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.     

Cell behaviour of Drosophila fat cadherin mutations in wing development

We have studied several cell behaviour parameters of mutant alleles of fat (ft) in Drosophila imaginal wing disc development. Mutant imaginal discs continue growing in larvae delayed in pupariation and can reach sizes of several times those of wild-type. Their growth is, however, basically allometric. Homozygous ft cells grow faster than their twin cells in clones and generate larger territories, albeit delimited by normal clonal restrictions. Moreover, ft cells in clones tend to grow towards wing proximal regions. These behaviours can be related with failures in cell adhesiveness and cell recognition. Double mutant combinations with alleles of other genes, e.g. of the Epidermal growth factor receptor (DER) pathway, modify ft clonal phenotypes, indicating that adhesiveness is modulated by intercellular signalling. Mutant ft cells show, in addition, smaller cell sizes during proliferation and abnormal cuticular differentiation, which reflect cell membrane and cytoskeleton anomalies, which are not modulated by the DER pathway.     

Modulo is a target of Myc selectively required for growth of proliferative cells in Drosophila

In Drosophila, the homologue of the proto-oncogene Myc is a key regulator of both cell size and cell growth. The identities and roles of dMyc target genes in these processes, however, remain largely unexplored. Here, we investigate the function of the modulo (mod) gene, which encodes a nucleolus localized protein. In gain of function or loss of function experiments, we demonstrate that mod is directly controlled by dMyc. Strikingly, in proliferative imaginal cells, mod loss-of-function impairs both cell growth and cell size, whereas larval endoreplicative tissues grow normally. In contrast to dMyc, over-expressing Mod in wing imaginal discs is not sufficient to induce cell growth. Taken together, our results indicate that mod does not possess the full spectrum of dMyc activities, but is required selectively in proliferative cells to sustain their growth and to maintain their specific size.     

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.     

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.     

When the control is lacking—the role of tumour suppressor genes in cancer development

The potential to genetically dissect tumorigenesis provides the major reason to study this process in the fruit flyDrosophila. Over the last 30 years genetic analysis has identified some 55 genes in which recessive mutations cause the appearance of specific tumours during development in tissues such as the imaginal discs, the brain hemispheres, the hematopoietic organs or the gonads, Since the normal allele acts dominantly over the mutated allele, these genes are designated as tumour suppressor genes. The estimate of the number of genes that can be mutated to tumour formation may be, however, much higher ranging between I00 to 200. The challenge before this field is how best to identify these genes and elucidate their function. Current molecular procedures, such as mutagenesis mediated by P-element transposon, provide new ways for tagging any gene of interest inDrosophila and thus for cloning it rapidly. Function of the gene product can be inferred by comparing its amino acid sequence with sequences of proteins with known function or can be determined by histochemical and biochemical investigations. Progress in the understanding of tumour suppression inDrosophila is most advanced in the case of genes regulating cell growth in imaginal discs. The imaginal discs are small groups of cells displaying a strong apical-basal polarity and form folded sacs of epithelia which grow throughout the larval life and give rise to the adult tegument during metamorphosis. Tumour suppressor genes regulating cell growth of imaginal discs, such as thelethal(2)giant larvae (l(2)g1),lethal(1)discs large-1 andexpanded genes, were found to encode proteins localized in domains of cell to cell contact on the plasma membrane and were thus thought to maintain cell adhesion. However, recent studies of l(2)gl have revealed that the l(2)gl protein is a component of the normal cytoskeleton which can participates to the cytoskeletal matrix underlaying the plasma membrane. These findings indicate that the changes in cell shape and the loss of apical-basal polarity in imaginal disc cells result primarily from alterations in the cytoskeleton structure. Furthermore the neoplastic growth of the mutated cells may be caused by the disorganization of an intracellular communication system that ultimately controls cell proliferation and/or cell differentiation.     

Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae)

During the last larval instar, the wing imaginal disks of Precis coenia grow continuously. The rate of disk growth is not disk-autonomous but closely matches the rate of somatic growth of the larva, so that the size of the disks is a function of the size of the body, irrespective of the growth rate of the larva. When larvae are starved, their wing disks cease growth within 4 h, which indicates the existence of an efficient coupling mechanism between the growth of the soma and growth of the imaginal disks. Disk growth is inhibited by juvenile hormone in a dose-dependent manner. In the presence of the hormone the wing disks stop growing even while the larva continues to grow normally. During the last larval instar the wing imaginal disks also undergo a complex differentiation, consisting of the development of the lacunae and tracheation that define the future adult wing venation system. In normally growing larvae, differentiation of the wing disk is tightly correlated with wing size. Differentiation and size can be dissociated by starvation. If larvae are starved at any time after differentiation has begun, differentiation continues at a normal rate, even though the wing disk does not grow. Differentiation does not begin spontaneously in larvae that are starved before differentiation has begun. These findings indicate that the initiation of differentiation and its continuation are controlled independently. Juvenile hormone inhibits differentiation in a dose-dependent manner. Upon treatment with juvenile hormone, the stage of differentiation becomes fixed. These findings indicate that continued differentiation of the wing disk can only occur in the absence of juvenile hormone. Although the circulating level of juvenile hormone may be elevated during starvation, it is unlikely that this elevation is responsible for the observed effect of starvation on growth and differentiation of the disk.     

Growth and cell survival are unevenly impaired in pixie mutant wing discs

It is largely unknown how growth slows and then stops in vivo. Similar to most organs, Drosophila imaginal discs undergo a fast, near-exponential growth phase followed by a slow growth phase before final target size is reached. We have used a genetic approach to study the role of an ABC-E protein, Pixie, in wing disc growth. pixie mutants, like mutants in ribosomal proteins genes (known as Minutes), show severe developmental delay with relatively mild alterations in final body size. Intriguingly, pixie mutant wing imaginal discs show complex regional and temporal defects in growth and cell survival that are compensated to result in near-normal final size. In S2 cells, Pixie, like its yeast homolog RLI1, is required for translation. However, a comparison of the growth of eukaryotic translation initiation factor eIF4A and pixie mutant clones in wing discs suggests that only a subset of translation regulators, including pixie, mediate regional differences in growth and cell survival in wing discs. Interestingly, some of the regional effects on pixie mutant clone growth are enhanced in a Minute background. Our results suggest that the role of Pixie is not merely to allow growth, as might be expected for a translation regulator. Instead, Pixie also behaves as a target of putative constraining signals that slow disc growth during late larval life. We propose a model in which a balance of growth inhibitors and promoters determines tissue growth rates and cell survival. An alteration in this balance slows growth before final disc size is reached.     

A gain-of-function screen identifying genes required for vein formation in the Drosophila melanogaster wing

The formation of the Drosophila wing involves developmental processes such as cell proliferation, pattern formation, and cell differentiation that are common to all multicellular organisms. The genes controlling these cellular behaviors are conserved throughout the animal kingdom, and the genetic analysis of wing development has been instrumental in their identification and functional characterization. The wing is a postembryonic structure, and most loss-of-function mutations are lethal in homozygous flies before metamorphosis. In this manner, loss-of-function genetic screens aiming to identify genes affecting wing formation have not been systematically utilized. As an alternative, a number of genetic searches have utilized the phenotypic consequences of gene gain-of-expression, as a method more efficient to search for genes required during imaginal development. Here we present the results of a gain-of-function screen designed to identify genes involved in the formation of the wing veins. We generated 13,000 P-GS insertions of a P element containing UAS sequences (P-GS) and combined them with a Gal4 driver expressed mainly in the developing pupal veins. We selected 500 P-GSs that, in combination with the Gal4 driver, result in modifications of the veins, changes in the morphology of the wing, or defects in the differentiation of the trichomes. The P-element insertion sites were mapped to the genomic sequence, identifying 373 gene candidates to participate in wing morphogenesis and vein formation.     

Refinement of wingless expression by a wingless- and notch-responsive homeodomain protein,defective proventriculus

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. Here, we show that a homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. Our results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.     

Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development

The Drosophila serum response factor (DSRF) is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the DSRF regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signalling pathway, is sufficient to induce DSRF expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signalling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory.     

Activation of knot (kn) specifies the 3-4 intervein region in the Drosophila wing

Hedgehog (Hh) plays an important role in Drosophila wing patterning by inducing expression of Dpp, which serves to organize the wing globally across the A-P axis. We show here how Hh signalling also plays a direct role in patterning the medial wing through the activation of the Hh-target gene, knot (kn). kn is expressed in Hh-responsive cells near the A-P compartment boundary, where its expression is dependent on fu, a component of Hh signalling. kn is required for the proper positioning of veins 3 and 4 and to prevent ectopic venation between them. Furthermore, the expansion anteriorly of the normal kn expression domain causes an associated anterior shift in the position of vein 3 in the resultant wing. Ectopic expression of kn elsewhere in the wing imaginal disc results in the failure to properly activate the vein initiation genes, rho and Dl. Expression of the gene encoding the EGF-receptor (EGFR), which is required for vein initiation and subsequent differentiation, is normally depressed in the 3-4 intervein region. This downregulation of EGFR in the medial portion of the imaginal disc is dependent on kn activity and ectopic expression of kn inactivates EGFR elsewhere in the wing primordium. We propose kn expression in Hh-responsive cells of the wing blade anlagen during the late third instar creates a zone of cells in the medial wing in which vein primordia cannot be induced. The primordia for veins 3 and 4 are laid down adjacent to the kn-imposed vein-free zone, presumably by a signalling factor (such as Vn) also synthesized in the medial region of the wing.