Article |
Address correspondence to William Chia, MRC Centre for Developmental Neurobiology, 4th Fl., New Hunts House, Guy's Campus, King's College London, London SE1 1UL, UK. Tel.: 44-207-8486544. Fax: 44-207-8486550. email: william.chia{at}kcl.ac.uk; or Xiaohang Yang, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609. Tel.: 65-687-47848. Fax: 65-677-91117. email: mcbyangn{at}imcb.nus.edu.sg
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Abstract |
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Key Words: neuroblast; asymmetric division; astral microtubules; heterotrimeric G proteins; Drosophila
Abbreviations used in this paper: baz, bazooka; CNN, centrosomin; DaPKC, Drosophila atypical PKC; insc, inscuteable; mira, miranda; NB, neuroblast; pins, partner of inscuteable; pon, partner of numb; pros, prospera; wt, wild type.
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Introduction |
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The well-characterized features of the NB asymmetric division are controlled by a complex of proteins that are apically localized in dividing NBs, which include the Drosophila homologues of the conserved Par3 (Bazooka [Baz])/Par6 (DmPar6)/aPKC (Drosophila atypical [DaPKC]) (Kuchinke et al., 1998; Schober et al., 1999; Wodarz et al., 1999, 2000; Petronczki and Knoblich, 2001) protein cassette first described in Caenorhabditis elegans (Kemphues, 2000; Matsuzaki, 2000; for review see Doe and Bowerman, 2001; Knoblich, 2001; Wodarz, 2002), the novel protein Inscuteable [Insc] (Kraut and Campos-Ortega, 1996; Kraut et al., 1996), and an subunit of the heterotrimeric G protein complex (G
i) (Schaefer et al., 2001) and an evolutionarily conserved molecule, Partner of inscuteable (Pins) (Parmentier et al., 2000; Schaefer et al., 2000; Yu et al., 2000) that acts as a guanine nucleotide dissociation inhibitor for G
i. Since Insc can directly interact with both Baz and Pins in vitro, this apical complex of proteins can be viewed as comprising of two conserved protein cassettes, Baz/DmPar6/DaPKC and Pins/G
i, that are held together by Insc. Loss of function mutations exist for all members of the NB apical complex genes except G
i. Loss of single members of the apical complex, such as baz, insc, and pins, results in defective basal protein localization and spindle misorientation in mitotic NBs up to metaphase, although these defects can be partially corrected late in mitosis, a phenomenon called telophase rescue (Ohshiro et al., 2000; Peng et al., 2000; Cai et al., 2001). However, unlike basal protein localization and spindle orientation, the generation of an asymmetry spindle and its displacement toward the basal cortex are largely unaffected, and NBs lacking one component of the apical complex usually produce two unequal size daughter cells like wild-type (wt) NBs.
Recent findings indicate that the apical proteins are also involved in daughter cell size determination and can be further subdivided into two redundant pathways that control mitotic spindle geometry and displacement late in NB divisions (Cai et al., 2003). Baz, DaPKC, Insc, and probably DmPar6 belong to one pathway and Pins and (probably) Gi belong to the other. Members of each pathway can asymmetrically localize when members of the other pathway are mutated, suggesting that localized spindle extension signals derived from either one of these two pathways are sufficient to generate asymmetric spindle geometry and spindle displacement, resulting in unequal size daughter cells. Simultaneous disruption of both pathways destroys the localized spindle extension and displacement signals. Consequently, the two half spindle arms remain identical in length and mutant NBs produce two daughter cells with equal size.
Heterotrimeric G protein signaling has been shown to be involved in controlling distinct microtubule-dependent processes in C. elegans P0 embryos (Gotta and Ahringer, 2001). Gß is important for correct centrosome migration around the nucleus and spindle orientation. G
is required for asymmetric spindle positioning in the one-cell embryos. In Drosophila, G protein signaling is also involved in microtubule-dependent processes such as the formation of an asymmetric spindle. When G
i is overexpressed (Schaefer et al., 2001) or when Gß13F function is abolished (Schaefer et al., 2001), the ability to generate an asymmetric spindle is disrupted and NBs frequently divide to produce two daughter cells with equal size (Fuse et al., 2003). However, it has not been possible to assess the relative roles of Gß13F and G
i in NB asymmetric divisions not only because G
i mutants are not available but also because in Gß13F mutants G
i is undetectable in all cell types (Schaefer et al., 2001).
In this study, we report the isolation and analysis of loss of function mutations in Gi and assessing the role of the apical complex components on NB astral microtubules and mitotic spindle geometry. Our findings indicate distinct roles for G
i and Gß13F in NB asymmetric divisions. Loss of G
i releases Pins from the apical cortex into the cytosol and exhibits a similar array of phenotypes seen in pins mutant NBs. Mutations in G
i and one of the genes in Baz/DaPKC/Insc pathway cause NB to generate symmetric spindles and two equal size daughter cells, suggesting that G
i and Pins act in same pathway with respect to mediating mitotic spindle geometry. Formally, Gß13F functions upstream of both Baz/DaPKC/Par6/Insc and Pins/G
i pathways and is required, at least in part, for the asymmetric localization and/or stability of all apical complex members. Mutation in Gß13F can disrupt the asymmetric localization of members of both apical pathways in NBs and results in the formation of symmetric spindles and equal size daughter cells. Strikingly, our analyses has also revealed that the two apical pathways act downstream of Gß13F to redundantly suppress the formation of basal astral microtubules during NB divisions.
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Results |
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Gi and Pins act in the same pathway to regulate asymmetric spindle geometry and unequal cell size divisions
Gi has been implicated previously in the generation of spindle asymmetry from overexpression and RNAi experiments (Schaefer et al., 2001; Cai et al., 2003). The availability of G
i loss of function alleles enables us to more definitively assess the role of G
i in NB spindle geometry and the generation of daughters of unequal cell size. In wt NBs, the mitotic spindle is symmetric until metaphase. Starting from anaphase, the differential extension of the apical half spindle arm results in an apically biased asymmetric spindle (Kaltschmidt et al., 2000): the distance from the midspindle to the apical centrosome is larger than that to the basal centrosome. In addition, the spindle is displaced basally: the apical centrosome is located away from the NB apical cortex, whereas the basal centrosome lies close to the basal cortex (Cai et al., 2003). Consequently, the future cleavage plane is located toward the basal side of the NBs. Similar to pins, the majority of G
i mutant NBs generate an asymmetric spindle and produce two daughter cells with different cell sizes; however, similar to pins NBs, 21% (n = 86) of G
i NBs produce a symmetric spindle and give rise to equal size daughters (Fig. 3 B).
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Apical functions are necessary to suppress basal astral microtubule formation
One striking observation seen with anti-tubulin staining of mitotic NBs that had not been noted before is the influence of the apical functions on the asymmetric nature of the astral microtubules associated with the two centrosomes. In wt NBs, astral microtubules are nucleated at the apical centrosome, and the intensity of this staining increases markedly during the later stages of mitosis from metaphase onwards (Fig. 4, AC), resulting in the formation of a prominent astral microtubule cap structure associated with the apical centrosome. In contrast, little astral microtubules can be seen near the basal centrosome. Although this preferential formation and association of astral microtubules with only the apical centrosome is not affected in single mutants of apical complex genes or double mutants affecting components of the same apical pathway (unpublished data), a dramatic change is observed in double mutants which affects both the Pins/G
i and Baz/DaPKC/Insc pathways. In these double mutant NBs, both centrosomes are associated with astral microtubules, with a cap structure forming over each centrosome from metaphase onwards (Fig. 4, JL, N, and O). In addition, overexpression of G
i, which can lead to the uniform cortical localization of all apical components, and the loss of Gß13F (see next section), also result in the production of prominent astral microtubules over both centrosomes (Fig. 4, GI). This symmetric astral microtubule association with both centrosomes is similar to the astral microtubule structure seen in dividing epithelial cells (Fig. 4, DF). These observations suggest that the presence of either of the asymmetric apical pathways is sufficient to suppress the formation of basal astral microtubules in NBs (see Discussion).
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These data suggest that Gß13F (presumably in association with G) can function upstream of both apical pathways and act to promote the asymmetric localization/stability of the Baz/DaPKC and Pins/G
i pathway members. In the absence of Gß13F, the functions of both apical pathways are compromised in the majority of NBs; they fail to generate an asymmetric mitotic spindle and consequently undergo equal size divisions. In the remainder of mutant NBs, although the function of the Pins/G
i pathway is compromised, Baz/DaPKC remain asymmetrically localized and functional; consequently asymmetric spindles and daughter cells of unequal size are produced. These findings support and extend on our earlier two pathway model (Cai et al., 2003) for the generation of an asymmetric mitotic spindle.
Dosage-dependent effects of Gi overexpression on equal size NB divisions
Our previous study (Cai et al., 2003) showed that the equal size NB divisions caused by overexpression of Gi driven by sca-gal4 was dependent on pins function. Our interpretation of these results was that both proteins need to be present in a complex in order for a signal to be generated. However the observations that overrepression of G
i, but not constitutively activated form of G
i, in NBs disrupted asymmetric divisions and produced two equal size daughter cells (Schaefer et al., 2001) suggest that it is the depletion of free Gß
(caused by an excess of GDP-G
i) that might be the cause for the equal size NB divisions; the equal size NB divisions seen in the Gß13F embryos provide further support for this view. If this were the case, then one would expect that under conditions in which G
i was in excess (with respect to all other molecules it can complex with like Pins and Gß
) free Gß
should be depleted whether Pins was present or not. How can these seemingly contradictory observations be reconciled?
One possible explanation is that under conditions that we used previously (sca-gal4 driving UAS-Gi) G
i is not overexpressed to excess. Under these conditions, the phenotypic effects produced are caused by uniform Pins/G
i signaling from the cortex, and not by the sequestration of Gß
due to excess G
i, and therefore are Pins dependent. To test whether the equal size division phenotype is dependent on Pins under circumstances in which G
i is overexpressed to higher levels, we used a stronger driver (mata-gal4 VP16 V32). This driver increases G
i levels by about fivefold (compared with wt) compared with a twofold increase by sca-gal4 as judged by Western blot analysis of embryonic extracts (Fig. 6 A). In immunofluorescence experiments using identical conditions, mata-gal4 VP16 V32 also drives a higher level of expression than sca-gal4 in NBs (Fig. 6 A). The increased levels of G
i overexpression leads to a high frequency of equal size NB divisions (83%, n = 55 [Fig. 6 B]) which is largely independent of Pins, since overexpression in the absence of Pins only marginally reduce the frequency of equal size NB divisions (62%, n = 62 [Fig. 6 B]).
Our interpretation of these observations is that overexpression of Gi can cause NBs to undergo equal size divisions via two different mechanisms. With the levels of overexpression obtained with sca-gal4, G
i binds primarily to Pins and recruits Pins uniformly to the NB cortex (Cai et al., 2003). The cortical Pins/G
i can, presumably through a signaling function, disrupt the Baz/DaPKC apical localization, resulting in equal size NB divisions. In the absence of Pins, although both endogenous and ectopic G
i molecules are uniformly cortical, G
i alone cannot or is less able to interfere with Baz/DaPKC asymmetric localization. With higher levels of ectopic G
i (mata-gal4 VP16 V32 driver), not only are Pins/G
i uniformly cortical but the excess G
i can also bind to and deplete free Gß
. With limiting levels of free Gß
, both apical pathways can be disrupted as seen in the Gß13F mutants. In the presence of higher levels of G
i, Pins is not required for the majority of the equal size NB divisions since its absence would not affect the ability of G
i to sequester free Gß
.
Overexpression of Go causes equal size NB divisions
If the depletion of free Gß can disrupt asymmetric NB divisions, we might expect that other G
molecules that can interact with Gß
may also be able to reproduce the G
i overexpression phenotypes when ectopically expressed in NBs. One such molecule, G
o47A, which shares high homology with G
i, is able to bind/complex Gß13F in vivo as indicated by the observation that it coimmunoprecipitates with Gß13F when it is overexpressed (Fig. 6 C). Anti-G
o47A staining shows a weak cortical localization of the protein in NBs (unpublished data; Schaefer et al., 2001). However, removal of both maternal and zygotic G
o47A does not affect any aspect of NB asymmetric division, indicating that G
o47A is not normally required in wt NBs. When G
o47A is overexpressed, we observe a high frequency of NB equal size divisions (85%, n = 41 [Fig. 7, H, I, and K]), similar to that seen with G
i overexpression (Fig. 4 I). In metaphase NBs overexpressing G
o, it shows a strong uniform cortical signal (Fig. 7 A); G
i levels are reduced dramatically (100%, n = 76 [Fig. 7 B]); Pins is cortical (Fig. 7 C); Insc is delocalized (100%, n = 23 [Fig. 7 D]); DaPKC becomes uniformly cortical or undetectable (100%, n = 36 [Fig. 7 F]); and spindle geometry late in mitosis remains symmetric (Fig. 7, I and K), suggesting the disruption of both apical pathways. In addition, Mira is delocalized and can segregate into both daughter cells (75%, n = 40 [Fig. 7, G and H]).
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Discussion |
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Gi is required to target Pins to the NB cortex
Our results indicate that Pins and Gi apical localization are mutually dependent. In pins NBs, G
i is evenly distributed to the NB cortex, and in G
i mutant NBs, Pins localizes to the cytosol. We have provided evidence previously that Pins asymmetric localization to the apical cortex of the NBs is a two-step process (Yu et al., 2002): Pins need to be targeted to the cortex first, which requires the COOH-terminal Goloco motifs that can bind G
i before it can be recruited to the apical cortex in a process which requires its NH2-terminal TPR that can interact with Insc. Our current results therefore suggest that Pins cortical targeting is most likely mediated by G
i, which cannot only bind Pins but is also able to localize to the plasma membrane through lipid modifications (Casey, 1994).
However, in Gß13F mutant NBs, although the levels of Pins are drastically reduced, the residual Pins is localized both to the cytosol and to the cell cortex. This poses a problem since in the Gß13F mutant NBs not only is Gß13F absent but Gi also is undetectable with an anti-G
i antibody. One possible explanation is that although G
i is undetectable, there is still some G
i remaining in the Gß13F NBs which may account for the low level residual uniform cortical distribution of Pins. Alternatively, we cannot formally rule out the possibility that the cortical Pins in Gß13F NBs is due to some unknown molecule that can recruit Pins to cortex in the absence of both G
i and Gß13F.
Gß13F acts upstream of the apical components to mediate their asymmetric localization
The analysis of Gß13F function is complicated by the fact that in the Gß13F mutant NBs, Gi levels are also down-regulated presumably due to the instability of the protein in the absence of Gß13F. Although loss of either G
i or Gß13F causes aberrations in localization of the basal components and orientation of the mitotic spindle, it is clear that at least some of the defects associated with the loss of Gß13F cannot be attributable solely to the depletion of G
i. In the great majority of G
i mutant NBs, DaPKC and Baz still localize asymmetrically to a subset of the cell cortex. And consistent with our proposal that spindle geometry and the size asymmetry of the NB daughters are mediated by two redundant apical pathways, Pins/G
i and Baz/DaPKC, the great majority (79%) of the G
i mutant NBs generate an asymmetric mitotic spindle and divide to produce unequal size daughters. In contrast, in Gß13F NBs not only do Pins/G
i always fail to become asymmetrically localized but the majority of mutant NBs (71%) also fail to asymmetrically localize Baz/DaPKC; consequently
65% of NBs fail to generate an asymmetric mitotic spindle and divide to produce equal size daughters. Therefore, at least formally, Gß13F acts upstream of the two apical pathways (Fig. 8 A).
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Apical pathways act redundantly to prevent basal astral microtubule formation
The apical centrosome associates with prominent astral microtubules, whereas the basal centrosome connects to few if any astral microtubules in wt NBs and in mutants in which one of the two apical pathways is compromised. In contrast, in NBs that lack both apical pathways a symmetric mitotic apparatus is established that features extensive arrays of astral microtubules at both centrosomes. Therefore, either of the two apical pathways appears sufficient to prevent formation of basal astral microtubules. It is not clear how this might be accomplished at a mechanistic level. However, one might speculate that there exists an asymmetrically localized molecule, which can act to promote the formation of astral microtubules. When either of the apical pathways is functional, this molecule is asymmetrically localized and promotes the formation of astral microtubules only over the centrosome it overlies. However, when both apical pathways are mutated, or when Gß13F is mutated or when all apical components become uniformly cortical, e.g., when Gi is overexpressed, then the hypothetical molecule becomes uniformly cortical and can promote the formation of astral microtubules over both centrosomes (Fig. 8 B). This type of model can readily explain why either loss or uniform cortical localization of both apical pathways leads to symmetric astral microtubule formation over both centrosomes.
In summary, our results demonstrate that for NB asymmetric divisions Gi and Gß13F play distinct roles. G
i and Pins are members of one of the two apical pathways and Baz/DaPKC/Insc forms the other. Loss of G
i function results in defects in NB asymmetry that are essentially indistinguishable from those seen in pins mutants. Gß13F (Gß
) functions upstream of both Pins/G
i and Baz/DaPKC/Insc pathways to mediate their stability and/or asymmetric localization (and function). Without Gß13F, the function of both apical pathways are attenuated; G
i levels are dramatically reduced and Pins/G
i pathway is defective; in addition, the asymmetric localization of members of the Baz/DaPKC/Insc pathway is often defective. Consequently, loss of Gß13F function yields phenotypes which are similar to those seen when both apical pathways are disrupted by mutations. A schematic summary depicting the hierarchical relationship between Gß13F and the apical pathways and our speculative model of how the apical pathways might act to "suppress" the formation of basal astral microtubules are depicted in Fig. 8.
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Materials and methods |
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Mobilization of P element
KG01907 carrying a P element derivative that contains the white gene is inserted near the 5' end of the Gi transcription unit at cytological location 65D6. The P element in this stock was mobilized using P(ry
23)(99B) as a transposase source. 300 independent w- revertant lines were established. These were analyzed on Southern blots using various portions of the Gai cDNA as hybridization probes. Several small deletion events which resulted in deletions that removed some or all of the Gai coding region were recovered.
Germline transformation, overexpression studies, and RNAi experiments
Transgenes were expressed in NBs using either the maternal GAL4 driver V32 (obtained from D. St. Johnston, Wellcome/CRC Institute, Cambridge, UK) or scabrous-gal4 (Brand and Perrimon, 1993). UAS-Gao and UAS-Gao Q205L were created by cloning the full-length Gao cDNA (Fremion et al., 1999) or a mutant version in which glutamine 205 had been replaced with leucine into pUAST (Brand and Perrimon, 1993). Rescue experiments were performed by driving the expression of the UAS-Gai transgene with a sca-gal4 driver in Gi mutant background.
A 0.8-kb PstI fragment of baz cDNA (from Andreas Wodarz, University of Duesseldorf, Duesseldorf, Germany) was used as a template for RNAi experiments and subcloned into a modified pBluescript vector (pKS-ds-T7) (Cai et al., 2001) for double strand RNA synthesis.
Immunocytochemistry and confocal microscopy
Embryos were collected and fixed according to Yu et al. (2000); for -tubulin and ß-tubulin stainings, embryos were fixed with 38% formaldehyde for exactly 1 min. Rabbit anti-Asense (provided by Y.-N. Jan, University of California, San Francisco, San Francisco, CA), rabbit anti-Baz (provided by F. Matsuzaki, Center for Developmental Biology, RIKEN, Kobe, Japan), mouse anti-Eve (Kai Zinn, Caltech, Pasadena, CA), rabbit anti-Insc, rabbit and rat anti-Pins, rabbit anti-G
i (aa 327355; provided by J.A. Knoblich, IMP), guinea pig anti-G
o (provided by M. Forte, Oregon Health Sciences University, Portland, OR), rabbit anti-PKC
C20 (Santa Cruz Biotechnology, Inc.), rabbit anti-Gß13F (provided by J.A. Knoblich), rabbit anti-Mira (provided by F. Matsuzaki), rabbit anti-Pon (provided by Y.-N. Jan), rabbit anti-Numb (provided by Y.-N. Jan), mouse anti-
tubulin (DM1A; Sigma-Aldrich), rabbit anti
-tubulin (provided by D. Glover, University of Cambridge, Cambridge, UK), rabbit anti-CNN (provided by T.C. Kaufman, Indiana University, Bloomington, IN), anti-Pros MR1A (provided by C.Q. Doe, University of Oregon, Eugene, OR), mouse anti-ß gal (Chemicon), antiß-tubulin E7 (Developmental Studies Hybridoma Bank [DSHB]) and anti-Nrt BP106 (DSHB) were used in this study. Cy3- or FITC-conjugated secondary antibodies were obtained from Jackson Laboratories. Stained embryos were incubated with ToPro3 (Molecular Probes) for chromosome visualization and mounted in Vectashield (Vector Laboratories). Embryos were analyzed with laser scanning confocal microscopy (Bio-Rad Laboratories MRC 1024 and Zeiss LSM510 [Carl Zeiss MicroImaging, Inc.]). Images were processed with Adobe Photoshop®.
Coimmunoprecipitation and Western blot
Embryos overexpressing Go using the maternal Gal4 driver V32 were ground in liquid nitrogen and mixed with fives times volume of the lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail from Roche) for 30 min at 4°C. The embryo lysate was centrifuged at maximum speed in a microcentrifuge for 20 min. The supernatant (embryo extract) was used to immunoprecipitate with anti-Gß13F antibody and the protein A/G beads (Amersham Biosciences). Beads were washed three times (10 min each) in lysis buffer. Bound proteins were analyzed by Western blots with anti-G
o and anti-Gß13F.
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Acknowledgments |
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X. Yang is an adjunct staff, Department of Anatomy, National University of Singapore. W. Chia is a Wellcome Trust Principal Research fellow. This work was supported by A*STAR Singapore and the Wellcome Trust.
Submitted: 26 March 2003
Accepted: 7 July 2003
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brand, A.H., and N. Perrimon. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118:401415.
Cai, Y., W. Chia, and X. Yang. 2001. A family of snail-related zinc finger proteins regulates two distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions. EMBO J. 20:17041714.
Cai, Y., F. Yu, S. Lin, W. Chia, and X. Yang. 2003. Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions. Cell. 112:5162.[Medline]
Campos-Ortega, J.A. 1995. Genetic mechanisms of early neurogenesis in Drosophila melanogaster. Mol. Neurobiol. 10:7589.[Medline]
Casey, P.J. 1994. Lipid modifications of G proteins. Curr. Opin. Cell Biol. 6:219225.[Medline]
Chia, W., and X. Yang. 2002. Asymmetric division of Drosophila neural progenitors. Curr. Opin. Genet. Dev. 12:459464.[CrossRef][Medline]
Doe, C.Q., and B. Bowerman. 2001. Asymmetric cell division: fly neuroblast meets worm zygote. Curr. Opin. Cell Biol. 13:6875.[CrossRef][Medline]
Fremion, F., M. Astier, S. Zaffran, A. Guillen, V. Homburger, and M. Semeriva. 1999. The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila. J. Cell Biol. 145:10631076.
Fuse, N., K. Hisata, L.A. Katzen, and F. Matsuzaki. 2003. Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast division. Curr. Biol. 13:947954.[CrossRef][Medline]
Giansanti, M.G., M. Gatti, and S. Bonaccorsi. 2001. The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts. Development. 128:11371145.
Gotta, M., and J. Ahringer. 2001. Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos. Nat. Cell Biol. 3:297300.[CrossRef][Medline]
Jan, Y.N., and L.Y. Jan. 2001. Asymmetric cell division in the Drosophila nervous system. Nat. Rev. Neurosci. 2:772779.[CrossRef][Medline]
Kaltschmidt, J.A., C.M. Davidson, N.H. Brown, and A.H. Brand. 2000. Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nat. Cell Biol. 2:712.[CrossRef][Medline]
Kemphues, K. 2000. PARsing embryonic polarity. Cell. 101:345348.[Medline]
Knoblich, J.A. 2001. Asymmetric cell division during animal development. Nat. Rev. Mol. Cell Biol. 2:1120.[CrossRef][Medline]
Kraut, R., and J.A. Campos-Ortega. 1996. inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeleton adaptor protein. Dev. Biol. 174:6581.[CrossRef][Medline]
Kraut, R., W. Chia, L.Y. Jan, Y.N. Jan, and J.A. Knoblich. 1996. Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature. 383:5055.[CrossRef][Medline]
Kuchinke, U., F. Grawe, and E. Knust. 1998. Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. 8:13571365.[Medline]
Matsuzaki, F. 2000. Asymmetric division of Drosophila neural stem cells: a basis for neural diversity. Curr. Opin. Neurobiol. 10:3844.[CrossRef][Medline]
Ohshiro, T., T. Yagami, C. Zhang, and F. Matsuzaki. 2000. Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast. Nature. 408:593596.[CrossRef][Medline]
Parmentier, M.L., D. Woods, S. Greig, P.G. Phan, A. Radovic, P. Bryant, and C.J. O'Kane. 2000. Rapsynoid/partner of inscuteable controls asymmetric division of larval neuroblasts in Drosophila. J. Neurosci. 20:RC84.[Medline]
Peng, C.Y., L. Manning, R. Albertson, and C.Q. Doe. 2000. The tumour-suppressor genes lgl and dlg regulate basal protein targeting in Drosophila neuroblasts. Nature. 408:596600.[CrossRef][Medline]
Petronczki, M., and J.A. Knoblich. 2001. DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3:4349.[CrossRef][Medline]
Schaefer, M., A. Shevchenko, and J.A. Knoblich. 2000. A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila. Curr. Biol. 10:353362.[CrossRef][Medline]
Schaefer, M., M. Petronczki, D. Dorner, M. Forte, and J.A. Knoblich. 2001. Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system. Cell. 107:183194.[Medline]
Schober, M., M. Schaefer, and J.A. Knoblich. 1999. Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature. 402:548551.[CrossRef][Medline]
Siderovski, D.P., M. Diverse-Pierluissi, and L. De Vries. 1999. The GoLoco motif: a Galphai/o binding motif and potential guanine-nucleotide exchange factor. Trends Biochem. Sci. 24:340341.[CrossRef][Medline]
Wodarz, A. 2002. Establishing cell polarity in development. Nat. Cell Biol. 4:E39E44.[CrossRef][Medline]
Wodarz, A., A. Ramrath, U. Kuchinke, and E. Knust. 1999. Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature. 402:544547.[CrossRef][Medline]
Wodarz, A., A. Ramrath, A. Grimm, and E. Knust. 2000. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts. J. Cell Biol. 150:13611374.
Yu, F., X. Morin, Y. Cai, X. Yang, and W. Chia. 2000. Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization. Cell. 100:399409.[Medline]
Yu, F., C.T. Ong, W. Chia, and X. Yang. 2002. Membrane targeting and asymmetric localization of Drosophila partner of inscuteable are discrete steps controlled by distinct regions of the protein. Mol. Cell. Biol. 22:42304240.
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