S5 In addition to rescue experiments using DNA co-injection, specificity of the atp4aMO was confirmed by (a) atp4a knockdown using a second non-overlapping translation blocking MO (atp4aMO-2; Fig. S5G, H and K), (b) a splice-site MO (atp4a-SplMO; Fig. S5I–K) targeting the second exon/intron boundary of atp4a pre-mRNA, which lead to atp4a intron 2 retention as confirmed by PCR (see (Walentek et al. 2015, Fig. 4)), and (c) specific pharmacological inhibition of ATP4 using SCH28080 (Fig. S5A, B and K; Walentek et al., 2012). Each of these treatments caused MCC ciliation defects comparable to atp4aMO injections, without significant decrease in tubulin-enriched epidermal cells ( Fig. S5K). ... Our experimental results argue strongly for a role of ATP4 during development of the Xenopus mucociliary epidermis, thereby suggesting a potential role for ATP4 in mucociliary clearance in the mammalian airway epithelium as well. In contrast to our early interventions in the embryo, PPIs are administered to adult patients after development of the airway epithelium. In order to address how pharmacological inhibition of ATP4 in mature mucociliary epithelia could influence mucociliary clearance, we performed experiments in which SCH28080 was applied at different stages of epidermal development and function. Both, MCC ciliation and serotonin deposition were analyzed as readout (Figs. S5A–F and K and 7). When applied early (stage 5–30), SCH28080 incubations had essentially the same effects on MCCs and SSCs as MO-mediated knockdown of atp4a ( Figs. S5A, B, G–J and K and 3), while later application of SCH28080 during epidermal development, starting at stages 12 or 22, had statistically significant but less pronounced effects on both MCC ciliation (p<0.001; Fig. S5C–F and K) and number of SSCs (p<0.01/p>0.05; Fig. S5C–F and K). These data indicated that ATP4 mainly plays a developmental role in MCC ciliation and specification of SSCs in the embryonic epidermis. In line with this assumption, SCH28080-mediated inhibition of ATP4 starting at stage 29, i.e. when functional MCCs and SSCs were already present, did not significantly affect ciliation or serotonin deposition in MCCs and SSCs, respectively (Figs. S5K and 7A and B). Nevertheless, a quantitative analysis of extracellular fluid flow (Walentek et al., 2014) revealed significantly reduced velocities of automatically tracked fluorescent beads at stage 35 in tadpoles treated with SCH28080 from stage 29 onwards (p<0.05; Fig. 7E). Prolonged incubation of tadpoles in SCH28080-containing media until stage 41 further decreased extracellular fluid flow velocities, resulting in an almost complete loss of mucociliary clearance (p<0.001; Fig. 7E). Interestingly, tadpoles treated with SCH28080 from stage 30–41 (~72 h) displayed a massive loss of MCCs (ciliated MCCs as well as tubulin-enriched cells) in the epithelium, while serotonin-containing SSCs were not affected (Fig. 7C and D). Analysis of epidermal morphology using high-resolution confocal microscopy further revealed loss of cells containing a MCC-specific apical actin meshwork, apical enlargement of ISC-like cells as well as SSCs, and accumulations of actin at cell junctions (Fig. 7F and G). This phenotype suggested that MCCs were lost in the epithelium, leading to a decrease in apical surface tension and, therefore, allowing other intercalating cell types to expand their apical surface area.
Image published in: Walentek P et al. (2015)
Copyright © 2015. Image reproduced with permission of the Publisher, Elsevier B. V.
| Gene | Synonyms | Species | Stage(s) | Tissue |
|---|---|---|---|---|
| tuba4b.L | alpha tubulin, alpha-tubulin, tuba4, tuba4a | X. laevis | Throughout NF stage 29 and 30 | ciliated epidermal cell |
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