Comparison of Klf6^-/-, Klf6^+/-, and Klf6^+/+ embryos. (A) Ten random E 10.5 embryos derived from Klf6^+/- × Klf6^+/- crosses were classified according to size and color and genotypes by PCR for Klf6 and neomycin. Klf6^-/- embryos are small and pale (see also E-F), whereas no significant differences were detected between Klf6^+/- and Klf6^+/+ embryos. Compared with the appearance at E 10.5 of the Klf6^+/+ yolk sac (B) and embryo (D), Klf6^-/- yolk sac (C) and embryos (E) are smaller and paler, and vascular structures in the Klf6^-/- yolk sac are poorly developed. The Klf6^-/- embryo lacks a definable liver (arrow), although a beating heart was observed at the time of death (not shown). (Scale: bar = 1 mm.) Sections of yolk sacs reveal that Klf6^-/- yolk sacs (G) have fewer hematopoietic cells than Klf6^+/+ yolk sacs (F). Whole-mount immunohistochemistry of yolk sacs using anti-PECAM1 demonstrates that vascular formation in Klf6^-/- yolk sacs (I) is much less organized than KLlf6^+/- yolk sacs (H), while expression of PECAM1 is apparent. Images in Figure 2B-E were obtained using a Leica MZ APO stereomicroscope (Leica Microsystems, Bannockburn, IL) equipped with a 1 ×/0.09 objective lens at 12 × (B,D) or 25 × (C,E) total original mangification. A Sony 3CCD DXC-970MD camera (Sony, New York, NY) and NIH Scion public domain software (National Institutes of Health, Bethesda, MD) were used to photograph and capture images. Images in Figure 2F-I were obtained using a Nikon Eclipse E300 microscope equipped with a 40 ×/0.75 objective lens (Nikon Instruments, Melville, NY); they were photographed with a Spot RT Slider camera and then processed with Spot Image Capture for Mac OS 3.5.4 software (Diagnostic Instruments, Sterling Heights, MI).