Targets of Activity (part 3/3)

Notch function in the eye disc

In the Drosophila eye, Notch antagonizes the basic helix-loop-helix (bHLH) protein Atonal, which is required for R8 photoreceptor determination. Similar antagonism between Notch and proneural bHLH proteins regulates most neural cell determination, however, it is uncertain whether the mechanisms are similar in all cases. Analysis of the sensitivity of atonal expression to Notch signaling used a temperature-sensitive Notch allele to monitor either the expression of activated Notch or the ligand Serrate, as well as the expression of the atonal-dependant gene scabrous and the Notch-dependent Enhancer of split genes. The atonal expression pattern evolves from general prepattern expression, through transient intermediate groups to R8 precursor-specific expression. Successive phases of atonal expression differ in sensitivity to Notch. Prepattern expression of atonal is not inhibited. Inhibition begins at the intermediate group stage, corresponding to the period when atonal gene function is required for its own expression. At the transition to R8 cell-specific expression, Notch is activated in all intermediate group cells except the R8 cell precursor. R8 cells remain sensitive to inhibition in columns 0 and 1, but become less sensitive thereafter; non-R8 cells do not require Notch activity to keep atonal expression inactive. Thus, Notch signaling is coupled to atonal repression for only part of the atonal expression pattern. Accordingly, the Enhancer-of-split mdelta protein is expressed reciprocally to atonal at the intermediate group and early R8 stages, but is expressed in other patterns before and after. It is concluded that, in eye development, inhibition by Notch activity is restricted to specific phases of proneural gene expression, beginning when the prepattern decays and is replaced by autoregulation. It is suggested that Notch signaling inhibits atonal autoregulation, but not expression by other mechanisms, and that a transition from prepattern to autoregulation is necessary for patterning neural cell determination. Distinct neural tissues might differ in their proneural prepatterns, but use Notch in a similar mechanism (N. E. Baker, 1996).

The Notch signaling pathway is involved in many processes where cell fate is decided. Previous work has shown that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signaling represses atonal expression during lateral specification. Once ato autoregulation is established, lateral specification starts to limit ato expression to R8 precursor cells. Thus Notch signaling is required at successive steps during R8 specification, initially to promote neural potential and later to suppress it through lateral specification. Consequently the phenotype of loss of Notch gene function varies with time. If Notch function is removed conditionally once ato expression has been enhanced, supernumerary R8 cells differentiate because lateral specification is affected. If N function is absent from the outset, such as in a clone of cells lacking N, little R8 specification can occur. For this reason clones of N null mutant cells in the eye disc almost completely lack neural differentiation, contrasting with the neurogenic phenotype of null mutant embryos. Using clonal analysis it is shown that Delta, a ligand of Notch, is required along with Notch for both proneural enhancement and lateral specification (Ligoxygakis, 1998).

E(spl) bHLH genes have been shown to be transcriptionally activated as a direct consequence of Notch signaling and, along with the corepressor protein Groucho, to mediate inhibition of proneural genes in the nucleus. In the eye, Notch-dependent expression of mdelta and mgamma accompanies repression of ato expression, suggesting that at least these two of the E(spl) bHLH genes contribute to R8 patterning during lateral specification. In addition, mdelta and perhaps mgamma are also transiently expressed prior to lateral specification, and the m7, m8 and mbeta genes are transcribed in distinct patterns that remain uncharacterized in detail for lack of specific antibodies. Thus, particular E(spl) bHLH proteins might mediate proneural Notch signaling as well as or instead of lateral specification. Clones of cells deleted for portions of the E(spl) complex were used to define its role more precisely. The E(spl)b32.2 deficiency deletes all seven bHLH genes and m4. Partial gro function was supplemented in the experiment by a linked gro + transgene. E(spl)b32.2 gro + homozygous cells display a cell autonomous neurogenic phenotype quite unlike that of N or Dl mutant clones. Antibodies against Boss or Elav proteins each label a much greater number of cells within the clone than in the surrounding wild-type tissue. Some clones were difficult to photograph because neurogenic regions often seem to fold in on themselves and crease the eye disc. Because neurogenesis can still occur, it appears that the proneural function of Notch can proceed without any E(spl)-C bHLH genes, whereas N function in lateral specification is severely impaired. Clones of cells homozygous for E(spl)BX22 are also neurogenic in phenotype. E(spl)BX22 affects gro and the bHLH genes m5, m7 and m8. It follows that gro is also dispensable for the proneural function of Notch, although it is probably required in lateral specification (Ligoxygakis, 1998).

Forced expression experiments were performed to further define the role of particular bHLH proteins. The hairy H10 enhancer trap was used to drive GAL4-dependent transgene expression anterior to and within the morphogenetic furrow. The double homozygote for both h H10 and UASmdelta was expressed during the requirement for ato. In the resulting phenotype, the eyes contain few facets and are greatly reduced in size. Eye imaginal discs contain few ommatidia. The defect is associated with reduction or absence of ato expression in the morphogenetic furrow. These findings indicate that mdelta protein is capable of repressing ato expression, as occurs during lateral specification. Not all E(spl)bHLH proteins repressed ato. Eye disc patterning occurred almost normally in h H10 /h H10;UASm5/UASm5 homozygotes and in h H10 /h H10; UASmbeta/UASmbeta homozygotes. Both of these homozygotes die as pupae without differentiating adult structures. It is concluded that the mdelta protein is qualitatively distinct from m5 and mbeta proteins in its ability to inhibit ato expression (Ligoxygakis, 1998).

Recent studies have identified Su(H) as a common component in the Notch signal transduction pathway. Ligand binding (Delta or Serrate) to Notch activates Su(H), which can shuttle between the cytoplasm and the nucleus and act as a transcription factor. Activated Su(H) turns on a number of downstream target genes mediating Notch signaling in lateral specification or inductive processes. In order to investigate the role of Su(H), clones of cells homozygous for an apparent null allele of Su(H) were generated by FLP-mediated recombination. In the eye imaginal disc Su(H)- mutant cells are associated cell autonomously with neural hypertrophy. Many of the ectopic neural cells are R8 photoreceptors, based on expression of the R8- specific protein Boss. It appears that, like the E(spl)-C, Su(H) is required for lateral specification but not for R8 differentiation. To confirm this conclusion ato expression was examined. In wild type, initial broad expression of Ato protein is replaced by R8-specific expression that persists for 6-8 hours (3-4 columns of ommatidia) and then fades. Whereas ato expression begins normally in Su(H) mutant cells, ato expression is maintained in many more R8 cells than in wild type, indicating failure of lateral specification. Expression of ato then fades from Su(H) mutant R8 cells at the same time as from wild-type cells. Thus, like the E(spl)-C, Su(H) is required for lateral specification but not for the proneural function of Notch in the retina. Interestingly, although many extra R8 precursors form in Su(H) mutant clones, not all Su(H) mutant cells maintain ato expression or subsequently express the R8-specific Boss protein. Instead clusters of R8-like cells often seem interspersed with non-R8 neurons. ato expression in wild type first becomes patterned into regular ‘intermediate groups’ of about ten ato-expressing cells before resolving to individual R8 precursors. These results support the conclusions that initial spacing of intermediate groups is not part of the N-dependent lateral specification process, and so does not depend on E(spl) or Su(H) (Ligoxygakis, 1998).

The Drosophila eye, a paradigm for epithelial organization, is highly polarized with mirror-image symmetry about the equator. The R3 and R4 photoreceptors in each ommatidium are vital in this polarity; they adopt asymmetrical positions in adult ommatidia and are the site of action for several essential genes. Two such genes are frizzled (fz) and dishevelled (dsh), the products of which are components of a signaling pathway required in R3, and which are thought to be activated by a diffusible signal. The transmembrane receptor Notch is required downstream of dsh in R3/R4 for them to adopt distinct fates. By using an enhancer for the Notch target gene Enhancer of split mdelta, it is shown that Notch becomes activated specifically in R4. Analyzing the regulation of E(spl)mdelta, it has been found that this target of Notch is expressed specifically in R4. Transiently reducing Notch activity for 6 hours in late-third-instar larvae, using temperature sensitive Notch, leads to a loss of E(spl)mdelta expression, whereas transient activation of a constitutively active Notch derivitive has the converse effect. Genetic experiments show the importance of Dsh in the establishment of eye polarity. The mutant protein coded for in dishevelled mutants has impaired signaling, but the phenotype can be partially rescued by overexpressing downstream components. When E(spl) proteins are expressed ectopically in dsh mutant discs, the eyes are less roughened and more ommatidia have correct rotation and chirality, compared to when E(spl) proteins are not ectopically expressed. It is proposed that Fz/Dsh promotes expression of the Notch ligand Delta and inhibits Notch receptor activity in R3, creating a difference in Notch signaling capacity between R3 and R4. Subsequent feedback in the Notch pathway ensures that this difference becomes amplified. This interplay between Fz/Dsh and Notch indicates that polarity is established through local comparisons between two cells and explains how a signal from one position (for example, the equator in the eye) could be interpreted by all ommatidia in the field. Additional targets of Notch needed to specify R4 identity may include strabismus. Ommatidial polarity also requires spiney-legs (sple). Sple is normally inhibitory to Notch in R3, because high levels of E(spl)mdelta expression are found in sple mutants. The inhibitory effect of Sple provides a second mechanism for polarizing Notch signaling in R3/R4 that could be coordingated by Fz/Dsh (Cooper, 1999).

The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signaling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signaling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. The function and regulation of four-jointed (fj) during eye patterning has been analyzed. Loss-of-function clones or ectopic expression of four-jointed results in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicate that localized four-jointed expression is required at the time during development when ommatidial polarity is being determined. In contrast, complete removal of four-jointed function results in only a mild ommatidial polarity defect. four-jointed expression has been found to be regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. It is concluded that the clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal (Zeidler, 1999).

Both in situ hybridization for fj transcripts and the lacZ activity patterns revealed by enhancer traps in the fj locus indicate that fj is normally expressed most strongly in a broad domain around the dorsoventral midline of the eye imaginal disc). To determine whether this localized expression is functionally significant, fj was ectopically expressed during eye development. Ectopic expression of fj was driven at the poles of the eye during eye patterning using an optomotor-blind driver. This results in dorsoventral inversions of ommatidial polarity at both the dorsal and ventral poles of the eye, often with three or more rows of ommatidia inverted (Zeidler, 1999).

The expression pattern of fj, and the phenotypes that were observed for loss-of-function and gain-of-function of fj activity, indicate a role for fj function in ommatidial polarity determination along the dorsoventral axis. Recent studies have revealed functions for the N, JAK/STAT and Wg pathways as regulators of ommatidial polarity determination, with the current model suggesting that Notch and Upd are positive regulators of a graded signal that is highest at the equator, whereas Wg is a negative regulator of such a factor (or factors). The fj gene is therefore a good candidate for being a downstream target of regulation by one or more of these pathways. Consistent with this, fj is regulated by the JAK/STAT and Wg pathways. In clones mutant for the Drosophila JAK homolog hop, which lack JAK function, a reduction in fj expression is observed. Although JAK is a cell-autonomously acting signal-transduction component, the effect on fj expression is not cell-autonomous, with greatest downregulation being observed in the center of the clone. In accordance with downregulation in hop clones, clones of cells ectopically expressing the JAK ligand Upd result in activation of fj expression. Conversely, ectopic expression of Wg (which is predicted to be a negative regulator) results in downregulation of fj expression. Activated N can nonautonomously activate fj expression. Taken together, these results indicate that fj is regulated by all three of these pathways in a manner consistent with mediating their functions in dorsoventral polarity determination (Zeidler, 1999).

One of the noteworthy aspects of fj regulation by the Notch and JAK/STAT pathways is that it is non-autonomous, even when it is studied using cell-autonomously acting signaling components such as the intracellular domain of N, Nintra. One possible explanation for this non-autonomy would be that fj is able to activate its own expression via an autoregulatory loop. To test this hypothesis, fj was ectopically expressed in the presence of a fj enhancer trap and it was found that fj was indeed able to activate its own expression. The activation of fj expression by ectopic expression of fj is non-autonomous, again consistent with the proposed secreted nature of the fj gene product. In addition to the N, JAK/STAT and Wg pathways, the only other gene reported to non-autonomously influence ommatidial polarity is frizzled (fz). A possible mechanism for non-autonomy of fz function would be via regulation of fj expression. The expression of fj was examined in fz loss-of-function clones and in clones of cells ectopically expressing fz, but in neither case is there any change in fj expression (Zeidler, 1999).

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Together with the transcription factor Lozenge, the nuclear effectors of the Egfr and Notch signaling pathways directly regulate D-Pax2 (shaven) transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of shaven expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. It is expected that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication (Flores, 2000).

shaven is the Drosophila homolog of the vertebrate Pax2 gene. This locus is represented by at least two classes of mutant alleles: shaven (sv) and sparkling (spa). spa mutants show cone cell defects resulting from mutations in the fourth intron of the gene, which have led to the identification of a 926 bp SpeI fragment within this intron that includes the eye-specific enhancer (Flores, 2000).

In Nts third-instar larvae raised at 29°C for 20 hr prior to dissection, Shaven expression is eliminated from cone cell precursors. Similarly, expression of a dominant-negative form of N under lz-Gal4 control causes a loss of Shaven expression in cone cell precursors without perturbing neuronal development. Shaven expression is also reduced in discs mutant for Delta (Dl), which encodes a N ligand. Moreover, expression of a dominant-negative form of Dl (DlDN) under lz-Gal4 control causes a loss of Shaven expression in cone cell precursors, while neuronal patterning occurs in a wild-type fashion. A further reduction in Shaven expression is seen when DlDN is driven by GMR-Gal4. A loss of Shaven expression is also seen upon ectopic expression of Hairless (H), a direct antagonist of Su(H) function. These results together suggest that N/Dl signaling via Su(H) is required for proper shaven expression in cone cell precursors. This is an inductive rather than lateral inhibitory function of the N signaling pathway in cone cell development that has not been previously analyzed with molecular markers. A reporter gene under the transcriptional control of Su(H) binding sites is expressed in cone cell precursors, which demonstrates that Su(H) is activated by the N pathway in cone cells (Flores, 2000).

The Su(H) binding sites in the minimal shaven eye promoter (SME) were altered to determine whether the N pathway directly regulates shaven transcription. The SME contains eight putative Su(H) binding sites. EMSAs show that the Su(H) consensus binding sequence is not strictly followed, since three sites with one mismatch can bind Su(H). Su(H) binding is eliminated when the central 5'-GRG-3' sequence is mutated to 5'-CCC-3' in all eight sites. A construct containing these mutations in the context of SME-lacZ was transformed into flies. In these transgenic flies, ß-galactosidase expression is lost in cone cell precursors. These in vitro and in vivo results together demonstrate that Su(H) directly controls shaven expression in cone cell precursors by binding to the SME (Flores, 2000).

Mutating Su(H) and ETS binding sites eliminates expression of the target gene in the cone cells, which demonstrates a direct role for these pathways in transcriptional activation of shaven. Clonal analysis was undertaken to establish the requirement of the Notch and Egfr pathways in shaven expression. Unfortunately, these pathways are necessary for proliferation and have many layers of function. Therefore a flip-out strategy was used to inhibit N and Egfr function in GFP-labeled single-cell clones. This was best achieved in clones induced by GMR-flp. The GMR enhancer is only active behind the furrow and only a single cell division takes place in this population of cells. As a result, the clone size is very small. In a wild-type background, single cells marked with GFP express Shaven. However, when these single cells also express EGFRDN or NECN, they do not express Shaven. Thus, cone cells need functional Notch and Egfr receptors in order to express Shaven (Flores, 2000).

The results described so far suggest that shaven expression is limited to cells which (1) express Lz; (2) receive a sufficiently strong Egfr signal to both alleviate Yan-imposed repression and stimulate PntP2 activation, and (3) receive a N signal able to stimulate Su(H) activation. The tripartite control of shaven expression in the cone cell precursors requires that they receive all three inputs at the proper time in their development. Lz expression in cone cell precursors has been documented. Consistent with their reception of the Egfr signal, activated MAPK is detected in cone cell precursors at the time when they initiate Shaven expression. Dl is expressed in developing photoreceptor clusters at the time when the cone cell precursors express Shaven. Thus, the neuronal clusters signal through an inductive Dl/N pathway to activate shaven expression in the neighboring cone cell precursors. These results suggest that, in addition to expressing Lz, the cone cell precursors receive the Egfr and N signals at the time of fate acquisition and Shaven expression. Presumably, at least one of these three activation mechanisms is lacking in cells that do not express shaven. This hypothesis was tested through genetic manipulation of the system (Flores, 2000).

Undifferentiated cells immediately posterior to the furrow receive the N signal and express Lz, but they do not express Shaven. It is hypothesized that the absence of Shaven expression in these cells is caused by a lack of the Egfr signal. This hypothesis is consistent with the observation that Egfr signaling causes these cells to differentiate. Indeed, Shaven is ectopically expressed in undifferentiated cells that express an activated form of Egfr. Loss-of-function yane2D/yanpokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SMEmETSx6-lacZ, in which the six ETS sites in the SME are mutated, ß-galactosidase is also expressed in undifferentiated cells. Presumably, relief of Yan repression is sufficient to activate some shaven in undifferentiated cells. In SMEmETS(1,6)-lacZ,where the Pnt binding sites are eliminated but two of the Yan binding sites are still intact, there is no expression of ß-galactosidase in the undifferentiated cells. These results suggest that while the undifferentiated cells posterior to the furrow express Lz and receive the N signal, they fail to express Shaven because they do not receive the Egfr signal and are therefore unable to relieve the Yan-imposed repression of shaven (Flores, 2000).

The R7 precursors express Lz and receive RTK signals, yet they do not express Shaven. It is hypothesized that this is due to the lack of the N signal at the time of R7 determination. Indeed, expression of an activated form of N (Nact), leads to ectopic Shaven expression in R7 precursors, which suggests that Shaven is not normally expressed in R7 because this cell does not receive the N signal. These results are consistent with the previous observation that the R7 cell loses its neuronal characteristics upon expression of Nact (Flores, 2000).

Thus far, this study has focused on cells that express Lz. However, the regulation of shaven expression can also be tested in cells that lack Lz, such as the R3/R4 precursors. These cells receive the Egfr signal but receive the N signal after their initial fate specification, during ommatidial rotation. Ectopic expression of either Lz or Nact in the R3/R4 precursors fails to activate shaven expression in these cells. However, when Lz and Nact are coexpressed in the R3/R4 precursors, Shaven is expressed in these cells. These results demonstrate that the lack of both N signaling and Lz during the proper time window prevents R3/R4 cells from expressing shaven (Flores, 2000).

The eight photoreceptors in each ommatidium of the Drosophila eye are assembled by a process of recruitment. First, the R8 cell is singled out, and then subsequent photoreceptors are added in pairs (R2 and R5, R3 and R4, R1 and R6) until the final R7 cell acquires a neuronal fate. R7 development requires the Sevenless receptor tyrosine kinase, which is activated by a ligand from R8. The specification of R7 requires a second signal that activates Notch. A Notch target gene is expressed in R7 shortly after recruitment. When Notch activity is reduced, the cell is misrouted to an R1/R6 fate. Conversely, when activated Notch is present in the R1/R6 cells, it causes them to adopt R7 fates or, occasionally, cone cell fates. In this context, Notch activity appears to act co-operatively, rather than antagonistically, with the receptor tyrosine kinase/Ras pathway in R7 photoreceptor specification. Two models are proposed: a ratchet model in which Notch would allow cells to remain competent to respond to sequential rounds of Ras signaling, and a combinatorial model in which Notch and Ras signaling would act together to regulate genes that determine cell fate (Cooper, 2000).

Transcription of the Enhancer of split [E(spl)] genes is promoted by Notch activity and is therefore a good indicator of cells where the Notch receptor is activated. Through analysis of E(spl)mDelta cis-regulatory elements, a 500bp fragment, mDelta0.5, has been identified that directs expression in the R4 photoreceptor, and reveals a role for Notch in selecting R4-type fates. In more mature ommatidia, however, mDelta0.5 is active in a second cell, albeit at much lower levels than in R4. This represents a subset of the endogenous E(spl) protein expression patterns, and indicates a second focus of Notch activity in the developing ommatidia (Cooper, 2000).

In the eye imaginal disc, ommatidial development is initiated at the morphogenetic furrow, which moves across the disc from posterior to anterior. The clusters behind the furrow are therefore at progressively more mature stages of differentiation. Expression of mDelta0.5 in R4 appears in clusters close to the furrow, shortly after R3/R4 are recruited. Expression in the second cell is first detected in clusters 4-5 rows posterior, around the stage when R7 acquires its neural character. Although Notch is involved in the development of the cone cells, which join the cluster after R7, mDelta0.5 activity is clearly in one of the neuronal photoreceptors and not in the surrounding Cut-expressing cone cells. The position of the mDelta0.5-expressing cell was further clarified by staining for markers of different photoreceptor types, such as Rough (R2,3,4,5), Dachshund (R1,6,7) and BarH1 (R1,6), which confirms that this cell was the presumptive R7. The mDelta0.5 fragment contains two binding sites for Suppressor of Hairless [Su(H)], a DNA-binding protein that is pivotal in Notch signal transduction; the enhancer activity of mDelta0.5 is dependent on both Su(H) and Notch. Expression from mDelta0.5 in R7 suggests therefore that Notch is activated in this cell (Cooper, 2000).

If the expression of mDelta0.5 is indicative of an important function for Notch in R7, R7 specification might be subject to change by manipulating Notch activity. Previous experiments targeting expression of an activated Notch (Nact) to photoreceptors did give rise to ommatidia with extra R7-like cells and fewer 'outer' photoreceptors; note that R2,R5, R3,R4, R1,R6 are outer receptors; R7 and R8 are the central inner photoreceptors. At the time, there was no indication that Notch normally had a specific role in R7 and the initial interpretation was that ectopic Notch activity in R3/R4 hindered their differentiation, causing them to be redirected towards later fates. Subsequently it became clear that R3/R4 photoreceptors persist under these conditions, although the normal distinction between R3 and R4 is lost. Nevertheless, the phenotypes of extra R7 cells could clearly be reproduced using both limited Nact expression in R3,R4, R1,R6 and R7 (via the sevenless enhancer; sev-N?ecd or sev-Gal4/UAS-Nicd) and ubiquitous Nact expression (induced by heat-shock enhancer; hs-Nicd). These manipulations gave rise to adult eyes containing ommatidia with 4-5 outer cells and up to three R7-like cells although some ommatidia had only one or two R7 photoreceptors. Mutant ommatidia with fewer photoreceptors could have arisen because some cells with high levels of Nact become cone cells, as indicated by the co-expression of mDelta0.5 and Cut in a few cells. Reducing Notch activity causes the opposite transformation. In eyes from Nts animals that had been exposed transiently to the non-permissive temperature, several ommatidia lacked an R7 cell and had an extra outer photoreceptor. Therefore, the phenotypes of reduced and ectopic Notch activity both indicate that Notch activity is involved in R7 specification, transforming it from an outer cell fate (Cooper, 2000).

To investigate which outer cell fates are transformed by Notch activity, an examination was made of the effects of ectopic Notch (sev-N?ecd or sev-Gal4/UAS-Nicd) on proteins that are expressed in a subset of photoreceptors. Expression of BarH1 (R1,6) was lost from most ommatidia but was not changed in the undifferentiated cells of the epithelium. In 17.5%±9 of clusters from sev-Gal4/UAS-Nicd eye discs, only one of the two R1/6 photoreceptors had lost BarH1 expression, and it often corresponded to a cell with higher levels of mDelta0.5 expression. This negative regulation of BarH1 was recapitulated by ectopic expression of E(spl)MDelta itself, which resulted in 43%±7.5 ommatidia with only one BarH1-expressing cell, although the ommatidia contained the normal complement of photoreceptors. The effects on BarH1 suggest that the role of Notch is to distinguish R7 from R1,R6, which implies that there should be extra BarH1-expressing cells when Notch activity is reduced, indicating a transformation of R7 to R1/6. This is indeed the case; a row of ommatidia with three BarH1-expressing cells (27%±3.5) is formed when Notch activity is perturbed in Nts larvae (Cooper, 2000).

For Notch to be activated in R7, there needs to be a nearby source of ligand. Delta distribution is consistent with it being a ligand for Notch activation in R7, as it appears in photoreceptors R1/6 and 7 at the appropriate stage. Delta transcripts are initially higher in R1/6 than in R7. This differential would influence the direction of signaling. When the distribution of Delta was altered experimentally, expression of mDelta0.5 was detected ectopically in R1/6 and most clusters lacked one or both BarH1-expressing cells. This is in agreement with the adult phenotypes observed for Delta misexpression, where ommatidia have fewer outer photoreceptors. Conversely, reductions in Delta function result in extra outer photoreceptors and loss of R7 (Cooper, 2000).

The expression of mDelta0.5 and the effects observed from manipulating Notch and Delta expression indicate that Notch activity is required in R7, and that it transforms R7 from R1/6 type fates. Further support for the latter comes from observations of two other genes that are expressed in specific photoreceptors: seven up (svp, R3,4,1,6), and prospero (pros, R7 and cone cells). Ectopic Nact has been shown to inhibit svp expression in R1/6 and to activate pros expression in two extra cells, consistent with a change in cell fates from R1/6 to R7, although no specific role for Notch in R7 has been extrapolated from these results. Given the stage at which manipulations in Notch affect R7 fates, and the effects on BarH1, svp and pros, it appears that Notch is normally activated in R7 shortly after recruitment where it promotes differences between R7 and R1/R6, regulating svp, pros and BarH1 either directly or indirectly (Cooper, 2000).

The role of sevenless and the Ras signaling pathway in specifying the R7 photoreceptor is well established. Notch activation is also necessary for R7 formation. This combined role of Notch and Ras signaling appears different from other characterized intersections between these pathways, where Notch is most commonly involved in antagonizing the Ras pathway to limit the number of cells that adopt a particular fate. However, there are two ways that Notch might operate, referred to here as the ratchet and combinatorial models. It is likely that the recruitment of R1,6 and 7 is mediated through activation of the Drosophila epidermal growth factor (EGF) receptor/Ras signaling. One possibility is that activation of Notch shortly after recruitment influences whether or not the cells are competent to receive a second Ras-mediated signal, via Sevenless activation (ratchet model). Notch activation has been proposed to prevent cells from differentiating, which would be compatible with this model if Notch activity in R7 were to delay its differentiation and so that it could respond to Sevenless. If Notch is acting in this manner, it would in fact be antagonizing Ras signaling, even though the final outcome in terms of cell fates involves a combination of the two signals. An analogous ratchet mechanism might operate in vertebrate eye development, where Notch activity appears to regulate whether cells adopt earlier or later cell fates (Cooper, 2000).

Alternatively, Notch might act cooperatively with the Sevenless-dependent Ras signal in R7 (combinatorial model). One prediction of this more proactive role for Notch signaling would be that certain R7-specific genes are direct targets of both Notch and Ras signaling, in which case their enhancers should contain binding sites for the transcription factors that mediate the activity of these pathways [that is, Su(H) and Pointed, respectively]. Although the data clearly demonstrate that Notch activity is required in R7, and is sufficient to misdirect the fates of R1/6, it is not yet possible to distinguish between these two models for its function. This can only fully be resolved when the promoters of genes, such as pros and klingon, which are expressed in R7 but not R1/6, are examined (Cooper, 2000).

Localized Notch signal acts through eyg and upd to promote global growth in Drosophila eye

Notch (N) signal is activated at the dorsoventral (DV) border of the Drosophila eye disc and is important for growth of the eye disc. In this study, the Pax protein Eyg is shown to be a major effector mediating the growth promotion function of N. eyg transcription is induced by N signaling occurring at the DV border. Like N, eyg controls growth of the eye disc. Loss of N signaling can be compensated by overexpressing eyg, whereas loss of the downstream eyg blocks the function of N signaling. In addition, N and eyg can induce expression of upd, which encodes the ligand for the Jak/STAT pathway and acts over long distance to promote cell proliferation. Loss of eyg or N can be compensated by overexpressing upd. These results suggest that upd is a major effector mediating the function of eyg and N. The functional link from N to eyg to upd explains how the localized Notch activation can achieve global growth control (Chao, 2004).

Notch is activated at the DV boundary of the early eye disc. This equatorial N signal then activates eyg expression at the transcriptional level. When N signal is reduced, eyg expression is reduced. When N signal is elevated, eyg expression is induced. Induction of eyg expression occurs at the DV border between the dorsal Dl-expressing and the ventral Ser-expressing cells. Furthermore, when the upstream N signal is blocked, overexpression of eyg can rescue the growth defect in the eye, whereas increasing N signaling cannot rescue the eye-growth defect caused by the downstream eyg gene. This analysis shows that the induction of eyg by N is dependent on the ligands Dl and Ser, and involves the effector Su(H) and the antagonist Hairless. Thus, the localized activation of N signal is transmitted to the induction of a transcription factor, Eyg, which then promotes cell proliferation. The Chao study confirms the work of Dominguez (2004), who showed that Notch promotes growth in eyes by acting through Eyegone (Chao, 2004).

Eyg is a transcription factor, so must activate the transcription of some genes that promote cell proliferation. Upd is reported to act through the Jak/STAT signaling pathway to promote cell proliferation. upd expression is dependent on eyg and N signaling. Furthermore, when the upstream N signaling or eyg is reduced, overexpression of upd can rescue the growth defect. The overgrowth effect due to overexpression of the upstream N or eyg is blocked when the downstream upd is defective. The results suggest that upd is a major effector for the growth promotion by N and eyg (Chao, 2004).

These results have demonstrated the functional link from Notch to eyg to upd in the promotion of eye growth. The link to upd solved a long-standing problem. N signaling is activated locally at the border between the dorsal Dl-expressing cells and the ventral Ser-expressing cells. How does a localized activation of N signal promote cell proliferation throughout the entire eye disc? The finding of eyg as the major mediator of N function did not solve the problem, since Eyg is a transcriptional factor and is expected to affect target gene expression autonomously. The link from eyg to upd provided a solution, because Upd is a diffusible signaling molecule. Upd protein can distribute over a long distance and exert long-range non-autonomous effect to promote cell proliferation (Tsai, 2004). So the localized N activation can locally activate eyg, which then turns on upd expression, probably through a short-range signal. The Upd signal then acts over a long range to promote cell proliferation in the early eye disc (Chao, 2004).

Although N activates eyg, and eyg activates upd, these transcriptional activation may be direct or indirect. When novel DV borders were created by ectopic expressing Dl or Ser, eyg is induced non-autonomously at the border of these clones. It is also noted that in Su(H) mutant clones, mutant cells at the border of the clone can still express eyg-lacZ. These observations suggest that N may induce a short-range signal, which then activates eyg expression. Alternatively, the apparent non-autonomous induction may be due to perdurance of the reporter protein in cells that were once close to the clone border. The induction of upd by eyg also may be indirect. Clonal expression of eyg also induced upd expression non-autonomously. In addition, based on RNA in situ hybridization, eyg expression in the eye disc does not extend to the posterior margin, so does not overlap with the expression domain of upd (Tsai, 2004). These observations suggested that the effect of eyg on upd expression may be indirect. However, an eyg enhancer trap line showed reporter expression extending to the posterior margin (Dominguez, 2004). Thus, the possibility that Eyg can directly activate the expression of upd cannot be excluded (Chao, 2004).

The activation of eyg and upd are context dependent. Nact does not induce eyg expression in antenna and wing discs. In the eye disc, Nact induces eyg expression only in the region anterior to the MF, and not within the wg expression domain in the lateral margin. Similarly, Nact and eyg can only induce upd expression at the margin, but not in the center of the eye disc. Nact induces upd at the posterior margin but not lateral margins, while eyg can induce upd in the lateral margins but not in the posterior margin. The context dependence indicates that additional factors are involved to determine the specificity of induction (Chao, 2004).

In a late third instar eye disc, eyg is expressed in an equatorial domain that does not overlap with the disc margin, so eyg cannot induce upd. In early eye disc, eyg expression domain comes closer to the posterior margin. Thus, the induction of upd by eyg is likely at second instar, which is consistent with the timing of upd expression (Chao, 2004).

Although eyg plays an important role in mediating the growth-promoting N signal, it is probably not the only effector. In the eygM3-12 null mutant, ey>Nact does not rescue the endogenous eye field, but can still induce proliferation to provide the antennal disc and an extra eye field. Thus, N can induce proliferation by an eyg-independent mechanism. The effect on antenna and on eye seems to be separate, because ey>Nact can induce a large antenna disc with duplicate or triplicate antennal field without rescue of the eye disc. Because N can induce upd, but not eyg, in the posterior margin, the induction of upd can also be through an eyg-independent mechanism (Chao, 2004).

Nact can induce overgrowth in the central domain of the eye disc. In this case, eyg, but not upd, is induced. In addition, the overgrowth does not extend much beyond the clone. Ectopic eyg in the central domain also induces proliferation without inducing upd. In eyg1 mutant, there is no upd-lacZ expression in eye disc, but the eye is only slightly reduced. These results suggest that the N signaling and eyg can induce local proliferation independent of upd (Chao, 2004).

Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye

Two types of basic helix-loop-helix (bHLH) family transcription factor have functions in neurogenesis. Class II bHLH proteins are expressed in tissue-specific patterns, whereas class I proteins are broadly expressed as general cofactors for class II proteins. The Drosophila class I factor Daughterless (Da) is upregulated by Hedgehog (Hh) and Decapentaplegic (Dpp) signalling during retinal neurogenesis. The data suggest that Da is accumulated in the cells surrounding the neuronal precursor cells to repress the proneural gene atonal (ato), thereby generating a single R8 neuron from each proneural cluster. Upregulation of Da depends on Notch signalling, and, in turn, induces the expression of the Enhancer-of-split proteins for the repression of ato. It is proposed that the dual functions of Da--as a proneural and as an anti-proneural factor--are crucial for initial neural patterning in the eye (Lim, 2008).

Da is upregulated in the furrow region. Surprisingly, however, it was found that there are two distinct patterns of Da upregulation. The first pattern is a broad, low-level upregulation in the furrow (hereafter referred to as basal level). The second pattern is a stronger expression of Da (hereafter referred to as high level) selectively in the non-neural cells surrounding the Ato-positive R8 cells between proneural clusters. Tests were perfomed to see whether this previously unrecognized pattern of expression of Da is specific by examining eye discs containing da loss-of-function (LOF) clones. Both the basal and high-level expressions of Da in the furrow were lost in the LOF clones of da3, a null allele, showing the specificity of the pattern of Da expression (Lim, 2008).

The basal level of Da upregulation overlaps with the domain of Ato expression near the furrow, where they function together to regulate neurogenesis. As the furrow progression and expression of Ato are controlled by Hh and Dpp signalling, it was reasoned that regulation of Da expression in the furrow might be linked to these signalling pathways (Lim, 2008).

To test whether Hh signalling is required for the expression of Da, Da expression was examined in hh1 mutant eye discs in which the production of Hh ceases after the mid-third instar stage, resulting in reduced expression of Ato and arrest of furrow progression. The expression of Da was downregulated in hh1 mutant eye discs. LOF clones of smoothened (smo), a crucial component for Hh signal transduction, were generated. Da expression was significantly reduced in smo mutant clones spanning the furrow, suggesting that Hh signalling is required for the expression of Da. However, the expression of Da was not completely eliminated in hh1 mutant eye discs or in smo LOF clones. As Dpp signalling is partly required for the expression of Ato, whether Dpp signalling is also necessary for the expression of Da was tested by analysing LOF clones of mad (mothers against dpp), an essential factor for Dpp signalling transduction. Da expression showed little reduction in mad mutant clones, indicating that Dpp signalling by itself is not essential for Da expression. By contrast, the expression of Da was almost completely abolished in LOF clones of smo and mad double-mutant cells in the furrow region. Thus, the Hh and Dpp signalling pathways are crucial but partly redundant for the expression of Da. It was also found that loss of function of Ato reduced the level of Da expression in the furrow. Therefore, several factors, including Ato, coordinate the accumulation of Da in the furrow (Lim, 2008).

To test whether the upregulation of Da in the furrow has a function in neurogenesis, da3 LOF clones were generated and the effects of da mutation on the expression of Ato and neuronal differentiation were examined. Loss of da resulted in ectopic expansion of Ato expression in the mutant clone, suggesting that Da is crucial for repressing the expression of Ato (Lim, 2008).

Despite ectopic expression of Ato, most of the cells in da LOF mutant clones could not differentiate into photoreceptor cells, as indicated by the lack of neuronal markers such as Senseless (R8 marker) and Elav (pan-neural marker). Hence, the expression of ectopic Ato is insufficient to induce retinal differentiation in the absence of Da. However, local differentiation was occasionally detected near the posterior end of some clones. This might be due to the perdurance of Da in LOF clones, although other possibilities, such as partial non-autonomy or partial independence of photoreceptor differentiation from Da in the posterior region of the eye disc, cannot be excluded (Lim, 2008).

To support the idea that a high level of Da expression is required for the repression of Ato, a temperature-sensitive allele of da (dats) was examined that causes conditional partial loss of function of Da at the restrictive temperature. In dats mutant eye discs, Ato was expressed in several cells rather than a single R8 cell per proneural cluster. In addition, the effects of conditional expression of Da was tested by temperature shifts of heat-shock (hs)-da flies. Ato was repressed by the overexpression of Da after a longer heat shock but not after a shorter heat shock. These observations support the idea that enriched Da expression in the cells surrounding each R8 cell is required for generating a single R8 cell by the inhibition of Ato expression (Lim, 2008).

The expanded expression of Ato in da mutant clones might, in part, be due to the failure of da mutant cells to induce lateral inhibition of Ato expression. It is also possible that Da might be involved in the cell-autonomous repression of Ato expression. To test this possibility, Da was overexpressed in the dorsoventral margin of the eye disc using the optomotor blind (omb)-Gal4 driver. The overexpression of Da downregulated Ato expression in the expression domain of omb. Furthermore, the overexpression of Da in the antenna disc using the dpp-Gal4 driver resulted in Ato repression in the expression domain of dpp. Taken together, these data from LOF and overexpression analyses suggest that the high-level expression of Da is necessary and sufficient for the cell-autonomous repression of Ato during the selection of R8 (Lim, 2008).

Both Da and Notch (N) are essential for the selection of R8 by repressing Ato expression in non-R8 precursors within proneural clusters. Hence, Da might be involved in N-dependent lateral inhibition. Furthermore, the overexpression of ASC proneural factors, together with Da, can synergize with Suppressor of hairless and N to activate the expression of Enhancer-of-split (E(spl)) in cultured cells. Since E(spl) is expressed complementary to the expression of Ato in the same cells expressing a high level of Da, whether Da alone could regulate the expression of E(spl) was tested in vivo. The expression of E(spl) proteins was reduced in da3 mutant cells, showing that Da is required for the expression of E(spl) in vivo. Furthermore, the overexpression of Da with dpp-Gal4 could induce the expression of ectopic E(spl) in the dpp domain of the antenna disc. These results indicate that a high level of Da expression is necessary and sufficient for the activation of E(spl) expression (Lim, 2008).

Since E(spl) is the main mediator of N signalling, Ato repression by a high level of Da might be dependent on the expression of E(spl). To test this possibility, the MARCM method was used to generate E(spl) LOF clones in which the expression of Da is induced by tubulin (tub)-Gal4. Da overexpression in E(spl) LOF clones did not show a significant repression of Ato. Similarly, overexpression of E(spl)mδ in da LOF clones did not show noticeable repression of Ato. These data suggest that both Da and E(spl) are required for positive feedback regulation and for repression of Ato during lateral inhibition. However, it is also possible that other bHLH family genes of the E(spl) complex loci might be required, or that the overexpression of E(spl) or Da by tub-Gal4 in MARCM assays might not be strong enough to repress the expression of ato. By contrast, Da expression by dpp-Gal4 induces the expression of E(spl), even in the proximal sector of the antenna disc where Ato is not expressed. amos, the proneural gene for olfactory sensilla, is not expressed in the antenna disc at this time. Thus, a high level of Da can induce E(spl) in the absence of Ato, although Da might act with other class II proteins to promote the expression of E(spl) (Lim, 2008).

Since N signalling is activated in the same cells surrounding R8 founder neurons, whether Da expression is affected was examined by removing the function of N using a temperature-sensitive allele, Nts. The loss of function of N at the restrictive temperature resulted in several Ato-positive cells per proneural cluster. Furthermore, the transient loss of N activity abolished the high-level of Da expression between the proneural clusters but did not eliminate the basal level of Da expression in the same cells. This suggests that N signalling is essential for the high-level upregulation of Da expression. Since the expression of da is regulated by Hh and Dpp signalling, as well as Ato, it is possible that the regulation of Da by Hh and Dpp might be mediated by Ato-dependent N signalling in the non-R8 precursor cells (Lim, 2008).

To investigate further the role of N signalling in the expression of Da, whether E(spl) proteins mediate the function of N in inducing a high level of Da expression was examined. Loss of E(spl) caused ectopic expression of Ato in E(spl) mutant clones because of the lack of N-mediated lateral inhibition. Interestingly, the high level of Da expression was suppressed, but the basal level of Da expression was still detected in E(spl) mutant clones, as seen in Nts mutant eye discs. Thus, E(spl) is required for the high level but not for the basal level of Da expression. In contrast to da3 LOF mutant cells that fail to differentiate in spite of ectopic Ato expression, E(spl) LOF mutant cells not only expressed ectopic Ato but also differentiated into ectopic photoreceptors. Thus, the basal level of Da expression remaining in E(spl) LOF clones is sufficient for the formation of a functional complex with Ato to induce neural differentiation (Lim, 2008).

On the basis of the above observations, a model is proposed in which Da has dual functions as a proneural and as an anti-proneural factor depending on the expression level during early retinal neurogenesis . The anti-proneural function of Da proposed in this model provides an explanation for the abnormal upregulation of Ato in da mutant cells in the furrow, although the LOF experiments are also consistent with the pre-existing view that Da promotes the function of Ato. In Ato-positive neural precursors, low levels of Da expression are sufficient to form heterodimers with Ato to function as a proneural factor. In neighbouring cells, the N-E(spl) pathway further upregulates the expression of Da, which, in turn, induces more expression of E(spl). This putative feedback regulation might provide a mechanism for more effective lateral inhibition of Ato expression for the selection of R8. Interestingly, Da can form a homodimer and bind to DNA in vitro. Thus, in Ato-negative cells surrounding the R8 precursors, a high level of Da expression might enforce the formation of Da homodimers and/or heterodimers with other unknown bHLH proteins to repress the expression of ato. It would be interesting to see whether mammalian type I bHLH proteins such as E proteins might also be specifically regulated to have distinct developmental functions as seen in the case of Da (Lim, 2008).

Notch-dependent expression of the archipelago ubiquitin ligase subunit in the Drosophila eye

archipelago (ago)/Fbw7 encodes a conserved protein that functions as the substrate-receptor component of a polyubiquitin ligase that suppresses tissue growth in flies and tumorigenesis in vertebrates. Ago/Fbw7 targets multiple proteins for degradation, including the G1-S regulator Cyclin E and the oncoprotein dMyc/c-Myc. Despite prominent roles in growth control, little is known about the signals that regulate Ago/Fbw7 abundance in developing tissues. This study used the Drosophila eye as a model to identify developmental signals that regulate ago expression. It was found that expression of ago mRNA and protein is induced by passage of the morphogenetic furrow (MF), and the hedgehog (hh) and Notch (N) pathways were identified as elements of this inductive mechanism. Cells mutant for N pathway components, or hh-defective cells that express reduced levels of the Notch ligand Delta, fail to upregulate ago transcription in the region of the MF; reciprocally, ectopic N activation in eye discs induces expression of ago mRNA. A fragment of the ago promoter that contains consensus binding sites for the N pathway transcription factor Su(H) is bound by Su(H) and confers N-inducibility in cultured cells. The failure to upregulate ago in N pathway mutant cells correlates with accumulation of the SCF-Ago target Cyclin E in the area of the MF, and this is rescued by re-expression of ago. These data suggest a model in which N acts through ago to restrict levels of the pro-mitotic factor Cyclin E. This N-Ago-Cyclin E link represents a significant new cell cycle regulatory mechanism in the developing eye (Nicholson, 2011).

The ago gene and its vertebrate ortholog Fbw7 have well established roles in controlling cell division, cell growth and apoptosis in developing tissues, yet only a few studies have investigated pathways that regulate ago activity in cells. It is known that dimerization of Fbw7 enhances its ability to degrade Cyclin E, and that ago and Fbw7 are both transcriptionally induced by p53/dp53 via a checkpoint pathway that responds to either energy starvation. This study shows that the N and hh pathways are necessary for the proper regulation of Ago levels in the developing Drosophila eye, specifically by increasing ago transcription in the region within and immediately behind the MF. This effect correlates with the presence of Su(H) binding sites in the ago promoter, and can be enhanced by co-expression of the N-terminal activation domain of Da. DaN can bind to Su(H) and drive elevated expression of the N-target E(spl)m8, and the requirement for da in Ago expression at the MF suggests that a similar mechanism might occur here. Interestingly, mutating the six putative Su(H) sites in the ago2kb-luc reporter does not completely abolish transactivation by NICD and DaN, suggesting that they have a secondary effect on ago transcription that is independent of the Su(H) sites. Finally, it was found that the defect in the MF-associated pulse of ago expression in N pathway-defective cells results in hyperaccumulation of the SCF-Ago target protein Cyclin E, indicating that this novel transcriptional link between N and ago is an important mechanism through which N regulates the G1-S phase transition in eye disc cells (Nicholson, 2011).

Although these data shed light on the initial inductive phase of Ago expression at the MF, additional mechanisms must operate immediately posterior to the MF to refine the pattern of Ago expression. The regular pattern of Ago expression posterior to the MF appears to overlap with gaps in Ato expression between the R8 equivalence groups, arguing that these expression patterns might share some regulatory inputs. Interestingly, N is required both for induction of Ato within MF cells and for restriction of Ato expression in cells immediately posterior to the MF via a lateral inhibition mechanism. Thus, although Ago does not display precisely the same pattern as that of Ato restriction posterior to the MF (e.g. expression only in the presumptive R8), it seems possible that N might play a similar dual activator/inhibitor role upstream of ago. The effect of the Nts allele on Ago patterning behind the MF supports such a model. Moreover, since Fbw7 can target mammalian NICDs for proteasomal degradation, the pattern of Ago expression posterior to the MF might also reflect a requirement for SCF-Ago to inhibit NICD activity in differentiating neurons. In support of this type of feedback model, expression of the N pathway reporter E(spl)mß-CD2 is elevated in ago mutant cells posterior to the MF, indicating that Ago might also regulate N activity. As overall N protein levels are not obviously altered in ago mutant eye cells, as assessed by both indirect immunofluorescence and immunoblotting with the C17.9C6 antibody that recognizes the cytoplasmic tail of the N receptor, it remains to be established whether or not this potential feedback loop involves changes in N protein turnover (Nicholson, 2011).

In addition to the potential for a cell-autonomous feedback mechanism between Ago and N, ago expression is subject to N pathway-mediated, non-cell-autonomous effects at the MF. Expression of ago>Gal4 is rescued in Dl,Ser mutant cells by adjacent wild-type cells, and Su(H) mutant cells appear to be able to upregulate ago expression in adjacent wild-type cells, probably via increased Dl expression. Alleles of other N pathway components might thus be expected to exhibit effects on ago expression at clonal boundaries. However, expression of Ago in Psn mutant clones that span the MF is not obviously rescued by adjacent wild-type cells, and, reciprocally, these cells do not induce higher levels of Ago in adjacent wild-type cells, indicating that the cross-border induction of ago transcription might be restricted to alleles of genes required for repression of Dl (Nicholson, 2011).

Depending on the developmental context, N can be either pro- or anti-mitotic. Exactly how N fulfils these roles is not fully understood. The finding that ago is regulated by N, and that N appears to act through ago to control Cyclin E levels in a subset of eye disc cells, provide a novel link between N and the core cell cycle machinery. This link could explain certain cell cycle phenotypes described in N mutant disc cells. For example, N-deficient cells in the second mitotic wave (SMW) hyperaccumulate Cyclin E but also simultaneously fail to enter the SMW properly. Su(H) mutant cells also accumulate Cyclin E, which is consistent with a role for the N pathway in promoting ago expression. The latter pro-mitotic effect of N has been attributed to a requirement for N for relief of Rbf-mediated repression of dE2f1 activity, but the former role of the N pathway in antagonizing Cyclin E levels is not well understood. Based on data presented in this study that place the N pathway upstream of ago, it is proposed that this phenotype is due to defective turnover of Cyclin E resulting from insufficient ago expression. The identity of the N target that promotes SMW entry has remained controversial. Interestingly, high Cyclin E activity can downregulate levels of the pro-S-phase transcription factor dE2f1 in the developing wing and eye. Thus, N-induced ago transcription may promote Cyclin E turnover following S-phase, but might also ensure that cells are only able to enter the SMW with an appropriate amount of Cyclin E present (Nicholson, 2011).

Interestingly, the reduced Ago expression in N pathway mutants has no discernible effect on the levels of dMyc. One logical explanation for this is that the threshold of SCF-Ago activity required to degrade dMyc is lower than that required for Cyclin E. Such a mechanism would imply that the N-ago link selectively regulates some SCF-Ago targets but not others; alternatively, N might have a second role in this pathway by protecting dMyc from SCF-Ago activity, although there is no clear evidence of such a role (Nicholson, 2011).

The role for N upstream of Ago and Cyclin E could theoretically be significant in mediating N effects in tissues outside of the eye, and it will thus be important to examine whether N is required for optimal ago expression in other developing tissues. However, available data suggest that the N-ago link might be fairly context specific and not generalized to N signaling in all cell types. First, the pattern of Ago protein in developing larval discs (including the eye) does not generally mirror that of N pathway activity in each organ. Other signals, present at the MF but not elsewhere, must thus cooperate with N to induce ago within the MF. The proneural transcription factor Da is a clear candidate to fulfill this role: Da expression peaks in the MF and is required for the pulse of Ago protein expression at the MF. Thus, it seems likely that N signals synergize with Da, and perhaps with additional unidentified factors, to pattern ago expression. Given the role that dp53 plays in ago induction and in the control of Cyclin E protein under conditions of metabolic stress, it will be interesting to determine how pathways involving N, da and dp53 interact on the ago promoter under various stresses and developmental conditions. Furthermore, it will important to determine whether additional signaling and checkpoint mechanisms, especially those that interact functionally with the N pathway, act through the ago promoter to pattern Cyclin E levels in developing tissues (Nicholson, 2011).

Notch controls cell adhesion in the Drosophila eye

Sporadic evidence suggests Notch is involved in cell adhesion. However, the underlying mechanism is unknown. This study has investigated an epithelial remodeling process in the Drosophila eye in which two primary pigment cells (PPCs) with a characteristic 'kidney' shape enwrap and eventually isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that in the developing Drosophila eye the ligand Delta is transcribed in cone cells and Notch is activated in the adjacent PPC precursors. In the absence of Notch, emerging PPCs fail to enwrap cone cells, and hibris (hbs) and sns, two genes coding for adhesion molecules of the Nephrin group that mediate preferential adhesion, are not transcribed in PPC precursors. Conversely, activation of Notch in single IOCs leads to ectopic expression of hbs and sns. By contrast, in a single IOC that normally transcribes rst, a gene coding for an adhesion molecule of the Neph1 group that binds Hbs and Sns, activation of Notch leads to a loss of rst transcription. In addition, in a Notch mutant where two emerging PPCs fail to enwrap cone cells, expression of hbs in PPC precursors restores the ability of these cells to surround cone cells. Further, expression of hbs or rst in a single rst- or hbs-expressing cell, respectively, leads to removal of the counterpart from the membrane within the same cell through cis-interaction and forced expression of Rst in all hbs-expressing PPCs strongly disrupts the remodeling process. Finally, a loss of both hbs and sns in single PPC precursors leads to constriction of the apical surface that compromises the 'kidney' shape of PPCs. Taken together, these results indicate that cone cells utilize Notch signaling to instruct neighboring PPC precursors to surround them and Notch controls the remodeling process by differentially regulating four adhesion genes (Bao, 2014).

Notch function during oogenesis; mirror links EGF signaling to embryonic dorso-ventral axis formation through Notch activation

Recent studies in vertebrates and Drosophila have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. This may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis. Ectopic Notch activation has a similar long-range effect on pip. These results suggest that Mirror and Notch induce secretion of diffusible morphogens; a TGF-beta (encoded by dpp) has been identified as one such molecule in the germarium. mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and mirr then represses pipe expression in all but the ventral follicle cells, connecting Egfr activation in the dorsal follicle cells to repression of pipe in the dorsal and lateral follicle cells. These results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signaling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation (Jordan, 2000).

In a number of developmental systems, regulation of fng by a homeobox gene has a role in establishing a domain in which Notch is activated. Thus the phenotypes observed in mirr and Notch (N) mutants during oogenesis have been compared. In oogenesis, Notch activity is required in the germarium and for the formation of the termini at stage 6. A test was performed to see whether Notch function is also required for dorsal-ventral patterning of follicle cells by analysing the eggs laid by Nts females at the restrictive temperature. The strongest phenotype observed in eggs laid by Nts females is similar to that observed in eggs laid by mirr loss-of-function females: a complete loss of the dorsal appendages. In addition, the ventral pip expression domain is defective in Nts females and restricted due to expression of constitutively active Notch. Thus Notch, like Mirr, functions to restrict pipe expression to the ventral region and to organize dorsal structures; loss of either Mirr or Notch function affects follicle cells on both sides of the Mirr-Fng expression border (Jordan, 2000).

Activation of Notch at a fng expression border has been observed in wing and eye development. In the wing this border acts as an organizing center by producing a morphogen, Wingless, that acts on cells on both sides of the border. At stage 9 in oogenesis the mirr-fng expression border and a region of localized Notch activation are approximately 10 cell diameters from the ventral pip expression border. Nevertheless, reduction of mirr expression expands the pip domain laterally. If a Mirr-Fng border activates Notch locally to produce a morphogen that represses pip, a reduction of pip expression should be seen upon expansion of the mirr expression domain or ectopic activation of Notch. To examine this, mirr was expressed ectopically in anterior follicle cells. pip repression occurs 5-7 cell diameters beyond the mirr expression domain, showing that the effect of Mirr on pip is non-cell autonomous and supporting the idea that a Mirr-Fng border generates a pip-repressing agent. To further test the effect of ectopic Mirr expression, Mirr was expressed in the posterior follicle and the effect on pip and rho, which is normally expressed as two stripes on the dorsal region at stage 10, was tested. Such ectopic mirr expression induces a ring of rho expression and represses pip at a distance. Expression of constitutively active Notch in the posterior follicle cells also represses pip expression at a distance. These results suggest that Mirr and Notch induce secretion of a diffusible molecule that represses pip. Although it is not known what the Notch-dependent diffusible molecule is at stage 9, it was found that dpp is expressed in follicle cells in the mid-germarium near a stripe of cells showing localized Notch activity in a Notch-dependent manner. Furthermore, in follicle cell clones of MAD or MEDEA (downstream effectors of the Dpp pathway), encapsulation defects of 16-cell cysts are seen. This phenotype is similar to Notch- and mirr-mutant phenotypes in the germarium, suggesting that Dpp may be a morphogen induced by Notch activity in the germarium (Jordan, 2000).

Results from several developmental systems have led to the idea that the trio of a homeobox gene, FNG and Notch are fundamental to organogenesis. It is suggested that Mirr, Fng and Notch are part of a conserved mechanism for dividing epithelial cell layers into domains; it is thought that such a mechanism is not restricted to organogenesis. Furthermore, the data suggest that Mirr integrates the Egfr and Notch pathways in oogenesis: mirr transcription is induced by the Egfr pathway, and Mirr in turn spatially regulates fng expression leading to a Notch activation border. Finally, it is proposed that the link between Egfr pathway signaling in the dorsal follicle cells and the differentiation of the ventral follicle cells suggested by genetic studies is mediated by Mirr. The Egfr pathway induces mirr expression, which leads to creation of a Notch-Fng border in lateral follicle cells from which molecules are secreted that repress pipe expression. Pipe regulates the activity of a protease cascade that activates Toll and ultimately determines the dorsal-ventral pattern of the Drosophila embryo. These data show that expression of pip in the ventral follicle cells is not a direct consequence of a graded germline signal by Gurken, but depends on Mirr-dependent long-range signals from dorsal follicle cells. Mirr therefore connects the well-studied events in early and late Drosophila dorsal-ventral axis formation (Jordan, 2000).

Notch-Delta signaling induces a transition from mitotic cell cycle to endocycle in Drosophila follicle cells

In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. The transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway. Loss of Notch function in follicle cells or loss of Delta function in the underlying germline disrupts the normal transition of the follicle cells from mitotic cycle to endocycle: mitotic cycling continues, leading to overproliferation of these cells. The regulation is at the transcriptional level, since Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase that activates Cdc2 by dephosphorylation. String is normally repressed in the follicle cells just before the endocycle transition, but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just before the onset of polyploidy in wild-type follicle cells but well beyond this stage in Notch mutant follicle cells. This suggests that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, which explains why Notch, while active at both stages, represses String only at the mitotic cycle-endocycle transition (Deng, 2001).

To investigate the possibility that the Notch pathway is involved in the regulation of the mitotic cycle-to-endocycle transition, the expression of the Notch ligand, Delta, was examined in oogenesis. Weak Delta expression is observed in follicle cells and germline from the germarium to stage 5 in oogenesis. At around stage 6, a dramatic upregulation of Delta expression is observed in the germline cells reaching the highest level at stage 7. This upregulation of Delta in the germline coincides with the transition from mitotic cell cycle to endocycle in the follicle cells. To determine if Delta plays a role in regulating the mitotic-to-endocycle-transition, a mosaic analysis was undertaken. Mitotic recombination was employed to create clones of cells lacking Delta function, then cell cycle stage was analyzed by examining Phospho-Histone 3 (PH3) and CycB. The mutant cell clones were marked by lack of green fluorescence protein (GFP) expression, while the wild-type cells were labeled by GFP. Since wild-type follicle cells lack PH3 and CycB after stage 6, owing to the transition into the endocycle, the expression of these markers was analyzed in follicle cells adjacent to Delta germline clones after stage 6. This analysis revealed defects in cell cycle regulation in follicle cells surrounding the Delta germline clones. In almost all cases, follicle cells surrounding the Delta germline clones expressed PH3 and CycB beyond stage 7, suggesting that these cells remain in the mitotic cell cycle program. Although dividing, these follicle cells appeared to maintain their apical-basal polarity, as shown by normal apical localization of Armadillo. In Delta follicle cell clones, no PH3 and CycB staining was detected in follicle cells after stage 6, suggesting that the germline but not the follicle cell contribution of Delta is essential for the follicle cell mitotic cycle transition into the endocycle program (Deng, 2001).

In Drosophila, mutations in three neoplastic genes, discs large (dlg), lethal(2)giant larvae (lgl) and scribble (scrib), cause loss of apical-basal polarity accompanied by hyperproliferation. It is not understood how loss of apical-basal polarity results in hyperproliferation in these mutants. One attractive hypothesis, however, is that overproliferation is caused by mislocalization of critical plasma membrane proteins that are involved in cell cycle control. The Notch protein is indeed mislocalized throughout lgl mutant cells; this defect could reduce responsiveness to Delta from the germline and thus lead to overproliferation. In addition, many lgl mutant follicle cells existed in multilayered structures where they are less likely to receive the Delta signal because of lack of direct contact with the germline. By contrast, in Notch mutant follicle cells, the cell cycle defect is observed before any obvious polarity defect; normal Armadillo localization is observed in Notch but not in lgl clones. Additional factors probably also contributed to the overproliferation in lgl mutant clones because significantly more proliferation is observed in lgl than in Notch clones (Deng, 2001).

Mitosis in most Drosophila cells is triggered by brief bursts of transcription of string, which encodes a Cdc25-type phosphatase that activates the mitotic kinase, Cdk1 (Cdc2). Transcriptional control is an important mode of regulation for the activity of this G2/M controller. string is expressed in a patchy fashion when follicle cells are in the mitotic cell cycle but is repressed when the follicle cells are in the endocycle. This repression requires Notch activity, since String mRNA is observed in egg chambers with Notch clones beyond the normal stage 6 in oogenesis (Deng, 2001).

To test whether String is essential for follicle cell mitotic cycles, string mutant clones were generated. The mutant clones had far fewer cells than sister clones and showed no expression of G2/M stage markers, suggesting that String is required for the follicle cell mitotic cycle (Deng, 2001).

Previous studies have revealed that many cis-acting elements distributed over >30 kb upstream of string control string transcription in different cells and tissue types. To map the transcriptional control elements that regulate string in follicle cells, advantage was taken of gene-fusion constructs in which the string locus was dissected into fragments and fused to lacZ reporter genes containing the basal 0.7 kb string promotor. Of 11 different gene fusions tested, two showed specific expression in follicle cells. These constructs define the regions of the string promoter that are required for string expression and therefore for controlling the cell cycle in follicle cells. Interestingly, R4.9 (4.9 kb region) exhibits expression in follicle cells of the germarium to around stage 2 in oogenesis, while R6.4 (6.4 kb region) shows expression beginning in stage 3 and abruptly terminating at the mitotic-to-endocycle transition in stage 6 (Deng, 2001).

If the two enhancer elements (4.9 and 6.4) are required for controlling two different bursts of cell divisions in follicle cells, then a rescue construct that covers only the first element should only partially rescue the division defects in string clones. That is exactly what was observed: when string clones were produced in the background of the 15.3 kb rescue construct, mutant clones of half the size of sister clones were detected. This result suggests that the 4.9 kb element completely contained within the 15.3 kb rescue construct supports only the early expression and rescues the first but not the second burst of cell divisions in the follicle cell layer (Deng, 2001).

These data suggest that Notch pathway activity impinges on the 6.4 kb region enhancer element. To test this, both the R6.4-lacZ and R4.9-lacZ gene fusions were crossed to the line mutant for Delta and the expression of these elements was examined in egg chambers with germline mutant for Delta. In controls, the expression of R6.4 element was observed in stage 5 but not in stage 9 egg chambers. However, in Delta germ line clones, expression of R6.4 element was observed in domains of 5-20 cells through the follicle cell layer past stage 6. By contrast, when R4.9-element was analyzed in Delta germ line clones, no expression beyond the normal early expression was observed. These data suggest that Notch pathway activity does control String at the 6.4 kb element but not at the 4.9 kb element (Deng, 2001).

It is plausible that Notch also regulates other components of the cell cycle in addition to String, because the transition to the endocycle requires not only the repression of a mitosis promoter (string) but the upregulation during G2 of an S phase promoter, such as CycE and/or CycD (for comparison, during early embryogenesis cells that lack String pause in G2 rather than undergoing endocycles). However, when string was restored to clones under the control of the 15.3 kb rescue construct, which contains the control element active in the germarium and stage 1 but not the control element active between stage 3 and stage 6, some cell divisions occurred before premature stopping of the mitotic cycle, and interestingly, the nuclei were 2.2 fold bigger in the mutant clones than in the sister clones or in the neighboring wild type cells, suggesting that the mutant cells that stopped dividing too early also entered the endocycle too early. Thus, in contrast to embryonic cells, cessation of string expression may lead the follicle cells to endocycle rather than simply arrest, and thus Notch might act solely on string to stop mitosis and the subsequent transition to the endocycle could be due to other factors constitutively present in follicle cells. However, the possibility exists that Notch also acts on other cell cycle components, and it will be interesting to examine the role of Cyclin D in the follicle cell transition (Deng, 2001).

The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/CdhFzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

The exit from mitosis and/or progression through G1 requires the inactivation of cyclin-dependent kinases, mediated by the APC/C-dependent destruction of cyclins. APC/C is regulated by multiple mechanisms, such as phosphorylation and by spindle checkpoints. Key factors for APC/C function and regulation are the WD proteins Cdc20 and Hec1/Cdh. These proteins seem to bind directly to substrates and recruit them to the APC/C core complex. Importantly, Cdc20 and Hec1/Cdh bind and activate APC/C in a sequential manner during mitosis. APC/C-Cdc20 is activated at the metaphase/anaphase transition, and gets replaced by APC/C-Hec1/Cdh in telophase. This second complex remains active in the subsequent G1 phase. In Drosophila the homolog of Hec1/Cdh, Fzr, also induces the APC/C-complex-dependent proteolysis of CycA and B and is required for the G1-phase progression. Fzr is required for cyclin removal during G1 when the embryonic epidermal cell or follicle epithelial proliferation stops and the cells enter endocycles. Premature Hec1/CdhFzr transcription in follicle cells is sufficient to block mitosis and initiate precocious endocycling. This suggests that Fzr is a powerful player in the mitotic-to-endocycle switch, yet regulation of other components is also required for the efficiency of this process. Regulators of G1-S transition, such as Dacapo/CIP/KIP, which also turns out to be a Notch-regulated component, possibly abort premature attempts by follicle cells to enter the endocycle (Shcherbata, 2004).

The data suggest that a component regulating growth and thereby the kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene instead and independent of CycD. In mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size. Interestingly, overexpression of Myc in follicle cells does not affect the mitotic cycles but induces, instead, extra endocycles. Because the timing for entering and exit from the endocycles has not changed, however, increased ploidy is observed; therefore, it is suggested that the rate of endocycles is increased because of the overexpression of Myc. This finding is in accordance with recent loss-of-function analysis on myc in follicle cells, suggesting that myc mutant follicle cells can make the transition from mitosis to the endocycle, but that they can only very inefficiently support the endocycle. Therefore, both loss-of-function and overexpression experiments suggest that Myc is an essential component for the proper rate of endocycles in follicle cells (Shcherbata, 2004).

In addition to Myc and Cyclin D, Cyclin E also plays an important role in the regulation of the G1/S-transition. Cyclin E binds to and activates the cyclin-dependent kinase Cdk2, and thereby promotes the transition from G1 to S. Oscillation of Cyclin E activity is a mechanism responsible for the timely inactivation of this G1 cyclin/Cdk complex and an arrest in cell proliferation. The oscillation of Cyclin E level is controlled partly by a SCF-ubiquitin-dependent proteolysis. Fluctuations of Cyclin E are critical for multiple rounds of endocycles. Cyclin E is critical for endocycles in follicle cells as well, and this analysis shows that the CycE level is controlled by an SCF-regulator, F-box protein, Ago/hCdc4/Fbw7. Fbw7 (Ago) associates specifically with phosphorylated Cyclin E, and catalyzes Cyclin E ubiquitination in vitro. Depletion of Ago leads to accumulation and stabilization of Cyclin E in vivo in human and D. melanogaster. This leads to increased mitosis in certain mammalian and Drosophila cell types. In addition, ago loss-of-function clones in the germ line will cause extra mitotic divisions or, in contrast, cell cycle arrest and polyploidy. However, increased Cyclin E levels observed in ago loss-of-function mutant clones do not affect the mitotic cycles in follicle cells but do halt the transition to endocycles that normally occurs at stage 6 (Shcherbata, 2004).

Why is the function of Ago/hCdc4/Fbw7 critical to endocycles but not to mitotic cycles in follicle epithelial cells? A potential answer might reside in Dacapo, a CIP/KIP-type inhibitor of Cyclin E/Cdk2 complexes that is regulated in the mitotic to endocycle transition by activation of Notch pathway. dacapo is downregulated at mitotic-to-endocycle transition because of Notch activation and ectopic expression of dacapo represses endocycle progression. It is plausible that during mitotic phases Ago and Dacapo share a redundant role for regulating the Cyclin E activity level, however, dacapo is downregulated by Notch pathway at the time of mitotic-to-endocycle transition and at that point Ago gains the critical role of sole regulator of Cyclin E protein activity level. However, downregulation of Dacapo does not readily explain the reduction of CycE levels observed in mitotic-to-endocycle transition. Elevation of CycE protein level is detected in response to Dacapo overexpression, pointing out that this CKI may stabilize CycE in an inactive form. One possibility therefore is that less CycE protein is observed after the Dacapo downregulation because Dacapo is no longer stabilizing it (Shcherbata, 2004).

Why is Dacapo downregulated at the time of endocycle transition? Expression of Dacapo is important for proper cell cycle regulation. For example, during vertebrate development, members of the CIP/KIP family of CKIs are often upregulated as cells exit the mitotic cycle and begin to terminally differentiate. Also, reduced expression of p27Kip1 is frequently shown to correlate with a poor prognosis in various cancers, and in the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S-phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Also, p21 elimination causes centriole overduplication and polyploidy in human hematopoietic cells. In the Drosophila germ line Dap is differentially regulated in the nurse cells versus the oocyte. High Dap levels in the oocyte are critical to the maintenance of the prophase I meiotic arrest and ultimately to later events of oocyte differentiation, and in the nurse cells the oscillations of Dap drive the endocycle. In contrast to all these examples, in endocycling follicle cells reduction of p21/Dacapo is a requirement for normal endocycle progression. Similarly, in a megakaryocytic cell line, differentiation is correlated with a downregulation of p27. It is proposed that the downregulation of Dacapo is a reasonable strategy to bypass the G1/S transition and to enter endocycling when mitosis is not completed, however, how these endocycling cells escape possible centrosome amplification and apoptosis that could be consequences of the lack of Dacapo/p21-activity is not clear. This diversity in the processes, that allow cells to exit from mitotic cell cycle, is generating or representing regulatory multiplicity that might be reflected in the ways eukaryotic cells acquire tumor formation capacity (Shcherbata, 2004).

Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. The homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut is expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression is controlled by the Notch pathway and is essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells enter the endocycle and differentiate prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut causes defects in the mitotic cycle/endocycle switch. These cells continue to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promotes Cyclin A expression by negatively regulating Fizzy-related. These data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells (Sun, 2005).

The switch of cell-cycle programs in Drosophila follicle cells provides an excellent opportunity to study how developmental signals control the intrinsic cell-cycle machinery. A switch from the mitotic cycle to the endocycle in follicle cells is induced by the Notch pathway. Cell-cycle regulators such as CycA, CycB, Stg and Fzr are regulated by Notch during this process. The homeodomain gene cut acts between the Notch pathway and some of these cell cycle regulators. Its expression is downregulated by the Notch pathway in main-body follicle cells during the mitotic cycle/endocycle switch. Cut function was required cell-autonomously for maintenance of the mitotic cycle and an immature cell fate in main-body follicle cells. Cut downregulation by Notch signaling is a key step allowing proper entry into the endocycle and cell differentiation. Fzr, an adaptor of the APC/C that promotes endocycle, is negatively regulated by Cut, but Stg is not regulated by Cut (Sun, 2005).

Several roles for cut during Drosophila oogenesis have been described. (1) cut negatively interacts with Notch for partitioning of individual germline cysts into egg chambers. (2) cut defines a signaling pathway from the follicle cells to the oocyte to maintain the germline integrity. In addition, the rarely produced ctC145 mutant follicle-cell clones have fewer, but larger, cells. This last phenotype agrees with findings that (3) cut is required to maintain the follicle cells in mitotic cycle and that ctdb7 mutation results in premature entry into the endocycle. Study of the involvement of cut in this process required generation of clones after the egg chamber exited the germarium, so that defects caused by germarium requirements of cut would not interfere. Interestingly, during both egg-chamber encapsulation and cell-cycle switch, cut function is related to the Notch pathway. Cut expression is downregulated by Notch signaling during the cell-cycle switch at stages 6-7. In the germarium, cut negatively interacts with Notch; heterozygous cut mutation suppresses the Notch phenotype. Whether Cut acts as a target of Notch signaling at this stage is unclear. Gain- or loss-of-function clones of Notch prior to stage 6 were studied and no obvious change of Cut expression was detected; this argues that Cut is not a downstream target of Notch during early oogenesis (Sun, 2005).

The interaction between Notch and cut is not restricted to the follicle cells during Drosophila development. In wing imaginal discs, cut also interacts with Notch, but the Notch pathway positively regulates Cut expression in DV boundary formation. Clones of Notch eliminate Cut expression cell-autonomously at the DV border, a pattern opposite that of the follicle-cell-cycle switch. Ectopic expression of constitutively active Notch causes Cut to be ectopically activated in the disc. Notch downstream genes, such as strawberry notch and Enhancer of split (E(spl)), also positively regulate Cut expression in the disc, suggesting Notch-regulated Cut expression is indirect. In follicle cells, downregulation of Cut expression by Notch signaling seems to be indirect as well, because NICD/Su(H) usually functions as a transcriptional activator. Downregulation of Cut is probably achieved by a transcriptional repressor activated by NICD/Su(H). E(spl) is unlikely to be the mediator in this process, because loss of E(spl) has no effect on follicle-cell cycle transition and follicle-cell differentiation. Two other nuclear proteins, bHLH transcription factor dMyc (Dm, encoded by diminutive - FlyBase) and bHLH protein Emc (encoded by extra macrochaetae), are both required in follicle cells for proper entry into the endocycle. Removal of the function of dMyc results in lack of endocycle in follicle cells, but whether dMyc expression is regulated by Notch is uncertain, because the expression of dMyc is uniform throughout oogenesis. Emc expression is detected in main-body follicle cells from the germarium to stage 8 of oogenesis; its expression after stage 6 depends on Notch signaling. Loss of emc function results in upregulated CycB and FasIII in follicle cells after stage 6, a phenotype similar to that of loss of Notch. Emc could function as a transcriptional repressor, and it is known to be involved in promoting differentiation, so it is a good candidate for mediating Notch-dependent downregulation of Cut during the mitotic cycle/endocycle switch. No change of Cut expression however, was found in emc loss-of-function clones generated by a null allele, emcAP6, thus excluding the possibility that cut is repressed by emc during the mitotic/endocycle switch. Another transcription factor may therefore be involved (Sun, 2005).

The known role of Cut in Drosophila is mostly related to cell differentiation. During neurogenesis, Cut is involved in cell-fate determination in sensory-organ cells. Loss of cut function causes transformation of the external sensory organ into the chordotonal sensory organ, whereas overexpression of Cut has the opposite effect. In main-body follicle cells, overexpression of Cut maintains expression of immature cell-fate markers FasIII and Eya, whereas loss of cut function in early stages represses their expression. Although Cut is involved in cell differentiation in these two developmental processes, its roles in the two are significantly different. In sensory organs, the role of Cut is post-mitotic, whereas in main-body follicle-cell differentiation, it appears to be correlated with Cut function in the mitotic cycle. The requirement for Cut in main-body follicle-cell differentiation may be related to its function in cell-cycle regulation (Sun, 2005).

The role of cut in polar-cell differentiation is intriguing. Cut expression is normally retained in these specialized cells while its expression in main-body cells is decreased. Consistent expression of Cut leads follicle cells to take the immature main-body cell fate, but these cells eventually take the polar-cell fate. Main-body cell fate and polar/stalk-cell fate are separated in the germarium, which requires Notch activity. Continuous Cut activity seems able to reverse this differentiation process (Sun, 2005).

In contrast to the role of Drosophila Cut in cell differentiation, mammalian Cut has mainly been shown to be involved in regulating cell-cycle progression in some cell types. CDP, the mammalian homolog of Cut, has been shown to be physically associated with the complex regulating the G1/S progression. Cut can functionally replace E2F in forming a complex with RB in regulating cell-cycle progression. The requirement for Cut in maintaining the mitotic cell cycle in Drosophila follicle cells echoes its role in mammalian systems. Whether Drosophila E2F has a function in follicle cell proliferation is not known: weak alleles of E2F1 and E2F2 affect gene amplification, whereas no defect appears in the mitotic cycle. Cut may functionally replace E2F for cell-cycle progression in proliferating follicle cells, but it is not an essential regulator of the cell cycle machinery because the mitotic cycle did not seem to be affected in cut germline clones. In addition, cut function has been extensively studied during embryogenesis and in imaginal discs, but no reported function is related to cell-cycle regulation in these developmental stages. The requirement for Cut in cell-cycle regulation is therefore probably specific to follicle cells in Drosophila (Sun, 2005).

Notch-dependent String and Fizzy-related expression is required for the mitotic-to-endocycle transition in follicle cells

During Drosophila oogenesis, Notch function regulates the transition from mitotic cell cycle to endocycle in follicle cells at stage 6. Loss of either Notch function or its ligand Delta (Dl) disrupts the normal transition; this disruption causes mitotic cycling to continue and leads to an overproliferation phenotype. In this context, the only known cell cycle component that responds to the Notch pathway is String/Cdc25 (Stg), a G2/M cell cycle regulator. Prolonged expression of string is not sufficient to keep cells efficiently in mitotic cell cycle past stage 6, suggesting that Notch also regulates other cell cycle components in the transition. By using an expression screen, such a component was found: Fizzy-related/Hec1/Cdh1 (Fzr), a WD40 repeat protein. Fzr regulates the anaphase-promoting complex/cyclosome (APC/C) and is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner. Mutant clones of Fzr have revealed that Fzr is dispensable for mitosis but essential for endocycles. Unlike in Notch clones, in Fzr mutant cells mitotic markers are absent past stage 6. Only a combined reduction of Fzr and ectopic Stg expression prolongs mitotic cycles in follicle cells, suggesting that these two cell cycle regulators, Fzr and Stg, are important mediators of the Notch pathway in the mitotic-to-endocycle transition (Schaeffer, 2004).

In Drosophila, nurse and follicle cells in the adult ovary endocycle in a regulated manner. It has been suggested that endocycling requires the loss of M-phase cyclin-dependent kinase (Cdk) activity and oscillations in the activity of S-phase Cdk. In Drosophila follicle cells, the function of the Notch pathway in the mitotic-to-endocycle transition has been well established. Lack of Notch activity in Drosophila follicle cells leads to prolonged mitosis at the expense of endocycles, suggesting that Notch functions in this context as a tumor suppressor. Because very few signaling pathways that stop the mitotic cell cycle have been identified, it is important to understand the relationship between the Notch pathway and known cell cycle regulators in more detail (Schaeffer, 2004).

String encodes the Drosophila homolog of the yeast cell cycle regulator Cdc25, a phosphatase whose role is to activate the Cdk-cyclin complex at the G2/M transition by dephosphorylating the inhibitory sites. As a consequence, cells are propelled into mitosis. Notch signaling downregulates string at stage 6 of oogenesis to allow the cells to transit into the endocycle. The 4.9 kb and 6.4 kb elements found in the 50 kb-long string promoter drive string expression in follicle cells from germarium to stage 3 and from stage 4 to stage 6, respectively. The Notch-Delta cascade achieves the tight downregulation of the 6.4 kb element at stage 6, when the mitotic-to-endocycle transition takes place. A string rescue construct that contains 15.3 kb of the string promoter restores only the early string expression pattern between germarium and stage 1-2 egg chambers (because of the 4.9 kb element) but does not contain the control element active between stages 3 and 6 (the 6.4 kb element). Although stg clones produce cells arrested in G2, the mutant nuclei were larger than in the wild-type cells when stg clones were produced in the background of the 15.3 kb rescue construct. Furthermore, the mutant clones are half the size of sister clones, suggesting that the mutant cells stop division and possibly enter endocycle too early. If downregulating String leads the follicle cells to enter an endocycle rather than to completely arrest, then the sole role of Notch, which downregulates string expression at the switch, is to act on string to promote endocycling. If this is the case, string expression is the only limiting factor in the mitotic-to-endocycle transition. Also, because ectopic expression of stg in Drosophila embryos and discs is capable of driving cells blocked in G2 into mitosis, continuous string expression should keep most cells in the mitotic phase (Schaeffer, 2004).

stg (either with a heat-shock-inducible promoter or with one or two copies of the UAS-stg transgene) was overexpressed via the flip-out Gal4 system to analyze whether String is sufficient to prolong division of follicle cells past stage 6. Overexpression of string with a transgene driven by a heat-shock promoter did not show any ectopic Cyclin B or Phospho-Histone 3 (PH3) expression. With one copy of the UAS string transgene, prolonged mitotic divisions were rarely observed in follicle cells past stage 6, except in the posterior region, where 10% of the clones that overexpressed string showed ectopic Cyclin B or PH3 expression. When two copies of the UAS-stg construct were present, leading to higher string expression levels, a higher incidence of Cyclin B and PH3 expression was seen in posterior clones. However, only in a few cases did the ectopic expression of string in lateral and anterior follicle cells prolong their mitotic state. In addition, in most cells, overexpression of string did not affect endocycling (Schaeffer, 2004).

Because the String protein is not enough to create extra cell divisions, except in the highly sensitized posterior area, it was proposed that the mitotic-to-endocycle transition is regulated by a combination of String and other Notch-controlled components yet to be uncovered. Lack of String generally arrests cells in G2, when high levels of mitotic cyclins can be found. Cyclin A and Cdc2 have been implicated in inhibiting the assembly of prereplication complexes in G2. Furthermore, when Cdc2 or Cyclin A activity is eliminated, mutant cells enter endocycles in Drosophila because the assembly of prereplication complexes is then allowed. The hypothesis here is that the Notch signaling pathway allows cells to bypass this inhibition by activating a specific gene/genes that would allow cells to continue to cycle without undergoing the mitotic phase. Expression of such a gene would be activated after/during the mitotic-to-endocycle transition and possibly act on mitotic cyclin regulation and/or the mitotic cyclin-associated kinase, Cdc2. In order to find these genes, an expression screen was performed for genes differentially expressed before and after the transition (Schaeffer, 2004).

400 lethal X chromosome P element enhancer trap lines were screened for changes in expression levels at stage 7 by using the β-gal reporter gene. Three interesting functional groups were obtained from this screen: adhesion molecules, transcriptional control proteins, and cell cycle regulators. Premature expression of fzr caused formation of enlarged nuclei, a potential indication of precocious endocycles. Therefore the cell cycle regulator Fzr was analyzed in more detail in the mitotic-to-endocycle transition (Schaeffer, 2004).

The lines fzrG0326 and fzrG0418 have the P{lacW} element inserted in the first intron and at the 5'-end of the Fizzy-related gene, respectively, and are hypomorphic alleles of fzr. These constructs drive expression of the reporter gene after the transition, from stage 6-7 onward. This expression is tightly correlated with the end of mitotic cycles; no fzr expression is observed in follicle cells that show PH3 staining. A similar expression pattern of Fzr was observed with the Fzr specific antibody, and the fzr mRNA pattern in follicle cells reflects this pattern as well (Schaeffer, 2004).

Fzr, also known as Retina aberrant in pattern, is a conserved WD domain protein that is required during G1 for proteolysis of mitotic regulators such as Aurora-A kinase and Cyclins A, B, and B3 in an APC/C-dependent manner. Loss of Fzr in Drosophila causes cells to progress through an extra division cycle in the epidermis and inhibits endoreduplication in the salivary gland cells, whereas fzr overexpression inhibits mitosis and transforms mitotic cycles into endoreduplication cycles. This finding suggests that, in at least some cell types, the Fzr protein is essential for the mitotic-to-endocycle transition. Because fzr expression is upregulated in follicle cells when the Notch cascade is activated, whether the fzr expression was responsive to Notch activity was tested by using the fzrG0326 (fzr-LacZ) enhancer trap line to analyze fzr expression levels in follicle cells that surround the Dl germline clones. A clear reduction of fzr expression was observed in all Dl germline clones past stage 6, demonstrating that fzr expression is dependent on Notch activity in the mitotic-to-endocycle transition (Schaeffer, 2004).

Mitotic-cyclin protein levels are downregulated at the mitotic-to-endocycle transition. Cyclin A protein levels are reduced at the end of mitotic cycles in the follicle cells. Similarly, Cyclin B is downregulated at the protein level at the mitotic-to-endocycle transition. In situ hybridization studies indicated that neither gene was regulated at the transcriptional level during or after the transition but showed mRNA expression in the follicle cells throughout oogenesis until stage 10. Thus, both the Cyclin A and the Cyclin B protein levels are regulated posttrancriptionally at the mitotic-to-endocycle transition. This regulation is critical for the mitotic-to-endocycle transition because continuous expression of cyclin A in posterior follicle cells results in small nuclei and a reduced DNA level, indicative of a defect in the transition to endocycles. This supports previous reports showing that overexpression of cyclin A inhibits the progression of endoreplication cycles in Drosophila salivary glands (Schaeffer, 2004).

Because the downregulation of Cyclin A and B expression coincides with the upregulation of Fzr and because Fzr is required for proteolysis of Cyclin A and Cyclin B in embryonic epidermal cells, clones for a fzr null allele (fzrie28) were generated to test whether Fzr might be responsible for the mitotic-to-endocycle transition by downregulating the mitotic cyclin levels. Initially, the ovaries were immunostained with antibodies against Cyclin B. The clonal cells lacking Fzr function showed a limited but consistent increase of Cyclin B level after stage 6, when Cyclin B is normally absent. In a similar manner, fzrie28 mutant cells strongly upregulated Cyclin A after the mitotic-to-endocycle transition. It is therefore concluded that, as seen in other systems, Fzr function in follicle cells is to degrade mitotic cyclins (Schaeffer, 2004).

Upregulation of the mitotic Cyclin A during endocycles has been shown to inhibit endoreplication. The ovaries bearing fzrie28 mutant cells were stained with DAPI to observe nuclei size and shape. In addition to showing a failure of Cyclin A and B removal, the fzrie28 mutant cells showed phenotypes that indicated endocycle inhibition. The small nuclei size and reduced DNA level seen in the fzrie28 mutant cells are reminiscent of the Notch phenotype and thereby show that Fzr is required for the mitotic-to-endocycle transition. Unlike the Notch clones in which Cyclin B and PH3 expression were detected after stage 6, the fzrie28 mutant cells do not shown signs of overproliferation. No PH3 staining is ever observed in mutant clones after the transition, suggesting that the fzrie28 mutant cells do not continue to divide past stage 6. The 6.4 kb Stg-LacZ transgene, abruptly downregulated by Notch at the mitotic-to-endocycle transition, did not show any prolonged expression in fzrie28 mutant cells after stage 6, indicating that cells are not in a mitotic phase. The number of cells in mutant and sister clones (two copies of GFP) were counted. The same number of cells was observed in mutant clones as in the associated twin spot; the ratio varied from 0.64 to 3, with a mean of 1.01). All these clues led to the idea that despite mitotic cyclins' upregulation in fzr-/- mutant cells, those cells do not divide past stage 6 (Schaeffer, 2004).

Because the number of cells in the mutant follicle cell clones and the associated twin spots are the same and because the expression of the 6.4 kb stg-LacZ transgene is normal prior to the switch to endocycling, it is concluded that, even though Fzr is required for endocycles, it is dispensable for the mitotic stages in the Drosophila ovary. Similarly, it has been shown that completion of mitosis does not require Fzr in embryos (Schaeffer, 2004).

Although fzrie28 mutant cells show upregulation of the mitotic cyclins, these cells do not divide. In all dividing cells, the G2/M transition depends on String to activate the kinase activity of the mitotic cyclin-Cdc2 complexes. Because the 6.4 kb stg-LacZ transgene is downregulated by the Notch pathway after the transition, the fzrie28 mutant cells might require Stg to prolong mitosis. To test this, string was overexpressed by using a heat shock promoter in the fzrie28 follicle cell clones. The flies were heat shocked twice, once to promote the formation of fzrie28 mutant clones and again, 12 hr prior to dissection, in order to induce stg expression. In a wild-type fzr background, this low level of string expression alone is insufficient for prolonging mitosis past the mitotic-to-endocycle transition (no upregulation of Cyclin B and PH3 markers). However, this prolonged stg expression is enough to push the fzrie28 mutant cells into mitosis, as shown by the PH3 staining and mitotic figures in egg chambers at stage 9. More strikingly, PH3-positive cells as well as mitotic figures were seen in nonclonal areas heterozygous for fzrie28, which prompted a test to see whether reducing the level of Fzr to one copy while overexpressing stg by heat shock is sufficient to produce the PH3-positive cells. In order to demonstrate a direct stg effect, the flies were examined 2 hr after heat shock. The fzrG0326 enhancer trap line (fzr LacZ) was used to reduce the level of Fzr and to mark the stages precisely. It was found that 39% of ovarioles of the experimental group fzrG0326;;Hs Stg displayed PH3-positive cells and mitotic figures at stage 7-8, whereas 0%-2% did so in the control groups. In order to further determine whether the ratios of cells in a mitotic stage in mutant and control situations were similar, the number of PH3-positive cells observed in a single focal plan was quantified before and at stage 7-8, in the experimental group fzrG0326;;Hs Stg as well as in three control groups. On average, 12%-15% of the cells showed PH3 staining at mitotic stages before the transition (before stage 7), but none did so after the transition (stage 7-8). In contrast, ovaries with reduced fzr and prolonged stg expression showed PH3 staining after stage 7, whereas the control groups did not. In comparison, 8.5% of cells in the egg chamber did exhibit PH3 staining. It is possible that the percentage of mitotic cells observed in mutant egg chambers past the transition was somewhat lower than the percentage of mitotic cells observed in wild-type egg chambers before the transition because of the low level of string expression given by the heat shock construct or subtle effects of yet-unraveled components in the transition. However, these data strongly suggest that reducing the Fzr level in combination with prolonged stg expression can prolong the mitotic stage in follicle cells (Schaeffer, 2004).

In Drosophila, loss of Fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreplication in the salivary glands, in addition to the upregulation of mitotic cyclins. In follicle cells loss of Fzr causes an inhibition of endoreplication as well as an upregulation of the mitotic cyclins, particularly Cyclin A, but no prolonged mitosis. This difference might be due to the lack of String in follicle cells. It is possible that in the epidermis, residual String might dephosphorylate and therefore activate the mitotic cyclin/Cdk complexes and allow an extra mitosis to proceed, whereas in follicle cells the absence of String might result in G2-arrest. This is supported by the fact that overexpressing a string transgene under the control of a heat shock promoter rescues cell division in a fzr mutant (Schaeffer, 2004).

Notch mutant cells are mitotic: in those cells, Stg is upregulated, and Fzr is not activated. Those two events (upregulation of String and downregulation of Fzr) are able to keep the cells in mitotic cycle in 39% of stage 7-8 egg chambers. It is therefore possible that Notch controls the mitotic to endocycle transition by repressing String to block mitosis and by activating Fzr to allow endocycle progression (Schaeffer, 2004).

Based on earlier studies, it has been proposed that endocycle is induced by lack of M-phase Cdk activity. However, the regulation and exact manifestation of this task has not been previously uncovered. This study shows that in Drosophila follicle cells the Notch pathway executes the task by first freezing the mitotic cyclin/Cdk complex in an inactive, phosphorylated form and thereafter inducing the degradation of the mitotic cyclins to allow progression to S phase. Further studies will reveal whether Notch action is also required for G1-to-S-phase transition or whether these two alterations, lack of String, and expression of Fzr are sufficient to transform mitotic cells to endocycling cells (Schaeffer, 2004).

Notch and Apoptosis

An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).

Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notchts1 allele was used. However, when Notchts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notchts1/Notch55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).

Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).

In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).

Notch synergizes with axin to regulate the activity of armadillo in Drosophila

Cell fate decisions require the integration of various signalling inputs at the level of transcription and signal transduction. Wnt and Notch signalling are two important signalling systems that operate in concert in a variety of systems in vertebrates and invertebrates. There is evidence that the Notch receptor can modulate Wnt signalling and that its target is the activity and levels of Armadillo/β-catenin. This function of Notch has been characterized in relation to Axin, a key element in the regulation of Wnt signalling that acts as a scaffold for the Shaggy/GSK3beta-dependent phosphorylation of Armadillo/beta-catenin. While Notch can regulate ectopic Wingless signalling caused by loss of function of Shaggy, it can only partially regulate the ectopic Wnt signalling induced by the loss of Axin function. The same interactions are observed in tissue culture cells where a synergy is observed in between Axin and Notch in the regulation of Armadillo/β-catenin. These results provide evidence for a function of Axin in the regulation of Armadillo that is different from its role as a scaffold for GSK3β (Hayward, 2006).

Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers

The CSL [CBF1/Su(H)/Lag2] proteins [Su(H) in Drosophila] are implicated in repression and activation of Notch target loci. Prevailing models imply a static association of these DNA-binding transcription factors with their target enhancers. Analysis of Su(H) binding and chromatin-associated features at 11 E(spl) Notch target genes before and after Notch revealed large differences in Su(H) occupancy at target loci that correlated with the presence of polymerase II and other marks of transcriptional activity. Unexpectedly, Su(H) occupancy was significantly and transiently increased following Notch activation, suggesting a more dynamic interaction with targets than hitherto proposed (Krejcí, 2007).

To investigate changes in chromatin that accompany Notch activation, it was necessary to establish conditions where receptor activation could be temporally controlled. It has been reported that exposing cells to EDTA stimulates shedding of the Notch ectodomain. This renders the residual transmembrane fragment a substrate for γ-secretase cleavage and, hence, results in Notch activation. Despite results suggesting that cell surface Notch in Drosophila would not be susceptible, it was found that EDTA causes robust activation of E(spl) Notch-target genes in a Notch-expressing Drosophila S2 cell line (S2-N). No effect was seen when S2 cells that do not express Notch were treated with EDTA (Krejcí, 2007).

All 11 E(spl) genes were induced following EDTA treatment. Most were expressed at very low levels before activation and, although stimulated following EDTA treatment, their absolute levels of expression remained low. One gene, m3, was expressed at intermediate levels prior to activation and was induced 50 times by EDTA treatment to expression levels that were ~100 times higher than other E(spl) genes. There was no change in expression of the housekeeping genes or nontarget loci analyzed. A qualitatively similar effect on E(spl) gene expression was obtained in S2 cells transfected with a plasmid expressing Nicd (Krejcí, 2007).

To ascertain whether EDTA activates E(spl) genes through its effects on Notch, cells were treated in the presence of γ-secretase inhibitors (e.g., DFK-167). This compromised the induction of m3 and m7 expression by EDTA. In addition, by immunoprecipitating Su(H) from cell extracts and probing for coprecipitation of Nicd, it was confirmed that there is a robust association of Nicd with Su(H) in EDTA-treated, but not in control cells (Krejcí, 2007).

In summary, EDTA treatment provides a method to rapidly activate Notch in a temporally controlled manner throughout a cell population (and is subsequently referred to here as Notch activation). By activating Notch in this a larger, more concerted burst of Notch activity may taking planc than occurs during normal signaling in the animal. Nevertheless, this makes it possible to analyze the chromatin state before and after Notch activation under carefully timed conditions (Krejcí, 2007).

The results demonstrate that there is a significant increase in Su(H) occupancy at target genes following EDTA/Notch activation. This increase is transient and correlates with the presence of Nicd, implying that the kinetics of binding differ when Su(H) is complexed with Nicd, and that the association between Su(H) and its cognate sites is much more dynamic than expected. It was also noted that there are differences in Su(H) occupancy between genes prior to activation, suggesting the possibility of gene-specific modes of regulation [i.e., at some there may be constitutive recruitment of Su(H), whereas at others Su(H) binding is only signaling induced] (Krejcí, 2007).

Several mechanisms could account for the increased Su(H) occupancy after Notch activation. One possibility is that the activation complex has a higher affinity for DNA. Structural analysis of the CSL/Nicd/Mam tertiary complex did not reveal any novel interactions between CSL and DNA that could account for an increase in affinity per se. However, the affinity could be increased by cooperative interactions between two activation complexes on the DNA. Recent analysis demonstrates that Nicd-containing complexes can bind cooperatively to DNA with appropriately arranged paired sites. This may therefore be a significant factor in the enhanced Su(H) occupancy that is detected after activation. However, not all the enhancer fragments analyzed contain paired Su(H) sites, suggesting that additional mechanisms are involved (Krejcí, 2007).

A second explanation is that interactions with other cofactors help to stabilize the Su(H) activation complex on the DNA. Studies of nuclear receptors suggest that in the resting state they rapidly exchange on and off the DNA, and that the formation of transcriptionally competent complexes slows this exchange. It is envisaged that the interactions of Su(H)/CSL with its cognate sites have similar change in dynamics; a fast exchange and low residency occurring when Su(H)/CSL is complexed with corepressors, a slow exchange and longer residency occurring when it is complexed with Nicd and competent to recruit productive transcription complexes and/or to make cooperative interactions (Krejcí, 2007).

Direct response to Notch activation: signaling crosstalk and incoherent logic

Notch is the receptor in one of a small group of conserved signaling pathways that are essential at multiple stages in development. Although the mechanism of transduction impinges directly on the nucleus to regulate transcription through the CSL [CBF-1/Su(H)/LAG-1] DNA binding protein, there are few known direct target genes. Thus, relatively little is known about the immediate cellular consequences of Notch activation. This study set out to determine the genome-wide response to Notch activation by analyzing the changes in messenger RNA (mRNA) expression and the sites of CSL occupancy within 30 minutes of activating Notch in Drosophila cells. Through combining these data, high-confidence direct targets of Notch were identified that are implicated in the maintenance of adult muscle progenitors in vivo. These targets are enriched in cell morphogenesis genes and in components of other cell signaling pathways, especially the epidermal growth factor receptor (EGFR) pathway. Also evident are examples of incoherent network logic, where Notch stimulates the expression of both a gene and the repressor of that gene, which may result in a transient window of competence after Notch activation. Furthermore, because targets comprise both positive and negative regulators, cells become poised for both outcomes, suggesting one mechanism through which Notch activation can lead to opposite effects in different contexts (Krejci, 2009).

The H3K27me3 demethylase dUTX is a suppressor of Notch- and Rb-dependent tumors in Drosophila

Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. A disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Mammalian UTX, UTY, and JmjD3 and Drosophila UTX (dUTX) are histone demethylases that specifically demethylate di- and trimethylated lysine 27 on histone H3 (H3K27me2 and H3K27me3, respectively). The catalytic domain of this activity is the Jumonji C (JmjC) domain, located at the C terminus of these proteins. The N-terminal domains of UTX, UTY, and dUTX contain several tetratricopeptide repeats (TPRs) thought to be required for protein-protein interactions (Herz, 2010).

H3K27me3 is a histone mark for Polycomb (Pc)-mediated genomic silencing and transcriptional repression and is associated with animal body patterning, X-chromosome inactivation, genomic imprinting, and stem cell maintenance. H3K27 methylation is catalyzed by Polycomb repressive complex 2 (PRC2), which in Drosophila is composed of the catalytic subunit enhancer of zeste [E(z)] (EZH2 in mammals), extra sex combs (Esc), suppressor of zeste 12 [Su(z)12], and nucleosome remodeling factor 55 (Nurf55). H3K27me3 is recognized by the chromodomain of Pc, which is a component of a different silencing complex, called PRC1, which, in addition to Pc, contains Polyhomeotic (Ph), posterior sex combs (Psc), and dRING. The wild-type function of UTX is to demethylate H3K27me3 and, thus, to antagonize Polycomb-mediated silencing (Herz, 2010).

UTX is also a component of mixed-lineage leukemia complex 3 (MLL3) and MLL4. MLL complexes are histone methyltransferases for H3K4. The function of UTX in MLL3 and MLL4 is unknown. However, it appears that UTX is not required for the H3K4 methyltransferase activity of MLL3 and MLL4. The best-characterized targets of H3K27me3/Pc-mediated silencing are homeotic genes, which are critical regulators of animal patterning. However, many other genes are also enriched for H3K27 methylation and Pc binding. Furthermore, elevated H3K27me3 levels due to an increased activity of the methyltransferase EZH2 could be a leading cause of certain human cancers. Recently, mutations that inactivate UTX, and which are thus expected to cause increased H3K27me3 levels, have been linked to the development and progression of human cancer. However, the precise mechanisms by which this occurs are largely unknown (Herz, 2010).

Notch is the receptor of a highly conserved signaling pathway involved in many biological processes, including lateral inhibition, stem cell maintenance, and proliferation control. The binding of Delta or Serrate, the two ligands in Drosophila melanogaster, triggers the proteolytic processing of Notch, resulting in the release and translocation of the Notch intracellular domain (NICD) into the nucleus, where it regulates gene expression. Aberrant, oncogenic Notch signaling has been linked to tumor development in humans, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. Thus, an improved understanding of Notch signaling will have significant implications for human health (Herz, 2010).

In Drosophila, the Notch signaling pathway also controls the growth of the eye primordium and wing margin formation during development. Although the mechanistic details are unclear, one way by which Notch signaling controls proliferation during Drosophila eye development is through the negative regulation of the Retinoblastoma (Rb) family member Rbf. Rbf inactivation has also been implicated in Notch-induced eye tumors in Drosophila. Rb is a tumor suppressor that negatively regulates cell cycle progression through the inhibition of the transcription factor E2F. Rb binds directly to E2F and represses its transcriptional activity. The release of Rb activates E2F to induce the transcription of cell cycle regulators such as cyclin E and PCNA. Therefore, the inactivation of Rbf by increased Notch signaling can trigger increased proliferation, which may lead to cancerous growth (Herz, 2010).

This study genetically characterizes loss-of-function mutations of dUTX. dUTX mutants display some of the characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. dUTX mutant tissue has an H3K27me3-dependent growth advantage over wild-type tissue due to increased proliferation in the developing eye. The growth advantage of dUTX mutant tissue is caused by increased Notch activity, demonstrating that dUTX is a Notch antagonist. The inactivation of Rbf contributes to the growth advantage of dUTX mutant tissue. Moreover, an excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Based on the enzymatic activity of the JmjC catalytic domain as H3K27me3 demethylases, UTX proteins are predicted to counteract Polycomb function. Consistently, it was found that dUTX mutants display genetic characteristics of Trithorax group genes. In vitro studies have shown that dUTX and UTX demethylate H3K27me2 and H3K27me3. However, dUTX mutants affect the global levels of only H3K27me3 but not of H3K27me2. Nevertheless, this observation does not mean that dUTX does not demethylate H3K27me2 in vivo. There may be fewer genes regulated by dUTX at the H3K27me2 level such that the global levels are not detectably altered in dUTX mutants (Herz, 2010).

Interestingly, dUTX mutants also affect global levels of H3K4me1, which are significantly reduced in mutant tissue. Mammalian UTX is a component of the MLL3 and MLL4 methyltransferase complexes, and based on the reduction of H3K4me1 levels, it is predicted that dUTX is also a component of the Drosophila equivalent of the MLL3/MLL4 methyltransferase complex, which contains Trithorax-related (Trr) as a histone methyltransferase. The function of UTX in MLL3 and MLL4 complexes is currently unknown. It was suggested previously that UTX is not required for H3K4 methylation, but in these studies, only H3K4me2 and H3K4me3 were investigated. Consistently, the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutant clones. The data demonstrate that dUTX is required for the monomethylation of H3K4. Interestingly, the JmjC demethylase domain of dUTX is not required for H3K4me1 methylation, suggesting that other domains of dUTX, such as the TPR domains, may be necessary for mediating this function. The finding that the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutants is also quite interesting, as it implies that the monomethylation of H3K4 is not required for the di- or trimethylation of H3K4 (Herz, 2010).\

The epigenetic control of gene expression has been best studied for the control of homeotic gene expression, which is established during embryogenesis and maintained throughout animal life. However, not only homeotic genes are regulated through epigenetic modifications. Other genes in different developmental processes are also subject to epigenetic control. In this study, by analyzing the dUTX mutant phenotype, a role was establised of H3K27me3 levels in cell cycle control. The data suggest that increased H3K27me3 levels in dUTX clones cause the epigenetic silencing of several genes involved in Notch signaling. This includes both positive and negative regulators of Notch signaling activity as well as target genes that are either positively or negatively regulated by the Notch pathway. Such an incoherent control of gene expression by the Notch pathway has been reported previously, suggesting that the final outcome of Notch activity may be determined by the relative expression levels of positive or negative regulators. Because this study determined that the overrepresentation phenotype of dUTX clones is caused by elevated levels of Notch signaling, it appears that the silencing of Notch inhibitors is dominant over the silencing of Notch activators, resulting in a net increase of Notch activity. However, this increased Notch activity may be specific for the cell cycle phenotype of dUTX mutants, since increased Notch activity was not found for other Notch-dependent paradigms, such as E(spl)m8-lacZ. This is also consistent with the finding that E(spl) genes contain increased H3K27me3 levels in dUTX mutants. Thus, the wild-type function of dUTX is to restrict the cell cycle through the negative control of Notch. Therefore, the data link H3K27me3-dependent Notch activity with enhanced tissue growth, implying that dUTX is a Notch antagonist regarding the cell cycle and explaining the overrepresentation phenotype of dUTX mutant clones (Herz, 2010).

However, this phenotype is subtle compared to that of mutants in growth control pathways such as the Hippo pathway. Nevertheless, the overgrowth of dUTX clones is strongly potentiated by the additional activation of Notch. The expression of Delta in dUTX clones causes a strong tumor-like growth phenotype. Thus, dUTX functions as a suppressor of Notch-induced tumors under normal conditions. This synergistic interaction between the loss of dUTX and increased Notch activity is a clear example that tumor development requires several hits for progression (Herz, 2010).

The overrepresentation phenotype of dUTX clones can be dominantly enhanced by the genetic loss of Rbf, suggesting that the reduction of Rbf contributes to the overrepresentation phenotype. However, the reduction of Rbf activity in dUTX clones is not caused by direct epigenetic silencing at the Rbf locus. No increased H3K27me3 levels was found at the Rbf locus in dUTX mutants, and mRNA levels of Rbf were unchanged. Instead, Rbf is negatively regulated by the Notch pathway during eye growth. Thus, the increased activity of Notch in dUTX clones leads to a partial inactivation of Rbf and increased proliferation, causing the overrepresentation phenotype. Currently, it is unknown how Notch regulates Rbf (Herz, 2010).

The control of cell cycle progression by UTX proteins is likely conserved in mammals. A parallel study performed by Wang showed that the loss of mammalian UTX also results in elevated levels of proliferation (Wang, 2010). Consistent with the current work, that study also implicated the inactivation of Rb function in increased proliferation in response to UTX knockdown. Similar to the current study, Rb itself is not subject to increased H3K27m3 silencing, but the promoters of several genes in the Rb network were found to be occupied and likely controlled by UTX (Wang, 2010). Thus, although the mechanisms of Rb control by UTX proteins (Notch in this study and the Rb network in the study reported previously by Wang are distinct, both studies established the control of the Rb pathway as a common element of cell cycle control by UTX proteins. Wang also demonstrated a link between UTX and Rb during vulval development in Caenorhabditis elegans. Thus, these studies combined suggest a well-conserved function of UTX proteins for Rb control (Herz, 2010).

Although these studies establish a link between UTX genes and Rb for cell cycle control, it should be noted that the loss of dUTX (and likely mammalian UTX) affects many genes. While the deregulation of individual genes may not cause a significant phenotype on its own, the combined deregulation may disrupt gene regulatory networks, which accounts for the growth phenotype of dUTX mutants. Thus, while aberrant Notch signaling was identified as an important element of the overrepresentation phenotype of dUTX mutants, other genes and signal transduction pathways may also contribute to this phenotype. For example, this study also identified genes involved in growth control by the Hippo pathway (four-jointed [fj] and warts) associated with increased H3K27me3 levels in dUTX mutants and showed reduced transcript levels for fj. Thus, it is possible that the Hippo pathway and other genes contribute to the overrepresentation phenotype of dUTX mutants (Herz, 2010).

These observations have important implications for the initiation and development of human tumors. Increased levels of H3K27me3 due to the elevated activity of the H3K27me3 methyltransferase EZH2 have been associated with human cancer. Furthermore, mutations that inactivate UTX have been linked to human cancer, and low UTX activity correlates with poor patient prognosis. This study establishes that increased levels of H3K27me3 affect Notch activity, which in turn affects Rbf activity. Rb is a well-known tumor suppressor, the loss of which causes human tumors. Therefore, tumors associated with the loss of UTX and, thus, increased H3K27me3 levels may be caused by decreased Rb activity. It should also be noted that aberrant Notch signaling is the cause of several human cancers, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. In summary, these data demonstrate that the appropriate control of H3K27 methylation is critical for normal tissue homeostasis, and increased H3K27me3 levels may contribute to cancer through the inactivation of Rb (Herz, 2010).

Return: Notch Targets of Activity part 1/3 | part 2/3

Notch continued: Biological Overview | Evolutionary Homologs | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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