Homeodomain TFs expressed in MNs fine tune wiring specificity in the neuromuscular system.Figure 3(A) Representative confocal image of a stage 17 embryonic head with Dfd expressing cells (purple), Myosin expressing muscles (blue) and FasII expressing axonal projections (green) highlighted. The MN2a is identified as a Dfd expressing neuron projecting a FasII expressing axon to the Dfd positive MHE muscle. This stereotypic innervation is the basis for identifying wiring defects after RNAi interference shown in (D). (B) Time course of RNAi induced effect using the pan-neural elav-GAL4 driver line, showing that efficient knock-down of gene expression is achieved only at embryonic stage 14, which represents the start of synaptic wiring. (C) Chart highlighting the expression of the three genes used for RNA interference, Dfd, hth and mirr, in MN2c, MN2a and MN3. (D) Representative confocal images of stage 17 embryonic heads highlighting expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and after RNA interference with three different homeodomain TF genes, Dfd, hth and mirr, which are all co-expressed in MN2a (see Fig 2E). A zoom on the projection (FasII staining) of MN2a to the MHE and MHD muscle of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a, which is in all cases identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) in control and perturbation conditions. (E) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern (magenta); MN2a axons projecting to muscles other than the MHE termed abnormal innervation (light grey); abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects (dark grey). Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. Each experiment was performed in triplicates, innervation rates were calculated from n=56 for elav>RFP, n=21 for elav>DfdRNAi, n=10 for elav>hthRNAi and n=7 for elav>mirrRNAi. In the case of Dfd knock-down, two different driver lines were used, the pan-neural elav-GAL4 and the motoneuron-specific OK6-GAL4 drivers, respectively. Both result in similar phenotypes, highlighting that the elav-GAL4 driven effects are specific to MNs. p-values between two genetic conditions were calculated by a two-sided Fisher test. ***: p < 0.005. (F) Correct MHE innervation is required for hatching of Drosophila embryos from the eggshell (Friedrich et al., 2016). Hatching rate was calculated based on the number of L1 larvae observed after 24h in genetic crosses, depleted of Dfd (UAS-DfdRNAi) in neurons (elav-GAL4; n=217) compared to crosses with control animals (mock = elav>RFP; n=156). P-values were calculated by a two-sided Fisher test, ***: p < 0.005.