Spindles and centrosomes during male meiosis in Drosophila melanogaster

We have studied the spatial distribution of chromosomes, spindle fibers and centrosomes throughout the first meiotic division in males of Drosophila melanogaster. There seem to be two different types of spindle fibers: those which connect the poles to the chromosomes, and others arranged as cup-shaped hemispheres that reach from the poles to an unstained area on the equator of the cell. These pole-equator fibers could be responsible for positioning the nucleus and distributing cytoplasmic organelles around the nucleus during prophase, so that after meiosis, the daughter cells are provided with equal amounts of preorganized cytoplasmic organelles. These fibers remain until after the daughter nuclei have formed during telophase. An antigen associated with the centrosomes of mitotic spindles appears during meiosis as dispersed particles surrounding the nucleus; these particles might provide the developing spermatids with microtubule-organizing centers.     

Comparative Analysis of Pdf-Mediated Circadian Behaviors Between Drosophila melanogaster and D. virilis

PAR proteins (partitioning defective) are major regulators of cell polarity and asymmetric cell division. One of the par genes, par-1, encodes a Ser/Thr kinase that is conserved from yeast to mammals. In Caenorhabditis elegans, par-1 governs asymmetric cell division by ensuring the polar distribution of cell fate determinants. However the precise mechanisms by which PAR-1 regulates asymmetric cell division in C. elegans remain to be elucidated. We performed a genomewide RNAi screen and identified six genes that specifically suppress the embryonic lethal phenotype associated with mutations in par-1. One of these suppressors is mpk-1, the C. elegans homolog of the conserved mitogen activated protein (MAP) kinase ERK. Loss of function of mpk-1 restored embryonic viability, asynchronous cell divisions, the asymmetric distribution of cell fate specification markers, and the distribution of PAR-1 protein in par-1 mutant embryos, indicating that this genetic interaction is functionally relevant for embryonic development. Furthermore, disrupting the function of other components of the MAPK signaling pathway resulted in suppression of par-1 embryonic lethality. Our data therefore indicates that MAP kinase signaling antagonizes PAR-1 signaling during early C. elegans embryonic polarization.ASYMMETRIC cell division, a process in which a mother cell divides in two different daughter cells, is a fundamental mechanism to achieve cell diversity during development. We use the early embryo of Caenorhabditis elegans as a model system to study asymmetric cell division. The C. elegans one-cell embryo divides asymmetrically along its anteroposterior axis, generating two cells of different sizes and fates: the larger anterior daughter cell will generate somatic tissues while the smaller posterior daughter cell will generate the germline (Sulston et al. 1983).A group of proteins called PAR proteins (partitioning defective) is required for asymmetric cell division in C. elegans (Kemphues et al. 1988). Depletion of any of the seven par genes (par-1 to -6 and pkc-3) leads to defects in asymmetric cell division and embryonic lethality (Kemphues et al. 1988; Kirby et al. 1990; Tabuse et al. 1998; Hung and Kemphues 1999; Hao et al. 2006). PAR-3 and PAR-6 are conserved proteins that contain PDZ-domains and form a complex with PKC-3 (Etemad-Moghadam et al. 1995; Izumi et al. 1998; Tabuse et al. 1998; Hung and Kemphues 1999). This complex becomes restricted to the anterior cortex of the embryo in response to spatially defined actomyosin contractions occurring in the embryo upon fertilization (Goldstein and Hird 1996; Munro et al. 2004). The posterior cortex of the embryo that becomes devoid of the anterior PAR proteins is occupied by the RING protein PAR-2 and the Ser/Thr kinase PAR-1 (Guo and Kemphues 1995; Boyd et al. 1996; Cuenca et al. 2003). Once polarized, the anterior and posterior PAR proteins mutually exclude each other from their respective cortices (Etemad-Moghadam et al. 1995; Boyd et al. 1996; Cuenca et al. 2003; Hao et al. 2006). Loss of function of the gene par-1, as opposed to loss of most other par genes, results in embryos that exhibit only subtle effects on the polarized cortical domains occupied by the other PAR proteins (Cuenca et al. 2003). However defects in this gene are associated with a more symmetric division in size, an aberrant distribution of cell fate specification markers, altered cell fates of the daughter cells of the embryo, and ultimately embryonic lethality (Kemphues et al. 1988; Guo and Kemphues 1995).PAR-1 controls asymmetric cell division and cell fate specification by regulating the localization of the two highly similar CCCH-type zinc-finger proteins MEX-5 and MEX-6 (referred to as MEX-5/6). MEX-5 and MEX-6 are 70% identical in their amino acid sequence and fulfill partially redundant functions in the embryo (Schubert et al. 2000). In wild-type animals, endogenous MEX-5 and GFP fusions of MEX-6 localize primarily to the anterior of the embryo while both proteins are evenly distributed in par-1 mutant embryos (Schubert et al. 2000; Cuenca et al. 2003). This suggests that in wild-type animals, PAR-1 acts in part by restricting MEX-5 and MEX-6 to the anterior of the embryo. The precise mechanism of this regulation is not known, but an elegant study performed for MEX-5 indicates that differential protein mobility in the anterior and posterior cytoplasm of the one-cell embryo contributes to this asymmetry (Tenlen et al. 2008). While increased mobility in the posterior of the one-cell embryo correlates with a par-1- and par-4-dependent phosphorylation on MEX-5, the kinase directly phosphorylating MEX-5 remains to be identified (Tenlen et al. 2008).Some of the phenotypes associated with loss of par-1 function are dependent on the function of mex-5 and mex-6. First, loss of function of par-1 leads to a decreased stability and aberrant localization of the posterior cell fate specification marker PIE-1, a protein that is usually inherited by the posterior daughter cell in wild-type animals and ensures the correct specification of the germline (Mello et al. 1996; Seydoux et al. 1996). This decreased stability is dependent on mex-5/6 function as PIE-1 levels are restored, albeit with symmetrical distribution, in mex-6(RNAi); mex-5(RNAi); par-1(b274) embryos (Schubert et al. 2000; Cuenca et al. 2003; Derenzo et al. 2003). Second, embryos lacking par-1 function exhibit decreased amounts of P granules in the one-cell embryo, while these markers are present in mex-6(pk440); mex-5(zu199); par-1(RNAi) embryos of comparable age (Cheeks et al. 2004). Third, in par-1(RNAi) one-cell embryos the posterior cortical domain occupied by the polarity protein PAR-2 is extended anteriorly, when compared to wild-type embryos (Cuenca et al. 2003). This anterior extension is rescued in embryos deficient for both par-1 and mex-5/6 (Cuenca et al. 2003). Taken together, these results indicate that par-1 acts in the embryo—at least in part—by regulating the localization and/or activity of the proteins MEX-5 and MEX-6. However, it remains unclear whether other proteins can modulate PAR-1 function to affect MEX-5/6 activity.To gain insight into the mechanisms of par-1 function in the embryo, we sought to identify genes that act together with par-1 during embryonic development. We performed an RNAi-based screen for genetic interactors of the temperature-sensitive allele par-1(zu310), using the embryonic lethal phenotype of this mutant as a readout. This method has proven successful in previous screens to identify genes involved in early embryonic processes (Labbé et al. 2006; O''Rourke et al. 2007). We were able to identify six genes that, upon disruption of their function, suppress the embryonic lethal phenotype of par-1 mutant embryos. One of these genes is mpk-1, the C. elegans homolog of the highly conserved MAP kinase ERK. Closer analysis subsequently showed that reduction of function of mpk-1 not only increases viability of par-1 mutant embryos, but also reverts several polarity phenotypes associated with loss of function of par-1. Our data indicate that mpk-1 antagonizes par-1 activity to regulate polarization and asymmetric cell divisions in the early embryo.     

Unusual prophase structures and multiple nucleoli in male meiosis of Drosophila species of the virilis group

With silver nitrate (Ag-NOR) staining, unusual fibrillar structures, apparently coupled to the nucleolus, were found is several species of the D. virilis group. In D. littoralis, beaded strings appear in connection with these structures, whereas the late prophase is characterized by the appearance of multiple nucleoli in the nucleoplasm. In D. virilis, the nucleus has a prominent pointed protrusion in the region of the nucleolus and often a fibril protrudes from this point. Small nucleoli are budding from the nucleolus during prophase. The multiple nucleoli at late prophase are smaller and fewer. A nucleolar body with black spots appears at prometaphase and persists through metaphase and anaphase. In D. lummei, the nucleolus becomes surrounded by fibrils, which are released into the nucleoplasm and on which multiple nucleoli are synthesized.These phenomena are similar to the events described in oocyte meiosis of many animals, where rDNA amplification, coupled to the synthesis of multiple nucleoli in late prophase, has been established.     

Mitochondrial DNA in Drosophila. An analysis of genome organization and transcription in Drosophila melanogaster and Drosophila virilis

Transfer RNAs of Escherichia coli were separated by two-dimensional polyacrylamide gel electrophoresis, and the relative abundance of each of the 26 known tRNAs thus separated was measured on the basis of molecular numbers in cells. Based on this relative abundance, the distributions of cognate codons in E. coli genes (lacI, rpA, asnA, recA, lpp and four ribosomal protein genes) and in coliphage (MS2, φX174 and λ) genes were examined. A strong positive correlation between the tRNA abundance and the choice of codons, among both synonymous codons and those corresponding to different amino acids, was found for all E. coli protein genes that had been sequenced completely. However, the correlation was less significant for the phage genes. The relationship between tRNA abundance and its usage (namely anticodon usage) was examined by regression analysis. The degree of the relationship found for individual E. coli genes differed from gene to gene: those of r-protein genes and recA were higher than those of trpA, lacI and asnA. The dependent relationship of tRNA usage on its content for the first two genes seems to be greater than that expected from the proportional relationship between the two variables; i.e. these genes selectively use codons corresponding to major tRNAs but nearly avoid using those of minor tRNAs.     

The genetic analysis of meiosis in female Drosophila melanogaster.

The three major features of meiosis are first synapsis, then exchange, and finally, disjunction of homologous chromosomes; these phenomena occur before pachytene, during pachytene, and after pachytene respectively. The effects of meiotic mutants, or other perturbations, either endogenous or exogenous, on the meiotic process may be assigned tentatively to one of these intervals, based on the earliest discernible abnormality. Thus mutants exhibiting abnormal disjunction and normal exchange affect post-pachytene functions; mutants exhibiting abnormal disjunction and exchange but with ultrastructurally normal appearing synaptonemal complex affect pachytene functions; and mutants with abnormal disjunction, exchange, and synaptonemal complex affect prepachytene functions. This rationale is applied to the temporal seriation of effects of meiotic mutants and chromosomal abnormalities on the meiotic programme.     

Electron microscopy of meiosis in Drosophila melanogaster females

Complete reconstruction of the synaptonemal complex in 12 pachytene (defined here as that stage in which the synaptonemal complex is continuous throughout the bivalents) nuclei from one wild-type germarium has permitted the following observations. 1) Drosophila melanogaster bivalents at pachytene exhibit a chromocentral arrangement; the pericentric heterochromatin of all bivalents lies in one region of the nucleus, the chromocenter. Telomeric ends do not appear to abutt the nuclear envelope. 2) Synaptonemal complex is present in the pericentric heterochromatin; however, it is morphologically distinct from that present in the euchromatic portion of the bivalents. 3) Length of the synaptonemal complex of the bivalent arms is greatest at early pachytene; the synaptonemal complex then becomes progressively shorter. Minimum length is approximately one-half of the maximum. 4) Decrease in length of synaptonemal complex is accompanied by an increase in thickness. Reconstruction of 20 pachytene nuclei from an additional 8 germaria suggests that these observations are typical. Correlations between these cytological observations and genetic observations (e.g., patterns of crossing-over) are discussed.     

Localization of genes affecting species differences in male courtship song between Drosophila virilis and D. littoralis

The males of six species of the Drosophila virilis group (including D. virilis) keep their wings extended while producing a train of sound pulses, where the pulses follow each other without any pause. The males of the remaining five species of the group produce only one sound pulse during each wing extension/vibration, which results in species-specific songs with long pauses (in D. littoralis about 300 ms) between successive sound pulses. Genetic analyses of the differences between the songs of D. virilis and D. littoralis showed that species-specific song traits are affected by genes on the X chromosome, and for the length of pause, also by genes on chromosomes 3 and 4. The X chromosomal genes having a major impact on pulse and pause length were tightly linked with white, apricot and notched marker genes located at the proximal third of the chromosome. A large inversion in D. littoralis, marked by notched, prevents more precise localization of these genes by classical crossing methods.     

Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis

The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.     

Purification and enzyme stability of alcohol dehydrogenase from Drosophila simulans, Drosophila virilis and Drosophila melanogaster adhS.

Three alcohol dehydrogenases from Drosophila simulans, Drosophila virillis and Drosophila melanogaster adhS (which possesses an alloenzyme with slow electrophoretic mobility) were purified essentially to homogeneity. The purification procedure involves a new step of affinity chromatography, which efficiently lowers the amount of contaminants in the final preparation, producing a very stable enzyme. The purification procedure developed consists of a salmine sulphate precipitation, two CM-Sepharose CL-6B colume-chromatography steps, an affinity-chromatography step and a Sephacryl gel filtration. A minimum of 30-fold purification is obtained and the yield is not less than 34%. The isoelectric points and molar absorption coefficients were determined.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Genetics of Drosophila simulans male mating discrimination in crosses with D. melanogaster

The genetic bases of sexual isolation between Drosophila melanogaster and D. simulans have been mainly studied in females, and there is little information about the role of the males in interspecific mating discrimination. Using D. simulans synthetic lines with compound chromosomes from a population of the Seychelles Islands (high frequency of interspecific mating) and a multimarker strain (low frequency), we show that D. simulans males play an important role in discriminating D. melanogaster females. The genetics of male discrimination fits well with the inheritance mode of a single locus, dominant for sexual isolation, located in chromosome II near the net mutation (2L-0.0). The heterospecific mating success of the male was not related to his sexual vigor. The specific load of male cuticular hydrocarbons was counted as a possible source of discrimination used by the D. melanogaster female.     

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.     

Genetic evidence that nonhomologous disjunction and meiotic drive are properties of wild-type Drosophila melanogaster male meiosis

We have followed sex and second chromosome disjunction, and the effects of these chromosomes on sperm function, in four genotypes: wild-type males, males deficient for the Y-linked crystal locus, males with an X chromosome heterochromatic deficiency that deletes all X-Y pairing sites, and males with both deficiencies. Both mutant situations provoke chromosome misbehavior, but the disjunctional defects are quite different. Deficiency of the X heterochromatin, consonant with the lack of pairing sites, mostly disrupts X-Y disjunction with a decidedly second-level effect on major autosome behavior. Deleting crystal, consonant with the cytological picture of postpairing chromatin-condensation problems, disrupts sex and autosome disjunction equally. Even when the mutant-induced nondisjunction has very different mechanics, however, and even more importantly, even in the wild type, there is strong, and similar, meiotic drive. The presence of meiotic drive when disjunction is disrupted by distinctly different mechanisms supports the notion that drive is a normal cellular response to meiotic problems rather than a direct effect of particular mutants. Most surprisingly, in both wild-type and crystal-deficient males the Y chromosome moves to the opposite pole from a pair of nondisjoined second chromosomes nearly 100% of the time. This nonhomologous interaction is, however, absent when the X heterochromatin is deleted. The nonhomologous disjunction of the sex and second chromosomes may be the genetic consequence of the chromosomal compartmentalization seen by deconvolution microscopy, and the absence of Y-2 disjunction when the X heterochromatin is deleted suggests that XY pairing itself, or a previously unrecognized heterochromatic function, is prerequisite to this macrostructural organization of the chromosomes.     

A single amino-acid substitution toggles chloride dependence of the alpha-amylase paralog amyrel in Drosophila melanogaster and Drosophila virilis species

In animals, most α-amylases are chloride-dependent enzymes. A chloride ion is required for allosteric activation and is coordinated by one asparagine and two arginine side chains. Whereas the asparagine and one arginine are strictly conserved, the main chloride binding arginine is replaced by a glutamine in some rare instances, resulting in the loss of chloride binding and activation. Amyrel is a distant paralogue of α-amylase in Diptera, which was not characterized biochemically to date. Amyrel shows both substitutions depending on the species. In Drosophila melanogaster, an arginine is present in the sequence but in Drosophila virilis, a glutamine occurs at this position. We have investigated basic enzymological parameters and the dependence to chloride of Amyrel of both species, produced in yeast, and in mutants substituting arginine to glutamine or glutamine to arginine. We found that the amylolytic activity of Amyrel is about thirty times weaker than the classical Drosophila α-amylase, and that the substitution of the arginine by a glutamine in D. melanogaster suppressed the chloride-dependence but was detrimental to activity. In contrast, changing the glutamine into an arginine rendered D. virilis Amyrel chloride-dependent, and interestingly, significantly increased its catalytic efficiency. These results show that the chloride ion is not mandatory for Amyrel but stimulates the reaction rate. The possible phylogenetic origin of the arginine/glutamine substitution is also discussed.     

A method for quantifying aggression in male Drosophila melanogaster

Aggressive behavior is a complex social behavior that is difficult to measure. Here, we describe a simple method for the quantitative analysis of aggression in male Drosophila melanogaster. Traditional measurements of aggressive behavior have relied on a territorial context with a food territory and a female as factors that induce or enhance aggression. The protocol described here is devoid of a food territory or a female, making it simpler than most existing methods used to measure aggressive behavior. Multiple pairs of males are tested simultaneously to obtain an average fighting score. Four parameters are used to quantify the behavior: frequency, index, latency and intensity of fighting based on unambiguous offensive fighting behaviors. The assay takes 15 min, during which time a frequency score is obtained for 20-35 pairs simultaneously. More in-depth analysis, including latency, index and intensity, can be performed on the videotaped record of the experiment. The assay is highly reproducible and requires limited resources in a simple setup.