EEC mitochondrial activity changes during development. (A) Schematic showing in vivo imaging to trace the EEC cytoplasmic and mitochondrial Ca2+ in the same zebrafish from 3 dpf to 6 dpf. The commensal microbiota colonizing conventionally raised zebrafish was used. EECs from nine zebrafish were traced and analyzed. (B-E″) Confocal projections of the same Tg(neurod1:Gcamp6f); Tg(neurod1:mitoRGECO) zebrafish at 3 dpf, 4 dpf, 5 dpf, and 6 dpf. The EEC cytoplasmic Ca2+ level is represented via the Gcamp6f fluorescence (green). The EEC mitochondrial Ca2+ level is displayed through mitoRGECO fluorescence (magenta). (F-H) EECs from the same zebrafish were analyzed from 3 dpf to 6 dpf. EEC mitochondria-to-cytoplasmic Ca2+ ratio, cytoplasmic Ca2+ and mitochondrial Ca2+ were quantified. (I-K) Pooled EECs from nine zebrafish were analyzed from 3 dpf to 6 dpf. Mitochondria-to-cytoplasmic Ca2+ ratio, cytoplasmic Ca2+ and mitochondrial Ca2+ were quantified. Each dot in F-K represents an individual EEC. Dashed lines indicate the changes of EEC cellular and mitochondrial calcium activity from 3 dpf to 7 dpf. Data are mean±s.d. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (one-way Anova followed by Tukey's post test). Scale bars: 50 μm.
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