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Enhancement of overgrowth by gene interactions in lethal(2)giant discs imaginal discs from Drosophila melanogaster.

Recessive lethal mutations of the lethal(2)giant discs (l(2)gd) and lethal(2)fat (l(2)ft) loci of Drosophila melanogaster cause imaginal disc hyperplasia during a prolonged larval stage. Imaginal discs from l(2)ft l(2)gd or Gl(2)gd double homozygotes show more extensive overgrowth than in either single homozygote, and double homozygous l(2)ft l(2)gd mitotic clones in adult flies show much more overgrowth than is seen in clones homozygous for either l(2)gd or l(2)ft alone. dachsous (ds) also acts as an enhancer of l(2)gd, producing dramatically overgrown discs and causing failure to pupariate in double homozygotes. The comb gap (cg) mutation, which also interacts with ds, greatly enhances the tendency of imaginal discs from l(2)gd larvae to duplicate as they overgrow. If l(2)gd homozygotes are made heterozygous for l(2)ft, then several discs duplicate, indicating that l(2)ft acts an a dominant enhancer of l(2)gd. l(2)ft also acts as a dominant enhancer of l(2)gd, and conversely l(2)gd acts as a dominant modifier of l(2)ft. The enhancement of overgrowth caused by various mutant combinations is accompanied by changes in expression of Decapentaplegic and Wingless. These results show that tumor suppressor genes act in combination to control cell proliferation, and that tissue hyperplasia can be associated with ectopic expression of genes involved in pattern formation.

Animals↗

Developmental analysis and squamous morphogenesis of the peripodial epithelium in Drosophila imaginal discs.

Imaginal discs of Drosophila provide an excellent system with which to study morphogenesis, pattern formation and cell proliferation in an epithelium. Discs are sac-like in structure and are composed of two epithelial layers: an upper peripodial epithelium and lower disc proper. Although development of the disc proper has been studied extensively in terms of cell proliferation, cell signaling mechanisms and pattern formation, little is known about these same processes in the peripodial epithelium. We address this topic by focusing on morphogenesis, compartmental organization, proliferation and cell lineage of the PE in wing, second thoracic leg (T2) and eye discs. We show that a subset of peripodial cells in different imaginal discs undergo a cuboidal-to-squamous cell shape change at distinct larval stages. We find that this shape change requires both Hedgehog and Decapentapelagic, but not Wingless, signaling. Additionally, squamous morphogenesis shifts the anteroposterior (AP) compartment boundary in the peripodial epithelium relative to the stationary AP boundary in the disc proper. Finally, by lineage tracing cells in the PE, we surprisingly find that peripodial cells are displaced into the disc proper during larval development and this movement leads to Ubx repression.

Animals↗

Pattern formation in imaginal discs.

Imaginal discs are sacs of folded epithelium arising during embryogenesis. They proliferate during the larval instars, and at metamorphosis secrete the adult cuticle, therefore being responsible for the characteristic surface patterning of this insect. Each disc has intrinsic growth controls and their cell lineage shows constraints known as compartments. Spatial patterns emerge through interaction between adjacent cells. Molecular genetic analysis of mutants with changed pattern has implicated transcription factors, secreted, membrane-bound and growth factor related proteins in the position-signalling mechanism. Their accessibility to contemporary cell biological techniques makes imaginal discs a model system for investigating patterning in animal tissues.

Animals↗

Genes expressed during imaginal discs morphogenesis: IMP-L2, a gene expressed during imaginal disc and imaginal histoblast morphogenesis.

We describe our analysis of IMP-L2, one of a set of six ecdysone-inducible genes in imaginal discs of Drosophila whose transcripts are associated with membrane-bound polysomes. The spatial and temporal patterns of expression of the IMP-L2 transcript were analyzed. This transcript is first expressed in imaginal discs in areas that are precursors of head and thoracic epithelium, particularly the peripodial epithelia. It is later expressed in the imaginal histoblasts, precursors of the adult abdomen. The appearance of IMP-L2 transcript in each of these tissues precedes the spreading and fusion of the separate imaginal anlagen to form the continuous adult epidermis.

Animals↗

Cell determination and transdetermination in Drosophila imaginal discs.

Drosophila imaginal discs, the precursors of the adult fly appendages, are an important system for studying mechanisms of cell determination. How the different imaginal discs acquire and maintain their appendage-specific determined states are problems that have been addressed using experimental embryology as well as genetic and molecular approaches. Here we discuss the concept of cell determination and describe what is known about how determination is established and maintained in imaginal disc cells. The phenomenon of imaginal disc transdetermination, originally discovered in the 1960s, has remained an intriguing problem for understanding imaginal disc cell determination. We review the topic of imaginal disc transdetermination and describe how recent results from molecular genetic approaches have provided new insights into imaginal disc transdetermination and determination. We also discuss how an understanding of imaginal disc transdetermination can aid our understanding of parallel phenomena in other organisms, including human metaplasias.

Animals↗

The genetic control of cell proliferation in Drosophila imaginal discs.

The imaginal discs of Drosophila provide a favorable system for the analysis of the mechanisms controlling developmental cell proliferation, because of the separation in time between cell proliferation and differentiation, and the facility with which controlling genes can be identified and characterized. Imaginal discs are established in the embryo, and grow by cell proliferation throughout the larval period. Proliferation terminates in a regular spatial pattern during the final stages of larval development and the first day of pupal development. Cell proliferation can be locally reactivated in growth-terminated imaginal discs by removing part of the disc and culturing the remaining fragment in an adult host. The pattern of proliferation in these fragments suggests that cell proliferation in imaginal discs is controlled by direct interactions between cells and their neighbors. Proliferation appears to be stimulated by positional information differences, and these differences are reduced by the addition of new cells during tissue growth. Genes involved in cell proliferation control have been identified by collecting and analyzing recessive lethal mutations which cause overgrowth of imaginal discs. In some of these mutants (fat, lgd, c43, dco) the overgrowing tissue is hyperplastic; it retains its single-layered epithelial structure and is capable of differentiating. In two of the hyperplastic mutants (dco and c43), the imaginal discs show a failure of gap-junctional cell communication, suggesting that this form of cell communication may be involved in termination of proliferation. In other mutants the overgrowing disc tissue is neoplastic: it loses its structure and ability to differentiate, becoming a tumorous growth. The two genes that give a neoplastic phenotype (dlg and lgl) have been cloned and cDNAs of one of them (lgl) sequenced. The lgl gene encodes a cell surface molecule with significant homology to calcium-dependent cell adhesion molecules (cadherins). The expression of lgl at the time of termination of cell proliferation suggests that there are changes in the way that cells interact with one another at these times, and that these changes may be implemented by cell adhesion molecules. Direct cell contact within the epithelium, as well as signalling through gap junctions, appears to be involved in the cell interactions needed for the termination of cell proliferation. Mutations in genes encoding the Drosophila homologs of growth factors, growth factor receptors and oncogenes usually show an effect on cell-fate decisions rather than cell proliferation control, but this may be because oncogenic mutations in these genes would be dominant lethals and would therefore not be identified by conventional genetic analysis.

Aging↗

Lumenal transmission of decapentaplegic in Drosophila imaginal discs.

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.

Animals↗

Wingless induces transdetermination in developing Drosophila imaginal discs.

Drosophila imaginal discs, the precursors of the adult fly appendages, have been the subject of intensive developmental studies, particularly on cell determination. Cultured disc fragments are recognized not only for the ability to maintain their determined state through extra cell divisions but also for the ability to transdetermine, or switch to the determined state of a different disc. An understanding of transdetermination at a molecular level will provide further insight into the requirements for maintaining cell determination. We find that ectopic expression of the Drosophila gene wingless induces transdetermination of foreleg imaginal disc cells to wing cells. This transdetermination occurs in foreleg discs of developing larvae without disc fragmentation. The in situ-transdetermining cells localize to the dorsal region of the foreleg disc. This wingless-induced transdetermination event is remarkably similar to the leg-to-wing switch that occurs after leg disc culture. Thus we have identified a new approach to a molecular dissection of transdetermination.

Animals↗

Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs.

Wing imaginal disc cells in Drosophila develop by using information received from a signaling center associated with the anterior/posterior compartment border. We show here that disc cells have thin, actin-based extensions (cytonemes) that project to this signaling center. Cytonemes can be induced when cells from the lateral flanks of a wing disc are cultured next to cells from the A/P border or next to a source of fibroblast growth factor. Mouse limb bud cells also grow projections during a brief culture period, indicating that cytonemes are an attribute of both vertebrate and invertebrate cells. We suggest that cytonemes may be responsible for some forms of long-range cell-cell communication.

Actins↗

Sequence and expression of IMP-L1, an ecdysone-inducible gene expressed during Drosophila imaginal disc morphogenesis.

Drosophila imaginal discs are induced by the steroid hormone 20-hydroxyecdysone to initiate morphogenesis leading to formation of the adult appendages and thoracic epidermis at the end of the third larval instar. Ecdysone-dependent transcriptional activation of a set of genes that encode imaginal disc transcripts found on membrane-bound polysomes precedes and may be responsible for some aspects of the cellular changes that mediate epithelial morphogenesis in this system. A 1.35 kb transcript from one of these genes, IMP-L1, is first observed in vivo at or just prior to pupariation, as ecdysone titers are peaking and beginning to decline. Expression is initiated in proximal areas of the antennal disc, later spreading to a more widespread but nonuniform distribution throughout other thoracic imaginal discs. IMP-L1 is not, however, expressed in other ecdysone target tissues such as salivary glands or fat body. The IMP-L1 gene encodes a novel protein product containing a signal peptide, a possible transmembrane domain, two highly charged domains and a proline rich C-terminal domain. We suggest that the delayed timing of expression of this secondary response gene is necessary for proper ordering of cellular events associated with disc morphogenesis.

Amino Acid Sequence↗

Analysis of an enhancer trap expressed in regenerating Drosophila imaginal discs.

In Drosophila, imaginal discs are the undifferentiated larval precursors of the pattern of epidermal and sensory neural cells in each adult segment. Although cell fates are already specified by late third instar, disc fragments can either regenerate or duplicate after growth in culture. The outcome depends on signaling between cells across the healed wound and involves a redeployment of the expression patterns of selector genes and other disc pattern genes. We recently used the enhancer-trap method to screen for such genes that are expressed ectopically at the wound-heal site in imaginal discs undergoing regeneration. Here we report the cloning by plasmid rescue of transcribed sequences adjacent to one such enhancer-trap insertion. Using Northern analysis and in situ hybridization we show that one transcript is expressed in the embryo and in imaginal discs in a pattern similar to that of the enhancer trap. We also, by imprecise excision of the enhancer-trap insertion, generated a series of flanking deletions that were mapped using Southern analysis and complementation.

Animals↗

Lethal P-lacZ insertion lines expressed during pattern respecification in the imaginal discs of Drosophila.

The imaginal discs of Drosophila are a useful experimental system in which we can study the origin and genetic determination of spatial patterns in development. This involves the separation of the disc-cell population into distinct lineage compartments, based on clonally transmitted expression states of a number of known selector genes. However, these commitments can be abrogated and the compartment boundaries redeployed, when repatterning occurs in cultured disc fragments. This has so far only been explained using the idea of positional information. The genetic basis of this property of the imaginal disc system and its relationship to compartments have not been identified. Here we have screened over 470 recessive lethal P-lacZ enhancer-trap insertions from the Berkeley Drosophila Genome Project for expression after cell death, which initiates pattern respecification in the imaginal discs. The positive lines obtained identify essential genes that may be important for pattern formation. Most show patterned imaginal disc expression, and many have maternal or zygotic effects on embryonic development. One is an allele of schnurri, a gene that encodes a component of the decapentaplegic (dpp) signal transduction pathway used for positional signalling in the embryo and in imaginal discs.

Animals↗

Ecdysteroid control of cell cycle and cellular commitment in insect wing imaginal discs.

The wing imaginal disc shows two different developmental responses to the steroid hormone 20-hydroxyecdysone (20E) in conjunction with cell cycle, and the responses depend on the developmental stage and the concentration to which it is exposed. The time of head capsule slippage (HCS) in late fourth instar, which signals the onset of molting process into the fifth instar, is the boundary for the appearance of 20E-dependency. Before HCS, 20E was not effective in maintaining the cell cycle, while, after HCS, 20E drove the cell cycle (20E-dependent cell cycle). In the 20E-dependent cell cycle, S phase was induced by 20E above a threshold concentration, whereas M phase was induced in a range of concentrations, above which it was suppressed. The 20E-dependent cell cycle first appeared concomitantly with the entrance of the discs into the reversible stage of pupal commitment. The composite data suggest that the control mechanism of cell cycle is altered in the pupally committed cells so that a high ecdysteroid titer to induce a stationary molt in young instars does not affect the cell division of uncommitted imaginal disc cells while that to induce pupal ecdysis suppresses it in the committed discs, which occurs before the onset of pupal differentiation.

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Dynamics of decapentaplegic expression during regeneration of the Drosophila melanogaster wing imaginal disc.

Regeneration of an imaginal disc involves highly ordered proliferation and pattern regulation of the newly formed tissue. Although the general principles of imaginal disc regeneration have been extensively studied, knowledge of the underlying molecular mechanisms is far from complete. Results from other model organisms suggest that regeneration is the result of local recapitulation of the normal patterning genes. To analyze the dynamics of one major Drosophila patterning gene, decapentaplegic (dpp), in wing imaginal disc regeneration, a vital GFP reporter together with iontophoretic cell labeling were used. Our observations reveal that the restoration of compartment-border-specific dpp expression is a common event in imaginal disc regeneration. However, we did not find evidence of an upregulation of dpp expression during the regeneration process.

Animals↗

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 disorganization 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.

Amino Acid Sequence↗

Expression profiling of Drosophila imaginal discs.

BACKGROUND: In the Drosophila larva, imaginal discs are programmed to produce adult structures at metamorphosis. Although their fate is precisely determined, these organs remain largely undifferentiated in the larva. To identify genes that establish and express the different states of determination in discs and larval tissues, we used DNA microarrays to analyze mRNAs isolated from single imaginal discs. RESULTS: Linear amplification protocols were used to generate hybridization probes for microarray analysis from poly(A)+ RNA from single imaginal discs containing between 10,000 and 60,000 cells. Probe reproducibility and degree of representation were tested using microarrays with approximately 6,000 different cDNAs. Hybridizations with probes that had been prepared separately from the same starting RNA pool had a correlation coefficient of 0.97. Expression-profile comparisons of the left and right wing imaginal discs from the same larva correlated with a coefficient of 0.99, indicating a high degree of reproducibility of independent amplifications. Using this method, we identified genes with preferential expression in the different imaginal discs using pairwise comparisons of discs and larval organs. Whereas disc-to-disc comparisons revealed only moderate differences, profiles differed substantially between imaginal discs and larval tissues, such as larval endodermal midgut and mesodermal fat body. CONCLUSIONS: The combination of linear RNA amplification and DNA microarray hybridization allowed us to determine the expression profiles of individual imaginal discs and larval tissues and to identify genes expressed in tissue-specific patterns. These methods should be widely applicable to comparisons of expression profiles for tissues or parts of tissues that are available only in small amounts.

Animals↗

Drosophila null slimb clones transiently deregulate Hedgehog-independent transcription of wingless in all limb discs, and induce decapentaplegic transcription linked to imaginal disc regeneration.

Drosophila Slimb (Slmb) is a F-box/WD40 protein which potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless (wg) and decapentaplegic (dpp), as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional mechanisms in which slmb is required, such as leg dorsal-ventral restriction. We have isolated new alleles of the slmb gene in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, we have determined that regulation of Dpp and Wg morphogens by Slmb could be different from what has already been published. We show here that in leg discs, lack of slmb function derepresses the transcription of wg independently of Hh signaling. We present evidence that ectopic legs resulting from slmb(-) clone induction only come from wg misexpression in the normal dpp domain, as ectopic proximo-distal axis are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. We also describe, in discs bearing amorphic slmb clones and in discs of two other slmb(-) contexts, a novel pattern of dpp expression consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb(-) cell elimination. We have investigated the fate of slmb(-) clones, which disappear before adulthood, and found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out which excludes all slmb(-) clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, as in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc.

Animals↗

A screen for genes expressed in Drosophila imaginal discs.

The development of Drosophila imaginal discs serves as a model system to understand how genes determine the shape and size of an organ. The identification of genes involved in this process is an important step towards this goal. Here we describe a P-element based enhancer trap screen for genes expressed in the larval imaginal discs. Our aim was to establish a large collection of enhancer trap lines each showing expression of Gal4 in imaginal discs. To this end, we improved the well established P-element vector pGawB in order to obtain higher in vivo transposition frequencies. In addition we chose an F1-screening approach using UAS-GFP as a reporter gene. This system permits the efficient screening of larval and pupal stages of living animals and the detection of imaginal gene expression patterns through the transparent cuticle. The procedure has been optimized for high-throughput. 2'000 P-element insertions have been established which exhibit expression in imaginal discs.

Animals↗