InteractiveFly: GeneBrief

lethal (2) giant discs 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - lethal (2) giant discs 1

Synonyms - lethal giant discs, lgd

Cytological map position - 32D1--4

Function - regulator of endocytosis

Keywords - imaginal discs, Notch pathway, tumor suppression

Symbol - l(2)gd1

FlyBase ID: FBgn0261983

Genetic map position - 2-[41]

Classification - conserved C2 domain protein

Cellular location - cytoplasmic



NCBI link: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

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 between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal (2) giant discs (lgd), a gene whose coding region has yet to be identified, 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 (Klein, 2003).

Imaginal disc development depends on the Drosophila tumor suppressor genes (TSGs). Fifty TSGs have been identified and the loss-of-function of many of these genes results in overproliferation of the imaginal discs. These genes can be divided into two groups based on the mutant phenotypes (Bryant, 1993; Watson, 1994). Deletion of genes belonging to the tumorous class causes cells to overproliferate and invade new regions so that eventually the epithelial and compartmental organization of the discs is lost. In contrast, the loss of genes of the hyperplastic group causes overproliferation, but does not disturb the epithelial and compartmental organization of the discs. l(2)giant discs belongs to this second group. The loss of lgd causes massive overproliferation of imaginal disc cells and extended larval life (Bryant, 1971; Klein, 2003).

It has also been observed that wingless (wg) is expressed ectopically in the pouch of lgd mutants during wing development (Buratovich, 1995). Similar phenotypes are observed, if the Notch pathway is ectopically activated during wing development, raising the possibility that the lgd mutant phenotype could stem from the ectopic activation of the Notch pathway. The Notch pathway is indeed ectopically active in lgd mutants, and hyperactivation as well as ectopic activation of the pathway accounts for the lgd phenotype during wing development. In lgd mutants, the expression of Notch target genes along the D/V boundary is expanded, indicating that Lgd is required for the restriction of Notch activity to the D/V boundary. Furthermore, the mutant phenotype of lgd is suppressed by concomitant loss of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the activation of the Notch pathway. Evidence is provided that the activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch by Lgd is not restricted to wing development and occurs during several other developmental processes, such as vein, eye, and bristle development, suggesting that Lgd suppresses the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).

Loss of lgd function leads to an overgrowth of the imaginal discs, clearly noticeable in the wing region of the wing disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a much broader domain that extends into the wing pouch (Buratovich, 1995). In addition, lgd mutant wing discs often develop a second wing pouch in the region of the anlage of the scutellum (Buratovich, 1995). Similar phenotypes are caused by gain-of-function alleles of N (for example, Abruptex) and are also observed upon expression of the activated intracellular form of Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing discs and precedes the visible morphological changes that occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The phenotype is always variable, but the overall phenotype of lgdd7 and lgdd10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgdd4 and lgdd1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in homozygotes, indicating that the observed phenotype is probably caused by the loss-of-function of the lgd gene (Klein, 2003).

The similarity between the loss of lgd function and ectopic N activation suggests that the phenotype of lgd could be caused by ectopic activation of the Notch pathway. To examine this possibility, the expression of E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the vg-boundary enhancer (vgBE) in mutant wing discs. The expression of all these markers is initiated in cells at the D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is activated in an additional stripe along the anteroposterior compartment boundary (A/P boundary), which is also dependent on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is more complex but always dependent on the activity of Notch in cells at the D/V boundary. In lgd mutant wing discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer, since a version lacking it shows no ectopic activity in the mutants. As in the case of wg, the expression of the vgBE is already expanded in early third larval wing discs. Altogether, these results show that the loss of lgd function leads to the ectopic expression of Notch target genes. This suggests that the Notch pathway is ectopically activated in lgd mutants (Klein, 2003).

All tested Notch-target genes are ectopically activated in lgd mutant wing discs or lgd mutant cell clones. The ectopic activation of Notch target genes as well as the observed overproliferation of lgd mutants is abolished in lgd;Psn double mutants. In addition, Notch target gene expression is also abolished in Psn or Su(H) mutant clones generated in lgd mutant wing imaginal discs. These data suggest that the Notch pathway becomes ectopically active in the absence of lgd function. Furthermore, the fact that Delta alone seems to provide sufficient Notch activity to sustain wing development in lgd mutants indicates that the pathway can be activated more efficiently in the mutant background. The activation of Notch is a consequence of loss of lgd function also in other developmental processes, such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).

Although most aspects of the mutant phenotype of lgd mutants can be explained by the inappropriate activation of the Notch pathway, the cell death observed during induction of lgd mutant clones has not been observed if activated forms of Notch are expressed in the wing pouch or in gain-of-function mutants of Notch, such as Ax. These facts would suggest that lgd function might also have another function for cell viability that is separable from its role in the regulation of Notch activity. However, inappropriate activation of the Notch pathway elicits apoptosis in wing pouch cells under certain circumstances. Hence, it is also possible that this aspect of the lgd mutant phenotype is a consequence of Notch activation (Klein, 2003).

The clonal analysis of lgd reveals several interesting effects. One effect is that Notch becomes activated at the boundary of Dl;Ser double mutant cell clones. At the moment, it is not clear how this activation is achieved. A likely explanation is that activation of Notch at the clone boundaries is caused by the removal of the negative effects of strong Dl and Ser expression observed during late wing development. During normal development, Dl and Ser are expressed in a dorsal and ventral band of cells adjacent to the cells at the D/V boundary in later stages of the third larval instar. Both ligands signal from there to the cells at the boundary to maintain expression of Wg and other genes. It has been shown that activation of Notch is blocked in the cells expressing the ligands because of their autonomous inhibitory effect on Notch signalling at high concentrations. Loss of Dl and Ser expression leads to the loss of the suppressive effect, and the mutant cells at the clone boundary activate expression of Notch target genes. In lgd mutants, the expression domains of Dl and Ser are expanded and the pathway can be activated more efficiently. Thus, the effect of activation of Notch at the boundary of Ser/Dl double mutant clones should also be comparably enhanced (Klein, 2003).

The analysis of the lgd mutant clones suggests that lgd acts in a cell-autonomous way. However, this autonomy is not complete, and in some cases, Notch target genes are activated in wild type cells at the boundary of lgd mutant clones. An explanation for this observation is the fact that the activation of Notch results in the expression of the ligands Dl or Ser. Clones of wing pouch cells expressing the activated form of Notch, Nintra, also activate Notch target gene expression in cells outside the clone, indicating a nonautonomous behavior of Nintra in this cases. This nonautonomous behavior is caused by the induction of the expression of the Notch ligands. The nonautonomy of Nintra is not observed in all situations. For example, if UAS Nintra is expressed with ptcGal4, activation of Notch target genes is cell-autonomous, although induction of ligand expression is observed. Hence, the nonautonomous activation of Notch target genes by Nintra is dependent on other criteria, such as the level of expression or the time span of signalling. It is likely that the observed weak nonautonomy of lgd in clones is caused by the activation of expression of Dl and Ser close to threshold levels of activity that are required to activate Notch in some cells outside the clone (Klein, 2003).

Several explanations for how the Notch pathway is activated in lgd mutants are possible. A very simple one would be that the expansion of Notch target genes in lgd mutant clones or wing discs is caused by an overproliferation of the mutant cells that cause an expansion of the expression domains of the Notch target genes. Thus, the effects on Notch signalling would be secondary. However, clones that are located in the wing pouch and do not have any contact with the normal domain of Notch activity at the D/V boundary are able to activate the expression of Notch target genes, indicating that the pathway is activated de novo. Furthermore, Notch is activated in mutant clones of wing discs of the early third instar. These discs do not show any visible overproliferation. Hence, it is very likely that the expansion of the target gene expression is not caused by a secondary effect, such as cell proliferation, but by the activation of the Notch pathway (Klein, 2003).

The expansion of Notch activity could also be caused by the loss of the suppressive effect on signalling of high concentrations of the ligands observed in the lgd mutants. Although this mode of regulation is important during the second half of the third larval instar stage, it cannot account for the ectopic activation of Notch targets in earlier wing discs observed here (Klein, 2003).

lgd could act in a parallel pathway that is required to restrict the activation of the target genes by Notch. An example of this is the Nubbin transcription factor that seems to bind to the regulatory region of at least some Notch target genes and represses their expression away from the D/V boundary. lgd could act in a similar way. However, there are important differences in the behavior of nub and lgd mutants. nub mutants do not show the overproliferation of the imaginal discs seen in lgd mutants and, in contrast to lgd, the effects of Nub on Notch target gene expression are restricted to the wing. These differences make it unlikely that both genes act in the same pathway. In agreement with these conclusions, it has been found that nub expression is not affected in lgd mutant wing imaginal discs (Klein, 2003).

A further possibility is that lgd could modulate the effectiveness of the Notch signal, e.g., by creating a threshold for Notch activity required for activation of the target genes or influencing the activity of a selector gene such as Vg for the wing. However, the activity of one target gene of Vg/sd, spalt, is not affected in lgd mutants, suggesting that the activity of the selector is not affected (Klein, 2003).

The comparison of the Ax and lgd mutant phenotype reveals a striking similarity: In Ax mutant wing discs, as in those of lgd mutants, Notch activity expands into the wing pouch. In addition, in Ax mutant wing discs, the dominant negative activity of the ligands is suppressed in a fashion similar to that observed in lgd mutants. The phenotype of both of these mutants requires the activity of the Notch ligands. Furthermore, in both mutants, the cell-autonomous suppressive effect of Fng on Notch signalling is strongly suppressed. Finally, the development of the veins and SOPs is suppressed in both mutants. The similarity of the phenotypes between lgd and Ax mutants raises the possibility that they are based by the interruption of the same process required to negatively regulate Notch activity. One argument against this conclusion is that the phenotype of the lgdd7,AxMI double mutant wing discs described here is synergistic. This suggests that the genes do not act in the same regulatory mechanism. The problem with this argument is that it is not clear whether any of the known Ax mutations are abolishing the affected function completely and thus does not rule out the possibility that lgd and Ax affect the same regulatory pathway (Klein, 2003).


REGULATION

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. This study identified the protein Lethal (2) giant discs (Lgd) as a critical regulator of Notch signaling in the SOP lineage. 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. These experiments suggest that Lgd is a critical regulator of endocytosis that is not present in yeast and acts in the degradative pathway after Hrs (Gallagher, 2006).

The phenotypes observe in lgd mutants are strikingly similar to those that have recently been described for Drosophila members of the ESCRT complexes. These complexes have been identified in yeast but are found in all animals. They are required for protein sorting in the degradative pathway and the formation of multivesicular bodies. Ubiquitinated internalized proteins are recognized by Hrs (Vps27 in yeast), a ubiquitin-binding protein targeted to early endosomes by its FYVE domain. Hrs binds to Vps23, a member of the ESCRT I complex, and these proteins recruit the other members of the ESCRT I complex. ESCRT I activates ESCRT II, leading to the recruitment of ESCRT III, the budding of vesicles into the endosomal lumen, and MVB formation. When MVBs fuse with lysosomes, these internal vesicles and their protein contents are degraded by lipases and hydrolases (Gallagher, 2006).

Although there is no yeast homolog of Lgd, three pieces of evidence suggest that Lgd might act in this pathway: (1) mutations in the Drosophila homologs of vps27 (hrs in flies and mammals), vps23 (erupted in Drosophila; tsg101 in mammals), and vps25 (another ESCRT II complex member) lead to accumulation of ubiquitinated transmembrane proteins in enlarged endosomes, a phenotype that is also observe in lgd mutants. Notch is found in enlarged, Hrs-positive compartments in both lgd and vps25 mutant cells. (2) In lgd mutants, just like in flies mutant for hrs, erupted, or vps25, signaling through transmembrane receptors is ectopically activated. (3) lgd was initially identified as a tumor suppressor gene, and recent papers describing the Drosophila homologs of ESCRT complex members show that they also have tumor suppressor properties (Gallagher, 2006).

Where in the pathway could Lgd act? Due to a paucity of markers for ESCRT complex members in Drosophila, it was not possible to precisely determine the point at which lgd is required. However, the results indicate that lgd acts after hrs in the pathway. Unlike mutants in ESCRT I (vps23, erupted) or ESCRT II (vps25), the Notch pathway is not ectopically activated in hrs mutants. Furthermore, hrs, lgd double mutant experiments suggest that the ectopic activation of Notch in lgd mutants requires the activity of hrs. Consistent with this, in lgd mutant cells, Hrs is recruited to vesicles, and these vesicles contain ubiquitinated proteins. An interpretation is that hrs mutants block Notch trafficking at an earlier step than lgd. In the double mutant, the early block in vesicle trafficking does not allow Notch to reach the later compartment, in which it would accumulate in lgd single mutants, thus preventing ectopic activation of the Notch pathway (Gallagher, 2006).

Is it possible to reconcile the protein-trafficking defect and Notch overactivation observed in lgd mutants? The final step in Notch activation is the Presenilin-dependent S3 cleavage. Since Presenilin has been shown to be required for ectopic Notch activation in lgd mutants, it is proposed that lgd leads to the accumulation of Notch in a compartment where it can be more easily cleaved by the protease. Presenilin localizes to the plasma membrane and to internal membranes and has been shown to be active both at the plasma membrane and in endosomes. Although it cannot be excluded that the S3 cleavage occurs at the cell surface, the data suggest that this proteolytic event can also occur to some level in endosomal compartments. Two reasons can be envisaged to explain the Notch overactivation phenotype in lgd mutants: either Notch is endocytosed to some level even if it has not encountered a ligand, and this pool of endocytosed Notch is activated over time when it accumulates in endosomes. Alternatively, ligand binding triggers the S2 cleavage at the cell surface, and it is the NEXT fragment that accumulates in endosomes and therefore can undergo a more complete S3 cleavage before being degraded in lysosomes. Although full-length Notch is not a good substrate for Presenilin and upregulation of Notch signaling in lgd mutants was thought to be ligand dependant, an accompanying paper (Jaekel, 2006) shows that ectopic Notch signaling in lgd mutants is ligand independent, favoring the first possibility (Gallagher, 2006).

It is puzzling that loss of lgd and loss of ESCRT I/II complex members leads to Notch overactivation but hrs mutations do not. Recent work has shown that accumulation of Notch is not always sufficient to activate Notch signaling, whether it is at the plasma membrane or in late endosomes. In hrs mutants, Notch colocalizes with the syntaxin Avalanche, while in vps25 mutants it does not. This finding indicates that although Notch accumulates in enlarged early endosomes in both cases, there are differences between these endosomes. One difference could be the presence or absence of Presenilin, although this remains to be tested (Gallagher, 2006).

Just as accumulation of Notch does not always lead to ectopic activation of signaling, activation of Notch signaling does not always have the same consequences for the cell. lgd mutant cells activate the Notch target gene Cut, whereas vps25 mutant cells do not. Loss of ESCRT I/II complex members leads to Notch-dependant activation of Unpaired, leading, in turn, to nonautonomous overproliferation, while lgd mutant cells themselves overproliferate. lgd mutant cells retain the capacity to differentiate, while ESCRT I/II mutant cells lose polarity, fail to differentiate, and undergo apoptosis. Clearly, further characterization of lgd and its homologs is required to define its functional relationship with the ESCRT complex (Gallagher, 2006).

All ESCRT complex members identified so far are conserved between yeast and humans. Given that lgd is not conserved in yeast, the phenotypic similarity to vps23 and vps25 mutations is surprising. It is possible that the more complex sorting requirements in multicellular organisms require modifications of the ESCRT machinery. Further study will be required to figure out exactly what evolutionary advantage this modification offers metazoa (Gallagher, 2006).

The tumour suppressor Lethal (2) giant discs is required for the function of the ESCRT-III component Shrub/CHMP4

Recent work indicates that defects in late phases of the endosomal pathway caused by loss of function of the tumour suppressor gene lethal (2) giant discs (lgd) or the function of the ESCRT complexes I-III result in the ligand-independent activation of the Notch pathway in all imaginal disc cells in Drosophila. lgd encodes a member of an uncharacterised protein family, whose members contain one C2 domain and four repeats of the DM14 domain. The function of the DM14 domain is unknown. This study reports a detailed structure-function analysis of Lgd protein, which reveals that the DM14 domains are essential for the function of Lgd and act in a redundant manner. Moreover, this analysis indicates that the DM14 domain provides the specific function, whereas the C2 domain is required for the subcellular location of Lgd. Lgd was found to interact directly with the ESCRT-III subunit Shrub through the DM14 domains. The interaction is required for the function of Shrub, indicating that Lgd contributes to the function of the ESCRT-III complex. Furthermore, genetic studies indicate that the activation of Notch in ESCRT and lgd mutant cells occurs in a different manner and that the activity of Shrub and other ESCRT components are required for the activation of Notch in lgd mutant cells (Troost, 2012).

This study reports the results of a detailed structure-function analysis of Lgd, a member of a recently discovered protein family whose hallmark is the possession of four tandem repeats of the uncharacterised DM14 domain. Although a recent study has reported a similar analysis for human Lgd2 in cell culture (Zhao, 2010), this is the first comprehensive analysis of a member of this uncharacterised protein family in an animal model. For the analysis a new assay system was developed that assured expression of the constructs at the level of endogenous lgd. This was necessary because it was found that the process of protein trafficking is very sensitive to overexpression of Lgd. Thus, data obtained by overexpression of Lgd proteins (e.g., in cell culture) must be interpreted with great caution. This notion can probably be extended to other elements of the endosomal pathway, because dramatic changes have be observed in endosome morphology if other endosomal proteins, such as FYVE-GFP, Rab5-GFP or Rab7-GFP, are expressed with the Gal4 system. Moreover, this study found that overexpression of these proteins suppresses the activation of Notch in lgd cells. These findings indicate that the overexpression of endosomal proteins induces significant changes in protein trafficking through the endosomal pathway (Troost, 2012).

This study found that the DM14 domains are important for the function of Lgd and that they constitute novel modules for direct interaction with a core member of the ESCRT-III complex during protein trafficking. Moreover, this analysis reveals that the DM14 domains provide the specific function of Lgd and function in a redundant manner. Using cell culture, Nakamura (2008) provided evidence that the fourth DM14 domain of Lgd2 is especially important for its function as a scaffold protein that is required for PDK1/Akt signalling activated by the EGF. However, no specific importance of the fourth DM14 domain could be detected in Drosophila. In the assay conditions used, any combination of two of the four domains appears to be sufficient for Lgd function and can rescue the lgd mutant phenotype (Troost, 2012).

However, this notion holds true only if the concentration of Shrub is normal. In situations where the activity of Shrub is reduced (shrub4-1/+), variants with four domains can provide more activity and assure sufficient interaction to maintain correct endosomal trafficking. This was already observed in animals that are hypomorphic for lgd (lgdd7/lgdSH495 shrub4-1). In other words, four DM14 copies enable the organism to tolerate the lgd shrub double heterozygous situation. Because almost all Lgd-like proteins discovered so far have four copies, it is likely that this ability endows members of the family with a functional robustness that is evolutionarily advantageous. The rescue experiments in the sensitized lgd +/lgd shrub4-1 backgrounds also suggest that the second DM14 domain is of greatest importance for the function of Lgd in Drosophila. This is in contrast to results of cell culture experiments for human Lgd2 (Nakamura, 2008). However, it is important to point out that most of the evidence for function in mammals is obtained with cell culture experiments, which often involve the overexpression of Lgd orthologues at levels way above endogenous levels. Given the great difficulties in gaining sensible results using the Gal4 system, these data should be interpreted carefully (Troost, 2012).

It has been previously shown that the C2 domain of Lgd can bind to certain phospholipids, such as phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, in an in vitro assay (Gallagher, 2006). Furthermore, cell fractionation experiments using cytosolic extracts from wild-type and the lgd08 mutant animals that encode a variant lacking the C2 domain, suggest that a small fraction is associated with the membrane in a C2-dependent manner. These biochemical data are contrasted by microscopy studies, which reported a cytosolic distribution of Lgd without any obvious association with membrane structures. In agreement, this study found that tagged Lgd variants expressed at the endogenous level are localised within the cytosol. Moreover, it was found that a lgd construct, encoding little more than the C2 domain and virtually identical to the Lgd fragment used in the in vitro phospholipid binding assay (Gallagher, 2006), is located in the cytosol similarly to Lgd. The discrepancy between the biochemical and microscopy data might be explained by the possibility that only a small fraction of Lgd (which cannot be detected in antibody staining) is associated with membranes. However, knowing that Lgd interacts with Shrub, it is surprising that no obvious association of Lgd was found even upon depletion of Vps4, although the ESCRT-III complex is locked on the endosomal membrane in this situation. One would expect that the membrane-associated fraction of Lgd should be increased in this situation. Thus, it is believed that Lgd is located within the cytosol. This notion is further supported by the fact that variants of Lgd that lack the C2 domain can rescue the lgd mutant phenotype to a high degree, although they are produced at a much lower level than the other constructs tested and than endogenous Lgd (Troost, 2012).

Three distinct functions were determined for the C2 domain. The first function is that it provides protein stability, because it was found that the constructs encoding variants without the C2 domain give rise to significantly lower amounts of protein than variants with the domain. The second function is the localisation of Lgd within the cytosol. This function provides an explanation for the discrepancy between the in vivo and biochemical studies, because variants without the C2 domains were found to be located in the nucleus. The reason for the mis-localisation of Lgd variants that lack the C2 domain is unclear at the moment. No cryptic nuclear localisation sequence (NLS) has been found within Lgd. Thus, it is possible that it is transported in the nucleus in complex with another protein that contains an NLS (Troost, 2012).

The presented results suggest a third function for the C2 domain, because it was found that LgdδDM14, which cannot provide any specific function in the rescue assay, can out-compete NESLgdδC2 in a C2-dependent manner and thereby prevent the partial rescue of lgd mutants. A likely possibility is that the C2 domain mediates an interaction with other proteins that results in concentration of Lgd at the site of action within the cytosol. In agreement with this possibility, recent reports have shown that the C2 domains of Nedd4L, PKC and PKCe mediate protein-protein interactions. Furthermore, human Lgd2/CC2D1A appears to interact via its C2 domain with the E2 enzyme Ubc13 during NF-kappaB signalling (Zhao, 2010). Therefore, the possibility is favored that the C2 domain of Lgd mediates protein-protein interactions instead of localising Lgd to a distinct membrane. It is possible that the cytosolic interaction prevents Lgd from migrating into the nucleus (Troost, 2012).

Recent results obtained in mammalian cell culture experiments suggest that human Lgd1 and Lgd2 might also act as transcriptional repressors (Hadjighassem, 2009; Ou, 2003). This study found that Lgd requires location within the cytosol for its function. Hence, the current results are not easily compatible with a function as a transcription factor, as suggested for human Lgd1 and Lgd2, and it is believed that a gene regulatory function for Lgd inDrosophila is unlikely (Troost, 2012).

Previous work has established that loss of function of ESCRT-I-ESCRT-III complexes results in non-autonomous and autonomous cell proliferation and activation of the Notch pathway. In addition, the mutant cells lose their epithelial organisation and eventually die. Although loss of function of lgd results in activation of the Notch pathway and overproliferation, these effects are cell-autonomous, and the mutant cells do not lose their polarity and survive well. Thus, the phenotypes of the two groups overlap, but are not identical. Nevertheless, this study has found an intimate relationship between the ESCRT-III component Shrub and Lgd. Both proteins physically interact and this direct interaction is important in vivo, as indicated by the strong genetic interactions uncovered between the two genes. Importantly, it was observed that the time of death for a hypomorphic allelic combination of lgd, which normally results in pharate adults, is earlier than that of lgd null mutants if the activity of shrub is reduced by half. The earlier time of death suggests that the function of shrub is impaired upon loss of lgd function. Thus, it appears that the physical interaction with Lgd is required for the proper function of Shrub. Because the loss-of-function phenotype of shrub is more deleterious and includes more aspects than that of lgd, it is likely that lgd contributes to, but is not absolutely required for, the function of shrub. Either loss of lgd results in the loss of one distinct aspect of Shrub function or it reduces its activity beyond a threshold that is required for complete function. The finding that overexpression of Shrub can rescue the lgd phenotype supports the second possibility. Recent work suggests that Shrub forms long homopolymers on the cytosolic surface of the endosomal membrane. This polymerisation is required for the abscission of vesicles into the lumen of the maturing endosome (Saksena, 2009). In order to polymerise, Shrub has to be converted from a closed cytosolic into the open form (Babst, 2002). After intraluminal vesicle (ILV) formation, Shrub becomes converted into the closed form by Vps4, with consumption of ATP. Because the data suggest that Shrub and Lgd interact in the cytosol, it is possible that Lgd somehow helps to prepare Shrub for the next round of polymerisation on the endosomal membrane (Troost, 2012).

The presented genetic studies suggest an antagonistic relationship between Lgd and several components of the ESCRT complexes with respect to Notch activation. This implies that activation of Notch in lgd cells depends on the function of the ESCRT complexes and therefore indicates that it must occur in a different manner in lgd cells to that in ESCRT-mutant cells. The results suggest that loss of lgd function somehow affects the activity of Shrub, which in turn results in the activation of Notch. It is important to point out that the antagonism between lgd and ESCRT is observed only with respect to activation of Notch signalling. With respect to endosome morphology, they appear to act synergistically because a reduction of shrub function by half results in a dramatic enlargement of endosomes of lgd hypomorphic cells, which normally do not exhibit such a defect. The results therefore reveal a complex relationship between Lgd and the ESCRT function and further work is required to resolve this relationship in detail (Troost, 2012).

Because activation of Notch is not possible without release of the Notch intracellular domain (NICD) into the cytosol, it is assumed that a fraction or all of Notch must somehow remain at the limiting membrane of the endosome and is not incorporated into ILVs in lgd cells. There are three possibilities for how this might be achieved: no ILVs form; Notch might not be efficiently incorporated into the ILVs; or ILVs might back-fuse with the limiting membrane of the maturing endosome. Back-fusion has been documented to occur in vertebrate cells. The current results suggest that loss of lgd function results in a reduction in the activity of Shrub. Therefore, the possibility is favored that the loss of lgd function results in a less efficient incorporation of Notch into the ILVs due to the reduced activity of Shrub (Troost, 2012).

How is Notch that remains in the limiting membrane activated (see Model of Notch activation in lgd cells - Supplemental Figure S4? Activation of Notch in lgd cells is independent of the ligands, but dependent on the γ-secretase complex. The S3 cleavage of Notch mediated by γ-secretase requires previous shedding of its ectodomain. This is normally performed by the ligand-dependent S2 cleavage through Kuz. Thus, it is thought that ectodomain shedding must occur in an alternative, ligand-independent manner in lgd cells. A possibility is that the ectodomain that reaches into the endosomal lumen might simply change its conformation because of the increasing acidification in the lumen. This conformational change might allow Kuz to access its normally hidden cleavage site in Notch to cleave the ectodomain independently of the ligands. Alternatively, the ectodomain might be degraded by the peptidases that become activated in the acidic environment of the late endosome or lysosome. The resulting NEXT-like intermediate could be cleaved by γ-secretase, and the intracellular domain would be released into the cytosol. In the second scenario, activation of Notch could be independent of Kuz. Thus, it is important to determine whether Kuz is required for the ectopic activation of Notch in lgd cells. It is also important to determine whether Notch is activated in the maturing-late endosome or in the lysosome: One possibility to explain the puzzling antagonism observed between Lgd and several components of the ESCRT complexes with respect to Notch activation could be explained by the synergistic endosomal morphology phenotype: if activation of Notch occurs in the lysosome and the synergism between lgd and ESCRT mutants on endosome morphology prevents fusion of the late endosome with the lysosome (e.g., through loss of association with the HOPS tethering complex), Notch activation would be suppressed in double mutant cells (Troost, 2012).

In any case, the data so far available suggest that Lgd is a general component of the endosomal machinery and that the activation of Notch in lgd cells is probably not caused by a specific defect in Notch regulation. It occurs because of a general defect in endosomal trafficking and because of the extraordinary mechanism of Notch activation (Troost, 2012).

So far no function in endosomal trafficking has been described for the mammalian orthologues. However, the current data provide overwhelming evidence that Lgd is involved in endosomal trafficking in Drosophila. In addition, work by Tsang (2006) suggests that the functional relationship between Lgd and Shrub is conserved in humans. That study consists of a yeast-two-hybrid screen in which proteins were sought that interacted with the ESCRT components. Among the identified proteins was human Lgd2/CC2D1A, which interacted with all three Shrub orthologues, CHMP4A, CHMP4B and CHMP4C (Troost, 2012).


DEVELOPMENTAL BIOLOGY

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. This study reports 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, these data identify Lgd as a novel tumor-suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling (Childress, 2006).

Lgd has been identified as a novel C2-domain protein, and the results indicate that it acts by regulating Notch trafficking. A model is proposed in which Lgd functions as a negative regulator of Notch through endosomal sorting of Notch downstream of Hrs function. Several lines of evidence support this model. The loss of Lgd resulted in the accumulation of Notch in early endosomes, and the results suggest that this triggered a signaling event that was distinct from normal activation of Notch signaling. Furthermore, the data indicate that Notch can be activated in a ligand-independent manner in lgd mutant cells, similarly to other mutations that affect Notch trafficking. Additionally, cells that lack both Hrs and Lgd did not display ectopically activated Notch signaling as measured by Cut expression. Interestingly, hrs lgd double-mutant cells at the wing margin were still able to express margin-specific genes. Therefore, Hrs is not required for normal (ligand-dependent) Notch signaling, but it is required for the ectopic activation of Cut expression found in lgd mutant cells (Childress, 2006).

lgd mutant cells display both similarities and differences compared with cells that are mutant for vps25, a known endosomal-trafficking component. Both mutations induce ectopic Notch signaling resulting in tissue overgrowth, and both mutations alter Notch trafficking. However, lgd mutant cells induce higher levels of Notch signaling than do vps25 mutant cells (Cut was not notably ectopically activated in vps25 mutant cells, do not induce apoptosis, and can survive into adulthood. Also unlike vps25 mutants, lgd mutant cells have no significant defects in cell polarity and do not accumulate increased levels of ubiquitylated proteins. It is thought that Vps25 is an endosomal component used to sort many different molecules, whereas Lgd might act specifically in the Notch pathway. A model is therefore propose where Lgd function is required to target full-length Notch for endosomal degradation or recycling. Removal of Lgd function might leave Notch in an optimal position or modification state for γ-secretase cleavage. The molecular mechanism by which Lgd affects Notch trafficking is currently not known, and no evidence was found of direct binding between Notch and Lgd by immunoprecipitation (Childress, 2006).

It is important to note that the subcellular location of the γ-secretase-complex cleavage of Notch (S3 cleavage) remains controversial. The traditional view is that the cleavage of Notch occurs at the plasma membrane. However, this view conflicts with the evidence that endocytosis is required for Notch signaling in Drosophila. When protein internalization is blocked by shibire mutations, Notch signaling is eliminated. A different view of the location of Notch S3 cleavage was recently developed when the γ-secretase enzyme Presenilin was shown to have a low optimal pH, suggesting that it could be active in the acidic endocytic compartments. It is possible that differentially processed Notch could be activated in separate cellular compartments. In accordance with the model proposed by Hori (2004), Notch activation in the ligand-dependent canonical pathway may occur at the plasma membrane or in endocytic vesicles, whereas Lgd-regulated activation of Notch may occur later, at Hrs-positive endosomes (Childress, 2006).


EFFECTS OF MUTATION

If the lgd phenotype is caused by the ectopic activation of Notch, inactivation of the Notch pathway should suppress the mutant phenotype of lgd. To test this prediction, examinations were performed to see whether the lgd mutant phenotype is present in mutants where Notch is not processed correctly, such as in Presenilin (Psn). In lgd; Psn double mutant wing discs, the overproliferation of the disc cells, as well as the ectopic expression of wg is abolished. Furthermore, the formation of ectopic wings in the notum is missing. This suggests that the Psn mutant phenotype is epistatic over that (functions downstream) of lgd mutants and that lgd acts through the Notch pathway. The slight rescue of the Psn phenotype is probably due to a residual activity of the Notch pathway, since a similar rescue of the Psn mutant phenotype is observed if the Hairless gene is concomitantly removed. This residual activity seems to be enhanced in the absence of lgd (Klein, 2003).

The phenotype of Ser;lgd double mutant wing discs was further analyzed to examine the effect of loss of one Notch ligand in lgd mutants. Loss of Ser function leads to the loss of most of the wing blade and the margin. The presence of a remnant of the wing pouch is due to the fact that the Notch pathway is active during early stages of wing development. This activation is achieved through a residual expression of Dl. Animals of the Ser;lgd double mutant phenotype develop very slowly, and only few larva survive until the third instar. The wing imaginal discs of the larva have expanded wing pouches and, in contrast to Ser-mutant discs, they express vg and Dl and wg in the wing blade. This shows, that in the absence of lgd function, the activity of Ser is not required to maintain Notch-dependent gene activity. In summary, the observed genetic interactions reveal a functional relationship between the Notch and lgd locus and support the conclusion that lgd is a negative regulator of the Notch pathway (Klein, 2003).

The observation that loss of lgd function can compensate for the loss of Ser function raises the possibility that Notch could be activated in a ligand-independent manner in the absence of lgd function. To test this possibility, Ser/Dl double mutant clones were generated in lgd-mutant wing discs. The clones were induced through combining the Flp/FRT and the targeted Gal4-System. In the experiments described here, the expression of UASFlp was activated with sdGal4. sdGal4 is active throughout wing development and therefore activates UAS Flp expression at all stages of development (Klein, 2003).

In the clones, the expression of the Notch-regulated genes wg and cut was interrupted in the center of the clone area, suggesting that the expression of these genes in lgd mutants depends on Notch ligands. However, several interesting additional effects were observed. (1) Surprisingly, wg and cut expression was induced on both sides of the clone boundary, which can be clearly seen in clones located outside the expanded expression domain normally observed in lgd mutants . The effect is observed in the dorsal as well as the ventral half of the pouch. This suggests that the removal of the ligands leads to the activation of Notch at the boundary of Dl/Ser-expressing and nonexpressing cells. (2) In several cases, the expression of cut and wg expands outside the clone, even far away from the clone boundary. This effect is biased, and the expansion toward the D/V boundary is stronger (Klein, 2003).

(3) The expression of the Notch targets is activated up to three-cell diameter into the clone in a graded manner. Since the ligands are membrane anchored and thought to signal to adjacent cells, an activation of Notch target gene expression beyond one-cell diameter into the clone is not expected. One possibility is that the induction of Cut by Notch is indirect and mediated by a diffusible factor that is induced at the clone boundary (Klein, 2003).

However, it was found that clones of Su(H) mutant cells in lgd mutant discs lose expression of Notch target genes, such as Cut, indicating that the cells require a functional Notch pathway to activate expression of its target genes. Similar results were obtained with Psn mutant clones, using Wg expression as a read out of Notch activity. These results rule out the possibility that the target genes of Notch are induced indirectly through a diffusible factor induced by the Notch pathway (Klein, 2003).

In summary, these results suggest that, in lgd mutant wing blades, all cells that express Notch-regulated genes require the activity of the signal cascade and receive a signal through Dl and/or Ser. In addition, they indicate that, in the Ser;lgd double mutant wing discs described above, Dl alone is sufficient not only to initiate, but also to maintain N-activity during wing development. Hence, it seems that Notch can be activated more efficiently by Dl in the absence of lgd (Klein, 2003).

To further characterize the function of lgd, lgd mutant clones were generated and the expression of Notch-regulated genes, such as cut, wg, and Dl, as well as the activity of the Gbe+Su(H)m8 reporter construct, was monitored. The Gbe+Su(H)m8 reporter construct consists of an ubiquitously expressing promoter of the grainyhead gene in which four copies of the Su(H) binding site, derived from the E(spl) m8 promoter, have been inserted (Klein, 2003).

This construct specifically detects Su(H)-dependent Notch activity in imaginal discs. The clones were generated by using the FLP/FRT system. In a first experiment, the clones were induced with help of an hsFlp construct. If lgd mutant clones are induced during the first larval instar stage [24-48 h after egg laying (ael)], they are rarely found in wing pouches of the late third larval instar stage. In most cases, the twin clone, containing two copies of the GFP marker, is present but the mutant counterpart is missing, indicating that the mutant cells are not able to compete with their wild type neighbors in the wing pouch. In contrast, outside the pouch, e.g., in the hinge region, mutant clones can be frequently recovered, indicating that, in these regions, the mutant cells do not have any growth disadvantage. In addition, scars are often found in wing pouches where lgd mutant clones are induced, indicating that the mutant cells probably have undergone apoptosis. Even if the clones are induced during the second larval instar stage, many 'orphan' wild type twin clones are found. However, in these cases, also some mutant clones are recovered. The mutant cells often express Notch target genes, such as wg and cut, even if they are located away from the D/V boundary and do not include the normal activity domain of Notch. Expression of Cut or Wg was not always activated in mutant clones (Klein, 2003).

In this first set of experiments, expression of the genes was always restricted to mutant territories, suggesting that lgd acts cell-autonomously. The mutant clones often had a round shape and seemed to try to minimize their contact to their normal neighbors. This suggests that the mutant cells have different adhesive properties than their normal neighbors. In a second set of experiments, lgd mutant clones were generated by using an UAS Flp construct, activated by vgBEGal4 or sdGal4. Using this method, large lgd mutant areas were induced in wing pouches. This was surprising because of the difficulties of recovering mutant clones in the hsFlp experiment. The explanation of this difference might be the continuous expression of UAS FLP during all stages of wing development (Klein, 2003).

Hence, clones are continuously induced, also beyond the phase of cell lethality of lgd mutant cells in early stages of wing development. In the large mutant territories, an expansion of the expression of Wg was often found within the clone area. The use of the Gbe+Su(H)m8 construct in these experiments allowed for the detection of Notch activity outside the wing pouch, where the expression of genes like wg and cut is not controlled by Notch. The activity of this construct was often strongly upregulated in mutant territories in and also outside the wing pouch, such as the pleura, in the notum, in regions of the leg disc and the peripodial-membrane of the wing imaginal disc. This suggests that ectopic activation of Notch is a consequence of loss of lgd function in the wing imaginal disc outside the wing pouch and also in other imaginal discs (Klein, 2003).

In the wing pouch, the activity of the Gbe+Su(H)m8 construct was often upregulated in mutant cells/regions that did not express Wg or Cut, indicating that Notch is activated in these cells but this activation is not sufficient for expression of Cut and Wg. Activation of the Gbe+Su(H)m8 construct is already observable in early wing discs. At this stage, no morphological alteration of the wing disc is observed. This suggests that the activation of Notch precedes the overproliferation of the disc (Klein, 2003).

In the set of experiments using UASFlp, expression of the Gbe+Su(H)m8 construct in some wild type cells was observed. This is especially clear if clones are located in the peripodial membrane. Although most of the normal cells at the clone boundary do not show activity of the Gbe+Su(H)m8 construct, a few cells do so. This result shows that cell-autonomy of lgd is not complete (Klein, 2003).

As expected, Dl is strongly activated in lgd mutant clones. This observation raises the possibility that lgd is a negative regulator of expression of Dl. Such a function of lgd would explain the ectopic activation of the Notch pathway in lgd mutant imaginal discs and clones. Alternatively, Dl is also a target of the Notch pathway, and hence the strong ectopic expression of Dl in the mutant clones could be a consequence of the activation of the Notch pathway rather than its initial cause. Two experiments argue for the second alternative. Clones double mutant for lgd and Su(H) fail to express Dl, indicating that a functional Notch pathway is required for expression of Dl in lgd mutant cells. Furthermore, Dl expression is strongly reduced in Su(H) mutant clones induced in lgd mutant wing imaginal discs. Both results indicate that the ectopic expression of Dl is not the cause but a consequence of the activation of the Notch pathway in the wing imaginal disc of lgd mutants. In agreement with this conclusion is the fact that Dl is not activated in lgd mutant clones located in the hinge region. This suggests that expression of Dl is not a consequence of loss of lgd function in all regions of the disc (Klein, 2003).

Expression of Ser with ptcGal4 during normal wing development results in interruption of the expression of Notch target genes, like wg, in the region where the ptc domain crosses the D/V boundary. The reason for this interruption is that the activity of the Notch pathway is suppressed in cells expressing high levels of Ser. In lgd mutants, this effect is not observed, and consequently, the expression of wg along the D/V-boundary is not interrupted. This observation suggests that the negative effect of strong Ser expression at the D/V boundary is absent in cells that lack lgd. To further support this conclusion, Ser was activated by sdGal4 throughout the wing during normal development. Continuous expression of UASSer in the wild type leads to the loss of the wing margin and a dramatic reduction of the size of the wing pouch. This negative effect is again absent in lgd mutants. The results raise the possibility that lgd might be involved in the inhibition of the Notch pathway through high concentration of its ligands (Klein, 2003).

A similar effect of loss of lgd function on the ability to suppress Notch signalling cell-autonomously is observed if Fng is ectopically expressed. Furthermore, clonal analysis of fng suggests that the loss of lgd seems to abolish the requirement of a boundary of Fng-expressing and nonexpressing cells for Notch activation (Klein, 2003).

If ectopic activation of Notch signalling is a general consequence of loss of lgd function, one would expect other Notch-related processes, other than that of wing development to be affected. To test this assumption, the effect was analyzed of loss of lgd function on other developmental processes that are dependent on Notch signalling. The selection of sensory organ precursors (SOP) out of the proneural clusters is one process regulated by the interactions between Notch and Dl. The function of Notch is to suppress neural development in the non-SOP cells of the proneural cluster by downregulating the activity of the proneural genes, such as achaete (ac). In lgd mutant discs, some of the proneural clusters are formed, but in contrast to the wild type, the cells do not accumulate high levels of proneural activity, and as a consequence, most of the SOPs do not form. This is indicated by the absence of most of the expression of the SOP-specific marker A101 in lgd mutant wing imaginal discs. A similar phenotype is also observed in Abruptex mutant wing imaginal discs and suggests that the Notch-pathway is hyperactive during SOP development in the absence of lgd function. The antineurogenic phenotype of lgd mutants is suppressed by concomitant loss of Psn function. lgd; Psn double mutant wing discs display a neurogenic phenotype similar to Psn mutant discs: clusters of large cells that strongly express Ac can be observed, and these cells express the neural differentiation marker. The neurogenic phenotype of the double mutants indicates that the mutant phenotype of Psn is epistatic over that of lgd and that the antineurogenic phenotype of lgd mutants is mediated by the activation of the Notch pathway. Hence, lgd is involved in the regulation of Notch activity during this process. Notch plays an important role in the establishment of the equator and in cell proliferation within the eye disc. Consequently, in Psn mutants, where the Notch pathway is inactivated, the eye disc remains small and poorly differentiated. In contrast to lgd mutants, the eye disc is enlarged (Bryant, 1971). lgd;Psn double mutants resemble the Psn mutant, and the eye disc is small, suggesting that the lgd mutant phenotype in the eye is also caused by overactivity of the Notch pathway (Klein, 2003).

Another process affected by the overactivation of the Notch pathway is the development of the wing veins. In flies, where lgd mutant clones have been generated, the veins are often interrupted. Furthermore, vein formation is strongly affected in lgd mutant wing discs as assessed by the expression of argos-lacZ. Although it is not clear that this loss is due to the activation of the Notch pathway, the similarity of the phenotype to that of the Ax alleles makes it very likely that this phenotype is caused by overactivation of Notch (Klein, 2003).

The involvement of lgd in regulation of Notch activity in these developmental processes and the activation of the Gbe+Su(H)m8 construct in mutant clones outside the wing imply that loss of lgd function causes the activation of the Notch pathway in many developmental processes and suggest that lgd might be a more general regulator of the Notch pathway during development of the adult fly (Klein, 2003).

Ectopic expression of the dpp gene has been reported to contribute to the phenotype of lgd mutant wing discs (Buratovich, 1995). In these experiments, expression of dpp was monitored with a lacZ-insertion in the dpp gene. The expression of dpp in lgd mutant discs was examined by in situ hybridization to see whether the insertion might reflect the expression of dpp incorrectly. A weak expression of dpp that seems to lie in the anterior compartment of the disc was detected, similar to that which has been reported by Buratovich using the P-lacZ insertion line. However, closer examination revealed that this stripe is located in the peri podial membrane, and it is likely that this 'ectopic' domain is the normal expression domain of dpp in the peripodial membrane that is visible in the mutant due to a slightly stronger expression. In contrast, expression of dpp in the wing pouch seems weaker than in normal discs, and a weaker expression in the pouch is also observed with dpp-lacZ (Buratovitch, 1995). It was further found that the expression of the gene spalt (sal), which is a target of the dpp signalling pathway, is not changed in lgd mutant discs. This suggests that dpp activity is normal in lgd mutant wing discs. Thus, ectopic dpp expression or overactivity of dpp does not appear to contribute to the phenotype caused by the loss of lgd function (Klein, 2003).

The activation of the Notch pathway in the wing along the D/V boundary depends on the presence of a boundary between cells that express and cells that do not express the Fng protein. Consistent with this model, expression of UASfng with ptcGal4 interrupts the expression of Notch-dependent genes along the D/V boundary and induces a new domain of expression along the posterior end of the ptc domain, where cells expressing high levels of Fng are juxtaposed to nonexpressing cells. In contrast, performing the same experiment in lgd mutant discs, Fng does not interrupt the expression of wg at the D/V boundary. This raises the possibility that establishment of a distinct boundary of cells that express fng and those that do not is not necessary in lgd mutant wing discs. To further confirm this conclusion, UAS fng was expressed throughout the wing blade with sdGal4 to remove a sharp expression boundary of fng throughout wing development. Expression of UASfng in this way during normal development results in the loss of the wing blade and distal hinge. However, in lgd mutant discs, the expression of UAS fng by sdGal4 has little effect on wing development, and the disc develops a wing blade similar to that of lgd mutants. This result supports the conclusion that a sharp boundary between fng-expressing and nonexpressing cells is not required in lgd mutant wing discs for wing development. To find more evidence for this conclusion, fng13 mutant clones were induced in lgd mutant wing discs. Dorsal clones induced by sdGal4 UAS FLP in wild type wing discs led to the ectopic activation of the Notch pathway and the activation of wg expression at the clone boundaries. Mutant clones located in the ventral half of the pouch have no effect since fng is not expressed there during early development, and hence no ectopic boundary of fng-expressing and nonexpressing cells is generated. In lgd mutant wing discs, fng mutant clones, which do not include the D/V boundary, behave like the clones in wild type discs and wg expression is activated at the clonal boundaries in the dorsal half of the blade. However, unlike in the wild type, dorsal clones that are located within the expanded expression domain lead only to a weakening of wg expression in the center of the clone but do not result in a loss of wg expression, as in the wild type. This result suggests that, in lgd mutant wing pouches, wg expression can be induced by Notch in the absence of Fng. Furthermore, clones that cross the D/V boundary do not lead to an interruption of wg expression at the D/V boundary within the mutant area, and clones that include parts of the ventral half of the expanded domain do not affect Wg expression at all, indicating that Fng has no function in the regulation of the ventral half of the expanded domain of Notch target genes. Altogether, the clonal analysis of fng13 confirms that, in the absence of lgd, a boundary of fng-expressing and nonexpressing cells is not necessary for activation of Notch. Nevertheless, an ectopic boundary of Fng-expressing and nonexpressing cells can activate Notch (Klein, 2003).

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 that 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. In contrast, 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. It is concluded 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 (Bryant, 1985).

The expression of segment polarity genes during the development of overgrowing and duplicating imaginal discs in the lethal overgrowth mutant lethal (2) giant discs [l(2)gd] of Drosophila was investigated in order to explore the molecular basis of hyperplasia and axis establishment in imaginal discs. The expression of wingless, detected using an enhancer trap, is initially restricted to a ventral sector of the leg disc, as in wild type, but expands toward the opposite end of the disc during overgrowth. In the third leg disc, the expanding wg expression stripe evolves to a new center of wg expression at the site where a duplicate leg is subsequently formed. Expression of decapentaplegic also begins normally in l(2)gd discs, but the dpp expression domain expands into the posterior region of the disc where it enlarges to eventually become the center of dpp expression in the duplicate. In l(2)gd homozygotes that are simultaneously homozygous for wg or dpp mutations the leg discs overgrow but do not duplicate. Thus ectopic wg and dpp expression is associated with and appears to be required for disc duplication. The wing discs of l(2)gd homozygotes also show expansion of the wg and dpp expression domains, but in this disc wg and dpp mutations inhibit overgrowth as well as pattern duplication. These results raise the possibility that hyperplasia in other mutants and in other systems may be caused by the misexpression of genes involved in the generation of positional information (Buratovich, 1995).

Lethal mutations in the giant discs (lgd) and fat (ft) tumor suppressor genes of Drosophila cause epithelial hyperplasia in all imaginal discs. By contrast, mutations in the vestigial gene adversely affect cell viability in the wing imaginal discs and consequently cause loss of pattern in the adult wings. However, combining homozygous lgd or ft mutations with homozygous vg1 increases the size of the wing imaginal discs and partially restores the bristle pattern in the wings of pharate adults. Comparable pattern restoration in vg1 wings is also induced by a newly isolated weak hypomorphic lgd3 allele. Further, mosaic analysis has revealed that whereas lgd clones generated by the Minute technique display abnormal differentiation, those induced in a homozygous vg1 background exhibit autonomous restoration of wing pattern. These results suggest that pattern restoration in vg1 wings can serve as an assay for hyperplasia induced by mutations in Drosophila tumor suppressor genes (Agrawal, 1995).


REFERENCES

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