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Figure 5 (A–B) Larvae were stained with sox2 antibody and atoh1a RNA probe at 6 hr post neo or LY+neo treatment. The number of atoh1a+sox2+ cells was significantly increased in LY+neo group. (C–D) The atoh1a:TdTomato larvae were used to trace atoh1a+ HC precursors in ctrl, LY or LY+neo. Results showed that Tomato+ cells labeled partial ET4+ HCs and ET4-sox2- HC precursors (yellow arrows). However, many atoh1a+ cells start to express sox2 from 12 hr post LY+neo and their numbers were significantly increased compared with normal larvae. The arrowheads in (C) pointed the atoh1a+sox2+ cells at 48 hr post LY+neo. (E) The atoh1a:TdTomato;hs:lin28a larvae was treated with LY+neo and verteporfin and collected for immunostaining with sox2 antibody. The cell number of Tomato+sox2+ is decreased in verteporfin and overexpression of lin28a could rescue the phenotype. (F–G) The LY-treated hs:lin28a larvae were heat-shocked to overexpress lin28a. Samples were collected for staining with sox2 antibody and atoh1a RNA probe. The number of atoh1a+sox2+ cells was significantly increased in hs:lin28a group, indicating that lin28a is sufficient to express sox2 in atoh1a+ HC precursors. Scale bar equals 10 μm.