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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02707

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Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Institute of Genetics, University of Mainz, Saarstrasse 21, D-55122 Mainz, Germany.

View larger version (104K): [in this window] [in a new window]   Fig. 1. The CNS of homozygous H99 embryos is not grossly deformed. (A,B). Axon tracts are visualized using the BP102 antibody in wt (A) and H99 (B) embryos. Their pattern is similar, although thickened junctions between the longitudinal connectives and the commissures (arrow), and a widened midline (bracket) are visible in H99. Note that the CNS of H99 embryos is generally wider than that of wt due to additional cells. (C,D) FasII staining of axons reveals a variably altered pattern. The H99 embryo shown in D has a more extreme phenotype in that the fascicles are somewhat disordered and fuzzy. (E,F) Glial cells, visualized with the anti-Repo antibody. A stack of scans was made throughout the CNS and the scans then projected together to show all Repo-positive cells. The positioning of glia is only slightly affected in H99. All images show four abdominal segments (A3 to A6) of late stage 16 embryos; anterior is up.

View larger version (11K): [in this window] [in a new window]   Fig. 2. CNS cell count comparison of mid-stage 16 and stage 17 wt and H99 embryos. White and black bars represent cell counts from wt and H99 embryos, respectively. For exact numbers see text. Asterisks mark statistically significant differences between wt and H99 (P<0.001, two-tailed t-test). The thoracic CNS shows a difference between wt and H99 only at stage 17. The abdominal part of the CNS of H99 embryos contains more cells already at mid-stage 16, and the difference increases further at stage 17. th, thorax; abd, abdomen.

View larger version (67K): [in this window] [in a new window]   Fig. 3. BrdU staining in late stage 17 wt and H99 embryos. All embryos were injected at early stage 17. (A) Wt VNC. Sporadic staining can be seen in the abdominal segments. In the thoracic region, BrdU-labeled cells are found only in the very lateral region of each hemisegment. (B,C) VNCs of two H99 embryos, representing the two classes of staining we found in H99 mutants. One shows a similar amount of staining as in wt (B), whereas the other has many more BrdU-positive cells (C). In both cases, there is a concentration of dividing cells in the lateral region of each abdominal hemisegment. The thoracic segments in B and C show reduced staining in the medial regions. Anterior is to the left in all images.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Profile of Caspase-dependent PCD in the embryonic CNS. Activated-Caspase-3-positive cells in the wt embryonic CNS were counted in abdominal hemisegments over the course of development. PCD in the CNS begins at stage 11 and increases until stage 14. The levels remain high until the end of embryonic development. Bars represent s.d.; n=36-51 (for all stages). Developmental stages are indicated above the bars.

View larger version (85K): [in this window] [in a new window]   Fig. 5. DiI-labeled clones of NB1-1a and NB7-3 in H99 embryos. Each plate shows a semischematic illustration of a wt and an H99 clone, and the corresponding image of the H99 clone. Glia are depicted in green, motoneurons in red. Dashed lines indicate the CNS midline. (A-C) No difference can be seen between NB1-1a in wt (A) and H99 (B,C). Shown is an H99 clone containing 11 cells (including three glial cells). Both the cell numbers and the axonal projection pattern are unchanged in H99. (D-F) NB7-3 consists of more cells in H99 (E,F) than in wt (D), including an additional motoneuron that projects its axon outside the CNS (arrow). The image shows an abdominal H99 clone with 9 cells.

View larger version (64K): [in this window] [in a new window]   Fig. 6. DiI-labeled clones of NB4-2, NB5-3, NB7-2 and NB7-4. Each plate shows an illustration of a wt and an H99 clone, and the corresponding image of the H99 clone. Glia are depicted in green, motoneurons in red. Dashed lines indicate the CNS midline. (A-C) The NB4-2 lineage has two motoneuronal projections in wt (A). In H99, there is a third motoneuronal axon (arrow in C) projecting ipsilaterally through the posterior root of the intersegmental nerve. The image shows an abdominal clone comprising 25 cells. (D-F) The wt NB5-3 shows an ipsilateral motoneuronal and two contralateral interneuronal projections (D). In H99 (E,F), there is an additional ipsilateral interneuronal projection extending anteriorly (arrow in F). Shown is an image of an abdominal clone containing 25 cells. (G-I) In the wt, NB7-2 interneurons form two fascicles, an ipsilateral one extending posteriorly, and a contralateral one projecting through the posterior commissure (G). The H99 clones (H,I) show another contralateral projection, extending alongside the wt one (arrow in I). Shown is an abdominal H99 clone with 21 cells. (J-L) In the wt, NB7-4 interneurons form one fascicle that traverses contralaterally through the posterior commissure (J). In H99 (K,L), NB7-4 clones exhibit an atypical projection that runs through the anterior commissure of the next segment (arrow in L). Shown is an abdominal clone with 29 cells.

View larger version (66K): [in this window] [in a new window]   Fig. 7. Caspase-dependent PCD of dHb9-expressing cells. (A,B) At late stage 16, H99 mutants (B) have more dHb9-positive cells than wt (A). (C) Activated-Caspase-3 staining reveals that some of these cells die at stage 14 (arrow). In this case, the dying cell lies in a position corresponding to the RP (1, 3, 4 or 5) neurons. dHb9 staining is in green, activated Caspase-3 in red. Anterior is up in all images.

View larger version (134K): [in this window] [in a new window]   Fig. 8. Caspase-dependent PCD of Lbe-, Eg- and Eve-expressing cells. (A-C) Lbe-positive cells, most likely from the NB5-3 lineage, are present in higher numbers in H99 mutants (B) than in wt (A). Apoptotic Lbe-positive cells can be seen at stage 14 (arrow in C). (D-F) The Egexpressing NB7-3 lineage also shows too many cells in H99 (E, compare with wt in D). The GW motoneuron undergoes apoptosis in segments T3 to A8 at late stage 16 (arrows in F). (G-I) Eve-positive U motoneurons are not present in segments A7 and A8 in wt embryos (G). In H99, these cells survive (H). U neurons in A7 and A8 die at stage 14 (arrows in I). Lbe staining in C, Eg in F and Eve in I are in green, activated Caspase-3 in red. A,B,D-H show late stage 16 embryos; C,I show stage 14 embryos. Lines in G-I demarcate segments A7 and A8 for clarity. Anterior is up in all images.

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