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.     

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.     

Evidence for cell-replacement repair of X-ray-induced teratogenic damage in male genital imaginal discs of Drosophila melanogaster

Male genital imaginal discs from old (late-third-instar) larvae of Drosophila that had been X-irradiated with appropriate doses developed into severely damaged adult genitalia when implanted into old larvae, but they developed into completely normal adult genitalia when transplanted into 2-day-younger larvae. The major cause of X-ray-initiated teratogenesis in the genital disc was DNA damage because development of the genital discs of strain mei-9a with defective DNA repair was twice as sensitive as that of the wild-type strain to impairment by X-irradiation, just as larvae of this strain were twice as sensitive to killing by X-irradiation as those of the wild-type strain. Complete repair of X-ray-induced teratogenic damage in the genital discs on transplantation into young host larvae was similar in the wild-type and mei-9a strains. These results are discussed in relation to the hypothesis that repair of X-ray-induced teratogenic damage depends not on DNA repair but on replacement of damage-bearing primordial cells by healthy ones after suicidal elimination of the former.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

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.     

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.     

Isolation and characterization of temperature-sensitivelethal(2)giant larva alleles

Summary In this paper we present an analysis of the behavior ofl(2)gl ^tsimaginal wing discs during culture in adult hosts. Thel(2)gl ^tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl ^tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl ^tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl ^tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl ^tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl ^tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl ^tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl ^tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl ⁺function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.     

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.     

Imaginal discs regulate developmental timing in Drosophila melanogaster

The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Despite the importance of critical size in regulating final body size, the developmental mechanisms regulating critical size are poorly understood. Here we demonstrate that the developing adult organs, called imaginal discs, are a regulator of critical size in larval Drosophila. We show that damage to, or slow growth of, the imaginal discs is sufficient to retard metamorphosis both by increasing critical size and extending the period between attainment of critical size and metamorphosis. Nevertheless, larvae with damaged and slow growing discs metamorphose at the same size as wild-type larvae. In contrast, complete removal of all imaginal tissue has no effect on critical size. These data indicate that both attainment of critical size and the timely onset of metamorphosis are regulated by the imaginal discs in Drosophila, and suggest that the termination of growth is coordinated among growing tissues to ensure that all organs attain a characteristic final size.     

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.     

Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.     

What are and what are not imaginal discs: reevaluation of some basic concepts (Insecta, Holometabola).

Some general aspects of the concept of imaginal discs in the Holometabola are reevaluated. Their monolayer character and continuity with the surrounding epidermis are confirmed. Studies on the imaginal discs of the silkworm (Bombyx mori) and data from the literature show that the discs and their peripodial cells produce cuticle during larval life, as well as at metamorphosis. In B. mori it is demonstrated that adult and larval antennae are produced by the same cells or their progeny. The results also suggest that segments of the typically three-segmented larval antenna of Holometabola are not scape, pedicel, and one-segmented flagellum; at least segments 2 and 3 are of flagellar origin. Based on these and some additional facts it is argued that: (1) No larval organs are "replaced" at metamorphosis, but strict "sequential homology" is always maintained. (2) Imaginal discs are not undifferentiated structures destined to form the adult after larval breakdown, cannot be unambiguously defined, and do not represent qualitatively different epidermal structures. Classical imaginal discs (invaginated and present also in pre-final larval instars) arose several times independently and were not present in the larvae of ancestral Holometabola. (3) Since the disc cells are not undifferentiated and "embryonic" (if these words have a defined meaning at all), it is unreasonable to expect that the processes taking place in discs at metamorphosis would differ fundamentally from those occurring in other diploid metamorphosing epidermal cells.     

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.     

Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain

We have previously cloned lethal(2)giant larvae, a tumor-suppressor gene of Drosophila that normally controls cell proliferation and/or differentiation in the optic centers of the brain and the imaginal discs. Here we describe the structure of the l(2)gl genes as determined by sequencing genomic and cDNA clones. The structure of the cDNAs indicates the use of alternative splicing, either in the 5' untranslated exons or in the 3' coding exons. Thus the gene encodes two putative proteins of 1161 and 708 amino acids, p127 and p78, respectively, differing at their C termini. A 3'-truncated l(2)gl transposon that leaves the coding sequence of p78 intact but deletes 141 residues of p127 was capable of suppressing tumor formation in l(2)gl-deficient animals. These results suggest that the putative p78 protein is effective in controlling cell proliferation and/or differentiation.     

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     

A DETAILED FATE MAP OF THE WING DISC OF SARCOPHAGA PEREGRINA

In order to construct an anlageplan of the imaginal disc, various fragments of the wing disc from mature larvae of Sarcophaga peregrina were implanted into host larvae of the same age. The implants were metamorphosed with the host and the differentiated adult structures were analyzed.
On the basis of the results obtained from the transplanted disc fragments, a fate map of the wing disc was satisfactorily constructed.
The fate map of S. peregrina is similar to an earlier study of Drosophila , but the PAA, PWP, and AP are located in different positions from those of Drosophila .