Extended Data Fig. 11: Genes expressed in L3 required for R8 wiring and model figure.
From: A global timing mechanism regulates cell-type-specific wiring programmes
a, R8 presynaptic sites (Brp, green) ± EcRDN expression in L3. Right, quantification of Brp puncta number under the conditions shown. Scale bar, 5 μm. (WT, 58 neurons; EcRDN, 47 neurons). b, Total number of presynaptic sites (Brp puncta) in R8 neurons with WT, NetB overexpressing, or EcRDN and NetB-expressing L3 neurons. NetB overexpression is unable to rescue the reduction in R8 presynaptic sites seen with expression of EcRDN in L3 neurons. a, b, p-value (two-sided Student’s t-test) is given. c, d, Distributions of R8 axon terminal depth in w RNAi (red) or with expression of other RNAi (as shown, blue) in L3 neurons. p-values (two-sided Kolmogorov-Smirnov test) are given and number of neurons/ conditions are given. ns = Not significant. c, RNAi against TFs in the Ecdysone-pathway. d, Results from RNAi screen showing genes that did not significantly increase R8 axon depth. Note that nrm RNAi yields a subtle reduction in R8 axon depth. Numbers of neurons/ condition are given. For all box-plots, solid line depicts median, while the upper and lower bounds of the box depict the third and first quantile of the data spread respectively. e, Expression of NetA and NetB in L3 with (blue) or without (red) EcRDN expression. *, fold change between WT and EcRDN > 2, p-value < 0.05, two-sided Wilcoxon rank sum test. f, Staining using anti-NetB antibody (magenta) at the indicated times in development. Marker for M6 medulla layer (24B10, grey) and L3 neurons labeled with GFP (green) are shown. g, Staining using anti-NetB antibody (magenta) ± EcRDN expression only in L3. L3 neurons labeled with GFP (green) are shown. f, g, Scale bar, 50μm. Yellow arrowhead, M3 medulla layer. For number of replicates and exact p-values, see Supplementary Table 10. h, Model for temporal regulation of wiring genes. Temporally-regulated TFs (grey triangle) controlled by various external cues such as neural activity, cell-cell interactions and endocrine signals (e.g. Ecdysone) work together with cell-type specific TFs (red and blue proteins). Their combined activity controls the timing and cell-type specificity of wiring gene (such as the ones coding for cell recognition molecules) expression.
