Delayed repression of non-neural transgene expression. (A-H) Representative time series confocal images of 4- and 7-dpf transgenic larvae showing gradual loss of reporter expression from non-neural tissues; a common phenotype of NRSE-delimited Gal4-VP16 driver lines. (A-D) Time series of a triple transgenic larva from a cross between an NRSE-delimited Gal4-VP16 (KalTA4) driver line (NRCK gmc607), an mCherry reporter line (Tg(14xUAS:nfsb-mCherry)c264), and a pan-neuronal marker for neuronal expression, Tg(elavl3:EGFP)knu3 line (formerly HuC:GFP ). Image magnifications of bordered regions in A and C show skeletal muscle expression fading over time (B and D, arrows), while neuronal expression is maintained (B and D, arrowheads). Expression within the gut (dashed line) is an enteric neuron subpopulation; note 100% overlap with the pan-neural elavl3:GFP reporter (co-expressing cells appears yellow). (E-H) Time series of a double transgenic larva from a cross between an NRSE-delimited Gal4-VP16 (KalTA4) driver line (NRCK gmc607) and the YFP-expressing reporter line (5xMY-HMY gmc930). Image magnifications of bordered regions in E and G show skeletal muscle expression also fades over time when driver lines are crossed to YFP reporter lines (F and H, arrows), thus this phenomenon is not a result of NRSE-independent silencing of the reporter (as has been shown for the Tg(14xUAS:nfsb-mCherry)c264 line [48,49]). YFP expression typically takes longer to decay than mCherry, possibly due to stronger basal expression and/or absence of methylation due to the reduced number of UAS elements  or barrier insulator sequences. Note that the membrane-tagged YFP reporter improves visualization of axonal and dendritic neuronal outgrowths, e.g., the labeled enteric neuron subpopulation in the gut.
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