H+-V-ATPase subunits and heterotaxia in zebrafish. (A-F) Immunohistochemistry for H+-V-ATPase subunits in zebrafish embryos: (A) subunit F in a two-cell embryo; (B) subunit c in a four-cell embryo; (C) subunit F in an eight-cell embryo. Brown (HRP-conjugated secondary) indicates a positive signal. At these stages, the cortical cytoplasm of all cells is positive for these two subunits, as is the cortical cytoplasm of the yolk cell. (D) Thirty-two-cell zebrafish embryo stained for subunit c. Staining (blue, AP-conjugated secondary) is heaviest in the marginal cells of this stage, but present in all cells. (E) Early in epiboly, subunit c staining is dark in the cells of the spreading blastoderm. In addition, staining is visible around a ring of yolk syncitial nuclei, probably representing vesicle staining (red arrowhead). (F) Once epiboly is more than 50% complete, immunohistochemistry for subunit c shows an even and heavy distribution in all the cells of the blastoderm. The antibodies used in this figure did not work in western blots on chick and fish extracts. (G-I) Heterotaxia in zebrafish larvae. Tricaine-anaesthetized 5- to 6-day larvae were examined on a Zeiss StemiSV11 dissecting microscope under 488/40 nm illumination, using a 510 nm barrier filter. An embryo was considered heterotaxic if either pancreas (orange arrowheads), gall bladder (red arrowheads) or both organs were on the side opposite normal. (G) Normal position of gall bladder (right) and pancreas (left). (H) Heterotaxia involving the pancreas. (I) Heterotaxia involving both organs. When scoring two organs, the top heterotaxia rate is 75% as, by chance, organ situs will appear to be wild type in 25% of fully randomized embryos. (J-L) Whole-mount in situ hybridization for the nodal-related gene southpaw (Spaw), which is expressed in the left lateral plate mesoderm (LPM) and tailbud of wild-type embryos from approximately 15-somite to 22-somite stage. (J) Untreated embryos at 20-22-somite stages showed wild-type Spaw expression in the left LPM (green arrow). Spaw expression in the tail bud (an internal positive control for the specificity of the treatment on Spaw asymmetry) is indicated by a blue arrowhead. Similarly staged embryos treated early with the H+-V-ATPase inhibitor lobatomide A16 exhibited reversed (K, red arrow) and bilateral (L, green and red arrows) Spaw expression (Long et al., 2003). See Table 5 for statistical analysis.
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