Lines w*; P{EP}drk^EP2477/CyO, Df(2R)CX1 wg¹² b¹ pr¹/SM1 and w^67c23 P{w^+MC = lacW}tlk^{G0113a, b, c} were obtained from the Szeged and Bloomington Stock Center. Tan and coworkers has reported that w*; P{EP}drk^EP2477/CyO is a hypomorph of mars, designated as mars^P 30. A drk hypomorph, drk^R1/CyO 31, y¹ w¹ sn³, P{w^+MC = UAST-tlk}#0 (abbreviated as UAST-tlk) and w tlk^Δ14/FM7 17 were generous gifts from Drs. E. Hafen, H.Y. Sun and F. Karch.

Starting with w^67c23 P{w^+MC = lacW}tlk^G0113a in which the other two P-element inserts were segregated by meiotic recombination, a duplication line, w^67c23 tlk²⁷, with another copy of P{w^+MC = lacW} inserted at the 5'end of Rala, was generated. Using P-element imprecise excision, a deficiency, w^67c23 tlk^27-9, with a deletion between the 5'end of Rala and 4^th exon of tlk-RB, was obtained. Transgenic fly lines carrying a P{w^+MC = UASP-tlk} transgene (abbreviated as UASP-tlk) were generated by inserting the tlk coding region in the GH07910 EST clone into pUASP 32 and then the resulting plasmid DNA was transformed into flies using P-element mediated germ-line transformation 3334. To express tlk in the germ line, females of a selected UASP-tlk line were crossed with GAL4-GCN4 males 35. The resulting females carrying both transgenes were crossed with UAST-tlk males for the collection of embryos, which are described here as GCN4>tlk embryos.

The w*; P{EP}drk^EP2477/CyO strain obtained from the stock center was homozygous lethal. To eliminate the possible existence of a second-site mutation, meiotic recombination was used 36. After five rounds of meiotic recombination, some newly generated lines were homozygous viable and P-element insertion was confirmed by PCR.

To determine the level of Mars protein in syncytial blastoderm embryos, a collection of metaphase embryos as described by Su (2000) was made and this collection was used to perform immunoblotting. In brief, embryos from 0 to 1 hour were collected and aged for 1.5 hour. The embryos were dechorionated in bleach, fixed in methanol for 5 minutes and stained with 40 pg/ml of Hoechst 33342 (Sigma/Aldrich, Inc) in 1× PBS. Syncytial blastoderm embryos at metaphase were picked out under an inverted fluorescence microscope (Leica Model DM Illinois, USA) 37. Protein pools from 30 embryos were separated using 8% SDS polyacrylamide gels. The proteins in the SDS gels were transferred onto PVDF membrane and Mars was detected using anti-Mars antibody (1:5000; generously provided by Dr. S.-S. Fan 38). After incubation with the secondary antibody, a chemilluminescent assay kit (Western lighting™, Blossom Biotechnologies, Inc, Taiwan) was used to detect the protein 39.

Embryos were fixed in 37% formaldehyde at room temperature (RT) for 5 min. Eye-antennal discs of late third instar larvae were dissected in 1× PBS and then transferred into 1× PBS containing 4% paraformaldehyde for 30 min. The embryos or eye-antennal discs were washed with 1× PBST (0.3% Triton X-100 in 1× PBS) and incubated with a blocking solution (1% BSA in 1× PBST) prior to incubation with various primary antibodies, anti-phosphohistone H3 (Upstate) (1:200 dilution), α-Tub (Sigma/Aldrich, Inc) (1:200 dilution), γ-Tub (Sigma/Aldrich, Inc) (1:200 dilution), or Mars (1:400 dilution), at either 4°C overnight or at RT for 2 hours. Localization of the proteins was detected by incubating with secondary antibodies conjugated with either Cy3 or FITC (Jakson ImmunoResearch Lab) (1:200 dilution) at 4°C overnight or at RT for 2 hours. The contours of the photoreceptor clusters in the eye discs were stained with Phalloidin-Tetramethylrhodamin B isothiocyanate (Phalloidin-TRITC) (Invitrogen Molecular Probe) (1:80 dilution) for 1 hour. Embryos were incubated with 4 ng/ml of Hoechst 33342 (Sigma/Aldrich, Inc) to stain their chromosomes. The embryos or eye-antennal discs were mounted in a mounting medium (20 mM Tris-HCl pH 8.8, 50% glycerol and 4% n-propyl gallate) and viewed under a Leica confocal microscope (Model TCS-SP2) 3940.

To determine the density of the mitotic spindles at metaphase, anaphase or telophase, as represented by the fluorescence intensity of the mitotic spindle, the green channel of the selected images was converted into grayscale and the gray value surrounding the mitotic spindle was adjusted to around 80% using Photoshop. For each mitotic phase, the intensity of one 10 by 10 pixel square of one spindle from each nucleus was measured using Image J http://rsb.info.nih.gov/ij. Six nuclei in twelve embryos at nuclear cycles 10 or 11 (as shown in Fig. 2D) were randomly chosen for the measurements, which were used to obtain an average intensity. The average value was then normalized against the background, which was the average value from ten 10 by 10 pixel squares outside of and surrounding the mitotic spindle. This produced values in arbitrary units (au) of fluorescence intensity. The length of the mitotic spindle from centrosome to centrosome of the same nuclei was also measured. The statistical significance of differences in the density and the length of the assessed mitotic spindles were determined using the Student's t-test.

Immunostaining was performed to reveal the localization of endogenous Mars protein. Throughout the syncytial blastoderm stage, a low level of punctate staining was observed, indicating that a low level of Mars was uniformly present in the cytoplasm of the embryos (data not shown). In addition to this low level and uniform distribution of Mars protein, at the onset of mitosis, when the centrosome is divided into two, a high level of Mars was detected in the nucleus (Fig. 3). During metaphase and anaphase, Mars was predominantly localized on the mitotic spindles, a cellular structure essential for faithful distribution of chromosomes into the two daughter cells 4142, but not on the centrosomes or the astral microtubules. At telophase, a high level of Mars colocalized with the de-condensing chromosomes. The subcellular localizations of Mars at M phase are consistent with other reports 3043, indicating that Mars functions on the mitotic spindles.

Located at 50A13-14, mars and downstream receptor kinase (drk) are transcribed in opposite directions and overlap by 36 bp. The insertion of P{EP} at 23 bp upstream of the putative start codon of mars in line mars^P/CyO results in a hypomorphic mutation of mars 30. Since the line obtained from the stock center is homozygous lethal, second-site lethal mutations were segregated out by meiotic recombination. The hatching rate of embryos from parents homozygous for mars^P (abbreviated as mars embryos hereafter) was 67.0%, while those of embryos from females homozygous for w¹¹¹⁸ and heterozygous for drk^R1 and mars^P were 91.7 or 96.4%, respectively (Table 1). The hatching rate was slightly higher than that reported by Tan et al. (49%, 30). Furthermore, the viability of the mars embryos was temperature-dependent and the hatching rate was further reduced to 39.2% at 28.5°C. These results supported the fact that mars^P is a hypomorph and indicated that lethality is due to a shortage of both maternal and zygotic mars activity.

An organism that has a mixture of male and female characteristics is called as gynandromorph. Before sex is determined in a female embryo (X/X), loss of one sex chromosome during mitosis results in X/O cells that eventually lead to a male phenotype at the adult stage 36. From mars^P parents, we found gynandromorphs formed approximately 0.05% of the progeny. Mars plays an important role to stabilize spindle microtubules, and its mutations thus result in the formation of abnormal spindle microtubules 3038. Similarly, it has been shown that nonclaret disjunctional loss-of-function causes defects in the mitotic spindle, which also results in gynandromorph 44. Therefore, based on the above results, we examined morphology of mitotic spindles in mars embryos.

Yang and Fan have shown that two bands are detected in extracts from Drosophila S2 cells by immunoblotting with anti-Mars antibody and that the upper band consists of Mars that is phosphorylated 38. To reveal whether both forms of Mars protein are differentially reduced in mars embryos, Mars protein in metaphase embryos was determined by immunoblotting. Using extracts from Drosophila embryos, reproducibly, two bands located close together and with equal intensity were able to be detected in this study (Fig. 4A); this differs from other studies, where a single band has been found 3043. In mars embryos, the amount of the unphosphorylated Mars, the lower band, was less than half of that in w¹¹¹⁸ embryos and the phosphorylated Mars was barely detectable (lane mars^P in Fig. 4A). These results plus the association of phosphorylated Mars with taxol-stabilized microtubules 38 indicated that the phosphorylated Mars, but not the unphosphorylated protein, is associated with mitotic spindles. The drastic reduction in phosphorylated Mars on the mitotic spindles might cause gynandromorphs to appear in progeny from parents homozygous for mars^P.

Mitotic spindles in mars embryos from 1 to 2.5 hours at 28.5°C were examined using immunostaining with anti-α-Tub, anti-γ-Tub and anti-phospho-histone H3 antibodies. In metaphase mars embryos, the length and the density of mitotic spindles (7.50 ± 0.17 μm and 28.53 ± 3.31 au) were significantly shorter and less, respectively, than those in w¹¹¹⁸ embryos (11.98 ± 0.16 μm and 53.71 ± 7.03 au) (compare Fig. 4B with 4E; p < 0.001 and 0.05, respectively). At anaphase and at telophase, neither the length nor the density of mitotic spindles was substantially affected in the mars embryos (Figs 4C, D, F and 4G). These results indicated that the polymerization of the mitotic spindles in mars embryos is delayed, but not entirely prevented.

Consistent with the fact that polymerization of mitotic spindles is affected in mars embryos, progression of mitotic events in patches of nuclei, each of which contained at least two nuclei, was obviously delayed comparing to those in adjacent nuclei (Figs 2C and 2D) in 13% of mars embryos (n = 32). A possible explanation for these results is that the mitotic spindles lacking phosphorylated Mars pull the chromosomes toward the spindle poles inefficiently, which results in both asynchronous chromosome segregation (Fig. 2D) and embryos pausing at anaphase (Table 2).

Without a proper genetic marker, identification of gynandromorphs is difficult if the X/O cells do not locate to the posterior end of adult females. Therefore, mars^P males that also carry yellow (y) and singed (sn) markers on the X chromosome were used to measure the gynandromorphy rate. Interestingly, the frequencies of non-disjunction, namely the ratios of y^- sn^- males to total males, in the male progeny from w¹¹¹⁸; mars^P females crossed with y¹w¹sn³; mars^P at 24°C and 28.5°C were 0.6% and 1.1%, respectively (Table 3), which are much higher than the gynandromorphy rate in the mars parent. Hereafter, we measured the non-disjunction rate instead of gynandromorphy because it was a simpler procedure.

To test whether mars interacts with tlk genetically, dosage-dependent interaction experiments were performed with two tlk alleles, Δ14 and 27-9. The hatching rate of embryos from tlk^27-9/+; mars^P females was 48%, a further reduction from the 67% for mars embryos (Table 1), indicating that mars genetically interacts with tlk.

The frequencies of non-disjunction in male progenies from tlk^27-9/+; mars^P and tlk^Δ14/+; mars^P females at 28.5°C were 6.1% and 4.4%; these rates were much higher than those from mars^P (1.1%) or tlk^Δ14/+ females (0.4%) (Table 3). This increase in frequency of non-disjunction was also temperature dependent, since there was no substantial increase in progeny from tlk^27-9/+; mars^P and tlk^Δ14/+; mars^P females (1.2% and 0.5%, respectively) at 24°C when compared to mars^P or tlk^Δ14/+ females. These results indicated that mars genetically interacts with tlk and that the interaction is involved in ensuring accurate chromosome segregation.

To reveal whether mitosis is severely affected in embryos with reductions in both mars and tlk activity, embryos from tlk^Δ14/+; mars^P females crossed to mars^P males (abbreviated as tlk/mars embryos) at 28.5°C; these embryos were then immunostained using anti-α-Tub, anti-γ-Tub and anti-phospho-histone H3 antibodies. A series of images along the z axis were stacked to observe the distribution of chromosomes and mitotic spindles. In embryos from females heterozygous for tlk^Δ14, the chromosomes before entering anaphase were aligned at the spindle midzone as indicated by a bracket in Fig. 2B and the morphology of the mitotic spindles appeared normal (tlk^Δ14/+; compare Fig. 2A with 4B). In almost all tlk/mars embryos at either metaphase or anaphase (n = 70), however, the asynchrony was so severe that it was impossible to distinguish what phase an embryo belonged to. In addition to the asynchrony that was observed in mars embryos (Fig. 2D), asynchronous chromosome segregation was observed in embryos where most of the nuclei were likely at anaphase (Fig. 2E). Despite the severe asynchrony during chromosome congression or segregation, the morphology of the mitotic spindles in tlk/mars embryos was not significantly different from that in mars embryos (compare Fig. 2E with 2C). These results suggested that mars acts in parallel to tlk.

To explore the parallel nature of the interaction between mars and tlk further, we tested whether the morphology of the mitotic spindles and Mars localization was affected in embryos overexpressing tlk. GCN4>tlk embryos at 24°C were immunostained with anti-Mars, anti-α-Tub and anti-γ-Tub antibodies. Images were processed as described above. Overexpression of tlk induced a delayed progression of mitotic events, which was manifest as several observable features similar to those seen in mars embryos. Firstly, the fraction of embryos at prophase was similar (Table 2). Secondly, patches of nuclei with delayed chromosome congression were seen in 30% of the metaphase embryos (n = 150) (Fig. 5B). Thirdly, at least one patch of nuclei exhibited delayed chromosome segregation with chromosome bridges in half of anaphase embryos (n = 32) (Fig. 5C). Despite the similarity of these effects to those observed in mars embryos, neither the length nor the density of most mitotic spindles at metaphase was substantially affected by tlk overexpression (compare Figs. 5A with 5B). In agreement with this, tlk overexpression did not either affect the localization of Mars protein to mitotic spindles (Fig. 6) or decrease the quantity of acetylated tubulin (data not shown) that exists in the stable microtubules 2. Taken together with the different subcellular localizations of Mars and Tlk, which localize to spindle microtubules and chromosomes respectively (this study; 173043), these results supported the notion that Mars functions in parallel to Tlk.

Our previous results have shown that mars overexpression induces metaphase arrest in eye discs, with chromosomes attached to spindle monotelicaly in some cases 29. Based on the role of Tlk-1 acting as a cofactor of Aur-B 31923, we next asked the question whether tlk overexpression could overcome the metaphase arrest. To test this, we counted M-phase cells in the three different domains behind morphogenetic furrow (MF) as classified by Baker and Yu (2001) 45. Reproducibly, mars overexpression resulted in cells in domain I being retained at interphase and in many cells in domains II and III being retained at M-phase (Figs. 7B and 7E); this should be compared with the fact that most M-phase cells appear in domain I of w¹¹¹⁸ discs (Fig. 7A and 7E). Similarly, tlk overexpression induced a delayed progression of mitosis, but to a lesser extent (Figs. 7C and 7E). When both genes were co-overexpressed, the number of M-phase cells in domain III was reduced to a level close to that of the wild-type, showing that the metaphase arrest induced by mars overexpression was suppressed by tlk overexpression (Fig. 7E). These results indicated that a balance between mars and tlk activities is required for cells to progress through mitosis correctly.