Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.     

Specific expression of the Drosophila midline-jumper retro-transposon in embryonic CNS midline cells

Here we describe of a novel Drosophila LTR-type retrotransposon that is expressed in the embryonic CNS midline glia and in the embryonic germ cells. The element is related to the gypsy and burdock retrotransposons and was termed midline-jumper. In addition to cDNA clones generated from internal retrotransposon sequences, we have identified one cDNA clone that appears to reflect a transposition event, indicating that the midline-jumper retrotransposon is not only transcribed but also able to transpose during Drosophila development.     

Postembryonic Hematopoiesis in Drosophila

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte.     

Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development

The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TORC1 proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. We found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. We found that ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, our genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from our data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development.     

Search for Drosophila genes encoding a conserved domain present in the achaete-scute complex and myc proteins

Summary Several genes of the achaete-scute complex (ASC) of Drosophila melanogaster encode a 60 amino acids long conserved domain which shares a significant homology with a region of the vertebrate myc proteins. Based on these results, the existence of a family of Drosophila genes that would share both this conserved domain and the neurogenic function of the AS-C has been postulated. To test this proposal, we have searched a D. melanogaster genomic library with a probe that encodes the conserved domain. Only under very low stringency hybridization conditions, clones not belonging to the AS-C cross-hybridized with the probe. Those that gave the strongest signals were characterized. Sequencing of the cross-hybridizing regions showed that they had no significant homology with the conserved domain, the sequence similarity extending at the most for 37 nucleotides. Although our results do not conclusively disprove the existence of a family of AS-C-like genes, they indicate that the conservation of the domain would be lower than that found for shared motifs in other families of Drosophila developmental genes.     

Functional interactions between the gene tetanic and the Shaker gene complex of Drosophila

Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII [l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K⁺ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.     

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.     

Utilization and tolerance of ethanol, acetic acid and acetaldehyde vapour by Asobara persimilis, a parasitoid of Drosophila

The tolerance and utilization of ethanol, acetic acid and acetaldehyde vapour was investigated in Asobara persimilis (Hymenoptera: Braconidae), a parasitoid of Drosophila. No significant utilization of ethanol or acetaldehyde occurred at low concentrations (< 1.5% and 0.1% respectively), however both female and male longevity was increased at concentrations of 1.0 and 1.5% acetic acid. All substances were toxic at higher concentrations, but there was sexual dimorphism in that females survived significantly longer than males.

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Résumé L'étude a porté sur la tolérance et l'utilisation de vapeurs d'éthanol, d'acide acétique et d'acétaldéhyde par Asobara persimilis (Hym. Braconidae), parasitoïde de Drosophila récemment découvert en Australie. Aux faibles concentrations, il n'y a pas d'utilisation significative d'éthanol et d'acide (respectivement moins de 1,5% et 0,1%), cependant la longévité des mâles et des femelles ont augmenté avec les concentrations d'acide acétique de 1,0 et 1,5%. Toutes ces substances sont toxiques à plus forte concentrations, bien qu'il y ait un dimorphisme sexuel et que les femelles survivent significativement plus longtemps que les mâles. La tolérance des braconides est inférieure à celle de leurs hôtes, les Drosophiles cosmopolites et endémiques à l'Australie. Ceci peut faire que ces parasites limitent l'exploitation de leurs hôtes aux habitats avec une faible concentration de produits de fermentation.

     

Retention of autonomous replicating plasmids in cultured Drosophila cells

Summary Six kinds of autonomously replicating sequences (ARSs) derived from Drosophila or tobacco were inserted into the vector pDSV, constructed with pSV2-gpt and the copia long terminal repeat (LTR). The resulting ARS-containing plasmids, pDSV-ARSs, were transfected into the cultured Drosophila cells of GM1 S1cl1. Most of the plasmids remained for about 2 weeks and some for about 1 month in these cells. The retention time of the plasmid was not directly correlated with autonomously replicating activity of ARSs detected in the yeast. Two plasmids, one carrying ARS of Drosophila nuclear DNA and the other carrying tobacco DNA, showed the longest retention time in transformed cells and replication was confirmed in these cells. Some of these long lived plasmids were recovered, however, as modified forms. Other plasmids had disappeared 1 month after transfection. Two months following transfection, none of plasmids were recovered but they were detected in nuclear DNA as the integrated form. The integration patterns in all the cells transformed by different kinds of ARS-containing plasmids were similar to each other, and to the distribution pattern of copia LTR in the genome. These results suggest that copia LTR sequences contained in the pDSV-ARSs may participate in the integration process of these plasmids into Drosophila DNA.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.     

Phenotypic suppression of the Drosophila mitochondrial disease-like mutant tko by duplication of the mutant gene in its natural chromosomal context

A mutation in the Drosophila gene technical knockout (tko^25t), encoding mitoribosomal protein S12, phenocopies human mitochondrial disease. We isolated three spontaneous X-dominant suppressors of tko^25t (designated Weeble), exhibiting almost wild-type phenotype and containing overlapping segmental duplications including the mutant allele, plus a second mitoribosomal protein gene, mRpL14. Ectopic, expressed copies of tko^25t and mRpL14 conferred no phenotypic suppression. When placed over a null allele of tko, Weeble retained the mutant phenotype, even in the presence of additional transgenic copies of tko^25t. Increased mutant gene dosage can thus compensate the mutant phenotype, but only when located in its normal chromosomal context.     

Metabolic pools differ among ecologically diverse Drosophila species

Studies of the genetic mechanisms underlying metabolic storage have focused on a few model organisms. Although very fruitful, these studies have not allowed for the examination of mechanisms across a phylogenetic spectrum. The exploration of natural patterns of metabolic pool size variation across species will help us to better understand the genetics of metabolic adaptation.We examined the metabolic pools size (triglyceride, glycogen and protein) at two ages in 12 Drosophila species with distinctly different ecologies for which complete genome sequences (for 11 of the 12 species) are known. Overall, there were significant differences across species for all three pools, while age and sex appear to affect some metabolic pools more than others. After correcting for the phylogenetic relatedness of the species used, we observed no association between triglyceride and glycogen content. Although within species these two pools sometimes are correlated, at a larger phylogenetic scale control of triglyceride and glycogen contents may have been shaped independently by natural selection.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

TRP gating is linked to the metabolic state and maintenance of the Drosophila photoreceptor cells

The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. In spite of much progress, the gating mechanism of TRP channels is still unknown. However, recent studies have shown multi-faceted functions of the Drosophila light-sensitive TRP channel that may shed light on TRP gating. Accordingly, metabolic stress, which leads to depletion of cellular ATP, reversibly activates the Drosophila TRP and TRPL channels in the dark in a constitutive manner. In several Drosophila mutants, constitutive activity of TRP channels lead to a rapid retinal degeneration in the dark, while genetic elimination of TRP protects the cells from degeneration. Additional studies have shown that TRPL translocates in a light-dependent manner between the signaling membranes and the cell body. This light-activated translocation is accompanied by reversible morphological changes leading to partial and reversible collapse of the microvillar signaling membranes into the cytosol, which allows turnover of signaling molecules. These morphological changes are also blocked by genetic elimination of TRP channels. The link of TRP gating to the metabolic state and maintenance of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death upon malfunction.     

In vivo modification of Na,K-ATPase activity in Drosophila

We have constructed and characterized transgenic Drosophila lines with modified Na⁺,K⁺-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na⁺,K⁺-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (α^D369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na⁺,K⁺-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.     

Genotoxicity is modulated by ascorbic acid: Studies using the wing spot test in Drosophila

The ability of ascorbic acid (Vitamin C) to modulate the genotoxic action of several mutagens was investigated in the wing spot test of Drosophila melanogaster. In this assay, 3-day-old transheterozygous larvae for the multiple wing hairs (mwh, 3-0.3) and flare (flr, 3-38.8) genes were treated with three reference mutagenic compounds, namely cobalt chloride (CoCl₂), 4-nitroquinoline 1-oxide (4-NQO) and potassium dichromate (K₂Cr₂O₇). The results obtained show that the three reference mutagens tested were clearly genotoxic in the Drosophila wing somatic mutation and recombination test (SMART). None of the three concentrations tested of ascorbic acid (25, 75 and 250 mM) induced significant increases in the frequency of the mutant clones recorded. When co-treatment experiments with ascorbic acid were carried out, different results were found. Thus, ascorbic acid was effective in reducing the genotoxicity of K₂Cr₂O₇ virtually to the control level; on the contrary, it did not show any antigenotoxic effect on the genotoxicity of 4-NQO. Finally, co-treatments with CoCl₂ and ascorbic acid show a significant increase in the frequency of mutant clones over the values obtained with CoCl₂ alone.     

Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis

During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places that these actin regulators shape oogenesis.