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doi: 10.1242/10.1242/dev.00198

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The glial cell undergoes apoptosis in the microchaete lineage of Drosophila

UMR 7622, CNRS-Université Paris VI, 9, Quai St. Bernard, 75252 Paris Cedex 05, France

View larger version (69K): [in a new window]   Fig. 1. Glial cells fragment in the microchaete lineage. (A,B) A live neuP72 UAS-H2B::YFP pupa was observed by time-lapse confocal microscopy. Time (hours:minutes) after puparium formation (APF) is shown at the bottom right of each panel. (A) A pI cell was followed from its initial division (at about 17:15 hours APF) until pIIIb division (at about 22:00 hours APF). Two hours later, the glial cell began to fragment (arrow at 23:53 hours APF). (B) Glial cell fragmentation in another bristle lineage from the same pupae as in A. Note that, in this case, the glial cell began to fragment before the division of pIIIb (at 21:33 hours APF and 22:23 hours APF, respectively). (C) Image taken from an in vivo observation of a scabrous-GAL4 UAS-nlsGFP pupae at 23:45 hours APF. The glial cell was identified by the size of its nucleus and lineage criteria. Note glial cell fragments (arrowhead). With this GFP construction, fragmentation was discernible only in rare situations. (D) Glial cells fragment in wild-type pupae. Sensory organ cells were stained with Senseless (green) and Repo (blue) antibodies to identify sensory and glial cells, respectively. Note that the TUNEL-positive cell (red) nearby a sensory cluster is also Senseless-immunoreactive (arrow in D). Also note that Repo-positive glial cells did not co-exist with other TUNEL-positive cells in the same cluster (arrowhead in D). In A and B, each image corresponds to the merging of 12 horizontal confocal optical sections. pI, primary precursor cell; pIIa and pIIb, secondary precursor cells a and b; pIIIb, tertiary precursor cell b; g, glial cell, n, neurone; so, socket cell; sf, shaft cell; st, sheath cell. Anterior is upwards and the view is horizontal.

View larger version (104K): [in a new window]   Fig. 2. The glial cell undergoes apoptosis. (A,B) Deletion of the pro-apoptotic genes rpr, grim and hid in H99-deficient clones blocked fragmentation of the glial cell. Clones were detected due to their lack of GFP staining (green); their limits are shown with a white line. Sensory organ cells were identified by anti-Cut immunoreactivity (also in green). Glial cells were immunostained with anti-Repo antibodies (red). Repo-labelled glial cells are observed in clusters inside H99 deficient clones at 24 hours APF (arrowheads in A). Note that, outside the clone, in the homozygous wild-type twin clone (GFP positive cells on the left of the figure), no glial cell was observed. (B) At 30 hours APF, some sensory organs in H99-deficient clones were associated with a glial cell (arrowheads). Note also organs in which the glial cell was not present (asterisk). (C,D) Overexpression of p35 represses glial cell fragmentation. neuP72 UAS-H2B::YFP UAS-p35pupae dissected at 24 hours APF (C) and 30 hours APF (D). Sensory cells are in green; Repo immunostaining is in red. Note that glial cells are still present at 24 hours APF (arrowheads in C) and have disappeared at 30 hours APF (D). Note also that no cell fragments are observed. Anterior is upwards and the view is horizontal.

View larger version (72K): [in a new window]   Fig. 3. Inhibition of apoptosis did not prevent the loss of the glial cell. Time-lapse confocal analysis of a neuP72 UAS-H2B::YFP UAS-p35 living pupae. Hours:minutes APF is shown at the bottom left of each panel. The glial cell (arrowheads) was maintained alive after overexpression of p35. (A-C) Horizontal views. (A'-C') Lateral views of the corresponding horizontal views. The glial cell remained near the cluster for about 4 hours after it was produced (A,A'). After this period, the glial cell moved away from the cluster and fell down towards the internal cavity (lateral view at 24:15 hours APF, B'). At about 25 hours APF, the glial cell could not be observed anymore. Note that fragmentation was never observed. Each image corresponds to the merge of 18 horizontal confocal optical sections. Lateral projections were reconstructed from the horizontal sections at the positions showed inside the brackets. Broken line in A'-C' denotes the dorsal surface. Anterior is upwards in A-C.

View larger version (97K): [in a new window]   Fig. 4. Ectopic glial cells were frequently associated with growing axons. Growing axons from neuP72 UAS-H2B::YFP UAS-p35 sensory clusters were immunostained with 22C10 antibodies (shown in green). YFP expressing sensory cells are depicted in red. Glial cells (arrowheads) were maintained alive after overexpression of p35. In A-C, typical positions of the ectopic glial cell are shown relative to the growing axon at 24 hours APF. The glial cell was associated with the growth cone (A), standing along the axon (B) or near the sensory cluster even when the axon was well developed (C). Clusters on the first row of microchaete are shown. Clusters in B and C are from the same notum. Another example of growth cone-associated glial cell is shown on the second row of microchates (D). Anterior is upwards and the view is horizontal.

View larger version (41K): [in a new window]   Fig. 5. Ectopic glial cells promote precocious axogenesis. (A,B) Camera lucida drawing of nota stained with 22C10 antibodies at different times of development. In each case, the entire region between the two dorsocentral macrochaetes is depicted. (A) Wild-type nota. At 24 hours APF, only axons from macrochaetes were present. Axogenesis of microchaetes starts at around 25 hours APF. (B) Nota from neuP72 UAS-H2B::YFP UAS-p35. Microchaete axogenesis initiates at about 23:30 hours APF. Note that in nota older than 27 hours APF, no major difference regarding the orientation of axonal processes could be discerned. (C) H99-deficient clone stained with 22C10 antibody (shown in green on the bottom overlay image). The clone was detected because of its lack of GFP staining (shown in red on the bottom overlay image), its medial limit is shown with a white line. Each channel is shown individually (22C10 top left and GFP top right). The midline is associated with a dotted 22C10 immunoreactivity of unknown nature. Note that axon projections are more developed in sensory organs inside the clone (arrowheads) than the counterpart axons in the contra-lateral region (H99 heterozygous). Limits of clones are indicated with a white line. Anterior is upwards and the view is horizontal.

View larger version (143K): [in a new window]   Fig. 6. The apoptosis of the glial cell is independent of glial identity and of pros expression. (A,B) gcme1/gcme1 somatic clones at 24 hours APF are shown. Clones were detected because of their lack of GFP staining (green), their limits are shown with a white line (in A). Sensory organs cells were detected with anti-Cut antibodies (also in green). Neurones were identified by Elav-immunoreactivity (red). (A) At 24 hours APF, two types of sensory clusters were observed inside the gcme1/gcme1 clone: four-cell clusters (arrow) and five-cell clusters (arrowhead). In these cases, an extra Elav-positive cell could be detected (arrowhead). Note that clusters with two Elav-positive cells were never observed outside the clone. (B) Detailed view from another gcme1/gcme1 clone. The arrowhead shows Elav-positive cell fragments next to a four-cell cluster. (C,D) pros17/pros17 somatic clones at 24 hours APF are shown. Clones were detected because of their lack of GFP staining (green), their limits are shown with a white line. Sensory organs cells were detected with anti-Senseless antibodies (in green, C,D) and with anti-Cut antibodies (also in green, D). (C) At 24 hours APF, some cell fragments co-labelled with Senseless and Repo (red) were detected within pros17 clones (arrow), other fragments did not show any Repo staining (arrowhead). (D) Glial cell fragmentation could be detected using TUNEL staining (red) inside pros17 clones as well as outside the clones (arrows). Anterior is upwards and the view is horizontal.

View larger version (53K): [in a new window]   Fig. 7. Cell fragments are englufed by macrophages. neuP72 UAS-H2B::YFP third instar larvae were injected with Indian Ink and dissected at pupal stage (23 hours APF). Macrophages which have incorporated Indian Ink depicted black inclusions of different sizes (red). Sensory organ cells expressing GFP are in green. To avoid reduction in green staining after superposition with a transmitted image, the Indian Ink image was depicted in negative. Note that GFP-positive fragments (arrowheads) are surrounded by an Indian Ink-labelled cell. Anterior is upwards and the view is horizontal.

View larger version (21K): [in a new window]   Fig. 8. Proposed core cell lineage of sensory organs in Drosophila and creation of diversity. Sensory diversity is generated when different processes are added to this basic pattern of cell division. Two processes may be distinguished: first, processes in which cell identity is changed without altering the configuration of the core pattern of cell divisions (red arrows); and, second, processes in which new complexity is added to this core without profound modifications in cell identity (blue arrows).

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