Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs

Imaginal discs contain a population of cells, known as peripodial epithelium, that differ morphologically and genetically from the rest of imaginal cells. The peripodial epithelium has a small contribution to the adult epidermis, though it is essential for the eversion of the discs during metamorphosis. The genetic mechanisms that control the identity and cellular morphology of the peripodial epithelia are poorly understood. In this report, we investigate the mechanisms that pattern the peripodial side of the wing imaginal disc during early larval development. At this time, the activities of the Wingless (Wg) and Epidermal growth factor receptor (Egfr) signalling pathways specify the prospective wing and notum fields, respectively. We show that peripodial epithelium specification occurs in the absence of Wingless and Egfr signalling. The ectopic activity in the peripodial epithelium of any of these signalling pathways transforms the shape of peripodial cells from squamous to columnar and resets their gene expression profile. Furthermore, peripodial cells where Wingless signalling is ectopically active acquire hinge identity, while ectopic Egfr activation results in notum specification. These findings suggest that suppression of Wg and Egfr activities is an early step in the development of the peripodial epithelium of the wing discs.     

The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. We show here that hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in discs morphogenesis, especially in the fusion of the two lateral wing discs. We show that these defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role of puc in discs morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role of puc in disc morphogenesis. Furthermore, we show that the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence for a role of the peripodial membrane in disc morphogenesis. We discuss a general model whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges.     

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.     

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.     

Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs

The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. We have reexamined the evagination of both the leg and wing discs and find that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreased as the tissue extended during evagination and we observed cell rearrangement to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. Our observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes.     

Tissue repair and regeneration in Drosophila imaginal discs

Exploring the mechanisms involved in tissue regeneration is one of the main challenges in biology and biomedicine. Multiple examples of tissue regeneration exist across the animal phyla, ranging from the recovery of the whole animal (e.g. flatworms) to the limited capability of the human liver. Studies performed in the 1960s showed that Drosophila imaginal discs are able to regenerate. This property, together with multiple genetic tools available, make fly an excellent model for the study of the regenerative process. Here we present an overview of the use of Drosophila for the study of regeneration and describe major recent advances in the understanding of this process. Current studies in Drosophila have unraveled some of the pathways and factors needed for a tissue to regenerate. Many observations point to the reuse of developmental programs and genetic reprogramming to drive regeneration. We discuss how this reprogramming could be orchestrated by the initial activity of the JNK pathway.     

Muscle connections between imaginal discs in Drosophila

A comparison of the protein content of different imaginal discs of Drosophila revealed that among more than 600 protein species that can be detected, only three show differences in concentration among different types of discs. Two of them form a doublet that can be resolved only by using extended electrophoresis conditions. This doublet was also reported to have a nonhomogeneous distribution within some discs. Here we show that these two proteins are tropomyosin components, and that they are associated not with the discs themselves but with a new type of muscle that connects some of the discs together.     

The hormonal coordination of cuticulin deposition and morphogenesis in Drosophila imaginal discs in vivo and in vitro

Cuticulin is the first layer of the insect cuticle to be deposited and is laid down as a continuous inelastic sheet over the apical surface of cuticle-secreting cells. During metamorphosis in Drosophila melanogaster, imaginal discs deposit the cuticulin layer of the pupal cuticle between 3 and 7 hr after puparium formation. This is a period of rapid morphogenesis involving cell shape changes and cell rearrangements. We have examined cuticulin deposition in vivo and in vitro with a view to understanding the coordination of cuticulin deposition with morphogenesis. We find that the optimum hormonal regimen (of the steroid hormone, 20-hydroxyecdysone) for the completion of both morphogenesis and cuticulin deposition in vitro parallels the changes in hormone titer observed in vivo. We also find that cuticulin is deposited last over cell boundaries, thereby allowing cell rearrangements to occur as cuticulin is laid down. We have identified in vitro conditions under which cuticulin deposition is completed precociously, inhibiting further morphogenesis. Cytochalasin B and colchicine do not inhibit cuticulin deposition and we therefore conclude that an intact cytoskeleton is not necessary for secretion of this extracellular structure. Finally, we present a preliminary protocol for the partial purification of cuticulin synthesized in vitro by mass isolated discs.     

Metamorphosis of imaginal discs of Drosophila melanogaster

Summary Imaginal discs ofDrosophila melanogaster larvae, 24–53 hrs after oviposition, were transplanted into mature immobile larval hosts. The transplants did not respond to the hormonal stimuli of metamorphosis, but instead completed their larval development. When reinjected into mature larval hosts, they now differentiated the full set of their presumptive imaginal structures. The process of acquiring competence for metamorphosis appears to be independent of the hormonal conditions.Supported by a credit of the Swiss National Foundation granted to Prof. Dr. E. Hadorn. I thank Dr. R. Nöthiger for his valuable criticism during this investigation.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.     

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.     

The embryonic origin of imaginal discs in Drosophila

The thoracic imaginal discs of Drosophila melanogaster can be observed during embryogenesis as clusters of cells with particular shapes, sizes and behaviours. These structures can be detected soon after germ band shortening and their development appears to be tightly linked to that of the larval epidermis.     

Establishment of imaginal discs and histoblast nests in Drosophila

In Drosophila the homeotic genes of the bithorax-complex (BX-C) and Antennapedia-complex (ANT-C) specify the identity of segments. Adult segment primordia are established in the embryo as the histoblast nests of the abdomen and the imaginal discs of the head, thorax and terminalia. We have used a molecular probe for the limb primordia and in vivo culture to describe the nature of the adult primordia in mutants in which the pattern of homeotic gene expression was altered. The results suggest that the histoblast or disc 'mode' of development is initiated by the extended germ band stage through activity of the BX-C and ANT-C and is relatively inflexible thereafter [corrected].     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Extracellular protease production by Drosophila imaginal discs

We are investigating the role of extracellular proteases in imaginal disc eversion to understand the mechanism that controls cell rearrangements within epithelia. We have identified three cation-dependent neutral proteases released by Drosophila leg discs everting in culture. Serine protease inhibitors block disc eversion and inhibit activity of disc proteases. The pattern of extracellular proteases changes when eversion is blocked with added protease inhibitors. Changes in protease activity occur when released disc proteases are treated with trypsin. Trypsin treatment of intact imaginal discs releases protease and inhibitor activities to the medium, indicating their presence on the cell surface before release. Our results suggest that extracellular proteases are required for imaginal disc morphogenesis and are regulated by more than one mechanism.     

Engrailed gene expression in Drosophila imaginal discs.

Genetic and molecular analyses indicate that the Drosophila engrailed gene is required to distinguish posterior from anterior compartments in each segment of the developing animal. Here, the patterns of engrailed expression in the imaginal discs and ventral ganglion of Drosophila larvae are examined, using an antiserum against the engrailed protein and a novel image processing method to reduce non-specific background. As expected, engrailed expression generally is restricted to cells in the posterior compartment of the discs, and the patterns of expression allow refinements in the fate maps of the discs to be made. More significant is the finding that expression of the gene is highly variable in different regions of posterior compartments, suggesting that engrailed may do more than simply specify 'posteriorness'. In the ventral ganglion engrailed appears to be expressed by a subset of cells, primarily in the posterior regions of each segment. In wing discs from animals that are homozygous for the en1 mutation, the pattern of expression of the gene is altered, as opposed to being simply reduced uniformly in the posterior cells.