Belle is a Drosophila DEAD-box protein required for viability and in the germ line

DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. Here, we report identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator. We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. We conclude that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development.     

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.     

E(nos)/CG4699 required for nanos function in the female germ line of Drosophila

The translational repressor Nanos is required in the germ line stem cells of the Drosophila ovary to maintain their capacity for self‐renewal. Following division of the stem cells, Nanos is inhibited in the daughters that differentiate into cysts and ultimately become mature oocytes. The control of Nanos activity is thus an important aspect of the switch from self‐renewal to differentiation. In this report, we describe a genetic interaction between nanos and Enhancer of nos, an allele of the previously uncharacterized locus CG4699. We find that E(nos) protein is required for normal accumulation of Nanos in the ovary and thus for maintenance of the germ line. The mechanism by which E(nos)/CG4699 protein acts is not clear, although it has been found in a complex with Mof acetylase. Consistent with the finding that E(nos) interacts with Mof, we observe that nanos and mof also interact genetically to maintain normal oogenesis. genesis 48:161–170, 2010. © 2010 Wiley‐Liss, Inc.     

Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster

Octopamine is a major monoamine in invertebrates and affects many physiological processes ranging from energy metabolism to complex behaviors. Octopamine binds to receptors located on various cell types and activates distinct signal transduction pathways to produce these diverse effects. We previously identified one of the Drosophila octopamine receptors named OAMB that produces increases in cAMP and intracellular Ca2+ upon ligand binding. It is expressed at high levels in the brain. To explore OAMB's physiological roles, we generated deletions in the OAMB locus. The resultant oamb mutants were viable without gross anatomical defects. The oamb females displayed normal courtship and copulation; however, they were impaired in ovulation with many mature eggs retained in their ovaries. RT-PCR, in situ hybridization, and expression of a reporter gene revealed that OAMB was also expressed in the thoracicoabdominal ganglion, the female reproductive system, and mature eggs in the ovary. Moreover, analysis of various alleles pinpointed the requirement for OAMB in the body, but not in the brain, for female fecundity. The novel expression pattern of OAMB and its genetic resource described in this study will help advance our understanding on how the neuromodulatory or endocrine system controls reproductive physiology and behavior.     

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.     

Drosophila Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval PNS. We report a role of scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis we find that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an “off-centred” manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality.     

Mutations in supernova,indicate that this gene is required for the division of germ line cells in Drosophila

Mutations in supernova, previously shown to uncouple chromosome replication from segregation during cleavage in Drosophila embryos, also sanctions extra divisions of cystoblasts and spermatoblasts. This leads either to the formation of egg chambers which contain more than fifteen nurse cells or testes which have an excess of spermatocytes. In maturing egg chambers two potential oocytes may be specified in which case they are often ectopically located and connected with surrounding nurse cells by four ring canals. However, a typical oocyte nucleus is not always present and these chambers usually become necrotic and degenerate. The nurse cells are of variable size, but are still interconnected by a system of ring canals. They all possess a polyploid nucleus. Sequestering of maternal mRNA's from the nurse cells into the potential oocyte(s) takes place but there is no localization of this maternal information within the oocyte probably because of defective microtubule assembly. Many spermatocytes fail to complete meiosis so that bundles of spermatids are reduced in size and the males have reduced fertility. It is proposed that this gene is indirectly involved in regulating the timing of mitotic divisions in both cystoblasts and spermatoblasts through its interference with microtubule assembly which is consistent with its role during embryogenesis.     

Genotoxicity of p-aminophenol in somatic and germ line cells of Drosophila melanogaster

p-Aminophenol (PAP; as a component of, e.g., hair dyes, photographic developers, as adsorbent in gas filters, as a metabolite of various fungicides, pesticides and drugs) has been tested for genotoxicity in Drosophila by means of the sex-linked recessive lethal test (SLRLT) and the somatic mutation and recombination test (SMART) of the wing. While the SLRLT was not significant, the SMART clearly indicated that the compound has genotoxic properties in this in vivo test in agreement with a majority of mammalian short-term tests in vitro and in vivo. The reducing agent dithiothreitol enhanced the genotoxic effects of PAP in the SMART; the reasons for this interaction remain to be elucidated.     

The development and function of the female germ line in Drosophila melanogaster: a cell lineage study.

X-ray-induced mitotic recombination was used to follow the development and function of the female germ line in Drosophila melanogaster. Clones marked by maternal effect mutations which alter the morphology of the egg [fs(1)K10] or the phenotype of the resulting progeny (maroonlike) were produced in trans-heterozygotes irradiated during embryonic, larval, or pupal development or as 5-day-old adults. Judging from the size of clones induced at the blastoderm stage, only five to ten of the pole cells observed on the surface of the embryo contribute to the germ line. Most of the K10 clones induced during embryonic and larval development were associated with mal twin spots, indicating that both daughters of the irradiated germ cell remained in the germ line and gave rise to eggs in the adult. During larval life the number of cells increases logarithmically and reaches a maximum of 110 at 24 hr after pupation. The same value was obtained for 5-day-old adults. In contrast to the mosaic females produced as embryos and larvae, mosaics obtained after pupal and adult irradiations were of two types, those laying only one K10 egg and those laying several K10 eggs distributed over the lifespan of the adult. This result indicates that the stem cell divisions characteristic of the adult period have begun shortly after pupation. About 9 to 11 days are required for an irradiated stem cell to produce its first clonal K10 egg, and two-thirds of this time is spent in the germarium. Each ovariole possesses on the average two to three functioning stem cells. This multiplicity of stem cells was confirmed by the recovery of mosaic ovarioles when mal heterozygotes irradiated as adults or late larvae were stained for aldehyde oxidase activity.     

Rab11 is required for tubulogenesis of Malpighian tubules in Drosophila melanogaster

Intracellular vesicular trafficking is one of the important tools in maintaining polarity, adhesion, and shape of epithelial cells. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases and a molecular marker of recycling endosomes, transports different components of plasma membrane. Here, we report that Rab11 affects tubulogenesis of Malpighian tubules (MTs). MTs are simple polarized epithelial tubular structures, considered as functional analogue of human kidney. Rab11 has pleiotropic effects on MTs development as down‐regulation of Rab11 in principal cells (PCs) of MTs from embryonic stages of development results in reduced endoreplication, clustering of cells, disorganized cytoskeleton, and disruption of polarity leading to shortening of MTs in third instar larvae. Rab11 is also required for proper localization of different transporters in PCs, essential for physiological activity of MTs. Collectively, our data suggest that Rab11 plays a key role in the process of tubulogenesis of MTs in Drosophila.     

Acrylamide is genotoxic to the somatic and germ cells of Drosophila melanogaster

The genotoxic effects of acrylamide, a recently detected carcinogen, have been studied in the somatic (wing primordia) and germ cells of Drosophila melanogaster by the wing mosaic assay and the sex-linked recessive lethal test respectively. Larvae, 72 +/- 4 h old, were exposed to 6 different concentrations of acrylamide ranging between 0.25 mM and 5.0 mM in instant medium for 48 h. It is observed that acrylamide is both mutagenic and recombinogenic in the wing disc cells and induces sex-linked recessive lethals.