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Tramtrack controls glial number and identity in the Drosophila embryonic CNS

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

View larger version (118K): [in a new window]   Fig. 1. Ttk69 is expressed after Repo in lateral glia. (A-E) Ttk69 (red) and Repo (green) expression in lateral glia of early Stage 11 to Stage 14 embryos. Panels show either a merge of Ttk69 and Repo (co-expression is denoted by a yellow overlap) or Ttk69 expression alone. Later stages show separate confocal sections through either outer or inner layers of the CNS, allowing overlying glial populations to be distinguished. A schematic (Halter et al., 1995) of Repo-expressing glia at every embryonic stage is shown. Anterior is upwards; open arrow, midline; lgb, longitudinal glioblast; lg, longitudinal glia (colored purple in schematic); tp, tracheal pits; asterisk, glioblast 3-1 (Schmidt et al., 1997).

View larger version (80K): [in a new window]   Fig. 2. Early ectopic expression of Ttk69 inhibits neural development. (A) UAS-Ttk69 was expressed ectopically in parasegments T2 to A4 (between arrowheads) under the control of Kr-Gal4. Ectopic expression levels were comparable with endogenous expression in the cephalic region (asterisk). Ectopic Ttk69 does not prevent the early neuroblast expression of Achaete (B), but does inhibit Asense expression (C) and the mature neural marker Elav (D). (E) Expression of a dpn-lacZ reporter (in green) is inhibited by ectopic expression of Ttk69 using sca-Gal4 (F, Ttk69 revealed in red).

View larger version (121K): [in a new window]   Fig. 3. Ectopic expression of Ttk69 in mature neurons disrupts axonal guidance. Late ectopic expression of Ttk69 in post-mitotic neurons, using the elav-Gal4 driver, does not repress the neural markers Elav (A), 22C10 (C) or Fasciclin II (E). It does, however, affect the behavior of mature neurons. (B-C) MAb 22C10 staining shows that the longitudinal tracts are disrupted (arrowheads) and the segmental and intersegmental nerves exhibit fasciculation and pathfinding abnormalities (arrows). (D-E) Anti-Fasciclin II (mAb 1D4) staining confirms that the longitudinal fascicles are disorganized after Ttk69 ectopic expression and that the intersegmental nerve is misrouted and often fails to exit the CNS, instead migrating back towards the midline (arrows). The elav-Gal4 driver also directs Ttk69 expression in neurons of the PNS. Strikingly, PNS organization was unaffected by this ectopic expression (data not shown).

View larger version (93K): [in a new window]   Fig. 4. Ectopic Ttk69 expression blocks glial development. Expression of the glial marker Gcm, revealed using the enhancer-trap line rA87 (A), and Repo (B) is inhibited by early Ttk69 expression using the Kr-Gal4 driver. Later expression of Ttk69 in the progeny of glioblasts using the sca-Gal4 driver also blocks glial development. (C) In stage 12 wild-type embryos, anti-Repo antibody staining (in green) stains between 13-15 glial cells per hemineuromere. (D) After ectopic expression of Ttk69 (revealed in red), the Repo-positive glial number is reduced to two to three cells per hemineuromere. The remaining glia are some longitudinal glia (arrows) and the glioblast 3-1 (asterisk) (E) Expression of a nuclear-targeted lacZ driven by sca-Gal4 initially is low in the longitudinal glia (arrowhead, stage 11) but later increases (arrowhead, stage 13). Other glia such as cell body glia (arrows) express high levels of lacZ from the first. (F-G) Anti-Repo staining shows that overexpression of Ttk69 using MZ1580-Gal4 reduces longitudinal glial number in the fully-formed stage 15 embryonic CNS (arrows in G; compare with control F). Anterior is upwards; open arrow, midline; lg, longitudinal glia.

View larger version (95K): [in a new window]   Fig. 5. Ttk69 is undetectable during DNA replication. DNA replication in glia was monitored by BrdU incorporation followed by triple labeling with anti-BrdU (green), anti-Repo (blue) and anti-Ttk69 (red) antibodies. Ttk69 is first expressed at low levels in the Repo-expressing longitudinal glioblast (LGB, arrowhead) at embryonic stage 11. The LGB divides and its progeny immediately enter S phase. During DNA replication, Ttk69 is not detected in the daughter cells (two-cell stage, arrowheads). But, once replication is complete, low level Ttk69 expression is again detected. Anti-Repo staining (red) of the egP289 lacZ enhancer trap line (green; shown in the lowest panel) confirms the identity of the Repo-positive longitudinal glia relative to the Repo- and Eg-positive cell body glia (arrows). At stage 12, the longitudinal glia divide again, and immediately enter S phase and incorporate BrdU (four cell stage). Again, Ttk69 is absent during DNA replication. Two to three Repo-positive cells near the longitudinal glia do not undergo replication express high levels of Ttk69. These glia do not derive from the LGB: anti-Repo (red) and anti-lacZ (green) staining of hkbAI7 enhancer-trap line shows that they are probably exit glia and sub-perineural glia (lowest panel, arrows). After this stage, BrdU is not incorporated in the longitudinal glia and Ttk69 is expressed in the four longitudinal glial progeny. These glia divide once more to generate eight glial cells, all of which express high levels of Ttk. Further replication is not detected in these glia. Anterior is upwards, the midline towards the right; cbg, cell body glia; eg, exit glia; lg, longitudinal glia; mg; midline glia; spg, sub-perineural glia; tp, tracheal pit; arrows in mature panels, midline.

View larger version (57K): [in a new window]   Fig. 6. Ectopic expression of Ttk69 inhibits replication and represses cyclin E. (A) Kr-Gal4-mediated expression of Ttk69 blocks BrdU incorporation in the domain of expression (flanked by arrowheads). Failure to replicate is due to repression of cyclin E. Expression of cyclin E in the wild-type CNS (B) is inhibited by ectopic expression of Ttk69 in heat-shocked hs-ttk69 embryos (C).

View larger version (91K): [in a new window]   Fig. 7. Glia undergo extra rounds of DNA replication in ttk mutant embryos. After BrdU incorporation, wild-type (A) and ttk1e11 mutant embryos (B) were stained with anti-Repo (red) and anti-BrdU (green) antibodies. In wild-type stage 12 embryos, four Repo-positive longitudinal glia in each hemi-neuromere incorporate BrdU. By contrast, six to seven glia undergo DNA replication in mutant embryos of the equivalent stage. The number of Repo-positive glia associated with the longitudinal connectives is increased from 9.9±0.76 in wild-type embryos (C) to 17.8±2.23 in ttk mutant embryos (D). Data are mean±s.d. of 20 determinations. Anterior is upwards; open arrows, midline.