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First published online 24 July 2008
doi: 10.1242/dev.024380

Development 135, 2883-2893 (2008)
Published by The Company of Biologists 2008
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Clonal analysis of Drosophila antennal lobe neurons: diverse neuronal architectures in the lateral neuroblast lineage

Sen-Lin Lai1,*, Takeshi Awasaki1,2, Kei Ito2 and Tzumin Lee1,{dagger}

1 Department of Neurobiology, University of Massachusetts, Worcester, MA 01605, USA.
2 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.


Figure 1
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  		Fig. 1. Brain atlas and mosaic analysis. (A) Schematic of the olfactory circuitry (left hemisphere) and brain structures (right hemisphere) in the adult Drosophila. The olfactory circuitry is exemplified by representative olfactory receptor neurons (green), antennal lobe neurons, which include projection neurons (blue) and interneurons (purple), and mushroom body neurons (orange). Various brain structures are outlined by solid or broken lines and are superimposed with different colors. The naming of brain structures follows Otsuna and Ito (Otsuna and Ito, 2006Go). Abbreviations: adPN, anterodorsal projection neuron; AN, antennal nerve; AL, antennal lobe; lPN, lateral projection neuron; LN, local interneuron; de, deutocerebrum other than the AL; GC, great commissure; LH, lateral horn; MB, mushroom body; MBN, mushroom body neuron; ORN, olfactory receptor neuron; pilpr, posterior inferior lateral protocerebrum; pimpr, posterior inferior medial protocerebrum; SOG, sub-esophageal ganglion; vlpr, ventrolateral protocerebrum. (B) The genetic basis of MARCM (top) and dual-expression-control MARCM (bottom). Mitotic recombination mediated by flipase (FLP) in the heterozygous mother cell leads to the loss of GAL80 in one of the two homozygous daughter cells. All GAL80-negative progenies are labeled by tubP-LG-driven lexop-rCD2::GFP (green outlined oval), and only GAL80-negative GAL4-positive cells are dually labeled by GAL4-controlled UAS-mCD8 (pink oval). (C) Clone size in dual-expression-control MARCM. The induction of multicellular Nb clone (green outlined circles in the upper panel) or single-cell clone (green outlined circle in the lower panel) depends on the occurrence of FLP-mediated mitotic recombination in the self-renewing progenitor or ganglion mother cell. Within the clone, only GAL4-positive cells can be labeled by GAL4 and LexA::GAD simultaneously (green outlined pink circles). Abbreviations: Nb, neuroblast; GMC, ganglion mother cell; N, neuron.

 

Figure 2
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  		Fig. 2. GAL4-GH146-positive projection neurons are subsets of the antennal lobe neuron lineages. (A-C) Illustrations of antennal lobe neuron lineages and cellular composition. (A) GAL4-GH146-positive projection pathways of PNs. Abbreviations: iACT, inner antennal-cerebral tract; mACT, middle ACT; LH, lateral horn; MB, mushroom body. (B,C) Proportion of GAL4-GH146- and Acj6-positive PNs in the AL adPN, lAL and vPN lineages. (D-F) Composite confocal images of three AL lineages (D1'',D2'',E1'',E2'',F1'',F2'') that generate GAL4-GH146-positive PNs (D1',D2',E1',E2',F1',F2'). The Nb clones are generated at early larval stage and labeled by dual-expression-control MARCM. D1, E1 and F1 show the projection patterns and are merged from D1',D1'', E1',E1'' and F1',F1'', respectively. D2, E2 and F2 are the single confocal images of cell bodies magnified from D1, E1 and F1 and are merged from D2',D2'', E2',E2'' and F2',F2'', respectively. (G-I) The composite confocal images of the location and composition of Acj6-positive neurons in the adPN, lAL and vPN lineages. The MARCM Nb clones are labeled with tubP-GAL4 (white) and counterstained with Acj6 and nc82 (blue). The broken white lines outline the AL. (J-J'') Projected confocal images of the PNs co-labeled by GAL4-GH146 (J') and LG-GH146 (J''). J shows the merged images from J' and J''. The GAL4 and LG drivers differ only in the intensity of some expression. Scale bars: 20 µm.

 

Figure 3
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  		Fig. 3. Anterodorsal lineage composed of uniglomerular projection neuron. (A-A'') Single confocal images of the cell bodies of adPN neuroblast clone dually labeled by acj6-GAL4 (A') and tubP-LG (A''). A is the merged image from A' and A''. (B,C) Single confocal images of a representative adPN Nb clone. B shows the superficial (anterior) layer and C shows the deep (posterior) layer. The yellow text indicates the glomeruli that are not innervated by GH146-PNs. (D,E) Projected confocal images of an adPN MARCM neuroblast clone (white) (D) and a representative single-cell clone (white) (E). (F-H) The axon terminals (white) of two different DL2d and one VA1lm-targeting adPNs at the MB (green circled areas) and LH (yellow circled areas). Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. Diverse uniglomeurular and multiglomerular projection neurons in the ventral lineage. (A-A'') Single confocal image of the AL neurons dually labeled by LG-GH146 (A) and GAL4-MZ699 (A''). A is the merged image from A' and A''. (B) Single confocal image of the vPN neuroblast clone labeled by dual-expression-control MARCM with GAL4-MZ699 (B') and tubP-LG (B'') counterstained with Acj6 (cyan). B is the merged image from B' and B''. (C-F) Projected confocal images of a vPN MARCM Nb clone (magenta) (C) and three representative single-cell clones (magenta) (D-F). Note the different dendrite and axon (white arrows) projection patterns of different vPNs in the AL (yellow circled areas) and lateral horn (white circled areas). Scale bars: 20 µm.

 

Figure 5
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  		Fig. 5. The lateral lineage covered by multiple enhancer trap GAL4 lines. (A) Schematic drawing of antennal lobe neurons labeled by different enhancer trap GAL4 lines in lAL lineage. (B-O) Composite confocal images of lAL neuroblast clones labeled by dual-expression-control MARCM and MARCM with different GAL4 lines. The name of each GAL4 driver is listed on the right (magenta text). (D,G,J,M) The single confocal images of GAL4 drivers (magenta) in the presence of LG-GH146 (yellow). (B-B'',E-E'',H-H'',K-K'',N-N'') Single confocal images of Nb clones labeled by the dual-expression-control MARCM with GAL4 drivers (B',E',H',K',N') and tubP-LG (B'',E'',H'',K'',N'') counterstained with the cell marker Acj6 (cyan). B, E, H, K and N are merged from B',B'', E',E'', H',H'', K',K'' and N',N'', respectively. (C,F,I,L,O) The morphologies of the neuroblast clones (magenta) labeled by MARCM with different GAL4 drivers and counterstained with nc82 (blue). Note the intensified DL3 glomerulus labeled by GAL4-NP6115 (L) and the thin tract that projects outward AL in the acj6-GAL4-labeled neuroblast clones (O). Abbreviations: GC, great commissure; in ant con, inter antennal connective; oACT, outer antennocerebral tract. Scale bars: 20 µm.

 

Figure 6
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  		Fig. 6. Diverse neuronal architectures in the lateral antennal lobe neuron lineage. (A-H) Each panel shows one representative single-cell MARCM clones (white) of distinct types of antennal lobe neurons in the lAL lineage. The white circled areas in B,C indicate the AL. The nomenclature of each type neuron is listed at the top of each panel. Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. Co-production of the temporally specified lateral antennal lobe neurons. (A) Illustration of two models of production of two different types of neurons (square and triangle) in the lAL lineage after clonal induction (red arrow). The labeled cells are outlined in green. Note the difference of labeled cellular composition in the Nb clones in two models. (B-C'') The early (B) and later (C) generated neuroblast clones labeled by dual-expression-control MARCM. The neuroblast clones are dually labeled with GAL4-GH298 (B',C') and LG-GH146 (B'',C''). B and C show the merged images from B',B'' and C',C'', respectively. Note the disappearance (arrow) or weak labeling (arrowhead) of glomeruli located at the ventral AL labeled by LG-GH146 (C').

 

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