20E-induced programmed cell death of putative RP2s in culture. Sister cultures prepared at 8 h-APF were treated with medium with 6 μg/ml 20E (+20E) or no hormone (-20E). On day 0, putative RP2 neurons were identified by GFP expression and a similar-appearing neuron in the same optical field was selected to serve as a control for each putative RP2. At 48 or 72 h (independent experiments with four and two pairs of sister cultures, respectively) neurons were scored as dead or alive by morphological criteria. (A, B) Photomicrographs show putative RP2s at 0 h (left panels) and 48 h (right panels) in culture, under phase contrast (left) and GFP epifluorescence (right) optics. When multiple neurons were present, arrows mark putative RP2s. At 0 h, all putative RP2s had smooth, ovoid somata with well-defined nuclei. (A1, A2). In the absence of 20E, putative RP2s were still alive at 48 h, with GFP labeling and neurite outgrowth (B1, B2). In the presence of 20E, putative RP2s were dead by 48 h, as determined by morphological criteria; the neuron in (B1) was shrunken and phase-bright but retained GFP fluorescence, while that in (B2) was shrunken, had a fragmented outline and faint GFP fluorescence, and the nucleus was reduced to a small spot. (C) Histograms show percentage of neurons alive at the time indicated; n given above each bar. (C1) The percentage of control neurons alive at 48 or 72 h did not differ significantly between +20 and -20E cultures. (C2) At both 48 and 72 h, significantly fewer putative RP2s survived in cultures containing 20E (*P < 0.05, **P < 0.01, two-tailed Chi square test with Yates continuity correction). Scale bars: 10 μm.