Drosophila Incenp is required for cytokinesis and asymmetric cell division during development of the nervous system

Abstract

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, we have performed a genetic screen looking for new alleles and interactors of Drosophila Incenp. We have isolated a new null allele of Incenp that has allowed us for the first time to study the functions of the chromosomal passengers during development. Homozygous incenp(EC3747) embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process.

Fig. 1

incenp^P(EP)2340 individuals show defects in chromosome segregation and cytokinesis. (A) Diagram of the Incenp genomic region showing insertion site of the P-element into the third exon of the Incenp gene in incenp^P(EP)2340. Black lines on either side of the P-element represent the genomic DNA fragments cloned by plasmid rescue and sequenced. Red boxes indicate the position of primers used for the PCR characterization of imprecise excision lines. Diagram of the Incenp transcript below shows the position of the point mutation (C to T) in incenp^EC3747. Below, diagrams of the predicted products of both Incenp mutants. (B) Northern blot, showing total RNA from wild-type and both Incenp mutants. Arrow points to truncated transcript present in heterozygous incenp^P(EP)2340/+ individuals. (C) Western blot analysis of wild-type embryos (WT), mutant embryos carrying a deficiency for the Incenp gene (DF), incenp³⁷⁴⁷ homozygous embryos (3747) and incenp^P(EP)2340 homozygous embryos [3747, [P(EP)]. Arrow points to wild-type Incenp protein. Lower panel shows α-tubulin loading control. (D) Phenotype of incenp^P(EP)2340/+ in male meiosis. (Left) Wild-type onion-stage cyst; arrow points to individual spermatid with equal-size nucleus and mitochondrial derivative (Nebenkern). (Right) Same stage in incenp^P(EP)2340/+ males; notice large Nebenkerns indicative of defective cytokinesis (arrowhead), as well as variable-size nuclei indicative of problems in chromosome segregation (arrow).

Fig. 2

Phenotypic analysis of the new genetic interactors of incenp^P(EP)2340. (A-B) Normal eye and wing morphology in control (w¹¹¹⁸) strain. (C-D) Representative examples of eye and wing phenotypes observed in trans-heterozygous combinations of EMS-induced mutations over incenp^P(EP)2340: (C) Reduced eyes in trans-heterozygous EC3959/incenp^P(EP)2340. (D) Irregular nicked wing-shape in trans-heterozygous EC2519/incenpP^(EP)2340 fly. (E,F) Defects in the primary spermatocytes in trans-heterozygous males as a result of abnormalities in the gonial mitoses. (E) Variation in the nuclear size in EC2519/incenpP^(EP)2340 is an indication of defective chromokinesis. Arrows point to micronuclei, arrowhead to polyploid nucleus. (F) The presence of giant primary spermatocytes (arrows) in EC2394/incenpP^(EP)2340 could be indicative of a defect in cytokinesis in gonial mitosis. (G) Wild-type primary spermatocyte cysts. (H,I) Abnormal (H) meiotic spindles and (I) tetrapolar spindles (arrows) in EC2394/incenpP^(EP)2340. (J) Wild-type telophase I spindles. Dotted lines in H-J delimit the meiotic cyst. Bar, 10 μm.

Fig. 3

Peripheral neuron system morphology incenp^EC3747 embryos. (A-D) Examples of 20-hour embryos stained for the neural marker Mab22C10. Small blue arrows indicate the position of the lateral cluster of chordotonal neurons. (A) incenp^EC3747/CyO KrGFP embryo. The wild-type PNS pattern is visible. (B-D) Homozygous incenp^EC3747 embryos. (B and C) Small blue arrows show examples of neural loss from the lateral cluster. (D) Green arrows show a lack of organization of neuron clusters and reduced number of neurons. In these images anterior is left, dorsal is top. The views in C and D are more ventral than those of A and B.

Fig. 4

Drosophila Incenp becomes undetectable at stage 13 of embryonic development in incenp^EC3747. Ventral view of stage-13 embryos. (A-A”) Wild-type embryos, showing mitotic cells staining positive for (A’) Drosophila Incenp and (A”) phosphorylated histone H3. (B-B”) Homozygous incenp^EC3747 embryos at the same stage show no (B’) Drosophila Incenp or (B”) phosphorylated histone H3. Insets in A and B show the DNA staining (DAPI). Notice that the embryos have developed normally up to this stage. In the merged figures, DAPI is shown in blue, phosphorylated histone H3 in red, Drosophila Incenp in green.

Fig. 5

incenp^EC3747enlarged CNS cells lack Drosophila Incenp and phosphorylated histone H3. (A-A’’’) Mitosis in a wild-type CNS cell showing Drosophila Incenp localized to the centromere at prometaphase. Histone H3 is phosphorylated in the mitotic chromosomes. (B-B’’’) Enlarged cells in the CNS of Incenp^EC3747 embryos do not show any Drosophila Incenp phosphorylated histone H3 staining. In the merged figures, DAPI is shown in blue, phosphorylated histone H3 in red, Drosophila Incenp in green. Enlarged CNS cells in incenp^EC3747 embryos are polyploid and show multiple centrosomes. (C-C’’’) Wild-type cell with Drosophila Incenp localized to centromeres at metaphase (arrowhead) and in the midzone at telophase (arrow). (D-E’’’) Enlarged CNS cells are polyploid and show either and abnormal accumulation of γ-tubulin in centrosomes or multiple centrosomes. The two centrosomal masses in D-D’’’ have not separated properly in this cell. Multiple γ-tubulin spots in E-E’’’. In the merged figures, DAPI is shown in blue, γ-tubulin in red, Drosophila Incenp in green. Bar, 10 μm.

Fig. 6

Localization of the cell-fate determinant Prospero in mitosis of wild-type and incenp^EC3747 embryos. (A-C) Localization of Prospero in mitosis of wild-type CNS cells. (A) In early prophase, Drosophila Incenp shows general association with chromatin, whereas Prospero localizes in discrete spots associated with chromatin. (B) By early prometaphase, both proteins become closer although they do not colocalise totally. (C) In metaphase, Drosophila Incenp remains associated with centromeres, whereas Prospero is localized in a crescent in the basal cell cortex. (D-D’) In incenp^EC3747 homozygous embryos, we observed enlarged polyploidy cells depleted of Drosophila Incenp in which Prospero localization is abnormal. All images are projections of series of deconvolved images. Bars, 5 μm.