Mutations in the alpha-tubulin 67C gene specifically impair achiasmate segregation in Drosophila melanogaster

Drosophila melanogaster oocytes heterozygous for mutations in the alpha-tubulin 67C gene (alphatub67C) display defects in centromere positioning during prometaphase of meiosis I. The centromeres do not migrate to the poleward edges of the chromatin mass, and the chromatin fails to stretch during spindle lengthening. These results suggest that the poleward forces acting at the kinetochore are compromised in the alphatub67C mutants. Genetic studies demonstrate that these mutations also strongly and specifically decrease the fidelity of achiasmate chromosome segregation. Proper centromere orientation, chromatin elongation, and faithful segregation can all be restored by a decrease in the amount of the Nod chromokinesin. These results suggest that the accurate segregation of achiasmate chromosomes requires the proper balancing of forces acting on the chromosomes during prometaphase.     

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.     

A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila.     

A screen for genes that interact with the Drosophila pair-rule segmentation gene fushi tarazu

The pair-rule gene fushi tarazu (ftz) of Drosophila is expressed at the blastoderm stage in seven stripes that serve to define the even-numbered parasegments. ftz encodes a DNA-binding homeodomain protein and is known to regulate genes of the segment polarity, homeotic, and pair-rule classes. Despite intensive analysis in a number of laboratories, how ftz is regulated and how it controls its targets are still poorly understood. To help understand these processes, we conducted a screen to identify dominant mutations that enhance the lethality of a ftz temperature-sensitive mutant. Twenty-six enhancers were isolated, which define 21 genes. All but one of the mutations recovered show a maternal effect in their interaction with ftz. Three of the enhancers proved to be alleles of the known ftz protein cofactor gene ftz-f1, demonstrating the efficacy of the screen. Four enhancers are alleles of Atrophin (Atro), the Drosophila homolog of the human gene responsible for the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Embryos from Atro mutant germ-line mothers lack the even-numbered (ftz-dependent) engrailed stripes and show strong ftz-like segmentation defects. These defects likely result from a reduction in Even-skipped (Eve) repression ability, as Atro has been shown to function as a corepressor for Eve. In this study, we present evidence that Atro is also a member of the trithorax group (trxG) of Hox gene regulators. Atro appears to be particularly closely related in function to the trxG gene osa, which encodes a component of the brahma chromatin remodeling complex. One additional gene was identified that causes pair-rule segmentation defects in embryos from homozygous mutant germ-line mothers. The single allele of this gene, called bek, also causes nuclear abnormalities similar to those caused by alleles of the Trithorax-like gene, which encodes the GAGA factor.     

The meiotic defects of mutants in the Drosophila mps1 gene reveal a critical role of Mps1 in the segregation of achiasmate homologs

The conserved kinase Mps1 is necessary for the proper functioning of the mitotic and meiotic spindle checkpoints (MSCs), which monitor the integrity of the spindle apparatus and prevent cells from progressing into anaphase until chromosomes are properly aligned on the metaphase plate. In Drosophila melanogaster, a null allele of the gene encoding Mps1 was recently shown to be required for the proper functioning of the MSC, but it did not appear to exhibit a defect in female meiosis. We demonstrate here that the meiotic mutant ald1 is a hypomorphic allele of the mps1 gene. Both ald1 and a P-insertion allele of mps1 exhibit defects in female meiotic chromosome segregation. The observed segregational defects are substantially more severe for pairs of achiasmate homologs, which are normally segregated by the achiasmate (or distributive) segregation system, than they are for chiasmate bivalents. Furthermore, cytological analysis of ald1 mutant oocytes reveals both a failure in the coorientation of achiasmate homologs at metaphase I and a defect in the maintenance of the chiasmate homolog associations that are normally observed at metaphase I. We conclude that Mps1 plays an important role in Drosophila female meiosis by regulating processes that are especially critical for ensuring the proper segregation of nonexchange chromosomes.     

A deficiency screen for dominant suppressors of telomeric silencing in Drosophila

Heterochromatin is a specialized chromatin structure in chromosomal regions associated with repeated DNA sequences and low concentrations of genes. Formation of heterochromatin is determined in large part by enzymes that modify histones and structural proteins that bind to these modified histones in a cooperative fashion. In Drosophila, mutations in genes that encode heterochromatic proteins are often dominant and increase expression of genes placed into heterochromatic positions. To find components of telomeric heterochromatin in Drosophila, we screened a collection of autosomal deficiencies for dominant suppressors of silencing of a transgene at the telomere of chromosome 2L. While many deficiency chromosomes are associated with dominant suppressors, in the cases tested on chromosome 2 the suppressor mapped to the 2L telomere, rather than the deficiency. We infer that background effects may hamper the search for genes that play a role in telomeric heterochromatin formation and that either very few genes participate in this pathway or mutations in these genes are not dominant suppressors of telomeric position effect. The data also suggest that the 2L telomere region plays a major role in telomeric silencing.     

A large-scale RNAi screen reveals that mitochondrial function is important for meiotic chromosome organization in oocytes

Chromosoma - In prophase of the first meiotic division, chromatin forms a compact spherical cluster called the karyosome within the enlarged oocyte nucleus in Drosophila melanogaster. Similar...     

Identification of two proteins required for conjunction and regular segregation of achiasmate homologs in Drosophila male meiosis

In Drosophila males, homologous chromosomes segregate by an unusual process involving physical connections not dependent on recombination. We have identified two meiotic proteins specifically required for this process. Stromalin in Meiosis (SNM) is a divergent member of the SCC3/SA/STAG family of cohesin proteins, and Modifier of Mdg4 in Meiosis (MNM) is one of many BTB-domain proteins expressed from the mod(mdg4) locus. SNM and MNM colocalize along with a repetitive rDNA sequence known to function as an X-Y pairing site to nucleolar foci during meiotic prophase and to a compact structure associated with the X-Y bivalent during prometaphase I and metaphase I. Additionally, MNM localizes to autosomal foci throughout meiosis I. These proteins are mutually dependent for their colocalization, and at least MNM requires the function of teflon, another meiotic gene. SNM and MNM do not colocalize with SMC1, suggesting that the homolog conjunction mechanism is independent of cohesin.     

A screen for modifiers of hedgehog signaling in Drosophila melanogaster identifies swm and mts

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. We performed an enhancer/suppressor screen in Drosophila to identify novel components of the pathway and identified 26 autosomal regions that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway-patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. We show that mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn(+) finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity.     

A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.     

A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila

The Dpp signaling pathway is essential for many developmental processes in Drosophila and its activity is tightly regulated. To identify additional regulators of Dpp signaling, we conducted a genetic screen for maternal-effect suppressors of dpp haplo-insufficiency. We screened approximately 7000 EMS-mutagenized genomes and isolated and mapped seven independent dominant suppressors of dpp, Su(dpp), which were recovered as second-site mutations that resulted in viable flies in trans-heterozygous with dpp(H46), a dpp null allele. Most of the Su(dpp) mutants exhibited increased cell numbers of the amnioserosa, a cell type specified by the Dpp pathway, suggesting that these mutations may augment Dpp signaling activity. Here we report the unexpected identification of one of the Su(dpp) mutations as an allele of the eukaryotic translation initiation factor 4A (eIF4A). We show that Su(dpp)(YE9) maps to eIF4A and that this allele is associated with a substitution, arginine 321 to histidine, at a well-conserved amino acid and behaves genetically as a dominant-negative mutation. This result provides an intriguing link between a component of the translation machinery and Dpp signaling.