Fig. 4

tUAS is not silenced across generations of two reporter lines. One adult from each F1-F4 generation of the Tg(CV,tUAS:tRFP)^w80 reporter line or F1-F3 generations of the Tg(CV,tUAS:nlsEos)^w81 reporter line was crossed to an F2 or F3 adult from Tg(CC,myo6b:nlsTrpR-G4AD)^w83. Progeny double positive for the lens markers were sorted out and stained for Parvalbumin, which labels mature hair cells. Head neuromasts, which consist of hair cell clusters, were scored for tRFP (magenta) and Parvalbumin (green) expression (A-D) or nlsEos (green) and Parvalbumin (magenta) expression (F-H). In each case, 5 neuromasts from each of 5 embryos at 5 dpf were analyzed producing a total of 25 total neuromasts. (A-D) A representative neuromast across four generations Tg(CV,tUAS:tRFP)^w80 showing tRFP and Parvalbumin (Parv) expression. In each case, the representative neuromast was located over the otic vesicle. (E) The ratio of tRFP/Parv positive hair cells was not statistically different across the generations of the reporter line, showing that tUAS is not silenced. One-way ANOVA, F=0.7655, R²=0.02336, P=0.5161. Tukey?s pairwise column comparison post-test shows no significant differences between any of the columns. (F-H) A representative neuromast showing nlsEos and Parv expression across three generations of Tg(CV,tUAS:nlsEos)^w81. (I) The ratio of nlsEos/Parv positive hair cells was always equal or above 100%, showing that across generations all mature cells (Parv positive) also expressed nlsEos. Because Parvalbumin is a late marker for mature hair cells, some nlsEos-positive cells were Parv negative, which most likely indicates the presence of immature hair cells in these samples. Error bars show s.e.m.