Figure 4.
ADM and its receptor RAMP2 are novel and conserved HSPC regulators. (A) Top left panel: cryosection of 35-day-old human embryo stained with anti-ADM (green) and anti-CD34 (red) antibodies and 4′,6-diamidino-2-phenylindole (DAPI; blue). (A′) ADM expression in the ventral aortic microenvironment. Top right panels: cryosection of E10.5 mouse embryo stained with anti-ADM (green) and anti-CD31 (red) antibodies and DAPI (blue). (A′′) Enlargement of the boxed area showing immunostaining for ADM in the ventral aortic microenvironment close to budding hematopoietic cells. Bottom panels: WISH showing the expression pattern of adm in 40-hpf zebrafish embryos (left panel). Post-ISH embryos cryosectioned to show the expression pattern of adm along the dorsal-ventral axis of the embryo trunks (right panel). (B) Top left (B1): cryosection of E10.5 mouse embryo stained with anti-RAMP2 (red) and anti-RUNX1 (green) antibodies and DAPI (blue). (B1′,B1′′,B1′′′) Enlargement of boxed areas showing immunostaining for RAMP2 in the ventral endothelium and clusters. (B2) Top right panels: ISH showing the expression pattern of RAMP2 in an E3 chicken embryo cryosection. (B2′,B2′′,B2′′′) Enlargement of boxed areas showing RAMP2 expression in the ventral aortic endothelium and clusters. Bottom panels: WISH showing the expression pattern of ramp2 in 40-hpf zebrafish embryos (left panel). Post-ISH embryos show the expression pattern of ramp2 along the dorsal-ventral axis of the embryo trunks (right panel). The number of embryos analyzed for WISH is indicated on the photos. (C-D) The number of runx1+ HSPC cells was counted per aorta after WISH in noninjected embryos, embryos injected with mismatch MOs (controls), and embryos injected with blocking MOs (ATG and/or SB) for ADM (C, right panels) and Ramp2 (D, right panels). Representative cases of WISH for runx1 expression in controls and adm (C) and ramp2 (D) injected MOs, analyzed at 40 hpf (left panels). Graphs represent the average number (±SEM) of runx1+ cells in the dorsal aorta in each condition (n = 3 independent experiments, n ≈ 50 zebrafish embryos each). (E-F) Number of cmyb+ HSPCs per aorta in embryos not injected or injected with mismatch MOs (MO_MM) or blocking MOs (MO_ATG or MO_SB) for adm (E) and ramp2 (F). Example of WISH for cmyb in controls and adm (E) and ramp2 (F) injected MOs, analyzed at 48-hpf (left panels). Graphs represent the average number (±SEM) of cmyb+ cells in the aorta (n = 3 independent experiments; n ≈ 30 embryos for each condition). (G-H) Number of cd41+ HSPCs per aorta in embryos not injected or injected with mismatch MOs (MO_MM) or blocking MOs (MO_ATG or MO_SB) for adm (G) and ramp2 (H). Fluorescent micrographs of controls and adm (G) and ramp2 (H) injected MOs in the Tg(kdrl:mCherry;cd41:eGFP) fish background, analyzed at 40 hpf (left panels). Graphs represent the average number (±SEM) of cd41+ cells in the aorta (n = 2 independent experiments, 11-18 embryos for each condition). (I-J) WISH for ephrinB2a (arterial marker) in controls and adm (I) and ramp2 (J) injected MOs, analyzed at 40 hpf. (K) Graph represents the percentage of CD45+ hematopoietic cells in E3 chicken trunks after intracardiac injection of MilliQ water (control), ADM, or ADM-Antag. n = 2 independent experiments. Dot: 2 to 3 trunks pooled. (L-M) In vitro clonogenic assay with cells isolated from E9.5 (L) or E10.5 (M) wild-type AGMs cultured as explants with MilliQ (control) or ADM-Antag. Top: the number of CFU-Cs per AGM embryo equivalent (ee). Bottom: the number of CFU-Cs per 100 000 AGM explant cells. One representative experiment of 2 independent experiments. Bars represent 100 µm (A-H) and 200 µm (I-J). *P < .05; **P < .01; ***P < .001; ****P < .0001; n.s., not significant, by Student t test. BFU-E, burst forming unit-erythroid; CFU-G, CFU-granulocyte; CFU-GEMM, CFU-granulocyte-erythroid-macrophage-megakaryocyte; CFU-GM, CFU-granulocyte-macrophage; CFU-M, CFU-macrophage.

ADM and its receptor RAMP2 are novel and conserved HSPC regulators. (A) Top left panel: cryosection of 35-day-old human embryo stained with anti-ADM (green) and anti-CD34 (red) antibodies and 4′,6-diamidino-2-phenylindole (DAPI; blue). (A′) ADM expression in the ventral aortic microenvironment. Top right panels: cryosection of E10.5 mouse embryo stained with anti-ADM (green) and anti-CD31 (red) antibodies and DAPI (blue). (A′′) Enlargement of the boxed area showing immunostaining for ADM in the ventral aortic microenvironment close to budding hematopoietic cells. Bottom panels: WISH showing the expression pattern of adm in 40-hpf zebrafish embryos (left panel). Post-ISH embryos cryosectioned to show the expression pattern of adm along the dorsal-ventral axis of the embryo trunks (right panel). (B) Top left (B1): cryosection of E10.5 mouse embryo stained with anti-RAMP2 (red) and anti-RUNX1 (green) antibodies and DAPI (blue). (B1′,B1′′,B1′′′) Enlargement of boxed areas showing immunostaining for RAMP2 in the ventral endothelium and clusters. (B2) Top right panels: ISH showing the expression pattern of RAMP2 in an E3 chicken embryo cryosection. (B2′,B2′′,B2′′′) Enlargement of boxed areas showing RAMP2 expression in the ventral aortic endothelium and clusters. Bottom panels: WISH showing the expression pattern of ramp2 in 40-hpf zebrafish embryos (left panel). Post-ISH embryos show the expression pattern of ramp2 along the dorsal-ventral axis of the embryo trunks (right panel). The number of embryos analyzed for WISH is indicated on the photos. (C-D) The number of runx1+ HSPC cells was counted per aorta after WISH in noninjected embryos, embryos injected with mismatch MOs (controls), and embryos injected with blocking MOs (ATG and/or SB) for ADM (C, right panels) and Ramp2 (D, right panels). Representative cases of WISH for runx1 expression in controls and adm (C) and ramp2 (D) injected MOs, analyzed at 40 hpf (left panels). Graphs represent the average number (±SEM) of runx1+ cells in the dorsal aorta in each condition (n = 3 independent experiments, n ≈ 50 zebrafish embryos each). (E-F) Number of cmyb+ HSPCs per aorta in embryos not injected or injected with mismatch MOs (MO_MM) or blocking MOs (MO_ATG or MO_SB) for adm (E) and ramp2 (F). Example of WISH for cmyb in controls and adm (E) and ramp2 (F) injected MOs, analyzed at 48-hpf (left panels). Graphs represent the average number (±SEM) of cmyb+ cells in the aorta (n = 3 independent experiments; n ≈ 30 embryos for each condition). (G-H) Number of cd41+ HSPCs per aorta in embryos not injected or injected with mismatch MOs (MO_MM) or blocking MOs (MO_ATG or MO_SB) for adm (G) and ramp2 (H). Fluorescent micrographs of controls and adm (G) and ramp2 (H) injected MOs in the Tg(kdrl:mCherry;cd41:eGFP) fish background, analyzed at 40 hpf (left panels). Graphs represent the average number (±SEM) of cd41+ cells in the aorta (n = 2 independent experiments, 11-18 embryos for each condition). (I-J) WISH for ephrinB2a (arterial marker) in controls and adm (I) and ramp2 (J) injected MOs, analyzed at 40 hpf. (K) Graph represents the percentage of CD45+ hematopoietic cells in E3 chicken trunks after intracardiac injection of MilliQ water (control), ADM, or ADM-Antag. n = 2 independent experiments. Dot: 2 to 3 trunks pooled. (L-M) In vitro clonogenic assay with cells isolated from E9.5 (L) or E10.5 (M) wild-type AGMs cultured as explants with MilliQ (control) or ADM-Antag. Top: the number of CFU-Cs per AGM embryo equivalent (ee). Bottom: the number of CFU-Cs per 100 000 AGM explant cells. One representative experiment of 2 independent experiments. Bars represent 100 µm (A-H) and 200 µm (I-J). *P < .05; **P < .01; ***P < .001; ****P < .0001; n.s., not significant, by Student t test. BFU-E, burst forming unit-erythroid; CFU-G, CFU-granulocyte; CFU-GEMM, CFU-granulocyte-erythroid-macrophage-megakaryocyte; CFU-GM, CFU-granulocyte-macrophage; CFU-M, CFU-macrophage.

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