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.     

Pattern formation in the imaginal discs of a temperature-sensitive cell-lethal mutant of Drosophila melanogaster

To explore the effects of cell death on pattern formation in the developing imaginal discs of Drosophila melanogaster, I have isolated a number of cell-autonomous temperature-sensitive lethal mutants. Sex-linked temperature-sensitive lethals were screened for cell-autonomy by scoring the survival of lethal-bearing clones in genetic mosaics. The mutant with the strongest effect on clone viability gave rise to a high frequency of structural deficiencies and duplications in the derivatives of the eye-antennal discs, when subjected to pulse-treatments at the nonpermissive temperature during the late second and third instars. The patterns produced were nonrandom, with some structures showing a tendency to become deficient, and others a tendency to duplicate. Duplicated structures were only found in heads in which other structures were missing. Genetic tests identified the lethal as a point mutation at the suppressor-of-forked locus. Recombination, and complementation tests with a small duplication of this region showed that a second mutational lesion is in all probability not involved in the generation of abnormal patterns in the imaginal discs. It is therefore proposed that the cell-lethal action of the mutant is sufficient to account for phenotypic effects described. According to this hypothesis, cell death primarily causes deficiencies, and duplications occur as a response of the discs to injury. In agreement with this, it was found that in gynandromorphs, pattern duplications can be found in wild-type tissue in the presence of lethal tissue in the same disc. Thus, a cell-autonomous lethal may affect the process of pattern formation in a nonautonomous way.     

ROS-Induced JNK and p38 Signaling Is Required for Unpaired Cytokine Activation during Drosophila Regeneration

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.     

Patterns of cell division in imaginal discs of Drosophila

A transmission electron microscopic study of cell division in serially sectioned imaginal discs of early third instar fruitfly larvae revealed that mitotic cells maintain a relationship with the basal surface of the disc through thin cytoplasmic extensions abutting on vesicular material. Two patterns of cell division were discerned. In one, cell divisions were isolated and usually found near the peripodial membrane-disc epithelium junction; in the other, cell divisions were clustered. Observations on cell death and cell division in the peripodial membrane are also reported.     

Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.     

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.     

Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner

The imaginal discs of Sarcophaga were found not to develop normally in the presence of galactose, a hapten sugar of Sarcophaga lectin, or anti-Sarcophaga lectin antibody. Wing and leg discs cultured with these substances became morphologically abnormal and no imaginal discs reached the stage of terminal differentiation, even in the presence of 20-hydroxyecdysone. The development of the imaginal discs was shown to be autonomously regulated in an autocrine manner by Sarcophaga lectin; namely Sarcophaga lectin was secreted by the imaginal discs in the presence of 20-hydroxyecdysone, and the stimulus of self-induced Sarcophaga lectin seemed to be indispensable for further development of the imaginal discs. Sarcophaga lectin was originally found as a defense protein, but these results show that it plays independent roles in both defense and development.     

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.     

Mitosis in neoplastic and hyperplastic imaginal discs ofDrosophila

Homozygosity for recessive mutations inDrosophila tumour suppressor genes likelethal giant larvae (Igl), lethal giant discs (Igd) orfat (ft) induce uncontrolled cell proliferations in the imaginal discs of the mutant larvae. Imaginal discs of larvae mutant forIgl tumour suppressor gene display neoplastic growths while those mutant forIgd orfat display hyperplastic growths. Results presented in this study reveal that mutant wing imaginal discs with neoplastic or hyperplastic overgrowths display high mitotic activity primarily during the extended period of larval life when their wild-type siblings have already pupariated. Both these categories of overgrowths show overall stability of the karyotypes and only low frequency of aneuploidy. The hyperplastic imaginal discs ofIgd orft mutant larvae displayed normal chromosome condensation. In contrast, the neoplastic imaginal discs ofIgl mutants showed high frequency of mitotic cells with undercondensed chromosomes. In this respect the neoplastic discs resemble malignant neuroblastomas of theIgl larvae which also display undercondensed chromosomes. These results thus suggest an indirect role of the cytoskeletal protein encoded byIgl tumour suppressor gene in aspects of normal chromosome condensation during mitosis.     

Ecdysteroid action in imaginal discs of Drosophila melanogaster does not involve cyclic AMP

A possible role for cAMP in the mechanism of ecdysteroid induction of morphogenesis in imaginal discs of Drosophila melanogaster was explored in a variety of experiments. Neither cAMP, supplied externally, nor theophylline at concentrations which increase internal cAMP levels 2–3 fold will substitute for the hormone in the induction of morphogenesis. Hormone action in imaginal discs is neither enhanced nor repressed by externally supplied cAMP or theophylline. Internal cAMP levels in discs, determined by kinase binding assays, are not altered by juvenile hormone or 20-hydroxy-ecdysone under incubation conditions which permit ecdysteroid stimulation of RNA synthesis and induction of morphogenesis. Adenylate cyclase activity in disc extracts is not stimulated or depressed by 20-hydroxy-ecdysone or juvenile hormone, but is stimulated by NaF. These findings suggest that ecdysteroid action in imaginal discs does not involve changes in the internal concentration or metabolism of cAMP.     

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.     

A method for estimating the number of blastoderm cells which give rise to Drosophila imaginal discs

Summary A mathematical method for calculating the number of blastoderm cells whose descendants form the various imaginal discs is described. The method differs from available approaches in two respects: (1) It is based only upon the frequency of mosaicism of the adult derivatives of a given imaginal disc and ignores the relative surface area of the two genetically marked cell populations which comprise these derivatives. (2) The method estimates the average number of cells at the blastoderm stage which give rise to a particular imaginal disc and not at the developmental stage at which restriction of the pool of cells which will form this imaginal disc occurs. Despite their methodological differences the estimates obtained from this method and from other approaches are of the same order of magnitude and thus provide further support to the currently available estimates and to the notion that restriction of whole imaginal discs occurs at the blastoderm stage. The proposed method also provides a quantitative approximation of the non-linear relationship that exists between the frequencies of mosaicism of different imaginal discs and the number of cells which comprise these discs.     

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.     

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.     

Radiosensitivity of somatic cells and imaginal disks of male and female Drosohila]

Primary chromosome damages as well as the frequency of spontaneous and X-rays induced chromosome aberrations in Drosophila melanogaster males and females are studied. It is found using cytofluorimetric method that primary chromosome damages in ganglia cells of females and males are the same. In these cells as well as in cells of imaginal discs the frequency of induced chromosome aberrations, except gaps, is considerably higher for females. Ganglia cells of females and males of Drosophila are found not to differ from each other in their proliferation activity. The frequency of morphoses for both sexes is also the same. The assumption is made concerning the role of the non-identical repair in the increased mutability of female somatic cells, which is more intensive in this sex, as well as concerning more intensive identical repair in imaginal discs of females.     

Ecdysteroid concentration inducing cell proliferation brings about the imaginal differentiation in the wing disc of Bombyx mori in vitro

This study was undertaken to evaluate the range of 20-hydroxyecdysone (20HE) concentrations which induce cell proliferation and imaginal differentiation in lepidopteran wing discs in vitro . Wing discs were cultured in medium containing various doses of 20HE. During imaginal differentiation in vitro , wing discs were observed histologically and the number of mitosis was counted every day. Wing discs differentiated adult features in medium containing 0.02–0.2 μg/mL 20HE, and these doses also increased the number of mitosis in disc cells. Wing discs developed the same in vitro as they do in vivo . The concentration of 20HE over 0.2 μg/mL inhibited both mitosis and imaginal differentiation. Cell proliferation, cuticle deposition and tissue elongation were successively observed in vitro the same as observed in vivo . These results suggest that a moderate concentration of ecdysteroid can induce cell proliferation followed by imaginal differentiation.     

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.