Fig. 6

Inhibition of Aldh1 enzymatic activity increases the number of Aldh1- and NeuroD-expressing endocrine progenitor cells and induces their premature differentiation.

Panels to the right of merged images represent corresponding single channel images. (A and B) NeuroD:eGFP/Tp1:hmgb-mCherry fish were treated with DMSO (A) or DEAB (B) at 72-120 hpf. DEAB treatment increased the number of Notch-responsive cells also expressing NeuroD:eGFP (red arrows). (C) Quantification of the number of eGFP^pos and/or mCherry^pos cells in 1000 µm³ of 2F11^pos tissue in NeuroD:eGFP/Tp1:hmgb-mCherry fish treated with DMSO or DEAB between 72-120 hpf. Inhibition of Aldh1a induced NeuroD:eGFP expression in Notch-responsive cells. Values indicate mean + SEM, with minimum of 20 larvae analyzed for each condition. (*) indicates p<0.05. (D) Quantification of the number of Aldh1-expresing cells in 100,000 μm³ of 2F11^pos tissue in NeuroD:eGFP/Tp1:hmgb-mCherry fish treated with DMSO or DEAB treatment between 72-120 hpf. Values indicate mean + SEM, with minimum of 20 larvae analyzed for each condition. (*) indicates p<0.05. (E and F) NeuroD:eGFP/Tp1:hmgb-mCherry fish were treated with DMSO (E) or DEAB (F) at 18-20 dpf. DEAB treatment increased the number of Notch-responsive cells also expressing NeuroD:eGFP (red arrows), and these cells were frequently adjacent to Aldh1-expressing cells (blue arrows). (G and H) Pax6b:eGFP/Tp1:hmgb-mCherry fish were treated with DMSO (G) or DEAB (H) between 18-20 dpf. DEAB treatment activated Pax6:eGFP expression in Aldh1-expressing cells. (I) Quantification of number of eGFP^pos and/or mCherry^pos cells in 1000 µm² of 2F11^pos tissue in NeuroD:eGFP/Tp1:hmgb-mCherry or Pax6b:eGFP/Tp1:hmgb-mCherry fish treated with DMSO or DEAB between 18-20 dpf. NeuroD:eGFP, but not Pax6:eGFP expression is activated within Notch responsive cells following DEAB treatment. In contrast, Pax6:eGFP but not NeuroD:eGFP expression is induced in cells not expressing Tp1:hmgb1-mCherry. (*) indicates p<0.05, with n=8 fish per condition and a minimum of 3 sections analyzed per fish. (J) Quantification of Aldh1-expressing cells also expressing either NeuroD:eGFP or Pax6:eGFP following treatment with either DMSO or DEAB between 18-20 dpf. DAEB treatment increases the number of cells Aldh1-expressing cells also expressing either NeuroD:eGFP or Pax6:eGFP. (*) and (**) indicates p<0.05 and p<0.01, respectively. n=10 fish per condition and a minimum of three sections analyzed per fish.