Mutational Analysis of the Drosophila Sister-Chromatid Cohesion Protein Ord and Its Role in the Maintenance of Centromeric Cohesion

The ord gene is required for proper segregation of all chromosomes in both male and female Drosophila meiosis. Here we describe the isolation of a null ord allele and examine the consequences of ablating ord function. Cytologically, meiotic sister-chromatid cohesion is severely disrupted in flies lacking ORD protein. Moreover, the frequency of missegregation in genetic tests is consistent with random segregation of chromosomes through both meiotic divisions, suggesting that sister cohesion may be completely abolished. However, only a slight decrease in viability is observed for ord null flies, indicating that ORD function is not essential for cohesion during somatic mitosis. In addition, we do not observe perturbation of germ-line mitotic divisions in flies lacking ORD activity. Our analysis of weaker ord alleles suggests that ORD is required for proper centromeric cohesion after arm cohesion is released at the metaphase I/anaphase I transition. Finally, although meiotic cohesion is abolished in the ord null fly, chromosome loss is not appreciable. Therefore, ORD activity appears to promote centromeric cohesion during meiosis II but is not essential for kinetochore function during anaphase.     

Meiotic mutations from natural populations ofDrosophila melanogaster

Two meiotic genes from natural populations are described. A female meiotic mutation,mei(1)g13, mapped to 17.4 on the X chromosome, causes nondisjunction of all homologs except for the fourth chromosomes. In addition, it reduces recombination by 10% in the homozygotes and causes 18% increased recombination in the heterozygotes. A male meiotic mutation,mei-1223 ^m144 , is located on the third chromosome. Although this mutation causes nondisjunction of all chromosomes, each chromosome pair exhibits a different nondisjunction frequency. Large variations in the sizes of the premature sperm heads observed in the homozygotes may reflect irregular meiotic pairing and the subsequent abnormal segregation, resulting in aneuploid chromosome complements.     

Paternal Loss (PAL): A Meiotic Mutant in DROSOPHILA MELANOGASTER Causing Loss of Paternal Chromosomes

The effects of a male-specific meiotic mutant, paternal loss (pal), in D. melanogaster have been examined genetically. The results indicate the following. (1) When homozygous in males, pal can cause loss, but not nondisjunction, of any chromosome pair. The pal-induced chromosome loss produces exceptional progeny that apparently failed to receive one, or more, paternal chromosomes and, in addition, mosaic progeny during whose early mitotic divisions one or more paternal chromosomes were lost. (2) Only paternally derived chromosomes are lost. (3) Mitotic chromosome loss can occur in homozygous pal⁺ progeny of pal males. (4) Chromosomes differ in their susceptibility to pal-induced loss. The site responsible for the insensitivity vs. sensitivity of the X chromosome to pal mapped to the basal region of the X chromosome at, or near, the centromere. From these results, it is suggested that pal⁺ acts in male gonia to specify a product that is a component of, or interacts with, the centromeric region of chromosomes and is necessary for the normal segregation of paternal chromosomes. In the presence of pal, defective chromosomes are produced and these chromosomes tend to get lost during the early cleavage divisions of the zygote. (5) The loss of heterologous chromosome pairs is not independent; there are more cases of simultaneous loss of two chromosomes than expected from independence. Moreover, an examination of cases of simultaneous somatic loss of two heterologs reveals an asymmetry in the early mitotic divisions of the zygote such that when two heterologs are lost at a somatic cleavage division, almost invariably one daughter nucleus fails to get either, and the other daughter nucleus receives its normal chromosome complement. It is suggested that this asymmetry is not a property of pal but is rather a normal process that is being revealed by the mutant. (6) The somatic loss of chromosomes in the progeny of pal males allows the construction of fate maps of the blastoderm. Similar fate maps are obtained using data from gynandromorphs and from marked Y chromosome (nonsexually dimorphic) mosaics.     

SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF^Slimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF^Slimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF^Slimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.     

Chromatin diminution and early cleavage in Parascaris univalens (Nematoda)

Summary In Parascaris developmental commitment to the germ line and somatic lineages is indicated by the orientation of the mitotic spindle in blastomeres, the topology of cells in the embryo, and chromatin diminution in presomatic blastomeres. Using three different experimental techniques: transient pressure treatment, application of cytochalasin B, and isolation of blastomeres, we have succeeded in uncoupling several developmental processes during cleavage of P. univalens. The following results were obtained: (1) Following mitotic nondisjunction we observed identical behavior of all chromatids in each blastomere. Thus chromosome differentiation by differential replication does not occur. (2) Chromosome fragments obtained by pressure treatment of egg cells underwent chromatin diminution. Thus this process does not require an intact germ-line chromosome. However, chromosomes immobilized on a monopolar spindle did not undergo chromatin diminution. Thus diminution appears to require segregation of chromatids. (3) Blastomeres that completely lacked chromosomes as a result of mitotic nondisjunction underwent normal early cleavage divisions. (4) Pressure treatment or prolonged treatment with cytochalasin B caused egg cells or germ line blastomeres to lose their germ line quality, as deduced from the coincident occurrence of symmetrical (presomatic-like) cleavage and chromatin diminution. (5) Isolated blastomeres from 2-cell embryos, i.e. 1/2 blastomeres, usually cleaved according to their prospective fates in the whole embryo. However, in some partial embryos derived from such blastomeres, chromatin diminution was delayed for either one or two cleavage mitoses. An activation model as an alternative to a prelocalization model is presented, which can account for early blastomere topogenesis and chromatin diminution.     

The cytology of paedogenesis in the gall midgeMycophila speyeri

In paedogenetically developing female eggs of the gall midgeMycophila speyeri only one equational meiotic division occurs. The primary cleavage nucleus contains 29 chromosomes. In the fourth cleavage division 23 chromosomes are eliminated from the future somatic nuclei while the primordial germ-line nucleus keeps the high chromosome number.—The paedogenetic development of male eggs begins with two meiotic divisions. The egg nucleus with 14 or 15 chromosomes fuses with two, sometimes only one, somatic nuclei (2n=6) of maternal origin (regulation). Thus the primary cleavage nucleus contains 26 or 27 chromosomes, sometimes only 20 or 21. Elimination in cleavage divisions V and VI leeds to somatic nuclei with 3 chromosomes while the primordial germ-line nucleus keeps the high chromosome number.—Differences between male and female eggs and the evolution of regulation in gall midges are discussed.     

Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster

The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. We show that the conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions similarly to somatic cells. To determine BubR1 function during embryogenesis, we generated a new hypomorphic semi-viable female sterile allele. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, we find that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. Our results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis.     

Die Zytologie der bisexuellen und parthenogenetischen Fortpflanzung von Heteropeza pygmaea Winnertz,einer Gallmücke mit pädogenetischer Vermehrung

Heteropeza pygmaea (syn. Oligarces paradoxus) can reproduce as larvae by paedogenesis or as imagines (Fig. 1). The eggs of imagines may develop after fertilization or parthenogenetically. The fertilized eggs give rise to female larvae, which develop into mother-larvae with female offspring (Weibchenmütter). Only a few of the larvae which hatch from unfertilized eggs become motherlarvae with female offspring; the others die. Spermatogenesis is aberrant, as it is in all gall midges studied to date. The primary spermatocyte contains 53 or 63 chromosomes. The meiotic divisions give rise to two sperms each of which contains only 7 chromosomes (Figs. 5–11). The eggs of the imago are composed of the oocyte and the nurse-cell chamber. In addition to the oocyte nucleus and the nurse-cell nuclei there are three other nuclei in the eggs (Figs. 15–17). They are called small nuclei (kleine Kerne). In prometaphase stages of the first cleavage division it could be seen that these nuclei contain about 10 chromosomes. Therefore it is assumed that these nuclei originate from the soma of the mother-larva. The chromosome number of the primary oocyte is approximately 66. The oocyte completes two meiotic divisions. The reduced egg nucleus contains approximately 33 chromosomes. The polar body-nuclei degenerate during the first cleavage divisions. The fertilized egg contains 2–3 sperms. The primary cleavage nucleus is formed by the egg nucleus and usually all of the sperm nuclei and the small nuclei (Figs. 21–29). The most frequent chromosome numbers in the primary cleavage nuclei are about 77 and 67. The first and the second cleavage divisions are normal. A first elimination occurs in the 3rd, 4th, and 5th cleavage division (Fig. 30). All except 6 chromosomes are eliminated from the future somatic nuclei. Following a second elimination (Figs. 33, 34), the future somatic nuclei contain 5 chromosomes. No elimination occurs in the divisions of the germ line nucleus. In eggs which develop parthenogenetically the primary cleavage nucleus is formed by the egg nucleus and 2–3 small nuclei. It's chromosome number is therefore about 53 or 63. After two eliminations, which are similar to the ones which occur in fertilized eggs, the soma contains 5 chromosomes. The somatic nuclei of male larvae which arrise by paedogenesis contain 5 chromosomes; while the somatic nuclei of female larvae of paedogenetic origin contain 10 chromosomes. It was therefore assumed earlier that sex was determined by haploidy or diploidy. But the above results show that larvae from fertilized as well as from unfertilized eggs of imagines have 5 chromosomes in the soma, but are females, and the female paedogenetic offspring of larvae from unfertilized eggs have either 5 or 10 chromosomes in their somatic cells. Therefore sex determination is not by haploidy-diploidy but by some other, unknown, mechanism. The cytological events associated with paedogenetic, bisexual, and parthenogenetic reproduction in Heteropeza pygmaea are compared (Fig. 37). The occurrence and meaning of the small nuclei which are found in the eggs of most gall midges are discussed. It has been shown here that these nuclei function to restore the chromosome number in fertilized eggs; it is suggested that they function similarity in certain other gall midges. Consideration of the mode of restoration of the germ-line chromosome number leads to the conclusion that in Heteropeza few, if any, of the chromosomes are limited to the germ-line, i.e. can never occur in somatic cells (p. 124).     

Subcellular Localization of Bacillus subtilis SMC, a Protein Involved in Chromosome Condensation and Segregation

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.     

A Chromatin-associated Kinesin-related Protein Required for Normal Mitotic Chromosome Segregation in Drosophila

The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles.     

Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1 ⁺ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1 ⁺ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.     

Perturbation Analysis of Heterochromatin-Mediated Gene Silencing and Somatic Inheritance

Repetitive sequences in eukaryotic genomes induce chromatin-mediated gene-silencing of juxtaposed genes. Many components that promote or antagonize silencing have been identified, but how heterochromatin causes variegated and heritable changes in gene expression remains mysterious. We have used inducible mis-expression in the Drosophila eye to recover new factors that alter silencing caused by the bw^D allele, an insertion of repetitive satellite DNA that silences a bw⁺ allele on the homologous chromosome. Inducible modifiers allow perturbation of silencing at different times in development, and distinguish factors that affect establishment or maintenance of silencing. We find that diverse chromatin and RNA processing factors can de-repress silencing. Most factors are effective even in differentiated cells, implying that silent chromatin remains plastic. However, over-expression of the bantam microRNA or the crooked-legs (crol) zinc-finger protein only de-repress silencing when expressed in cycling cells. Over-expression of crol accelerates the cell cycle, and this is required for de-repression of silencing. Strikingly, continual over-expression of crol converts the speckled variegation pattern of bw^D into sectored variegation, where de-repression is stably inherited through mitotic divisions. Over-expression of crol establishes an open chromatin state, but the factor is not needed to maintain this state. Our analysis reveals that active chromatin states can be efficiently inherited through cell divisions, with implications for the stable maintenance of gene expression patterns through development.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindle sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G₂ nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.Key words: mitotic chromosomes, Xenopus, egg extracts, intracellular scaling, spindle, embryogenesis, cell division     

Early embryonic requirement for nucleoporin Nup35/NPP-19 in nuclear assembly

Nuclear pore complexes (NPCs) are gateways for transport between the nucleus and cytoplasm of eukaryotic cells and play crucial roles in regulation of gene expression. NPCs are composed of multiple copies of ∼ 30 different nucleoporins (nups) that display both ubiquitous and cell type specific functions during development. Vertebrate Nup35 (also known as Nup53) was previously described to interact with Nup93, Nup155 and Nup205 and to be required for nuclear envelope (NE) assembly in vitro. Here, we report the first in vivo characterization of a Nup35 mutation, npp-19(tm2886), and its temperature-dependent effects on Caenorhabditis elegans embryogenesis. At restrictive temperature, npp-19(tm2886) embryos exhibit chromosome missegregation, nuclear morphology defects and die around mid-gastrulation. Depletion of Nup35/NPP-19 inhibits NE localization of Nup155/NPP-8, NPC assembly and nuclear lamina formation. Consequently, nuclear envelope function, including nucleo-cytoplasmic transport, is impaired. In contrast, recruitment of Nup107/NPP-5, LEM-2 and nuclear membranes to the chromatin surface is Nup35/NPP-19-independent, suggesting an uncoupling of nuclear membrane targeting and NPC assembly in the absence of Nup35/NPP-19. We propose that Nup35/NPP-19 has an evolutionary conserved role in NE formation and function, and that this role is particularly critical during the rapid cell divisions of early embryogenesis.     

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.     

Genome size and determination of DNA content of the X chromosomes, autosomes, and germ line-limited chromosomes of Sciara coprophila

The unique chromosome biology of the fungus fly Sciara coprophila has fascinated investigators for over 80 years. Male meiosis exhibits a monopolar spindle, nonrandom segregation of imprinted chromosomes and nondisjunction of the X chromosome. The unusual mechanism of sex determination requires selective elimination of X chromosomes in embryogenesis. Supernumerary (L) chromosomes are also eliminated from the soma during early cleavage divisions. Distinctive DNA puffs on the larval salivary gland chromosomes are sites of DNA amplification. As a foundation for future genome studies to explore these many unusual phenomena, we have used DNA-Feulgen cytophotometry to determine genome size from hemocyte nuclei of male (X0) and female (XX) larvae and adults. The DNA content of the X chromosome is approximately 0.05 pg DNA and the autosomal complement is approximately 0.45 pg DNA. Measurements of DNA levels for individual sperm from adults showed that the DNA contribution of the germ line-limited (L) chromosomes constitutes as much as 35% of the DNA of the male gamete. A parallel study using Sciara ocellaris, a related species lacking L chromosomes, confirmed the presence of two X chromosomes in the sperm of this species.     

mei-2, a mutagen-sensitive mutant of Neurospora defective in chromosome pairing and meiotic recombination

Summary A Neurospora crassa mutation, mei-2, affecting meiosis and mutagen sensitivity, was characterized for its effect on meiotic recombination and chromosome pairing. Results from homozygous mei-2 crosses involving distant markers on the same chromosome demonstrated a drastic reduction in meiotic recombination. However, mitotic recombination continued to occur. Cytological observations indicated that pairing of homologous chromosomes in zygotene was greatly reduced or absent, resulting in aberrant segregation at anaphase I and often at subsequent divisions as well. The few mature ascospores produced were frequently disomic for one or more chromosomes.     

Normal Segregation of a Foreign-Species Chromosome During Drosophila Female Meiosis Despite Extensive Heterochromatin Divergence

The abundance and composition of heterochromatin changes rapidly between species and contributes to hybrid incompatibility and reproductive isolation. Heterochromatin differences may also destabilize chromosome segregation and cause meiotic drive, the non-Mendelian segregation of homologous chromosomes. Here we use a range of genetic and cytological assays to examine the meiotic properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into D. melanogaster. These two species differ by ∼12–13% at synonymous sites and several genes essential for chromosome segregation have experienced recurrent adaptive evolution since their divergence. Furthermore, their chromosome 4s are visibly different due to heterochromatin divergence, including in the AATAT pericentromeric satellite DNA. We find a visible imbalance in the positioning of the two chromosome 4s in sim-IV/mel-IV heterozygote and also replicate this finding with a D. melanogaster 4 containing a heterochromatic deletion. These results demonstrate that heterochromatin abundance can have a visible effect on chromosome positioning during meiosis. Despite this effect, however, we find that sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females and does not experience elevated nondisjunction. We conclude that segregation abnormalities and a high level of meiotic drive are not inevitable byproducts of extensive heterochromatin divergence. Animal chromosomes typically contain large amounts of noncoding repetitive DNA that nevertheless varies widely between species. This variation may potentially induce non-Mendelian transmission of chromosomes. We have examined the meiotic properties and transmission of a highly diverged chromosome 4 from a foreign species within the fruitfly Drosophila melanogaster. This chromosome has substantially less of a simple sequence repeat than does D. melanogaster 4, and we find that this difference results in altered positioning when chromosomes align during meiosis. Yet this foreign chromosome segregates at normal frequencies, demonstrating that chromosome segregation can be robust to major differences in repetitive DNA abundance.     

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro⁷¹ and Chro⁶¹² have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.