Fig. 7. Late pharmacological inhibition of ATP4 causes loss of MCCs and decreased mucociliary clearance.

Immunofluorescent analysis (A–D) of MCC ciliogenesis (acetylated-α-tubulin, blue) and serotonin (5-HT; red) deposition in small secretory cells (SSCs) of the skin at stages 35 (AB) and 41 (C–D). Actin (phalloidin-Alexa488) staining in green. Embryos were treated with DMSO as control or SCH28080 during the indicated stages. Stage 35 (A) and stage 41 (C) DMSO treated controls displayed fully ciliated MCCs (A′, C′) and the presence of large numbers of serotonin positive SSCs (A″, C″). At stage 35 no apparent defects in MCC ciliation (B′) or SSC serotonin deposition (B″) were observed in SCH28080 treated embryos (cf. Quantification in Fig. S3K), although 50% of specimens contained MCCs with a relatively small apical surface area and 60% of specimens were abnormal in their epithelial morphology, i.e. enlarged cells were present (cf. Quantification in Fig. S3K). In contrast, SCH28080 treatment until stage 41 lead to a massive loss of MCCs (D′), but no effect on SSCs (D″). (E) Quantification of cilia-driven fluid flow velocities revealed a significantly reduced extracellular fluid flow at the epidermis at stages 35 and 41, as compared to DMSO treated controls of the same stage. (F–G) High-magnification confocal imaging on the same set of specimens depicted in (C and D) confirmed the lack of MCCs, but intact SSCs in SCH28080 treated embryos and further indicated apical expansion in ISC-like cells and SSCs as well as enriched actin staining at some cell junctions (G). MCCs and epithelial morphology appeared normal in DMSO treated controls (F). (H–I) Embryos were treated with DMSO or SCH28080 from stage 24–40 and foxj1 expression was analyzed by in situ hybridization. In comparison to controls (H), foxj1 staining was increased in SCH28080 treated tadpoles (I).

n, number of embryos; st., stage.