Fig. 6. In situ hybridization for sonic hedgehog and Nodal, and pH imaging, in concanamycin-treated chick embryos. Embryos exposed to vehicle alone exhibited the normal left-sided expression of Shh (A) and Nodal (C). When exposed to the H⁺-V-ATPase inhibitor concanamycin during early streak stages, the left-sided expression becomes destabilized (see Table 4). Examples include bilateral expression of Shh (B) or Nodal (D). Culture of chick embryos can lead to bending of the AP axis such as that of embryo in D. In our experiments, about 15% of both control and treated embryos show this bend. Green arrows indicate normal position of staining; red arrows indicate ectopic expression domain. (E-H) Embryos treated with the pH indicator cSNARF-5F. Anterior is upwards and left is leftwards in all images. White dots indicate the approximate boundaries of the primitive streak. (E,F) Transmitted light images of two embryos. (G) Pseudocolored image of ratiometric data representing pH, shown in control embryo E. In most images, pH of the primitive streak is somewhat lower than pH of the surrounding area pellucida (AP) cells; the degree of contrast varies and is somewhat low in the image shown. (H) In concanamycin-treated embryos, the primitive streak cells (which stain positively for H⁺-V-ATPase subunits; see Fig. 5E-H) are at a lower pH than control cells (purple compared to green; embryo F). The AP, by contrast, is at a higher pH than controls. The difference between pH of the primitive streak and pH of the AP is much more pronounced in concanamycin-treated embryos.