MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK
Present address: NCI Laboratory of Molecular Cell Biology, Building 37 Room 6066, NIH, Bethesda, MD 20892-4255, USA. Author for correspondence (e-mail: badenhop{at}pop.nci.nih.gov)
Accepted July 19, 2001
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SUMMARY |
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Key words: Tramtrack, gliogenesis, Drosophila, CNS
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INTRODUCTION |
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In the Drosophila embryonic CNS similar bipotential precursor cells have been identified. Each abdominal segment of the lateral CNS is derived from 31 progenitor cells. Lineage studies have shown that these precursor cells either produce only neurons (neuroblasts), exclusively glia (glioblasts) or both neurons and glia (neuroglioblasts) (Bossing et al., 1996; Ito et al., 1995; Schmidt et al., 1997). Two well-characterized glia-producing stem cells are the longitudinal glioblast (LGB) and the neuroglioblast 6-4. The longitudinal glioblast exclusively generates glia. It divides to produce glia that eventually ensheath the longitudinal axonal tracts of the CNS (Ito et al., 1995; Jacobs et al., 1989). However, the precise number of cell divisions that this progenitor undergoes is unclear, as are the mechanisms that regulate the final longitudinal glial number. In thoracic segments, the neuroblast 6-4 divides to generate a neural stem cell that produces four to six neurons, and a glioblast that generates three glial cells (Akiyama-Oda et al., 1999; Bernardoni et al., 1999; Schmidt et al., 1997).
Neural fate appears to be the default identity of all these progenitors. Both neuroblasts, and glioblasts and neuroglioblasts, transiently express neural stem cell markers such as the pan-neural genes asense and deadpan during their development (Bernardoni et al., 1999; Dominguez and Campuzano, 1993; Jarman et al., 1993). Further development into glia requires the activation of a glial-determining pathway. In glia of the lateral CNS, transient activation of the binary switch glial cells missing (gcm) induces glial differentiation (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996). Gcm expression is succeeded by expression of the Ets transcription factor pointedP1 (pntP1; pnt FlyBase) and the homeodomain protein reversed polarity (repo) both of which enforce the glial differentiation pathway initiated by gcm (Halter et al., 1995; Klaes et al., 1994; Klämbt, 1993).
The switch to glial fate requires also the simultaneous repression of the previous and alternative neuronal fate. The zinc-finger transcriptional repressor Tramtrack (Ttk) has been suggested to perform this function (Badenhorst et al., 1996; Giesen et al., 1997). The ttk locus encodes two DNA-binding proteins Ttk69 and Ttk88 which share a common N-terminal domain, the BTB (Bric-à-Brac-Tramtrack-Broad) motif, but have divergent C-terminal zinc-finger domains and, it is assumed, DNA-binding specificities (Harrison and Travers, 1991; Read and Manley, 1992). Ttk69 is expressed in all mature glia and ectopic expression can block neuron development (Giesen at al., 1997). I analyzed the timing of Ttk69 expression in glia and found that its expression succeeds that of existing markers of glial determination. I show that Ttk69 represses the neural stem cell-specific genes asense and deadpan. Ttk69 also can control glial proliferation. Thus, in proliferating glia, protein levels oscillate in a cell cycle-dependent manner. Expression is not detectable during DNA replication, but re-initiates after S-phase. Overexpression of Ttk69 prevents cell cycle progression by inhibiting expression of the S-phase cyclin, cyclin E and blocks glial development. Ttk69 controls glial development by both regulating glial cell number as well as identity.
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MATERIALS AND METHODS |
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Directed protein overexpression
Targeted expression of Ttk69 in the embryonic CNS used the Gal4-UAS system (Brand and Perrimon, 1993). Full-length Ttk69 cDNA was cloned into pUAST, placing it under the control of the Gal4 UAS. Several independent UAS-Ttk69 lines were established by P element-mediated transformation. Gal4 lines used were: Kr-Gal4 (expresses in T2-A4); sca-Gal4 (expresses in neuroblasts and their progeny) (Klaes et al., 1994); and MZ1580 (expresses in the longitudinal glia and its progeny, MP2 neurons and macrophages) (Hidalgo et al., 1995).
Immunocytochemistry and immunofluorescence
Embryo fixation and immunocytochemistry were performed as described in Halter et al. (Halter et al., 1995). For immunofluorescence, FITC-, Cy3- and Cy5-conjugated anti-IgG secondary antibodies (Jackson Immunoresearch) were used. Biotinylated anti-IgG secondary antibodies and FITC-, Cy5- or Cy3-conjugated streptavidin (Jackson Immunoresearch) were used where signal enhancement was required. Embryos were mounted in phosphate-buffered saline (PBS) containing 90% glycerol and 1 mg/ml phenylenediamine and viewed using an MRC 1024 confocal microscope. Digoxigenin-labeled ribroprobes were prepared and RNA in situ hybridization performed as described by Lehmann and Tautz (Lehmann and Tautz, 1994). DNAs used for probe preparation were a cDNA corresponding to cyclin E type II transcript.
Antibodies used were: mouse mAb 22C10 at 1:40 (Fujita et al., 1982); mouse mAb 9F8A9 and rat mAb 7E8A10 anti-Elav at 1:40 (ONeill et al., 1994); rabbit anti-Repo at 1:100 (Halter et al., 1995); mouse mAb 990E5F1 anti-Ac at 1:40 (Skeath and Carroll, 1992); rabbit anti-Ase at 1:1000 (Jarman et al., 1993); mouse mAb anti-BrdU at 1:50 (Becton Dickinson); rabbit anti-lacZ at 1:1000 (Cappel); rat anti-Ttk69 at 1:50; rat anti-Ttk88 at 1:50; and rabbit anti-Ttk69 (zinc fingers) at 1:100 (Lehembre et al., 2000). Embryos were staged according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985).
BrdU incorporation
Bromodeoxyuridine (BrdU) incorporation was performed as described by Lehner et al. (Lehner et al., 1991) with the following modifications. After fixation, embryos were washed four times in PBT (PBS + 0.1% Triton X-100) and then incubated with two changes of 2 N HCl for 30 minutes. Thereafter, embryos were washed with multiple changes of PBT for 1 hour and immunofluorescence performed as above.
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RESULTS |
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Confocal microscopy showed that Ttk69 is expressed late in glial development. In lateral glia, double labeling showed that Ttk69 is expressed after either Gcm, the ETS protein PointedP1 (not shown) or Repo (Fig. 1). For example, when Repo is first detected in the longitudinal glioblast, Ttk69 can not be detected. Later, however, low level expression of Ttk69 commences in this progenitor (Fig. 1A).
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In contrast, glia which express Repo and do not undergo further division express high levels of Ttk69 from the moment Repo can first be identified. An example is the cell body glia (Fig. 1B,C; arrows). In no case, was I able to detect expression of Ttk69 before that of Repo.
Neural targets repressed by Ttk
The timing of Ttk69 shows that it does not initiate glial determination. It has been proposed that Ttk69 is expressed in glia to repress neural identity genes (Badenhorst et al., 1996; Giesen et al., 1997). Lateral glioblasts transiently express neural stem cell markers during their development (Bernardoni et al., 1999) and can adopt the neuronal fate when the glial-determining pathway is not initiated in gcm mutants. Stable glial identity could require neuronal repression. To determine neuronal-specific genes repressed by Ttk69, I analyzed how ectopic expression of Ttk69 at various stages of nervous system development affected expression of the hierarchy of neuronal markers. This included the proneural genes of the achaete-scute complex, the pan-neural genes (for example asense) and the mature neuronal markers Elav and the antigen 22C10.
Ectopic expression of Ttk69 at any stage did not prevent neuroblast formation. Thus, expression of Ttk69 before neuroblast formation using Kr-Gal4 does not repress the proneural genes achaete (Fig. 2B) or lethal of scute (data not shown). Strikingly, however, it does inhibit the pan-neural genes asense (Fig. 2C), dpn and scratch. Consequently, further neuronal development is inhibited and expression of both mature neuronal markers Elav (Fig. 2D) and 22C10 (data not shown) is ablated. Equivalent results were obtained by ectopically expressing Ttk69 in neuroblasts and their progeny using the sca-Gal4 driver. Such expression almost completely inhibits the normal expression of dpn in the embryonic CNS (Fig. 2E,F; compare wild type and overexpressing lines).
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Ectopic expression of Ttk69 also blocks glial formation
In the Drosophila embryonic and larval PNS ectopic expression of Ttk69 can convert neurons into non-neuronal cells, while, conversely, loss of ttk leads to the opposite transformation (Guo et al., 1995; Guo et al., 1996). In the CNS, however, neuron loss induced by ectopic Ttk69 expression does not lead to increased glial number. In fact, ectopic expression of Ttk69 in the embryonic CNS also blocks glial development. I found that normal glial development is inhibited by early overexpression of Ttk69. Thus, ectopic expression of Ttk69 before precursor formation, using Kr-Gal4, extinguishes the lateral glial markers Gcm and Repo in the domain of expression (Fig. 4A,B).
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Ttk69 is not detected in glia undergoing DNA replication
To understand why early ectopic Ttk69 expression inhibits glial development, I re-examined the temporal profile of Ttk69 expression in the CNS. Specifically, I analyzed Ttk69 expression in the longitudinal glia relative to replication marked by BrdU incorporation. The gross expression profile in Fig. 1 illustrates the position of the longitudinal glia relative to the embryo co-ordinates and other glia. The longitudinal glioblast delaminates from the lateral edges of the neuroepithelium and as shall be seen, it divides symmetrically at least three times, the progeny migrating medially towards the ventral midline. The panels in Fig. 5 show high magnification of the longitudinal glia only allowing Ttk69 expression relative to DNA replication to be clearly visualized. Ttk69 protein is undetectable in longitudinal glial cells when they undergo DNA replication as indicated by BrdU incorporation. Although the LGBs express Ttk69, Ttk69 is absent from their daughters when undergoing replication (Fig. 5, two-cell stage). However, later when these glia exit S phase, Ttk69 is again expressed at low levels. The longitudinal glia divide synchronously once more and appear to enter S phase shortly thereafter. Again, during DNA replication Ttk69 is undetectable (Fig. 5, four-cell stage), but after S phase is complete, Ttk69 expression reinitiates (four-cell stage). After this division, glia do not incorporate BrdU, and consistently express high levels of Ttk69 (Fig. 5, mature).
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DISCUSSION |
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Glial versus neuronal specification
The widespread expression of Ttk69 and its timing are consistent with Ttk69 maintaining glial identity rather than initiating glial determination. Previous investigations have shown that the two main populations of Drosophila CNS glia, midline glia and lateral glia, are specified by distinct mechanisms. For example, lateral glia require Gcm to initiate development, while midline glia do not (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996). Yet Ttk69 is expressed in both populations the only transcription factor expressed in common. Giesen et al. (Giesen et al., 1997) have suggested that glial determination requires two independent steps: first, a proglial function, for example, Gcm (Hosoya et al., 1995; Jones et al., 1995; Vincent et al, 1996), that initiates glial development; and second, an anti-neural activity that downregulates the previous potential to form neurons. The ability of Ttk69 to repress to neuron development shows that it performs the later role and in this report I have identified the targets of neuronal repression.
In the Drosophila CNS, neural development requires the expression of a cascade of transcription regulators, initiating with high level expression of the proneural genes of the Achaete-Scute complex (AS-C), leading to expression of neural stem cell-specific genes asense and deadpan, and ultimately a battery of specific neural identity genes. Repressing any of these factors could inhibit neural development. Staged overexpression of Ttk69 shows that, in the CNS, Ttk69 inhibits expression of asense and deadpan. In this manner, the ability to adopt neuronal fate is blocked.
To date, in mammals, a master regulator of glial development analogous to Gcm has not been identified. Although mammalian homologs of the Drosophila gene gcm have been isolated (Kim et al., 1998), there is no evidence that they play a role in glial determination. However, recently, Notch signaling has been shown to allow some multipotent stem cells in the nervous system to differentiate preferentially into glia (Furukawa et al., 2000; Gaiano et al., 2000; Hojo et al., 2000; Morrison et al., 1999). Two downstream components of the Notch pathway have been implicated: the Hairy-E(Spl)-related bHLH repressors Hes1 and Hes5 (Furukawa et al., 2000; Hojo et al., 2000). Both are thought to act as repressors of the neuronal specific bHLH genes Mash1, NeuroD and Math3 (Morrow et al., 1999; Takebayashi, et al., 1997; Tomita et al., 1996). These studies, however, have not clarified whether Notch signaling acts instructively to promote glial development or whether repression of the neuronal specific bHLH genes permits multipotent stem cells to respond to other instructive signals that induce gliogenesis. If Hes1 and Hes5 act permissively, the parallels between vertebrate and Drosophila gliogenesis would be striking. In Drosophila, Ttk69 can be induced by Notch signaling (Guo et al., 1996). As seen above, Ttk69 represses the neuronal-specific bHLH gene asense. Ttk69 itself does not induce glial fate but blocks the alternative neuronal fate.
Control of proliferation
It is clear from the current study that, at least in some glial populations, Ttk69 has the ability to regulate proliferation. In the Drosophila glioblasts Ttk69 is expressed at low levels soon after glial specification, blocking neural genes. This expression is not constant, though, but oscillates during the cell cycle. Significantly, Ttk69 can not be detected when glia enter S phase and commence DNA replication. Like neuroblasts (Weigmann et al., 1997), glioblasts appear to delaminate in G2 of the cell cycle. The timing of BrdU incorporation indicates that immediately after mitosis they enter S phase and undergo DNA replication. Although Ttk69 is expressed in glia in G2, Ttk69 is not detected in glia that incorporate BrdU. As Ttk69 can repress cyclin E expression, the absence of Ttk69 allows replication to occur. Once replication is complete, Ttk69 is expressed again. The BrdU incorporation experiments indicate that the LGB undergoes three synchronous cell divisions to produce eight longitudinal glia. This agrees well with a recent estimate of between 7-9 longitudinal glia obtained by DiI labeling of the longitudinal glioblast (Schmidt et al., 1997). After the third mitosis, longitudinal glia do not undergo replication but instead express higher levels of Ttk. By inhibiting cyclin E, high levels of Ttk69 would keep glia in G1 of the cell cycle. Similarly, differentiation of oligodendrocytes (Durand et al., 1998) and Müller glia (Ohnuma et al., 1999) is accompanied by high levels of the cyclin-dependent kinase inhibitor p27, blocking re-entry into the cell cycle.
I determined longitudinal glial number in ttk mutant embryos at embryonic stage 14, as the longitudinal glia first extend along the longitudinal axonal tracts. At later stages of development, glia in ttk mutant embryos migrate inappropriately. The regular array of glia along the longitudinal connectives is lost as glia collapse towards the midline. Antibody staining against the nuclear glial antigen Repo indicates nuclear fragmentation, which is evidence of possible cell death. It is feasible that inappropriate proliferation triggers apoptosis. When Giesen et al. (Giesen et al., 1997) examined CNSs dissected from late-stage ttk mutant embryos, they reported that glial number (revealed by the enhancer trap-line rI50) is reduced. Differences between glial number reported here could be due to the stage at which determinations were made.
In its ability to repress pan-neural genes and control proliferation, Ttk69 resembles the homeodomain protein Prospero. Prospero is not detected in the nuclei of neuroblasts but is detected in the nuclei of their daughter ganglion mother cells (GMCs). GMCs divide once and terminally differentiate. In GMCs, Prospero appears to be required to repress the pan-neural genes asense and deadpan, thus enforcing the transition from neuroblast to GMC (Vaessin et al., 1991). Recently, it has been shown that loss of Prospero leads to ectopic proliferation in the CNS, suggesting that it is required for exit of GMCs from the cell cycle (Li and Vaessin, 2000). Ttk69 appears to play an analagous function in glia.
Dynamic regulation of Ttk69 levels
The oscillation in Ttk69 protein levels during the cell cycle demonstrates that there is dynamic control of Ttk expression. Several mechanisms may regulate Ttk69 protein levels. Ttk69 has PEST sequences characteristic of short-lived proteins (Harrison and Travers, 1991). Moreover, both isoforms of Ttk have been shown to be targeted for ubiquitin-dependent proteolysis by a complex containing the ring-finger proteins Sina and Phyllopod (Li et al., 1997). It is possible that Ttk69 is destroyed by regulated proteolysis prior to initiation of S-phase. It is interesting in this regard that Ebi, a modifier of over-proliferation phenotypes associated with E2F/DP overexpression, has been shown to interact with Sina and Phyllopod and targets destruction of at least one Ttk isoform in vitro (Boulton et al., 2000). Another dimension is added by the recent report that Ttk69 translation can be repressed by the RNA-binding protein Musashi (Okabe et al., 2001). Translational repression by Musashi is not constitutive but appears to be regulated, in part, by signaling through the Notch pathway. The intersection between regulated translational repression and targeted proteolysis of residual protein provides exquisite control of protein levels.
In conclusion, these results demonstrate that Ttk69 maintains glial differentiation. This is achieved in two ways. First, Ttk69 represses the neural stem cell-specific genes which prevents the reprogramming of glia into neurons. Second, Ttk69 inhibits inappropriate proliferation of glia. In this way CNS organization is preserved.
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ACKNOWLEDGMENTS |
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