Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis

The Notch signaling pathway plays a central role in animal growth and patterning, and its deregulation leads to many human diseases, including cancer. Mutations in the tumor suppressor lethal giant discs (lgd) induce strong Notch activation and hyperplastic overgrowth of Drosophila imaginal discs. However, the gene that encodes Lgd and its function in the Notch pathway have not yet been identified. Here, we report that Lgd is a novel, conserved C2-domain protein that regulates Notch receptor trafficking. Notch accumulates on early endosomes in lgd mutant cells and signals in a ligand-independent manner. This phenotype is similar to that seen when cells lose endosomal-pathway components such as Erupted and Vps25. Interestingly, Notch activation in lgd mutant cells requires the early endosomal component Hrs, indicating that Hrs is epistatic to Lgd. These data suggest that Lgd affects Notch trafficking between the actions of Hrs and the late endosomal component Vps25. Taken together, our data identify Lgd as a novel tumor-suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling.     

The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila

Drosophila sensory organ precursor (SOP) cells undergo several rounds of asymmetric cell division to generate the four different cell types that make up external sensory organs. Establishment of different fates among daughter cells of the SOP relies on differential regulation of the Notch pathway. Here, we identify the protein Lethal (2) giant discs (Lgd) as a critical regulator of Notch signaling in the SOP lineage. We show that lgd encodes a conserved C2 domain protein that binds to phospholipids present on early endosomes. When Lgd function is compromised, Notch and other transmembrane proteins accumulate in enlarged early endosomal compartments. These enlarged endosomes are positive for Rab5 and Hrs, a protein involved in trafficking into the degradative pathway. Our experiments suggest that Lgd is a critical regulator of endocytosis that is not present in yeast and acts in the degradative pathway after Hrs.     

The tumour suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule fringe (fng). The activity domain will be established symmetrically at each side of the boundary of Fng-expressing and non-expressing cells. Here, I present evidence that the Drosophila tumour-suppressor gene lethal (2) gaint discs (lgd) is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development.     

The Mammalian Orthologs of Drosophila Lgd,CC2D1A and CC2D1B,Function in the Endocytic Pathway,but Their Individual Loss of Function Does Not Affect Notch Signalling

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.     

Requirement for Cell-Proliferation Control Genes in Drosophila Oogenesis

Genes that are required for cell proliferation control in Drosophila imaginal discs were tested for function in the female germ-line and follicle cells. Chimeras and mosaics were produced in which developing oocytes and nurse cells were mutant at one of five imaginal disc overgrowth loci (fat, lgd, lgl, c43 and dco) while the enveloping follicle cells were normal. The chimeras were produced by transplantation of pole cells and the mosaics were produced by X-ray-induced mitotic recombination using the dominant female-sterile technique. The results show that each of the genes tested plays an essential role in the development or function of the female germ line. The fat, lgl and c43 homozygous germ-line clones fail to produce eggs, indicating a germ-line requirement for the corresponding genes. Perdurance of the fat+ gene product in mitotic recombination clones allows the formation of a few infertile eggs from fat homozygous germ-line cells. The lgd homozygous germ-line clones give rise to a few eggs with abnormal chorionic appendages, a defect thought to result from defective cell communication between the mutant germ-line and the nonmutant follicle cells. One allele of dco (dcole88) prevents egg development when homozygous in the germ line, whereas the dco18 allele has no effect on germ-line development. Fs(2)Ugra, a recently described follicle cell-dependent dominant female-sterile mutation, allowed the analysis of egg primordia in which fat, lgd or lgl homozygous mutant follicle cells surrounded normal oocytes. The results show that the fat and lgd genes are not required for follicle cell functions, while absence of lgl function in follicles prevents egg development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scabrous and Gp150 are endosomal proteins that regulate Notch activity

Notch and Delta are required for lateral inhibition during eye development. They prevent a tenfold excess in R8 photoreceptor cell specification. Mutations in two other genes, Scabrous and Gp150, result in more modestly increased R8 specification. Their roles in Notch signaling have been unclear. Both sca and gp150 are required for ectopic Notch activity that occurs in the split mutant. Similar phenotypes showed that sca and gp150 genes act in a common pathway. Gp150 was required for all activities of Sca, including inhibition of Notch activity and association with Notch-expressing cells that occur when Sca is ectopically expressed. Mosaic analysis found that the gp150 and sca genes were required in different cells from one another. Gp150 concentrated Sca protein in late endosomes. A model is proposed in which endosomal Sca and Gp150 promote Notch activation in response to Delta, by regulating acquisition of insensitivity to Delta in a subset of cells.     

Transdetermination: Drosophila imaginal disc cells exhibit stem cell-like potency

Drosophila imaginal discs, the primordia of the adult fly appendages, are an excellent system for studying developmental plasticity. Cells in the imaginal discs are determined for their disc-specific fate (wingness, legness) during embryogenesis. Disc cells maintain their determination during larval development, a time of extensive growth and proliferation. Only when prompted to regenerate do disc cells exhibit lability in their determined identity. Regeneration in the disc is mediated by a localized region of cell division, known as the regeneration blastema. Most regenerating disc cells strictly adhere to their disc-specific identity; some cells however, switch fate in a phenomenon known as transdetermination. Similar regeneration and transdetermination events can be induced in situ by misexpression of the signaling molecule wingless. Recent studies indicate that the plasticity of disc cells during regeneration is associated with high morphogen activity and the reorganization of chromatin structure. Here we provide both a historical perspective of imaginal disc transdetermination, as well as discuss recent findings on how imaginal disc cells acquire developmental plasticity and multipotency. We also highlight how an understanding of imaginal disc transdetermination can enhance an understanding of developmental potency exhibited by stem cells.     

The dachsous gene, a member of the cadherin family, is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of the canonical Wnt signal Wingless (Wg) during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis.     

Self-refinement of Notch activity through the transmembrane protein Crumbs: modulation of gamma-secretase activity

Cell interactions mediated by Notch family receptors have been implicated in the specification of tissue boundaries. Tightly localized activation of Notch is crucial for the formation of sharp boundaries. In the Drosophila wing imaginal disc, the Notch receptor is expressed in all cells. However, Notch activity is limited to a narrow stripe of cells along the dorsal–ventral compartment boundary, where it induces the expression of target genes. How a widely expressed protein becomes tightly regulated at the dorsal–ventral boundary in the Drosophila wing is not completely understood. Here, we show that the transmembrane protein Crumbs is involved in a feedback mechanism used by Notch to refine its own activation domain at the Drosophila wing margin. Crumbs reduces the activity of the γ-Secretase complex, which mediates the proteolytic intracellular processing of Notch. These results indicate a novel molecular mechanism of the regulation of Notch signal, and also that defects in Crumbs might be involved in similar abnormal γ-Secretase complex activity observed in Alzheimer's disease.     

Independent roles of Drosophila Moesin in imaginal disc morphogenesis and hedgehog signalling

The three ERM proteins (Ezrin, Radixin and Moesin) form a conserved family required in many developmental processes involving regulation of the cytoskeleton. In general, the molecular function of ERM proteins is to link specific membrane proteins to the actin cytoskeleton. In Drosophila, loss of moesin (moe) activity causes incorrect localisation of maternal determinants during oogenesis, failures in rhabdomere differentiation in the eye and alterations of epithelial integrity in the wing imaginal disc. Some aspects of Drosophila Moe are related to the activity of the small GTPase RhoA, because the reduction of RhoA activity corrects many phenotypes of moe mutant embryos and imaginal discs. We have analysed the phenotype of moesin loss-of-function alleles in the wing disc and adult wing, and studied the effects of reduced Moesin activity on signalling mediated by the Notch, Decapentaplegic, Wingless and Hedgehog pathways. We found that reductions in Moesin levels in the wing disc cause the formation of wing-tissue vesicles and large thickenings of the vein L3, corresponding to breakdowns of epithelial continuity in the wing base and modifications of Hedgehog signalling in the wing blade, respectively. We did not observe any effect on signalling pathways other than Hedgehog, indicating that the moe defects in epithelial integrity have not generalised effects on cell signalling. The effects of moe mutants on Hedgehog signalling depend on the correct gene-dose of rhoA, suggesting that the requirements for Moesin in disc morphogenesis and Hh signalling in the wing disc are mediated by its regulation of RhoA activity. The mechanism linking Moesin activity with RhoA function and Hedgehog signalling remains to be elucidated.     

Short-range cell interactions and cell survival in the Drosophila wing

During development of multicellular organisms, cells are often eliminated by apoptosis if they fail to receive appropriate signals from their surroundings. Here, we report on short-range cell interactions that support cell survival in the Drosophila wing imaginal disc. We present evidence showing that cells incorrectly specified for their position undergo apoptosis because they fail to express specific proteins that are found on surrounding cells, including the LRR transmembrane proteins Capricious and Tartan. Interestingly, only the extracellular domains of Capricious and Tartan are required, suggesting that a bidirectional process of cell communication is involved in triggering apoptosis. We also present evidence showing that activation of the Notch signal transduction pathway is involved in triggering apoptosis of cells misspecified for their dorsal-ventral position.     

Maintenance of imaginal disc plasticity and regenerative potential in Drosophila by p53

Following irradiation (IR), the DNA damage response (DDR) activates p53, which triggers death of cells in which repair cannot be completed. Lost tissue is then replaced and re-patterned through regeneration. We have examined the role of p53 in co-regulation of the DDR and tissue regeneration following IR damage in Drosophila. We find that after IR, p53 is required for imaginal disc cells to repair DNA, and in its absence the damage marker, γ-H2AX is persistently expressed. p53 is also required for the compensatory proliferation and re-patterning of the damaged discs, and our results indicate that cell death is not required to trigger these processes. We identify an IR-induced delay in developmental patterning in wing discs that accompanies an animal-wide delay of the juvenile-adult transition, and demonstrate that both of these delays require p53. In p53 mutants, the lack of developmental delays and of damage resolution leads to anueploidy and tissue defects, and ultimately to morphological abnormalities and adult inviability. We propose that p53 maintains plasticity of imaginal discs by co-regulating the maintenance of genome integrity and disc regeneration, and coordinating these processes with the physiology of the animal. These findings place p53 in a role as master coordinator of DNA and tissue repair following IR.     

Extracellular peptidases of imaginal discs of Drosophila melanogaster

The imaginal discs of Drosophila melanogaster give rise to the adult epidermis during metamorphosis. During this developmental period several peptidase genes are expressed in disc cells, but there is a paucity of biochemical information regarding substrate specificity. We have used peptides and peptidyl 7-amino-4-methylcoumarin (AMC) substrates to detect several peptidases either positioned on the surface of wing discs or secreted by the imaginal cells. Using [Leu(5)]enkephalin as a substrate, a captopril sensitive dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) and an amastatin-sensitive aminopeptidase were detected as prominent activities associated with intact discs. The formation of [Leu(5)]enkephalin-derived Phe was attributed to the concerted action of the D. melanogaster angiotensin I-converting enzyme (Ance) and a dipeptidase. The disc Ance also showed endopeptidic activity towards locust tachykinin-1 (LomTK-I) by cleaving the Gly-Val peptide bond, but this enzyme was not the sole endopeptidase activity associated with discs. Complete inhibition of the endopeptidic hydrolysis of the LomTK-1 by a disc homogenate required a combination of captopril and the neprilysin inhibitor, phosphoramidon, providing biochemical evidence for a neprilysin-like peptidase, in addition to Ance, in imaginal discs of D. melanogaster. Peptidyl AMC substrates for furin, prohormone convertase and tryptase provided evidence for trypsin-like serine endopeptidases in addition to the metalloendopeptidases. We conclude that imaginal discs are endowed with a variety of peptidases from different families that together are capable of hydrolyzing a broad range of peptides and proteins. Some of these peptidases might be responsible for the metabolic activation/inactivation of signaling peptides, as well as being involved in the production of dipeptides and free amino acids required for protein synthesis and osmotic balance during adult morphogenesis.     

The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye.

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. We have used loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) to determine the role of Hop in eye development. We find that Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously.     

Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor.

BACKGROUND: Class I(A) phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. Overexpression of the gene encoding the Drosophila class I(A) PI 3-kinase, Dp110, in imaginal discs, however, results in enlarged adult organs. These observations have led us to investigate the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size. RESULTS: Null mutations in Dp110 and p60 were generated and used to demonstrate that they are essential genes that are autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110 activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole. CONCLUSIONS: We conclude that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. Our results indicate that Dp110 and p60 signalling can affect growth in multiple ways, which has important implications for the function of signalling through class I(A) PI 3-kinases.     

Drosophila Ndfip is a novel regulator of Notch signaling

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch(+/-) wing phenotype and knockdown of dNdfip enhanced the Notch(+/-) wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate(+/-) wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation.