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First published online 21 February 2007
doi: 10.1242/dev.02814

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Cell lineage-specific expression and function of the empty spiracles gene in adult brain development of Drosophila melanogaster

Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

View larger version (76K): [in this window] [in a new window]   Fig. 1. ems is expressed in one cluster of clonally related cells per hemisphere. Frontal views of adult brains. Anti-Ems labelling is magenta; the neuropile marker Nc82 is white; GFP-labelled wild-type MARCM clones are green. (A,B) z-projection of optical sections. Dashed line indicates border between central brain and optic lobes. (C) Single optical section showing ems expression in cells between antennal lobes and suboesophageal ganglion (SOG). (D,F,G) Individual wild-type clone shows co-localization of GFP with Ems in adult-specific cells. Single optical section (higher magnification of selected area, boxed) reveals that all GFP-labelled cells express ems, whereas a small subset of ems-expressing cells lack GFP (arrowheads in F). At a deeper focal plane, the same clone as in F extends a projection medially into the superior medial protocerebrum (arrows in D,G) and arborizations into the adjacent SOG neuropile (asterisk in D,G). (E) Three-dimensional model of D, illustrating position of MARCM clone in relation to major neuropile compartments such as the SOG, antennal lobes, mushroom bodies and superior medial protocerebrum. Scale bars: 50 µm in A-D; 5 µm in F,G. AL, antennal lobe; CB, central brain; MB, mushroom body; OL, optic lobe; SMP, superior medial protocerebrum; SOG, suboesophageal ganglion.

View larger version (102K): [in this window] [in a new window]   Fig. 2. ems expression is restricted to eight neuroblast lineages in the larval brain. Ventral views of wandering stage larva; anterior to top. Anti-Ems labelling is magenta; green indicates anti-Neurotactin in B-E and membrane-bound GFP-labelled wild-type MARCM clones in G-L. (A) z-projection of optical sections. The dashed line indicates border between the central brain and the optic lobe. Analysis of Ems-positive clusters was performed on stacks of optical sections (B-E), and a 3D model (F) was generated. (B) z-projection of one brain hemisphere, showing selection (boxed) used for enlarged views of single optical sections (C-E). (C) Optical section close to surface of cortex, showing neuroblast (arrowhead) in close contact with small cells of medial cluster (dotted). (D) At a deeper focal plane, medial cluster cells (dotted) surround a neurite bundle (arrow). (E) Optical section close to neuropile surface showing neurite bundle of medial cluster (arrow). (F) Digital 3D-model illustrates eight Ems-positive clusters and their neurite projections. Neuropile (blue) based on ChAT-promoter driven GFP expression. The eight ems-expressing lineages could be tentatively assigned to the DAlv2, BAmas2, BAmv2, BAmv3, BAlp1, BAlp2, BAlp3, BAlc lineages of the late larval brain atlas (Pereanu and Hartenstein, 2006). Optical sections of wild-type MARCM clones recorded at different focal planes (G,J: superficial; H,K: intermediate) for ems expression analysis and 3D-modelling (I,L). In F, an arrowhead marks the lineage corresponding to the clone shown in G-I, whereas an asterisk indicates the EM lineage (J-L). White arrowheads indicate neuroblasts; black arrowheads indicate Ems-positive cells lacking GFP. Scale bars: 50 µm in A; 5 µm in C,G,J. CB, central brain; MB, mushroom body; OL, optic lobe; SMP, superior medial protocerebrum; SOG, suboesophageal ganglion.

View larger version (135K): [in this window] [in a new window]   Fig. 3. ems expression in EM lineage persists through metamorphosis. (A-D) Brains double stained with anti-Ems (magenta) and nc82 (white) from late third instar to 72 hours APF. Specimens selected for GFP-labelled wild-type EM clones (green). Reconstructions of optical sections. Insets show optical sections at plane where clones extend neurites (GFP only). Short arborizations arise close to cell bodies during early pupal stages (arrowheads). AL, antennal lobe; APF, after puparium formation; LAL, larval antennal lobe; SMP, superior medial protocerebrum; SOG, suboesophageal ganglion.

View larger version (105K): [in this window] [in a new window]   Fig. 4. Cell types in the EM lineage are not altered in ems mutant clones. (A-L) Single optical sections. Co-labelling of GFP-marked wild-type and ems mutant MARCM clones (green; for genotypes see Materials and methods) with antibodies against protein indicated on each panel (magenta). The anti-Ems immunoreactivity used for the identification of EM lineages is omitted for clarity. Neuroblasts encircled with dots; GMCs marked by arrowheads. Scale bar: 5 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Reduction of cell numbers in ems mutant EM clones in the late larval brain. (A) Average cell numbers of wild-type, ems mutant and rescued clones at late wandering larval stage (96 hours ALH) are indicated in bar graph (for genotypes see Materials and methods). Wild-type (B-D), mutant (E-G) and rescue (H-J) clones co-labelled with anti-Ems (magenta) and anti-ß-GAL (green in C,F) or GFP (green in I) and shown in z-projections. Note that in I a membrane-bound GFP marker results in weaker overlap with the nuclear anti-Ems signal compared with the nuclear anti-ß-GAL in C,F. Digital 3D models were generated to visualize clone size (white in D,G,J). (Ems-positive cells not co-labelled with clonal marker are shown in light magenta.) Neuroblast outlined with dots in confocal images and in green in the 3D models. Scale bars: 5 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 6. Wild-type and ems mutant EM clone size at different developmental stages. MARCM clone induction occurred at 0 hours ALH. Average number of cells is plotted against the time of analysis. Numbers of clones analysed indicated in brackets.

View larger version (108K): [in this window] [in a new window]   Fig. 7. ems is required for the formation of correct projections of the EM lineage. GFP-labelled MARCM clones (green) analysed in adult (A-H) or late wandering larval stage (I-P). Anti-Ems antibody (magenta) labels both wild-type protein and truncated form. Neuropile labelled with Nc82 (white); relevant compartments labelled as in Fig. 1. Only one hemisphere is shown in larva; double arrow indicates anterior (a) to posterior (p) axis. Merged images in left columns, clones (GFP channel only) in right columns. Protocerebral projection (arrow), ectopic neurites (arrowheads). Scale bars: 50 µm in A; 5 µm in I.

View larger version (45K): [in this window] [in a new window]   Fig. 8. ems is required for the formation of correct projections in postmitotic neurons of the EM lineage. (A-F) Single cell clone analysis. Viewed as in Fig. 7I-P. Single GFP-labelled cells (green) analysed in late wandering larval stage. MARCM clone induction at 48 hours ALH. GFP clones in right columns; merged images with anti-Ems signal (magenta) in left columns. Protocerebral projection (arrow), ectopic neurites (arrowheads). Scale bar: 5 µm.

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