Cellular diversity during development arises in part from asymmetric divisions, which generate two distinct cells by transmitting localized determinants from a progenitor cell into one daughter cell. In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell1,2. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex3,4, from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically5,6,7,8. Here we show that a tumour-suppressor protein, Lethal giant larvae (Lgl)9, is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumour-suppressor protein, Lethal discs large (Dlg)10, participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells.
In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch signalling, associate with their respective adapter proteins, Miranda11,12 and Partner of Numb (Pon)13, and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz) and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of the basal determinants5,6,7,8. However, the mechanisms that underlie the asymmetric protein sorting in neuroblasts are not known. To address this issue, we have searched for chromosomal deficiencies that affect the subcellular distribution of Miranda. Such screening identified the lgl tumour-suppressor gene9,14,15, which encodes a protein containing WD40 repeats. In wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the basal cortex upon mitosis after a transient spread into the cytoplasm11,12 (Fig. 1a). In germline clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda did not localize asymmetrically in mitotic neuroblasts, but rather was distributed uniformly throughout the cortex as well as in the cytoplasm, where it was concentrated along microtubule structures (Fig. 1b, e, f). Consequently, Miranda segregated into both the daughter neuroblast and the ganglion mother cell (GMC). Numb (Fig. 1g, h) and Pon (not shown) were also distributed uniformly at the cortex and in the cytoplasm.
Lgl is involved in epithelial polarity9,16, and neuroblasts inherit Baz as an apical polarity cue during the formation from neuroepithelia6,7. In epithelia lacking lgl function, Baz mislocalized as irregular patches (Fig. 1b). Baz as well as Insc and Pins normally localized as an apical crescent in the mutant neuroblasts (Fig. 1b; and data not shown), however, suggesting that the apical cue is inherited to neuroblasts in the absence of lgl. Moreover, neuroblast-specific expression of the lgl transgene could restore normal localization of Miranda (Fig. 1c; and see Methods). Thus, Lgl probably functions autonomously within neuroblasts for Miranda localization, and the phenotype in lgl neuroblasts would not result from the inheritance of an abnormal polarity cue. Together, we conclude that Lgl acts in the localization of the basal determinants in neuroblasts, but not of the apical Baz–Insc–Pins complex.
We next investigated whether other tumour-suppressor genes contribute to protein localization in neuroblasts. The tumour-suppressor gene dlg encodes a membrane-associated guanylate kinase homologue10. Germline clone embryos lacking both maternal and zygotic dlg activity (dlgGLC embryos) exhibited defective localization of Miranda (Fig. 1d) and Numb (data not shown) essentially identical to that of lglGLC embryos, suggesting that both tumour-suppressor proteins function in the same process in neuroblasts. To investigate the relationship between the roles of Dlg and Lgl, we compared their subcellular localization in neuroblasts. Both Lgl and Dlg were distributed mainly throughout the cortex, whereas the amount of Lgl in the cytoplasm appeared to be greater in mitosis than in interphase (Fig. 2a, b). The cortical localization of Lgl appears to be important for its function—whereas the mutant protein encoded by the temperature-sensitive allele lglts3 (ref. 16) was distributed normally at the permissive temperature (18 °C), it failed to localize cortically at the restrictive temperature (29 °C) (Fig. 2c; and ref. 17). The wild-type Lgl protein exhibited a similar, abnormal cytoplasmic distribution in dlgGLC embryos (Fig. 2d), whereas Dlg localization was not affected in lglGLC embryos (data not shown). Thus, the cortical localization of Lgl requires dlg activity, suggesting that Dlg may function in localization of cell-fate determinants in neuroblasts by positioning Lgl at the cortex.
To distinguish whether Lgl establishes or maintains determinant localization, we performed temperature-shift experiments with the lglts3 allele. At 18 °C, most (97%, n = 262) lglts3/lgl- embryos of females homozygous for lglts3 exhibited normal Miranda localization at the basal cortex during metaphase and telophase (Fig. 3a). Ten minutes after shifting to 29 °C, mislocalization of Miranda was apparent in 61% of metaphase or anaphase neuroblasts (n = 159) as well as in 43% of telophase neuroblasts (n = 124), whereas the apical localization of Miranda in interphase was not affected (Fig. 3b, c). On the basis of live recordings of basal crescent formation in cells that express a fusion protein comprising Miranda and green fluorescent protein (see Methods), we estimated that the time required for neuroblast mitosis is 14.3 ± 2.4 min (mean ± s.d., n = 13). Thus, a temperature shift during mitosis was able to induce mislocalization of Miranda, as apparent in telophase neuroblasts, indicating that Lgl functions during mitosis to determine Miranda localization. Metaphase arrest can be induced in lglts3/lgl- embryos by introduction of the fizzy (fzy) mutation18. The localization of Miranda in such metaphase-arrested neuroblasts was virtually insensitive to the shift to 29 °C (Fig. 3d). Thus, Miranda is not affected by the decrease in lgl function after it has localized to the basal cortex. These results therefore indicate that Lgl may act early during mitosis to recruit Miranda to the cortex but does not contribute to the maintenance of Miranda in this location.
Because Lgl is a component of cortical protein complexes that include nonmuscle Myosin II, or Zipper (Zip)19, we tested genetic interactions between lgl and zip in Miranda localization by examining embryos zygotically mutant for both lgl and zip (lgl-/lgl-, zip1/zip- (lgl–zip) embryos)20. The zip1 mutation did not affect Miranda localization throughout embryonic development and lgl–zip embryos showed no difference in Miranda localization from zygotic lgl- embryos until late embryonic stages (stage 16) owing to maternal contribution of zip. At stage 17 when maternal zip had been exhausted, however, lgl–zip embryos appeared to restore the basal crescent of Miranda in metaphase neuroblasts (62%, n = 150), whereas zygotic lgl- embryos at the same stage did not (2%, n = 138) (Fig. 3e, f). Thus, Lgl might act for Miranda localization in part by suppressing zip function directly or indirectly, consistent with a study on yeast21 that indicated negative genetic interactions between Lgl homologues and Myosin II. Alternatively, the asymmetric distribution of Pon requires myosin function in neuroblasts22, as revealed by the use of 2,3-butanedione monoxime (BDM) that generally inhibits myosin function23. We also examined the effect of BDM on Miranda localization. Treatment of wild-type embryos with BDM phenocopied lgl mutants, resulting in a partial redistribution of Miranda from the cortex to microtubules (Fig. 3g). The effect of BDM was more marked in lglGLC embryos: as the BDM concentration increased, the relocalization of Miranda to microtubules was synergistically enhanced in most BDM-treated neuroblasts and resulted in the complete exclusion of Miranda from the cortex at 50 mM BDM (Fig. 3h). The phenocopy and enhancement of lgl mutations by general inhibition of myosin function are in contrast with the suppressive effects of zip mutations, suggesting that Lgl cooperates with at least one type of Myosin other than Zip to anchor Miranda at the cell cortex. We thus infer that Lgl regulates negatively myosin II function and also positively the function of another Myosin isotype in cortical protein targeting in neuroblasts.
We would expect the abnormal distribution of Numb and Miranda in lgl mutant neuroblasts to result in incorrect determination of neural cell fate. Given the difficulty of monitoring neural cell fate in severely distorted lglGLC embryos, we tested this prediction by analysing the lineage of the external sensory organ in the notum, in which all cell divisions are asymmetric and sibling cells adopt distinct fates as a result of the asymmetric inheritance of Numb3,24,25 (Fig. 4a). Sensory organ precursor cells in this lineage segregate Numb into a daughter cell pIIb, which subsequently generates three inner cells (a glial cell, a neuron and a sheath cell). The sibling pIIa cell divides into two outer cells constituting the external sensory structure, a hair and a socket. Exposure of lglts3 mutant larvae to 29 °C during external sensory organ development mislocalized Numb in mitotic precursor cells, as observed in neuroblasts (data not shown), and often transformed inner cells into outer cells resulting in duplicated external sensory structures, a phenotype expected from loss of numb function3 (Fig. 4b, c). Indeed, this notum phenotype was enhanced by reducing the numb gene dosage by half (lglts3/lglts3, numb-/numb+ animals) (Table 1). Equal partition of Numb between sibling cells would result in numb gain of function phenotypes because the half dose of numb is enough for correct cell-fate decisions. The observed numb loss-of-function phenotype therefore suggests that a reduction in lgl activity does not only equalize Numb distribution between sibling cells but also attenuates numb function, consistent with our observation of cytoplasmic Numb in the lgl mutants. Conversely, the presence of an extra numb gene induced opposite phenotypes under the lgl mutant condition (Fig. 4d–g). The outer cells were frequently transformed into the inner cells, resulting in the loss of the external sensory structure (Fig. 4f, g). This appearance of the numb gain of function phenotypes is simply explained by the fact that the partition of additional Numb from the transgene into both sibling cells raises numb activities over the threshold necessary to suppress Notch function in both cells. Our data thus indicate that Lgl is essential in neural fate decisions through cortical targeting of cell-fate determinants.
There are two important processes associated with the asymmetric division1,2: first, the asymmetric localization of cell-fate determinants, which is achieved by specific adapter proteins that themselves localize asymmetrically to the cortex in neuroblasts3,4; and second, the orientation of the mitotic spindle and its coordination with the polarized localization of the determinants, which require the apical Baz–Insc–Pins complex5,6,7,8. Our study has revealed another important process mediated by Dlg, Lgl and Myosins, which is responsible for the cortical anchoring of the determinant–adapter complexes. This process occurs upstream of the first and independently or parallel to the second of those two aspects of asymmetric division, as the localization of Lgl and Dlg is independent of apical or basal components (data not shown). Both Lgl and Dlg contribute to the generation or maintenance of epithelial polarity9,16,17,26,27, and zygotic mutants of the corresponding genes develop epithelial cell tumours as well as brain tumours at late larval stages9,10,26,27. These previous observations with epithelial cells, together with our data on the roles of Lgl and Dlg in protein targeting in neuroblasts, suggest that aberrant sorting of intracellular proteins may be responsible for the tumour formation apparent in larval stages of lgl and dlg mutants.
Fly stocks and genetics
We used lgl1 and lgl4 as null alleles for deletion of the lgl transcriptional unit14, lglts3 as a temperature-sensitive allele16, zip1(a strong allele) and zipIIX62 (a deficiency uncovering zip and gooseberry) as zip alleles20, numb2 as a null allele (a gift from J. Skeath), dlgm52(ref. 10), fzy1 (ref. 18), the hs-numb transformant3, and the A101enhancer trap line28. Germline clone embryos for lgl1 (lglGLC embryos) and for dlgm52 (dlgGLC embryos) were produced by the FLP–DFS technique29. Genetic interactions between lgl and zip were examined for embryos of a genotype lgl4/lgl4, zip1/zipIIX62. All mutations were balanced by blue balancer chromosomes to distinguish heterozygous embryos.
The full-length lgl complementary DNA (LD06034 obtained from BDGP) was used to construct an UAS–lgl transgene. The lgl transgene was expressed by using the UAS–GAL4 system30 and a neuroblast-specific GAL4 driver, pros–GAL4 (Goto, S. and F.M., unpublished data).
Dechorionated embryos were gently shaken for 30 min in a 1:1 mixture of n-octane and Schneider's medium (Gibco BRL) containing various concentrations of BDM (Sigma). The medium was subsequently replaced by an equal volume of 4% paraformaldehyde for fixation.
of Miranda–GFP A UAS–Miranda–GFP fusion gene was constructed using a full-length miranda cDNA and a GFP gene (S65T/I167T, provided by A. Brand) and expressed in embryos produced from crosses of transformants carrying the UAS–Miranda–GFP gene with the daughterless–GAL4 driver (a gift from E. Knust). Formation of the basal crescent of the fusion protein was monitored in living embryos with a confocal microscope (BioRad MRC1024).
Sensory lineage analysis
lglts3 or lglts3, A101/TM6B females were separately mated with males of genotypes lglts3; lglts3, numb2/CyO; or lglts3, hs-numb/CyO. After laying, eggs were grown at 18 °C. Early third instar larvae were picked up and reared at 29 °C until 30 h after pupation for dissecting pupae or until eclosion for observing adult nota.
Generation of antibodies
The anti-Lgl-N antibody was raised against the amino-terminal portion (KGQQPSADRHRLQKD) of Lgl in rabbits and affinity purified for immunostaining; mouse monoclonal anti–Mira-81 against the carboxy-terminal portion (SPPQKQVLKARNI) of Miranda; rabbit antibodies to Numb against the polypeptide KQLSPDLPIPSTAR; and rabbit anti-Baz against the C-terminal PSQYGSAAGSQPHASKV. The specificity of all antibodies was confirmed by immunoblot analysis or by immunohistochemical staining of null mutants for the corresponding genes.
Embryos were fixed with 4% paraformaldehyde for 20 min or with 37% formaldehyde for 3 min (to visualize microtubules). Primary antibodies were as follows: rabbit anti-Mira-C11, mouse anti-Mira-81, rabbit anti-Numb, rabbit anti-Lgl-N, rabbit anti-Baz, mouse anti-α-tubulin DM1a (Sigma), guinea pig anti-Dlg (provided by P. Bryant), mouse anti-Cen 190 Bx63 (from D. Glover), rabbit anti-Insc5, mouse anti-Elav 9F8A9 (Developmental Studies of Hybridoma Bank, University of Iowa) and rat anti-Su (H) (ref. 28). Chromosomes were visualized with TOTO3 (Molecular Probes). Confocal images were obtained as described11.
Jan, L. Y. & Jan, Y. N. Asymmetric cell division. Nature 392, 775–778 (1998).
Matsuzaki, F. Asymmetric division of Drosophila neural stem cells: a basis for neural diversity. Curr. Opin. Neurobiol. 10, 38–44 (2000).
Rhyu, M. S., Jan, L. Y. & Jan, Y. N. Asymmetric distribution of Numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477–491 (1994).
Hirata, J., Nakagoshi, H., Nabeshima, Y. & Matsuzaki, F. Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature 377, 627–630 (1995).
Kraut, R., Chia, W., Jan, L. Y., Jan, Y. N. & Knoblich, J. A. Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383, 50–55 (1996).
Wodarz, A., Ramrath, A., Kuchinke, U. & Knust, E. Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402, 544–547 (1999).
Schober, M., Schaefer, M. & Knoblich, J. A. Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402, 548–551 (1999).
Yu, F., Morin, X., Cai, Y., Yang, X. & Chia, W. Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in Inscuteable apical localization. Cell 100, 399–409 (2000).
Gateff, E. Malignant neoplasms of genetic origin in Drosophila melanogaster. Science 200, 1448–1459 (1978).
Woods, D. F. & Bryant, P. J. The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451–464 (1991).
Ikeshima-Kataoka, H., Skeath, J. B., Nabeshima, Y., Doe, C. Q. & Matsuzaki, F. Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions. Nature 390, 625–629 (1997).
Shen, C. P., Jan, L. Y. & Jan, Y. N. Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila. Cell 90, 449–458 (1997).
Lu, B., Rothenberg, M., Jan, L. Y. & Jan, Y. N. Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 90, 225–235 (1998).
Mechler, B. M., McGinnis, W. & Gehring, W. J. Molecular cloning of lethal(2)giant larvae, a recessive oncogene of Drosophila melanogaster. EMBO J. 4, 1551–1557 (1985).
Jacob, L., Opper, M., Metzroth, B., Phannavong, B. & Mechler, B. M. Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain. Cell 50, 215–225 (1987).
Manfruelli, P., Arquier, N., Hanratty, W. P. & Semeriva, M. The tumor suppressor gene, lethal(2)giant larvae (l(2)gl), is required for cell shape change of epithelial cells during Drosophila development. Development 122, 2283–2294 (1996).
De Lorenzo, C., Strand, D. & Mechler, B. M. Requirement of Drosophila l(2)gl function for survival of the germline cells and organization of the follicle cells in a columnar epithelium during oogenesis. Int. J. Dev. Biol. 43, 207–217 (1999).
Dawson, I. A., Roth, S., Akam, M. & Artavanis-Tsakonas, S. Mutations of the fizzy locus cause metaphase arrest in Drosophila melanogaster embryos. Development 117, 359–376 (1993).
Strand, D. et al. The Drosophila lethal(2)giant larvae tumor suppressor protein forms homo-oligomers and is associated with nonmuscle myosin II heavy chain. J. Cell Biol. 127, 1361–1373 (1994).
Young, P. E., Richman, A. M., Ketchum, A. S. & Kiehart, D. P. Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev. 7, 29–41 (1993).
Kagami, M., Toh-e, A. & Matsui, Y. Sro7p, a Saccharomyces cerevisiae counterpart of the tumor suppressor l(2)gl protein, is related to myosins in function. Genetics 149, 1717–1727 (1998).
Lu, B., Ackerman, L., Jan, L. Y. & Jan, Y. N. Modes of protein movement that lead to the asymmetric localization of partner of Numb during Drosophila neuroblast division. Mol. Cell 4, 883–891 (1999).
Cramar, L. P. & Mitchison, T. J. Myosin is involved in postmitotic cell spreading. J. Cell Biol. 131, 179–189 (1995).
Wang, S., Younger-Shepherd, S., Jan, L. Y. & Jan, Y. N. Only a subset of the binary cell fate decisions mediated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless. Development 124, 4435–4446 (1997).
Gho, M., Bellaiche, Y. & Schweisguth, F. Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell. Development 126, 3573–3584 (1999).
Woods, D. F., Hough, C., Peel, D., Callaini, G. & Bryant, P. J. Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. J. Cell Biol. 134, 1469–1482 (1996).
Bilder, D., Li, M. & Perrimon, N. Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116 (2000).
Gho, M., Lecourtois, M., Geraud, G., Posakony, J. W. & Schweisguth, F. Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling. Development 122, 1673–1682 (1996).
Chou, T. B. & Perrimon, N. Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131, 643–653 (1992).
Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).
We thank A. Brand, P. Bryant, W. Chia, D. Glover, S. Hayashi, Y.N. Jan, E. Knust, C. Peng, F. Schweisguth, M. Semeriva, A. Shearn, J. Skeath, Umea Drosophila Stock Center and Bloomington Drosophila Stock Center for providing flies and reagents; K. Hisata for technical assistance; N. Fuse, Y. Izumi, A. Sehara-Fujisawa and W. Chia for comments on the manuscript. We also thank C. Peng and C. Doe for communicating unpublished results; we are indebted to them for first observing Miranda in lgl–zip double mutants. This work was supported (through F.M.) by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation.
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