The eyelessFLP/FRT system has been used to screen the Drosophila genome for genes that restrict tissue growth during eye development. Clones of eye cells homozygous for randomly induced mutations appear 'white' due to the absence of the mini-white+ (m-w+) gene, and wild-type 'twin spots', which carry two copies of m-w+, appear 'red'. Mutations that increased the relative representation of mutant over wild-type tissue were retained, as were those that increased the overall size of the eye. Several mutants had obviously enlarged eyes composed largely of wild-type ('red') tissue, suggesting that the mutations act non-cell-autonomously to deregulate organ growth. One such gene was named erupted (ept) (Moberg, 2005).
Most ept homozygotes die at or prior to the first larval instar, although rare corpses of mutant L2 larvae are sometimes observed. Eyes and heads of adult flies mosaic for the ept2 allele are dramatically enlarged and misshapen when compared to those of the wild-type flies. Significantly, these eyes are largely 'red' indicating that they are composed of mostly wild-type cells. The eye-antennal discs of ept2 mosaic third instar larvae are also enlarged, and they are composed of mostly wild-type cells. Thus, ept mutant cells appear compromised in their ability to contribute to adult eye tissue, but they seem to be able to stimulate the overgrowth of adjacent wild-type cells during larval stages of eye development (Moberg, 2005).
Adult retinal sections show that ept mosaic eyes have a disorganized cellular architecture, and they confirm that most cells are genetically wild-type, as indicated by the presence of pigment granules. Many ommatidia have missing or extra photoreceptors, and there is evidence of fusion events between adjacent clusters. ept mutant clones in the adult eye are small and have few recognizable cells. Rare ept mutant cells appear as 'bloated' cell bodies with lightly staining rhabdomeres. Thus, the Drosophila ept locus has two functions: a cell-autonomous role in cell viability and morphology, and a nonautonomous role in restricting eye size (Moberg, 2005).
The erupted gene was localized by complementation to the ~18 kb region of overlap of two deficiencies, Df(3L)Exel9002 and Df(3L)Exel9004. This region contains two predicted genes: CG9712, the Drosophila ortholog of the mammalian gene Tsg101, and CG9669, a small gene of unknown function. Mutations were not detected in the coding region of either gene in ept2, and no large-scale chromosomal rearrangements of the region were found. However, Southern blot analysis with a Tsg101-specific probe detects an ~8 kb increase in the size of restriction fragments derived from the 5' portion of the Tsg101 locus. PCR and polymorphism analysis indicate that sequences within the 5' end are present in ept2. However, attempts to amplify the entire 5' region from ept2 DNA were unsuccessful, despite the fact that this region is readily amplified from FRT80B DNA. These data indicate that ept2 contains a lesion that disrupts the continuity of sequences within the 5' portion of Tsg101. Since the probe used in Southern analysis flanks this apparent breakpoint, and detects a single larger fragment in both BamH1 and Nhe1 digests, it seems likely that the ept2 allele contains an ~8 kb insertion within the Tsg101 genomic locus. To confirm the identity of ept, a new lethal ept allele, eptP26, was generated by mobilization of a viable P element insertion located ~30 base pairs upstream of the Tsg101 transcriptional start site. eptP26 fails to complement the lethality of ept2, and it displays cellular and organismal phenotypes in the developing eye that are very similar to ept2. The strength of these phenotypes suggests that the eptP26 allele is weaker than ept2. Analysis of eptP26 revealed a duplication of the P element inserted into codon 163 (proline) of Tsg101 exon 5. To further confirm that ept alleles inactivate Tsg101, a full-length Tsg101 cDNA under the control of the heat-inducible hsp70 promoter (hs-Tsg101) was used to rescue the lethality of ept2/eptP26 trans-heterozygotes to viability. By these genetic data, it is concluded that ept mutations disrupt the function of the Tsg101 gene, and that Tsg101 is required for normal eye development. Consistent with this, RNA in situ analysis reveals that Tsg101 is expressed at low levels in the developing eye and antennal discs (Moberg, 2005).
Inactivation of mammalian Tsg101 and yeast Vps23p blocks the transit of certain cell surface receptors through the MVB pathway, leading to accumulation of ubiquitinated receptors in endosomes (Babst, 2000; Li, 1999). When eye discs carrying clones of ept mutant cells were stained with an anti-ubiquitin (Ub) antibody, little staining was observed in wild-type portions of the discs, but strong staining is observed in mutant cells and appears as bright 'puncta', suggesting that Ub accumulates in a specific intracellular compartment. This phenotype is consistent with a role for Tsg101 in the routing of cell surface proteins into the MVB pathway in Drosophila cells similar to that previously shown for mammalian Tsg101 and yeast Vps23p (Moberg, 2005).
Defects in the trafficking of membrane-associated proteins can affect many aspects of cell biology, including cell shape and polarity. Therefore the localization of two cell polarity markers was tested in ept mutant cells: Crumbs (Crb), a marker of the zonula adherens (ZA), and Discs large (Dlg), a protein that localizes to septate junctions and the basolateral membrane domain. Crb localization is significantly altered in ept mutant eye disc cells. An optical section through the middle of the disc epithelium reveals that Crb protein accumulates in a subapical domain in ept cells. A lateral section confirms that Crb in ept cells is not localized to the apical surface, as it is in adjacent wild-type cells. In contrast, ept cells display the normal localization of Dlg. The Dlg-positive lateral profile of ept cells indicates that they are more rounded than adjacent wild-type cells. Costaining for both Dlg and Crb in ept clones confirms these observations, and it reveals that, while some Crb protein localizes properly to the apical domain in ept cells, a significant amount of Crb is detected in nonnuclear, subapical aggregates, which show limited overlap with Ub-positive epitopes. Thus, loss of Tsg101 function in epithelial cells appears to cell-autonomously compromise cell shape coincident with defects in the compartmentalization of the cell polarity protein Crb (Moberg, 2005).
The Notch receptor has two properties that implicate it in a pathway by which ept mutations non-cell-autonomously promote tissue growth. (1) The restricted activation of Notch in cells along the dorsoventral (D/V) boundary of the eye imaginal disc is required for growth of the entire eye. (2) Ub-dependent endocytosis plays an important role in regulating Notch activity in vivo. In mammalian cells, ubiquitination and endocytosis contributes to Notch1 activation, and, in Drosophila, there is evidence to suggest that the ubiquitin ligase Deltex may be required for endocytosis-dependent Notch activation. Further, alleles of the endosomal sorting gene Hrs, the homolog of yeast Vps27, affect Notch localization in imaginal disc cells, indicating that Notch is a physiological target of the MVB pathway (Moberg, 2005).
In light of these observations, ept mosaic eye discs were stained with an antibody specific to the Notch cytoplasmic domain (anti-Ncyto). Notch protein is detected in wild-type eye discs most prominently in a stripe of cells within the morphogenetic furrow (MF) and is concentrated at the apical cell surface. In contrast, ept cells contain elevated levels of Notch. This increase occurs in ept clones throughout the eye disc, but it is most apparent in clones that lie within or posterior to the MF. Moreover, the Notch in ept cells accumulates in nonnuclear, intracellular puncta that also stain positive for Ub, and for the endosomal protein Hrs. Together, these data indicate that ept mutations block the routing of ubiquitinated cell surface proteins, among them Notch, in an Hrs-positive endosomal compartment (Moberg, 2005).
Notch is normally processed in cells by a series of cleavage events required for receptor maturation and presentation at the cell surface, and for ligand-stimulated activation of the Notch pathway. Because ubiquitination and endocytosis have been shown to affect Notch cleavage, attempts were made to determine if ept mutations also affect Notch processing. Eye-antennal discs composed of ept mutant cells [ept/M(3)] or FRT80B control cells (FRT80B/M(3)) were generated by the eyFLP/Minute technique (Moberg, 2004). Immunoblot of tissue extracts with the anti-Ncyto antibody confirms that Notch levels are increased considerably in eye-antennal discs composed of ept mutant cells, and shows that ept mutant cells are enriched in a ~120 kDa form of Notch. The molecular identity of this fragment has not been determined, but its size appears similar to certain processed forms of Notch. Indeed, while no one form of Notch predominates in wild-type cells, this species appears to be the most abundant Notch species in ept cells (Moberg, 2005).
To examine Notch activation, clones of ept mutant cells were generated in the presence of the Notch-inducible transgene E(spl)mβ-CD2, a Suppressor of Hairless (Su(H))-dependent transcriptional reporter that has been used to detect equatorial Notch activation in the developing eye. Posterior to the MF, CD2 expression is detected in the interommatidial cells, and outlines a single cell from each photoreceptor cluster in a mirror-image pattern along the equator. Thus, in addition to equatorial activation, the reporter detects Notch activation in postmitotic interommatidial cells, and in the R3-R4 cell fate choice. In ept mutant clones, reporter activity is strongly elevated. The degree of activation exceeds that observed in wild-type eye discs, and it does not appear to depend upon the location of ept cells within the disc, occurring on either side of the MF and in the antennal disc. Some ept cells within a single optical section appear not to activate the Notch reporter. However, in most of these cases, CD2, which localizes to cell membranes, can be detected in a focal plane slightly offset from that of the nuclear green fluorescent protein (GFP). Thus, these data show that defects in Notch regulation in ept cells are accompanied by ectopic and excessive activation of the Notch pathway (Moberg, 2005).
The requirement for Notch in eye disc growth has been linked to its ability to induce expression of the eyegone (eyg) gene at the D/V boundary of the eye disc. eyg encodes a Pax6-like transcription factor (Eyg) required for disc growth, and, like Notch, ectopic expression of eyg is able to induce growth nonautonomously. Consistent with its effect on Notch, it was found that ept mutant cells express elevated levels of Eyg compared to surrounding wild-type cells. Thus, Eyg may function downstream of Notch within ept cells to promote the growth of surrounding cells in a manner similar to its normal growth-promoting role at the D/V boundary (Moberg, 2005).
Recent work suggests that the unpaired (upd) gene may be an important growth regulatory target of Notch. upd encodes the secreted ligand (Upd) of the Domeless (Dome) receptor, which signals through the JAK-STAT pathway. JAK-STAT signaling is implicated in many processes during Drosophila development, including the control of cell proliferation, cell motility, stem cell renewal, and planar cell polarity. upd is required for normal growth of the eye, and ectopic expression of upd in the larval eye nonautonomously promotes cell proliferation and produces enlarged and misshapen eyes (Bach, 2003) similar to those observed in ept mosaics. Significantly, Notch is both necessary and sufficient to activate upd transcription (Chao, 2004) along the posterior margin of the eye disc (Moberg, 2005).
When ept mosaic eye discs were stained with an anti-Upd antiserum, a dramatic increase was observed in the level of Upd protein in ept mutant cells compared to adjacent wild type cells. Consistent with a transcriptional link between Notch and upd, Upd protein accumulation appears coincident with expression of the Notch reporter, and ept mosaic eye-antennal discs contain clones of cells expressing very high levels of upd mRNA. Together, these observations suggest that Notch, perhaps acting via Eyg, promotes ectopic upd expression in ept mutant cells (Moberg, 2005).
Clonal overexpression of upd induces localized tissue outgrowths and deregulates the division of surrounding cells (Chao, 2004; Tsai, 2004). This mitogenic activity is linked to induction of cyclin D (Tsai, 2004), and to accelerated progression through the G1 phase of the cell cycle (Bach, 2003). ept mutant clones can produce phenotypes quite similar to clonal overexpression of upd. In one example of an ept clone, lower half of the disc appeared morphologically normal, while the other half, despite being composed largely of wild-type cells, was misshapen and enlarged. This localized effect correlated with proximity to a large ept mutant clone expressing Upd. Similar hyperplastic growth was associated with clones of upd-expressing cells in the antennal disc. The patterns of BrdU incorporation in ept mosaic eye discs are disorganized, and the number of BrdU-labeled nuclei increases in proximity to Upd-expressing ept mutant cells in the eye and antenna. This aberrant cell proliferation occurs in GFP-positive wild-type cells. Hence, the growth-promoting activity of ept mutations is likely mediated by a diffusible extracellular signal like Upd (Moberg, 2005).
Receipt of the Upd signal via Domeless initiates a signaling cascade that activates a transcription factor encoded by the stat92E gene. stat92E encodes the Drosophila ortholog of the mammalian signal transducers and activators of transcription (STAT) family of transcriptional regulators, which function in diverse processes such as immunity and oncogenesis, and is the only member of this gene family in Drosophila. Heterozygosity for a stat92E loss-of-function allele (stat92E06346) strongly suppresses the nonautonomous eye overgrowth associated with mosaicism for ept mutations, such that ept-mosaic;stat92E06346/+ eyes are comparable in size to control FRT80B mosaic eyes. Thus, nonautonomous overgrowth elicited by ept mutations is sensitive to the genetic dosage of the Upd-responsive transcription factor stat92E. In light of the effect on Upd, these data strongly indicate that the growth-promoting activity of ept mutant cells requires Upd-dependent activation of the JAK-STAT pathway in adjacent tissue (Moberg, 2005).
ept mutant clones in mosaic eye discs are small and survive poorly into adulthood. It is possible that this is the result of cell competition, a process by which slow-growing cells in the vicinity of wild-type cells are eliminated. If so, then the poor survival of ept cells might be rescued by eliminating competing cells. Therefore the growth characteristics were examined of ept/M(3) discs, which are composed almost entirely of cells lacking Tsg101 function. ept/M(3) animals reach the larval 'wandering' stage 4 days later than control larvae, and, when they do, they are enlarged. A small fraction of these animals pupate and die before becoming pharate adults. The remainder die as giant larvae containing high levels of Upd (Moberg, 2005).
Allowing ept mutant cells to grow in epithelia lacking wild-type cells also uncovers a context-dependent cell-autonomous overgrowth phenotype. Rather than surviving poorly as they do in mosaic discs, ept/M(3) eye discs overgrow into large masses that lack normal disc morphology. These masses are composed of folded and convoluted sheets of cells fused together, and they often include a distended sac-like structure. The overgrowth phenotypes of ept/M(3) animals and discs do not reflect an increased rate of growth: control L3 larvae are the same size as ept/M(3) larvae of the same temporal age, and the ept/M(3) eye discs, while mispatterned, are not obviously increased in size. Thus, the ept/M(3) masses are the result of an extended larval phase, and a failure of the disc to stop growing when it reaches the appropriate size. Thus, cells lacking Tsg101 may be unable to respond to signals that normally sense and restrict organ size (Moberg, 2005).
Reference names in red indicate recommended papers.
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date revised: 15 January 2006
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