Figure 6.

MP partially restores basal levels of TUNEL+ cells and neurogenesis. (A) (a–l) Otic vesicles were isolated from HH18 chicken embryos and incubated for 17 h in the 0S condition. Then, they were cultured for 3 h in the presence or absence of 3-MA (10 mM), MP (10 mM) or a combination of both. Otic vesicles were incubated with 1 μM LTR (red) for the last 15 min of culture and apoptotic cell death was visualized by TUNEL (green). (c′, f′, i′ and l′) Higher magnification of the selected regions shown in the boxed areas of c, f, i and l, respectively. (m) TUNEL-positive cells and (n) LTR-positive cells were quantified with Image Analysis Software (Olympus, Tokyo, Japan) in at least five otic vesicles per condition. The results are shown as the mean±S.E.M. relative to the 0S condition. Statistical significance was estimated with the Student's t-test: *P<0.05, **P<0.01 and ***P<0.005 versus 0S and ^##P<0.01, ^###P<0.005 versus 3-MA. (B) (a–c) HH18 otic vesicles were incubated for 20 h in the 0S condition, with 3-MA (10 mM), MP (10 mM), or a combination of both. Whole otic vesicles were then immunostained for Islet-1 (green) and TuJ-1 (red) to label neuroblast nuclei and the neural processes, respectively. (d) AVG and OV areas were quantified (as described in the Materials and Methods section) and referred to the total area to compare the AVG size under the different culture conditions. The results were normalized to the 0S condition, which was given an arbitrary value of 100. The bars show the mean±S.E.M. of at least five otic vesicles from any of the conditions shown. Statistical significance was estimated with the Student's t-test: **P<0.01, and ***P<0.005 versus 0S and ^###P<0.005 versus 3-MA. Compiled projections of confocal microscopy images from otic vesicles are shown. The images shown are representative of at least three independent experiments. Orientation: A, anterior; D, dorsal. Bars=150 μm