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 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).
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).
Agrawal, N., et al. (1995). Epithelial hyperplasia of imaginal discs induced by mutations in Drosophila tumor suppressor genes: growth and pattern formation in genetic mosaics. Dev. Biol 169: 387-398. 7781886
Bryant, P. J. and Schubiger, G. (1971). Giant and duplicated imaginal discs in a new lethal mutant of Drosophila melanogaster. Dev. Biol. 24: 233-263. 4994924
Bryant, P. J. and Levinson, P. (1985). Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth. Dev. Biol. 107: 355-363. 3918894
Bryant, P. J., Watson, K. L., Justice, R. W., Woods, D. F. (1993). Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila. Dev. Suppl. 239-249. 8049479
Buratovich, M. A. and Bryant, P. J. (1995). Duplication of l(2)gd imaginal discs in Drosophila is mediated by ectopic expression of wg and dpp. Dev. Biol. 168: 452-463. 7729581
Childress, J. L., Acar, M., Tao, C. and Halder, G. (2006). Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis. Curr. Biol. 16(22): 2228-33. PubMed citation: 17088062
Gallagher, C. M. and Knoblich. J. A. (2006). The conserved c2 domain protein Lethal (2) giant discs regulates protein trafficking in Drosophila. Dev. Cell. 11(5): 641-53. Medline abstract: 17084357
Hori, K., et al. (2004). Drosophila deltex mediates suppressor of Hairless-independent and late-endosomal activation of Notch signaling, Development 131: 5527-5537. PubMed citation: 15496440
Jaekel, R. and Klein, T. (2006). The Drosophila Notch inhibitor and tumor suppressor gene lethal (2) giant discs encodes a conserved regulator of endosomal trafficking, Dev. Cell 11: 655-669. Medline abstract: 17084358
Klein, T., et al. (2003). The tumor suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development. Dev. Bio. 255: 313-333. 12648493
Watson, K. L., Justice, R. W. Bryant, P. J. (1994). Drosophila in cancer research: the first fifty tumor suppressor genes. J. Cell Sci. Suppl. 18: 19-33. 7883789
date revised: 25 June 2008
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.