Figure 4.

MID coordinates expression of key axon guidance genes. (A) Levels of mRNA in the salivary gland of normal third instar larvae (lane 1) or upon MID misexpression (sgP[Gal4]/+; UAS-mid/+) (lane 2). Shown are levels of transcripts normalized to the corresponding levels of tubulin mRNA in each sample, as determined by quantitative real time PCR. (B) Schematic representation of the MID-binding sites on the genomic sequence of slit, frazzled, and robo. The black boxes indicate the MID-binding site and the gray boxes indicate regions without the MID-binding site adopted as negative controls. Only the sites with perfect match with the consensus sequence were considered. The arrows represent transcription start sites and the numbers in base pairs are distance from the start sites. (C) ChIP with the antibody against MID revealed occupancy of MID at sliU, fraU, fraI, roboU1, roboU2, and roboU3, but not at the control regions sliC, fraC, and roboC (shown in lanes 3,6). The input was 0.1% (lanes 1,4). No primary antibody was used for mock (lanes 2,5). (D,E) Staining of stage 16 embryos with an anti-β-galactosidase antibody. (D) The reporter fraPlacZ was expressed in CNS and the mutant reporter fraMPlacZ exhibited significantly reduced expression in the CNS. (E) The reporter sliPlacZ but not the reporter sliMPlacZ was expressed in the midline glia and lateral cells.