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. 2018 Jun 8;9:2232. doi: 10.1038/s41467-018-04675-x

Fig. 3.

Fig. 3

Larval LNs undergo pruning or cell death during metamorphosis. a (Top) Scheme for Kaede photoactivation. At 24 h APF, stimulated LNs appear with both newly synthesized 488 nm-excited and 560 nm-excited Kaede (yellow). (Bottom) Images from live 24 h APF pupal brains are shown. LNs without 380 nm stimulation only express 488 nm-excited Kaede (100%, n = 8 ALs, left panels), while 380 nm-stimulated larval LNs expressed both 488 and 560 nm-excited Kaede (90% analyzed ALs) or only 560 nm-excited Kaede (10%) (n = 10 ALs, right panels). Arrowheads indicate LNs. Dashed lines indicate brain midlines. b Scheme of two binary systems that together drive myr::smGdP reporter expression exclusively in larval LNs in pupal brains. c (Top) Flies raised at 18 °C exhibited no GFP-labeled NP3056-LNs in larval and 24 h APF pupal brains (n = 37 and 44 ALs, respectively). (Bottom) Flies raised at 29 °C from embryonic stages and then shifted to 18 °C at puparium formation exhibited up to 2 or 3 NP3056-positive larval LNs in larval (1.1 ± 0.1 smGdP+ LNs, n = 52 ALs) and 24 h APF pupal brains (2.0 ± 0.1 smGdP+ LNs, n = 30 ALs). d Compared to 24 h APF control brains (n = 23 brains), blocking ecdysone signal in NP3056-positive larval LNs leads to pruning failure (n = 35 brains). e 670-positive larval LNs. (Top) 12 h APF (n = 10 brains) and 36 h APF (n = 20 brains) pupal brains of flies raised at 18 °C. (Bottom) 12 h APF (n = 17 brains) and 36 h APF (n = 15 brains) pupal brains of flies raised at 29 °C from embryonic stages and then shifted to 18 °C at puparium formation. f 24 h APF pupal brains were stained for cleaved Caspase 3 and are shown as a projection of stacks (upper panel) or a single confocal section (bottom panels). Arrows indicate 670-positive larval LNs that express cleaved Caspase 3. g 36 h APF pupal brains from control flies (n = 36 ALs) and 670 > p35 flies (n = 20 ALs). Presumably larval LNs (arrowheads) were only found in 670 > p35 brains. h Quantification of presumed larval LN numbers in g (mean ± s.e.m.). i Model of LN development