AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors
Figure 7
AKT inhibition decreases neuroblast populations.
(A) SOX2 expression upon PI3K-AKT inhibition. Otic vesicles were isolated from HH18 chicken embryos, made quiescent and cultured for 20 h in serum-free culture medium without additions (0S, a) or in the presence of IGF-I (10 nM, b), LY294002 (LY, 25 µM, c) or AKTi VIII (AKTi, 50 µM, d). Double immunostaining was carried out for SOX2 (magenta) and for the neuroblast nuclear marker Islet-1 (green). The quantification of SOX2-positive population was performed as described in Materials and Methods). SOX2 is expressed in the otic epithelial neuroblast population (a, arrowhead) and it is significantly increased in presence of IGF-I (b, arrowhead, e) but it is markedly reduced in the presence of either AKTi or LY (c and d, arrowheads, e). Representative images of compiled confocal microscopy projections are shown from at least six otic vesicles per condition from at least three independent experiments. All otic vesicles are orientated in the same way: A, anterior; D, dorsal. Scale bar, 75 µm. The data are shown as the mean ± SEM and the statistical significance between the different conditions was estimated by Student's t-test: *P<0.05, ***P<0.001, versus control (0S). (B) TUNEL labeling and PH3 expression upon PI3K-AKT inhibition. Otic vesicles were isolated from HH18 chicken embryos and they were cultured in the same conditions as in A. Apoptotic cell death was visualized by TUNEL (green) in cultured otic vesicles, and immunostaining for the mitotic marker phospho-Histone-3 was performed (PH3, magenta). Both TUNEL and PH3-positive cells were quantified (as described in Materials and Methods). PH3 levels did not vary with the different treatments, but TUNEL levels decreased markedly in the presence of IGF-I and increased dramatically with either inhibitor. Treatment with IGF-I restored low TUNEL-levels (e). The data are shown as the mean±SEM and the statistical significance between the different conditions was estimated by Student's t-test: *P<0.05, **P<0.01 and ***P<0.001 versus control (0S). (C) Acoustic-vestibular ganglia (AVG) explants were obtained from HH19 embryos and cultured in serum-free medium for 20 h with no additives (0S, a) or in the presence of LY294002 (LY, 25 µM, b) or AKTi VIII (AKTi, 50 µM, c). Immunostaining for TuJ-1 (magenta) and Islet-1 (green) were performed. Compared to 0S (a, arrowhead), the neuronal soma area of the LY and the AKTi-treated AVG is smaller (d). Areas were measured as described in Materials and Methods (d). The data are shown as the mean±SEM and the statistical significance between the different conditions was estimated by Student's t-test: *P<0.05, **P<0.01, versus control (0S). Scale bars, 75 µm. Representative images from compiled confocal microscopy projections of AVG are shown, from at least six otic vesicles or AVG explants per condition studied in at least three independent experiments.