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Fig. 8. Effect of early H⁺-V-ATPase inhibition on Kupffer's vesicle cilia and localization of serotonin. (A) Oblique confocal section through an 8-somite zebrafish larva doubly immunostained for H⁺-V-ATPase subunit A (green) and acetylated tubulin (red). Although subunit A is obvious in cells of the overlying epithelium (Ep), no H⁺-V-ATPase subunits are associated with KV cilia. (B,C) IHC for acetylated tubulin reveals the structure of KV cilia (green arrowheads) in five- to seven-somite stage zebrafish embryos. (B) Untreated embryo showing the characteristic circular field of long straight cilia, (C) KV cilia of five- to seven-somite embryos, soaked in H⁺-V-ATPase inhibitors from the one-cell to shield stage, are often reduced in number, altered in distribution or appear foreshortened relative to controls. (D,E) Immunohistochemistry for serotonin (5-HT, green arrows) in four-cell Xenopus embryos using an antibody previously shown to be specific for 5-HT (Levin, 2004); (D) untreated, (E) incubated in concanamycin from the one-cell stage. At this stage, there is no effect of H⁺-V-ATPase inhibition on 5HT localization (green arrows) or level. (F,G) Immunohistochemistry for 5HT in 32-cell Xenopus embryos; (F) normal pattern of 5HT staining in one cell in an untreated embryo (green arrows); (G) 5HT is absent from the H⁺-V-ATPase inhibited embryo.

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