Fig. 2. Localization of Robo receptors during olfactory lobe development. Lobes are outlined by broken lines and oriented with antennal nerve at the bottom of each picture (arrowheads in A,D,E,F,J,L). Confocal imaging was from anterior to posterior and a few 1 µm confocal sections at the relevant level are stacked in each picture. The lateral (L), medial (M), ventral (V) and dorsal (D) coordinates are as indicated in L. (A-D) Twenty-four hours APF. Robo (A) is localized uniformly in the axons within the antennal nerve (arrowheads). Robo2 (B) is enriched lateral to the commissure (red arrows) and is reduced within the midline (arrow). Robo3 (C) is localized in axons that lie medially within the outer nerve layer (red arrows). Expression is also detected at the midline (arrow). (D) Diagram summarizing expression pattern in the early pupal stages (18 to 30 hours APF). Arrowhead indicates antennal nerve; arrow indicates antennal commissure. (E) Pupal olfactory lobe from ato¹/Df(3R)p¹³ stained with anti-Robo3. In the absence of Ato-dependent neurons, the rest of the axons stall after their entry in the antennal nerve (arrowhead). Robo3 is expressed in several terminals (arrows). (F) Thirty-hour APF ato-Gal4/UAS-GFP pupa stained with anti-Robo3 (red). The Ato-dependent neurons (green) can be seen in the outer nerve layer around the olfactory lobes and cross over in the antennal commissure. Robo3-expressing axons are located medially in the outer nerve layer (arrows). (G-I). Robo and Robo3 expression at 60 hours APF. Glomeruli that express high Robo (G; indicated by broken red lines) can be distinguished from the lower level expression. Robo3 is localized at high levels (H; indicated by broken red lines) in a small subset of glomeruli (depicted in blue in I). Immunoreactivity is enriched at the sensory terminals, which lie on the periphery of the glomerulus (inset in H). (I) Diagram representing Robo (green) and Robo3 (blue) localization among `glomeruli'. Entire confocal stacks from several stained lobes were examined. The pictures in G,H are from only a few sections through the lobe and not all stained glomeruli have been shown. (J) z-stack of an olfactory lobe stained with mAbnc82 showing the positions of glomeruli in the adult. Gaps between glomeruli are normally occupied by glial processes. (K) Twenty-hour APF brain stained with anti-Slit (green) and anti-Repo (red). A row of cells at the midline (arrowheads) of the ventral sub-esophageal ganglion (oe, esophagus) as well as regions in the mid-brain (arrows) are recognized by anti-Slit. Olfactory lobes (outlined by broken lines) are demarcated by glial cells and the Slit immunoreactivity in this neuropil was estimated using the Image J software (L). Pixel intensities were estimated in 1 µm sections through the lobe and summed. Low and high intensity levels are pseudo-colored blue and yellow, respectively.