marker, red), anti-KIT (hematopoietic marker, green), and anti-RUNX1 (hemogenic endothelium and hematopoietic marker, blue) antibodies. (E-F) Number of cells per clusters composed of more than 5 cells in E10.5 (E) and E12.5 (F) Svep1+/+ and Svep1−/− aortas. (G-H) Number of clusters per E10.5 (G) and E12.5 (H) Svep1+/+ and Svep1−/− aortas. (I) Hematopoietic repopulation analyses after injection of AGM cells isolated from Svep1+/+ and Svep1−/− E11 embryos (n = 1-3 independent experiments). Numbers above bars indicate the number of mice repopulated/number of mice injected. Dose of injected cells is indicated as embryo equivalent (ee). (J) Percentage of chimerism in peripheral blood for each injected mouse in panel I, 4 months after transplantation. Dot: 1 transplanted recipient mouse. Red dots: mice used to perform secondary transplantations (labeled a-d, here and in subsequent panels). Dashed line: limit of positivity (>5% of chimerism by flow cytometry). Red line: chimerism average. (K) HSC frequency determined based on the transplantation results in panel I. HSC frequency per AGM was estimated by Poisson statistics applied to the percentage of nonrepopulated recipients (y-axis) and the number of injected cells (x-axis). (L) Long-term donor-derived lymphoid (T cells: CD4+, CD8+; B cells: B220+) and myeloid (CD11b+, GR1+) contribution in the blood of reconstituted recipients in panel J. Each bar represents a single recipient. (M) Percentage of chimerism in peripheral blood for each secondary recipient mouse transplanted with 3 × 106 bone marrow cells isolated from the primary reconstituted recipients (a-d, indicated in panel J) at 4 months after transplantation. Dot: 1 transplanted repicient mouse. Dashed line: limit of positivity (>5% of chimerism by flow cytometry). Red line: chimerism average. (N) Long-term donor-derived lymphoid (T cells: CD4+, CD8+; B cells: B220+) and myeloid (CD11b+, GR1+) contribution in the blood of secondary reconstituted recipients shown in panel M. Each bar represents a single recipient. (O-P) Number of runx1+ (O) and cmyb+ (P) HSPCs per aorta at 40 and 48 hpf, respectively, were determined after WISH in svep1+/+ and svep1−/− zebrafish embryos. Representative cases of WISH for runx1 and cmyb expressions in svep1+/+ and svep1−/− zebrafish embryos (left side of each graph). Graphs represent the average number (±SEM) of runx1+ and cmyb+ cells in the dorsal aorta (n ≈ 50 zebrafish embryos each). (Q) Fluorescent micrographs of 2 svep1−/− (fli1a:GFP;flt1enh:RFP) embryos analyzed at 40 hpf. Global view (top panel, embryo 1) and enlargement of the dorsal aorta area (bottom panel, embryo 2). (R) WISH for ephrinb2a in svep1+/+ (top panel) and svep1−/− (bottom panel) embryos, analyzed at 40 hpf. The total number of embryos analyzed for WISH is indicated on the photographs. (S) The rag1+ thymic areas at 4 dpf were determined after WISH in svep1+/+ and svep1−/− zebrafish embryos. Representative cases of WISH for rag1 expression in svep1+/+ and svep1−/− zebrafish embryos (left panels). Graphs represent the average of rag1+ thymic area (±SEM) (n ≈ 10 zebrafish embryos). *P < .05; **P < .01; ***P < .001; ****P < .0001; n.s., not significant, by Student t test. Bars represent 50 µm (C-D), 100 µm (O-Q), and 200 µm (R-S).

Svep1 regulates the cellularity and fate of clusters. (A-B) Number of cluster cells per E10.5 (A) and E12.5 (B) Svep1+/+ and Svep1−/− aortas (at E10.5: n = 5 and 9 Svep1+/+ and Svep1−/− embryos, respectively; at E12.5: n = 7 and 5 Svep1+/+ and Svep1−/− embryos, respectively; n = 3 independent experiments). Bars represent means ± SEM. (C-D) Enlarged examples of clusters after maximal projection of E10.5 (C) and E12.5 (D) Svep1+/+ (top panels) and Svep1−/− (bottom panels) aortas stained with anti-CD31 (endothelial and hematopoietic marker, red), anti-KIT (hematopoietic marker, green), and anti-RUNX1 (hemogenic endothelium and hematopoietic marker, blue) antibodies. (E-F) Number of cells per clusters composed of more than 5 cells in E10.5 (E) and E12.5 (F) Svep1+/+ and Svep1−/− aortas. (G-H) Number of clusters per E10.5 (G) and E12.5 (H) Svep1+/+ and Svep1−/− aortas. (I) Hematopoietic repopulation analyses after injection of AGM cells isolated from Svep1+/+ and Svep1−/− E11 embryos (n = 1-3 independent experiments). Numbers above bars indicate the number of mice repopulated/number of mice injected. Dose of injected cells is indicated as embryo equivalent (ee). (J) Percentage of chimerism in peripheral blood for each injected mouse in panel I, 4 months after transplantation. Dot: 1 transplanted recipient mouse. Red dots: mice used to perform secondary transplantations (labeled a-d, here and in subsequent panels). Dashed line: limit of positivity (>5% of chimerism by flow cytometry). Red line: chimerism average. (K) HSC frequency determined based on the transplantation results in panel I. HSC frequency per AGM was estimated by Poisson statistics applied to the percentage of nonrepopulated recipients (y-axis) and the number of injected cells (x-axis). (L) Long-term donor-derived lymphoid (T cells: CD4+, CD8+; B cells: B220+) and myeloid (CD11b+, GR1+) contribution in the blood of reconstituted recipients in panel J. Each bar represents a single recipient. (M) Percentage of chimerism in peripheral blood for each secondary recipient mouse transplanted with 3 × 106 bone marrow cells isolated from the primary reconstituted recipients (a-d, indicated in panel J) at 4 months after transplantation. Dot: 1 transplanted repicient mouse. Dashed line: limit of positivity (>5% of chimerism by flow cytometry). Red line: chimerism average. (N) Long-term donor-derived lymphoid (T cells: CD4+, CD8+; B cells: B220+) and myeloid (CD11b+, GR1+) contribution in the blood of secondary reconstituted recipients shown in panel M. Each bar represents a single recipient. (O-P) Number of runx1+ (O) and cmyb+ (P) HSPCs per aorta at 40 and 48 hpf, respectively, were determined after WISH in svep1+/+ and svep1−/− zebrafish embryos. Representative cases of WISH for runx1 and cmyb expressions in svep1+/+ and svep1−/− zebrafish embryos (left side of each graph). Graphs represent the average number (±SEM) of runx1+ and cmyb+ cells in the dorsal aorta (n ≈ 50 zebrafish embryos each). (Q) Fluorescent micrographs of 2 svep1−/− (fli1a:GFP;flt1enh:RFP) embryos analyzed at 40 hpf. Global view (top panel, embryo 1) and enlargement of the dorsal aorta area (bottom panel, embryo 2). (R) WISH for ephrinb2a in svep1+/+ (top panel) and svep1−/− (bottom panel) embryos, analyzed at 40 hpf. The total number of embryos analyzed for WISH is indicated on the photographs. (S) The rag1+ thymic areas at 4 dpf were determined after WISH in svep1+/+ and svep1−/− zebrafish embryos. Representative cases of WISH for rag1 expression in svep1+/+ and svep1−/− zebrafish embryos (left panels). Graphs represent the average of rag1+ thymic area (±SEM) (n ≈ 10 zebrafish embryos). *P < .05; **P < .01; ***P < .001; ****P < .0001; n.s., not significant, by Student t test. Bars represent 50 µm (C-D), 100 µm (O-Q), and 200 µm (R-S).

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