Fig. 3: The NetB pathway controls asymmetric development of the H-neurons.
From: Asymmetric activity of NetrinB controls laterality of the Drosophila brain
a RNAi screen strategy and validated candidates. The tester line allows co-expression in H-neurons of both RNAi and a fluorescent reporter in the asymmetric circuit. b Among 61 candidate genes known to mediate axon guidance and/or midline crossing, 10 showed a statistically significant increase in the proportion of SYM flies when their function is blocked (n ≥ 20 brains/condition. P-values are calculated with Pearson’s Chi-squared test and Benjamini and Yekutieli multiple comparison correction. Significance threshold are: *, <0.05; **, <0.01; ***, <0.001). c GO term analysis with String database (v11) indicates that GO terms GO:0005042 “netrin receptor activity” and GO:0038007 “netrin-activated signalling pathway” are the most enriched terms for Molecular function and Biological process respectively (strength = 3.14 and 2.97). Red dots on the diagram indicate Netrin receptors, orange dots indicate Netrin pathway effectors and black lines indicate evidence for protein-protein interaction in Drosophila. d Confocal images of representative adult brains displaying most frequently observed phenotype (see e) following netB, unc-5 and fra RNAi mediated loss-of-function in period-Gal4 neurons or in netB, netA and unc-5 mutants. Brain neuropils are labelled with the Nc82 antibody (cyan) and H-neurons are labelled with the 72A10-lexA driver (magenta). Scale bars, 30 µm. Insets show enlargements of the AB region and the yellow dotted line represent the brain midline. e Domain requirement and phenotype of Netrin pathway genes. A collection of Gal4 lines were used to drive expression of RNAi lines targeting Netrin pathway genes. Confocal brain images illustrate the expression pattern of each Gal4 line. Scale bars, 30 µm. unc-5 and fra but not netB are required in H-neurons. Bars represent frequencies of ASYM (blue) and SYM (green) phenotypes in adult flies. Black triangles represent control SYM frequencies for each set of experiment. n represent the number of brains analysed. P-values are calculated with Pearson’s Chi-squared test and Benjamini and Yekutieli multiple comparison correction. P-values are: wildtype vs NetBΔ: 3,31E-08; wildtype vs unc-5 MI05371: 3,31E-08; wildtype vs Elav>NetB RNAi: 3,01E-08; wildtype vs Elav>unc-5 RNAi: 3,01E-08; wildtype vs Elav>fra RNAi: 1,48E-03; per>GFP vs per>NetB RNAi: 3,01E-08; per>GFP vs per>unc-5 RNAi: 3,01E-08; per>GFP vs per>fra RNAi: 3,94E-03; 72A10 ∩ VT017124 > GFP vs 72A10 ∩ VT017124 > unc-5 RNAi: 3,70E-04; 72A10 ∩ VT017124 > GFP vs 72A10 ∩ VT017124 > fra RNAi: 1,36E-04. Significance threshold are: *<0.05; **<0.01; ***<0.001. P-values in yellow correspond to comparisons between Gal4 controls and mutant control. Source data are provided as a Source Data file.
