Development Capacities of Mixed Normal and Neoplastic 1(3)gl Imaginal Discs and Neuroblast Tissue in Drosophila hydei

The pleiotropic mutant lethal(3)giant larvae [l(3)gl] of Drosophila hydei exhibits among other anatomical defects, hypertrophy of the larval brain and imaginal discs. Both hypertrophic tissues when transplanted into wild-type female flies behave as fast growing and lethal neoplasms. Implanted into mature wild-type larvae they fail to metamorphose. When l(3)gl neoplastic brain tissue or imaginal discs were mixed with normal imaginal discs, cultured in vivo in the abdomen of adult females and transplanted into mature wild-type larvae, the following results were obtained. The invasive l(3)gl brain neoplasm, while fatal for adult hosts, had no effect on the metamorphosis of normal imaginal disc tissue. On the other hand, the noninvasive l(3)gl imaginal disc neoplasms when mixed with normal imaginal disc tissue inhibited its development and metamorphosis in the wild-type host. This inhibitory effect was not observed when the tissues were injected as separate implants into the same host.     

Analysis of the autonomy of imaginal disc defects in a small-disc mutant of Drosophila melanogaster.

Lethal mutations which cause imaginal disc abnormalities in Drosophila melanogaster identify genes whose function is necessary for normal disc development, and these mutant genes may be used as probes of the role of their wild-type alleles in normal development. It is crucial to the interpretation of the disc phenotype of such mutants to know which abnormalities are autonomous (caused by expression of the mutant gene in imaginal cells) and which are nonautonomous (indirectly caused, for example, by expression of the mutant gene in larval cells). We chose for study l(3)c21R (3-67.8), a late-larval lethal mutation with a complex phenotype, to test the adequacy of available techniques for assessing autonomy. We employed surgical and genetic techniques to determine the imaginal cell autonomy of the defects in cell viability, growth, and differentiation in c21R discs. The imaginal cell viability defect is nonautonomous. The disc growth and differentiation defects are autonomous; however, in genetic mosaics these two autonomous defects are separable. These results show that c21R belongs to the class of mutations which affect both larval and imaginal cells. In combination, the available methods were adequate to resolve the issue of autonomy in this complex case. However, in isolation several of the methods could have led to incomplete or misleading interpretations. This emphasizes that to analyze any developmental mutant it is necessary to examine the issue of autonomy from several points of view.     

Isolation of temperature sensitive mutations blocking clone development inDrosophila melanogaster,and the effects of a temperature sensitive cell lethal mutation on pattern formation in imaginal discs

Summary A method of isolating temperature-sensitive (ts) mutations blocking clone development, based on the analysis of twin spots produced by X-ray induced somatic recombination is reported. From this screen 10 ts mutations were recovered which caused an absence of the lethal-bearing clone at the restrictive temperature. Eight of these mutations were analyzed. Seven proved to be autonomous ts cell lethals and one was an autonomous ts mutation which reversibly affected cell division and growth of imaginal disc cells and growth of larval cells. The effects on development of one of the cell lethal mutations,l(1)ts-504, are described. Heat pulses (29°C) 24–72 hrs long caused a high frequency (up to 90%) of morphologically abnormal animals. The abnormalities observed were of two major kinds: deficiencies and duplications of imaginal disc derivatives. In addition, alterations of tarsal segmentations occurred. Heat pulses to larvae also delayed pupariation and eclosion by as much as four days. In general, longer pulses led to a greater delay in pupariation and eclosion and a higher frequency of deficiencies and duplications than shorter pulses. Exposure to restrictive temperature early in larval development delayed pupariation and resulted in mostly normal animals; exposure during the second and early third larval instar also delayed pupariation and led to a high frequency of duplications; exposure later in larval life, i.e. mid and late third larval instar, caused no delay in pupariation but led to a high frequency of deficiencies. These results can be explained by the occurrence of areas of cell death, which can be seen in the imaginal discs of larvae exposed to restrictive temperature by staining with trypan blue. This conclusion is further supported by the observation in gynandromorphs of duplications of female nonmutant tissue. These results are discussed in relation to current theories of pattern formation.     

Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster: a comparison between different discs and stages

High-resolution two dimensional gel electrophoresis has been used to study the patterns of protein synthesis in imaginal discs of Drosophila melanogaster. In this paper we first compare the patterns of protein synthesis in wing, haltere, leg 1, leg 2, leg 3 and eye antenna imaginal discs of late third instar larvae. We have detected only quantitative changes: differences in 17 proteins among the different imaginal discs. In addition, we have analysed the variations in pattern of proteins in the wing disc of the last larval stage and early pupae as well as in wing discs cultured in vivo for 6 days. Variations in these patterns affect more than 20% of the proteins and involve both qualitative and quantitative changes. Some of the changes may correspond to protein phosphorylation. Correlations of these changes between discs and through development are also discussed. Correspondence to: F. Santaren     

Isolation and characterization of X-linked lethal mutants affecting differentiation of the imaginal discs in Drosophila melanogaster

Summary Twenty-seven late larval or early pupal lethal mutations were isolated for the X-chromosome, some of which showed structural and/or functional deficiencies of the imaginal discs. The mutants were grouped according to the size and morphology of their discs as follows: 1. discs normal: 18 mutants. 2. discs small: 2 mutants. 3. discs degenerate: 4 mutants. 4. discless: 1 mutant. 5. discs heterogeneous: 2 mutants. Preliminary characterization of the mutants included a study of disc morphology, puparium formation and pupal molt, in vivo and in vitro evagination of the imaginal discs, autonomy of the mutation in the disc tissue (differentiation after transplantation and gynander mosaicism test). Possible relations between disc morphology and the former characteristics are discussed.     

Analysis of molting and metamorphosis in the ecdysteroid-deficient mutant L(3)3DTS of Drosophila melanogaster

The dominant temperature-sensitive mutation L(3)3^DTS (DTS-3) in Drosophila melanogaster causes lethality of heterozygotes during the third larval instar at the restrictive temperature (29°C). Temperature-shift experiments revealed two distinct temperature-sensitive periods, with lethal phases during the third larval instar (which may persist for 4 weeks) and during the late pupal stage. At 29°C mutant imaginal discs are unable to evert in situ, but did evert normally if cultured in the presence of exogenous ecdysterone or when implanted into wild-type larval hosts. The only morphologically abnormal tissue present in the lethal larvae is the ring gland, the prothoracic gland being greatly hypertrophied in third instar DTS-3 larvae. Injection of a single wild-type ring gland rescued these mutant larvae, indicating that the mutant gland is functionally, as well as morphologically, abnormal. Finally, the mutant larvae were shown to have less than 10% of the wild-type ecdysteroid levels. These results are all consistent with a proposed lesion in ecdysteroid hormone production in DTS-3 larvae. A comparison with the phenotypes of other “ecdysone-less” mutants is presented.     

Genetic analysis of transdetermination in Drosophila. I. The effects of varying growth parameters using a temperature-sensitive mutation

The heat-sensitive mutation of Drosophila melanogaster l(3)c4(3)hs1, causes mutant larvae raised at a restrictive temperature to have abnormally large wing discs. The large size of these discs is a disc-autonomous property and results from an increase in the number rather than the size of wing disc cells. We have used wing discs from this mutant to further investigate properties of transdetermination which had previously been investigated with nonmutant discs. Transdetermination can occur in nonmutant discs when the proliferative phase of imaginal disc development is extended by wounding discs and culturing them in vivo. The results indicate that additional proliferation in the absence of wounding does not lead to transdetermination. There is a correlation between the extent of growth of a cultured disc and the probability that it will undergo transdetermination. The results suggest that this correlation does not depend on a differential rate of cell division. Finally, the results indicate that the cells which give rise to transdetermination are at an equivalent developmental stage no later than that characteristic of eye-antenna disc cells before the third larval instar.     

Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in specific tumor suppressor genes

 More than 50 genes have been identified in Drosophila by loss-of-function mutations that lead to overgrowth of specific tissues. Loss-of-function mutations in the lethal giant larvae, discs large, or brain tumor genes cause neoplastic overgrowth of larval brains and imaginal discs. In the present study, the growth and metastatic potential of tumors resulting from mutations in these genes were quantified. Overgrown brains and imaginal discs were transplanted into adults and β-galactosidase accumulation was used as a marker to identify donor cells. Mutations in these three genes generated tumors with similar metastatic patterns. For brain tumors, the metastatic index (a measure we defined as the fraction of hosts that acquired secondary tumors normalized for the amount of primary tumor growth) of each of the three mutants was similar. Analysis of cell proliferation in mutant brains suggests that the tumors arise from a population of several hundred cells which represent only 1–2% of the cells in third instar larval brains. For imaginal disc tumors from lethal giant larvae and brain tumor mutants, it is shown for the first time that they can be metastatic and invasive. Primary imaginal disc tumors from lethal giant larvae and brain tumor mutants formed secondary tumors in 43 and 53% of the hosts, respectively, although the secondary tumors were, in general, smaller than the secondary tumors derived from primary brain tumors. Received: 18 August 1997 / Accepted: 16 October 1997     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster : clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed. Received: 19 June 1997 / Accepted: 4 August 1997     

Actinomycin D-induced phenocopies in Drosophila melanogaster and their relevance to the time of gene action

Drosophila melanogaster larvae of a wild-type and a mutant stock, cultured in an axenic, chemically defined medium, were treated for one day with different concentrations of actinomycin D at different stages of development. Phenocopies affecting various organs of the adult occurred in different frequencies and in different patterns depending on the age at treatment. Assuming that the induced phenocopies were due primarily to the inhibition of DNA-dependent RNA synthesis by actinomycin D, the differential phenocopy effect indicates that: (1) Many genes which affect the differentiation of imaginal discs are activated in the third larval instar. (2) The developmental timing of gene activation in the third instar differs for various genes within a imaginal disc and in different imaginal discs.Submitted in partial fulfillment of the requirements for the Ph. D. degree. Supported by U.S. Public Health Service Grant GM1 1537 to I. H. Herskowitz.     

Structure and development of the larval visual system in embryos of Lytta viridana leconte (coleoptera,meloidae)

At hatching (252–264 hr. at 25 ± 0.5°C), the visual system in larvae of Lytta viridana consists of paired stemmata, stemmatal nerves, optic neuropiles, and inner and outer imaginal optic lobe anlagen. It originates between 64 and 72 hr. with invagination of an optic lobe primordium in the side of each protocephalic lobe. These primordia later differentiate into protocerebral ganglion cells and the imaginal optic lobe anlagen. Each stemma arises at 72 hr. from epidermis below and behind the optic lobe invagination and subsequently becomes cupshaped, closes over, and differentiates. At hatching, it consists of a planoconvex corneal lens, a corneagenous layer, and an everse retina of numerous, pigmented retinular cells, each with a terminal rhabdomere. Between 96 and 104 hr, proximal ends of the retinular cells grow posteromedially into a transverse, horizontal fold in the posterior wall of each optic lobe invagination and along its length to the protocerebral neuropile, which they contact by 112 hr. As the brain withdraws posteriorly within the head, these axons elongate correspondingly. Sheath cells of stemmata and stemmatal nerves descend either from protocerebral perineurium or the optic lobe primordia. Structure and development of the larval visual system in L. viridana are compared with those of other insects and its various components are shown to be homologous throughout the Insecta. However, the stemmata of this insect more closely resemble the atypical imaginal eyes of male scale insects than the photoreceptors of other holometabolous larvae–a similarity arising through convergence.     

Effects of the ecdysteroid agonists RH 5849 an RH 5992, alone and in combination with a juvenile hormone analogue, pyriproxyfen, on larvae ofSpodoptera exigua

Biological activity assays with RH 5849 and RH 5992 indicated that both compounds affected growth and development of last-instar larvae ofSpodoptera exigua (Hübner) (Lepidoptera: Noctuidae) in a dose-dependent manner. Within the first 24 h after treatment by continuously offering leaves dipped in a water solution of ≥50 mg/l RH 5849 and ≥0.5 mg/l RH 5992, symptoms of a prematurely induced larval moult and head capsule apolysis were visible. Intoxicated larvae died shortly afterwards, showing signs of unsuccessful ecdysis. LC₅₀-values of RH 5849 and RH 5992 for fifth-instarS. exigua larvae were 110 and 2.5 mg/l, respectively. Pyriproxyfen alone affected the larval stage and disturbed normal metamorphosis. One supernumerary larval instar occurred occasionally. LC₅₀-value for pyriproxyfen was 1.7 mg/l. Larvae simultaneously treated with RH 5849 or RH 5992 and pyriproxyfen, continued to grow until they attained a size and weight about 2–3 times that of the controls. This growth was accompanied by at least one and sometimes two supernumerary moults. Concerning thein vivo imaginal wing disc growth and development, only in larvae treated with 10 and 50 mg/l RH 5849 or 0.5 mg/l RH 5992, tracheole migration was observed earlier than in the controls. When applying 300 mg/l RH 5849 or 3–7 mg/l RH 5992, the discs remained small and no signs of tracheole migration were observed. In larvae simultaneously treated with RH 5849 or RH 5992 and pyriproxyfen, tracheole migration was not prematurely induced and a pupal cuticle was produced in the discs of larvae, undergoing a supernumerary moult. No clear signs of evagination were observed.     

Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid)

Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histological preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor, and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4-6.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.     

Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues

The major cell death pathways are apoptosis and autophagy-type cell death in Drosophila. Overexpression of proapoptotic genes in developing imaginal tissues leads to the activation of caspases and apoptosis, but most of them show no effect on the polytenic cells of the fat body during the last larval stage. Surprisingly, overexpression of Hid induces caspase-independent autophagy in the fat body, as well as in most other larval tissues tested. Hid mutation results in inhibition of salivary gland cell death, but the disintegration of the larval midgut is not affected. Electron microscopy shows that autophagy is normally induced in fat body, midgut and salivary gland cells of homozygous mutant larvae, suggesting that Hid is not required for autophagy itself. Constitutive expression of the caspase inhibitor p35 produces identical phenotypes. Our results show that the large, post-mitotic larval cells do not react or activate autophagy in response to the same strong apoptotic stimuli that trigger apoptosis in small, mitotically active imaginal disc cells.     

Post-larval development of compound eyes and stemmata of Chaoborus crystallinus (De Geer, 1776) (Diptera : Chaoboridae): Stage-specific reconstructions within individual organs of vision

During metamorphosis, the dioptric apparatus of the larval compound eye of Chaoborus crystallinus (Diptera : Nematocera) is radically reconstructed. The thin larval cornea of the ommatidia is replaced by strongly curved corneal lenses, and the eucone larval cone is replaced by an imaginal cone of the acone type. Curvature of the future lens is already apparent in very young pupae, in which the cornea consists only of a thin epicuticle with corneal nipples. Fibrillary cuticle is secreted by cone and primary pigment cells throughout pupal development. Lens formation is accompanied by movement of the nuclei of the accessory pigment cells. The larval cone disintegrates unexpectedly late in young, images. During late pupal development, 7 cone cell projections emerge. In contrast to the dioptric apparatus, the retinula cells and rhabdom remain almost unchanged during metamorphosis. The main refractive element of the larval ommatidium appears to be the cone, while that of the imaginal ommatidium is the corneal lens. In addition to the compound eyes, the pairs of stemmata are retained during the whole post-larval development. Pupal stemmata show no structural differences from the larval stemmata. The stemmata are still present in 2-day-old images (“retained stemmata”), but the primary stemma loses its dioptric apparatus and is proximally relocated to the basal region of the compound eye. The reconstructions in the visual system of Chaoborus, which occur during ontogeny, are probably connected with the change from aquatic living larvae to aerial adults, and appear to fulfill stage-specific needs of vision.     

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.     

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 disorganisation 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. Received: 22 August 1997 / Accepted: 9 September 1997