patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster : clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed. Received: 19 June 1997 / Accepted: 4 August 1997     

Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster

Fragments from the imaginal wing disc of Drosophila melanogaster were cultured in vivo for periods up to 28 days. One type of edge fragment first duplicated and then ceased to grow, but others often continued to grow following initial duplication and regenerated structures characteristic of other areas of the disc. After 28 days of culture, about 50% of fragments from the presumptive ventral hinge region of the disc grew extensively and produced regenerated as well as original structures. The regenerated structures in some implants were produced at the line of mirror-image symmetry. Regeneration was associated with fragment growth and in many cases was accompanied by loss of duplicate structures. Fragments which were only duplicated after the culture period could in some cases be stimulated to grow by additional culture in fresh hosts, but the results of coculturing two fragments in each host show that culture conditions alone do not control growth and regulation in the fragments. The large, normally regenerating fragment, complementary to the ventral fragment, did not appear to grow following regeneration and only occasionally produced supernumerary structures during prolonged culture. Intact wing discs cultured under similar conditions never produced supernumerary structures. Our results suggest that a duplicated pattern is less stable than a complete, regenerated pattern, which in turn is less stable than an intact disc. We propose that the growth of duplicated disc fragments is stimulated by polarity reversals present at lines of mirror-image symmetry.     

Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase

The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this self-dependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selD _HI) in an E. coliΔselD strain. Gene selD _HI gave rise to a selenium-containing gene product and also supported – via its activity – the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGA_Sec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif. Received: 8 July 1997 / Accepted: 3 September 1997     

Wound healing and regeneration in the imaginal wing disc ofDrosophila

Summary When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In 06 fragments (Bryant 1975a) wound healing has occurred after 1–2 days of culture and the regeneration of missing structures after 2–4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to³H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.     

Expression and localization of clathrin heavy chain in Drosophila melanogaster

Clathrin-coated vesicles mediate cellular endocytosis of nutrients and molecules that are involved in a variety of biological processes. Basic components of the vesicle coat are clathrin heavy chain (Chc) and clathrin light chain molecules. In Drosophila melanogaster the chc gene function has been analyzed in a number of previous studies mainly using genetic approaches. However, the chc mRNA and protein expression patterns have not been studied systematically. We have generated an antibody that specifically recognizes Chc and we have analyzed chc RNA and protein expression patterns throughout embryonic and larval stages. We found that chc mRNA and protein are highly expressed from early stages of embryogenesis onwards, consistent with genetic studies predicting a maternal contribution of the gene function. During subsequent stages mRNA and protein are co-expressed in all embryonic cells; however we found an up-regulation in specific tissues including the gut, the salivary glands, tracheal system and the epidermis. In addition the central nervous system and the nephrocyte-like garland cells show strong Chc expression at late embryogenesis. In larvae Chc is highly expressed in garland cells, imaginal discs, fat body, salivary glands and the ring gland. Subcellularly, we found Chc protein in a vesicle-like pattern within the cytoplasm and at the plasma membrane. Co-labeling studies show that Chc is partially in contact with the trans-Golgi network and co-localizes with markers for early endocytosis. Together, the antibody may serve as a new tool to study the function of Chc in clathrin-dependent cellular processes, such as endocytosis.     

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

The dominant Drop eye mutations of Drosophila melanogaster define two loci implicated in normal eye development

The three existing dominant gain-of-function Drop alleles, Dr ¹, Dr ^Mio and Dr ^We , previously assumed to define a single locus, severely disrupt eye development. Genetic analysis of ethylmethanesulphonate (EMS) and irradiation-induced revertants revealed that the Drop mutations define two loci: the Drop locus, which is defined by the Dr ¹ and Dr ^Mio mutants, and a separate locus defined by the Dr ^We mutation, which has been renamed Wedge. The majority of the Dr ¹ and Dr ^Mio revertants are embryonic lethal in trans, mutant embryos exhibiting trachea that fail to join the Filzkörper, thus revealing a role for the Drop gene in embryogenesis. Clonal analysis of lethal revertant alleles suggests a role for both genes in eye development. In the Drop homozygous mutant clones, the outer photoreceptor cells R1–R6 develop aberrantly. Wedge, however, is not required by the developing photoreceptor cells but its absence does disrupt normal ommatidial alignment. Although the Drop and nearby string loci were shown to be genetically distinct, both Dr ¹ and Dr ^Mio were found to interact in trans with lesions at the string locus, causing loss and derangement of bristles and loss of neuromuscular coordination.     

The behavior during the initial phase of in vitro aggregation of dissociated imaginal disc cells from Drosophila melanogaster

Aspects of the initial phase of aggregation in vitro of dissociated imaginal disc cells from Drosophila melanogaster are described. Using the methods described certain interdisc differences in the percent decrease in single cell units during the first hour of aggregation can be demonstrated. In addition it is shown that prospective notum cells isolated from the dorsal mesothoracic disc show less of a decrease than do prospective wing cells. This difference shows up in a variety of different wild-type stocks and in several mutant stocks as well. Prospective notum cells from the mutation fu59 show only a limited ability to adhere to one another, while the percentage of single cell units from prospective wing blade cells from r9 larvae grown on pyrimidine-poor medium decrease less compared to cells from the same stock grown on RNA-supplemented medium. There is a significantly greater decrease in the percent single cell units in cultures of prospective eye cells than in cultures of prospective antennal cells. Furthermore, cells from the antennal disc of larvae bearing the homeotic mutation ssa show a significantly lower decrease in single cell units when grown at restrictive temperatures. In contrast, antennal disc cells from the homeotic mutation ophthalmoptera; eyeless Dominant, a mutation which affects the eye disc, are unaffected, while cells from the eye disc are slightly less able to reassociate with one another.     

Differentiation capacities of the labial imaginal disc ofDrosophila melanogaster

Summary The mature labial disc, when implanted into a larva of the same age, undergoes metamorphosis along with the host and produces one lateral half of the medi- and distiproboscis. On the basis of results obtained from transplanted disc halves (including the separate peripodial membrane) a tentative fate map of the labial disc was constructed, which shows most of the presumptive mediproboscis to be located in the dorsal, and most of the presumptive distiproboscis in the ventral part of the disc. The distal protion of the peripodial membrane also contains imaginal anlagen, viz. part of the mediproboscis, prementum, and labellar cap anlagen. The involvement of this part of the peripodial membrane was checked by a careful histological analysis of labial disc development during the first ten hours after prepupation. The results were compared with the situation described forCalliphora imaginal discs.In addition, a detailed morphological analysis was made of the proboscis of the homoeotic mutantproboscipedia (pb). At 27°C,pb changes the distiproboscis into a telopodite (leg segments distal to the coxa); the (unchanged) prementum may therefore correspond to the coxa. At 15° C, the tarsus of this homoeotic telopodite is replaced to a greater or lesser extent by an arista. The present analysis thus confirms (a) the fundamental morphological correspondence of the medi- and distiproboscis with the labium of other insects, and (b) the fundamental developmental correspondence of the labial, antennal, and leg discs.K. K. was a member of the 8th International Research Group in Developmental Biology, and was the recipient of a UNESCO travel grant.)     

A further characterization of the cinnamon gene in Drosophila melanogaster

Summary Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2⁺:2^- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.The mutations map on the leaft arm of chromosome I and form a single complementation group with five other pyruvate kinase mutations in the pyk1 gene that was earlier suggested to be a regulatory locus controlling the synthesis of this enzyme. A comparative study of these mutants has been made with the structural mutants described here.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Growth is required for cell competition in the imaginal discs ofDrosophila melanogaster

Summary Imaginal wing discs from late third-instar larvae were gammairradiated to induce clones of rapidly growingMinute ^– cells in a background of slowly growingMinute cells and culturedin vivo for periods up to 18 days. Clones in discs cultured for 16 to 18 days did not grow significantly larger than clones in uncultured controls, indicating that competition between populations of cells having potentially different mitotic rates does not occur in imaginal discs after their growth is completed.     

Orphan nuclear receptor betaFTZ-F1 is required for muscle-driven morphogenetic events at the prepupal-pupal transition in Drosophila melanogaster

In Drosophila melanogaster, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. Our previous studies have supported the hypothesis that betaFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. We are examining the role of betaFTZ-F1 in morphogenesis. We have made a detailed study of morphogenetic events during metamorphosis in control and betaFTZ-F1 mutant animals. We show that leg development in betaFTZ-F1 mutants proceeds normally until the prepupal-pupal transition, when final leg elongation is delayed by several hours and significantly reduced in the mutants. We also show that betaFTZ-F1 mutants fail to fully extend their wings and to shorten their bodies at the prepupal-pupal transition. We find that betaFTZ-F1 mutants are unable to properly perform the muscle contractions that drive these processes. Several defects can be rescued by subjecting the mutants to a drop in pressure during the normal time of the prepupal-pupal transition. Our findings indicate that betaFTZ-F1 directs the muscle contraction events that drive the major morphogenetic processes during the prepupal-pupal transition in Drosophila.     

Tissue-specific expression of the acetylcholinesterase gene in Drosophila melanogaster embryos

Summary Acetylcholinesterase activity is present in both particulate and soluble forms in wild-type Drosophila melanogaster embryos. The particulate form of the enzyme is localized in the CNS, while the soluble forms are non-CNS-specific. Deletion mapping studies show that all AChE activity is abolished if the cytological region between 87E1-2 and 87E4 is missing. An additional region mapping to the proximal part of the 87E4 band is needed for CNS-specific AChE activityAbbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - ChE pseudocholinesterase (acetylcholine acylhydrolase, EC 3.1.1.8) - BAP 1,5-bis(allyldimethylammoniumphenyl)-pentan-3-one dibromide - i-OMPA tetraisopropylpyrophosphoramide - CNS central nervous system     

The role of the peripodial membrane in the morphogenesis of the eye-antennal disc ofDrosophila melanogaster

Summary The early morphogenesis of the eye-antennal disc ofDrosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial membrane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.