Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex

The partially conserved Mad3/BubR1 protein is required during mitosis for the spindle assembly checkpoint (SAC). In meiosis, depletion causes an accelerated transit through prophase I and missegregation of achiasmate chromosomes in yeast [1], whereas in mice, reduced dosage leads to severe chromosome missegregation [2]. These observations indicate a meiotic requirement for BubR1, but its mechanism of action remains unknown. We identified a viable bubR1 allele in Drosophila resulting from a point mutation in the kinase domain that retains mitotic SAC activity. In males, we demonstrate a dose-sensitive requirement for BubR1 in maintaining sister-chromatid cohesion at anaphase I, whereas the mutant BubR1 protein localizes correctly. In bubR1 mutant females, we find that both achiasmate and chiasmate chromosomes nondisjoin mostly equationally consistent with a defect in sister-chromatid cohesion at late anaphase I or meiosis II. Moreover, mutations in bubR1 cause a consistent increase in pericentric heterochromatin exchange frequency, and although the synaptonemal complex is set up properly during transit through the germarium, it is disassembled prematurely in prophase by stage 1. Our results demonstrate that BubR1 is essential to maintain sister-chromatid cohesion during meiotic progression in both sexes and for normal maintenance of SC in females.     

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.     

Heterochromatin-Mediated Association of Achiasmate Homologs Declines With Age When Cohesion Is Compromised

Normally, meiotic crossovers in conjunction with sister-chromatid cohesion establish a physical connection between homologs that is required for their accurate segregation during the first meiotic division. However, in some organisms an alternative mechanism ensures the proper segregation of bivalents that fail to recombine. In Drosophila oocytes, accurate segregation of achiasmate homologs depends on pairing that is mediated by their centromere-proximal heterochromatin. Our previous work uncovered an unexpected link between sister-chromatid cohesion and the fidelity of achiasmate segregation when Drosophila oocytes are experimentally aged. Here we show that a weak mutation in the meiotic cohesion protein ORD coupled with a reduction in centromere-proximal heterochromatin causes achiasmate chromosomes to missegregate with increased frequency when oocytes undergo aging. If ORD activity is more severely disrupted, achiasmate chromosomes with the normal amount of pericentric heterochromatin exhibit increased nondisjunction when oocytes age. Significantly, even in the absence of aging, a weak ord allele reduces heterochromatin-mediated pairing of achiasmate chromosomes. Our data suggest that sister-chromatid cohesion proteins not only maintain the association of chiasmate homologs but also play a role in promoting the physical association of achiasmate homologs in Drosophila oocytes. In addition, our data support the model that deterioration of meiotic cohesion during the aging process compromises the segregation of achiasmate as well as chiasmate bivalents.     

Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes

Chiasmata established by recombination are normally sufficient to ensure accurate chromosome segregation during meiosis by physically interlocking homologs until anaphase I. Drosophila melanogaster female meiosis is unusual in that it is both exceptionally tolerant of nonexchange chromosomes and competent in ensuring their proper segregation. As first noted by Puro and Nokkala [Puro, J., Nokkala, S., 1977. Meiotic segregation of chromosomes in Drosophila melanogaster oocytes. A cytological approach. Chromosoma 63, 273-286], nonexchange chromosomes move precociously towards the poles following formation of a bipolar spindle. Indeed, metaphase arrest has been previously defined as the stage at which nonexchange homologs are symmetrically positioned between the main chromosome mass and the poles of the spindle. Here we use studies of both fixed images and living oocytes to show that the stage in which achiasmate chromosomes are separated from the main mass does not in fact define metaphase arrest, but rather is a component of an extended prometaphase. At the end of prometaphase, the nonexchange chromosomes retract into the main chromosome mass, which is tightly repackaged with properly co-oriented centromeres. This repackaged state is the true metaphase arrest configuration in Drosophila female meiosis.     

The Genetic Analysis of Achiasmate Segregation in Drosophila Melanogaster. III. the Wild-Type Product of the Axs Gene Is Required for the Meiotic Segregation of Achiasmate Homologs

The regular segregation of achiasmate chromosomes in Drosophila melanogaster females is ensured by two distinct segregational systems. The segregation of achiasmate homologs is assured by the maintenance of heterochromatic pairing; while the segregation of heterologous chromosomes is ensured by a separate mechanism that may not require physical association. Axs(D) (Aberrant X segregation) is a dominant mutation that specifically impairs the segregation of achiasmate homologs; heterologous achiasmate segregations are not affected. As a result, achiasmate homologs frequently participate in heterologous segregations at meiosis I. We report the isolation of two intragenic revertants of the Axs(D) mutation (Axs(r2) and Axs(r3)) that exhibit a recessive meiotic phenotype identical to that observed in Axs(D)/Axs(D) females. A third revertant (Axs(r1)) exhibits no meiotic phenotype as a homozygote, but a meiotic defect is observed in Axs(r1)/Axs(r2) females. Therefore mutations at the Axs(D) locus define a gene necessary and specific for homologous achiasmate segregation during meiosis. We also characterize the interactions of mutations at the Axs locus with two other meiotic mutations (ald and ncd). Finally, we propose a model in which Axs(+) is required for the normal separation of paired achiasmate homologs. In the absence of Axs(+) function, the homologs are often unable to separate from each other and behave as a single segregational unit that is free to segregate from heterologous chromosomes.     

Identification of novel Drosophila meiotic genes recovered in a P-element screen.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.     

Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. Here, we ask how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. We show that the knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, we observed bipolar spindles in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4^th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis.     

The spindle-associated transmembrane protein Axs identifies a membranous structure ensheathing the meiotic spindle

Mutations in the aberrant X segragation (Axs) gene disrupt the segregation of achiasmate chromosomes during female meiosis in Drosophila melanogaster. We show that Axs encodes the founding member of an eukaryotic family of transmembrane proteins. Axs protein colocalizes with components of the endoplasmic reticulum and is present within a structure ensheathing the meiotic spindle. In both meiotic and mitotic cells, Axs is recruited to the microtubules of assembling spindles. We propose that Axs and the sheath represent novel mediators of meiotic spindle assembly and chromosome segregation.     

A model system for increased meiotic nondisjunction in older oocytes

For at least 5% of all clinically recognized human pregnancies, meiotic segregation errors give rise to zygotes with the wrong number of chromosomes. Although most aneuploid fetuses perish in utero, trisomy in liveborns is the leading cause of mental retardation. A large percentage of human trisomies originate from segregation errors during female meiosis I; such errors increase in frequency with maternal age. Despite the clinical importance of age-dependent nondisjunction in humans, the underlying mechanisms remain largely unexplained. Efforts to recapitulate age-dependent nondisjunction in a mammalian experimental system have so far been unsuccessful. Here we provide evidence that Drosophila is an excellent model organism for investigating how oocyte aging contributes to meiotic nondisjunction. As in human oocytes, nonexchange homologs and bivalents with a single distal crossover in Drosophila oocytes are most susceptible to spontaneous nondisjunction during meiosis I. We show that in a sensitized genetic background in which sister chromatid cohesion is compromised, nonrecombinant X chromosomes become vulnerable to meiotic nondisjunction as Drosophila oocytes age. Our data indicate that the backup pathway that normally ensures proper segregation of achiasmate chromosomes deteriorates as Drosophila oocytes age and provide an intriguing paradigm for certain classes of age-dependent meiotic nondisjunction in humans.     

Genetic dissection of heterochromatin in Drosophila: The role of basal X heterochromatin in meiotic sex chromosome behaviour

We have examined the female meiotic behaviour of three X chromosomes which have large deletions of the basal heterochromatin in Drosophila melanogaster. We find that most of this heterochromatin can be removed without substantially altering pairing and segregation of the two Xs. To compare the role of heterochromatin in male meiosis we have constructed individuals which carry two extra identical heterochromatic mini X chromosomes. These minis behave as univalents even though their heterochromatin is known to contain satellite DNA. We conclude therefore that this satellite DNA is not sufficient to allow effectively normal meiotic behaviour. In all other respects our results in the male extend and confirm Cooper's postulate that there exist specific pairing sites in the X heterochromatin. Thus we find no support in either female or male meiosis for the concept that satellite DNA is involved in meiotic chromosome pairing of either a chiasmate or an achiasmate kind.     

There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology.

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle.     

A cell division mutant of Drosophila with a functionally abnormal spindle

Normal distribution of chromosomes to daughter cells is insured by the proper functioning of the spindle. Homozygosity for a semi-lethal mutation of Drosophila melanogaster (abnormal spindle) altering this structure has the following effects: the mitotic cycle is arrested in metaphase, leading to a high frequency of polyploid cells; sex chromosome disjunction during male meiosis is severely affected, as revealed by the resulting exceptional (diplo and nullo) gametes (microscopic examination of spermiogenesis confirms this aberrant segregation); meiotic spindles of living cells are morphologically abnormal; and tubulins extracted from mutant larvae are normal in amount, electrophoretic mobility, and ability to form microtubules in vitro. The results suggest that the mutant phenotype is due to an altered structural component of the spindle other than tubulins.     

Evidence that the spindle assembly checkpoint does not regulate APC(Fzy) activity in Drosophila female meiosis

The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APC(Fzy)-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APC(Fzy) inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APC(Fzy)-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APC(Fzy) to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.     

A deficiency screen of the major autosomes identifies a gene (matrimony) that is haplo-insufficient for achiasmate segregation in Drosophila oocytes

In Drosophila oocytes, euchromatic homolog-homolog associations are released at the end of pachytene, while heterochromatic pairings persist until metaphase I. A screen of 123 autosomal deficiencies for dominant effects on achiasmate chromosome segregation has identified a single gene that is haplo-insufficient for homologous achiasmate segregation and whose product may be required for the maintenance of such heterochromatic pairings. Of the deficiencies tested, only one exhibited a strong dominant effect on achiasmate segregation, inducing both X and fourth chromosome nondisjunction in FM7/X females. Five overlapping deficiencies showed a similar dominant effect on achiasmate chromosome disjunction and mapped the haplo-insufficient meiotic gene to a small interval within 66C7-12. A P-element insertion mutation in this interval exhibits a similar dominant effect on achiasmate segregation, inducing both high levels of X and fourth chromosome nondisjunction in FM7/X females and high levels of fourth chromosome nondisjunction in X/X females. The insertion site for this P element lies immediately upstream of CG18543, and germline expression of a UAS-CG18543 cDNA construct driven by nanos-GAL4 fully rescues the dominant meiotic defect. We conclude that CG18543 is the haplo-insufficient gene and have renamed this gene matrimony (mtrm). Cytological studies of prometaphase and metaphase I in mtrm hemizygotes demonstrate that achiasmate chromosomes are not properly positioned with respect to their homolog on the meiotic spindle. One possible, albeit speculative, interpretation of these data is that the presence of only a single copy of mtrm disrupts the function of whatever "glue" holds heterochromatically paired homologs together from the end of pachytene until metaphase I.     

Chiasmata promote monopolar attachment of sister chromatids and their co-segregation toward the proper pole during meiosis I

The chiasma is a structure that forms between a pair of homologous chromosomes by crossover recombination and physically links the homologous chromosomes during meiosis. Chiasmata are essential for the attachment of the homologous chromosomes to opposite spindle poles (bipolar attachment) and their subsequent segregation to the opposite poles during meiosis I. However, the overall function of chiasmata during meiosis is not fully understood. Here, we show that chiasmata also play a crucial role in the attachment of sister chromatids to the same spindle pole and in their co-segregation during meiosis I in fission yeast. Analysis of cells lacking chiasmata and the cohesin protector Sgo1 showed that loss of chiasmata causes frequent bipolar attachment of sister chromatids during anaphase. Furthermore, high time-resolution analysis of centromere dynamics in various types of chiasmate and achiasmate cells, including those lacking the DNA replication checkpoint factor Mrc1 or the meiotic centromere protein Moa1, showed the following three outcomes: (i) during the pre-anaphase stage, the bipolar attachment of sister chromatids occurs irrespective of chiasma formation; (ii) the chiasma contributes to the elimination of the pre-anaphase bipolar attachment; and (iii) when the bipolar attachment remains during anaphase, the chiasmata generate a bias toward the proper pole during poleward chromosome pulling that results in appropriate chromosome segregation. Based on these results, we propose that chiasmata play a pivotal role in the selection of proper attachments and provide a backup mechanism that promotes correct chromosome segregation when improper attachments remain during anaphase I.     

Mps1p regulates meiotic spindle pole body duplication in addition to having novel roles during sporulation

Sporulation in yeast requires that a modified form of chromosome segregation be coupled to the development of a specialized cell type, a process akin to gametogenesis. Mps1p is a dual-specificity protein kinase essential for spindle pole body (SPB) duplication and required for the spindle assembly checkpoint in mitotically dividing cells. Four conditional mutant alleles of MPS1 disrupt sporulation, producing two distinct phenotypic classes. Class I alleles of mps1 prevent SPB duplication at the restrictive temperature without affecting premeiotic DNA synthesis and recombination. Class II MPS1 alleles progress through both meiotic divisions in 30-50% of the population, but the asci are incapable of forming mature spores. Although mutations in many other genes block spore wall formation, the cells produce viable haploid progeny, whereas mps1 class II spores are unable to germinate. We have used fluorescently marked chromosomes to demonstrate that mps1 mutant cells have a dramatically increased frequency of chromosome missegregation, suggesting that loss of viability is due to a defect in spindle function. Overall, our cytological data suggest that MPS1 is required for meiotic SPB duplication, chromosome segregation, and spore wall formation.     

Heterochromatic Threads Connect Oscillating Chromosomes during Prometaphase I in Drosophila Oocytes

In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We observed that achiasmate homologs display dynamic movements on the meiotic spindle during mid-prometaphase. An analysis of living prometaphase oocytes revealed both the rejoining of achiasmate X chromosomes initially located on opposite half-spindles and the separation toward opposite poles of two X chromosomes that were initially located on the same half spindle. When the two achiasmate X chromosomes were positioned on opposite halves of the spindle their kinetochores appeared to display proper co-orientation. However, when both Xs were located on the same half spindle their kinetochores appeared to be oriented in the same direction. Thus, the prometaphase movement of achiasmate chromosomes is a congression-like process in which the two homologs undergo both separation and rejoining events that result in the either loss or establishment of proper kinetochore co-orientation. During this period of dynamic chromosome movement, the achiasmate homologs were connected by heterochromatic threads that can span large distances relative to the length of the developing spindle. Additionally, the passenger complex proteins Incenp and Aurora B appeared to localize to these heterochromatic threads. We propose that these threads assist in the rejoining of homologs and the congression of the migrating achiasmate homologs back to the main chromosomal mass prior to metaphase arrest.     

Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.     

The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1-8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.     

The Arabidopsis ATK1 gene is required for spindle morphogenesis in male meiosis

The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis, called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.