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First published online 19 November 2008
doi: 10.1242/dev.023366

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Neuronal programmed cell death induces glial cell division in the adult Drosophila brain

Kentaro Kato^1,2,,*, Takeshi Awasaki^1,3, and Kei Ito^1,2

¹ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
² Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Corporation (JST), Chiyoda-ku, Tokyo 102-0081, Japan.
³ Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation (JST), Kawaguchi 332-0012, Japan.

View larger version (79K): [in this window] [in a new window]   Fig. 1. Programmed cell death around the antennal nerve after adult eclosion. All images show frontal views of the fly brain [wild-type Canton-S (CS) after 6 hours of eclosion] with dorsal up. (A) Cells labelled with anti-cleaved caspase 3 antibodies are observed in various brain regions, including the area around the antennal nerve (an, arrowheads). Projection of confocal optical sections of the frontal half of the brain. Dashed lines outline the brain. The rectangle indicates the area shown in B,D-G. (B,D-G) A merged image (left) and single-channel images (insets) around the area of the labelled cells (arrowheads). A caspase-positive cell (B, arrowhead) was also TUNEL-positive and had a condensed nucleus. A TUNEL-positive cell (D, arrowhead) expressed the neural marker ELAV, but not the glial marker REPO. A caspase-positive ELAV-positive neuron (E, white arrowhead) sends its neurite towards the antennal nerve (yellow arrowhead). Projection of ten confocal sections. The cells that strongly expressed EcRA were also TUNEL-positive (F, arrowheads). A grim-expressing cell (G, whole-mount in situ hybridisation of grim mRNA) expressed the neural marker ELAV, but not the glial marker REPO (arrowhead). (C,H) Time course of the percentage of the antennal nerves with TUNEL-positive (C) and strongly EcRA-positive (H) cells. Colour coding of each bar indicates the percentage of antennal nerves with the indicated numbers of positive cells. The sample number (n) is indicated above each bar. an, cross-section of the root of the antennal nerve; al, antennal lobe; SOG, suboesophageal ganglion; vlpr, ventrolateral protocerebrum. Scale bars: 50 µm in A; 10 µm in B,D-G.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Glial division around the antennal nerve after eclosion. (A,B) An adult fly brain of the wild-type (CS) strain after 10 days of BrdU treatment. Projection of confocal optical sections of the frontal half of the brain (A) and high-magnification single section of the right hemisphere around the antennal nerve (B). BrdU-positive cells were found predominantly around the antennal nerve (arrowheads). (C) Percentage of the antennal nerves associated with BrdU-positive cells. The colour coding indicates the percentage of antennal nerves with the indicated numbers of labelled cells. The sample number (n) is indicated above each bar. (D) Pulse-chase labelling experiment. Blue and orange bars show the percentage of antennal nerves with the indicated numbers of BrdU-positive cells. (E) Percentage of antennal nerves with lacZ-positive cells induced by MARCM. -, flies without heat shock; +, flies with heat shock to induce MARCM clones. **, P<0.01 (Fisher's exact test). (F) A MARCM-labelled cell (expressing β-galactosidase, arrowhead) also incorporated BrdU. Flies were heat shocked 1 day after eclosion and fixed 4 days later. Frontal optical section around an antennal nerve. A merged image (left) and single-channel images (insets) are shown. Scale bars: 50 µm in A; 10 µm in B,F.

View larger version (103K): [in this window] [in a new window]   Fig. 3. Glial cell division induced by neural PCD. (A) The percentage of cells that are REPO-positive or -negative among the BrdU-positive cells observed around the antennal nerve after a 3-hour BrdU treatment (0-3h BrdU). The sample number (n) is indicated above the bar. (B,C) Frontal optical section around an antennal nerve. Dorsal is up. A merged image (left) and single channel images (insets) are shown. (B) A BrdU-positive cell expressed the glial marker REPO, but not the neural marker ELAV (arrowhead). (C) Morphology of a BrdU-positive REPO-positive glial cell visualised by MARCM (expressing GFP, arrowhead). Flies were heat shocked 1 day after eclosion and fixed 4 days later. (D,E) Specimens after 10 days of BrdU treatment. Projection of confocal optical sections around the antennal nerves (left), and a single optical section of the ovary (right). (D) Wild-type (CS) flies. BrdU-positive cells were observed both in the brain (arrowheads) and ovary. (E) The elav-p35 strain, in which neuronal PCD was inhibited by continuous ectopic expression of p35 in neurons. BrdU incorporation in the brain was suppressed specifically. Scale bars: 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Neural injury-associated glial cell division. (A) Schematic of the antennal ablation. An antenna on the right side was ablated from the first antennal segment. (B) Antennae were ablated soon after eclosion, and the flies were fed BrdU for 3 days. Antennal ablation induced BrdU incorporation into glial cells (REPO-positive, arrowhead) even though neuronal PCD was inhibited by p35. (C) Percentage of the antennal nerves with BrdU-positive cells. Colour coding indicates the percentage of antennal nerves with the indicated numbers of labelled cells. int, antennal nerves with intact antennae; abl, antennal nerves with antennae ablated just after eclosion. elav-p35, 3-day BrdU treatment; +/+, wild type (CS), 5-day BrdU treatment; eiger1, homozygous eiger mutant, 5-day BrdU treatment. **, P<0.01 (Fisher's exact test). The sample number (n) is indicated above each bar. (D) Schematic of the needle stab. After feeding the flies with BrdU for 1 day, a needle was inserted into the brain, and the flies were fed BrdU for another day before fixation. (E) Projection of confocal optical sections of the frontal half of the brain. Dashed lines outline the brain. BrdU-positive cells were found around the region of injury. (F) An optical section of the area indicated in E (boxed). A montage of confocal bright-field and immunostaining images. BrdU-positive cells were REPO-positive but ELAV-negative, showing that they were glia (arrowheads). Asterisk indicates the site of injury. Scale bars: 50 µm in E; 10 µm in B,F.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Involvement of Eiger in glial division. (A) Time course of the percentage of antennal nerves with active caspase-positive cells. *, P<0.05 (Fisher's exact test). (B) Percentage of antennal nerves with BrdU-positive cells after 5-day BrdU treatment. +/+, wild type (CS); eiger1 and eiger3, homozygous eiger mutants; glial GAL4, GAL4-NP577. Genotypes that are responsible for significant differences are marked by ++ and - - above the bars, indicating high and low levels of BrdU incorporation, respectively (P<0.01, adjusted residual test after Fisher's exact test extended to r x c performed for the seven genotypes analysed). *, P<0.05 and -, P>0.05 (Bonferroni test). The number of BrdU-incorporating cells in the flies carrying UAS-eiger alone was slightly lower than in the wild type, possibly because of the difference in genetic background. Colour coding shows the percentage of antennal nerves with the indicated numbers of labelled cells. The sample number (n) is indicated above each bar.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Competence of glial division during adult life. (A) Time course of the percentage of antennal nerves with BrdU-positive cells after a 10-day-long feeding of BrdU until 50 days after adult eclosion. (B) Time course of the percentage of antennal nerves with BrdU-positive cells after antennal ablation. Antennae were ablated at the beginning of each period shown on the x-axis, and the flies were fed BrdU until the end of the period indicated. (C) Percentage of the brain with ectopic BrdU-positive cells around the site of injury. A needle was inserted on the day shown on the x-axis. The sample number (n) is indicated above each bar.

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