Figure 6. Commensal microbiota colonization alters the resting EEC cytoplasm and mitochondria calcium activity.

(A) In vivo imaging to analyze the 7dpf GF and CV zebrafish EEC cellular and mitochondrial activity. (B-C”) Confocal projection of the GF and CV Tg(neurod1:Gcamp6f); Tg(neurod1:mitoRGECO) zebrafish. Note that the absolute EEC Gcamp fluorescence is relatively higher in GF EECs. The EEC mitoRGECO/Gcamp ratio is lower in GF EECs. The white arrows in B” and C” indicates the EECs with higher mitochondrial activity near the base membrane. (C-D) zoom in view shows representative EECs in GF (C) and CV (D) zebrafish mitochondrial calcium activity. The CV EEC in D displayed high mitochondrial calcium near the base membrane (white arrow). (D-F) Quantification of absolute Gcamp, mitoRGECO, and mitoRGECO/Gcamp ratio in GF and CV zebrafish proximal intestinal EECs. Each dot represents an EEC. More than five zebrafish were analyzed in each condition. (G-M) Analyze the relative EEC Gcamp, EEC mitoRGECO, and EEC mitoRGECO/Gcamp ratio in GF and CV zebrafish on a temporal scale. The EEC Gcamp, EEC mitoRGECO, and EEC mitoRGECO/Gcamp ratio at each time point were normalized to t0. These quantification data sets reveal the dynamic change of EEC cellular and mitochondrial activity in GF and CV conditions. Each line in G-M represents an individual EEC. The red circles in G-M indicate the EECs that exhibit Gcamp fluorescence fluctuation. These EECs that display dynamic Gcamp fluorescence fluctuation were also referred to as active EECs in J and N. (J-N) Quantification of the percentage of the quiet and active EECs in GF and CV zebrafish. Student T-test was used in F for statistical analysis. * P<0.05, ** P<0.01.