Localized cell death caused by mutations in a Drosophila gene coding for a transforming growth factor-beta homolog

Recessive mutations (dppdisk) in one region of the decapentaplegic (dpp) gene of Drosophila, which codes for a transforming growth factor-beta homolog, cause loss of distal parts from adult appendages. Different dppdisk alleles cause effects of different severity, the milder alleles removing distal parts and the more severe alleles removing progressively more proximal structures. In the wing disc derivatives, the most extreme dppdisk genotype removes the entire wing and leaves only a thorax fragment. We show that structures are lost in these mutants as a result of massive apoptotic cell death in the corresponding regions of the imaginal discs during the mid-third larval instar. The remaining disc fragments do not regenerate when cultured alone in the growth-permissive environment of the adult abdomen, but they can be made to regenerate by coculturing them with appropriate fragments of wild-type wing discs. This nonautonomous development is interpreted as showing that a product of dpp+, presumably the TGF-beta homolog, is secreted by the normal cells and can rescue the mutant cells in the mixed tissue.     

Localized cell death in Drosophila imaginal wing disc epithelium caused by the mutation apterous-blot

Drosophila melanogaster carrying the mutation apterous-blot have blistered wings. Trypan blue stains a patch of dead cells localized to the wing pouch of imaginal discs and the same area shows acid phosphatase (AcPase) activity suggesting that the cell death is lysosomal. Autophagic vacuoles and other secondary lysosomes show AcPase activity within the disc epithelium and enzyme activity is found in fragments of dead cells which have been extruded basally. The cell death, although extensive and confined to the presumptive wing region, does not result in loss of adult structures.     

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.     

Truncated products of the vestigial proliferation gene induce apoptosis.

The vestigial (vg) gene in D. melanogaster, whose mutant phenotype is characterized by wing atrophy, encodes a novel nuclear protein involved in cell proliferation. The original vg mutant (vgBG) displays massive apoptosis in the wing imaginal disc. Here we tested the hypothesis that the vg mutant phenotype could be due: (i) to lack of cell proliferation in null mutants due to the absence of the Vg product and, (ii) to apoptosis in vgBG and other mutants due to the presence of a major Vg truncated product. In agreement with our hypothesis no cell death was observed in null vg mutants, and the anticell death baculovirus P35 product is unable to rescue the mutant phenotype caused by absence of the Vg product. In addition, expression of the antiproliferative gene dacapo, the homolog of p21, induces a mutant wing phenotype without inducing cell death. In contrast the wing phenotype of the original vg mutant could be reproduced by the ectopic expression of the reaper cell death gene when expressed by vg regulatory sequences. In agreement with the hypothesis, the classic vg mutant spontaneously displays an increase in reaper expression in the wing disc and its phenotype can be partially rescued by the P35 product. Finally, we showed that ectopic expression of a truncated Vg product is able on its own to induce ectopic cell death and reaper expression. Our results shed new light on the function of the vg gene, in particular, they suggest that the normal and truncated products affect vg target genes in different ways.     

Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster

Summary A number of mutants of Drosophila melanogaster are characterized by the absence of structures present in the wild type. Imaginal discs from the wing mutants vestigial, apterous-Xasta, Beadex and cut and from the eye mutants Bar, eyeless and lozenge were examined by light and electron microscopy. In all these mutants, with the exception of lozenge, clear evidence of degeneration was found. The onset and duration of degeneration and the number and distribution of dying cells were specific characteristics of each mutant. In most cases the degenerate areas of the disc could be correlated with the missing parts of the adult wing or eye. In contrast, in wild type wing and eye discs and in wing discs from the mutant miniature, which has a wing reduced in size but fully formed, extensive cell death was not observed.The ultrastructural features of the degenerating areas weresimilar in all the mutants studied. Conspicuous aspects of the cytolytic process included condensation and fragmentation of the dying cells followed by phagocytosis of the cell fragments by neighboring disc cells.The results indicate that localized cell death during development is a widespread occurrence among Drosophila mutants which exhibit structural deficiències.     

Regulative interactions between cells of wing discs from different dipteran species

The mechanism by which patterns are produced appears to be repeated in each segment of an animal, and it has been proposed that it may even have been conserved in evolution so that different species would have the same system of positional information. This idea has been tested by mixing cells of a defined fragment of the wing disc of Drosophila melanogaster with wing disc fragments of five other dipteran species to assay the ability of these disc fragments to stimulate intercalary regeneration of the D. melanogaster cells. The genetically marked (y; mwh) D. melanogaster fragment was mechanically mixed with wing discs or wing disc fragments of four drosophilids (D. melanogaster as a control, D. virilis, D. hydei, Zaprionus vittiger), of Musca domestica, and of Piophila casei. The mixed aggregates were cultured in vivo for 7 days, then metamorphosed in D. melanogaster larval hosts. The D. melanogaster fragments were only stimulated to regenerate when combined with complementary fragments from D. melanogaster or D. virilis wing discs. In the combination between D. melanogaster and D. hydei, the tissue formed integrated mosaic patterns, but no regeneration ensued. The one positive result (D. melanogaster mixed with D. virilis) shows that positional cues can be exchanged and correctly interpreted between cells of different species. The negative results do not prove that the mechanism for establishing patterns is different in the tested species, but may be due to incompatibilities that are not related to pattern formation.     

A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. Here we show that wg activates the gene homothorax (hth) in the hinge and that hth is necessary for hinge development. Further, we demonstrate that hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. We also show that the gene teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. Our results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Disruption of positional fields in apterous imaginal discs of Drosophila

Certain combinations of alleles at the apterous locus generate wings with extra copies of wing margin structures, some of which are located far from the normal margin. We have examined wing imaginal discs from these mutants, using position-specific antibodies as probes for two-dimensional patterning in the discs. Our results indicate that the adult phenotypes arise from unprecedented disruptions in the two-dimensional pattern of the disc epithelia. Examination of other apterous mutants suggest that pattern alterations may be a general consequence of lesions at this locus.     

Protein synthesis during imaginal disc pattern regulation

We have examined the pattern of protein synthesis during wing disc pattern regulation. Although in vivo culture dramatically alters the pattern of abundant protein synthesis in wing discs, only one protein--RG38--changes specifically in response to pattern regulation. This polypeptide, previously identified as being nonuniformly distributed in wing and haltere discs, is synthesized in a graded distribution across the wing disc. During wing disc pattern regulation, it acts as a molecular marker for regeneration of particular wing disc regions. Thus, the rate of RG38 synthesis increases during regeneration (by fragments with initial low levels) with kinetics that parallel those for regeneration as scored by the presence of adult cuticular structures.     

Transdetermination of imaginal wing disc fragments of Drosophila melanogaster

Fragments of the imaginal wing disc of Drosophila melanogaster were cultured in adult hosts before transfer to larvae for metamorphosis. Transdetermination occurred only after at least 2 weeks of culture in vivo, producing structures of the leg, antenna, head, and thoracic spiracle. Details of the transdetermined structures and their locations with respect to normal wing disc structures are reported. We present evidence suggesting that regulation can occur between the wing and the second leg imaginal discs, and we propose that many transdeterminations which involve neighboring discs may result from such interdisc regulation.     

Wingless mutation inDrosophila melanogaster

A temperature sensitive lethal allele of thewingless locus ofDrosophila melanogaster together with previously studied lethal and viable alleles in this locus, has been used to study some properties of this locus. These studies show the existence of two lethal phases for thewingless lesion; one during embryogenesis and another during pupation. By growing embryos with temperature sensitivewingless lesion at the permissive temperature and letting the larvae develop at non-permissive temperature, a large-scale cell death and subsequent regeneration were seen to occur in the mutant wing discs. This cell death followed by regeneration alters the normal developmental potential of the wing disc. Disc transplantation experiments show that these discs are incapable of differentiating into wing blade structures.     

Chironomus tentans epithelial cell lines sensitive to ecdysteroids, juvenile hormone, insulin and heat shock

A new culture medium, ZW, and the preparation of an extract of adult Drosophila, FX, are described, which for the first time allow the in vitro proliferation of normal Drosophila cells in the absence of undefined heterologous components. Cells from 6-hour-old Drosophila embryos can extensively differentiate and/or proliferate in ZW supplemented with FX and insulin. Cells isolated from wing discs of 90–120-hour-old larvae require ecdysterone for proliferation in ZW, in addition to FX and insulin. Explanted ovaries, testes, genital discs and intact or halved wing discs of 100-hour-old larvae grow in the same medium, at least in part due to cell proliferation. High concentrations of ecdysterone prevent differentiation and/or proliferation of cells from embryos and from wing discs and cause the lysis of most isolated imaginal disc cells grown in vitro, while cuticular differentiations are induced in wing discs and disc fragments grown in vitro.     

Ultrastructure of cell death in gamma- or X-irradiated imaginal wing discs of Drosophila

When Drosophila larvae were irradiated with 1300-1500 R of gamma rays both apoptotic and necrotic cell death were observed in imaginal wing discs. The ultrastructure of cell death by apoptosis was characterized by fragmentation of dead cells into highly condensed, membrane-bound particles. The ultrastructure of cell death by necrosis was characterized by cell lysis and organelle degeneration. Marked contrast was also seen in the distribution of the two types of cell death: apoptosis was universal in irradiated discs and affected widely distributed single cells, or small groups of cells, whereas necrosis formed lesions by afflicting large numbers of contiguous cells. It was noted that even where there were large lesions in the epithelial cell layer, which is the primary component of imaginal discs, the basement membrane associated with this epithelium always remained intact. Lesions could be identified in freshly extirpated discs by staining with trypan blue and were found in 50-70% of irradiated discs (depending on the larval age at the time of irradiation). Lesions were seen in all regions of the wing disc and varied greatly in size. In spite of extensive necrotic cell death wing discs developed into normal adult wings. Regenerative growth in this case would appear to require significant reorganization of cells. Implications of this for the appropriate interpretation of clonal analysis are discussed.     

Regenerative interactions between Drosophila imaginal discs of different types.

We have tested the ability of fragments of one type of imaginal disc to stimulate regeneration of another type. It has been shown by others that, when extreme proximal and distal fragments of the wing disc are combined, intercalary regeneration of the missing tissue ensues. Each fragment, if cultured alone, will merely duplicate its structures. We now find that distal fragments of other thoracic discs, haltere and leg, while retaining their autonomy for differentiation, also interact with proximal wing tissue to promote regeneration of more distal wing structures. The proximal wing tissue used in these experiments was the wingless abnormal wing disc which, in the absence of interaction, yields only proximal wing structures. These results suggest that spatial organization is controlled by similar systems in the various thoracic discs. In contrast, head and genital disc material provided no regenerative stimulus to the mutant wing disc tissue.