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First published online 11 June 2008
doi: 10.1242/dev.022210

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empty spiracles is required for the development of olfactory projection neuron circuitry in Drosophila

Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

View larger version (105K): [in this window] [in a new window]   Fig. 1. ems expression and olfactory projection neurons in the Drosophila late larval brain. (A,B) z-projection of central brain expressing GAL4-GH146 UAS-CD8::GFP (green) or labeled for Ems (magenta). ems-positive cells and GAL4-GH146-positive cells are both located close to antennal lobes. (C) Digital model of 3D reconstructed optical sections providing an overview of GAL4-GH146-positive adPNs and lPNs (green) and adjacent ems-expressing cell body clusters (magenta, arrowheads). (D,E) ems-expressing cell bodies (magenta) are in close proximity to the GAL4-GH146-positive adPNs and lPNs (green); ems-expressing cells are never GAL4-GH146-positve. Cell bodies of PNs are delimited by dotted lines. Single optical sections. (F,G) Two GFP-labeled MARCM clones (green) that extend axons in the antenno-cerebral tract, coexpress ems (magenta) in a subset of their cell bodies. z-projections of optical sections. Insets show that ems is expressed in the neuroblast (asterisk) and in cells located close to the neuroblast. Brackets indicate position of cells shown in insets. Single optical sections. Ventral views, anterior to the top. adPN, anterodorsal projection neuron; lPN, lateral projection neuron; ACT, antenno-cerebral tract; AL, antennal lobe; LH, lateral horn; MB, mushroom body calyx; OL, optic lobe. Dashed lines outline the brain; dotted lines outline the labeled anatomical structures within the brain. Scale bars: 10 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 2. ems-expressing cell types in the adPN and lPN lineages at the late third instar larval stage. (A-D''') MARCM clones induced in early first instar Drosophila larvae. (A,C) Lineage as shown in Fig. 1F. (B,D) Lineage as shown in Fig. 1G. MARCM clones were co-immunostained with (A,B) anti-Ems (blue) and anti-Grh (red) or (C,D) anti-Ems (blue) and anti-Elav (red). Single channels are shown separately. Dotted outline, ems-expressing neuroblasts; arrow, ganglion mother cells (GMCs); arrowhead, postmitotic neurons. Scale bars: 5 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 3. ems expression in adPN and lPN lineages. (A-D') Dual-expression MARCM. tubP-lexA-positive cells are green, GAL4-GH146-positive cells are red, coexpressing cells appear yellow; Ems-expressing cells are blue. Clones were induced during embryogenesis and analyzed 48 and 96 hours after larval hatching (ALH). Confocal images show single optical sections from four preparations. (A-D) Optical sections taken at superficial cortex layers include the ems-positive neuroblast (arrowhead). (A'-D') Optical sections taken at deeper cortical layers close to the antennal neuropile. (E) Summary of ems expression (blue) in larval PNs (see text for details). Scale bars: 10 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 4. ems is required for the dendritic targeting of adPN-specific glomeruli. Anti-GFP-labeled GAL4-GH146 UAS-mCD8::GFP MARCM clones (green); neuropile marker anti-Nc82 (magenta). Clones were induced in early first instar Drosophila larvae and analyzed in adult. Dotted lines mark the outline of single glomeruli. (A-H) Single confocal sections of antennal lobes with dorsal to top and medial to left showing dendritic innervation by wild-type (A,C,E,G) or ems mutant (B,D,F,H) clones. Loss of innervation of VA3, VA1lm (B) and VM2 (D) glomeruli by ems mutant clones as compared with wild-type clones (A,C). Ectopic innervation of inappropriate glomeruli DA2, VA6 (F) and DL2, DL5, VL2 (H) by ems mutant clones as compared with wild-type clones (E,G). (I,J) z-projections of frontal optical sections showing antennal lobes and subesophageal gangion (SOG). No innervation of SOG is observed in the wild type (I), whereas ems mutant adPN clones extend ectopic projection into SOG (J).

View larger version (94K): [in this window] [in a new window]   Fig. 5. Misexpression of ems in mature adPNs causes dendritic targeting defects. Anti-GFP-labeled GAL4-GH146 UAS-mCD8::GFP MARCM clones (green); anti-Nc82 (magenta). Clones were induced in early first instar Drosophila larvae and analyzed in adult. Single confocal sections of single antennal lobes; dorsal to top, medial to left showing dendritic innervation by wild-type clones (A,C,E) or ems gain-of-function (GOF) clones misexpressing ems (B,D,F). Ectopic innervation of inappropriate glomeruli DA2 (B) and DC1 (D) by ems-misexpressing clones as compared with wild-type clones (A,C). Loss of innervation of the DL1 glomerulus (F) by ems-misexpressing clones as compared with wild-type clones (E). Dotted lines mark the outline of single glomeruli.

View larger version (109K): [in this window] [in a new window]   Fig. 6. ems-misexpressing adPN clones but not ems mutant clones show axon terminal defects. Anti-GFP-labeled GAL4-GH146 UAS-mCD8::GFP MARCM clones (green); anti-Nc82 (magenta). Wild-type clones (A-C), ems mutant clones (D-F), and ems gain-of-function (GOF) clones misexpressing ems (G-I) were induced in early first instar Drosophila larvae. At this time, only neuroblast clones and DL1 class single-cell clones can be induced in the adPN lineage. Analysis was in adult. (A,D,G) z-projection of confocal sections of lateral horn with anterior to top and medial to left. Neuroblast clones of all three genotypes show two main arborization areas in lateral (asterisk) and dorsal (arrowhead) lateral horn. In the ems GOF clones, ectopic innervation is seen between two main arborization areas (arrow in G). Wild-type and ems mutant DL1 class single-cell clones innervate the DL1 glomerulus of antennal lobe (B,E, respectively) and bifurcate into a dorsal (arrowhead) and lateral (asterisk) process in lateral horn (C,F). By contrast, ems-misexpressing clones ectopically innervate DA2 and DM6 glomeruli (H) and show ectopic terminal branches (arrow) between dorsal terminal process (arrowhead) and main lateral terminal process (asterisk) in the lateral horn (I). Dotted lines mark the outline of single glomeruli.

View larger version (48K): [in this window] [in a new window]   Fig. 7. ems and acj6 expression in the adPN lineage at the late third instar larval stage. MARCM clones induced in early first instar Drosophila larvae. (A'-A'''') MARCM clone (continous outline) co-immunostained with anti-Ems (blue) and anti-Acj6 (red). Single optical section. Single channels shown separately. The neuroblast (dotted outline) and adjacent cells (white arrowhead) express ems alone. Whereas only a few cells coexpress ems and acj6 (open arrowhead), most cells express exclusively acj6 (arrow). (B) Summary of ems and acj6 expression in larval adPNs with average cell numbers indicated for each expression class (see text for details). (C) Comparison of the number of cells expressing ems, acj6 and elav in wild-type versus ems mutant MARCM clones at the late third instar larval stage. In the absence of ems, the number of acj6-expressing cells is reduced. Scale bar: 5 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 8. Misexpression of acj6 in the ems mutant adPN lineage rescues the loss of innervation phenotype in the VA1lm glomerulus. Anti-GFP-labeled GAL4-GH146 UAS-mCD8::GFP MARCM clones (green); anti-Nc82 (magenta). Clones were induced in early first instar Drosophila larvae and analyzed in adult. Single confocal sections of single antennal lobes (dorsal to top, medial to left) showing dendritic innervation by wild-type clones (A), ems loss-of-function (LOF) clones (B) and ems mutant clones (GOF) misexpressing acj6 (C). Loss of innervation of VA1lm glomeruli by ems mutant clones is significantly rescued by the misexpression of acj6, whereas no significant rescue could be found for the VA3 and VM2 glomeruli. Dotted lines mark the outline of single glomeruli.

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