A genetic screen for new Incenp alleles and interactors

To obtain new alleles or interactors of Incenp, we carried out a mutagenesis screen using a previously identified allele of Incenp (incenp^P(EP)2340) (Rorth, 1996). incenp^p(EP)2340 is a P-element insertion into the third exon of the Drosophila Incenp gene (Fig. 1A). The presence and location of the P-element was confirmed by plasmid rescue and sequencing of the neighbouring genomic region (Fig. 1A). incenp^P(EP)2340 is a homozygous lethal mutation and was described originally as a female sterile dominant (Rorth, 1996). The chromosome carrying the insertion was recombined over a wild-type chromosome to get rid of possible second-site mutations. The P-element insertion is lethal over a chromosomal deletion uncovering the Incenp gene (defined by six overlapping chromosomal deficiencies of the region - see Materials and Methods for further details). incenp^P(EP)2340 heterozygous individuals were shown to produce a smaller transcript of around 600 bp, which is not present in the wild-type control (Fig. 1B). This is predicted to potentially produce a 91 aa N-terminal peptide, however, we could not detect any truncated peptide in protein extracts from homozygous embryos by western blot (Fig. 1C).

Male flies carrying the incenp^P(EP)2340 insertion show a dominant meiotic phenotype that results in low fertility. The main defects observed include misshapen meiotic spindles, tetrapolar or multipolar spindles and defects in onion stage spermatids consistent with problems both in chromosome disjunction and cytokinesis (Fig. 1D). A detailed analysis of the role of Drosophila Incenp in meiosis is the subject of a separate study (T. Resnick, D. L. Satinover, F.M., T. Stukenberg, W.E., T.O.-W. and M.C., unpublished data). Heterozygous individuals also show defects in larval mitosis consistent with those shown by S2 cells depleted of Drosophila Incenp by double-stranded RNA interference (dsRNAi) (Adams et al., 2001c). Additionally, heterozygous adults show mild external defects consistent with abnormalities during imaginal mitosis, including slightly roughened eyes, nicked wings and defects in the abdominal terguites (data not shown). The Incenp locus is not haplo-insufficient: individuals heterozygous for a deficiency uncovering the Incenp gene do not show any of the phenotypes described above (M.C., unpublished data).

The precise excision of the P-element, using an exogenous source of transposase, reverted the lethality and the dominant phenotypes, demonstrating that the insertion is responsible for both effects. Precise excision was confirmed by sequencing the genomic region of the excised chromosome. Twenty-one imprecise excisions obtained were lethal and also showed dominant meiotic phenotypes. We isolated genomic DNA from six of these lines and devised a PCR strategy to characterize them at the molecular level (Fig. 1A). All the lines analysed (Δ8, Δ9, Δ14, Δ17, Δ18 and Δ24) were internal deletions in the P-element, and are predicted to produce the same truncated peptide as the original insertion.

To date, no mutations in any of the genes coding for passenger complex proteins have been described in Drosophila. Therefore, to isolate new recessive alleles of Incenp and also possible new interactors, we performed a second-site non-complementor screen (see Halsell et al., 2000; Hays et al., 1989; White-Cooper et al., 1996) using ethyl methanesulfonate (EMS) as mutagen. We chose EMS because it is more likely to induce point mutations that can disrupt specific protein-protein interactions. The Incenp gene is located on the second chromosome (43A04), so to simplify the screen, we only isolated mutations in the second chromosome. We sought to isolate and analyze mutations that - when in trans to incenp^P(EP)2340 - were lethal, male sterile or showed an enhancement of the dominant visible phenotype.

A total of 5000 individual mutagenised chromosomes were generated and analyzed. This screen yielded 16 homozygous lethal mutations corresponding to 13 complementation groups. Most of the mutants were isolated on the basis of lethality in trans with incenp^P(EP)2340, except for five (EC282, EC2388, EC2394, EC2519, EC3959) that showed defects in the eyes, bristles or wings (Fig. 2A-D). Two of the mutants had a dominant female sterile phenotype (EC3322, EC4330). When analyzed carefully on a larger scale, some of the trans-heterozygote combinations turned out to be semi-lethal or viable (Table 1). The same results were obtained when we analyzed trans-heterozygote combinations with imprecise excisions of P(EP)2340 (Δ9, Δ24). However, when in trans with a precise excision of P(EP)2340 (Δ11) the EC mutations show no phenotype.

Whenever the lethal phase allowed the analysis, we studied the phenotype in male meiosis of the trans-heterozygous combinations (EMS mutant/P(EP)2340). Some combinations showed defects in primary spermatocytes (Fig. 2E,F) as well as in the postmeiotic onion-stage spermatids (Fig. 2H,I). This is consistent with disruption of a function required for both mitosis and meiosis. In other cases, i.e. EC666/P(EP)2340, the defects were found exclusively in the meiotic divisions, which raises the possibility of identifying meiotic specific functions of the chromosomal passenger proteins.

We next used deficiency and, in some cases, meiotic-recombination mapping to further characterize the new mutations. For the deficiency mapping we used a second chromosome deficiency kit (a set of overlapping deletions of chromosome 2). Four of the mutants, corresponding to two complementation groups (EC1650/EC2388 and EC2364/EC2374) are semi-lethal over a chromosomal deficiency for the Drosophila Aurora B kinase gene [Df(2L)1469]. In addition, two of these mutations (EC1650/EC2388) do not complement a chromosomal deficiency for the Drosophila borealin gene - Df(2L)2892.

These experiments showed that the chromosomes typically contained more than a single EMS hit. Further analysis of the mutants requires the separation of the different mutations by recombination. As yet, the molecular identity of only one of the new mutants has been determined (see below). A detailed description of the new mutants and their phenotypes will be the subject of a future study.

Identification of a new allele of Incenp

One of the newly generated mutants (EC3747) did not complement Df(2R)pk78k (42E03-43C03), a chromosomal deficiency uncovering the Incenp gene. EC3747 was therefore selected for further characterization as a putative new allele of Incenp. Genetic recombination with a multiply marked chromosome was used to minimize the presence of other spurious EMS mutations on the chromosome containing the EC3747 mutation (Materials and Methods). To further refine the mapping of EC3747 we used six strains carrying chromosomal deficiencies with breakpoints in the Incenp gene region (Materials and Methods). EC3747 was mapped to region 43A04, the same location as the Incenp gene. In addition, EC3747 failed to complement any of the imprecise excisions generated by the mobilization of the P-element. Therefore, all of the available genetic evidence suggested that EC3747 is a new allele of Incenp.

Molecular characterization confirmed that EC3747 was indeed a new allele of Incenp. To identify the mutation in EC3747, we extracted genomic DNA from single EC3747-homozygous embryos, used PCR to amplify the entire coding region of the Incenp gene, and then sequenced the PCR products directly. Genomic DNA from four single embryos was prepared as template to perform four separate PCR reactions. Comparison of the sequences revealed a point mutation in exon 4 of the Incenp gene. This single base change created a stop codon in position 457 of the open reading frame (Fig. 1A). The predicted molecular weight of the putative truncated gene product is 61 kDa.

Although the sequence analysis of EC3747 predicted that the product of this mutated gene would be a truncated version of Drosophila Incenp, when we analyzed EC3747 homozygous embryos by immunoblotting we failed to detect a product of the predicted size (Fig. 1C). The expected full-length protein with molecular weight of 110 kDa was detected in wild type, but was absent from embryos homozygous for Df(2R)pk78k, EC3747 and incenp^P(EP)2340. This suggests that the truncated mutant protein is unstable. The antibody used for immunoblots, Rb801, was raised against the N-terminal 348 amino acids of Drosophila Incenp (Adams et al., 2001c), which is wholly contained within the 457 aa polypeptide encoded by EC3747.

The new recessive null allele of Incenp shows a genetic interaction with several of the mutants generated in our screen: incenp^EC3747 is semi-lethal over EC666 and EC2519, and male sterile over EC549, EC666, and EC1608. This confirms the genetic interaction between these as-yet-unidentified genes and Incenp, and excludes the notion that the interaction is specific to the dominant P-element insertion.

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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.

incenp^EC3747 embryos show defects in late embryonic development

Analysis of the lethal phase of homozygous incenp^EC3747 individuals showed that they died late in embryonic development. The rare first larval instar escapers show reduced mobility and abnormal wandering behavior (data not shown) consistent with defects in the nervous system. To investigate this further, late-stage embryos were stained for the marker Mab22C10 that stains neurons and their processes throughout the nervous system (Zipursky et al., 1984). Homozygous mutants were distinguished from heterozygous embryos using the CyOKrüppel-GFP balancer marker. Focusing on the peripheral nervous system (PNS), we observed that the heterozygous animals had a wild-type neural pattern (Fig. 3A). However, homozygous mutant embryos exhibited a range of defects, from severe cases of neuronal loss to perturbation of the neural clusters and missing subsets of neurons (Fig. 3B-D).

To determine the time in which the first defects in mitosis arose in these individuals, we fixed embryos at different stages and stained with anti-Drosophila Incenp and anti-tubulin antibodies. During the first 13 rapid synchronous cycles we did not observe any difference between wild-type and incenp^EC3747 embryos. Since Incenp has been proposed to be required for completion of cytokinesis, we then paid special attention to the process of blastodermal cellularization. This process, which occurs in mitotic cycle 14 when membranes grow inward between the blastoderm nuclei, in some ways resembles the process of cytokinesis. incenp^EC3747 embryos showed no defect in cellularization and Incenp could still be observed to be localized properly. This reflects perdurance of the wild-type maternal product. We could not observe significant defects in early-stage embryos, although from mitotic cycle 15, we did observe a very low percentage of cells with a reduced amount of Drosophila Incenp and chromosome segregation defects (data not shown).

By embryonic stage 13 (Ashburner et al., 2005; Campos-Ortega and Hartenstein, 1985) incenp^EC3747 Drosophila Incenp was no longer detectable by immunostaining in incenp^EC3747 embryos (Fig. 4B). Consistent with this, phosphorylation of histone H3 on Ser10 (a known Aurora B kinase substrate) was also no longer detected (compare Fig. 4A” with B”). This phenotype confirms that Incenp is required for Aurora B kinase to function as a histone H3 Ser10 kinase in an developing multicellular organism. At this stage, cells of the central nervous system (CNS) in incenp^EC3747 embryos showed enlarged nuclei (Fig. 5B) compared with wild-type (Fig. 5A) as observed by DAPI staining for DNA. Staining with anti-γ-tubulin antibody showed that these enlarged cells contain bigger than normal centrosomes (Fig. 5D') or multiple centrosomes (Fig. 5E'). This phenotype, detectable in mutant embryos following the complete disappearance of the Drosophila Incenp, can be explained as the result of failure of cytokinesis in the previous division and is consistent with a lack of Aurora B kinase function (Adams et al., 2001c; Oegema et al., 2001) in the early development of the CNS.

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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.

Localization of the cell-fate determinant Prospero is abnormal in incenp^EC3747 neuroblast divisions

Asymmetric cell division is key to the development of the Drosophila nervous system (for a review, see Bardin et al., 2004). Each dividing neuroblast produces one large daughter cell that remains a multipotent neuroblast and continues to divide, and a smaller daughter cell that becomes a ganglion mother cell that divides once more asymmetrically to produce neurons or glia cells. This cell-fate decision hinges on the segregation of Prospero, a homeodomain transcription factor that is segregated largely, if not exclusively, into the ganglion mother cell. This is accomplished by sequestering Prospero into a basal cortical crescent in the dividing neuroblast from prophase onwards (Spana and Doe, 1995).

In wild-type embryos, we observed the expected asymmetric distribution of Prospero at the basal cell surface of dividing cells (Fig. 6C). However, we were surprised to notice that, in early prophase, Prospero transiently associates with the condensing chromatin on entry into mitosis (Fig. 6A,B). The distribution of Prospero was abnormal in neuroblasts lacking detectable Incenp. Abnormalities observed in neuroblasts of incenp^EC3747 embryos included defects in the shape and orientation of the basal Prospero crescent (Fig. 6D). We also observed mitotic neuroblasts with Prospero distributed all around the cell cortex, and not restricted to a basal crescent (Fig. 6D).

These results reveal that Drosophila Incenp and, therefore, presumably the chromosomal passenger complex, is required for the correct localization of Prospero during asymmetric cell division in the developing Drosophila nervous system.

EC3747 is a recessive allele of Incenp

One of the new mutants, EC3747, contains a point mutation that generates a stop codon just before the coiled-coil region of Drosophila Incenp. The predicted molecular mass of the 456 aa protein product truncated at the C-terminal is approximately 61 kDa. Immunoblotting analysis of extracts from homozygous mutant embryos failed to detect any truncated Incenp polypeptide. This is probably due to instability of the polypeptide. Unlike incenp^P(EP)2340 heterozygous individuals, EC3747 heterozygous individuals do not show any defects in mitosis or meiosis (data not shown), suggesting that EC3747 is a recessive null allele of Incenp. Incenp is an essential gene in Drosophila, and individuals homozygous for EC3747 died late during embryogenesis. As discussed below, one probable factor contributing to this embryonic lethality was abnormal development of the nervous system.

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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.

Incenp is required for phosphorylation of histone H3 and cytokinesis in the embryonic CNS divisions

A maternal stockpile of Drosophila Incenp protein allowed incenp^EC3747 homozygotes to survive to late embryogenesis, therefore enabling us to study for the first time the role of this protein in development. During our phenotypic analysis of incenp^EC3747, we observed that maternal Incenp protein became generally undetectable at stage 13 of embryonic development. The only cells dividing at this stage are those of the nervous system, where we observed abnormally large mitotic cells without any phosphorylated histone H3. This is the first demonstration in a developing organism that Incenp is essential for the activity of Aurora B kinase.

Mutant neuroblasts were polyploid, and had multiple centrosomes, a phenotype that can result as a consequence of failure in chromosome segregation and cytokinesis. However, a recent study has shown that the new chromosomal passenger borealin, which is found in a complex with Aurora B kinase, Incenp and survivin, is required for maintenance of bipolar spindle in mitosis (Gassmann et al., 2004). In addition, we previously observed an elevated frequency of monopolar and multipolar spindles in S2-phase cells following dsRNAi knockdown of Drosophila Incenp (M.C., unpublished observations). Therefore it is possible that the centrosomal abnormalities observed in stage 13 embryos reflect a requirement for Incenp in maintenance of bipolar spindle integrity.

Incenp is required for asymmetric distribution of cell-fate determinants in the embryonic CNS

Our results reveal that Incenp is required for the asymmetric distribution of Prospero. This is likely to be a consequence of the involvement of Aurora B kinase in the regulation of asymmetric cell division rather than simply a consequence of defects in mitosis and cytokinesis. Importantly, mutations in the gene encoding pebble, a Rho GTP exchange factor that is essential for cytokinesis (Hime and Saint, 1992; Lehner, 1995), do not disrupt the polarized localization of cell-fate determinants (Barros et al., 2003). Furthermore, the prometaphase delay resulting from the loss of Incenp-Aurora B kinase complex function, does probably not in itself alter Prospero localization because colchicine treatment does not have an effect on this process (Knoblich et al., 1995; Spana and Doe, 1995). It has been reported that cortical Prospero is highly phosphorylated and that this modification could be relevant for its localization (Srinivasan et al., 1998). This raises the possibility that Prospero is an Aurora B kinase substrate. Our observation of the partial colocalisation of Prospero with Incenp on chromatin in mitosis would agree with this.

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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.

An alternative role for the passenger complex in asymmetric cell division could be through regulation of myosin. At late prophase of neuroblast asymmetric mitosis, localization of Prospero and Numb in the basal crescent is coordinated with the orientation of the mitotic spindle and depends on two protein subcomplexes acting together with the actin cytoskeleton. Recently, it has been shown that myosin motors are involved in localization of fate determinants in different ways. The myosin VI jaguar (Jar) is required for localization of Miranda (Petritsch et al., 2003) and myosin II - restricted to the apical cortex by lethal giant larva (Lgl) - is necessary for apical exclusion of fate determinants (Barros et al., 2003). Myosin II regulatory light-chain has been described as a substrate of Aurora B kinase (Murata-Hori et al., 2000). Thus a potential role of the chromosomal passenger complex is to regulate myosin-motor activity essential for localization of cell-fate determinants.

The Drosophila Incenp mutant incenp^EC3747 has allowed us to study the role of passenger proteins in development for the first time. This has revealed a previously unsuspected requirement for Incenp in the regulation of the asymmetric distribution of cell-fate determinants during CNS development in Drosophila embryos. Thus, the chromosomal passenger complex appears to have an essential role not only in regulating basic functions of normal mitosis, but also in regulating the more elaborate process of asymmetric cell division, which is essential for metazoan development.

Drosophila strains, mutagenesis and mapping methods, and embryo collection

Fly stocks were maintained at 18°C or room temperature on standard cornmeal-agar medium. The original P(EP)2340 strain was generated in a misexpression screen (Rorth, 1996). A w¹¹¹⁸ strain isogenic for chromosome 2 was used for the mutagenesis screen. Ethyl methanesulfonate (EMS) mutagenesis was perfomed according to standard protocols (see Grigliatti, 1998). The new mutants obtained from the screen were named EC (Edinburgh Chang) followed by the number of the mutagenised chromosome. Flies carrying mutagenised chromosomes were crossed with a `deficiency kit' for chromosome 2 (overlapping deficiencies uncovering most of the chromosome). Multiple EMS hits were separated by meiotic recombination over a multiple-marked chromosome 2 (al dp pr b cn cu sp).

We refined the mapping of EC3747 using the following deficiencies: Df(2R) Drl1 (42E01-43C03), Df(2R)cn-S3 (43B01/02-43B07/09), Df(2R) pk78s (43F08-59F05/08), Df(2R) nap19 (41E02/F01-43A02), Df(2R) p32a (43A03-43F06) and Df(2R) ST1 (41B03/05-43E15/18). All the stocks used for meiotic recombination and deficiency mapping of the new mutants were obtained from the Bloomington Drosophila Stock Center. Embryo staging, collection and fixation was performed according to standard protocols (see Rothwell and Sullivan, 2000).

To select homozygous embryos we used the second chromosome balancer CyOKr-GFP. Homozygous embryos were selected by hand, using a dissecting-microscope with a fluorescence illuminator that enabled visualization of the GFP signal.

Extraction of genomic DNA

Genomic DNA from adult flies was obtained using the Berkeley Drosophila Genome Project protocol (http://www.fruitfly.org/p_disrupt/inverse_pcr). Genomic DNA from single embryos for sequencing was obtained as follows: embryos of the desired genotype were collected in 0.5 ml tubes (1 per tube) and homogenized in 10 μl of Gloor-Engel's extraction buffer (10 mM Tris pH 8.2, 1 mM EDTA, 25 mM NaCl, 200 μg/ml proteinase K) by using a pipette tip. The homogenate was incubated at 37°C for 30 minutes, moved to 95°C for 2 minutes, then stored at 4°C.

Northern blotting

Total RNA from w¹¹¹⁸, incenp³⁷⁴⁷ and incenp^P(EP)2340 individuals was isolated using the RNAeasy kit (Qiagen), and 25-30 μg of RNA were loaded per lane. A 400 bp SalI restriction fragment from Incenp cDNA, expanding over the P-element insertion site was radioactively labelled to use as probe.

Antibodies

Antibodies used for immunostaining and western blotting were as follows: rabbit polyclonal anti-Drosophila Incenp (Rb801) (Adams et al., 2001c); anti-phosphorylated histone H3 Ser10 (rabbit polyclonal, used 1:200; Upstate Biotechnology; mouse monoclonal, used 1:200; NEB); mouse monoclonal anti-α-tubulin B-512 (used 1:2000, Sigma); mouse monoclonal anti-γ-tubulin GTU-88 (1:50, Sigma); mouse monoclonal anti-Prospero MR1A (1:5, developed by Chris Doe, obtained from Developmental Studies Hybridoma Bank); mouse monoclonal 22C10 (1:200, gift of Andrew Jarman, University of Edinburgh, UK); rabbit polyclonal anti-GFP (1:1000, Molecular Probes). All fluorescently-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (anti-rabbit conjugated-Texas Red, anti-rabbit conjugated-FITC, anti-mouse conjugated-Texas Red, anti-rabbit conjugated-FITC, all used 1:200).

Preparation of protein extracts from homozygous embryos and western blotting

Embryos (selected as described previously) were placed in a fresh 0.5 ml microcentrifuge tube; 50 μl of 1× SDS-PAGE sample buffer were added and the embryos were homogenized for 5 seconds three times with a sonicator. After homogenization, the mixture was placed at 100°C for 5 minutes. The heated sample was spun in a table-top centrifuge at 10,000 g for 3 minutes at 4°C. The supernatant was transferrred to a fresh tube and the sample was ready for loading on an SDS-PAGE gel. Samples were run on 10% SDS-PAGE gel and corresponding blots were probed with rabbit polyclonal anti-Incenp (Rb801-1) (Adams et al., 2001c) and monoclonal anti-α-tubulin antibody B-512 (Sigma).

Immunostaining of embryos

Fixation of Drosophila embryos for immunofluorescence and immunohistochemistry was performed in 4% paraformaldehyde for 30 minutes. Devitellinisation and staining was done according to standard protocols (Rothwell and Sullivan, 2000). For immunohistochemistry, after the incubation with secondary antibody and subsequent washes, embryos were stained using diaminobencidine (Vectastain DAB peroxidase substrate kit, Vector Laboratories) and then mounted in Vectashield (Vector).

Microscopy

Imaging was performed using Olympus IX-70 microscope controlled by Delta Vision SoftWorx (Applied Precision, Isswqua, WA, USA). Differential interference microscopy (DIC) was used to collect images on an Olympus Provis AX70 microscope for immunohistochemical samples. Images were deconvolved, projected and saved as tiff files to be processed with Adobe Photoshop.