Halving the selenophosphate synthetase gene dose confers hypersensitivity to oxidative stress in Drosophila melanogaster

Several lines of evidence indicate that selenoproteins mainly act as cellular antioxidants. Here, we test this idea comparing the sensitivity to oxidative stress (paraquat and hydrogen peroxide) between wild type and heterozygous flies for the selenophosphate synthetase selD(ptuf) mutation. Whereas under normal laboratory conditions no difference in life span is observed, a significant decrease is seen in heterozygous flies treated with oxidant agents. In contrast, overexpression of the selD gene in motoneurons did not extend longevity. Our results strongly suggest that selD haploinsufficiency makes heterozygous flies more sensitive to oxidative stress and add further evidence to the role of selenoproteins as cellular antioxidants.     

Modulation of the Ras/MAPK signalling pathway by the redox function of selenoproteins in Drosophila melanogaster.

Modulation of reactive oxygen species (ROS) plays a key role in signal transduction pathways. Selenoproteins act controlling the redox balance of the cell. We have studied how the alteration of the redox balance caused by patufet (selD(ptuf)), a null mutation in the Drosophila melanogaster selenophosphate synthetase 1 (sps1) gene, which codes for the SelD enzyme of the selenoprotein biosynthesis, affects the Ras/MAPK signalling pathway. The selD(ptuf) mutation dominantly suppresses the phenotypes in the eye and the wing caused by hyperactivation of the Ras/MAPK cassette and the activated forms of the Drosophila EGF receptor (DER) and Sevenless (Sev) receptor tyrosine kinases (RTKs), which signal in the eye and wing, respectively. No dominant interaction is observed with sensitized conditions in the Wnt, Notch, Insulin-Pi3K, and DPP signalling pathways. Our current hypothesis is that selenoproteins selectively modulate the Ras/MAPK signalling pathway through their antioxidant function. This is further supported by the fact that a selenoprotein-independent increase in ROS caused by the catalase amorphic Cat(n1) allele also reduces Ras/MAPK signalling. Here, we present the first evidence for the role of intracellular redox environment in signalling pathways in Drosophila as a whole organism.     

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.     

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. Received: 22 August 1997 / Accepted: 9 September 1997     

Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth

Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs (l(2)gd) which remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. On the other hand, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. We conclude that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ.     

double-time is identical to discs overgrown, which is required for cell survival, proliferation and growth arrest in Drosophila imaginal discs.

We have isolated the discs overgrown gene of Drosophila and shown that it encodes a homolog of the Casein kinase I(delta)/(epsilon) subfamily and is identical to the double-time gene. However, in contrast to the weak double-time alleles, which appear to affect only the circadian rhythm, discs overgrown alleles, including bona fide null alleles, show strong effects on cell survival and growth control in imaginal discs. Analysis of their phenotypes and molecular lesions suggests that the Discs overgrown protein is a crucial component in the mechanism that links cell survival during proliferation to growth arrest in imaginal discs. This work provides the first analysis in a multicellular organism of Casein kinase I(delta)/(epsilon) functions necessary for survival. Since the amino acid sequences and three-dimensional structures of Casein kinase I(delta)/(epsilon) enzymes are highly conserved, the results suggest that these proteins may also function in controlling cell growth and survival in other organisms.     

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.     

Effect of mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> on proliferation and differentiation of wing cells

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.     

The Drosophila tumor suppressor expanded regulates growth, apoptosis, and patterning during development

The Drosophila expanded (ex) gene encodes a protein thought to play a role in signaling at apical junctions of epithelial cells. Previous studies have characterized this gene as a tumor suppressor involved in regulating the growth of a subset of Drosophila imaginal discs (Boedigheimer, M., Laughon, A., 1993. expanded: a gene involved in the control of cell proliferation in imaginal discs, Development 118, 1291-1301); although ex negatively regulates cell proliferation in the developing wing, it appeared to have a conflicting role in the eye. In contrast, our analysis of the loss-of-function phenotype indicates that ex does, in fact, regulate growth in the eye. We also show that this gene plays a role in patterning of the eye, mainly at the level of planar polarity. Our studies further demonstrate that, contrary to what was expected based on loss-of-function data, the tissue reduction phenotypes resulting from Ex overexpression are attributable to the induction of apoptotic cell death. Taken together, our data suggest that Ex is a versatile molecule that plays a role in most of the processes that govern disc development.     

Developmental expression and tissue distribution of the lethal (2) giant larvae protein of Drosophila melanogaster

Recessive mutations in the Drosophila tumor gene lethal (2) giant larvae affect the growth and tissue specificity of determined cells in imaginal discs and presumptive optic centers of the brain. To analyse the function of the l (2) gl gene during development, we have raised monoclonal antibodies against the l (2) gl protein. These antibodies detect a 130-kd protein in wild-type tissue which is absent in homozygous mutant tissues. The protein is detected in increasing amounts up to mid-embryonic stages. Antibody binding to embryo sections and indirect immunofluorescence labeling indicate that the protein is localized at the cellular membranes or in the intercellular matrix of the embryonic cells. The primordia of all larval tissues are labeled in the embryo. Much less labeling is found in the neural primordia of the central nervous system, except that within the supraoesophageal ganglion the regions of the presumptive optic centers are distinctly labeled. Moreover, the axon bundles of the ventral cord are labeled in the embryo, apparently a reflection of the accumulation of cell membranes here. After embryogenesis the l (2) gl protein is found at a low level until the end of the 3rd larval instar, when it is preferentially seen in the brain and imaginal discs. The protein distribution in embryonic and larval tissues correlates with already known proliferation patterns, which could indicate that the l (2) gl protein is involved in proliferation arrest of cells.     

Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain

We have previously cloned lethal(2)giant larvae, a tumor-suppressor gene of Drosophila that normally controls cell proliferation and/or differentiation in the optic centers of the brain and the imaginal discs. Here we describe the structure of the l(2)gl genes as determined by sequencing genomic and cDNA clones. The structure of the cDNAs indicates the use of alternative splicing, either in the 5' untranslated exons or in the 3' coding exons. Thus the gene encodes two putative proteins of 1161 and 708 amino acids, p127 and p78, respectively, differing at their C termini. A 3'-truncated l(2)gl transposon that leaves the coding sequence of p78 intact but deletes 141 residues of p127 was capable of suppressing tumor formation in l(2)gl-deficient animals. These results suggest that the putative p78 protein is effective in controlling cell proliferation and/or differentiation.     

The BEAF insulator regulates genes involved in cell polarity and neoplastic growth

Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB

Organismal size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organismal size. Here we show that genetic manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (protein kinase B) during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner. Ectopic expression of Akt does not affect cell-fate determination, apoptosis or proliferation rates in imaginal discs. Thus, Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independently of cell proliferation in vivo.     

The distribution of mitoses in the imaginal disks of third-instar Drosophila melanogaster larvae

Development of Drosophila imaginal discs is accompanied by a high-ordered cell proliferation. However, the distinctions in the topographic distribution of mitoses at different developmental stages are insufficiently studied. In this work, we have analyzed the distribution of mitoses in the wing disc of third-instar larvae and determined the regions where mitotic clustering. The results obtained demonstrate that the proliferation rate is region-specific, which is determined by the location of cell cycle regulators and/or the location of growth factors. A comparison of the topography of mitoses with the activity patterns of the regulatory regions of gene string (stg), a known regulator of the mitotic M phase, has demonstrated a similarity between the topography and the activity pattern of one of these regions. The similarity between mitotic distributions in the left and right discs of the same larva (compared with the similarity of gene neuralized expression patterns is considered, and the degree of histone H3 phosphorylation at various mitotic stages is analyzed.     

Analysis of the autonomy of imaginal disc defects in a small-disc mutant of Drosophila melanogaster.

Lethal mutations which cause imaginal disc abnormalities in Drosophila melanogaster identify genes whose function is necessary for normal disc development, and these mutant genes may be used as probes of the role of their wild-type alleles in normal development. It is crucial to the interpretation of the disc phenotype of such mutants to know which abnormalities are autonomous (caused by expression of the mutant gene in imaginal cells) and which are nonautonomous (indirectly caused, for example, by expression of the mutant gene in larval cells). We chose for study l(3)c21R (3-67.8), a late-larval lethal mutation with a complex phenotype, to test the adequacy of available techniques for assessing autonomy. We employed surgical and genetic techniques to determine the imaginal cell autonomy of the defects in cell viability, growth, and differentiation in c21R discs. The imaginal cell viability defect is nonautonomous. The disc growth and differentiation defects are autonomous; however, in genetic mosaics these two autonomous defects are separable. These results show that c21R belongs to the class of mutations which affect both larval and imaginal cells. In combination, the available methods were adequate to resolve the issue of autonomy in this complex case. However, in isolation several of the methods could have led to incomplete or misleading interpretations. This emphasizes that to analyze any developmental mutant it is necessary to examine the issue of autonomy from several points of view.     

Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs

Cells employ a diverse array of signaling mechanisms to establish spatial patterns during development. Nowhere is this better understood than in Drosophila, where the limbs and eyes arise from discrete epithelial sacs called imaginal discs. Molecular-genetic analyses of pattern formation have generally treated discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. Here we demonstrate that during development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development.     

The suppressor of forked gene of Drosophila, which encodes a homologue of human CstF-77K involved in mRNA 3'-end processing, is required for progression through mitosis.

The Suppressor of forked (Su(f)) protein of Drosophila melanogaster is a homologue of the 77K subunit of human cleavage stimulation factor required for cleavage of pre-mRNAs before addition of poly(A). We have previously shown that the Su(f) protein is not ubiquitously distributed: it accumulates in dividing cells at various stages of Drosophila development. In this paper, we show that phenotypes of su(f) temperature-sensitive mutants result from a defect in cell proliferation. Analysis of the mitotic phenotype of su(f) temperature-sensitive alleles in larval brain and in imaginal discs reveals an increase in the number of metaphases with overcondensed chromosomes and asymmetric or reduced mitotic spindles. In contrast, neural differentiation in eye imaginal discs of the same mutant flies does not appear to be affected. These results indicate that su(f) is required during cell division for progression through metaphase. Taken together, these data suggest that a decrease in su(f) activity preferentially affects 3'-end formation of particular mRNAs, some of which are involved in mitosis, and are in agreement with a role of su(f) in the regulation of poly(A) site utilization.