Fig. 3: A targeted RNAi screen reveals Orb2 as a codon-specific regulator in the brain.

A Schematic indicating how screen targets were chosen. 21 genes were NB-enriched, and 24 genes were neuron-enriched. B Quantification of primary screen results, with average GFP-rare^b fluorescence intensity values (arb. units=arbitrary units) in target gene deficient brains (RNAi driven by insc-GAL4) ordered from highest to lowest mean. Ordinary one-way ANOVA compares all values to control (expressing GFP-rare^b and no RNAi) WL3 brain fluorescence value followed by Dunnett’s test for multiple comparisons. Knockdown of elp1, cg2021, not1, ewg and orb2 significantly decreased average GFP-rare^b WL3 brain fluorescence relative to the control. P values for significant comparisons with control as follows: cg2021: p value = 0.0437; not1: p value = 0.0203; elp1: p value = 0.0139; ewg: p value = 0.0018; orb2: p value = 0.0015. Mean ± SEM plotted. C Graphical demonstration of primary and secondary screen results. N = 35 RNAis with no change in GFP-rare^b fluorescence, N = 6 RNAis were lethal by larval stage, N = 5 RNAis significantly changed fluorescence of both GFP-rare^b and GFP=common, and N = 1 RNAi, (orb2), significantly changed only GFP-rare^b fluorescence (=”hit”). D Quantification of secondary screen results. Knockdown of primary screen hits driven by insc-GAL4 results in significant changes to GFP-common fluorescence in all but orb2 RNAi knockdown. Ordinary one-way ANOVA followed by Dunnett’s test for multiple comparisons. Compared to control: elp1 p = 0.034; cg2021, p = 0.0011; not1, p = 0.0142; ewg, p = 0.0040. Each data point = 1 animal. Mean ± SEM shown. Three biological replicates. E–H Representative images of control (white (w) RNAi) (E, G) and orb2 RNAi (F, H) WL3 brains with GFP-rare^b (E, F) or -common (G, H) fluorescence (green). I Quantification of fluorescence intensity change in insc-GAL4>w RNAi (WT) or orb2 RNAi WL3 brains compared to average fluorescence in WT, expressing either GFP-rare^b or GFP-common. Two separate orb2 RNAi constructs were tested (see Supplemental Dataset 4 for details). Mean ± SEM plotted, two biological replicates. Ordinary one-way ANOVA followed by Šídák’s multiple comparisons test. P value for GFP-rare^b vs RNAi#1 = 0.0096; GFP-rare^b vs RNAi#2 = 0.0087. J orb2 transcript expression in NBs and neurons from Yang et al. 2016⁸². Precise n values in source data files for B, D and I.