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 lgd^d7 and lgd^d10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgd^d4 and lgd^d1 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 lgd^d7,Ax^MI 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).

PROTEIN STRUCTURE

Structural Domains

lgd encodes a conserved C2 domain protein that binds to phospholipids present on early endosomes (Gallagher, 2006).

date revised: 25 June 2007

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