Fig. 3. rho3 is expressed by the ventral unpaired group of midline neurons (VUM). (A) Ventral view of late stage 16 inga heterozygote embryo triple stained to reveal the tracheal lumen (mAb2A12 in blue), a subset of PNS and CNS axons (mAb22C10 in green), and the expression of ß-Gal in the rho3^inga enhancer trap (anti-ß gal in red). The panel shows a 3D reconstructions deriving from a confocal stack, anterior is towards the left. Strong ß-gal expression is detected in each segment in ventral clusters of cells at the midline. (B) A similar expression pattern is detected by in situ hybridisation with a specific rho3 cDNA probe at stage 15, in clusters of cells at the VNC ventral midline (B, arrow; B-E are lateral views of the VNC, anterior towards the left, ventral downwards; B is a stage 15 embryo, C-E are late stage 16). (C-E) 3D confocal reconstruction allows the identification of the rho3-expressing cells (in red, stained by anti-ß-gal) as the VUM neurons. VUM cell bodies are readily stained by mAb22C10 (C, in green), which also allows identification of the characteristic VUM axonal tract (C, arrowhead). Slit staining (E, in green) and Wrapper staining (D, in green) of midline glia, shows little overlap with ß-gal expression in rho3^inga (D,E, in red). Scale bars: 20 µm.