VEGF and VEGFR2 expression at stage HH6: comparison with ACE. Transverse sections, in the posterior half of a blastodisc, at 24 hours of development (stage HH6) were used both for in situ hybridization with ACE, VEGF, or VEGFR2 ³⁵S riboprobes (A-C, E-H) and for immunohistochemistry with antibodies against ACE (D). (A-B) ACE mRNA levels in the extraembryonic area (ea). (C) ACE expression is restricted to the endoderm (en). Ectoderm (ec) and mesoderm (mes) lateral to the primitive streak (are not labeled. (D) Immunohistochemistry with antibodies against ACE, on the adjacent section, as shown in panel C. (E-F) Pattern of ACE expression in the yolk sac. For comparison, panels G and H show the expression of VEGF and its receptor VEGFR2, respectively. Unlike ACE mRNA, the VEGF and VEGFR2 mRNAs are restricted to the mesoderm both in the extraembryonic and embryonic area. (A,C-E) Bright-field illumination. (B,F-H) Dark-field illumination. (A-B) Original magnification, × 9; type of the objective lenses, EF × 4; numerical aperture, 0.12. (C-D) Original magnification, × 60; type of the objective lenses, PL APO × 25; numerical aperture, 0.70. (E) Original magnification, × 22; type of the objective lenses, EF × 10; numerical aperture 0.26. (F-H) Original magnification, × 22; type of the objective lenses, EF × 10; numerical aperture 0.26. Scale bar, 500 μm (A-B), 100 μm (G-H), 200 μm (C-F).