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The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division

The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division DEVELOPMENT AND DISEASE RESEARCH ARTICLE 2183 Development 134, 2183-2193 (2007) doi:10.1242/dev.02848 The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division 1 1 1 2 2 Silvia Bonaccorsi , Violaine Mottier , Maria Grazia Giansanti , Bonnie J. Bolkan , Byron Williams , 2 1, Michael L. Goldberg and Maurizio Gatti * We have isolated lethal mutations in the Drosophila lkb1 gene (dlkb1), the homolog of C. elegans par-4 and human LKB1 (STK11), which is mutated in Peutz-Jeghers syndrome. We show that these mutations disrupt spindle formation, resulting in frequent polyploid cells in larval brains. In addition, dlkb1 mutations affect asymmetric division of larval neuroblasts (NBs); they suppress unequal cytokinesis, abrogate proper localization of Bazooka, Par-6, DaPKC and Miranda, but affect neither Pins/Gi localization nor spindle rotation. Most aspects of the dlkb1 phenotype are exacerbated in dlkb1 pins double mutants, which exhibit more severe defects than those observed in either single mutant. This suggests that Dlkb1 and Pins act in partially redundant pathways to control the asymmetry of NB divisions. Our results also indicate that Dlkb1 and Pins function in parallel pathways controlling the stability of spindle microtubules. The finding that Dlkb1 mediates both the geometry of stem cell division and chromosome segregation provides novel insight into the mechanisms underlying tumor formation in Peutz-Jeghers patients. KEY WORDS: Lkb1, Neuroblasts, Asymmetric division, Spindle formation, Drosophila INTRODUCTION apical complex by the Inscuteable (Insc) protein that binds both Pins Drosophila neuroblasts (NBs) are one of the best model systems for and Baz (reviewed by Betschinger and Knoblich, 2004; Wodarz, the study of the control of cell polarity and asymmetric cell division. 2005). During Drosophila embryogenesis, NBs delaminate basally from Recent genetic analyses have shown that the Baz/Par-6 the neuroectodermal epithelium and divide asymmetrically along subcomplex is mainly involved in the control of proper basal the apical/basal axis to produce another NB and a smaller ganglion localization of Pros/Mira and Numb/Pon. The Pins/Gi subcomplex mother cell (GMC). The newly generated apical NB divides is instead required for spindle orientation during NB divisions. Both repeatedly in an asymmetric fashion, whereas the basal GMC complexes, however, cooperate in controlling cleavage furrow divides symmetrically just once to generate equally sized daughter positioning during asymmetric NB divisions. Mutations that disrupt cells that differentiate into neurons or glia (reviewed by Betschinger either the Baz/Par-6 or the Pins/Gi pathway have little or no effect and Knoblich, 2004; Wodarz, 2005). on asymmetric cytokinesis. However, mutations that disrupt both The asymmetric division of Drosophila NBs is regulated by pathways completely abrogate spindle displacement during several proteins that concentrate at the cell cortex. The basal cortex telophase, leading to symmetric cytokinesis (Cai et al., 2003; Izumi is enriched in the cell fate determinants Prospero (Pros) and Numb, et al., 2004; Shaefer et al., 2000; Yu et al., 2000; Yu et al., 2003). as well as in their respective adaptor proteins Miranda (Mira) and In this study, we have addressed the role of Drosophila Lkb1 Partner of Numb (Pon). These proteins are preferentially segregated (Dlkb1), the homolog of LKB1 kinase (STK11 – Human Gene into the GMC following NB cytokinesis. Localization of Pros-Mira Nomenclature Database) in NB division. LKB1 kinase is mutated in and Numb-Pon at the basal cortex is mediated by a large the Peutz-Jeghers syndrome, an autosomal dominantly inherited multiprotein complex that concentrates at the apical cortex. This disorder characterized by the formation of intestinal polyps and a complex includes two functionally distinct subcomplexes. One of high incidence of various cancer types. Somatic mutations in the them contains Bazooka (Baz; PAR-3 in C. elegans), aPKC LKB1 gene have also been detected in sporadic adenocarcinomas (Drosophila atypical protein kinase C, DaPKC) and Par-6; this (reviewed by Alessi et al., 2006; Baas et al., 2004b). There is assembly is hereafter referred to as the Baz/Par-6 subcomplex. The evidence that LKB1 plays a conserved role in the control of cell other subcomplex includes the Gi subunit of the heterotrimeric G- polarity. Recent work has unambiguously shown that activation of protein complex and Partner of inscuteable (Pins; Raps – Flybase), LKB1 leads to rapid and complete polarization of human intestinal and is hereafter referred to as the Pins/Gi subcomplex. The epithelial cells (Baas et al., 2004a). Similarly, PAR-4, the C. elegans Baz/Par-6 and Pins/Gi subcomplexes are integrated in a larger homolog of LKB1, is required for correct polarity and asymmetric division of one-cell embryos (Watts et al., 2000). Furthermore, Dlkb1 mediates determination of anterior/posterior polarity of egg chambers and embryos, as well as the proper polarity of follicle cells (Martin and St Johnston, 2003). Here, we demonstrate that Dlkb1 Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia controls many asymmetries that characterize the mitotic division of Molecolari del CNR, Dipartimento di Genetica e Biologia Molecolare, Università di larval NBs. dlkb1 mutations also disrupt mitotic spindle assembly, Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA. leading to the frequent formation of polyploid cells. Thus, in addition to cell polarity and the geometry of cell division, Dlkb1 *Author for correspondence (e-mail: [email protected]) directly or indirectly regulates the stability of spindle microtubules (MTs). Accepted 12 March 2007 DEVELOPMENT 2184 RESEARCH ARTICLE Development 134 (11) MATERIALS AND METHODS RESULTS Fly strains and genetic manipulations Isolation and characterization of mutations in the The dlkb1 mutant allele was isolated from a collection of 1600 third dlkb1 gene chromosome late lethals induced by ethylmethanesulfonate (EMS) in C. In the course of a screen aimed at the isolation of mitotic mutants Zuker’s laboratory (Koundakjian et al., 2004). The dlkb1 allele is (see Materials and methods), we identified a lethal mutation that associated with the chromosome carrying Df(3R)su(Hw)7. This and all the causes frequent polyploid cells in larval brains (see below). Animals deficiencies used for mapping were obtained from the Bloomington Stock homozygous for this mutation die at the larval/pupal transition, as P62 2 Center. The pins null allele and the asl mutation have been described do most mitotic mutants; most probably, they exploit maternally 315 P62 previously (Bonaccorsi et al., 1998; Yu et al., 2000); dlkb1 pins and supplied products to survive until late larval stages (Gatti and Baker, 315 2 dlkb1 asl double mutants were generated by recombination. All 1989). Deficiency mapping showed that this mutation is uncovered mutations were maintained over the TM6B balancer, and mutant larvae by both Df(3R)urd and Df(3R)26c, which define a map interval that were identified based on their non-tubby phenotype. Genetic markers and contains only 16 annotated genes. During these mapping studies, we special chromosomes are described in FlyBase (http://www.flybase.org/). also identified another mutant allele of this same mitotic gene. Germline transformation was performed as previously described (Vernì et This allele is associated with the chromosome that carries al., 2004). Df(3R)su(Hw)7, but is independent of the deficiency. We next Antibodies and immunoblotting sequenced the candidate genes and found that both mutant stocks The anti-Dlkb1 antibody was generated in guinea pig using a maltose- carry lesions in the Drosophila lkb1 gene (dlkb1, also known as binding protein (MBP)/Dlkb1 fusion protein. Expression of the fused protein CG9374). This gene encodes a 567 amino acid serine/threonine in Escherichia coli and the production and purification of antibodies against kinase homologous to the PAR-4 kinase of C. elegans and the this fusion were according to Vernì et al. (Vernì et al., 2004). Immunoblotting human LKB1 kinase mutated in Peutz-Jeghers syndrome (Martin was performed as previously described (Vernì et al., 2004); the anti-Dlkb1, and St Johnston, 2003). The dlkb1 mutant allele isolated in our anti-Pins and anti-Giotto (Giansanti et al., 2006) antibodies were diluted screen carries a frameshift mutation resulting in a truncated Dlkb1 1:2000, 1:1000 and 1:5000, respectively. protein of 234 amino acids; the dlkb1 allele, associated with Cytology Df(3R)su(Hw)7, has a stop codon that truncates Dlkb1 to a 346 Brains from third instar larvae were dissected and fixed according to amino acid polypeptide (Fig. 1A). A genomic fragment including Bonaccorsi et al. (Bonaccorsi et al., 2000). After several rinses in PBS, brain sequences that extend roughly 1 kb on either side of dlkb1 (Fig. 1A) preparations were incubated overnight at 4°C with a monoclonal anti-- rescued both the lethality and the mitotic phenotypes of tubulin antibody (Sigma-Aldrich), diluted 1:1000 in PBS-T (PBS with 0.1% 315 315 315 7 dlkb1 /dlkb1 and dlkb1 /dlkb mutants. Triton X-100), and any of the following rabbit antibodies, also diluted in PBS-T: anti-Centrosomin (1:300; gift of T. Kaufman, Indiana University, Mutations in the dlkb1 gene affect spindle Bloomington, IN, USA), anti-Deadpan (1:400; gift of Y. Jan, Howard formation Hughes Medical Institute, University of California, CA, USA), anti-Mira Drosophila brains contain mostly two types of dividing cells: NBs (1:500; gift of Y. Jan), anti-Bazooka (1:50; gift of F. Matsuzaki, Japan and GMCs (Goodman and Doe, 1993). Wild-type larval NBs are Science and Technology Corporation, Kobe, Japan); anti-Gi (1:200; gift of characterized by many asymmetries that develop during the course J. Knoblich, Austrian Academy of Sciences, Vienna, Austria); anti-Par-6 (1:1000; gift of J. Knoblich); anti-DaPKC (1:100; Santa Cruz of mitosis. Prometaphases and metaphases of larval NBs exhibit Biotechnology) and anti-Mud (1:200; gift of F. Matsuzaki). After two rinses centrosomes and asters of similar sizes at the two cell poles. in PBS, primary antibodies were detected by a 1-hour incubation at room However, as NBs progress through anaphase and telophase, the MTs temperature with FITC-conjugated anti-mouse IgG+IgM (1:20; Jackson of the basal aster shorten dramatically, whereas those of the apical Laboratories) and Alexa 555-conjugated anti-rabbit IgG (1:300; Molecular asters elongate slightly (Fig. 1B). Concomitantly, the basal Probes), diluted in PBS. centrosome becomes smaller than the apical one (Bonaccorsi et al., For double Centrosomin/Pins immunostaining, brains were incubated 2000) (see Fig. 4A below). These changes in aster and centrosome overnight at 4°C with the rabbit anti-Centrosomin antibody (1:300) and a rat morphology are accompanied by a progressive displacement of the anti-Pins antibody (1:100; gift of W. Chia, The National University of central spindle towards the basal pole, resulting in unequal Singapore, Singapore) diluted in PBS-T. Detection was performed by 1-hour cytokinesis (Giansanti et al., 2001). GMCs display equally sized incubation at room temperature with Alexa 555-conjugated anti-rabbit IgG centrosomes and very small asters throughout mitosis, and divide (Molecular Probes) and FITC-conjugated anti-rat IgG (Jackson symmetrically (Fig. 1B) (Bonaccorsi et al., 2000). Laboratories) diluted 1:300 and 1:20 in PBS, respectively. To determine the mitotic defect leading to polyploid cell For Dlkb1 immunostaining, brain preparations were incubated overnight formation in dlkb1 mutants, we examined larval brain preparations at 4°C with the anti-Dlkb1 antibody (1:100 in PBS-T) and, after rinsing in 315 315 315 315 7 PBS, were incubated 1 hour at room temperature with Alexa 555-conjugated from dlkb1 /dlkb1 , dlkb1 /Df(3R)urd and dlkb1 /dlkb1 anti-guinea pig IgG diluted 1:500 in PBS. larvae stained for both tubulin and DNA. These mutant In all cases, immunostained preparations were mounted in Vectashield combinations showed identical mitotic aberrations. Most strikingly, medium H-1200 (Vector Laboratories) containing the DNA-dye DAPI (4,6- mutant spindles showed an overall MT density substantially lower diamidino-2-phenylindole). Preparations were examined with a Zeiss than that seen in wild-type spindles (Fig. 1B). In approximately 80% Axioplan microscope, equipped with an HBO100W mercury lamp and a of mutant spindles, asters were absent or severely reduced; in control cooled charged-coupled device (CCD camera; Photometrics CoolSnap HQ). brains, the frequency of spindles without asters, or with very small Grayscale images were collected separately, converted to Photoshop (Adobe asters, was 49% (Fig. 1B; Table 1). In addition, most mutant Systems), pseudocolored and merged. prometaphase and metaphase figures were characterized by low Spindle measurements were taken on enlarged digital images and scaled densities of both kinetochore and interpolar MTs, and ana-telophases down to their size in m. In preparations stained for Centrosomin, displayed central spindles thinner than their wild-type counterparts measurements were taken from centrosome-to-centrosome. In the absence (Fig. 1B and Fig. 3D below). Mutant brains also showed an increase of Centrosomin staining, measurements were taken from pole-to-pole in in the relative frequency of metaphase figures with respect to wild anastral spindles; in the presence of asters, measurements were taken from type, suggesting that dlkb1 mutations lengthen metaphase duration the center of the astral MT array. DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2185 Fig. 1. Mutations in the dlkb1 gene disrupt spindle organization of both NBs and GMCs. (A) Map of the Drosophila lkb1 (dlkb1) gene and its genomic region. P[dlkb1 ] designates the genomic fragment that rescues both the lethality and the cytological defects associated with the dlkb1 mutation. Black boxes correspond to protein-coding exons, and the arrows indicate the direction of transcription. The positions of the stop codons 315 7 causing the dlkb1 and dlkb1 mutations are indicated by vertical lines. (B) Mitotic spindle morphology of NBs and GMCs from wild-type (wt) and dlkb1 brains. Cells were stained for tubulin (Tub, green) and DNA (blue). (C) Spindle morphology of wild-type (wt), dlkb1, asl,and asl dlkb1 metaphases stained for tubulin (green), DNA (blue) and Centrosomin (red). Note that the spindle density in asl dlkb1 double mutants is substantially lower than in asl single mutants. Scale bar (all panels): 5 m. (Table 1). Finally, mutant brains displayed approximately 20% of spindle MTs was substantially reduced with respect to wild type polyploid cells (not shown); in wild-type brains, the frequency of and similar to that observed in dlkb1 single mutants (Fig. 1C). These polyploid cells was virtually zero (Table 1). The phenotype of results strongly suggest that the low density of spindle MTs observed dlkb1 homozygotes was qualitatively and quantitatively similar in dlkb1 mutants does not depend on centrosome dysfunction. Thus, to that observed in dlkb1 /Df(3R)urd hemizygotes, indicating that the spindle phenotype of dlkb1 mutants is likely to be attributable to dlkb1 is a null mutation (Table 1). either a decreased rate of MT growth or an increased MT instability. The spindle phenotypes observed in dlkb1 mutants could be due either to a defect in MT elongation and/or stability, or to a defect in dlkb1 mutations disrupt the asymmetry of NB centrosome function. To distinguish between these possibilities, we division leading to a reduction in NB size sought to eliminate centrosome function in dlkb1 mutants. We have Observation of mitotic divisions stained for tubulin and DNA previously shown that brain cells of asterless (asl) mutants fail to revealed that the spindles of dlkb1 mutant cells are generally smaller assemble functional centrosomes and nucleate astral MTs. than in wild type (Fig. 1). In addition, the frequency of asymmetric Nonetheless, asl NBs and GMCs manage to form robust anastral telophases in mutant brains (35-37%) was significantly lower than spindles that are able to mediate chromosome segregation in wild-type brains (65%) (Table 1). These phenotypes could reflect (Bonaccorsi et al., 2000; Giansanti et al., 2001). We thus constructed a partial loss of morphological asymmetry during NB division, dlkb1 asl double mutants and compared their phenotype with those resulting in smaller than normal daughter NBs. We thus examined exhibited by dlkb1 and asl single mutants. The anastral spindles in greater detail the pattern of cell division in dlkb1 /Df(3R)urd from asl single mutants displayed a MT density comparable to wild brains, and compared this pattern with those observed in wild-type type (Fig. 1C). By contrast, in dlkb1 asl double mutants, the density pins and asl brains. Comparison between dlkb1 and pins mutants DEVELOPMENT 2186 RESEARCH ARTICLE Development 134 (11) Table 1. Mitotic parameters in larval brains from Drosophila lkb1 (dlkb1) mutants † ‡ § Genotype* # of cells Metaphases (%) Anaphases (%) Telophases (%) Sym. telo (%) No asters (%) Polyploid cells (%) Oregon R 1,095 68.0 16.3 15.6 35 49 0.2 315/315 423 83.5 10.9 6.6 68 82 20.2 315/Df 1,120 81.3 13.0 5.7 65 81 22.5 315/7 547 79.7 13.9 6.4 63 78 18.6 315 315 315 315 7 *Oregon R, wild-type stock used as control; 315/315, dlkb1 /dlkb1 ; 315/Df, dlkb1 /Df(3R)urd; 315/7, dlkb1 /dlkb1 . The numbers of cells scored refer only to diploid mitotic figures; polyploid cells were recorded but not used for calculation of the frequencies of different types of mitotic figures. Sym. telo, relative frequencies of symmetric telophases. No asters, diploid cells without asters or with very small asters. was prompted by two previous findings. First, mutations in pins that the NBs from dlkb1 and pins mutants divide more partially suppress the asymmetry of NB divisions, leading to a symmetrically than those of either asl or wild type. Collectively, progressive reduction in NB size (Cai et al., 2003; Parmentier et al., these results indicate that mutations in either dlkb1 or pins 2000). Second, the Drosophila and the human LKB1 kinases partially suppress the asymmetry of NB division, leading to a interact with the orthologous proteins Pins and AGS3 (GPSM1 – reduction in the NB size at each cell division cycle. Human Gene Nomenclature Database), respectively (Blumer et al., To ask whether mutations in dlkb1 and pins affect centrosome 2003). In addition, more detailed comparisons between dlkb1 and size, brain preparations were stained for Centrosomin (Cnn), an asl mutants would allow us to assess more precisely the role of astral integral component of Drosophila centrosomes (Megraw et al., MTs in asymmetric NB divisions. 2001). Observations were restricted only to those cells that, To unambiguously distinguish between NB and GMC spindles, according to our analysis of spindle size distribution (Fig. 3B,C), we immunostained preparations from control and mutant brains for were likely to be NBs (wild-type, dlkb1 and pins metaphases longer both tubulin and the NB marker Deadpan (Dpn) (Bier et al., 1992) than 12 m, and ana-telophases longer than 16 m). This analysis (Fig. 3A below). The analysis of Dpn-positive cells showed that the (Fig. 4A,B) revealed that 88% (n=76) of wild-type NBs display dlkb1 and pins NBs are indeed defective in aster formation. centrosomes of different sizes at their poles. By contrast, only in However, the two mutants displayed different patterns of spindle 34% (n=180) of dlkb1 NBs and 39% (n=100) of pins NBs was the P62 defects. In brains homozygous for the pins null mutation (Yu et centrosome at the apical pole larger than that at the basal pole. These al., 2000), most NB prometaphases and metaphases showed normal results indicate that dlkb1 and pins control asymmetry in centrosome asters but most ana-telophases were characterized by an abnormally size during NB division. small apical aster (Fig. 2A-D). Despite this defect in astral MTs, the density of the spindle MTs in pins NBs was comparable to that observed in their wild-type counterparts (compare Fig. 2A-D with Fig. 1B). By contrast, the spindles of dlkb1 NBs not only showed a reduction in MT density, but also displayed small asters in both metaphase and ana-telophase figures (Fig. 1B and Fig. 2E). In pins and asl mutants, the spindles of Dpn-negative GMCs displayed a normal morphology and were indistinguishable from their wild-type counterparts (data not shown). However, in dlkb1 mutants, GMC spindles were characterized by low MT density just as were those of the NBs (Fig. 1B). Thus, the wild-type function of dlkb1 is required for proper spindle formation in both NBs and GMCs. We next measured the spindle length of metaphase and ana- telophase figures in both NBs (Dpn-positive) and GMCs (Dpn- negative). In dlkb1 and pins mutant brains, the average sizes of NB spindles were substantially smaller than those measured in either asl or wild-type brains. This is mainly due to the absence of large NBs, as both dlkb1 and pins mutants lacked NB metaphases and telophases longer than 19 and 26 m, respectively; these large NBs represented approximately 20% of the NBs found in wild-type or asl mutant brains. By contrast, the average sizes of the GMC spindles observed in dlkb1, asl and pins mutants were very similar and comparable to those of wild-type controls (Fig. 3B,C). An explanation for these results is that in Fig. 2. Mutations in the Drosophila pins gene affect aster both dlkb1 and pins mutants, NBs divide more symmetrically than formation without altering the density of spindle MTs. Cells were in either asl or wild type. To test this possibility, we directly stained for tubulin (green), DNA (blue) and Deadpan (not shown, but evaluated the degree of asymmetry of NB telophases showing see Fig. 3A below) to identify NBs. (A) Prometaphase, (B) metaphase, P62 strong Dpn staining. The asymmetry index was determined using (C) anaphase and (D) telophase from pins mutant brains. the formula a-b/a+b, in which a is the long axis of the spindle and (E) Frequencies of NBs displaying normal asters in wild-type, dlkb1 and b its short axis (Fig. 3D). This analysis (Fig. 3E) clearly shows pins brains. Scale bar: 5 m. DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2187 Fig. 3. Mutations in the Drosophila lkb1 gene disrupt unequal cytokinesis and reduce the average size of NB population within mutant brains. (A) Wild-type and dlkb1 metaphases stained for tubulin (Tub) DNA and Deadpan (Dpn). Note that the NBs are more intensely stained by the anti-Dpn antibody than GMCs. Scale bar: 5 m. (B,C) Size distribution of metaphase (B) and ana-telophase (C) spindles in wild-type, 315 P62 P62 2 2 dlkb1 /Df(3R)urd, pins /pins and asl /asl brains. Dpn-positive (NB) and Dpn-negative (GMC) spindles are depicted in red and green, respectively. Size (m) classes in B: A, 4.7-6.9; B, 7.0-9.2; C, 9.3-11.5; D, 11.6-13.8; E, 13.9-16.1; F, 16.2-18.4; G, 18.5-20.7; H, 20.8-23.0; I, 23.1- 31.7. Size (m) classes in C: J, 6.7-11.2; K, 11.3-15.8; L, 15.9-20.4; M, 20.5-25.0; N, 25.1-29.6; O, over 29.7. The numbers above each column indicate the number of cells observed in each size class. (D) Criterion used for measuring the asymmetry index of NB divisions. The difference between the length of the long (a) and the short (b) spindle axis (a-b) was divided by the total length of the two axes (a+b). Scale bar: 5 m. 315 P62 P62 2 2 (E) Asymmetry indexes in wild-type, dlkb1 /Df(3R)urd, pins /pins and asl /asl NBs of different sizes. The bars correspond to the s.e.m. Size (m) classes: P, 11.3-15.8; Q, 15.9-20.4; R, 20.5-25.0; S, 25.1-29.6; T, over 29.7. dlkb1 mutations affect Mira and Baz/Par-6/DaPKC mutant NBs lacking a Mira crescent displayed a diffuse cytoplasmic but not Pins/Gi localization in dividing NBs localization of Mira. However, although the majority (67%) of pins We examined whether dlkb1 and pins mutations affect the mutant NBs without a Mira crescent had this same pattern, a distribution of Mira in dividing NBs. Larval brain preparations were substantial minority (33%) of these cells showed a diffuse cortical simultaneously stained for both tubulin and Mira and analyzed for distribution of Mira (Fig. 5B). Mira localization (Fig. 5A). We again restricted our analysis to large We next determined whether dlkb1 mutations affect the mitotic figures that are likely to be NBs by the criteria employed localization of Pins and Gi at the apical cortex of dividing NBs above. Both dlkb1 and pins mutant NBs revealed abnormal Mira (NBs were again identified by their size). A regular Pins signal was distribution, but the patterns of Mira mislocalization were different observed in 95% (n=60) of the wild-type NBs and in 84% (n=145) (Fig. 5A,B). In wild type, 93% of NB metaphases and ana- of the dlkb1 NBs (Fig. 6A,B). Gi formed a crescent at the apical telophases displayed a clear Mira crescent at the basal pole, whereas pole of 96% of wild-type NBs (n=61) and 74% of dlkb1 mutant NBs the remaining 7% showed diffuse Mira staining. By contrast, in (n=75) (Fig. 6A,B). Consistent with previous results (Cai et al., dlkb1 and pins mutants the frequencies of NBs with a basal Mira 2003; Schaefer et al., 2001), we observed a Gi apical crescent only crescent were 47% and 26%, respectively. Most (97%) of the dlkb1 in 4% (n=50) of pins mutant NBs (data not shown). Thus, although DEVELOPMENT 2188 RESEARCH ARTICLE Development 134 (11) Fig. 4. Mutations in the Drosophila lkb1 gene affect centrosome size during NB division. (A) Metaphases and telophases from wild- type and dlkb1 /Df(3R)urd mutant brains stained for tubulin (green), DNA (blue) and Centrosomin (red). Note the differently sized and the equally sized centrosomes at the poles of wild-type and dlkb1 mutant cells, respectively. Scale bar: 5 m. (B) Frequency of NBs displaying differently sized centrosomes in wild-type (wt), dlkb1 /Df(3R)urd and P62 P62 pins /pins brains. dlkb1 mutations affect Mira localization at the basal cortex, they have little or no effect on Pins and Gi localization at the apical cortex. We also analyzed Baz, DaPKC and Par-6 localization in both wild-type and dlkb1 mutant NBs. In wild-type larval brains, the Baz signal was rather weak and only 48% (n=63) of the dividing NBs displayed a clear Baz crescent at the apical pole. However, in dlkb1 mutant brains, only five of the 106 NBs scored showed a discernible Baz crescent (Fig. 6A,B). The DaPKC and Par-6 apical crescents were observed in 96% (n=24) and 71% (n=35) of wild-type NBs, respectively, but most dlkb1 NBs did not show apical accumulations of these proteins: DaPKC and Par-6 crescents were detected only in 7% (n=30) and 12% (n=33) of dlkb1 mutant NBs. These results suggest that the wild-type function of dlkb1 is required for the localization of Baz, DaPKC and Par-6 at the NB apical pole. Recent work has suggested that DaPKC delocalization from the apical cortex can result in NB overproliferation (Lee et al., 2006a). Consistent with this idea, the brains from third instar larvae of dlkb1- Fig. 5. Mutations in the Drosophila lkb1 gene affect Mira null mutants exhibit a dramatic hyperplasia of both the hemispheres localization at the NB basal pole. (A) Mira localization in wild-type, P62 P62 315 315 pins /pins and dlkb1 /dlkb1 NBs. Cells in metaphase and and the ventral ganglion; this phenotype has been attributed to a telophase were stained for tubulin (Tub), DNA and Mira. Scale bar: reduction in developmental apoptosis during embryogenesis (Lee et 5 m. (B) Distribution of Mira in dividing NBs from wild-type (wt), al., 2006b). We observed a clear brain overgrowth in all our dlkb1 315 315 P62 P62 dlkb1 /dlkb1 and pins /pins brains. Regular, regular Mira mutant alleles, confirming that Dlkb1 regulates Drosophila brain crescent at the basal pole; cortical, Mira associated with the entire cell size (data not shown). It is likely that the brain hyperplasia elicited cortex; diffuse, Mira dispersed in the cytoplasm. by dlkb1 mutations results from both defective apoptosis and DaPKC-related NB overproliferation. Dlkb1 is not required for NB spindle rotation Recent work has shown that that spindle rotation is regulated by We examined 128 metaphases of dlkb1 mutant NBs stained for Mud (Mushroom body defect), a protein related to vertebrate NuMA Mira; 53 of them displayed a Mira crescent, but only in one case was (also known as Numa1) that interacts with both Pins and the spindle this crescent incorrectly oriented with respect to the spindle axis MTs. In embryonic NBs, Mud forms an apical crescent and (Fig. 5B). By contrast, this crescent was misoriented with respect to accumulates at the spindle poles; in larval NBs, the cortical the spindle axis in nine of the 29 pins NB metaphases with a Mira localization of Mud is weak or undetectable but the protein remains crescent (Fig. 5B). These results confirm that Pins is required for enriched at the spindle poles (Bowman et al., 2006; Izumi et al., proper spindle rotation during NB division and indicate that the 2006; Siller et al., 2006). Immunostaining for Mud revealed that the Dlkb1 kinase is not involved in this process. protein is enriched at the centrosomes and the astral MTs in 93% DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2189 (n=45) of prophase and early prometaphase NBs (Fig. 7A). With progression through mitosis, Mud localization became more diffuse and 62% (n=45) of NB metaphase figures did not exhibit clear Mud accumulations at the spindle poles (Fig. 7A); however, Mud relocalized at the pericentrosomal regions of most anaphases and telophases (83%, n=30; data not shown). In dlkb1 mutant NBs, Mud accumulated at the centrosomes/asters in 91% (n=35) of prophase and early prometaphase NBs (Fig. 7C), and remained associated with the spindle poles in 78% (n=37) of the metaphases (Fig. 7D) and 85% (n=20) of the ana-telophases (not shown). Thus, mutations in dlkb1 do not affect Mud localization during metaphase and ana- telophase, but appear to increase Mud concentration at the spindle poles during metaphase. Subcellular localization of Dlkb1 To determine the subcellular localization of Dlkb1, we raised a guinea pig antibody against the entirety of Dlkb1. Western blot analysis showed that this antibody recognizes a band of the expected size (~63 kDa) in larval, embryonic and S2 cell extracts. This band 315 315 7 was absent from both dlkb1 /Df(3R)urd and dlkb1 /dlkb1 larvae (Fig. 8A), demonstrating that it corresponds with Dlkb1. Since the 315 7 truncated forms of Dlkb1 encoded by the dlkb1 and dlkb1 mutant alleles were not observed in mutant animals, either the mutant transcripts or the truncated proteins are unstable. These findings provide strong support for the genetic data (Table 1), indicating that the dlkb1 mutant allele is functionally null. Immunolocalization experiments revealed that Dlkb1 is dispersed in both the nucleus and the cytoplasm of interphase larval brain cells, and in the cytoplasm of both NBs and GMCs undergoing mitotic division. Immunostaining of dlkb1 /Df(3R)urd mutant cells did not reveal any clear cytoplasmic signal, confirming the specificity of the antibody (Fig. 8B and data not shown). The diffuse localization of Dlkb1 in brain cells is not consistent with its cortical localization in Drosophila oocytes (Martin and St Johnston, 2003). However, the Dlkb1 localization pattern in brain cells does not reflect the quality of our antibody, as the same antibody revealed a cortical accumulation of Dlkb1 in oocytes (data not shown). Dlkb1 and Pins function in different pathways controlling NB division The finding that Dlkb1 and Pins co-precipitate (Blumer et al., 2003), and that dlkb1 and pins mutations cause similar (but not identical) phenotypes, prompted us to perform an epistasis analysis. We thus 315 P62 compared the phenotype of the dlkb1 pins double mutant with those of the single mutants by examining brain preparations stained for tubulin, Dpn and DNA. In dlkb1 pins mutant brains, the spindles of both NBs and GMCs were much more defective than those observed in either of the single mutants (compare Fig. 9 with Figs 1 and 2). In addition to cells with severely defective spindles (Fig. 9A,C,F,G,I), we also observed many (50%, n=300) mitotic figures in which the spindle morphology was barely recognizable (Fig. 9B,D,E,H); the frequency of the latter type of cells was only 4% (n=201) in the dlkb1 single mutant. In dlkb1 pins metaphases, the MT density was extremely low, the spindle poles had a characteristic Fig. 6. Mutations in the Drosophila lkb1 gene disrupt Baz/Par- pointed appearance and the asters were completely absent (Fig. 9A). 6/DaPKC but not Pins/Gi localization at the NB apical pole. The ana-telophases were also devoid of asters and displayed few and (A) Pins, Gi, Baz, Par-6 and DaPKC localization in wild-type and sparse central spindle MTs, which were never pinched in the middle 315 315 dlkb1 /dlkb1 mutants. NBs in the first row were simultaneously (Fig. 9C,F). These results indicate that dlkb1 and pins function in stained for Pins, Centrosomin (Cnn) and DNA (blue). Cells shown in the parallel pathways to control spindle formation. subsequent rows were stained for tubulin (Tub), DNA (blue) and either The absence of central spindle pinching, which suggests an Gi, Baz, Par-6 or DaPKC. Scale bar: 5 m. (B) Frequencies of NBs with accompanying failure of cytokinesis, prevented a reliable Pins, Gi, Baz, Par-6 and DaPKC crescents in wild-type and 315 315 dlkb1 /dlkb1 mutant brains. assessment of the degree of asymmetry of NB divisions. However, DEVELOPMENT 2190 RESEARCH ARTICLE Development 134 (11) Fig. 8. Expression and intracellular localization of the Dlkb1 kinase. (A) Western blot showing that the anti-Dlkb1 antibody recognizes a band of approximately 63 kDa. This band is absent in 315 315 7 extracts from either dlkb1 /Df(3R)urd or dlkb1 /dlkb1 mutant Drosophila larvae. -Tubulin was used as a loading control (LC). (B) Immunostaining for Dlkb1 in metaphase and telophase of wild-type dividing NBs. Note that Dlkb1 is diffuse in the cytoplasm. Scale bar: 5 m. and spindle formation. Whether the latter pathways are the same as those that control the asymmetry of NB mitosis remains to be determined. Fig. 7. Mud localization in wild-type and dlkb1 mutant Drosophila NBs. Cells were stained for Mud, tubulin (Tub) and DNA (blue). (A,B) Wild-type prophase (A) and metaphase (B). DISCUSSION 315 315 (C,D) dlkb1 /dlkb1 prophase (C) and metaphase (D). Scale bar: Dlkb1 controls the stability of spindle MTs 5 m. Our results indicate that mutations in the dlkb1 gene disrupt spindle formation in both NBs and GMCs. In addition, the finding that the NB spindles of the double mutant were smaller than in wild type imaginal discs of dlkb1 mutant larvae are small and misshapen (Fig. 9J). In addition, the analysis of centrosome size in large suggests a defect in imaginal cell mitosis. Previous studies have metaphase figures (longer than 14 m), most of which are likely to shown that late larval lethality and small imaginal discs are be NBs, revealed that 90% (n=106) of them had equally sized diagnostic of abnormalities in mitotic divisions (Gatti and Baker, centrosomes. In wild type, dlkb1 and pins, the frequencies of NB 1989). Thus, the dlkb1 phenotype strongly suggests that the Dlkb1 metaphases with centrosomes of equal size were 12%, 72% and kinase plays an important mitotic role not only in NBs, but also in 61%, respectively (Fig. 4B). Finally, only 3% (n=120) of the dlkb1 other somatic cell types. Despite the low density of spindle MTs, pins NB metaphases were characterized by a Mira crescent; in the most mutant metaphases enter anaphase (the frequency of anaphases remaining cells, Mira was either diffuse in the cytoplasm (85%) or in dlkb1 mutants and in wild-type controls was 10-13% and 16%, associated with the entire cell cortex (12%). Thus, the Mira respectively; see Table 1), suggesting that in a substantial fraction of mislocalization phenotype observed in dlkb1 pins double mutants is mutant cells the spindle checkpoint is either not induced or only stronger than that seen in the single mutants (see Fig. 5B). transiently activated. However, the defects in NB spindles are likely 315 P62 Although the dlkb1 and pins alleles are both functionally to lead to the formation of polyploid cells. These cells could arise 315 P62 null, it cannot be excluded that the brains of the dlkb1 pins through two different mechanisms. Cells blocked in metaphase double mutants retain residual amounts of the maternally supplied owing to either reduced MT density or activation of the spindle Dlkb1 and Pins proteins. We thus performed a western blotting checkpoint could revert to interphase and become polyploid after an 315 P62 analysis of extracts from third instar larval brains of dlkb1 , pins additional round of DNA replication. Alternatively, cells that enter 315 P62 315 and dlkb1 pins mutants. As shown in Fig. 9K, dlkb1 and anaphase but assemble an abnormally thin central spindle might be 315 P62 dlkb1 pins brains did not exhibit detectable amounts of the unable to undergo cytokinesis and in consequence produce Dlkb1 kinase, consistent with the results shown in Fig. 8A. polyploid cells (Vernì et al., 2004). Similarly, the Pins protein appeared to be completely absent from The precise function of Dlkb1 in spindle formation and/or P62 315 P62 pins and dlkb1 pins brains. Thus, the phenotypes observed maintenance is currently unclear. However, the finding that the in the single and double mutants reflect a complete loss of the wild- spindles of dlkb1 asl double mutants display a lower MT density type function of either Dlkb1 or Pins, or both. than asl single mutants argues for a defect in MT stability and not in Collectively, our results suggest that dlkb1 and pins act in centrosome function. Studies in mammalian cells have shown that different pathways to control the asymmetry of NB division. These LKB1 is a master kinase that phosphorylates at least 14 kinases, all genes also function in parallel pathways involved in MT stability of which are related to AMP-activated kinases (AMPK). Kinases of DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2191 Fig. 9. dlkb1 and pins function in different pathways controlling the stability of spindle MTs in Drosophila. Mitotic figures 315 P62 from brains of dlkb1 pins double-mutants were stained for tubulin (Tub, green), DNA (blue) and Centrosomin (red). (A-F) NBs; (G-I) GMCs. (A,B) Metaphases; (C-E) anaphases; (F) telophase; (G) metaphases; (H) anaphase; (I) telophase. The arrow in E points to a lagging X chromosome with unseparated sister chromatids. Note the extremely defective spindle structures of the NBs shown in B,D,E,H. Scale bar: 5 m. (J) Size distribution of metaphase spindles in wild-type and dlkb1 pins brains. Dpn-positive (NB) and Dpn-negative (GMC) spindles are depicted in red and green, respectively. Size (m) classes: A, 4.7-6.9; B, 7.0-9.2; C, 9.3-11.5; D, 11.6-13.8; E, 13.9- 16.1; F, 16.2-18.4; G, 18.5-20.7. (K) Expression of Pins and Dlkb1 in brains from third instar larvae of dlkb1, pins and dlkb1 pins mutants. Note that the Dlkb1 protein is undetectable in larval brain extracts of both dlkb1 315 P62 homozygotes and dlkb1 pins double mutants. Similarly, Pins cannot be detected in P62 P62 brain extracts of both pins /pins mutants 315 P62 and dlkb1 pins double mutants. The Giotto protein (Giansanti et al., 2006) was used as a loading control (LC). the AMPK family include regulators of cellular energy levels, as discs, suggesting an underlying mitotic defect. Given that the 315 P62 well as four Microtubule affinity regulating kinases (MARKs) dlkb1 and pins alleles used in the analysis are both functionally (reviewed by Alessi et al., 2006; Baas et al., 2004b). The MARK null and that the corresponding proteins were undetectable in mutant enzymes are the mammalian homologs of C. elegans and brains (Yu et al., 2000) (this study), these results indicate that Pins Drosophila Par-1. However, Drosophila Par-1, which controls MT and Dlkb1 function in different pathways for the control of MT stability in oocytes (Shulman et al., 2000), appears to act upstream stability. The observation that the spindles of pins mutants display a of Dlkb1 (Martin and St Johnston, 2003). It is therefore unlikely that normal MT density further suggests that Pins plays a redundant role the Dlkb1 substrate required for the stability of spindle MTs is Par- in the maintenance of MT stability. A role for Pins in spindle 1. Further evidence that Dlkb1 does not act via Par-1 formation and/or stability has never been demonstrated in phosphorylation comes from RNAi experiments showing that Dlkb1 Drosophila. However, the mammalian homolog of Pins binds depletion, but not Par-1 depletion, causes defects in spindle NuMA and regulates mitotic spindle organization and positioning morphology (Bettencourt-Dias et al., 2004). Thus, current data (Du et al., 2001). indicate that the Dlkb1 kinase regulates the activity of an unknown factor required for the stability of the spindle MTs; this factor could Dlkb1 controls the asymmetry of NB division either be a direct substrate of Dlkb1 or a substrate for one of the We have analyzed the phenotypic consequences of dlkb1 mutations kinases acting downstream of Dlkb1. in larval brain NBs. In contrast to embryonic NBs that display Our cytological analyses have shown that the spindles of dlkb1 small, regularly sized spindles (their metaphase spindles are pins double mutants display a MT density that is substantially lower approximately 5 m long), brain NBs exhibit spindles of very than that observed in either single mutant. Here again, highly different sizes (ranging from 5 to 32 m for metaphase spindles). defective spindles were observed in both NBs and GMCs. In Nonetheless, dividing brain NBs exhibit the same asymmetries as addition, double-mutant larvae showed extremely reduced imaginal their embryonic counterparts, including asymmetries in aster and DEVELOPMENT 2192 RESEARCH ARTICLE Development 134 (11) centrosome size, localization of specialized protein complexes and in the Baz/Par-6 pathway, we cannot exclude the possibility that this positioning of the cleavage furrow (Bowman et al., 2006; Giansanti kinase functions in both the Baz/Par-6 and Pins/Gi pathways, or in et al., 2001; Lee et al., 2006a; Parmentier et al., 2000; Rolls et al., a third pathway different from either. 2003; Siller et al., 2006) (this study). However, the degree of In this context, it is important to note that our results exclude the asymmetry of brain NB division is directly related to the cell size, possibility that dlkb1 acts via Pins phosphorylation. Previous studies so that large NBs divide more asymmetrically than small NBs (Fig. have shown that mammalian LKB1 co-precipitates and 3E). This is likely to render large brain NBs particularly sensitive phosphorylates AGS3, the mammalian ortholog of Pins (Blumer et to mutations that affect cleavage furrow positioning. Consistent al., 2003). Dlkb1 and Pins coimmunoprecipitate as well, but it is with this hypothesis, mutations in pins have mild effects on the currently unclear whether Pins is phosphorylated by Dlkb1 (Blumer asymmetry of embryonic NB divisions (Cai et al., 2003), but et al., 2003). Regardless of whether Pins is a substrate of Dlkb1, the disrupt unequal cytokinesis in most larval brain NBs (Parmentier phenotypes elicited by dlkb1 mutations cannot be the consequence et al., 2000) (Fig. 3E). dlkb1 larval NBs also divide more of an impairment of Pins function. dlkb1 and pins mutant NBs do in symmetrically than their wild-type counterparts, leading to larval fact differ in a number of phenotypic traits, including spindle brains devoid of large NBs. In addition, most dlkb1 NBs display organization and the pattern of Mira localization, and do not belong centrosomes of equal size and very small asters at both poles. to the same epistasis group. However, the symmetric cytokinesis of dlkb1 NBs cannot result from their short astral MTs, as asl NBs divide asymmetrically in the Dlkb1 is not required for NB spindle rotation complete absence of asters (Fig. 3E). In vivo imaging has shown that the spindles of embryonic NBs dlkb1 mutant NBs are also characterized by the abnormal rotate during metaphase to become aligned with the center of the distribution of several components of the apical and basal Pins apical crescent (Kaltschmidt et al., 2000). By contrast, the complexes. In dlkb1 mutant brains, most NBs display normal Pins spindles of larval NBs align with the Pins crescent at prophase (Siller and Gi crescents at their apical pole but fail to accumulate Baz, et al., 2006). Failure of proper rotation of larval NB spindles results DaPKC and Par-6 at the same pole. In addition, most dlkb1 in spindles that are misoriented with respect to the apical (Pins) and mutant NBs fail to exhibit a normal Mira crescent at the basal pole basal (Mira) crescents (Giansanti et al., 2001; Siller et al., 2006). cortex. A normal localization of Pins and Gi has been observed There is also evidence that proper positioning of larval NB spindles in most embryonic NBs defective in the Baz/Par-6 pathway (Cai depends on astral MTs, because in approximately 50% of asl NB et al., 2003; Izumi et al., 2004; Schaefer et al., 2000; Yu et al., metaphases the Mira crescent is misoriented with respect to the 2000; Yu et al., 2003). Moreover, studies on embryonic NBs have spindle axis (Giansanti et al., 2001). suggested that Baz, Par-6 and DaPKC function as a complex, are Our results indicate that spindle rotation occurs normally in dlkb1 interdependent for their localization at the NB apical pole, and are mutant NBs. In addition, we have shown that prophase/ required for the formation of the Mira crescent at the basal pole prometaphase larval NBs of dlkb1 mutants normally accumulate the (Petronczki and Knoblich, 2000; Wodarz et al., 2000). However, Mud protein, which mediates proper spindle alignment in both subsequent work on second instar larval NBs has shown that these embryonic and larval NBs (Bowman et al., 2006; Izumi et al., 2006; proteins are not mutually dependent for the formation of the Siller et al., 2006). Together, these results indicate that the Dlkb1 Baz/Par-6/DaPKC apical crescent; they accumulate at the apical kinase is not required for spindle rotation and that the short astral cortex in a hierarchical fashion, with Baz and Par-6 mediating MTs of dlkb1 mutant NBs can mediate proper spindle positioning. proper DaPKC localization (Rolls et al., 2003). Mutations that These results are consistent with the idea that the Pins/Gi, but not disrupt the Pins/Gi pathway prevent asymmetrical localization the Baz/Par-6, pathway is involved in spindle rotation (Izumi et al., of either Pins or Gi in embryonic NBs but do not substantially 2004; Siegrist and Doe, 2005) and provide further support for the affect Mira accumulation at the basal pole (Cai et al., 2003). hypothesis that Dlkb1 functions in the latter pathway. However, it should be noted that mutations in pins partially Recent work has shown that in the absence of the Baz/Par-6 disrupt asymmetric Mira localization in larval brain NBs pathway, astral MTs can mediate the localization of Pins/Gi at the (Parmentier et al., 2000) (this study), suggesting that larval NBs apical cortex (Siegrist and Doe, 2005). Assuming that Dlkb1 acts in differ from embryonic NBs in some aspects of the control of Mira the Baz/Par-6 pathway, the finding that this kinase is not required for localization. Thus, taking into account the differences between the formation of Pins/Gi crescents indicates that the short astral embryonic and larval NBs, our results indicate that mutations in MTs of dlkb1 NBs retain the ability to mediate Pins/Gi cortical the dlkb1 gene and those that disrupt the Baz/Par-6 pathway affect localization. similar aspects of NB mitotic division. Our analyses have shown that in dlkb1 pins double mutants, the Conclusions and perspectives NBs divide more symmetrically than in the corresponding single Our results indicate that Dlkb1 and Pins function in partially mutants. This indicates that the dlkb1 and pins genes act in different redundant pathways controlling the stability of spindle MTs. These pathways that mediate unequal cytokinesis. Previous studies have proteins are also required for the asymmetry of NB divisions and, shown that the asymmetry of NB cytokinesis depends on the here again, they appear to function in different pathways. Pins acts Baz/Par-6 and Pins/Gi redundant pathways. When only one of in a common pathway with Gi, whereas Dlkb1 is likely to function these pathways is impaired, NBs still divide asymmetrically, but in the Baz/Par-6 pathway. Intriguingly, recent work has shown that they divide symmetrically when both are disrupted (Cai et al., 2003). simultaneous loss of pins and baz functions results in the formation The simplest interpretation of our findings is that dlkb1 acts in the of abnormally small embryonic NB spindles that lack astral MTs at Baz/Par-6 pathway. In addition, the observation that Dlkb1 is both poles (Fuse et al., 2003). Thus, the embryonic NBs of baz pins required for proper localization of Baz, Par-6 and DaPKC suggest double mutants have a spindle phenotype reminiscent of that that this kinase acts at the top of the hierarchical mechanism that observed in dlkb1 larval NBs. These findings raise the question of mediates accumulation of the Baz/Par-6 complex at the apical whether the Pins/Gi and Baz/Par-6 pathways redundantly control cortex. However, although we favor the hypothesis that Dlkb1 acts spindle organization as they do for the asymmetry of NB divisions. DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2193 The extant data do not provide a clear answer to this question. The Giansanti, M. G., Bonaccorsi, S., Kurek, R., Farkas, R. M., Dimitri, P., Fuller, M. T. and Gatti, M. (2006). The class I PITP Giotto is required for Drosophila analysis of the roles of the two pathways in spindle formation and cytokinesis. Curr. Biol. 16, 195-201. their precise relationships with the Dlkb1 kinase are interesting Goodman, C. S. and Doe, C. Q. (1993). Embryonic development of the issues to be addressed in future studies. Drosophila central nervous system. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez Arias), pp. 1131-1206. New York: Previous studies in Drosophila and mammalian cells have led to Cold Spring Harbor Laboratory Press. the suggestion that loss of epithelial cell polarity is ultimately Izumi, Y., Ohta, N., Itoh-Furuya, A., Fuse, N. and Matsuzaki, F. (2004). responsible for the Peutz-Jeghers cancer syndrome (Martin and St Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J. Cell Biol. 164, 729-738. Johnston, 2003; Baas et al., 2004b). Here, we have shown that Dlkb1 Izumi, Y., Ohta, N., Hisata, K., Raabe, T. and Matsuzaki, F. (2006). Drosophila plays an essential mitotic role and is required for the asymmetry of Pins-binding protein Mud regulates spindle-polarity coupling and centrosome NB division. These results lead us to propose that tumor organization. Nat. Cell Biol. 8, 586-593. development in Peutz-Jeghers patients depends on the impairment Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. and Brand, A. H. (2000). Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in of multiple processes, including cell polarity, the asymmetry of stem the developing central nervous system. Nat. Cell Biol. 2, 7-12. cell division and the fidelity of chromosome segregation during Koundakjian, E. J., Cowan, D. M., Hardy, R. W. and Becker, A. H. (2004). The mitosis. Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster. Genetics 167, 203-206. We thank W. Chia, Y. Jan, T. Kaufman, J. Knoblich and F. Matsuzaki for Lee, C.-Y., Robinson, K. J. and Doe, C. Q. (2006a). Lgl, Pins, and aPKC regulate antibodies and fly stocks. This work was supported by grants from Centro di neuroblast self-renewal versus differentiation. Nature 439, 594-598. Lee, J. H., Koh, H., Park, J., Lee, S. Y., Lee, S. and Chung, J. (2006b). JNK Eccellenza di Biologia e Medicina Molecolare (BEMM) to M.G. and by NIH pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in grant GM48430 to M.L.G. Drosophila. Cell Death Differ. 13, 1110-1122. References Martin, S. G. and St Johnston, D. (2003). A role for Drosophila LKB1 in anterior- Alessi, D. R., Sakamoto, K. and Bayascas, J. R. (2006). LKB1-dependent posterior axis formation and epithelial polarity. Nature 421, 379-384. signaling pathways. Annu. Rev. Biochem. 75, 137-163. Megraw, T. L., Kao, L. R. and Kaufman, T. C. (2001). Zygotic development Baas, A. F., Kuipers, J., van der Wel, N. N., Batlle, E., Koerten, H. K., Peters, P. without functional mitotic centrosomes. Curr. Biol. 11, 116-120. J. and Clevers, H. C. (2004a). Complete polarization of single intestinal Parmentier, M. L., Woods, D., Greig, S., Phan, P. G., Radovic, A., Bryant, P. epithelial cells upon activation of LKB1 by STRAD. Cell 116, 457-466. and O’Kane, C. J. (2000). Rapsynoid/partner of inscuteable controls asymmetric Baas, A. F., Smit, L. and Clevers, H. (2004b). LKB1 tumor suppressor protein: division of larval neuroblasts in Drosophila. J. Neurosci. 20, RC84. PARtaker in cell polarity. Trends Cell Biol. 14, 312-319. Petronczki, M. and Knoblich, J. A. (2000). DmPar-6 directs epithelial polarity Betschinger, J. and Knoblich, J. A. (2004). Dare to be different: asymmetric cell and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43- division in Drosophila, C. elegans and vertebrates. Curr. Biol. 14, R674-R685. 49. Bettencourt-Dias, M., Giet, R., Sinka, R., Mazumdar, A., Lock, W. G., Balloux, Rolls, M. M., Albertson, R., Shih, H.-P., Lee, C.-Y. and Doe, C. Q. (2003). F., Zafiropoulos, P. J., Yamaguchi, S., Winter, S., Carthew, R. W. et al. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and (2004). Genome-wide survey of protein kinases required for cell cycle epithelia. J. Cell Biol. 163, 1089-1098. progression. Nature 432, 980-987. Schaefer, M., Shevchenko, A., Shevchenko, A. and Knoblich, J. A. (2000). A Bier, E., Vaessin, H., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (1992). protein complex containing Inscuteable and the Ga-binding protein Pins orients deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix asymmetric cell divisions in Drosophila. Curr. Biol. 10, 353-362. protein similar to the hairy gene product. Genes Dev. 6, 2137-2151. Schaefer, M., Petronczki, M., Corner, D., Forte, M. and Knoblich, J. A. (2001). Blumer, J. B., Bernard, M. L., Peterson, Y. K., Nezu, J., Chung, P., Dunican, D. Heterotrimeric G proteins direct two modes of asymmetric cell division in the J., Knoblich, J. A. and Lanier, S. M. (2003). Interaction of activator of G- Drosophila nervous system. Cell 107, 183-194. protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell Shulman, J. M., Benton, R. and St Johnston, D. (2000). The Drosophila polarity and cell cycle progression: phosphorylation of the G-protein regulatory homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with oskar mRNA localization to the posterior pole. Cell 101, 377-388. Galphai. J. Biol. Chem. 278, 23217-23220. Siegrist, S. E. and Doe, C. Q. (2005). Microtubule-induced Pins/Galphai cortical Bonaccorsi, S., Giansanti, M. G. and Gatti, M. (1998). Spindle self-organization polarity in Drosophila neuroblasts. Cell 123, 1323-1335. and cytokinesis during male meiosis in asterless mutants of Drosophila Siller, K. H., Cabernard, C. and Doe, C. Q. (2006). The NuMA-related Mud melanogaster. J. Cell Biol. 142, 751-761. protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Bonaccorsi, S., Giansanti, M. G. and Gatti, M. (2000). Spindle assembly in Nat. Cell Biol. 8, 594-600. Drosophila neuroblasts and ganglion mother cells. Nat. Cell Biol. 2, 54-56. Vernì, F., Somma, M. P., Gunsalus, K. C., Bonaccorsi, S., Belloni, G., Bowman, S. K., Neumuller, R. A., Novatchkova, M., Du, Q. and Knoblich, J. Goldberg, M. L. and Gatti, M. (2004). Feo, the Drosophila homolog of PRC1, A. (2006). The Drosophila NuMA Homolog Mud regulates spindle orientation in is required for central-spindle formation and cytokinesis. Curr. Biol. 14, 1569- asymmetric cell division. Dev. Cell 10, 731-742. 1575. Cai, Y., Yu, F., Lin, S., Chia, W. and Yang, X. (2003). Apical complex genes Watts, J. L., Morton, D. G., Bestman, J. and Kemphues, K. J. (2000). The C. control mitotic spindle geometry and relative size of daughter cells in Drosophila elegans par-4 gene encodes a putative serine-threonine kinase required for neuroblast and pI asymmetric divisions. Cell 112, 51-62. establishing embryonic asymmetry. Development 127, 1467-1475. Du, Q., Stukenberg, P. T. and Macara, I. G. (2001). A mammalian Partner of Wodarz, A. (2005). Molecular control of cell polarity and asymmetric cell division Inscuteable binds NuMa and regulates mitotic spindle organization. Nat. Cell in Drosophila neuroblasts. Curr. Opin. Cell Biol. 17, 1-7. Biol. 3, 1069-1075. Wodarz, A., Ramrath, A., Grimm, A. and Knust, E. (2000). Drosophila Atypical Fuse, N., Hisata, K., Katzen, A. L. and Matsuzaki, F. (2003). Heterotrimeric G Protein Kinase C associates with Bazooka and controls polarity of epithelia and proteins regulate daughter cell size asymmetry in Drosophila neuroblast neuroblasts. J. Cell Biol. 150, 1361-1374. divisions. Curr. Biol. 13, 947-954. Yu, F., Morin, X., Cai, Y., Yang, X. and Chia, W. (2000). Analysis of partner of Gatti, M. and Baker, B. S. (1989). Genes controlling essential cell-cycle functions inscuteable, a novel player of Drosophila asymmetric divisions, reveals two in Drosophila melanogaster. Genes Dev. 3, 438-453. distinct steps in inscuteable apical localization. Cell 100, 399-409. Giansanti, M. G., Gatti, M. and Bonaccorsi, S. (2001). The role of centrosomes Yu, F., Cai, Y., Kaushik, R., Yang, X. and Chia, W. (2003). Distinct roles of Gi and astral microtubules during asymmetric division of Drosophila neuroblasts. and G13F subunits of the heterotrimeric G protein complex in the mediation of Development 128, 1137-1145. Drosophila neuroblast asymmetric divisions. J. Cell Biol. 162, 623-633. DEVELOPMENT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division

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0950-1991
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10.1242/dev.02848
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Abstract

DEVELOPMENT AND DISEASE RESEARCH ARTICLE 2183 Development 134, 2183-2193 (2007) doi:10.1242/dev.02848 The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division 1 1 1 2 2 Silvia Bonaccorsi , Violaine Mottier , Maria Grazia Giansanti , Bonnie J. Bolkan , Byron Williams , 2 1, Michael L. Goldberg and Maurizio Gatti * We have isolated lethal mutations in the Drosophila lkb1 gene (dlkb1), the homolog of C. elegans par-4 and human LKB1 (STK11), which is mutated in Peutz-Jeghers syndrome. We show that these mutations disrupt spindle formation, resulting in frequent polyploid cells in larval brains. In addition, dlkb1 mutations affect asymmetric division of larval neuroblasts (NBs); they suppress unequal cytokinesis, abrogate proper localization of Bazooka, Par-6, DaPKC and Miranda, but affect neither Pins/Gi localization nor spindle rotation. Most aspects of the dlkb1 phenotype are exacerbated in dlkb1 pins double mutants, which exhibit more severe defects than those observed in either single mutant. This suggests that Dlkb1 and Pins act in partially redundant pathways to control the asymmetry of NB divisions. Our results also indicate that Dlkb1 and Pins function in parallel pathways controlling the stability of spindle microtubules. The finding that Dlkb1 mediates both the geometry of stem cell division and chromosome segregation provides novel insight into the mechanisms underlying tumor formation in Peutz-Jeghers patients. KEY WORDS: Lkb1, Neuroblasts, Asymmetric division, Spindle formation, Drosophila INTRODUCTION apical complex by the Inscuteable (Insc) protein that binds both Pins Drosophila neuroblasts (NBs) are one of the best model systems for and Baz (reviewed by Betschinger and Knoblich, 2004; Wodarz, the study of the control of cell polarity and asymmetric cell division. 2005). During Drosophila embryogenesis, NBs delaminate basally from Recent genetic analyses have shown that the Baz/Par-6 the neuroectodermal epithelium and divide asymmetrically along subcomplex is mainly involved in the control of proper basal the apical/basal axis to produce another NB and a smaller ganglion localization of Pros/Mira and Numb/Pon. The Pins/Gi subcomplex mother cell (GMC). The newly generated apical NB divides is instead required for spindle orientation during NB divisions. Both repeatedly in an asymmetric fashion, whereas the basal GMC complexes, however, cooperate in controlling cleavage furrow divides symmetrically just once to generate equally sized daughter positioning during asymmetric NB divisions. Mutations that disrupt cells that differentiate into neurons or glia (reviewed by Betschinger either the Baz/Par-6 or the Pins/Gi pathway have little or no effect and Knoblich, 2004; Wodarz, 2005). on asymmetric cytokinesis. However, mutations that disrupt both The asymmetric division of Drosophila NBs is regulated by pathways completely abrogate spindle displacement during several proteins that concentrate at the cell cortex. The basal cortex telophase, leading to symmetric cytokinesis (Cai et al., 2003; Izumi is enriched in the cell fate determinants Prospero (Pros) and Numb, et al., 2004; Shaefer et al., 2000; Yu et al., 2000; Yu et al., 2003). as well as in their respective adaptor proteins Miranda (Mira) and In this study, we have addressed the role of Drosophila Lkb1 Partner of Numb (Pon). These proteins are preferentially segregated (Dlkb1), the homolog of LKB1 kinase (STK11 – Human Gene into the GMC following NB cytokinesis. Localization of Pros-Mira Nomenclature Database) in NB division. LKB1 kinase is mutated in and Numb-Pon at the basal cortex is mediated by a large the Peutz-Jeghers syndrome, an autosomal dominantly inherited multiprotein complex that concentrates at the apical cortex. This disorder characterized by the formation of intestinal polyps and a complex includes two functionally distinct subcomplexes. One of high incidence of various cancer types. Somatic mutations in the them contains Bazooka (Baz; PAR-3 in C. elegans), aPKC LKB1 gene have also been detected in sporadic adenocarcinomas (Drosophila atypical protein kinase C, DaPKC) and Par-6; this (reviewed by Alessi et al., 2006; Baas et al., 2004b). There is assembly is hereafter referred to as the Baz/Par-6 subcomplex. The evidence that LKB1 plays a conserved role in the control of cell other subcomplex includes the Gi subunit of the heterotrimeric G- polarity. Recent work has unambiguously shown that activation of protein complex and Partner of inscuteable (Pins; Raps – Flybase), LKB1 leads to rapid and complete polarization of human intestinal and is hereafter referred to as the Pins/Gi subcomplex. The epithelial cells (Baas et al., 2004a). Similarly, PAR-4, the C. elegans Baz/Par-6 and Pins/Gi subcomplexes are integrated in a larger homolog of LKB1, is required for correct polarity and asymmetric division of one-cell embryos (Watts et al., 2000). Furthermore, Dlkb1 mediates determination of anterior/posterior polarity of egg chambers and embryos, as well as the proper polarity of follicle cells (Martin and St Johnston, 2003). Here, we demonstrate that Dlkb1 Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia controls many asymmetries that characterize the mitotic division of Molecolari del CNR, Dipartimento di Genetica e Biologia Molecolare, Università di larval NBs. dlkb1 mutations also disrupt mitotic spindle assembly, Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA. leading to the frequent formation of polyploid cells. Thus, in addition to cell polarity and the geometry of cell division, Dlkb1 *Author for correspondence (e-mail: [email protected]) directly or indirectly regulates the stability of spindle microtubules (MTs). Accepted 12 March 2007 DEVELOPMENT 2184 RESEARCH ARTICLE Development 134 (11) MATERIALS AND METHODS RESULTS Fly strains and genetic manipulations Isolation and characterization of mutations in the The dlkb1 mutant allele was isolated from a collection of 1600 third dlkb1 gene chromosome late lethals induced by ethylmethanesulfonate (EMS) in C. In the course of a screen aimed at the isolation of mitotic mutants Zuker’s laboratory (Koundakjian et al., 2004). The dlkb1 allele is (see Materials and methods), we identified a lethal mutation that associated with the chromosome carrying Df(3R)su(Hw)7. This and all the causes frequent polyploid cells in larval brains (see below). Animals deficiencies used for mapping were obtained from the Bloomington Stock homozygous for this mutation die at the larval/pupal transition, as P62 2 Center. The pins null allele and the asl mutation have been described do most mitotic mutants; most probably, they exploit maternally 315 P62 previously (Bonaccorsi et al., 1998; Yu et al., 2000); dlkb1 pins and supplied products to survive until late larval stages (Gatti and Baker, 315 2 dlkb1 asl double mutants were generated by recombination. All 1989). Deficiency mapping showed that this mutation is uncovered mutations were maintained over the TM6B balancer, and mutant larvae by both Df(3R)urd and Df(3R)26c, which define a map interval that were identified based on their non-tubby phenotype. Genetic markers and contains only 16 annotated genes. During these mapping studies, we special chromosomes are described in FlyBase (http://www.flybase.org/). also identified another mutant allele of this same mitotic gene. Germline transformation was performed as previously described (Vernì et This allele is associated with the chromosome that carries al., 2004). Df(3R)su(Hw)7, but is independent of the deficiency. We next Antibodies and immunoblotting sequenced the candidate genes and found that both mutant stocks The anti-Dlkb1 antibody was generated in guinea pig using a maltose- carry lesions in the Drosophila lkb1 gene (dlkb1, also known as binding protein (MBP)/Dlkb1 fusion protein. Expression of the fused protein CG9374). This gene encodes a 567 amino acid serine/threonine in Escherichia coli and the production and purification of antibodies against kinase homologous to the PAR-4 kinase of C. elegans and the this fusion were according to Vernì et al. (Vernì et al., 2004). Immunoblotting human LKB1 kinase mutated in Peutz-Jeghers syndrome (Martin was performed as previously described (Vernì et al., 2004); the anti-Dlkb1, and St Johnston, 2003). The dlkb1 mutant allele isolated in our anti-Pins and anti-Giotto (Giansanti et al., 2006) antibodies were diluted screen carries a frameshift mutation resulting in a truncated Dlkb1 1:2000, 1:1000 and 1:5000, respectively. protein of 234 amino acids; the dlkb1 allele, associated with Cytology Df(3R)su(Hw)7, has a stop codon that truncates Dlkb1 to a 346 Brains from third instar larvae were dissected and fixed according to amino acid polypeptide (Fig. 1A). A genomic fragment including Bonaccorsi et al. (Bonaccorsi et al., 2000). After several rinses in PBS, brain sequences that extend roughly 1 kb on either side of dlkb1 (Fig. 1A) preparations were incubated overnight at 4°C with a monoclonal anti-- rescued both the lethality and the mitotic phenotypes of tubulin antibody (Sigma-Aldrich), diluted 1:1000 in PBS-T (PBS with 0.1% 315 315 315 7 dlkb1 /dlkb1 and dlkb1 /dlkb mutants. Triton X-100), and any of the following rabbit antibodies, also diluted in PBS-T: anti-Centrosomin (1:300; gift of T. Kaufman, Indiana University, Mutations in the dlkb1 gene affect spindle Bloomington, IN, USA), anti-Deadpan (1:400; gift of Y. Jan, Howard formation Hughes Medical Institute, University of California, CA, USA), anti-Mira Drosophila brains contain mostly two types of dividing cells: NBs (1:500; gift of Y. Jan), anti-Bazooka (1:50; gift of F. Matsuzaki, Japan and GMCs (Goodman and Doe, 1993). Wild-type larval NBs are Science and Technology Corporation, Kobe, Japan); anti-Gi (1:200; gift of characterized by many asymmetries that develop during the course J. Knoblich, Austrian Academy of Sciences, Vienna, Austria); anti-Par-6 (1:1000; gift of J. Knoblich); anti-DaPKC (1:100; Santa Cruz of mitosis. Prometaphases and metaphases of larval NBs exhibit Biotechnology) and anti-Mud (1:200; gift of F. Matsuzaki). After two rinses centrosomes and asters of similar sizes at the two cell poles. in PBS, primary antibodies were detected by a 1-hour incubation at room However, as NBs progress through anaphase and telophase, the MTs temperature with FITC-conjugated anti-mouse IgG+IgM (1:20; Jackson of the basal aster shorten dramatically, whereas those of the apical Laboratories) and Alexa 555-conjugated anti-rabbit IgG (1:300; Molecular asters elongate slightly (Fig. 1B). Concomitantly, the basal Probes), diluted in PBS. centrosome becomes smaller than the apical one (Bonaccorsi et al., For double Centrosomin/Pins immunostaining, brains were incubated 2000) (see Fig. 4A below). These changes in aster and centrosome overnight at 4°C with the rabbit anti-Centrosomin antibody (1:300) and a rat morphology are accompanied by a progressive displacement of the anti-Pins antibody (1:100; gift of W. Chia, The National University of central spindle towards the basal pole, resulting in unequal Singapore, Singapore) diluted in PBS-T. Detection was performed by 1-hour cytokinesis (Giansanti et al., 2001). GMCs display equally sized incubation at room temperature with Alexa 555-conjugated anti-rabbit IgG centrosomes and very small asters throughout mitosis, and divide (Molecular Probes) and FITC-conjugated anti-rat IgG (Jackson symmetrically (Fig. 1B) (Bonaccorsi et al., 2000). Laboratories) diluted 1:300 and 1:20 in PBS, respectively. To determine the mitotic defect leading to polyploid cell For Dlkb1 immunostaining, brain preparations were incubated overnight formation in dlkb1 mutants, we examined larval brain preparations at 4°C with the anti-Dlkb1 antibody (1:100 in PBS-T) and, after rinsing in 315 315 315 315 7 PBS, were incubated 1 hour at room temperature with Alexa 555-conjugated from dlkb1 /dlkb1 , dlkb1 /Df(3R)urd and dlkb1 /dlkb1 anti-guinea pig IgG diluted 1:500 in PBS. larvae stained for both tubulin and DNA. These mutant In all cases, immunostained preparations were mounted in Vectashield combinations showed identical mitotic aberrations. Most strikingly, medium H-1200 (Vector Laboratories) containing the DNA-dye DAPI (4,6- mutant spindles showed an overall MT density substantially lower diamidino-2-phenylindole). Preparations were examined with a Zeiss than that seen in wild-type spindles (Fig. 1B). In approximately 80% Axioplan microscope, equipped with an HBO100W mercury lamp and a of mutant spindles, asters were absent or severely reduced; in control cooled charged-coupled device (CCD camera; Photometrics CoolSnap HQ). brains, the frequency of spindles without asters, or with very small Grayscale images were collected separately, converted to Photoshop (Adobe asters, was 49% (Fig. 1B; Table 1). In addition, most mutant Systems), pseudocolored and merged. prometaphase and metaphase figures were characterized by low Spindle measurements were taken on enlarged digital images and scaled densities of both kinetochore and interpolar MTs, and ana-telophases down to their size in m. In preparations stained for Centrosomin, displayed central spindles thinner than their wild-type counterparts measurements were taken from centrosome-to-centrosome. In the absence (Fig. 1B and Fig. 3D below). Mutant brains also showed an increase of Centrosomin staining, measurements were taken from pole-to-pole in in the relative frequency of metaphase figures with respect to wild anastral spindles; in the presence of asters, measurements were taken from type, suggesting that dlkb1 mutations lengthen metaphase duration the center of the astral MT array. DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2185 Fig. 1. Mutations in the dlkb1 gene disrupt spindle organization of both NBs and GMCs. (A) Map of the Drosophila lkb1 (dlkb1) gene and its genomic region. P[dlkb1 ] designates the genomic fragment that rescues both the lethality and the cytological defects associated with the dlkb1 mutation. Black boxes correspond to protein-coding exons, and the arrows indicate the direction of transcription. The positions of the stop codons 315 7 causing the dlkb1 and dlkb1 mutations are indicated by vertical lines. (B) Mitotic spindle morphology of NBs and GMCs from wild-type (wt) and dlkb1 brains. Cells were stained for tubulin (Tub, green) and DNA (blue). (C) Spindle morphology of wild-type (wt), dlkb1, asl,and asl dlkb1 metaphases stained for tubulin (green), DNA (blue) and Centrosomin (red). Note that the spindle density in asl dlkb1 double mutants is substantially lower than in asl single mutants. Scale bar (all panels): 5 m. (Table 1). Finally, mutant brains displayed approximately 20% of spindle MTs was substantially reduced with respect to wild type polyploid cells (not shown); in wild-type brains, the frequency of and similar to that observed in dlkb1 single mutants (Fig. 1C). These polyploid cells was virtually zero (Table 1). The phenotype of results strongly suggest that the low density of spindle MTs observed dlkb1 homozygotes was qualitatively and quantitatively similar in dlkb1 mutants does not depend on centrosome dysfunction. Thus, to that observed in dlkb1 /Df(3R)urd hemizygotes, indicating that the spindle phenotype of dlkb1 mutants is likely to be attributable to dlkb1 is a null mutation (Table 1). either a decreased rate of MT growth or an increased MT instability. The spindle phenotypes observed in dlkb1 mutants could be due either to a defect in MT elongation and/or stability, or to a defect in dlkb1 mutations disrupt the asymmetry of NB centrosome function. To distinguish between these possibilities, we division leading to a reduction in NB size sought to eliminate centrosome function in dlkb1 mutants. We have Observation of mitotic divisions stained for tubulin and DNA previously shown that brain cells of asterless (asl) mutants fail to revealed that the spindles of dlkb1 mutant cells are generally smaller assemble functional centrosomes and nucleate astral MTs. than in wild type (Fig. 1). In addition, the frequency of asymmetric Nonetheless, asl NBs and GMCs manage to form robust anastral telophases in mutant brains (35-37%) was significantly lower than spindles that are able to mediate chromosome segregation in wild-type brains (65%) (Table 1). These phenotypes could reflect (Bonaccorsi et al., 2000; Giansanti et al., 2001). We thus constructed a partial loss of morphological asymmetry during NB division, dlkb1 asl double mutants and compared their phenotype with those resulting in smaller than normal daughter NBs. We thus examined exhibited by dlkb1 and asl single mutants. The anastral spindles in greater detail the pattern of cell division in dlkb1 /Df(3R)urd from asl single mutants displayed a MT density comparable to wild brains, and compared this pattern with those observed in wild-type type (Fig. 1C). By contrast, in dlkb1 asl double mutants, the density pins and asl brains. Comparison between dlkb1 and pins mutants DEVELOPMENT 2186 RESEARCH ARTICLE Development 134 (11) Table 1. Mitotic parameters in larval brains from Drosophila lkb1 (dlkb1) mutants † ‡ § Genotype* # of cells Metaphases (%) Anaphases (%) Telophases (%) Sym. telo (%) No asters (%) Polyploid cells (%) Oregon R 1,095 68.0 16.3 15.6 35 49 0.2 315/315 423 83.5 10.9 6.6 68 82 20.2 315/Df 1,120 81.3 13.0 5.7 65 81 22.5 315/7 547 79.7 13.9 6.4 63 78 18.6 315 315 315 315 7 *Oregon R, wild-type stock used as control; 315/315, dlkb1 /dlkb1 ; 315/Df, dlkb1 /Df(3R)urd; 315/7, dlkb1 /dlkb1 . The numbers of cells scored refer only to diploid mitotic figures; polyploid cells were recorded but not used for calculation of the frequencies of different types of mitotic figures. Sym. telo, relative frequencies of symmetric telophases. No asters, diploid cells without asters or with very small asters. was prompted by two previous findings. First, mutations in pins that the NBs from dlkb1 and pins mutants divide more partially suppress the asymmetry of NB divisions, leading to a symmetrically than those of either asl or wild type. Collectively, progressive reduction in NB size (Cai et al., 2003; Parmentier et al., these results indicate that mutations in either dlkb1 or pins 2000). Second, the Drosophila and the human LKB1 kinases partially suppress the asymmetry of NB division, leading to a interact with the orthologous proteins Pins and AGS3 (GPSM1 – reduction in the NB size at each cell division cycle. Human Gene Nomenclature Database), respectively (Blumer et al., To ask whether mutations in dlkb1 and pins affect centrosome 2003). In addition, more detailed comparisons between dlkb1 and size, brain preparations were stained for Centrosomin (Cnn), an asl mutants would allow us to assess more precisely the role of astral integral component of Drosophila centrosomes (Megraw et al., MTs in asymmetric NB divisions. 2001). Observations were restricted only to those cells that, To unambiguously distinguish between NB and GMC spindles, according to our analysis of spindle size distribution (Fig. 3B,C), we immunostained preparations from control and mutant brains for were likely to be NBs (wild-type, dlkb1 and pins metaphases longer both tubulin and the NB marker Deadpan (Dpn) (Bier et al., 1992) than 12 m, and ana-telophases longer than 16 m). This analysis (Fig. 3A below). The analysis of Dpn-positive cells showed that the (Fig. 4A,B) revealed that 88% (n=76) of wild-type NBs display dlkb1 and pins NBs are indeed defective in aster formation. centrosomes of different sizes at their poles. By contrast, only in However, the two mutants displayed different patterns of spindle 34% (n=180) of dlkb1 NBs and 39% (n=100) of pins NBs was the P62 defects. In brains homozygous for the pins null mutation (Yu et centrosome at the apical pole larger than that at the basal pole. These al., 2000), most NB prometaphases and metaphases showed normal results indicate that dlkb1 and pins control asymmetry in centrosome asters but most ana-telophases were characterized by an abnormally size during NB division. small apical aster (Fig. 2A-D). Despite this defect in astral MTs, the density of the spindle MTs in pins NBs was comparable to that observed in their wild-type counterparts (compare Fig. 2A-D with Fig. 1B). By contrast, the spindles of dlkb1 NBs not only showed a reduction in MT density, but also displayed small asters in both metaphase and ana-telophase figures (Fig. 1B and Fig. 2E). In pins and asl mutants, the spindles of Dpn-negative GMCs displayed a normal morphology and were indistinguishable from their wild-type counterparts (data not shown). However, in dlkb1 mutants, GMC spindles were characterized by low MT density just as were those of the NBs (Fig. 1B). Thus, the wild-type function of dlkb1 is required for proper spindle formation in both NBs and GMCs. We next measured the spindle length of metaphase and ana- telophase figures in both NBs (Dpn-positive) and GMCs (Dpn- negative). In dlkb1 and pins mutant brains, the average sizes of NB spindles were substantially smaller than those measured in either asl or wild-type brains. This is mainly due to the absence of large NBs, as both dlkb1 and pins mutants lacked NB metaphases and telophases longer than 19 and 26 m, respectively; these large NBs represented approximately 20% of the NBs found in wild-type or asl mutant brains. By contrast, the average sizes of the GMC spindles observed in dlkb1, asl and pins mutants were very similar and comparable to those of wild-type controls (Fig. 3B,C). An explanation for these results is that in Fig. 2. Mutations in the Drosophila pins gene affect aster both dlkb1 and pins mutants, NBs divide more symmetrically than formation without altering the density of spindle MTs. Cells were in either asl or wild type. To test this possibility, we directly stained for tubulin (green), DNA (blue) and Deadpan (not shown, but evaluated the degree of asymmetry of NB telophases showing see Fig. 3A below) to identify NBs. (A) Prometaphase, (B) metaphase, P62 strong Dpn staining. The asymmetry index was determined using (C) anaphase and (D) telophase from pins mutant brains. the formula a-b/a+b, in which a is the long axis of the spindle and (E) Frequencies of NBs displaying normal asters in wild-type, dlkb1 and b its short axis (Fig. 3D). This analysis (Fig. 3E) clearly shows pins brains. Scale bar: 5 m. DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2187 Fig. 3. Mutations in the Drosophila lkb1 gene disrupt unequal cytokinesis and reduce the average size of NB population within mutant brains. (A) Wild-type and dlkb1 metaphases stained for tubulin (Tub) DNA and Deadpan (Dpn). Note that the NBs are more intensely stained by the anti-Dpn antibody than GMCs. Scale bar: 5 m. (B,C) Size distribution of metaphase (B) and ana-telophase (C) spindles in wild-type, 315 P62 P62 2 2 dlkb1 /Df(3R)urd, pins /pins and asl /asl brains. Dpn-positive (NB) and Dpn-negative (GMC) spindles are depicted in red and green, respectively. Size (m) classes in B: A, 4.7-6.9; B, 7.0-9.2; C, 9.3-11.5; D, 11.6-13.8; E, 13.9-16.1; F, 16.2-18.4; G, 18.5-20.7; H, 20.8-23.0; I, 23.1- 31.7. Size (m) classes in C: J, 6.7-11.2; K, 11.3-15.8; L, 15.9-20.4; M, 20.5-25.0; N, 25.1-29.6; O, over 29.7. The numbers above each column indicate the number of cells observed in each size class. (D) Criterion used for measuring the asymmetry index of NB divisions. The difference between the length of the long (a) and the short (b) spindle axis (a-b) was divided by the total length of the two axes (a+b). Scale bar: 5 m. 315 P62 P62 2 2 (E) Asymmetry indexes in wild-type, dlkb1 /Df(3R)urd, pins /pins and asl /asl NBs of different sizes. The bars correspond to the s.e.m. Size (m) classes: P, 11.3-15.8; Q, 15.9-20.4; R, 20.5-25.0; S, 25.1-29.6; T, over 29.7. dlkb1 mutations affect Mira and Baz/Par-6/DaPKC mutant NBs lacking a Mira crescent displayed a diffuse cytoplasmic but not Pins/Gi localization in dividing NBs localization of Mira. However, although the majority (67%) of pins We examined whether dlkb1 and pins mutations affect the mutant NBs without a Mira crescent had this same pattern, a distribution of Mira in dividing NBs. Larval brain preparations were substantial minority (33%) of these cells showed a diffuse cortical simultaneously stained for both tubulin and Mira and analyzed for distribution of Mira (Fig. 5B). Mira localization (Fig. 5A). We again restricted our analysis to large We next determined whether dlkb1 mutations affect the mitotic figures that are likely to be NBs by the criteria employed localization of Pins and Gi at the apical cortex of dividing NBs above. Both dlkb1 and pins mutant NBs revealed abnormal Mira (NBs were again identified by their size). A regular Pins signal was distribution, but the patterns of Mira mislocalization were different observed in 95% (n=60) of the wild-type NBs and in 84% (n=145) (Fig. 5A,B). In wild type, 93% of NB metaphases and ana- of the dlkb1 NBs (Fig. 6A,B). Gi formed a crescent at the apical telophases displayed a clear Mira crescent at the basal pole, whereas pole of 96% of wild-type NBs (n=61) and 74% of dlkb1 mutant NBs the remaining 7% showed diffuse Mira staining. By contrast, in (n=75) (Fig. 6A,B). Consistent with previous results (Cai et al., dlkb1 and pins mutants the frequencies of NBs with a basal Mira 2003; Schaefer et al., 2001), we observed a Gi apical crescent only crescent were 47% and 26%, respectively. Most (97%) of the dlkb1 in 4% (n=50) of pins mutant NBs (data not shown). Thus, although DEVELOPMENT 2188 RESEARCH ARTICLE Development 134 (11) Fig. 4. Mutations in the Drosophila lkb1 gene affect centrosome size during NB division. (A) Metaphases and telophases from wild- type and dlkb1 /Df(3R)urd mutant brains stained for tubulin (green), DNA (blue) and Centrosomin (red). Note the differently sized and the equally sized centrosomes at the poles of wild-type and dlkb1 mutant cells, respectively. Scale bar: 5 m. (B) Frequency of NBs displaying differently sized centrosomes in wild-type (wt), dlkb1 /Df(3R)urd and P62 P62 pins /pins brains. dlkb1 mutations affect Mira localization at the basal cortex, they have little or no effect on Pins and Gi localization at the apical cortex. We also analyzed Baz, DaPKC and Par-6 localization in both wild-type and dlkb1 mutant NBs. In wild-type larval brains, the Baz signal was rather weak and only 48% (n=63) of the dividing NBs displayed a clear Baz crescent at the apical pole. However, in dlkb1 mutant brains, only five of the 106 NBs scored showed a discernible Baz crescent (Fig. 6A,B). The DaPKC and Par-6 apical crescents were observed in 96% (n=24) and 71% (n=35) of wild-type NBs, respectively, but most dlkb1 NBs did not show apical accumulations of these proteins: DaPKC and Par-6 crescents were detected only in 7% (n=30) and 12% (n=33) of dlkb1 mutant NBs. These results suggest that the wild-type function of dlkb1 is required for the localization of Baz, DaPKC and Par-6 at the NB apical pole. Recent work has suggested that DaPKC delocalization from the apical cortex can result in NB overproliferation (Lee et al., 2006a). Consistent with this idea, the brains from third instar larvae of dlkb1- Fig. 5. Mutations in the Drosophila lkb1 gene affect Mira null mutants exhibit a dramatic hyperplasia of both the hemispheres localization at the NB basal pole. (A) Mira localization in wild-type, P62 P62 315 315 pins /pins and dlkb1 /dlkb1 NBs. Cells in metaphase and and the ventral ganglion; this phenotype has been attributed to a telophase were stained for tubulin (Tub), DNA and Mira. Scale bar: reduction in developmental apoptosis during embryogenesis (Lee et 5 m. (B) Distribution of Mira in dividing NBs from wild-type (wt), al., 2006b). We observed a clear brain overgrowth in all our dlkb1 315 315 P62 P62 dlkb1 /dlkb1 and pins /pins brains. Regular, regular Mira mutant alleles, confirming that Dlkb1 regulates Drosophila brain crescent at the basal pole; cortical, Mira associated with the entire cell size (data not shown). It is likely that the brain hyperplasia elicited cortex; diffuse, Mira dispersed in the cytoplasm. by dlkb1 mutations results from both defective apoptosis and DaPKC-related NB overproliferation. Dlkb1 is not required for NB spindle rotation Recent work has shown that that spindle rotation is regulated by We examined 128 metaphases of dlkb1 mutant NBs stained for Mud (Mushroom body defect), a protein related to vertebrate NuMA Mira; 53 of them displayed a Mira crescent, but only in one case was (also known as Numa1) that interacts with both Pins and the spindle this crescent incorrectly oriented with respect to the spindle axis MTs. In embryonic NBs, Mud forms an apical crescent and (Fig. 5B). By contrast, this crescent was misoriented with respect to accumulates at the spindle poles; in larval NBs, the cortical the spindle axis in nine of the 29 pins NB metaphases with a Mira localization of Mud is weak or undetectable but the protein remains crescent (Fig. 5B). These results confirm that Pins is required for enriched at the spindle poles (Bowman et al., 2006; Izumi et al., proper spindle rotation during NB division and indicate that the 2006; Siller et al., 2006). Immunostaining for Mud revealed that the Dlkb1 kinase is not involved in this process. protein is enriched at the centrosomes and the astral MTs in 93% DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2189 (n=45) of prophase and early prometaphase NBs (Fig. 7A). With progression through mitosis, Mud localization became more diffuse and 62% (n=45) of NB metaphase figures did not exhibit clear Mud accumulations at the spindle poles (Fig. 7A); however, Mud relocalized at the pericentrosomal regions of most anaphases and telophases (83%, n=30; data not shown). In dlkb1 mutant NBs, Mud accumulated at the centrosomes/asters in 91% (n=35) of prophase and early prometaphase NBs (Fig. 7C), and remained associated with the spindle poles in 78% (n=37) of the metaphases (Fig. 7D) and 85% (n=20) of the ana-telophases (not shown). Thus, mutations in dlkb1 do not affect Mud localization during metaphase and ana- telophase, but appear to increase Mud concentration at the spindle poles during metaphase. Subcellular localization of Dlkb1 To determine the subcellular localization of Dlkb1, we raised a guinea pig antibody against the entirety of Dlkb1. Western blot analysis showed that this antibody recognizes a band of the expected size (~63 kDa) in larval, embryonic and S2 cell extracts. This band 315 315 7 was absent from both dlkb1 /Df(3R)urd and dlkb1 /dlkb1 larvae (Fig. 8A), demonstrating that it corresponds with Dlkb1. Since the 315 7 truncated forms of Dlkb1 encoded by the dlkb1 and dlkb1 mutant alleles were not observed in mutant animals, either the mutant transcripts or the truncated proteins are unstable. These findings provide strong support for the genetic data (Table 1), indicating that the dlkb1 mutant allele is functionally null. Immunolocalization experiments revealed that Dlkb1 is dispersed in both the nucleus and the cytoplasm of interphase larval brain cells, and in the cytoplasm of both NBs and GMCs undergoing mitotic division. Immunostaining of dlkb1 /Df(3R)urd mutant cells did not reveal any clear cytoplasmic signal, confirming the specificity of the antibody (Fig. 8B and data not shown). The diffuse localization of Dlkb1 in brain cells is not consistent with its cortical localization in Drosophila oocytes (Martin and St Johnston, 2003). However, the Dlkb1 localization pattern in brain cells does not reflect the quality of our antibody, as the same antibody revealed a cortical accumulation of Dlkb1 in oocytes (data not shown). Dlkb1 and Pins function in different pathways controlling NB division The finding that Dlkb1 and Pins co-precipitate (Blumer et al., 2003), and that dlkb1 and pins mutations cause similar (but not identical) phenotypes, prompted us to perform an epistasis analysis. We thus 315 P62 compared the phenotype of the dlkb1 pins double mutant with those of the single mutants by examining brain preparations stained for tubulin, Dpn and DNA. In dlkb1 pins mutant brains, the spindles of both NBs and GMCs were much more defective than those observed in either of the single mutants (compare Fig. 9 with Figs 1 and 2). In addition to cells with severely defective spindles (Fig. 9A,C,F,G,I), we also observed many (50%, n=300) mitotic figures in which the spindle morphology was barely recognizable (Fig. 9B,D,E,H); the frequency of the latter type of cells was only 4% (n=201) in the dlkb1 single mutant. In dlkb1 pins metaphases, the MT density was extremely low, the spindle poles had a characteristic Fig. 6. Mutations in the Drosophila lkb1 gene disrupt Baz/Par- pointed appearance and the asters were completely absent (Fig. 9A). 6/DaPKC but not Pins/Gi localization at the NB apical pole. The ana-telophases were also devoid of asters and displayed few and (A) Pins, Gi, Baz, Par-6 and DaPKC localization in wild-type and sparse central spindle MTs, which were never pinched in the middle 315 315 dlkb1 /dlkb1 mutants. NBs in the first row were simultaneously (Fig. 9C,F). These results indicate that dlkb1 and pins function in stained for Pins, Centrosomin (Cnn) and DNA (blue). Cells shown in the parallel pathways to control spindle formation. subsequent rows were stained for tubulin (Tub), DNA (blue) and either The absence of central spindle pinching, which suggests an Gi, Baz, Par-6 or DaPKC. Scale bar: 5 m. (B) Frequencies of NBs with accompanying failure of cytokinesis, prevented a reliable Pins, Gi, Baz, Par-6 and DaPKC crescents in wild-type and 315 315 dlkb1 /dlkb1 mutant brains. assessment of the degree of asymmetry of NB divisions. However, DEVELOPMENT 2190 RESEARCH ARTICLE Development 134 (11) Fig. 8. Expression and intracellular localization of the Dlkb1 kinase. (A) Western blot showing that the anti-Dlkb1 antibody recognizes a band of approximately 63 kDa. This band is absent in 315 315 7 extracts from either dlkb1 /Df(3R)urd or dlkb1 /dlkb1 mutant Drosophila larvae. -Tubulin was used as a loading control (LC). (B) Immunostaining for Dlkb1 in metaphase and telophase of wild-type dividing NBs. Note that Dlkb1 is diffuse in the cytoplasm. Scale bar: 5 m. and spindle formation. Whether the latter pathways are the same as those that control the asymmetry of NB mitosis remains to be determined. Fig. 7. Mud localization in wild-type and dlkb1 mutant Drosophila NBs. Cells were stained for Mud, tubulin (Tub) and DNA (blue). (A,B) Wild-type prophase (A) and metaphase (B). DISCUSSION 315 315 (C,D) dlkb1 /dlkb1 prophase (C) and metaphase (D). Scale bar: Dlkb1 controls the stability of spindle MTs 5 m. Our results indicate that mutations in the dlkb1 gene disrupt spindle formation in both NBs and GMCs. In addition, the finding that the NB spindles of the double mutant were smaller than in wild type imaginal discs of dlkb1 mutant larvae are small and misshapen (Fig. 9J). In addition, the analysis of centrosome size in large suggests a defect in imaginal cell mitosis. Previous studies have metaphase figures (longer than 14 m), most of which are likely to shown that late larval lethality and small imaginal discs are be NBs, revealed that 90% (n=106) of them had equally sized diagnostic of abnormalities in mitotic divisions (Gatti and Baker, centrosomes. In wild type, dlkb1 and pins, the frequencies of NB 1989). Thus, the dlkb1 phenotype strongly suggests that the Dlkb1 metaphases with centrosomes of equal size were 12%, 72% and kinase plays an important mitotic role not only in NBs, but also in 61%, respectively (Fig. 4B). Finally, only 3% (n=120) of the dlkb1 other somatic cell types. Despite the low density of spindle MTs, pins NB metaphases were characterized by a Mira crescent; in the most mutant metaphases enter anaphase (the frequency of anaphases remaining cells, Mira was either diffuse in the cytoplasm (85%) or in dlkb1 mutants and in wild-type controls was 10-13% and 16%, associated with the entire cell cortex (12%). Thus, the Mira respectively; see Table 1), suggesting that in a substantial fraction of mislocalization phenotype observed in dlkb1 pins double mutants is mutant cells the spindle checkpoint is either not induced or only stronger than that seen in the single mutants (see Fig. 5B). transiently activated. However, the defects in NB spindles are likely 315 P62 Although the dlkb1 and pins alleles are both functionally to lead to the formation of polyploid cells. These cells could arise 315 P62 null, it cannot be excluded that the brains of the dlkb1 pins through two different mechanisms. Cells blocked in metaphase double mutants retain residual amounts of the maternally supplied owing to either reduced MT density or activation of the spindle Dlkb1 and Pins proteins. We thus performed a western blotting checkpoint could revert to interphase and become polyploid after an 315 P62 analysis of extracts from third instar larval brains of dlkb1 , pins additional round of DNA replication. Alternatively, cells that enter 315 P62 315 and dlkb1 pins mutants. As shown in Fig. 9K, dlkb1 and anaphase but assemble an abnormally thin central spindle might be 315 P62 dlkb1 pins brains did not exhibit detectable amounts of the unable to undergo cytokinesis and in consequence produce Dlkb1 kinase, consistent with the results shown in Fig. 8A. polyploid cells (Vernì et al., 2004). Similarly, the Pins protein appeared to be completely absent from The precise function of Dlkb1 in spindle formation and/or P62 315 P62 pins and dlkb1 pins brains. Thus, the phenotypes observed maintenance is currently unclear. However, the finding that the in the single and double mutants reflect a complete loss of the wild- spindles of dlkb1 asl double mutants display a lower MT density type function of either Dlkb1 or Pins, or both. than asl single mutants argues for a defect in MT stability and not in Collectively, our results suggest that dlkb1 and pins act in centrosome function. Studies in mammalian cells have shown that different pathways to control the asymmetry of NB division. These LKB1 is a master kinase that phosphorylates at least 14 kinases, all genes also function in parallel pathways involved in MT stability of which are related to AMP-activated kinases (AMPK). Kinases of DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2191 Fig. 9. dlkb1 and pins function in different pathways controlling the stability of spindle MTs in Drosophila. Mitotic figures 315 P62 from brains of dlkb1 pins double-mutants were stained for tubulin (Tub, green), DNA (blue) and Centrosomin (red). (A-F) NBs; (G-I) GMCs. (A,B) Metaphases; (C-E) anaphases; (F) telophase; (G) metaphases; (H) anaphase; (I) telophase. The arrow in E points to a lagging X chromosome with unseparated sister chromatids. Note the extremely defective spindle structures of the NBs shown in B,D,E,H. Scale bar: 5 m. (J) Size distribution of metaphase spindles in wild-type and dlkb1 pins brains. Dpn-positive (NB) and Dpn-negative (GMC) spindles are depicted in red and green, respectively. Size (m) classes: A, 4.7-6.9; B, 7.0-9.2; C, 9.3-11.5; D, 11.6-13.8; E, 13.9- 16.1; F, 16.2-18.4; G, 18.5-20.7. (K) Expression of Pins and Dlkb1 in brains from third instar larvae of dlkb1, pins and dlkb1 pins mutants. Note that the Dlkb1 protein is undetectable in larval brain extracts of both dlkb1 315 P62 homozygotes and dlkb1 pins double mutants. Similarly, Pins cannot be detected in P62 P62 brain extracts of both pins /pins mutants 315 P62 and dlkb1 pins double mutants. The Giotto protein (Giansanti et al., 2006) was used as a loading control (LC). the AMPK family include regulators of cellular energy levels, as discs, suggesting an underlying mitotic defect. Given that the 315 P62 well as four Microtubule affinity regulating kinases (MARKs) dlkb1 and pins alleles used in the analysis are both functionally (reviewed by Alessi et al., 2006; Baas et al., 2004b). The MARK null and that the corresponding proteins were undetectable in mutant enzymes are the mammalian homologs of C. elegans and brains (Yu et al., 2000) (this study), these results indicate that Pins Drosophila Par-1. However, Drosophila Par-1, which controls MT and Dlkb1 function in different pathways for the control of MT stability in oocytes (Shulman et al., 2000), appears to act upstream stability. The observation that the spindles of pins mutants display a of Dlkb1 (Martin and St Johnston, 2003). It is therefore unlikely that normal MT density further suggests that Pins plays a redundant role the Dlkb1 substrate required for the stability of spindle MTs is Par- in the maintenance of MT stability. A role for Pins in spindle 1. Further evidence that Dlkb1 does not act via Par-1 formation and/or stability has never been demonstrated in phosphorylation comes from RNAi experiments showing that Dlkb1 Drosophila. However, the mammalian homolog of Pins binds depletion, but not Par-1 depletion, causes defects in spindle NuMA and regulates mitotic spindle organization and positioning morphology (Bettencourt-Dias et al., 2004). Thus, current data (Du et al., 2001). indicate that the Dlkb1 kinase regulates the activity of an unknown factor required for the stability of the spindle MTs; this factor could Dlkb1 controls the asymmetry of NB division either be a direct substrate of Dlkb1 or a substrate for one of the We have analyzed the phenotypic consequences of dlkb1 mutations kinases acting downstream of Dlkb1. in larval brain NBs. In contrast to embryonic NBs that display Our cytological analyses have shown that the spindles of dlkb1 small, regularly sized spindles (their metaphase spindles are pins double mutants display a MT density that is substantially lower approximately 5 m long), brain NBs exhibit spindles of very than that observed in either single mutant. Here again, highly different sizes (ranging from 5 to 32 m for metaphase spindles). defective spindles were observed in both NBs and GMCs. In Nonetheless, dividing brain NBs exhibit the same asymmetries as addition, double-mutant larvae showed extremely reduced imaginal their embryonic counterparts, including asymmetries in aster and DEVELOPMENT 2192 RESEARCH ARTICLE Development 134 (11) centrosome size, localization of specialized protein complexes and in the Baz/Par-6 pathway, we cannot exclude the possibility that this positioning of the cleavage furrow (Bowman et al., 2006; Giansanti kinase functions in both the Baz/Par-6 and Pins/Gi pathways, or in et al., 2001; Lee et al., 2006a; Parmentier et al., 2000; Rolls et al., a third pathway different from either. 2003; Siller et al., 2006) (this study). However, the degree of In this context, it is important to note that our results exclude the asymmetry of brain NB division is directly related to the cell size, possibility that dlkb1 acts via Pins phosphorylation. Previous studies so that large NBs divide more asymmetrically than small NBs (Fig. have shown that mammalian LKB1 co-precipitates and 3E). This is likely to render large brain NBs particularly sensitive phosphorylates AGS3, the mammalian ortholog of Pins (Blumer et to mutations that affect cleavage furrow positioning. Consistent al., 2003). Dlkb1 and Pins coimmunoprecipitate as well, but it is with this hypothesis, mutations in pins have mild effects on the currently unclear whether Pins is phosphorylated by Dlkb1 (Blumer asymmetry of embryonic NB divisions (Cai et al., 2003), but et al., 2003). Regardless of whether Pins is a substrate of Dlkb1, the disrupt unequal cytokinesis in most larval brain NBs (Parmentier phenotypes elicited by dlkb1 mutations cannot be the consequence et al., 2000) (Fig. 3E). dlkb1 larval NBs also divide more of an impairment of Pins function. dlkb1 and pins mutant NBs do in symmetrically than their wild-type counterparts, leading to larval fact differ in a number of phenotypic traits, including spindle brains devoid of large NBs. In addition, most dlkb1 NBs display organization and the pattern of Mira localization, and do not belong centrosomes of equal size and very small asters at both poles. to the same epistasis group. However, the symmetric cytokinesis of dlkb1 NBs cannot result from their short astral MTs, as asl NBs divide asymmetrically in the Dlkb1 is not required for NB spindle rotation complete absence of asters (Fig. 3E). In vivo imaging has shown that the spindles of embryonic NBs dlkb1 mutant NBs are also characterized by the abnormal rotate during metaphase to become aligned with the center of the distribution of several components of the apical and basal Pins apical crescent (Kaltschmidt et al., 2000). By contrast, the complexes. In dlkb1 mutant brains, most NBs display normal Pins spindles of larval NBs align with the Pins crescent at prophase (Siller and Gi crescents at their apical pole but fail to accumulate Baz, et al., 2006). Failure of proper rotation of larval NB spindles results DaPKC and Par-6 at the same pole. In addition, most dlkb1 in spindles that are misoriented with respect to the apical (Pins) and mutant NBs fail to exhibit a normal Mira crescent at the basal pole basal (Mira) crescents (Giansanti et al., 2001; Siller et al., 2006). cortex. A normal localization of Pins and Gi has been observed There is also evidence that proper positioning of larval NB spindles in most embryonic NBs defective in the Baz/Par-6 pathway (Cai depends on astral MTs, because in approximately 50% of asl NB et al., 2003; Izumi et al., 2004; Schaefer et al., 2000; Yu et al., metaphases the Mira crescent is misoriented with respect to the 2000; Yu et al., 2003). Moreover, studies on embryonic NBs have spindle axis (Giansanti et al., 2001). suggested that Baz, Par-6 and DaPKC function as a complex, are Our results indicate that spindle rotation occurs normally in dlkb1 interdependent for their localization at the NB apical pole, and are mutant NBs. In addition, we have shown that prophase/ required for the formation of the Mira crescent at the basal pole prometaphase larval NBs of dlkb1 mutants normally accumulate the (Petronczki and Knoblich, 2000; Wodarz et al., 2000). However, Mud protein, which mediates proper spindle alignment in both subsequent work on second instar larval NBs has shown that these embryonic and larval NBs (Bowman et al., 2006; Izumi et al., 2006; proteins are not mutually dependent for the formation of the Siller et al., 2006). Together, these results indicate that the Dlkb1 Baz/Par-6/DaPKC apical crescent; they accumulate at the apical kinase is not required for spindle rotation and that the short astral cortex in a hierarchical fashion, with Baz and Par-6 mediating MTs of dlkb1 mutant NBs can mediate proper spindle positioning. proper DaPKC localization (Rolls et al., 2003). Mutations that These results are consistent with the idea that the Pins/Gi, but not disrupt the Pins/Gi pathway prevent asymmetrical localization the Baz/Par-6, pathway is involved in spindle rotation (Izumi et al., of either Pins or Gi in embryonic NBs but do not substantially 2004; Siegrist and Doe, 2005) and provide further support for the affect Mira accumulation at the basal pole (Cai et al., 2003). hypothesis that Dlkb1 functions in the latter pathway. However, it should be noted that mutations in pins partially Recent work has shown that in the absence of the Baz/Par-6 disrupt asymmetric Mira localization in larval brain NBs pathway, astral MTs can mediate the localization of Pins/Gi at the (Parmentier et al., 2000) (this study), suggesting that larval NBs apical cortex (Siegrist and Doe, 2005). Assuming that Dlkb1 acts in differ from embryonic NBs in some aspects of the control of Mira the Baz/Par-6 pathway, the finding that this kinase is not required for localization. Thus, taking into account the differences between the formation of Pins/Gi crescents indicates that the short astral embryonic and larval NBs, our results indicate that mutations in MTs of dlkb1 NBs retain the ability to mediate Pins/Gi cortical the dlkb1 gene and those that disrupt the Baz/Par-6 pathway affect localization. similar aspects of NB mitotic division. Our analyses have shown that in dlkb1 pins double mutants, the Conclusions and perspectives NBs divide more symmetrically than in the corresponding single Our results indicate that Dlkb1 and Pins function in partially mutants. This indicates that the dlkb1 and pins genes act in different redundant pathways controlling the stability of spindle MTs. These pathways that mediate unequal cytokinesis. Previous studies have proteins are also required for the asymmetry of NB divisions and, shown that the asymmetry of NB cytokinesis depends on the here again, they appear to function in different pathways. Pins acts Baz/Par-6 and Pins/Gi redundant pathways. When only one of in a common pathway with Gi, whereas Dlkb1 is likely to function these pathways is impaired, NBs still divide asymmetrically, but in the Baz/Par-6 pathway. Intriguingly, recent work has shown that they divide symmetrically when both are disrupted (Cai et al., 2003). simultaneous loss of pins and baz functions results in the formation The simplest interpretation of our findings is that dlkb1 acts in the of abnormally small embryonic NB spindles that lack astral MTs at Baz/Par-6 pathway. In addition, the observation that Dlkb1 is both poles (Fuse et al., 2003). Thus, the embryonic NBs of baz pins required for proper localization of Baz, Par-6 and DaPKC suggest double mutants have a spindle phenotype reminiscent of that that this kinase acts at the top of the hierarchical mechanism that observed in dlkb1 larval NBs. These findings raise the question of mediates accumulation of the Baz/Par-6 complex at the apical whether the Pins/Gi and Baz/Par-6 pathways redundantly control cortex. However, although we favor the hypothesis that Dlkb1 acts spindle organization as they do for the asymmetry of NB divisions. DEVELOPMENT The role of Drosophila Lkb1 RESEARCH ARTICLE 2193 The extant data do not provide a clear answer to this question. The Giansanti, M. G., Bonaccorsi, S., Kurek, R., Farkas, R. M., Dimitri, P., Fuller, M. T. and Gatti, M. (2006). The class I PITP Giotto is required for Drosophila analysis of the roles of the two pathways in spindle formation and cytokinesis. Curr. Biol. 16, 195-201. their precise relationships with the Dlkb1 kinase are interesting Goodman, C. S. and Doe, C. Q. (1993). Embryonic development of the issues to be addressed in future studies. Drosophila central nervous system. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez Arias), pp. 1131-1206. New York: Previous studies in Drosophila and mammalian cells have led to Cold Spring Harbor Laboratory Press. the suggestion that loss of epithelial cell polarity is ultimately Izumi, Y., Ohta, N., Itoh-Furuya, A., Fuse, N. and Matsuzaki, F. (2004). responsible for the Peutz-Jeghers cancer syndrome (Martin and St Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J. Cell Biol. 164, 729-738. Johnston, 2003; Baas et al., 2004b). Here, we have shown that Dlkb1 Izumi, Y., Ohta, N., Hisata, K., Raabe, T. and Matsuzaki, F. (2006). Drosophila plays an essential mitotic role and is required for the asymmetry of Pins-binding protein Mud regulates spindle-polarity coupling and centrosome NB division. These results lead us to propose that tumor organization. Nat. Cell Biol. 8, 586-593. development in Peutz-Jeghers patients depends on the impairment Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. and Brand, A. H. (2000). Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in of multiple processes, including cell polarity, the asymmetry of stem the developing central nervous system. Nat. Cell Biol. 2, 7-12. cell division and the fidelity of chromosome segregation during Koundakjian, E. J., Cowan, D. M., Hardy, R. W. and Becker, A. H. (2004). The mitosis. Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster. Genetics 167, 203-206. We thank W. Chia, Y. Jan, T. Kaufman, J. Knoblich and F. Matsuzaki for Lee, C.-Y., Robinson, K. J. and Doe, C. Q. (2006a). Lgl, Pins, and aPKC regulate antibodies and fly stocks. This work was supported by grants from Centro di neuroblast self-renewal versus differentiation. Nature 439, 594-598. Lee, J. H., Koh, H., Park, J., Lee, S. Y., Lee, S. and Chung, J. (2006b). JNK Eccellenza di Biologia e Medicina Molecolare (BEMM) to M.G. and by NIH pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in grant GM48430 to M.L.G. Drosophila. Cell Death Differ. 13, 1110-1122. References Martin, S. G. and St Johnston, D. (2003). A role for Drosophila LKB1 in anterior- Alessi, D. R., Sakamoto, K. and Bayascas, J. R. (2006). LKB1-dependent posterior axis formation and epithelial polarity. Nature 421, 379-384. signaling pathways. Annu. Rev. Biochem. 75, 137-163. Megraw, T. L., Kao, L. R. and Kaufman, T. C. (2001). Zygotic development Baas, A. F., Kuipers, J., van der Wel, N. N., Batlle, E., Koerten, H. K., Peters, P. without functional mitotic centrosomes. Curr. Biol. 11, 116-120. J. and Clevers, H. C. (2004a). Complete polarization of single intestinal Parmentier, M. L., Woods, D., Greig, S., Phan, P. G., Radovic, A., Bryant, P. epithelial cells upon activation of LKB1 by STRAD. Cell 116, 457-466. and O’Kane, C. J. (2000). Rapsynoid/partner of inscuteable controls asymmetric Baas, A. F., Smit, L. and Clevers, H. (2004b). LKB1 tumor suppressor protein: division of larval neuroblasts in Drosophila. J. Neurosci. 20, RC84. PARtaker in cell polarity. Trends Cell Biol. 14, 312-319. Petronczki, M. and Knoblich, J. A. (2000). DmPar-6 directs epithelial polarity Betschinger, J. and Knoblich, J. A. (2004). Dare to be different: asymmetric cell and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43- division in Drosophila, C. elegans and vertebrates. Curr. Biol. 14, R674-R685. 49. Bettencourt-Dias, M., Giet, R., Sinka, R., Mazumdar, A., Lock, W. G., Balloux, Rolls, M. M., Albertson, R., Shih, H.-P., Lee, C.-Y. and Doe, C. Q. (2003). F., Zafiropoulos, P. J., Yamaguchi, S., Winter, S., Carthew, R. W. et al. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and (2004). Genome-wide survey of protein kinases required for cell cycle epithelia. J. Cell Biol. 163, 1089-1098. progression. Nature 432, 980-987. Schaefer, M., Shevchenko, A., Shevchenko, A. and Knoblich, J. A. (2000). A Bier, E., Vaessin, H., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (1992). protein complex containing Inscuteable and the Ga-binding protein Pins orients deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix asymmetric cell divisions in Drosophila. Curr. Biol. 10, 353-362. protein similar to the hairy gene product. Genes Dev. 6, 2137-2151. Schaefer, M., Petronczki, M., Corner, D., Forte, M. and Knoblich, J. A. (2001). Blumer, J. B., Bernard, M. L., Peterson, Y. K., Nezu, J., Chung, P., Dunican, D. Heterotrimeric G proteins direct two modes of asymmetric cell division in the J., Knoblich, J. A. and Lanier, S. M. (2003). Interaction of activator of G- Drosophila nervous system. Cell 107, 183-194. protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell Shulman, J. M., Benton, R. and St Johnston, D. (2000). The Drosophila polarity and cell cycle progression: phosphorylation of the G-protein regulatory homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with oskar mRNA localization to the posterior pole. Cell 101, 377-388. Galphai. J. Biol. Chem. 278, 23217-23220. Siegrist, S. E. and Doe, C. Q. (2005). Microtubule-induced Pins/Galphai cortical Bonaccorsi, S., Giansanti, M. G. and Gatti, M. (1998). Spindle self-organization polarity in Drosophila neuroblasts. Cell 123, 1323-1335. and cytokinesis during male meiosis in asterless mutants of Drosophila Siller, K. H., Cabernard, C. and Doe, C. Q. (2006). The NuMA-related Mud melanogaster. J. Cell Biol. 142, 751-761. protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Bonaccorsi, S., Giansanti, M. G. and Gatti, M. (2000). Spindle assembly in Nat. Cell Biol. 8, 594-600. Drosophila neuroblasts and ganglion mother cells. Nat. Cell Biol. 2, 54-56. Vernì, F., Somma, M. P., Gunsalus, K. C., Bonaccorsi, S., Belloni, G., Bowman, S. K., Neumuller, R. A., Novatchkova, M., Du, Q. and Knoblich, J. Goldberg, M. L. and Gatti, M. (2004). Feo, the Drosophila homolog of PRC1, A. (2006). The Drosophila NuMA Homolog Mud regulates spindle orientation in is required for central-spindle formation and cytokinesis. Curr. Biol. 14, 1569- asymmetric cell division. Dev. Cell 10, 731-742. 1575. Cai, Y., Yu, F., Lin, S., Chia, W. and Yang, X. (2003). Apical complex genes Watts, J. L., Morton, D. G., Bestman, J. and Kemphues, K. J. (2000). The C. control mitotic spindle geometry and relative size of daughter cells in Drosophila elegans par-4 gene encodes a putative serine-threonine kinase required for neuroblast and pI asymmetric divisions. Cell 112, 51-62. establishing embryonic asymmetry. Development 127, 1467-1475. Du, Q., Stukenberg, P. T. and Macara, I. G. (2001). A mammalian Partner of Wodarz, A. (2005). Molecular control of cell polarity and asymmetric cell division Inscuteable binds NuMa and regulates mitotic spindle organization. Nat. Cell in Drosophila neuroblasts. Curr. Opin. Cell Biol. 17, 1-7. Biol. 3, 1069-1075. Wodarz, A., Ramrath, A., Grimm, A. and Knust, E. (2000). Drosophila Atypical Fuse, N., Hisata, K., Katzen, A. L. and Matsuzaki, F. (2003). Heterotrimeric G Protein Kinase C associates with Bazooka and controls polarity of epithelia and proteins regulate daughter cell size asymmetry in Drosophila neuroblast neuroblasts. J. Cell Biol. 150, 1361-1374. divisions. Curr. Biol. 13, 947-954. Yu, F., Morin, X., Cai, Y., Yang, X. and Chia, W. (2000). Analysis of partner of Gatti, M. and Baker, B. S. (1989). Genes controlling essential cell-cycle functions inscuteable, a novel player of Drosophila asymmetric divisions, reveals two in Drosophila melanogaster. Genes Dev. 3, 438-453. distinct steps in inscuteable apical localization. Cell 100, 399-409. Giansanti, M. G., Gatti, M. and Bonaccorsi, S. (2001). The role of centrosomes Yu, F., Cai, Y., Kaushik, R., Yang, X. and Chia, W. (2003). Distinct roles of Gi and astral microtubules during asymmetric division of Drosophila neuroblasts. and G13F subunits of the heterotrimeric G protein complex in the mediation of Development 128, 1137-1145. Drosophila neuroblast asymmetric divisions. J. Cell Biol. 162, 623-633. DEVELOPMENT

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Development – The Company of Biologists

Published: Jun 1, 2007

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