Figure 3.
Figure 3. 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.

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.

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