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First published online 31 March 2004
doi: 10.1242/dev.01083

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Positioning sensory terminals in the olfactory lobe of Drosophila by Robo signaling

¹ Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India
² National Centre for Biological Sciences, TIFR, GKVK PO, Bangalore 560065, India

View larger version (90K): [in a new window]   Fig. 1. Development of the olfactory lobe in Drosophila. (A) Olfactory lobes (encircled with broken red lines) are visualized in the brain (marked with broken white lines) by expression of GFP driven by SG18.1-Gal4. Neurons enter the lobe through the antennal nerve (AN) and cross over to the contralateral side through the antennal commissure (AC). Scale bar: 100 µm. (B) Olfactory lobes of Or47b-Gal4/+;UAS-NsybGFP (UNG12)/+ stained with mAbnc82 (red). Projections to the glomerulus VA1 can be seen (arrow indicates the AC). Scale bar: 50 µm. (C,D). Schematic diagrams summarizing cellular events occurring during lobe development in the early (C) and late pupa (D). In the early pupa (18-30 hours APF), sensory neurons enter the brain via the antennal nerve (arrowhead) and traverse in the outer nerve layer in close association with glial cells (red). Ato-derived `pioneer' neurons arrive at the lobe first (purple) and guide the rest of the neurons (green) to the antennal commissure (arrow). At this time, the projection neurons (blue) are already present within the lobe. Sensory neurons invade the lobe from about 25 hours APF. (D) Termination of sensory neurons (green) and their contact with appropriate projection neurons (blue) is followed by ensheathing of the neural elements to form glomeruli.

View larger version (119K): [in a new window]   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.

View larger version (137K): [in a new window]   Fig. 3. Robo receptors regulate targeting of olfactory neurons to specific glomeruli. Lobes are oriented as indicated in C,F: M, medial; D, dorsal; V, ventral. Imaging was carried out from anterior to posterior and a few 1 µm confocal sections are stacked in each picture. (A) Olfactory lobes of or22a-Gal4;UAS-N-SybGFP stained with mAbnc82 (red). Or22a-expressing neurons project to a single glomerulus DM2 and also cross over in the inter-antennal commissure (arrow). (B,D,E) The MARCM method was used to positively mark neurons mutant for robo21 (B) or robo31 (D,E). Axons lacking Robo2 function terminate within the midline creating a `roundabout' appearance (arrowheads in B; diagram in C). Ectopic axonal arbors are immunoreactive for mAbnc82 (arrow in B). Axons lacking Robo3 function terminate aberrantly (broken lines in D,E; diagram in F). (G-L) Sensory neuron terminals in the olfactory glomeruli are visualized using a SG18.1-UAS-GFP recombinant strain in different conditions. (G,J) Appropriate confocal sections have been selected to visualize normal glomeruli (G, *) and antennal commissure (J, arrow). (H) SG18.1 UAS-GFP/UAS-comm. (I) SG18.1 UAS-GFP/UAS-abl. In both H and I, glomerular patterning is significantly disrupted while the antennal commissure is present (arrow), although axons appear somewhat loosely fasciculated. (K,L) SG18.1 UAS-GFP/UAS-Dcdc42v12 (constitutively active). Glomeruli cannot be discerned within the lobes (K). The commissural region (boxed in K) is examined at better resolution in L. Stacking of fewer sections shows a clear glomerular-like organization (*) within the commissure.

View larger version (123K): [in a new window]   Fig. 4. Robo/Slit-dependent signaling regulates glomerular patterning. Orientation is as described for previous figures. (A-F).The SG18.1-Gal4 UAS-GFP recombinant line was used to visualize sensory neuron within the lobe and also to misexpress genes under UAS control. (A) SG18.1-Gal4 UAS-GFP/UAS-robo;UAS-robo(2X)/+ (three copies). Glomerular formation is abrogated but the commissure is still present (arrow). (B) SG18.1-Gal4 UAS-GFP/UAS-robo3. Positions of most terminals are shifted to the dorsomedial location within the lobe (indicated with broken red lines). Commissure is unaffected (arrow) (C) SG18.1-Gal4 UAS-GFP/UAS-robo2.? indicates the position of the commissure that is absent. (D) SG18.1 UAS-GFP UAS-robo2/Df(2R) WMG (Df(2R)Slit). Commissure (arrow) formation is restored when dose of the Slit ligand is reduced by half. Glomerular morphology is also restored (asterisks). (E) SG18.1 UAS-GFP UAS-robo2/UAS-comm: partial restoration of glomerular morphology (*) and complete rescue of the commissure (arrow). (F) SG18.1 UAS-GFP UAS-robo2/UAS-Dcdcv12. The commissure is present but sensory axons are much more loosely packed than in the normal (arrows). (G-I) Adult olfactory lobes stained with the synaptic marker mAbnc82. (G) Normal controls. (H,I) loco-Gal4/UAS-Slit: ectopic glomeruli are circled by broken red lines. Only a few glomeruli are recognizable when Slit is ectopically expressed (marked by an asterisk).

View larger version (122K): [in a new window]   Fig. 5. Robo misexpression shifts location of sensory arbors in the olfactory lobe. (A-P) Orientation of the olfactory lobes is as described in the previous figures. Relevant 1 µm confocal sections are stacked in each picture (green). B,C,G,O are also labeled with the synaptic marker mAbnc82 (red). The diagrams in D,H,L,P depict the positions of sensory terminals depicted from several preparations. Colors indicate the anterior to posterior location of the terminals as observed in confocal imaging of the lobe. Violet is most anterior, followed by blue, green, yellow, orange with red appearing most posteriorly. Arrows indicate the antennal commissure. Quantitative results are presented in Table 2. (A-D) lz-Gal4/+;UAS-GFP/+. Axons project to two glomeruli (arrowheads in A) and cross over in the antennal commissure (arrow in A). Staining with mAbnc82 (B,C) allowed identification of these glomeruli as DM6 and DL3 (diagram in D). (E-H) lz-Gal4/+;UAS-GFP/UAS-robo. Sensory terminals located centrally within the lobe (arrowheads). (I-L) lz-Gal4/+;UAS-GFP;UAS-robo2. Targets to only one lobe were frequently observed (arrowheads in I). (M-P) lz-Gal4/+;UAS-GFP/UAS-robo3. Sensory neurons terminate at ectopic positions and often show altered morphologies (arrowheads). (Q-V) Cytoarchitecture of individual glomeruli. (Q,S) Visualization of SG18.1-Gal4;UAS-GFP show that sensory arbors (green) are located in a `doughnut-like' arrangement on the periphery of the glomerulus. (Q',S') staining with the synaptic marker mAbnc82 (red) shows presence of synapses within the outer region (yellow) as well as the core of the glomerulus. (R,R'). GH146 UAS-GFP (green) shows position of projection neurons within the glomerulus. These neurons arborize within the entire glomerulus as can be seen with the overlap with mAbnc82 staining (red, R'). (T-V) Three examples of ectopic glomeruli generated by misexpression of Robo receptors show comparable organization of sensory neurons and interneurons. The sensory neurons are located on the peripheries (T,U,V) and the staining with mAbnc82 highlights both peripheral and central regions of the glomerulus (T',U',V').

View larger version (43K): [in a new window]   Fig. 6. Model for the role of Slit-dependent Robo signaling in glomerular patterning. (A) Sensory neurons enter the lobe via the antennal nerve (AN) and radiate over the olfactory lobe. Neurons expressing Robo (green) and Robo3 (purple) segregate from those expressing Robo alone and decussate onto the medial side. Robo2 (pink) expression is elevated after neurons have crossed in the commissure. Regions of Slit expression at the midline and in the brain are indicated by yellow bars. The yellow shading indicates the proposed Slit gradient. (B) Sensory neurons invade the lobe starting at 25 APF. The positioning of terminals within the lobe is based on the Robo code (green, Robo; purple, Robo3) that they express and the differential response to Slit levels (yellow). The targeting of sensory neurons to specific positions allows short-range interactions with the projection neurons which are already present in the lobe. This leads to consolidation of the glomeruli, which become ensheathed by glial cell processes. The ventral, medial and lateral axis are indicated.