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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02848

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

¹ Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Genetica e Biologia Molecolare, Università di Roma "La Sapienza", P.le A. Moro 5, 00185 Roma, Italy.
² Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA.

View larger version (91K): [in this window] [in a new window]   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 causing the dlkb1315 and dlkb17 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 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Mutations in the Drosophila pins gene affect aster formation without altering the density of spindle MTs. Cells were stained for tubulin (green), DNA (blue) and Deadpan (not shown, but see Fig. 3A below) to identify NBs. (A) Prometaphase, (B) metaphase, (C) anaphase and (D) telophase from pinsP62 mutant brains. (E) Frequencies of NBs displaying normal asters in wild-type, dlkb1 and pins brains. Scale bar: 5 µm.

View larger version (61K): [in this window] [in a new window]   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 µm. (B,C) Size distribution of metaphase (B) and ana-telophase (C) spindles in wild-type, dlkb1315/Df(3R)urd, pinsP62/pinsP62 and asl2/asl2 brains. Dpn-positive (NB) and Dpn-negative (GMC) spindles are depicted in red and green, respectively. Size (µm) 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 (µm) 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 µm. (E) Asymmetry indexes in wild-type, dlkb1315/Df(3R)urd, pinsP62/pinsP62 and asl2/asl2 NBs of different sizes. The bars correspond to the s.e.m. Size (µm) 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.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Mutations in the Drosophila lkb1 gene affect centrosome size during NB division. (A) Metaphases and telophases from wild-type and dlkb1315/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 µm. (B) Frequency of NBs displaying differently sized centrosomes in wild-type (wt), dlkb1315/Df(3R)urd and pinsP62/pinsP62 brains.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Mutations in the Drosophila lkb1 gene affect Mira localization at the NB basal pole. (A) Mira localization in wild-type, pinsP62/pinsP62 and dlkb1315/dlkb1315 NBs. Cells in metaphase and telophase were stained for tubulin (Tub), DNA and Mira. Scale bar: 5 µm. (B) Distribution of Mira in dividing NBs from wild-type (wt), dlkb1315/dlkb1315 and pinsP62/pinsP62 brains. Regular, regular Mira crescent at the basal pole; cortical, Mira associated with the entire cell cortex; diffuse, Mira dispersed in the cytoplasm.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Mutations in the Drosophila lkb1 gene disrupt Baz/Par-6/DaPKC but not Pins/Gi localization at the NB apical pole. (A) Pins, Gi, Baz, Par-6 and DaPKC localization in wild-type and dlkb1315/dlkb1315 mutants. NBs in the first row were simultaneously stained for Pins, Centrosomin (Cnn) and DNA (blue). Cells shown in the subsequent rows were stained for tubulin (Tub), DNA (blue) and either Gi, Baz, Par-6 or DaPKC. Scale bar: 5 µm. (B) Frequencies of NBs with Pins, Gi, Baz, Par-6 and DaPKC crescents in wild-type and dlkb1315/dlkb1315 mutant brains.

View larger version (80K): [in this window] [in a new window]   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). (C,D) dlkb1315/dlkb1315 prophase (C) and metaphase (D). Scale bar: 5 µm.

View larger version (60K): [in this window] [in a new window]   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 extracts from either dlkb1315/Df(3R)urd or dlkb1315/dlkb17 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 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 9. dlkb1 and pins function in different pathways controlling the stability of spindle MTs in Drosophila. Mitotic figures from brains of dlkb1315 pinsP62 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 µm. (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 (µm) 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 dlkb1315 homozygotes and dlkb1315 pinsP62 double mutants. Similarly, Pins cannot be detected in brain extracts of both pinsP62/pinsP62 mutants and dlkb1315 pinsP62 double mutants. The Giotto protein (Giansanti et al., 2006) was used as a loading control (LC).