Fig. 1.

Impaired recovery of acidic-lysosome function and mucin production after repeated DSS injury in zebrafish intestines. (A) Dose-dependent mortality observed after repeated DSS injury (N=1739 from 11 clutches). Compared to the single DSS injury, which has a 98-100% survival rate (day 6), repeated DSS injury induces a high mortality rate of 63% in 0.25% DSS and 29% in 0.1% DSS. (B) Hematoxylin and eosin (H&E) staining of a longitudinal section of zebrafish larvae intestine. A total of 20 larvae per experimental condition were embedded in paraffin and sectioned at 10 μm per slide. Scale bars: 100 μm. (C,D) Quantification of Neutral Red images (top panels) and Neutral Red accumulation (bottom panel) for single (C) and repeated (D) DSS-injured zebrafish. Neutral Red accumulation in the intestine indicates normally functioning acidic lysosomes. The damage to lysosomal function is fully recovered within 2 days with the single DSS injury (C; N=253 from four clutches) but is impaired with the repeated DSS injury (D; N=137 from three clutches; day 2 N=5 out of 13 from single clutch due to high mortality rate of 61.5%). Neutral Red images for single (C) and repeated (D) injury are shown as control, and 0.1% and 0.25% (w/v) DSS treatments at treatment times of 1 day and 2 days after injury. Scale bars: 100 µm. ***P<0.001, **P<0.01, *P<0.05. Error bars, mean±s.e.m. (E) Images (left panel) and quantification (right panel) of Alcian Blue staining of intestine with single and repeated injury with control and 24 h after removal from DSS treatment. The quantification of total Alcian Blue intensity from single (N=160 from three clutches) and repeated (N=154 from three clutches) injury is shown. There was an increase in mucin production after single DSS injury, but mucin production was impaired after repeated DSS injury. Scale bars: 100 µm.