Enhancer of split

Regulation of other Enhancer of Split Complex Genes

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).

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homolog (Egfr) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. This phenotype suggests a possible role for Egfr in specifying the founder R8 photoreceptor cells for each ommatidium. Ellipse mutations have been used to probe the role of Egfr in eye development: Elp mutations result from a single amino acid substitution in the kinase domain, which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirm that the mutation is hypermorphic in vivo, but the Egfr function is elevated less than by ectopic expression of the ligand Spitz. Ectopic Spi promotes photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of Egfr activation were assessed to explore the basis of these distinct outcomes. Elp mutations cause overexpression of the Notch target gene E(spl) mdelta and require the function of Notch to suppress ommatidium formation. E(spl) mdelta is known to be expressed in cell clusters in the morphogenetic furrow of wild type eyes. The Elp phenotype also depends on the secreted protein Argos and therefore, in Elp;aos double mutants, the phenotype is reverted. Complete loss of Egfr function in clones of null mutant cells leads to delay in R8 specification and subsequently to the loss of mutant cells. Argos is required for loss of ommatidia in Elp. It is suggested that nonautonomy of Elp mutations suggests that aos acts nonautonomously to inhibit ommatidium formation (Lesokhin, 1999).

The role of the Notch signaling pathway has been examined in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes. Using a reporter assay system in Drosophila tissue culture cells, a significant induction of E(spl) m gamma and m delta expression is observed after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. Su(H) binding sites are present in the upstream regions of both E(spl) genes. Notch-inducible expression of E(spl) m gamma and m delta, both in cultured cells and in vivo, is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter. The behavior of Drosophila Notch is consistent with a mechanism whereby N activates Su(H) by covalent modification. It is unlikely that N functions to sequester Su(H) in the cytoplasm, since Su(H) is nuclear. There also are no apparent differences in strength in reporter gene activation in Drosophila between nuclear and membrane bound forms of activated Notch. In the covalent modification hypothesis, N-Dl binding could result in the binding and/or activation of a modifying enzyme (such as a kinase or methylase) which could act on Notch-bound Su(H) (Eastman, 1997).

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 selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).

The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).

The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).

To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999).

Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999).

Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).

Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologs in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes were analyzed and comparisons to E(spl)m8 were made. Although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. It is concluded that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators; since the activation of target genes is critical in determining the developmental consequences of Notch activity, these results provide a framework for understanding Notch function in different developmental contexts (Cooper, 2000a).

E(spl)m8 is transcribed in all sensory organ clusters: E(spl)mdelta and E(spl)mgamma in a subset of sensory clusters but strongly in the developing eye, and E(spl)mbeta in the intervein regions of the wing primordium, at the dorsal/ventral boundaries of the wing and eye, and in the presumptive leg joints. To identify the regions responsible for conferring the specific expression patterns, 1- to 2-kb fragments from the region encompassing E(spl)mdelta, E(spl)mgamma, E(spl)mbeta were fused to a minimal promoter upstream of the lacZ gene to test for enhancer activity. For each of the three genes, the fragment adjacent to the promoter (mdelta1.9, mgamma1.1, and mbeta1.5) confers a pattern of expression that largely recapitulates the endogenous genes, although there are some notable exceptions: (1) neither mdelta1.9 nor mgamma1.1 generates the strong expression associated with the morphogenetic furrow that is observed with both genes; (2) the mdelta1.9 fragment fails to confer the tegula expression normally associated with E(spl)mdelta. Given the close proximity of the genes in the complex, it is possible that adjacent genes could share regulatory elements. Because mgamma1.1 confers strong expression in the tegula domain, it might account for the tegula expression of E(spl)mdelta as well as E(spl)mgamma. To test whether there is an insulator within mgamma1.1 that would prevent it acting on the adjacent E(spl)mdelta transcription unit, mgamma1.1 was inserted between the lacZ and CD2 coding sequences. Both proteins have similar patterns of expression, indicating that mgamma1.1 is able to regulate an upstream transcription unit and so could mediate the tegula expression of the upstream E(spl)mdelta. Further indirect support for the hypothesis that enhancers can act on neighboring genes comes from analysis of a P-element (K33) inserted at the E(spl)mgamma locus. When the sequences proximal to the P-element are deleted, as occurs in Df(3R)NF1P1, the inserted lacZ gene is now expressed in a pattern weakly resembling the distal E(spl)mbeta gene, even though none of the intervening sequences have been altered. These results indicate that the E(spl)mbeta enhancer has the potential to act on the E(spl)mgamma region, but in the wild-type chromosome it must be prevented by the sequences adjacent to the E(spl)mgamma promoter (Cooper, 2000a).

Thus E(spl)mbeta, E(spl)mgamma, and E(spl)mdelta patterns can largely be recapitulated by DNA fragments of ~400-500 bp located close to the transcription start site. As expected, these fragments contain Su(H) binding sites, consistent with their responsiveness to Notch signaling. However, they are also sufficient to generate quite diverse patterns of expression. The fact that this activity resides in such localized enhancers contrasts with the organization of other genes expressed in similar complex patterns in the disc, such as proneural and intervein genes. These are regulated by an array of enhancers, each of which responds to a different combination of patterning genes. The comparative simplicity of the identified E(spl) enhancers suggests that they are unlikely to be regulated by a similar array, but are more likely to be responding to the next level in the hierarchy, i.e., to the factors that are themselves expressed in complex patterns (Cooper, 2000a).

The suggestion that E(spl) genes are regulated by intermediates in the patterning hierarchy is consistent with the proneural proteins contributing to their regulation. However, this also presents an inconsistency, because the E(spl) products are not detected in the neural precursor cells where proneural proteins accumulate to highest levels. This study demonstrates that proneural proteins work synergistically with Su(H)/Nicd (the complex between Suppressor of hairless protein and the intracellular domain of Notch) to activate transcription from E(spl)m8 and E(spl)mgamma enhancers in cultured cells. For E(spl)m8, this synergy can also be demonstrated in vivo, as a combination of proneural proteins and Nicd leads to higher levels of m8-lacZ expression than either component alone. This combined regulation can explain why E(spl) genes are activated in the cells surrounding the sensory organ precursors, since these are cells where both proneural proteins and Notch activity would be present. In this respect the regulation of some E(spl) genes, in particular E(spl)m8, fits with a combinatorial model, which suggests that the activation of genes in response to signaling pathways involves the transcriptional response factor for the signaling pathway acting in combination with specific patterning genes (Cooper, 2000a).

The combinatorial synergy between Notch and proneural proteins may be sufficient to explain E(spl)m8 regulation, but it is not sufficient to account for the expression of some other E(spl) genes. Two key points are highlighted by the different enhancers and tissues that have been analysed. The first is that there must be factors equivalent to the proneural proteins that synergise with Notch on the E(spl)mbeta enhancer. The second is that the competence of the E(spl) enhancers to respond to Su(H)/Nicd is spatially restricted by more than just the availability of an appropriate synergising activator. Unlike the other enhancers analysed, mbeta1.5 is highly sensitive to activated Notch and Su(H)VP16 throughout the wing pouch. Intriguingly, the E(spl)mbeta fragments confer much higher levels of expression than any of the other fragments tested, even though one of the two Su(H) sites in mbeta1.5 does not conform fully to a consensus binding site. The widespread activation of mbeta1.5 in the wing pouch and its poor response to proneural proteins suggest that the E(spl)mbeta enhancer responds to other activators. This explains why it is still possible for ectopic Nicd to promote increased levels of E(spl) proteins in scute^10-1 discs. Under these conditions transcription of E(spl)mbeta [and possibly E(spl)m3] could still be increased in the wing pouch, even if E(spl)m8, E(spl)mgamma and E(spl)mdelta could not. These investigations have not yet identified specific activators that account for the activity of mbeta1.5, although there are binding sites for a variety of factors including two proteins expressed in the wing, Scalloped and Caupolican (Cooper, 2000a).

The differences in the responses of mgamma1.1 and mdelta1.9 compared to E(spl)m8 argue that there is an additional level of regulation that limits the accessibility of mgamma1.1 and mdelta1.9 proneural proteins/Su(H). Thus, although mgamma1.1 and mdelta1.9 are targets for proneural proteins and Su(H)/Nicd, based on effects in tissue culture and/or in vivo, they cannot be activated very effectively within the wing pouch even when high levels of certain proneural proteins and/or Nicd are expressed ectopically. Likewise, mgamma1.1 and mdelta1.9 are largely resistant to activation by Su(H) VP16 in the wing pouch, although weak activation of mdelta1.9 is sometimes detected. Similar restrictions have been observed when an E(spl)m5 enhancer, whose Su(H) binding sites had been replaced with Gal4 UAS sites, was exposed to ubiquitous Gal4. This transgene could only be activated in a limited domain, indicating that Gal4 activity can also be influenced by E(spl) regulatory sequences (Cooper, 2000a).

The factors that modulate the responsiveness of the enhancers to Su(H)/Nicd and activators such as proneural proteins also act through the small 180- to 500-bp enhancer fragments, and several different mechanisms can be envisioned that might account for this modulation. One is that there is a 'prefactor' that is necessary to initially modify the chromatin and allow entry of Su(H) and proneural proteins. Recent analyses of the mechanisms involved in gene activation demonstrate that there may be sequential stages in chromatin remodelling. If an earlier step of chromatin modification is needed before Su(H) and other activators can access the enhancers, the differential response of E(spl)m8 and E(spl)mgamma fragments to Su(H) VP16 in the wing pouch would arise from a requirement for different factors to implement this initial step. An alternative model is that the enhancer fragments are also targets for specific repressors, for example, mdelta1.9 and mgamma1.1 could be specifically repressed throughout most of the wing pouch. However, none of the truncations or site-specific mutations of the mgamma1.1 and mdelta1.9 fragments have ever led to ectopic activity, as would be indicative of loss of a repressor binding region (Cooper, 2000a).

Su(H)VP16 mimics phenotypes produced by activated Notch both in Drosophila and in Xenopus consistent with the evidence that Su(H) is essential for activation of target genes, via its association with Nicd. Results from mammalian tissue culture cells, however, indicate that CBF/Su(H) also functions as a repressor, interacting with histone deacetylase (HDAC). There is as yet no evidence to support this model in Drosophila, but the low levels of residual expression from E(spl) enhancers in Su(H) mutant discs might be explained by this mechanism. If in wild-type discs, Su(H) is bound to E(spl) enhancers in association with HDAC, it could prevent any activation from proneural proteins until Nicd is present. In animals that lack Su(H), this repression would no longer occur, so that high levels of proneural proteins could activate the enhancers. In support of this reasoning it is found that in tissue culture cells some activation is elicited by proneural proteins alone, particularly of the E(spl)m8 reporter. Furthermore, the residual expression from mdelta1.9 and mgamma1.1 enhancers is greatest in the oldest discs, where the levels of proneural proteins are highest and residual maternal Su(H) protein would be lowest. The dual repressor/activator roles proposed for Su(H) are like those put forward for TCF/Pangolin, which becomes a transcriptional activator of Wnt/Wingless responsive genes upon binding to beta-catenin, but appears to act as a repressor in the absence of Wnt signalling (Cooper, 2000a).

Previous studies of E(spl) regulation in the embryo suggested an element of autoregulation since expression of m8-lacZ is elevated in E(spl) mutant embryos. Similar effects are also seen with HES expression in tissue culture cells, where the levels of transcription decline after their initial activation. The data suggest that this is likely to be a general mechanism, since all four E(spl) enhancers are responsive to ectopic E(spl) proteins in vivo, especially mbeta1.5. Furthermore, in cells where the repressive function of E(spl) proteins is compromised, their expression levels increase. Both these results are compatible with autoregulatory negative feedback by E(spl) proteins, so that once a critical amount is produced these proteins inhibit their own expression. This negative feedback regulation could help to keep cells in a pliable state, for example, during neurogenesis, when the balance between proneural and E(spl) proteins is critical in determining whether a cell adopts the neural fate (Cooper, 2000a).

Several results indicate that the individual enhancers are able to influence more than one E(spl) gene. (1) The fragment between E(spl)mdelta and E(spl)mgamma (mgamma1.1) confers strong tegula cluster expression and contains no insulator to prevent it from acting on the 5' E(spl)mdelta gene, suggesting that it normally acts on both transcription units and accounts for the tegula expression of both genes (although the possibility that there is an insulator within E(spl)mdelta itself has not been ruled out). (2) In the Df(3R)NF1P1 deletion, the E(spl)mbeta enhancers acts on the lacZ gene inserted at E(spl)mgamma, demonstrating that the regulatory elements have the potential to act on adjacent genes. Other evidence suggests that the complex E(spl) expression patterns involve a combination of shared and redundant elements. For example, although E(spl)mgamma and E(spl)mdelta are both expressed in the ommatidial field, only mdelta1.9 confers a high level of ommatidial expression: mgamma1.1 is much less robust. In the native E(spl) complex, these two elements could act in concert to give strong E(spl)mgamma expression in ommatidia (Cooper, 2000a).

The sharing of regulatory elements means that there is significant overlap in the expression patterns of adjacent genes, which accounts for some of their redundancy. In addition the effects of deleting one gene could be rescued by residual elements influencing the expression of neighboring genes. The fact that there is some interdigitation of regulatory elements may also help to explain the conservation of the E(spl) complex, as has been argued for the paralogous Hox clusters in mammals where sharing of regulatory elements has been documented and is proposed to have helped constrain the organization of the clusters (Cooper, 2000a).

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, 2000b).

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, 2000b).

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, 2000b).

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 (N^act) 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 N^act expression in R3,R4, R1,R6 and R7 (via the sevenless enhancer; sev-N^?ecd or sev-Gal4/UAS-N^icd) and ubiquitous N^act expression (induced by heat-shock enhancer; hs-N^icd). 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 N^act 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 N^ts 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, 2000b).

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-N^icd) 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-N^icd 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 N^ts larvae (Cooper, 2000b).

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, 2000b).

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 N^act 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, 2000b).

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, 2000b).

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, 2000b).

Deadpan contributes to the robustness of the Notch response

Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation, this study has identified several Notch responsive enhancers in the bHLH hairy and Enhancer of split (E^spl) family gene dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several E^spl genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from E^spl gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits E^spl gene expression following Notch activation. Thus the combined actions of dpn and E^spl appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets (Babaoglan, 2013).

HES genes are well-known targets of Notch activity. However, in Drosophila only the bHLH genes within the E(spl) Complex were originally thought to be directly downstream of Notch. The expression of another HES genes, dpn, appeared independent of Notch and indeed was associated with cells where Notch activity is considered to be down-regulated (embryonic neuroblasts). More recently it has emerged that dpn expression is under Notch regulation in some contexts (San Juan, 2012; Zacharioudaki, 2012). The current results extend these findings by demonstrating that dpn is directly bound by Su(H) in vivo. As the Su(H) occupied regions differ according to the tissue-type, it appears that dpn contains several Notch responsive enhancers, and the results demonstrate that these direct Notch-dependent expression in different subsets of tissues. Nevertheless, it is striking that a single dpn enhancer, dpn[b] exhibits Notch related expression in both the eye and the wing discs yet these patterns are characteristic of distinct genes/enhancers from the E(spl) Complex (Babaoglan, 2013).

Despite the clear regulation by Notch, there is however relatively few phenotypes resulting from loss of dpn in many tissues. For example, both the wing and eye disc exhibit robust expression of dpn but neither exhibit phenotypes when dpn function was ablated. However, genetic interactions demonstrate that dpn function is related to Notch and both the current evidence, and that from recent studies (San Juan, 2012; Zacharioudaki, 2012), indicate that it has partially redundant functions with the E(spl) genes. This is exemplified by the fact that absence of dpn or of E(spl) Complex alone has little effect on the D/V boundary, but the combined knock down leads to loss of key gene expression (Babaoglan, 2013).

What then is the relevance of dpn in these contexts, especially given that there are 7 E(spl)bHLH genes that also appear to have largely redundant functions? Two components to dpn function are proposed to explain its importance in the Notch response. Clues for the first come from the fact that subtle phenotypes were detected from reductions in dpn when early developmental stages were analyzed. Thus the absence of dpn led to a delay in the ability of Notch to up-regulate cut. Earlier studies also demonstrated a subtle decrease in cut expression in cells lacking E(spl) genes. These results could be explained if both E(spl) and dpn make a contribution to cut regulation. It is suggested that this must be indirect, via the inhibition of a repressor, since both dpn and E(spl)bHLH are thought to be dedicated repressors. So far, no other repressor has been identified that could act as an intermediary (Babaoglan, 2013).

The second component of dpn function is suggested by the observation that Dpn can repress the enhancers derived from E(spl)bHLH genes but not vice versa. Futhermore, it was observed that cells with high levels of Dpn often had lower levels of E(spl)bHLH on a cell by cell level. It is therefore proposed that there is a direct regulatory relationship between dpn and E(spl)bHLH, whereby dpn represses E(spl)bHLH expression. This could set a maximum threshold for E(spl) gene expression since, in previous studies, it was found that dpn shows a less rapid up-regulation following Notch activation than the E(spl) genes. This is reminiscent of the differences seen between HES gene responses in the oscillatory clock involved in somitogenesis and suggests that similar HES gene cross-regulatory network may underpin other Notch dependent processes (Babaoglan, 2013).

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