Neural development in an imaginal disc mutant of Drosophila melanogaster

The neural phenotype of an imaginal disc degenerate mutant l(1)d deg-3 was studied in histological sections. The mutant larvae showed severe abnormalities in the imaginal neural development. Gynandromorphs, which are composed of genetically mutant and nonmutant cells, were generated and analyzed as late larvae. The results of mosaic analysis were consistent with l(1)d deg-3 gene acting autonomously in the imaginal disc and imaginal neural cells. The optic lobe development patterns observed in the larval mosaics provided evidence for an eye disc-optic lobe interaction during the late third instar larval stage.     

The roles of miRNAs in wing imaginal disc development in Drosophila

During development, it is essential for gene expression to occur in a very precise spatial and temporal manner. There are many levels at which regulation of gene expression can occur, and recent evidence demonstrates the importance of mRNA stability in governing the amount of mRNA that can be translated into functional protein. One of the most important discoveries in this field has been miRNAs (microRNAs) and their function in targeting specific mRNAs for repression. The wing imaginal discs of Drosophila are an excellent model system to study the roles of miRNAs during development and illustrate their importance in gene regulation. This review aims at discussing the developmental processes where control of gene expression by miRNAs is required, together with the known mechanisms of this regulation. These developmental processes include Hox gene regulation, developmental timing, growth control, specification of SOPs (sensory organ precursors) and the regulation of signalling pathways.     

Ecdysones and imaginal disc development during the last larval instar of Pieris brassicae

Ecdysone haemolymph levels and in vivo development of imaginal wing discs have been studied during the last larval instar of Pieris brassicae.During this period, β-ecdysone variations show two successive peaks, the first one related to the induction of wandering stage, and the second (main) one to pupal cuticle synthesis. The observed situation is very similar to that of Manduca sexta. Imaginal wing disc growth is composed of several genetically programmed steps that need the presence of ecdysone, but do not appear very closely linked to circulating hormone levels. It seems that ecdysone haemolymph peaks should be considered as periods where ecdysone levels are above a threshold value.     

The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. We present evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. We present evidence that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. We suggest that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development.     

Compartments and distal outgrowth in theDrosophila imaginal wing disc

Summary Peripheral tissue of the imaginal wing disc gives rise to the proximal mesothoracic structures of the adult. Pieces of peripheral tissue, which have no regenerative capacity when cultured as intact fragments, are capable of distal outgrowth (regeneration) after dissociation and reaggregation. This ability depends on the region of the disc periphery from which the fragment is taken. Extensive distal outgrowth occurs in reaggreages of a fragment containing equal proportions of tissue from anterior and posterior developmental compartments. The extent of outgrowth decreases as the proportion of posterior tissue is reduced, so that a fragment containing only anterior tissue shows no regeneration after dissociation. Limited distal outgrowth occurs in reaggregates of a wholly posterior fragment, but the regenerative capacity is increased greatly when a small amount of anterior tissue is included. It is concluded that distal outgrowth in the wing disc requires an interaction between cells of the anterior and posterior compartments.     

Dissociation and sorting out of Drosophila imaginal disc cells

Previous attempts to study sorting out of Drosophila imaginal disc cells have been hampered by an inability to thoroughly dissociate these cells and the need to use cuticular markers which require several days of in vivo culture. This study overcomes these limitations by using a new dissociation procedure and a genetic marker for undifferentiated cells, the succinate dehydrogenase8 (sdh8) mutation. Dissociated and reaggregated cells from wing and leg imaginal discs segregated or "sorted out" from one another after only 24 hr of in vivo culture. It was also found that leg cells from different body segments may sort out, but to a lesser degree than wing and leg cells. Mixtures of wing and haltere cells did not sort out, in contrast to previous reports. These results constitute the first unambiguous study of sorting out with Drosophila imaginal disc cells and indicate that dorsally situated imaginal cells share a recognition specificity which is different from that of ventral imaginal cells.     

The eye imaginal disc as a model to study the coordination of neuronal and glial development

A complex nervous system comprises two distinct cell types, neurons and glial cells, whose development, differentiation and function is mutually interdependent. Many studies contributed to uncovering the basic mechanisms determining neuronal and glial fate and we are progressing enormously towards an understanding of how neurons interconnect to form intricate neuronal networks. However, the mechanisms used to couple neuronal and glial development remain largely obscure. Here we advocate the usefulness of the developing Drosophila compound eye as a new model to study the complex relationship between glial and neuronal cells.     

Drosophila myt1 is the major cdk1 inhibitory kinase for wing imaginal disc development

Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Wee1) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dWee1. Although dWee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these myt1 mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes.     

Regulative interaction of mature imaginal disc Fragments with embryonic and immature disc tissues inDrosophila

Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.     

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.     

Cell-cell and cell-substrate adhesion in cultured Drosophila imaginal disc cells

Summary Drosophila imaginal disc cell lines were used to investigate various aspects of cellular adhesion in vitro. The distribution of PS integrins and their involvement in cell-cell and cell-substrate adhesion were assessed with the monoclonal antibody aBG-1 against the βPS subunit, and both forms of adhesion were found to be impeded by the presence of the antibody. Adhesion to a number of extracellular matrix components was investigated, and the cells were found to adhere to human fibronectin. This adhesion was inhibited by aBG-1. The adhesion molecule fasciclin III was also found in these cells. Given that the cells are competent to perform cell-cell and cell-substrate adhesion, it was thought that apical basal polarity might be restored when other suitable conditions were provided, i.e., an artificial basement layer with feeder cells to provide nutrients basally to the cells, and some features of apical-basal morphology were seen in cells cultured under these conditions.     

The Werner syndrome helicase/exonuclease (WRN) disrupts and degrades D-loops in vitro

The loss of function of WRN, a DNA helicase and exonuclease, causes the premature aging disease Werner syndrome. A hallmark feature of cells lacking WRN is genomic instability typified by elevated illegitimate recombination events and accelerated loss of telomeric sequences. In this study, the activities of WRN were examined on a displacement loop (D-loop) DNA substrate that mimics an intermediate formed during the strand invasion step of many recombinational processes. Our results indicate that this model substrate is specifically bound by WRN and efficiently disrupted by its helicase activity. In addition, the 3' end of the inserted strand of this D-loop structure is readily attacked by the 3'-->5' exonuclease function of WRN. These results indicate that D-loop structures are favored sites for WRN action. Thus, WRN may participate in DNA metabolic processes that utilize these structures, such as recombination and telomere maintenance pathways.     

Function and regulation of homothorax in the wing imaginal disc of Drosophila

The gene homothorax (hth) is originally expressed uniformly in the wing imaginal disc but, during development, its activity is restricted to the cells that form the thorax and the hinge, where the wing blade attaches to the thorax, and eliminated in the wing pouch, which forms the wing blade. We show that hth repression in the wing pouch is a prerequisite for wing development; forcing hth expression prevents growth of the wing blade. Both the Dpp and the Wg pathways are involved in hth repression. Cells unable to process the Dpp (lacking thick veins or Mothers against Dpp activity) or the Wg (lacking dishevelled function) signal express hth in the wing pouch. We have identified vestigial (vg) as a Wg and Dpp response factor that is involved in hth control. In contrast to its repressing role in the wing pouch, wg upregulates hth expression in the hinge. We have also identified the gene teashirt (tsh) as a positive regulator of hth in the hinge. tsh plays a role specifying hinge structures, possibly in co-operation with hth.     

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.     

Compartments and the control of growth in the Drosophila wing imaginal disc

The mechanisms that control organ growth are among the least known in development. This is particularly the case for the process in which growth is arrested once final size is reached. We have studied this problem in the wing disc of Drosophila, the developmental and growth parameters of which are well known. We have devised a method to generate entire fast-growing Minute(+) (M(+)) discs or compartments in slow developing Minute/+ (M/+) larvae. Under these conditions, a M(+) wing disc gains at least 20 hours of additional development time. Yet it grows to the same size of Minute/+ discs developing in M/+ larvae. We have also generated wing discs in which all the cells in either the anterior (A) or the posterior (P) compartment are transformed from M/+ to M(+). We find that the difference in the cell division rate of their cells is reflected in autonomous differences in the developmental progression of these compartments: each grows at its own rate and manifests autonomous regulation in the expression of the developmental genes wingless and vestigial. In spite of these differences, ;mosaic' discs comprising fast and slow compartments differentiate into adult wings of the correct size and shape. Our results demonstrate that imaginal discs possess an autonomous mechanism with which to arrest growth in anterior and posterior compartments, which behave as independent developmental units. We propose that this mechanism does not act by preventing cell divisions, but by lengthening the division cycle.     

Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates

Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA. Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication. Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy. The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif. The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 (approximately 9 A) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn33 as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity. We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.