1. Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin, PECAM-1, Tie2, endoglin, and CD34 in the mouse embryo
by Masatsugu Ema, Tomomasa Yokomizo, Asami Wakamatsu, Tsumoru Terunuma, Masayuki Yamamoto, and Satoru Takahashi
Blood
Volume 108(13):
December 15, 2006
©2006 by American Society of Hematology

2. Anatomic locations of primitive hematopoiesis and vasculogenesis.
Anatomic locations of primitive hematopoiesis and vasculogenesis. (A) Transverse section of an E8.5 (6sp) embryo. Flk1 (red) and ϵyglobin (green) are expressed exclusively. Note that Flk1 marks endothelial cells, whereas ϵyglobin marks hematopoietic cells. The domain of the vascular plexus and the blood island region are clearly regionalized. (B-E) High-magnification image of the blood island region stained with a nuclear staining dye, Hoechst (B), anti-ϵyglobin antibody (C), anti-Flk1 antibody (D), and the merged image (E). (F) Merged image of PECAM-1 (red), ϵyglobin (green), and Hoechst dye staining (blue) of a transverse section of an E9.5 embryo proper. Scale bar represents 100 μm. (G-J) Higher magnification of the boxed area in panel F. (G) Hoechst staining. (H) Anti–ϵyglobin antibody staining. (I) Anti–PECAM-1 antibody staining. (J) Merged image of panels G-I. (K-O) Merged image of anti-ϵyglobin antibody staining and Hoechst staining in combination with staining for VE-cadherin (K), Tie2 (L), CD34 (M), endoglin (N), or Flk1 (O). Endothelial markers are expressed in endothelial but not in hematopoietic cells. (P) Merged image of CD34 (red), ϵyglobin (green), and Hoechst dye staining (blue) of a transverse section of an E9.5 embryo proper. (Q-T) Higher magnification of the boxed area in panel P. (Q) Hoechst staining. (R) Anti-ϵyglobin antibody staining. (S) Anti-CD34 antibody staining. (T) Merged image of panels Q-T. (arrow) Clump of cells at the ventral side of OA. ex indicates extra-embryonic region; em, embryonic region; DA, dorsal aorta; VE, visceral endoderm; EPC, ectoplacental cone; HF, head fold; NT, neural tube; OA, omphalomesenteric artery. All images were captured by a Leica DC500 CCD camera with IM50 Imaging Manager through a Leica DMRXA microscope using a 5×/0.15 NA objective (panel A) or a 20×/0.50 NA objective (panels B-T).
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology

3. PECAM-1 expression in globin-producing cells and in the extraembryonic region as early as the streak stage.
PECAM-1 expression in globin-producing cells and in the extraembryonic region as early as the streak stage. (A) Whole mount anti–PECAM-1 antibody staining of E8.5 (4sp) embryo. Note that most blood vessels, including arteries and veins, are well stained with the anti–PECAM-1 antibody and that there are densely stained cells within the blood island region. (B) Transverse section showing that blood island cells are PECAM-1 positive. Nuclei are visualized by methyl green staining. (C) High magnification of the boxed area in panel B. (D-G) High magnification of the blood island region stained with Hoechst (D), anti-ϵyglobin antibody (E), anti–PECAM-1 antibody (F), and the merged image (G). PECAM-1 is expressed in globin-positive cells. (H-L) Whole mount PECAM-1 antibody staining of embryo at late-streak stage (H), no-bud (OB) stage (I), early-bud (EB) stage (J), early–head fold (EHF) stage (K), and late–head fold (LHF) stage (L). PECAM-1 is expressed in the extraembryonic region, presumably corresponding to the mesodermal layer as early as the late-streak stage, and is expressed continuously in the extraembryonic regions including the blood island region until E8.5. (H, inset) High magnification of PECAM-1–positive cells in the extraembryonic region. (arrow) Forming dorsal aortae. hc indicates heart crescent; HF, head fold; ec, endocardium; VE, visceral endoderm. Images in panels A and H-L were captured by Leica DC500 CCD camera with IM50 Imaging Manager through Leica MZ FLIII microscope a PLAN APO lens. A Leica DC500 CCD camera with IM50 Imaging Manager through Leica DMRXA microscope was used to capture the images using a PLAN APO 1.0/0.125 NA lens (panel A; total magnification, ×32), a 5×/0.15 NA objective (panel B) or a 20×/0.50 NA objective (panels C-G).
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology

4. Gastrulating embryos express PECAM-1, VE-cadherin, CD34, endoglin, and Tie2 in the extraembryonic mesoderm layer.
Gastrulating embryos express PECAM-1, VE-cadherin, CD34, endoglin, and Tie2 in the extraembryonic mesoderm layer. (A) GFP expression in Flk1-GFP knock-in embryos at the late-streak stage. Line shows a transverse section used for immunohistochemistry (PLAN APO lens, 1.0×/0.125; final magnification ×80). (B-D) Immunohistochemical analysis of a transverse section as shown in panel A using anti-GFP antibody (B, green), anti-Flk1 antibody (C, red), and the merged image (D). Note that GFP expression driven by the endogenous Flk1 locus recapitulates endogenous Flk1 protein expression. (Arrow) Boundary between the extraembryonic and embryonic regions. Boxed area in panel D is magnified in panel E. (F) Schematic representation of PECAM-1, GATA1, and Flk1 expression. (G-J) Immunohistochemical staining of a transverse section as shown in panel A using anti-GFP antibody (G, green), anti–PECAM-1 antibody (H, red), and the merged image (I). Note that PECAM-1 is expressed in extraembryonic mesoderm cells contacting the visceral endoderm layer from the boundary between the extraembryonic and embryonic regions. Boxed area in panel I is magnified in panel J. (K) Control embryo section stained with secondary antibodies alone. (L-O) Immunohistochemical staining of a transverse section as shown in panel A using anti-GFP antibody (L, green), anti-GATA1 antibody (M, red), and the merged image (N). (O) Higher magnification of GATA1-positive cells in panel N. Note that GATA1 is expressed in a small subset of extraembryonic mesoderm cells, in contrast to the broader spectrum of PECAM-1 expression. (P) CD34 expression in the extraembryonic mesoderm layer. (Q) Tie2 expression in the extraembryonic mesoderm layer. (R) Endoglin expression in the extraembryonic mesoderm and ectoderm layers. (S) VE-cadherin expression in the extraembryonic mesoderm layer. Arrowheads indicate Flk1-positive cells expressing either PECAM-1, GATA-1, CD34, Tie2, endoglin, or VE-cadherin. Images in panel A were captured with a Leica MZ FLIII microscope and a Leica DC500 CCD camera with IM50 Imaging Manager, with a PLAN APO 1.0 lens (1.0×/0.125 NA). Images in panels B-S were captured by Leica DC500 CCD camera with IM50 Imaging manager and a Leica DMRXA microscope using a 20×/0.50 NA objective.
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology

5. GATA1-positive cell population exists as a subset of EC marker–positive cell.
GATA1-positive cell population exists as a subset of EC marker–positive cell. (A) GFP expression in GATA1-GFP embryos at no-bud (OB) stage. Line shows a transverse section used for immunohistochemistry (PLAN APO lens, 1.0×/0.125; final magnification, ×63). (B) Immunohistochemical analysis of a transverse section as shown in panel A using anti-GFP antibody shown in green and anti–PECAM-1 antibody shown in red. Merged image is shown. (C) High magnification of the blood island region shown in panel B, Hoechst staining, anti–PECAM-1 antibody, anti-GFP antibody, and the merged image. (white arrows) Cells expressing GFP but not PECAM-1. (green arrows) Cells expressing PECAM-1 but not GFP. Note that GFP (GATA1) is expressed in a subset of extraembryonic mesoderm cells, in contrast to the broader spectrum of PECAM-1 expression. (D) Flow cytometric analysis of GATA1-GFP embryos (LS-OB) with GFP and cell surface markers VE-cadherin, PECAM-1, or Tie2. Note that GATA1 (GFP)–positive cells coexpress VE-cadherin and Tie2. Images were captured by a Leica DC500 CCD camera with IM50 Imaging Manager. Images in panel A were captured through a Leica MZ FLIII microscope using a PLAN APO 1.0 lens. Images in panels B and C were taken with a Leica DMRXA microscope using a 5×/0.15 NA objective (B) or 20×/0.50 NA objective (C).
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology

6. Primitive erythropoietic progenitors coexpress PECAM-1, VE-cadherin, Tie2, CD34, and endoglin.
Primitive erythropoietic progenitors coexpress PECAM-1, VE-cadherin, Tie2, CD34, and endoglin. (A) Lateral view of a mouse embryo at the EHF stage. Line shows a transverse section (PLAN APO lens, 1.0×/0.125; final magnification, ×40). (B-E) Immunohistochemical staining of a transverse section as shown in panel A using Hoechst (B blue), anti-Flk1 antibody (C, red), anti-ϵyglobin antibody (D, green), and the merged image (E). (F-K) High magnification of the blood island region stained with Hoechst (blue), anti-ϵyglobin antibody (green), anti-Flk1 antibody (F, red), anti–VE-cadherin antibody (G, red), anti–PECAM-1 antibody (H, red), anti-Tie2 antibody (I, red), anti-CD34 antibody (J, red), and antiendoglin antibody (K, red). Note that the expression of VE-cadherin, PECAM-1, Tie2, endoglin, and CD34 is observed in globin-producing cells but that the expression of Flk1 is decreased in globin-producing cells. The scale bar indicates 50 μm. Images were captured with a Leica DC500 CCD camera with IM50 Imaging Manager. The image in panel A was taken through a Leica MZFLIII microscope using a PLAN APO 1.0 lens. Images in panels B-K were taken with a Leica DMRXA microscope using a 5×/0.15 NA objective (panels B-E) or a20×/0.50 NA objective.
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology

7. Primitive erythropoietic progenitors are enriched in PECAM-1– and Tie2-positive cells.
Primitive erythropoietic progenitors are enriched in PECAM-1– and Tie2-positive cells. (A) Flow cytometric analysis of a 7.75 dpc wild-type embryo at the late–head fold (LHF) stage using anti–PECAM-1 or Tie2 antibody. (B) Primitive hematopoietic colony assay. Cells from fractions positive or negative for the expression of endothelial markers shown in panel A were subjected to the primitive hematopoietic colony assay.
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology

8. Tie2 and GATA1 expression in transgenic mice
Tie2 and GATA1 expression in transgenic mice. β-Galactosidase activities driven by the transgene recapitulate endogenous Tie2 and GATA1 protein expression, respectively, in the Tie2-LacZ and GATA1-LacZ transgenic mice. ex indicates extraembryonic region; em...
Tie2 and GATA1 expression in transgenic mice. β-Galactosidase activities driven by the transgene recapitulate endogenous Tie2 and GATA1 protein expression, respectively, in the Tie2-LacZ and GATA1-LacZ transgenic mice. ex indicates extraembryonic region; em, embryonic region. Note that the pattern of Tie2 expression visualized by β-gal activity is similar to the pattern observed after immunohistochemical analysis using an anti-Tie2 antibody. Scale bars indicate 50 μm. All images were captured with a Leica DC500 CCD camera with IM50 Imaging Manager through a Leica MZ FLIII microscope using a PLAN APO 1.0×/0.125 lens (final magnification, ×32 for embryos at somite stages; ×40 for embryos at headfold stages; and ×63 for embryos at streak and neural plate stages).
Masatsugu Ema et al. Blood 2006;108:
©2006 by American Society of Hematology