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1 Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604; 2 Department of Anatomy, University of California, San Francisco, San Francisco, California 94158, USA; 3 University of Utah, Salt Lake City, Utah 84112, USA
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[Keywords: Neuroblast; stem cells; asymmetric division; tumor suppressor; self-renewal]
Received August 29, 2006; revised version accepted October 31, 2006.
; Betschinger and Knoblich 2004; Wodarz 2005; Yu et al. 2006). The asymmetric division of NBs involves the asymmetric localization and segregation of cell fate determinants Numb, Prospero (Pros), Brat, and their adaptor proteins Partner of Numb (Pon) and Miranda (Mira) into the basal GMC (Rhyu et al. 1994; Knoblich et al. 1995; Ikeshima-Kataoka et al. 1997; Shen et al. 1997; Lu et al. 1998). During NB asymmetric divisions, the mitotic spindle is oriented along an axis perpendicular to the epithelial layer and an asymmetric spindle is generated to give rise to two unequal-sized daughter cells with distinct cell fates. NB asymmetric divisions are controlled by an apically localized complex of proteins that include the Drosophila homologs of the conserved Par3 (Bazooka, Baz)/Par6 (DmPar6)/atypical protein kinase C(DaPKC) proteins (Kuchinke et al. 1998; Wodarz et al. 2000; Petronczki and Knoblich 2001), Inscuteable (Insc) (Kraut et al. 1996), and heterotrimeric G proteins G
i (Schaefer et al. 2001; Yu et al. 2003) and their regulators Partner of Insc (Pins) (Yu et al. 2000), Locomotion defects (Loco) (Yu et al. 2005), and a Pins-interacting protein mushroom body defective (Mud) (Bowman et al. 2006; Izumi et al. 2006; Siller et al. 2006). The asymmetric localization of Gi requires G
(Schaefer et al. 2001; Yu et al. 2003) and G
(Fuse et al. 2003) and its membrane localization requires Ric-8 (Hampoelz et al. 2005; Wang et al. 2005). Basal protein localization and segregation are mediated by apical proteins through cortically localized tumor suppressors, Discs large (Dlg) and Lethal (2) giant larvae (Lgl) (Ohshiro et al. 2000; Peng et al. 2000). Phosphorylation of Lgl by apically localized aPKC leads to Lgl inactivation, while nonphosphorylated Lgl at the basal cortex is active and allows the localization of basal proteins (Betschinger et al. 2003).
Drosophila larval NBs have emerged as a model system for studying stem cell self-renewal as larval NBs, unlike embryonic NBs, share many features of stem cells as they can undergo growth and self-renewal for extended periods and produce a large number of progeny (see reviews Jan and Jan 2001; Betschinger and Knoblich 2004; Wodarz 2005; Yu et al. 2006). There is increasing support for the idea that tumors can arise from tumor stem cells in which the normal control of self-renewal versus differentiation is disturbed (Passegue 2006). The regulation of self-renewal versus differentiation, associated with asymmetric cell divisions, is tightly controlled during Drosophila larval brain development. Perturbation of the asymmetric division process can lead to uncontrolled proliferation and aberrant terminal differentiation. When transplanted into wild-type adults, mutant larval brain tissue from pins, mira, numb, or pros can form malignant tumors that rapidly kill the host (Caussinus and Gonzalez 2005). Recent studies have also suggested that NBs utilize the asymmetric localization/segregation machinery to distribute "proliferation factors" to the NB daughter and "differentiation factors" to the GMC daughter during asymmetric cell division. aPKC, which is inherited by NBs, is necessary to promote NB self-renewal, and when a membrane targeted version is overexpressed and localized to the entire cortex, leads to NB overgrowth (Lee et al. 2006a). In contrast, "differentiation factors" such as tumor suppressors Brain tumor (Brat) and Prospero (Pros), which are asymmetrically segregated into the GMC daughter act to suppress self-renewal and promote differentiation in GMC. Loss of Brat or Pros results in massive production of NBs at the expense of neurons (Bello et al. 2006; Betschinger et al. 2006; Lee et al. 2006b). Delocalization of aPKC and Brat/Pros in lethal giant larvae (lgl) mutants can lead to tumor formation in larval brains (Betschinger et al. 2006; Lee et al. 2006a, b). However, the mechanism of self-renewal versus differentiation is still largely unknown. For example, aPKC is not required for proliferation in brat mutant and the downstream target(s) of aPKC for the regulation of NB proliferation remains unclear. It appears that additional novel mechanisms might act to suppress excessive NB self-renewal.
Here we show that Aurora-A (AurA) acts as a tumor suppressor by suppressing NB self-renewal and promoting neuronal differentiation during larval brain development. We show that AurA acts upstream of aPKC to regulate NB self-renewal. AurA is required for the asymmetric localization of aPKC and preventing it from localizing to the basal cortex. We also suggest that Numb is one major target of AurA and aPKC in regulating NB self-renewal. The overproliferation phenotype of aurA or numb mutants is due to the up-regulation of Notch activity, but appears to be largely independent of Brat and Pros. Thus, our data provides evidence for a novel pathway that involves AurA, aPKC, Numb, and Notch for the regulation of neuroblast (NB) self-renewal versus neuronal differentiation.
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We screened a collection of pupal lethal mutants (L. Wang, J. Evans, H. Andrews, R. Beckstead, C.S. Thummel, and A. Bashirullah, in prep.) to isolate mutants that are defective in asymmetric cell division or self-renewal of larval NBs. Three recessive EMS-induced pupal lethal mutants (Fig. 1A; data not shown) belonging to a single complementation group were identified. In these mutants, supernumerary NBs that express both Mira and Inscuteable (Insc) {Fig. 1A [wild type], A' [l(3)LL-8839 (aurA8839)]; data not shown} were produced. Genetic analyses and sequencing of these mutations, aurA8839 (Lys 377 to a stop codon), aurA14641 (Val 302 to Glutamate) and aurA17961 (Asp 344 to Asparagine), revealed that they each harbor either a single missense or a nonsense mutation in the conserved C-terminal kinase domain of aurora-A (aurA) (Fig. 1B). An available strong hypomorphic allele, aur87Ac-5, also shows a similar tumorous larval brain phenotype (data not shown). These alleles of aurA represent an allelic series, with aurA8839 showing the strongest phenotype. The aurA8839 tumor phenotype and lethality are fully rescued by an aurA-GFP transgene expressed with a NB-specific driver, worniu-Gal4 (wor-Gal4) (data not shown). In addition, AurA protein, which normally localizes on both centrosomes in wild-type metaphase NBs (Fig. 1C), is undetectable on the centrosomes of aurA8839 larval NBs (Fig. 1C'). These data indicate that loss of AurA function is responsible for the overproliferation phenotype, and that aurA8839 is either a strong hypomorphic or null allele.
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We quantified central brain NB numbers in aurA8839 mutant larvae from 24 h to 96 h after larval hatching (ALH). Larval NBs can be identified unambiguously by the expression of the markers Worniu, Deadpan (Dpn) and Mira (which is also transiently present in newborn GMCs) and the absence of the neuronal marker, Elav (Betschinger et al. 2006; Lee et al. 2006a, b). Wild-type larval central brains have 31 ± 6 NBs at 24 h ALH and the NB number increases to 89 ± 16 by 96 h ALH just prior to metamorphosis (Figs. 1D, 2A,B; Lee et al. 2006a). In aurA8839 mutant larvae, NBs start with a similar number at 24 h ALH. However, NB number increases dramatically to 371 ± 85 at 96 h ALH (Figs. 1D, 2A',B') and to more than a thousand at 120 h ALH during an extended larval life (data not shown). Before puparium formation, the aurA8839 larval brains (Fig. 1E', arrow) can grow up to 10 times the wild-type size (Fig. 1E, arrow). BrdU incorporation experiments indicate a large increase in the number of S-phase cells in aurA mutant (Fig. 2C') compared with wild-type brains (Fig. 2C). A significant increase of mitotic cells labeled by phospho-Histone H3 was also observed in aurA brains (Fig. 2D [wild type], D'). There is also a significant decrease in cells expressing the neuronal markers, Elav (Fig. 2E') and nuclear Pros (Fig. 2F'), in aurA compared with wild-type brains (Fig. 2E,F), suggesting that the increase in NB-like cells occurs at the expense of differentiated neurons.
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; Meraldi et al. 2002). Human AurA phosphorylates P53, a tumor suppressor that regulates apoptosis, leading to its degradation and thus facilitating the oncogenic transformation of cells (Katayama et al. 2004). However, overexpression of AurA with wor-Gal4 did not affect NB number in the larval brains (data not shown). Apoptosis also appears not to be affected in aurA8839 mutant larval brains, as the numbers of cells labeled by Caspase-3 (Supplementary Fig. 1A') or DIAP-1 (data not shown) in aurA8839 are similar to those of wild-type brains (Supplementary Fig. 1A; data not shown). These data suggest that AurA acts via a novel mechanism in regulating Drosophila larval NB proliferation.
CycE/CDK2 is an important cell cycle regulator and CycE levels were shown to be up-regulated in imaginal discs of tumor suppressor mutants including warts, salvador, and hippo (Vidal and Cagan 2006). We also observed a dramatic increase in the number of CycE-positive cells in aurA larval brains (Fig. 2G') compared with wild type (Fig. 2G). To investigate whether this is a causal effect, we overexpressed Dacapo, a member of the P21/P27 family of CDK inhibitors (Lane et al. 1996), by wor-Gal4 in the aurA8839 mutant. The tumor phenotype of aurA8839 is largely suppressed (Fig. 2I–L) by overexpressing Dacapo. However, overexpression of CycE alone does not cause overgrowth of larval NBs (Betschinger et al. 2006; data not shown), suggesting that elevated levels of CycE are necessary but not sufficient to induce tumor formation. In addition to CycE, dMyc, another important growth factor, is also up-regulated in aurA8839 mutant (Fig. 2H') compared with wild-type larval brains (Fig. 2H). aurA mutant also generate weak NB overproliferation phenotype in mitotic clones by the Mosaic Analysis with a Repressible Cell Marker (MARCM) system (Supplementary Fig. 2; Lee et al. 2000), presumably due to protein perdurance in the clone.
Asymmetric localization of aPKC and Numb requires AurA function
Tumor suppressors Brat (96%, n = 27, Fig. 3A'; wild type, 100%, n = 19, Fig. 3A) and Pros (96%, n = 25, Fig. 3B'; wild type, 100%, n = 16, Fig. 3B) remain asymmetrically localized in aurA8839 mutants. Consistently, asymmetric localization (94%, n = 36, Fig. 3C'; wild type, 100%, n = 20, Fig. 3C) and segregation (100%, n = 18, Fig. 3D'; wild type, 100%, n = 20, Fig. 3D) of Mira, an adaptor protein of Brat and Pros, is also unaffected in aurA mutants. Localization of Inscuteable (Insc) (97%, n = 32, Fig. 1A'; wild type, 100%, n = 21, Fig. 1A) and Bazooka (97%, n = 34, Fig. 3E'; wild type, 100%, n = 15, Fig. 3E), remain asymmetric in aurA mutants.
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, b). We tested whether AurA regulates NB self-renewal by modulating aPKC function. Indeed, in aurA8839 larval metaphase NBs, asymmetric localization of aPKC is strongly affected and it is delocalized to the entire cortex with punctuate staining (58%, n = 33 at 96 h ALH; Fig. 3F'). This phenotype is first observed at 48 h ALH (31.8%, n = 22) and is more severe in NBs from 120-h-ALH larvae (88.4% of delocalization, n = 69) in which most of maternal AurA had been presumably depleted. In this context, Mira remains largely asymmetrically localized (76.1% normal crescent, 15.2% reduced crescent, and 8.6% missing or cytoplsmic; n = 46), presumably because Baz and Insc remain asymmetrically localized. Overexpressed aPKC-CAAX (a membrane-targeted version of aPKC) causes ectopic localization of aPKC to both the apical and basal cortex and results in tumor formation (Lee et al. 2006a). Delocalization of aPKC in aurA mutants raises the interesting possibility that overproliferation of aurA may result from ectopic localization of aPKC on the cortex. To examine whether this is the case, we generated an aPKC;aurA double mutant. We found that aPKC significantly suppresses the aurA overproliferation phenotype at 68 h ALH (number of NB per brain lobe for each genotype: wild type, 67.2 ± 10.6; aPKC, 53.2 ± 13.5; aur8839, 187.9 ± 16.4; aPKC;aur8839 double mutant, 109 ± 17.1) (Fig. 7C–G, below). Thus, AurA may suppress self-renewal by preventing aPKC from localizing to the basal cortex.
We also found that in aurA8839 larval metaphase NBs (at 96 h ALH), Numb levels are strongly reduced, and its asymmetric localization is also compromised (24.7% weakly cortical localization; 28.8% strongly reduced crescent, n = 73) (data not shown) compared with wild-type NBs (100% crescents, n = 16, Fig. 3G,H). Delocalization of Numb has been observed at 48 h ALH (32% weakly cortical or strongly reduced crescent, n = 25) in aurA8839, and these defects were more prominent (66.7% weakly cortical with punctate staining, Fig. 3G'; 29.8% strongly reduced crescent, Fig. 3G''; n = 57) in aurA8839 metaphase NBs from 120-h-ALH larvae. During anaphase/telophase, Numb is often missegregated to both daughter cells (57.9%, n = 19, Fig. 3I') in aurA larval NBs, whereas segregation is exclusively to one daughter in wild type (100%, n = 12, Fig. 3I). These are consistent with the previous finding that AurA is required for the asymmetric localization of Numb in sensory organ precursors (SOPs) of the Drosophila peripheral nervous system (Berdnik and Knoblich 2002). Pon, an adaptor protein required for the asymmetric localization of Numb (Lu et al. 1998), was also often delocalized to the entire cortex of aurA metaphase NBs (34.1%, n = 41, 96 h ALH, Fig. 3J'; 58.6%, n = 29, 120 h ALH) in contrast to wild-type metaphase NBs (100% crescents, n = 11, Fig. 3J). We then tested to see whether delocalization of aPKC to the entire NB cortex might affect asymmetric localization of Numb. Under conditions in which aPKC-CAAX is overexpressed, where it localizes uniformly to the NB cortex and causes overproliferation (Lee et al. 2006a), Numb also delocalized to the entire cortex (86.2%, n = 29, Fig. 3H). These data further suggest that AurA acts upstream of aPKC in controlling NB self-renewal, probably by regulating Numb function.
AurA is required for proper spindle orientation
aurA has a function in centrosome separation and chromosome segregation (Glover et al. 1995; Giet et al. 2002). We investigated whether AurA might be required for proper orientation of the mitotic spindle. In wild-type metaphase NBs, the mitotic spindle, as judged by the positions of centrosomes, orients perpendicular to the Mira crescent (Fig. 4A,B). In 60% of aurA metaphase NBs, the spindle appears to be misaligned relative to the Mira crescent (Fig. 4A',B'). Centrosomin (CNN) is absent in the majority of metaphase NBs and appears cytoplasmic (Fig. 4A''). The centrosome number in aurA mutants is also abnormal, and NBs with one (data not shown) to three (Fig. 4A''') centrosomes can be observed. mud NBs also exhibit spindle orientation and centrosome organization defects (Bowman et al. 2006; Izumi et al. 2006; Siller et al. 2006) similar to those seen for aurA. Mud is required for proper spindle orientation in NBs and it is normally localized to both centrosomes and forms an apical crescent in wild-type NBs (Fig. 4C). Given that both AurA and Mud localize to the centrosomes and exhibit similar defects in spindle orientation, they may act together to regulate spindle orientation. Indeed, in aurA8839 larval NBs Mud cortical localization was no longer asymmetric, and was uniformly cortical (Fig. 4C', arrow), while its centrosomal localization was unaffected (Fig. 4C'). These observations suggest that AurA may act through Mud to regulate spindle orientation in larval NBs.
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To examine whether Numb might be a downstream target of AurA and aPKC in regulating NB self-renewal, we generated numb15 NB MARCM clones in larval brains. numb15 clones contained a greater number of cells compared with wild-type clones (Fig. 5E, numb15, 342 ± 82; wild type, 38 ± 18) and most of them express Dpn (Fig. 5B) and Mira (Supplementary Fig. 3B) in contrast to wild-type NB clones in which only a single cell expresses Dpn (Fig. 5A) and Mira (Supplementary Fig. 3A). numb15 clones contain very few neurons expressing Elav (Fig. 5C [wild type], D) and nuclear Pros (Supplementary Fig. 3C,D [wild type]). Thus, similar to AurA, Numb acts as a tumor suppressor to suppress self-renewal and promote differentiation during larval brain development. Ectopic expression of Numb significantly suppresses the aurA overproliferation phenotype (178 ± 40 NBs per brain lobe at 96 h ALH and 302 ± 63 NBs per brain lobe at 120 h ALH, Fig. 5F',G) compared with control aurA8839 mutant (352 ± 94 NBs per brain lobe at 96 h ALH, and >1000 at 120 h ALH, Fig. 5F,G), suggesting that Numb acts downstream from AurA to regulate NB self-renewal. In this case, Numb expressed at high levels remains mislocalized on the cortex in a significant proportion of aurA mutant NBs (25%, n = 24 at 96 h ALH) (data not shown), indicating that the level of Numb has probably exceeded the threshold necessary to suppress proliferation in a proportion of these cells.
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There is increasing evidence that Notch—a transmembrane receptor that is cleaved to release its intracellular domain, which directly participates in transcriptional regulation (Louvi and Artavanis-Tsakonas 2006)—is involved in the control of proliferation versus differentiation (Go et al. 1998; Androutsellis-Theotokis et al. 2006; Ferres-Marco et al. 2006; Micchelli and Perrimon 2006; Ohlstein and Spradling 2006). Numb can antagonize Notch signaling to specify distinct sibling cell fates during asymmetric divisions of various progenitor cells (Rhyu et al. 1994; Spana and Doe 1996; Skeath and Doe 1998). Overexpression of a Notch dominant-active form (Nact, the intracellular domain of Notch) in NB clones also leads to the overproliferation of larval NBs (Fig. 6B,B'). These mutant clones (O/E Nact, 306 ± 99 cells per clone) (Fig. 6C) were of much larger size than control clones (53 ± 11 cells per clone) (Fig. 6C) and contained mostly NBs that expressed Mira (Fig. 6A [control], B) and Dpn (Supplementary Fig. 4A [control], B), and were essentially devoid of cells expressing Elav (Supplementary Fig. 4C [control], D). A previous study (Almeida and Bray 2005) suggested that Notch does not play a role in controlling NB proliferation in larval ventral nerve cord (VNC). The discrepancies in our findings may be due to differences in which proliferation of VNC and central brain NBs are regulated. To test whether Notch might be hyperactivated in aurA mutants, we examined the membrane localization of Sanpodo (Spdo), which is required to activate Notch but is inhibited by Numb during GMC and SOP asymmetric divisions (O’Connor-Giles and Skeath 2003; Le Borgne et al. 2005). In wild-type interphase larval NBs, Spdo displayed a weak cortical staining and in punctate structures throughout the cytoplasm (100%, n = 38; Fig. 6D). In both numb15 (93%, n = 15; Fig. 6D') and aurA8839 (57%, n = 21; Fig. 6D'') larval NBs, Spdo was localized strongly to the cortex. In addition, aurA larval NBs expressed elevated levels of Notch (Fig. 6E') compared with wild type (Fig. 6E) using antibodies against Notch intracellular domain or extracellular domain (data not shown). The number of cells expressing CycE and dMyc are drastically increased when Nact is overexpressed (Supplementary Fig. 4E–G' and legend).
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). Mammalian AurA when overexpressed can be oncogenic. However, future studies on its possible role as a tumor suppressor will be particularly interesting.
Our data suggest that aurA negatively regulates aPKC function to regulate NB self-renewal. aPKC appears to act as a NB proliferation factor since overexpression of a modified membrane-targeted version, aPKC-CAAX, which exhibits ectopic cortical localization throughout the NB cortex, leads to overproliferation and tumor formation (Lee et al. 2006a), similar to loss of aurA. AurA is required for the asymmetric localization of aPKC and restrict aPKC to the cortical region associated with the future NB daughter and loss of aurA results in delocalization of aPKC to the entire cortex. Consistent with and supporting this notion, we have shown that loss of aPKC can suppress, albeit partially, the aurA mutant overgrowth phenotype.
In contrast to the well-studied role of Numb as a cell fate determinant during asymmetric divisions of embryonic GMCs, SOPs, or muscle progenitors (Rhyu et al. 1994; Spana and Doe 1996; Carmena et al. 1998), a role for Numb during NB asymmetric divisions has not been described. We show here that Numb also acts as a tumor suppressor in Drosophila larval brains, and that Numb is a key downstream target of AurA and aPKC in the regulation of NB self-renewal. In both aurA mutant NBs or NBs overexpressing aPKC-CAAX, the asymmetric localization of Numb is compromised and the resultant overgrowth phenotype is consistent with that of numb loss-of-function. numb and aurA mutant NBs also share several common features including excessive self-renewal at the expense of neuronal differentiation as well as the membrane enrichment of Spdo, a positive regulator of Notch signaling. Our data suggest that AurA positively regulates Numb function. Genetic analysis is consistent with the notion that this is achieved through the negative regulation of aPKC that in turn negatively regulates Numb.
Numb is known to be a negative regulator of Notch signaling (Rhyu et al. 1994; Spana and Doe 1996; Carmena et al. 1998). Our findings indicate that Notch is necessary and sufficient for promoting larval NB proliferation and suppressing neuronal differentiation. Our genetic epistasis studies suggest that an AurA–aPKC–Numb–Notch genetic hierarchy acts to regulate self-renewal of Drosophila neural progenitor cells (Fig. 7H). During a wild-type larval NB asymmetric division, aurA acts to negatively regulate aPKC and restrict its localization to the cortical region associated with the future NB daughter; aPKC negatively regulates Numb and ensures that its localization/activity is restricted to the future GMC where Numb acts to antagonize Notch. The net effect is that Notch is asymmetrically activated in the NB daughter where it acts to promote self-renewal and suppress differentiation. Although our data suggest that aurA acts through the aPKC/Numb/Notch pathway, given the partial suppression seen in the double mutants aPKC;aurA and Notchts-1;aurA, we cannot exclude the possibility that additional mechanisms may be involved.
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Fly strains used were aurA8839 [l(3)LL8839], aurA14641 [l(3)LL14641], aurA17961 [l(3)LL17961], worniu-Gal4, and brat11 (C.Q. Doe); aPKC (A. Wodarz and D. Bilder); UAS-aurA-GFP (J. Knoblich); UAS-CycE (P. O’Farrells); UAS-Dacapo (P. O’Farrells); UAS-aPKC-CAAX (C.Q. Doe); numb15 (J. Knoblich); Notchts-1, UAS-Numb-GFP (Y.N. Jan); UAS-actNotch (S. Artavanis-Tsakonas); yw, hs-Flp; act-FRT-y+-FRT-Gal4, UAS-LacZ(nls) (J. Knoblich); and Tub-Gal80ts and UAS-Sanpodo (J. Skeath).
Clonal analysis
To generate NB clones, 24-h-ALH larvae were heat-shocked at 37°C for 90 min and further aged for 3 d at 25°C.
Immunohistochemistry
For larval brain immunofluorescence stainings, larvae were dissected in PBS and fixed for 20 min in 4% formaldehyde, and processed similar to embryo stainings as described (Yu et al. 2000). Antibodies used were rabbit anti-Insc (1:1000), mouse anti-Mira (1:50, F. Matsuzaki), guinea pig anti-Dpn (1:1000, J. Skeath), mouse anti-Worniu (1:500), rabbit anti-phospho-Histone H3 (1:1000, Sigma), rat anti-Elav (1:10, DSHB), mouse anti-CycE (1:10, H. Richadson), mouse anti-dMyc (1:5, B. Edgar), rabbit anti-AurA (1:100, D. Glover), mouse anti-Pros (1:10, DSHB), rabbit anti-Brat (1:100, J. Knoblich), rat anti-Brat (1:100, R.P. Wharton), guinea pig anti-Numb (1:1000, J. Skeath), rabbit anti-Pon (1:500, Y.N. Jan), rabbit anti-Mud (1:200, F. Matusuzaki), rabbit anti-GFP (1:500, Molecular Probes), guinea pig anti-Spdo (1:1000, J. Skeath), rabbit anti-cleaved-Caspase-3 (1:75, Cell Signaling Technology), mouse anti-BrdU (1:20, Roche), mouse anti-Diap-1 (1:200, B. Hay), rabbit anti-CNN (1:5000, E. Schejter), and rabbit and mouse anti--Gal (1:1000, Promega).
BrdU labeling
Proliferating cells within whole brains were detected as previously described (Ceron et al. 2001). Dissected larval tissue was given a 40-min pulse of 37.5 µg/mL BrdU in Shields and Sang 3M insect medium. Tissue was then fixed for 15 min in 3.7% formaldehyde, and DNA denatured with 2N HCl for 40 min, before washing in PBS and incubating with anti-BrdU.
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E-MAIL wanghy{at}tll.org.sg, FAX (415) 476-0526. 
5 E-MAIL wchia{at}tll.org.sg; FAX 65-68727007.
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1487506
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Androutsellis-Theotokis, A., Leker, R.R., Soldner, F., Hoeppner, D.J., Ravin, R., Poser, S.W., Rueger, M.A., Bae, S.K., Kittappa, R., and McKay, R.D. 2006. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 442: 823–826.[CrossRef][Medline]
Badano, J.L., Teslovich, T.M., and Katsanis, N. 2005. The centrosome in human genetic disease. Nat. Rev. Genet. 6: 194–205.[Medline]
Bello, B., Reichert, H., and Hirth, F. 2006. The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila. Development 133: 2639–2648.
Berdnik, D. and Knoblich, J.A. 2002. Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis. Curr. Biol. 12: 640–647.[CrossRef][Medline]
Betschinger, J. and Knoblich, J.A. 2004. Dare to be different: Asymmetric cell division in Drosophila, C. elegans and vertebrates. Curr. Biol. 14: R674–R685.[CrossRef][Medline]
Betschinger, J., Mechtler, K., and Knoblich, J.A. 2003. The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl. Nature 422: 326–330.[CrossRef][Medline]
Betschinger, J., Mechtler, K., and Knoblich, J.A. 2006. Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells. Cell 124: 1241–1253.[CrossRef][Medline]
Bowman, S.K., Neumuller, R.A., Novatchkova, M., Du, Q., and Knoblich, J.A. 2006. The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division. Dev. Cell 10: 731–742.[CrossRef][Medline]
Carmena, A., Murugasu-Oei, B., Menon, D., Jimenez, F., and Chia, W. 1998. Inscuteable and numb mediate asymmetric muscle progenitor cell divisions during Drosophila myogenesis. Genes & Dev. 12: 304–315.
Caussinus, E. and Gonzalez, C. 2005. Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster. Nat. Genet. 37: 1125–1129.[CrossRef][Medline]
Ceron, J., Gonzalez, C., and Tejedor, F.J. 2001. Patterns of cell division and expression of asymmetric cell fate determinants in postembryonic neuroblast lineages of Drosophila. Dev. Biol. 230: 125–138.[CrossRef][Medline]
Ferres-Marco, D., Gutierrez-Garcia, I., Vallejo, D.M., Bolivar, J., Gutierrez-Avino, F.J., and Dominguez, M. 2006. Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing. Nature 439: 430–436.[CrossRef][Medline]
Fuse, N., Hisata, K., Katzen, A.L., and Matsuzaki, F. 2003. Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions. Curr. Biol. 13: 947–954.[CrossRef][Medline]
Giet, R., McLean, D., Descamps, S., Lee, M.J., Raff, J.W., Prigent, C., and Glover, D.M. 2002. DrosophilaAurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules. J. Cell Biol. 156: 437–451.
Glover, D.M., Leibowitz, M.H., McLean, D.A., and Parry, H. 1995. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81: 95–105.[CrossRef][Medline]
Go, M.J., Eastman, D.S., and Artavanis-Tsakonas, S. 1998. Cell proliferation control by Notch signaling in Drosophila development. Development 125: 2031–2040.[Abstract]
Hampoelz, B., Hoeller, O., Bowman, S.K., Dunican, D., and Knoblich, J.A. 2005. Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G proteins. Nat. Cell Biol. 7: 1099–1105.[Medline]
Ikeshima-Kataoka, H., Skeath, J.B., Nabeshima, Y., Doe, C.Q., and Matsuzaki, F. 1997. Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions. Nature 390: 625–629.[CrossRef][Medline]
Izumi, Y., Ohta, N., Hisata, K., Raabe, T., and Matsuzaki, F. 2006. Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization. Nat. Cell Biol. 8: 586–593.[CrossRef][Medline]
Jan, Y.N. and Jan, L.Y. 2001. Asymmetric cell division in the Drosophila nervous system. Nat. Rev. Neurosci. 2: 772–779.[CrossRef][Medline]
Katayama, H., Sasai, K., Kawai, H., Yuan, Z.M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R.B., Czerniak, B.A., and Sen, S. 2004. Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat. Genet. 36: 55–62.[CrossRef][Medline]
Knoblich, J.A., Jan, L.Y., and Jan, Y.N. 1995. Asymmetric segregation of Numb and Prospero during cell division. Nature 377: 624–627.[CrossRef][Medline]
Kraut, R., Chia, W., Jan, L.Y., Jan, Y.N., and Knoblich, J.A. 1996. Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383: 50–55.[CrossRef][Medline]
Kuchinke, U., Grawe, F., and Knust, E. 1998. Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. 8: 1357–1365.[CrossRef][Medline]
Lane, M.E., Sauer, K., Wallace, K., Jan, Y.N., Lehner, C.F., and Vaessin, H. 1996. Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 87: 1225–1235.[CrossRef][Medline]
Le Borgne, R., Bardin, A., and Schweisguth, F. 2005. The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132: 1751–1762.
Lee, T., Winter, C., Marticke, S.S., Lee, A., and Luo, L. 2000. Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25: 307–316.[CrossRef][Medline]
Lee, C.Y., Robinson, K.J., and Doe, C.Q. 2006a. Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation. Nature 439: 594–598.[CrossRef][Medline]
Lee, C.Y., Wilkinson, B.D., Siegrist, S.E., Wharton, R.P., and Doe, C.Q. 2006b. Brat is a miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev. Cell 10: 441–449.[CrossRef][Medline]
Louvi, A. and Artavanis-Tsakonas, S. 2006. Notch signalling in vertebrate neural development. Nat. Rev. Neurosci. 7: 93–102.[CrossRef][Medline]
Lu, B., Rothenberg, M., Jan, L.Y., and Jan, Y.N. 1998. Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 95: 225–235.[CrossRef][Medline]
Meraldi, P., Honda, R., and Nigg, E.A. 2002. Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53–/– cells. EMBO J. 21: 483–492.[CrossRef][Medline]
Micchelli, C.A. and Perrimon, N. 2006. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439: 475–479.[CrossRef][Medline]
O’Connor-Giles, K.M. and Skeath, J.B. 2003. Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila. Dev. Cell 5: 231–243.[CrossRef][Medline]
Ohlstein, B. and Spradling, A. 2006. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439: 470–474.[CrossRef][Medline]
Ohshiro, T., Yagami, T., Zhang, C., and Matsuzaki, F. 2000. Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast. Nature 408: 593–596.[CrossRef][Medline]
Passegue, E. 2006. Cancer biology: A game of subversion. Nature 442: 754–755.[CrossRef][Medline]
Peng, C.Y., Manning, L., Albertson, R., and Doe, C.Q. 2000. The tumour-suppressor genes lgl and dlg regulate basal protein targeting in Drosophila neuroblasts. Nature 408: 596–600.[CrossRef][Medline]
Petronczki, M. and Knoblich, J.A. 2001. DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3: 43–49.[CrossRef][Medline]
Rhyu, M.S., Jan, L.Y., and Jan, Y.N. 1994. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76: 477–491.[CrossRef][Medline]
Schaefer, M., Petronczki, M., Dorner, D., Forte, M., and Knoblich, J.A. 2001. Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system. Cell 107: 183–194.[CrossRef][Medline]
Shen, C.P., Jan, L.Y., and Jan, Y.N. 1997. Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila. Cell 90: 449–458.[CrossRef][Medline]
Siller, K.H., Cabernard, C., and Doe, C.Q. 2006. The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Nat. Cell Biol. 8: 594–600.[CrossRef][Medline]
Skeath, J.B. and Doe, C.Q. 1998. Sanpodo and Notch act in opposition to Numb to distinguish sibling neuron fates in the Drosophila CNS. Development 125: 1857–1865.[Abstract]
Spana, E.P. and Doe, C.Q. 1996. Numb antagonizes Notch signaling to specify sibling neuron cell fates. Neuron 17: 21–26.[CrossRef][Medline]
Vidal, M. and Cagan, R.L. 2006. Drosophila models for cancer research. Curr. Opin. Genet. Dev. 16: 10–16.[CrossRef][Medline]
Wang, H., Ng, K.H., Qian, H., Siderovski, D.P., Chia, W., and Yu, F. 2005. Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins. Nat. Cell Biol. 7: 1091–1098.[CrossRef][Medline]
Wodarz, A. 2005. Molecular control of cell polarity and asymmetric cell division in Drosophila neuroblasts. Curr. Opin. Cell Biol. 17: 475–481.[CrossRef][Medline]
Wodarz, A., Ramrath, A., Grimm, A., and Knust, E. 2000. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts. J. Cell Biol. 150: 1361–1374.
Yu, F., Morin, X., Cai, Y., Yang, X., and Chia, W. 2000. Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization. Cell 100: 399–409.[CrossRef][Medline]
Yu, F., Cai, Y., Kaushik, R., Yang, X., and Chia, W. 2003. Distinct roles of Gi and G13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions. J. Cell Biol. 162: 623–633.
Yu, F., Wang, H., Qian, H., Kaushik, R., Bownes, M., Yang, X., and Chia, W. 2005. Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions. Genes & Dev. 19: 1341–1353.
Yu, F., Kuo, C.T., and Jan, Y.N. 2006. Drosophila neuroblast asymmetric cell division: Recent advances and implications for stem cell biology. Neuron 51: 13–20.[CrossRef][Medline]
Zhou, H., Kuang, J., Zhong, L., Kuo, W.L., Gray, J.W., Sahin, A., Brinkley, B.R., and Sen, S. 1998. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat. Genet. 20: 189–193.[CrossRef][Medline]
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