|
Fig. 6 deltaA expression and AA in mother NSCs predict NSC decisions independently of Notch signaling. (A) LY was added to the fish water for 7 days. Fish were imaged at days 1, 3, 5, 6, and 7 of treatment. (B) Merged and split snapshots, maximum projections (dorsal views) of a live-imaged 3-mpf casper;Tg(deltaA:eGFP);Tg(gfap:hZO1-mKate2) fish showing progressive NSC reactivation upon LY treatment (gfap:ZO1-mKate2: yellow, deltaA:GFP: cyan). Scale bars, 20 μm2. (C) Number of cells per segmented region (n = 3 fish, mean and error bars for SEM), normalized to the value at day −2, as a function of time [see (A)]. (D) Electroporation of deltaA-MO in Tg(gfap:dtTomato) shows no effect on the AA of GFAPpos cells (control MO: n = 123 cells, deltaA-MO: n = 55 cells). Statistics: Nonparametric t test (Mann-Whitney), P value ns, median in red. (E) Time window of reliable NSCs tracking upon LY treatment. (F) Logistic regression: Probability of NSC division as a function of AA for each MC deltaA status; compared untreated (CTRL) and LY conditions. Notch inhibition abolishes the differences in activation rate between deltaApos and deltaAneg NSCs (P = 0.16, χ2 test), but the AA effect persists. Predictions for CTRL: Rescaling of the rates over 4 days from all divisions over the duration of the movies; predictions for LY: estimated using all divisions over 4 days (see fig. S6A). Individual regressions pooled for this analysis: see (E). (G) Merged and split time-lapse images of a live-imaged 3-mpf casper;Tg(deltaA:eGFP);Tg(gfap:hZO1-mKate2) fish highlighting dividing NSCs (colored stars to MCs and their progeny) and nondividing NSCs (white dots) during the first and third days of LY treatment. Scale bars, 10 μm2. (H) Division modes based on deltaA expression in DCs, for divisions occurring after 3 days of LY (n = 3 fish) or in CTRL (n = 4 fish).