Fig. 3

Immunohistochemistry of H⁺-V-ATPase subunit A. Immunohistochemistry with an alkaline phosphatase conjugated secondary antibody for subunit A of the ⁺-V-ATPase. Embryos were oriented based on pigmentation and cleavage patterns - a technique that consistently reveals biased asymmetry among the L and R blastomeres with respect to a number of properties (Fukumoto et al., 2005b). Positive signals are blue to purple; LR orientation of embryos is only possible in four- and eight-cell embryos. (A) The molecular motor (Gross et al., 2002) myosin V (AV section i.e. parallel to AV axis) and (B) the ciliary protein (Bonnafe et al., 2004) RFX3 (flat section, i.e. perpendicular to the AV axis) are examples of symmetrically distributed proteins. (C) Western blot showing that the antibody detects a single band of approximately the right size (predicted: 69 kDa) for subunit A. Green and white arrows indicate positive and a lack of signal, respectively. (D-H) Immunostaining for subunit A. (D) Two-cell embryo, AV section, showing one common staining pattern: `fingers' reaching animal-ward from the pool in the vegetal cytoplasm. Although this pattern is not exclusive to H⁺-V-ATPase subunits (Qiu et al., 2005), it is not found for many proteins (e.g. compare with A). (E) Flat section of a two-cell embryo showing another common staining pattern in which one blastomere is more heavily stained than its contralateral counterpart. (F) The asymmetry in staining seen in the flat sections is still visible at the four-cell stage when it is right-sided. (G) A flat section through a latrunculin-treated (actin depolymerized) embryo fixed at the four-cell stage, showing disruption of the normal pattern and loss of asymmetry in subunit A staining (compare with F). (H) A flat section through a nocodazole-treated (microtubules depolymerized) embryo showing that localization of subunit A can appear relatively unchanged by depolymerization of microtubules (compare with F).