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.     

Tumor suppression in Drosophila is causally related to the function of the lethal(2)tumorous imaginal discs gene,a dnaJ homolog

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) causes in homozygotes malignant growth of cells of the imaginal discs and the death of the mutant larvae at the time of puparium formation. We describe the molecular cloning of the 1(2)tid+ gene and its temporal expression pattern in the wild-type and mutant alleles. Germ line rescue of the tumor phenotype was achieved with a 7.0 kb Hindlll-fragment derived from the polytene chromosome band 59F5. The l(2)tid+ gene spans approximately 2.5 kb of genomic DNA. The protein coding region, 1,696 bps long, is divided by an intron into two exons. The predicted Tid⁵⁶ protein contains 518 amino acids and possesses a theoretical molecular weight of 56 kDa. It shows significant homology to all known DnaJ related proteins from bacteria, yeast, and man. The possible function of the Tid⁵⁶ protein in tumor suppression is delineated. © 1995 Wiley-Liss, Inc.     

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.     

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.     

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.     

A comparison of protein synthesis in imaginal wing discs of wild-type and vestigial of Drosophila melanogaster

In vitro incorporation of labelled amino acids into TCA insoluble protein in the imaginal wing discs of vg mutant and wild-type Drosophila melanogaster is compared and correlated with the extent of cellular degeneration during development. (1) With 96–100-hr-old larvae, wing discs isolated from vg mutant incorporate radiolabelled amino acids at a higher rate than wild-type discs, although cellular degeneration is more extensive in vg discs than wild-type discs. This may reflect the larger size of the vg discs since they have more protein than the wild-type discs. (2) The vg discs of 105–110-hr-old larvae have a lower rate of incorporation than the wild-type disc, although both types of discs have the same protein content. This may be due to the presence of more degenerating cells in the vg discs. (3) The rate of incorporation in vg discs of 115–118-hr-old larvae is lower than that of wild-type discs; the vg discs have less protein but more degenerative cells than the wild-type discs. The rates of protein degradation are similar in both discs. Electrophoretic comparison reveals a general reduction in protein synthesis in vg discs as compared with wild-type discs.     

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.     

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to β-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr^xF9) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr⁺. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.     

Analysis of metamorphosis in Drosophila melanogaster: Characterization of giant,an ecdysteroid-deficient mutant

The mutant allele giant of Drosophila melanogaster affects the timing and the level of increase in ecdysteroid titer normally occurring at puparium formation. The third larval instar is extended by 4 days in phenotypically “giant” individuals during which the imaginal discs mature slower than normal and finally take on the folding pattern characteristic of maturity at a time when normal individuals have already formed puparia. After puparium formation, development occurs at the same rate in giant and wild-type animals. Feeding 20-hydroxyecdysone at 94 hr after oviposition allows giant larvae to develop at the same rate as wild-type larvae and to produce normal-sized adults (although at 94 hr the imaginal discs of giant lack much of the folding pattern of mature discs). Radioimmunological determination of ecdysteroid titers in giant and normal individuals indicates that the peak of ecdysteroid activity associated with puparium formation is lower in giant and occurs 4 days later than normal. These results indicate that giant is an ecdysteroid-deficient mutant with major effects on metamorphosis. Unlike previously reported ecdysteroid-deficient mutants, however, giant larvae eventually develop into adults and may be induced to undergo complete metamorphosis at the same time as wild type by feeding 20-hydroxyecdysone.     

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.     

The nonmuscle myosin phosphatase PP1beta (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1β (PP1β). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw¹, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw¹ and puc^A251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw¹ enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.     

Autonomous cellular differentiation of homoeotic bithorax mutants of Drosophila melanogaster

Various mixtures of imaginal disc cells from wild-type and from homoeotic bithorax mutants have been studied in an in vivo Drosophila cell culture system. These mutants effect specific types of segmental transformations, e.g., bithorax-3 (bx³) transforms the anterior region of the metathorax (MT) into a region resembling the anterior mesothorax (MS), while postbithorax (pbx) transforms the posterior MT into a posterior MS-like region. In cell mixtures, wild-type haltere-disc cells segregate from wild-type wing-disc cells. On the other hand, bx³ and pbx haltere-disc cells integrate with wild-type cells derived from anterior and posterior regions, respectively, of wing discs. The behavior of these and other tested mutants of the bithorax series indicates in all cases studied that (1) the effects of the mutants are cell-autonomous, and (2) cellular affinities are determined by the genetic constitution rather than the segmental origin of the cells.     

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(M) phenotype in Drosophila ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.     

Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl⁻ clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl⁻ tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl⁻ mutants are separable. Furthermore, lgl⁻ mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl⁻ and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl⁻ tissue shows less apoptosis.     

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.     

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.     

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.     

Htid-1, the human homolog of the Drosophila melanogaster l(2)tid tumor suppressor, defines a novel physiological role of APC

Htid-1, the human counterpart of the Drosophila tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) encodes three splice forms translated into three cytosolic - Tid50, Tid48 and Tid46 - and three mitochondrial - Tid43, Tid40 and Tid38 - proteins. Here we provide evidence for the association of the endogenous Tid50/Tid48 proteins with the adenomatous polyposis coli (APC) tumor suppressor in normal colon epithelium, colorectal cancer cells and mouse NIH3T3 fibroblasts. Using the Glutathione S-transferase binding assay we show that the N-terminal region including the Armadillo domain (ARM) of APC is sufficient to bind the Tid molecules. Using immunoprecipitation and confocal microscopy we show that the two molecular partners complex at defined areas of the cells with further proteins such as Hsp70, Hsc70, Actin, Dvl and Axin. Our data implicate that the formation of the complex is not associated with APC's involvement in beta-Catenin degradation. Furthermore, though it is linked to Actin it is neither associated with regulation of Actin cytoskeleton due to APC's binding to Asef nor to Tid's binding to Ras-GAP. We suggest that the novel complex acts in maintaining APC's availability for its distinct roles in the Wnt signaling important for the cell to take the right decision, either to switch the cascade OFF or ON, thus, to regulate the onset of proliferation of the cells.     

Understanding human cancer using Drosophila: Tid47, a cytosolic product of the DnaJ-like tumor suppressor gene l2Tid,is a novel molecular partner of patched related to skin cancer

Recessive mutations of the Drosophila gene lethal(2)-tumorous imaginal discs (l(2)tid) cause neoplastic growth of the anlagen of the adult organs, the imaginal discs. Here we report that the three proteins encoded by this evolutionarily conserved gene, Tid50, Tid47, and Tid40, identified as members of the DnaJ cochaperone family, are destined for different cellular compartments, build complexes with many proteins in a developmental stage-specific manner, and are likely to be involved in different cellular processes. We show that the cytosolic Tid47 molecule is a novel component of the Hedgehog (Hh)-Patched (Ptc) signaling regulating cell/tissue polarity and spatial patterning during development and is associated with human tumors such as basal cell carcinoma (BCC) and medulloblastoma. We provide functional evidence for its direct in vivo interaction with the Hh-bound Ptc receptor during signal transmission. Because loss of l(2)tid causes neoplastic transformation of Hh-responsive cells, we suggest that Tid47 may at least act as a guardian of the Hh signaling gradient by regulating Ptc homeostasis in the tissue. Finally, we show that the expression of htid-1, the human counterpart of l(2)tid, is altered in human BCCs. We demonstrate that in BCCs loss of htid expression correlates with loss of differentiation capacity of the neoplastic cells similar to that found in the Drosophila tumor model.     

Developmental expression and tissue distribution of the lethal (2) giant larvae protein of Drosophila melanogaster

Recessive mutations in the Drosophila tumor gene lethal (2) giant larvae affect the growth and tissue specificity of determined cells in imaginal discs and presumptive optic centers of the brain. To analyse the function of the l (2) gl gene during development, we have raised monoclonal antibodies against the l (2) gl protein. These antibodies detect a 130-kd protein in wild-type tissue which is absent in homozygous mutant tissues. The protein is detected in increasing amounts up to mid-embryonic stages. Antibody binding to embryo sections and indirect immunofluorescence labeling indicate that the protein is localized at the cellular membranes or in the intercellular matrix of the embryonic cells. The primordia of all larval tissues are labeled in the embryo. Much less labeling is found in the neural primordia of the central nervous system, except that within the supraoesophageal ganglion the regions of the presumptive optic centers are distinctly labeled. Moreover, the axon bundles of the ventral cord are labeled in the embryo, apparently a reflection of the accumulation of cell membranes here. After embryogenesis the l (2) gl protein is found at a low level until the end of the 3rd larval instar, when it is preferentially seen in the brain and imaginal discs. The protein distribution in embryonic and larval tissues correlates with already known proliferation patterns, which could indicate that the l (2) gl protein is involved in proliferation arrest of cells.