Suppressor of Hairless
Suppressor of Hairless [Su(H)]/Lag-1/RBP-Jkappa/CBF1 is the only known transducing transcription factor for Notch receptor signaling.
Su(H) has three distinct functions in the development of external mechanosensory organs in Drosophila: Notch-dependent transcriptional activation and a novel
auto-repression function (both of which direct cell fate decisions), and a novel auto-activation function required for normal socket cell differentiation. This third phase
of activity, the first known Notch-independent activation function for Su(H) in development, depends on a cell type-specific autoregulatory enhancer that is active
throughout adult life and is required for proper mechanoreception. These results establish a direct link between a broadly deployed cell signaling pathway and an
essential physiological function of the nervous system (Barolo, 2000).
It is concluded that Su(H), a core component of the ubiquitous N signaling pathway, is recruited -- via a discrete transcriptional enhancer module in the Su(H) gene itself -- for a fundamental differentiative role specifically in the socket cell. It is suggested that the autoregulatory activity of Su(H) in the socket cells of external sensory organs represents an unusually direct link between initial cell fate specification and the physiological function of a differentiated cell type (Barolo, 2000).
In nearly all of the many developmental settings (both embryonic and post-embryonic) in which Su(H) acts as the key transducing transcription factor for the N pathway, Su(H) is expressed generally, at a similar low-to-moderate level, in all cells of each tissue in which it acts. The discovery that Su(H) transcript and protein levels are greatly elevated specifically in the socket cells of all external sensory organs of the larval and adult PNSs posed two questions. (1) What is the developmental function of this elevated expression, given that the much lower Su(H) protein levels in all other cell types of the fly are sufficient for transduction of the N signal? (2) How is this cell type-specific elevation of Su(H) transcript and protein accumulation achieved (Barolo, 2000)?
This study of the transcriptional regulation of Su(H) has identified two distinct cis-regulatory modules: a promoter-proximal region that drives moderate general and maternal Su(H) expression, and a downstream enhancer, the ASE, which is responsible for strong socket cell-specific transcriptional activation. Because these two regulatory modules are physically separable, the socket cell activation of Su(H) could be selectively eliminated by deleting or mutating the ASE, while retaining the general and maternal Su(H) expression that is essential for viability and fertility. The loss of socket cell-specific Su(H) activation has no significant effect on N-mediated specification of the socket cell fate, or on any other cell fate decision. Since Su(H) is genetically required for socket cell fate determination, it is concluded that moderate levels of Su(H) protein are both necessary and sufficient for transduction of the N signal in the socket cell (and all other N-responsive cell types of the fly) (Barolo, 2000).
The characteristic high level of Su(H) in the socket cell is not only dispensable for the initial specification of the tormogen fate -- several major aspects of tormogen differentiation, such as significant cell growth, envelopment of neighboring sensory organ cells, and generation of a cuticular socket structure, also proceed normally in its absence. However, in other respects, autoregulation-deficient tormogens appear to be defective, leading to a failure of mechanoreception. Taken together, these results support a relatively simple developmental model in which the moderate levels of Su(H) protein present in the newly born tormogen are sufficient to transduce the incoming N signal and implement the socket cell fate, while socket cell-specific upregulation of Su(H), initiated in response to N signaling, reflects a specific differentiative role for Su(H) in the socket cell that is required for normal mechanoreception (Barolo, 2000).
Socket cell-specific transcriptional elevation of Su(H) expression depends on direct auto-activation through eight Su(H) binding sites in the ASE. This is the first known autoregulatory activity for Su(H) in any species. It is suggested that the ASE-mediated Su(H) autoregulatory loop is initiated during the socket-shaft cell fate decision in the early pupa by the direct binding of NIC/Su(H) activating complexes. Maintenance of this loop does not require continued N signaling activity. When does Su(H) auto-activation become N signal-independent? Shifting Nts1/Nnull flies to the restrictive temperature after 72 hr of pupal development at 18°C has no detectable effect on either the cuticular morphology of the socket or ASE activity; since temperature-shifting 36 hr (at 18°C) pupae can inhibit both the socket fate and ASE expression, it can be estimated that Su(H) auto-activation becomes signal-independent within 36 hr (at 18°C) after the birth of the tormogen (Barolo, 2000).
It is conceivable that NIC generated during pupal-stage N signaling perdures in the tormogen and acts as a coactivator throughout adult life. While this remains a formal possibility, it would require that NIC molecules in sufficient numbers to strongly activate Su(H) persist in the tormogen for several weeks. At present, the notion that Su(H) utilizes a distinct coactivator in the adult tormogen is favored (Barolo, 2000).
CBF1, a mammalian Su(H) homolog, is well-documented as a transcriptional repressor. Two vertebrate corepressor proteins, Silencing Mediator for Retinoid and Thyroid receptor (SMRT) and CBF1 Interacting coRepressor (CIR), have been shown to act as bridges between CBF1 and a histone deacetylase (HDAC) complex, which exerts transcriptional repression through chromatin remodeling. Studies of CBF1 function in vertebrate cells have led to a 'transcriptional switch' model in which (1) CBF1 directly represses target genes by recruiting HDAC and (2) NIC (or the viral protein EBNA-2) binds to CBF1, displacing the corepressor complex and acting as a transcriptional coactivator. Mutating the autoregulatory Su(H) binding sites in the Su(H) gene causes a gain-of-function cell fate conversion phenotype in which shaft cells inappropriately assume the socket cell fate. This result, in which the loss of Su(H)-mediated repression leads to a cell fate transformation, lends support to the 'transcriptional switch' model for Su(H)/CBF1 activity, and suggests that this model applies to Drosophila Su(H), not only in gene activation at cell boundaries, but also in N-mediated asymmetric cell fate decisions (Barolo, 2000 and references therein).
When all
eight Su(H) sites in the ASE have been mutated, the enhancer (ASEm) is no longer active in adult socket cells. One aspect of ASEm-GFP expression is potentially quite informative with respect to ASE regulation. ASEm is active in the tormogen in the early pupa, although its expression is substantially weaker than that of the wild-type enhancer, and unlike the wild-type ASE it is not active in adult tormogens. This early activity of the mutant enhancer indicates that other transcriptional activator(s), in addition to Su(H), bind directly to the ASE. These activator(s) are evidently present or active only in the socket and shaft cells, since these are the only cells in which ASEm-GFP is expressed. For the sake of brevity, the activity of this ASE binding factor or factors will be referred to as 'A', with the understanding that 'A' may stand for either expression or activity modulation (e.g., by phosphorylation) of one or more transcriptional activators. The activity 'A,' then, is sufficient to activate ASEm in the socket and shaft cells, but only during pupal stages. This provides an important clue to the necessity for Su(H) auto-repression. If 'A' is sufficient to activate Su(H) in the developing shaft cell (and perhaps to initiate a positive autoregulatory loop), the ASE must somehow be repressed in that cell in order to prevent inappropriate Su(H) activation. However, this repression must not occur in the socket cell, where Su(H) auto-activation is necessary for proper differentiation. Hence Su(H), which acts as a repressor only in the absence of N signaling, is an ideal repressor for the Su(H) ASE (Barolo, 2000).
The results obtained in this study lead to the following model for Su(H)-mediated transcriptional regulation during mechanosensory bristle development. Following the division of the precursor cell pIIA to give rise to the presumptive trichogen and tormogen, both daughter cells express the ligand Dl and the receptor N. However, the more anterior sister cell inherits from pIIA the N pathway inhibitor protein Numb, while the more posterior sister does not. In the absence of activated N (NIC), Su(H) in the anterior cell acts as an auto-repressor via the ASE, preventing activation of Su(H) by the activity 'A.' The posterior sister cell, however, contains nuclear NIC, which acts as a coactivator for Su(H) and induces the expression of socket-specific Su(H) target genes, including Su(H) itself. While this Su(H) auto-activation loop is initiated by N signaling, it is a consequence, rather than a determinant, of the socket cell fate. In the absence of N-stimulated activation of Su(H) target genes (and in the absence of N-dependent activities that repress the trichogen differentiation program), the anterior cell adopts the shaft cell fate. In the socket cell, Su(H) auto-activation becomes N signaling-independent during the course of pupal development, perhaps through an interaction with an alternative coactivator. By the time of eclosion of the adult fly, both Su(H) and 'A' are required to activate the ASE, but neither is sufficient. Su(H) auto-activation continues in the socket cell throughout pupal and adult life, where it is essential for specific aspects of late tormogen differentiation and for normal mechanoreception in the adult PNS (Barolo, 2000).
Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. dBre1 has been identified as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. These results indicate that histone modification is critical for the transcription of Notch target genes (Bray, 2005).
The lethal allele E132 was fortuitously identified among a collection of mutants that modify the wing notching phenotype caused by Armadillo depletion. Genetic mapping of the lethality associated with E132 placed this at 64E8, and it was found to be allelic to an existing mutation, l(3)01640, caused by the P element insertion P1541. Using plasmid rescue of the P element, the site of insertion was localized to the first intron of the open reading frame CG10542, which encodes a predicted protein of 1044 amino acids. The insertion site is 48 nucleotides upstream of the translation initiation codon. Precise excision of P1541 restores viability, confirming that the P element insertion and, by inference, E132 are lethal alleles of CG10542. In support of this, ubiquitous overexpression of the full-length protein encoded by CG10542 rescues the lethality of E132 or P1541 mutant embryos and sustains development to give essentially normal adult flies (with a few minor defects including slightly reduced bristles). CG10542 encodes a conserved protein with close relatives in mammals, C. elegans, plants, and fungi. The Drosophila protein has been named dBre1, after its relative Bre1p in the yeast S. cerevisiae (Bray, 2005).
The hallmarks of the Bre1 proteins are a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by ~15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p. RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (ΔRING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Bray, 2005).
To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-ΔRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a ΔRING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).
To investigate the role of dBre1 in the fly, homozygous mutant clones were generated in the imaginal disc precursors of the adult structures. Surprisingly, it was found that the majority of defects were similar to those caused by defects in Notch signaling. Thus, adult flies bearing E132 or P1541 mutant clones show notches in the wing margin and aberrant spacing of wing margin bristles, wing blistering and vein defects, fusions of leg segments, and loss of notal bristles and rough eyes. Most of these phenotypes are characteristic of reduced Notch signaling and are distinct from those produced by loss-of-function of other signaling pathways, such as Wingless, Dpp, or Hedgehog signaling that also operate during imaginal disc development. The phenotypic data suggest therefore that dBre1 has a role in promoting Notch signaling (Bray, 2005).
To confirm this, the expression of Notch target genes was examined in dBre1 mutant clones. Since dBre1 mutant clones are considerably smaller than their matched wild-type twin clones, the Minute technique was used to compensate for the growth defect of the mutant clones. In wing imaginal discs, cut and Enhancer of split [E(spl)] are expressed along the prospective wing margin, and their expression depends directly on Notch signaling. Cut expression is absent in large E132 mutant clones, and is lost (3/11) or reduced (6/11) in most P1541 mutant clones. Likewise, E(spl) expression is lost cell autonomously from all E132 mutant clones in the wing. Conversely, expression of spalt, a target of Dpp signaling in the wing, is not reduced in P1541 and E132 mutant cells, indicating that the effects of dBre1 mutation are relatively specific. Similar results are obtained in the eye, where E(spl) expression is also disrupted in E132 clones. Expression in the neurogenic region at the furrow is lost, and elsewhere it is absent or severely reduced, except in the basal layer of undifferentiated cells where expression is independent of Notch. In addition, a derepression of the neuronal cell marker Elav was observed in eye disc clones. The latter indicates excessive neuronal recruitment due to diminished Notch-mediated lateral inhibition (note, however, that the phenotypes are not identical to those produced by complete absence of Notch, which in the eye results in loss of neuronal markers because Notch is needed to promote neural development by alleviating Su(H)-mediated repression. These results demonstrate that dBre1 functions in multiple developmental contexts and, specifically, that it is required for the subset of Notch functions that involve Su(H)-dependent activation of Notch target genes (Bray, 2005).
To further confirm the importance of dBre1 during Notch signaling, it was asked whether any genetic interactions could be detected between overexpressed dBre1 or ΔRING and mutations in Notch (N) or its ligand Delta (Dl). Indeed, overexpression of either protein in the wing disc results in adult phenotypes. In each of 5 ΔRING-expressing lines, mild if consistent mutant phenotypes were observed in both males and females, namely upward-curved wings (due to stronger expression in the dorsal wing compartment), tiny vein deltas, and a significant decrease in wing size. These defects are more severe after overexpression of ΔRING in dBre1 heterozygotes, indicating that ΔRING acts as a weak dominant-negative. Consistent with this, excess ΔRING significantly enhances the phenotypes of N/+ and Dl/+ heterozygotes, resulting in increased vein thickening and additional vein material and, in the case of N/+, also in more frequent wing notching. These genetic interactions support the link between dBre1 and Notch signaling (Bray, 2005).
Excess full-length dBre1 in wing discs causes vein defects whose strength, however, varies considerably between different dBre1-expressing lines, and between males and females (probably because the ms1096.GAL4 driver produces higher expression levels in males). In most lines (4/6), vein thickening and additional vein material were observe only in males, while female wings appear normal. These vein defects in male wings are suppressed to almost normal in dBre1 heterozygotes, suggesting that they are due to increased levels of functional dBre1 protein. The remaining 2 lines produce similar vein defects also in females. Unexpectedly, these defects are enhanced in N/+ and Dl/+ heterozygotes, suggesting that the overexpressed dBre1 interferes with Notch signaling, rather than enhancing it as might have been expected. This anomalous result could be explained if dBre1 is part of a multiprotein complex, in which case its overexpression might interfere with the function of this complex by titrating one of its components. Nevertheless, the genetic interactions between overexpressed dBre1 and Notch and Delta further underscore the link between dBre1 and Notch signaling (Bray, 2005).
To test whether dBre1 directly influences Notch-dependent transcription, Drosophila S2 cells were transfected with Flag-tagged or untagged dBre1, and the activity of a Notch-specific reporter containing 4 Su(H) binding sites (NRE, a luciferase derivative of Gbe+Su(H)m8) was measured in the presence or absence of low levels of NICD. As a control, a reporter was used with mutant Su(H) binding sites [NME, or Gbe+Su(H)mut]. These experiments reveal a significant stimulation of the NRE reporter by dBre1, especially in the presence of NICD. The degree of stimulation is similar to that observed when the ubiquitin ligase Hdm2 is added to transcription assays of Tat activity. dBre1 also elicits a slight stimulation of NME. The fact that overexpressed dBre1 has stimulatory effects on Notch in the transfection assays but not in imaginal discs presumably reflects differences either in the levels of dBre1 or in the amounts of other limiting factors in the two cell contexts. Nevertheless, the transfection assays reveal an intrinsic potential of dBre1 in stimulating the transcription mediated by Su(H) and its coactivator NICD (Bray, 2005).
All these results point to a role of dBre1 in promoting Notch signaling. Since other ubiquitin ligases have been shown to influence the levels of specific protein components of the Notch pathway, whether there were any alterations to Notch, Delta, or Su(H) levels in dBre1 mutant clones was investigated. While there are no detectable changes in Notch or Delta staining in dBre1 mutant cells, the levels of Su(H) staining are enhanced slightly but consistently, and cell autonomously, in mutant clones of both dBre1 alleles, regardless of the location of these clones within the disc. This is also obvious in clones induced early in larval development in a non-Minute background in which the mutant dBre1 clones remain small. As an aside, these clones reveal that individual dBre1 mutant cells are enlarged, reminiscent of the yeast bre1p mutant which also shows a 'large cell'phenotype. This phenotype has not been observed in cells lacking Notch signaling, so this aspect of dBre1 function appears distinct from its role in the Notch pathway, and suggests that there are additional molecular targets. Nevertheless, the elevated levels of Su(H) in the dBre1 mutant clones identify Su(H) as one molecular target of dBre1 and suggest that, in the wild-type, dBre1 may expose Su(H) to ubiquitin-mediated degradation. The effects on Su(H) are consistent with the cell-autonomous action of dBre1 on Notch target gene expression, but the fact that removal of dBre1 has a stabilizing effect on Su(H) appears to contradict its stimulating effect on Notch-dependent transcription. Since Su(H) functions as both a repressor and an activator, this may be explained if loss of dBre1 specifically stabilizes the repressor complex. Alternatively, the effect of dBre1 mutations on Su(H) may reflect an indirect bystander activity of dBre1 (Bray, 2005).
Finally, it was asked whether dBre1 has a similar molecular function as its relative yeast Bre1p. The latter is required for the monoubiquitination of histone H2B, which is a prerequisite for the subsequent methylation of histone H3 at K4 by SET1-containing complexes. H3K4 methylation appears to be a chromatin mark for transcriptionally active genes, and yeast bre1p mutants show defects in the transcription of inducible genes that have been ascribed to the lack of H2B ubiquitination and H3K4 methylation at the promoters of these genes. Since there are no in vitro assays for H2B ubiquitination and no antibodies that detect this modified form of H2B, effects of dBre1 mutations on the linked H3K4 methylation were investigated. Wing discs bearing dBre1 mutant clones were stained with an antibody specific for trimethylated H3K4 (H3K4m). This revealed a significant reduction of H3K4m in P1541 mutant clones. More strikingly, in clones of the stronger E132 allele, H3K4m is barely detectable. In contrast, staining of these clones with an antibody against H3K9m does not show any changes in the mutant territory, indicating that the effect in dBre1 mutant clones on the methylation of H3K4 is relatively specific. It is noted that, in wild-type wing discs, there is slight modulation of trimethylated H3K4, with higher levels at the dorsoventral boundary where Notch is activated. However, Notch mutant cells retain robust H3K4m staining, although occasionally show slightly lowered levels compared to adjacent wild-type cells. Thus, the reduced H3K4m staining in dBre1 mutant cells is primarily due to an activity loss of dBre1 rather than due to loss of Notch signaling. Based on its effects on tri-methylated H3K4, it is concluded that dBre1 is indeed the functional homolog of yeast Bre1p. Furthermore, it appears that the activity of dBre1 is essential for the bulk of trimethylated H3K4 in imaginal disc cells (Bray, 2005).
In yeast, H2B ubiquitination and H3K4 methylation are associated with sites of active transcription, but the only identified natural target gene is GAL1. In Drosophila, the target genes of dBre1 evidently include genes regulated by Notch, given the requirement of dBre1 for their transcription. It is therefore conceivable that Su(H) may have a role in targeting dBre1 to their promoters (although it was not possible to detect direct binding or coimmunoprecipitation between dBre1 and Su(H). It is puzzling that dBre1 has a slight destabilizing effect on Su(H), despite being an activating component of Notch signaling. It is believed that this could be a bystander effect of dBre1: evidence suggests that the Bre1p-mediated monoubiquitination of H2B leads to a transient recruitment of proteasome subunits to chromatin, and that the subsequent methylation of H3K4 depends on the activity of these proteasome subunits. Their transient presence at specific target genes may have a destabilizing effect on nearby DNA binding proteins, and the mildly increased levels of Su(H) in dBre1 mutant cells could therefore reflect a failure of proteasome recruitment due to loss of H2B monoubiquitination (Bray, 2005).
Perhaps the most interesting implication of the results is that the dBre1-mediated monoubiquitination of H2B and methylation of H3K4 may be critical steps in the transcription of Notch target genes. Indeed, it appears that the Notch target genes belong to a group of genes whose transcription is particularly susceptible to the much reduced levels of H3K4m in dBre1 mutant cells. Based on the dBre1 mutant phenotypes, there are likely to be other genes in this group, including for example genes controlling cell survival and cell size. Nevertheless, it would appear that the transcription of Notch target genes is particularly reliant on the activity of dBre1. Other examples are emerging where the transcriptional activity of a subset of signal responsive genes is particularly sensitive to the function of a particular chromatin modifying and/or remodelling factor. This sensitivity presumably reflects the molecular mechanisms used by signaling pathways to activate transcription at their responsive enhancers. Understanding why Notch-induced transcription is particularly susceptible to loss of dBre1 function will require knowledge of these underlying molecular mechanisms (Bray, 2005).
The consensus binding sequence for Su(H) C/T GTGG/AGAAC/A mediates transcriptional activation of the Enhancer of split complex triggered by Notch signaling. Mutants in Su(H) fail to express m5 and m8 genes from the Enhancer of split complex (Lecourtois, 1995) (Bailey,1995). Enhancer of split complex genes regulate proneural genes achaete and scute.
Notch signal transduction appears to involve the ligand-induced
intracellular processing of Notch, and the formation of a processed
Notch-Suppressor of Hairless complex that binds DNA and activates the
transcription of Notch target genes. This suggests that loss of either
Notch or Su(H) activities should lead to similar cell
fate changes. However, previous data indicate that, in the Drosophila blastoderm embryo, mesectoderm specification
requires Notch but not Su(H) activity. The
determination of the mesectodermal fate is specified by Single-minded
(Sim), a transcription factor expressed in a single row of cells
abutting the mesoderm. The molecular mechanisms by which the
dorsoventral gradient of nuclear Dorsal establishes the single-cell
wide territory of sim expression are not fully understood. Notch activity is required for sim
expression in cellularizing embryos. In contrast, at this stage,
Su(H) has a dual function. Su(H) activity is required to up-regulate sim expression in the mesectoderm, and to
prevent the ectopic expression of sim dorsally in the
neuroectoderm. Repression of sim
transcription by Su(H) is direct and independent of Notch
activity. Conversely, activation of sim transcription by Notch
requires the Su(H)-binding sites. Thus, Notch signaling appears to
relieve the repression exerted by Su(H) and to up-regulate sim
transcription in the mesectoderm. A model is proposed in which
repression by Su(H) and derepression by Notch are essential to allow
for the definition of a single row of mesectodermal cells in the
blastoderm embryo. This is the first demonstration of the functional switch for Su(H) from a repressor to an activator (Morel, 2000).
To gain insight into the molecular mechanisms by which Su(H) and
Notch regulate sim expression, an examination was carried out to see whether Su(H) regulates sim expression in a direct manner. The regulatory elements necessary for mesectodermal expression of sim are contained within a 2.8-kb genomic DNA region. Sequence analysis has
identified 10 putative Su(H)-binding sites, with 6 of these exactly
matching the GTGRGAA consensus binding sites (Su4, Su5, Su7, Su8, Su9, and Su10). In gel shift experiments, Su(H)
binds strongly to oligonucleotides corresponding to each of these sites. Two additional sites, Su2 and Su6, match the consensus RTGRGAR that accomodates
nearly all sites that have been shown to bind Su(H) in vitro. These two sites bind weakly to Su(H), both
in direct binding assays and in competition
experiments. The ability of two noncanonical sites, Su1 and Su3, to bind Su(H) in vitro was also examined. Both Su1 and Su3 bind weakly to Su(H). Other sequences that differ
from the RTGRGAR at a single position are not known to bind Su(H) in
vitro. Thus, the sim regulatory sequences contain at least 10 binding sites for Su(H). Eight of these sites are clustered in a 500-bp
region that contains functional binding sites
for Dorsal, Twist, and Snail. Moreover, the organization of this regulatory region has been
conserved throughout evolution between D. melanogaster and
D. virilis. Together, these data
strongly suggest that Su(H) regulates sim transcription
directly (Morel, 2000).
Su(H) not only mediates the Notch-dependent activation of sim transcription, but also acts as a transcriptional repressor. This latter conclusion is supported by
the following two findings: (1) a complete loss of Su(H) activity leads to weak ectopic expression of sim in the
neuroectoderm; (2) the deletion of all of the Su(H)-binding sites
from the sim regulatory region also results in ectopic
activation of the sim promoter in the ventral neuroectoderm.
In Notch mutant embryos, repression by Su(H) is observed not
only in the neuroectoderm, but also in the mesectoderm. Because
Su(H) is expressed maternally,
it is speculated that uniformly localized Su(H) might repress the
activation of sim transcription in all of the cells in which
Notch is not activated (Morel, 2000).
This study provides the first evidence that Su(H) can act as a
transcriptional repressor in Drosophila, and that its
repression activity is inhibited by the activation of the Notch
receptor. In mammals it has been suggested that the binding of
processed Notch to CBF1 competes with the binding of corepressors to
CBF1 to promote the formation of an activation complex. The results presented here suggest that Su(H) might mediate such a transcriptional switch at the sim promoter in mesectodermal cells (Morel, 2000).
This regulatory mechanism, in which transcriptional repression is
inhibited by a signaling input, may be a general feature of
Notch-mediated gene regulation. Consistent with this view, repression
by Su(H) might contribute to the difference seen between Notch
and Su(H) mutant cuticular phenotypes. Similarly, the
cuticular phenotype associated with a deletion removing all of the
bHLH-Enhancer of split genes, but not groucho, also
appears to be more severe than the one associated with a complete loss
of Su(H) function. Because the
bHLH-Enhancer of split genes are direct transcriptional
targets of Su(H) during neurogenesis, it has been suggested that Su(H) might also
act as a transcriptional repressor of the Enhancer of split genes. The finding that Su(H) can repress a Notch target gene
indicates that phenotypic differences between Notch and
Su(H) mutations do not necessarily imply that Notch signals in
a Su(H)-independent manner (Morel, 2000).
How is a single-cell wide territory of sim expression
established on the basis of the nuclear gradient of Dorsal? The data presented here, together with previous studies, suggest the following model. In the mesoderm, transcriptional activation of sim by Dorsal and Twist is inhibited by Snail. Whether Su(H)
and/or Notch play any role in these cells is not known.
In more dorsal cells that do not accumulate Snail, it is proposed that
positive regulation of sim by low levels of Dorsal and Twist
is antagonized by Su(H). However, in cells bordering the mesoderm,
negative regulation by Su(H) would be relieved locally by Notch
signaling. This would lead to the specific expression of sim
in these cells, which will then form the mesectoderm (Morel, 2000).
An important feature of this model is that Notch signaling overcomes
repression by Su(H) only in the single row of cells abutting the
mesoderm. One possible explanation for this is that Notch participates
in the contact-dependent reception of a mesodermal signal. Results from
nuclear transplantation experiments support the existence of a
mesodermal signal. When transplanted into
snail/twist double mutant embryos that do not
express sim, wild-type nuclei can induce the expression of
sim in neighboring mutant cells. This
result suggests that, in wild-type embryos, mesodermal cells may
produce an inductive signal that activates sim transcription
in the mesectoderm. Although the molecular nature of this signal is not
known, it is speculated that this mesodermal signal might participate in
the activation of Notch (Morel, 2000).
Consistent with the view that Notch is specifically activated in
ventral cells, changes in the subcellular distribution of both Notch
and Delta have been observed ventrally in stage 5 embryos. (1) Lower
levels of Notch are found in ventral cells as the ventral furrow forms. (2) In cellularized embryos, Delta is found at
the cell membrane, except in ventral cells, in which it predominantly
accumulates in vesicles. Both down-regulation of
Notch and vesicular accumulation of Delta are consistent with Delta
activating Notch in ventral cells in stage 5 embryos (because Snail
represses sim transcription, activation of Notch in the
mesoderm may have no effect on sim transcription). It will
thus be of interest to determine whether these changes in the
subcellular distribution of Notch and Delta can be observed in both
mesodermal and mesectodermal cells, but not in the more dorsal
neuroectodermal cells (Morel, 2000).
In conclusion, repression by Su(H) can be viewed as a refining
mechanism ensuring that Notch target genes are expressed only in cells
reaching a high threshold of Notch activation. In the early embryo,
repression of sim expression allows for the definition of a
single row of mesectodermal cells. In these cells, a high level of
Notch activity might be induced by a juxtacrine (contact-dependent) inductive signal produced by the mesoderm. In view of this hypothesis, the sharp mesodermal boundary defined by snail expression
would be shifted dorsally by one cell, thereby defining a single row of
mesectodermal cells (Morel, 2000).
The activity of Notch is required
for the transcriptional activation of the sim gene in the
mesectoderm, and Su(H) directly regulates sim expression.
However, both the sim gene and the
simmut-lacZ construct that does not respond to activated
Su(H) are expressed in mesectodermal cells in the complete absence of
Su(H) activity. These results
might suggest that Notch signals, at least in part, in a
Su(H)-independent manner to activate sim expression in the
mesectoderm. Alternatively, the observation that Su(H) acts to repress
sim expression raises the possibility that Notch might be
required to antagonize repression by Su(H). To distinguish between
these two possibilities, the expression of
simmut-lacZ was examined in Notch mutant embryos. simmut-lacZ is expressed at a low level
both in the mesectoderm and ectopically in the dorsal neuroectoderm. This pattern is very similar to that observed for
simmut-lacZ in wild-type embryos, and dramatically
differs from the complete loss of sim-lacZ expression seen in
Notch mutant embryos. This shows that the Su(H)-binding sites
are required to repress sim transcription in the mesectoderm
as well as in the neuroectoderm in the absence of Notch signaling.
Furthermore, this demonstrates that repression of sim
expression by Su(H), both in ventral neuroectodermal and mesectodermal
cells, does not require Notch activity. It is concluded that Su(H)
acts as a Notch-independent repressor. Thus, no evidence has been found for a
Su(H)-independent function of Notch in the regulation of sim expression (Morel, 2000).
It has been suggested that wingless expression at the dorsal-ventral boundary of the wing disc depends on a signal from dorsal to ventral cells mediated by Serrate and Notch. Wingless expression is lost from the wing margin and the size of the wing is significantly reduced when Notch activity is removed from the third instar larva using a temperature sensitive allele of Notch. In addition, clones of cells mutant for Serrate can cause extensive non-autonomous loss of wing tissue, but only when the clone includes the cells that abut the dorsal compartment boundary. Therefore, it is likely that wingless is regulated by the Notch pathway acting through Suppressor of Hairless (Diaz-Benjumea, 1995).
Several different mechanisms have been proposed to account for the activation of Su(H) by Notch.
To further investigate how Su(H) activity is regulated, misexpression assays were used with wild-type Su(H) and with
modified forms of Su(H) containing either a nuclear localization signal [Su(H)NLS], a transcriptional activation domain
[Su(H)VP16], or a deletion of the domain required for interaction with the antagonist Hairless [Su(H)DH]. Only Su(H)VP16
is able to mimic Notch activation effectively in the Drosophila wing, in agreement with the model that Notch activity
normally confers coactivator function on Su(H). Neither nuclear localization nor elimination of Hairless binding is
sufficient for activation. The phenotypes produced by overexpression of Su(H)wt and Su(H)NLS indicate a mixture of both
increased and reduced Notch pathway activity and point to a role for Su(H) in both activation and repression of gene
expression, as has been proposed for the mammalian homolog CBF1. Some phenotypes are equivalent to Notch
loss-of-function, with wing-nicks and inhibition of a subset of target genes, which is most consistent with the ectopic
proteins displacing a Su(H)-coactivator complex. Conversely, other phenotypes are equivalent to Notch gain-of-function,
with wing-overgrowths and ectopic target-gene expression. These effects can be explained by the ectopic Su(H)/Su(H)NLS titrating a repressor complex. The wing-overgrowth phenotype is sensitive to the dose of Hairless and the phenotypes produced by coexpressing Su(H) and Hairless suggest that Hairless could form a component of this repressive complex (Furriols, 2000).
The phenotypes produced by misexpressing Su(H)wt and
Su(H)NLS appear to combine activation and repression of
Notch activity. To confirm whether this interpretation is
correct, the effects on genes whose expression
at the dorsal/ventral boundary is dependent on Notch were analyzed. Two
assays used fragments that are directly responsive to Su(H)
and Nicd: mbeta1.5 (a fusion between the E(spl)mbeta regulatory
sequences and lacZ) and vgBE-lacZ (a
fusion between the vestigial boundary enhancer and lacZ). Expression of wingless and the entire vestigial gene, which may involve
indirect as well as direct regulation by the Notch pathway, were also examined. All are ectopically activated by Su(H)VP16, consistent with it mimicking the effects of Nicd. As anticipated, the wing-nick phenotypes produced by
the modified Su(H) proteins in combination with ptc-Gal4
correlate with a reduction in the levels of mbeta1.5 and
wingless expression at the d/v boundary. mbeta1.5
appears to be more sensitive and is strongly repressed by
all three proteins, with Su(H)NLS the most
effective as suggested by the wing-nicking phenotypes. The effects on wingless were milder and only Su-(H)NLS strongly represses expression.
In contrast, vgBE-lacZ shows a very different response
and is ectopically activated by both Su(H)wt and Su-(H)NLS. The activation of vgBE-lacZ is also observed when the levels of misexpressed proteins are lower (using dpp-Gal4) although in
this combination Su(H)NLS and Su(H)wt still repress
mbeta1.5. The two enhancers therefore appear
intrinsically different in the way they respond to Su(H),
suggesting that their regulation may involve different
thresholds of activating and repressing Su(H) containing complexes (Furriols, 2000).
The mixed loss and gain of Notch function phenotypes
produced in the wing by ectopic expression of Su(H)wt and
Su(H)NLS suggests that Su(H) has a dual function, acting in
some contexts as an activator and in others as a repressor.
The simplest model is that Su(H) can exist in at least
two complexes -- one where it interacts with a coactivator(s)
and the other where it interacts with a corepressor(s). In cells
where there is no/low Notch signaling the primary function
of Su(H) would be to keep the target genes repressed by interacting with a corepressor complex. One of these corepressors could be Hairless; another could be the HDAC complex described in vertebrates. In contrast, in the cells where Notch is active, Su(H) would be complexed with coactivator(s) (e.g., Nicd so that the transcription of Notch target genes would be initiated). Depending on the relative levels or activity of the components, the equilibrium would shift in favor of one or the other complex (Furriols, 2000).
The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to
different signaling pathways. The obligate integration of selector and
signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory
networks during development (Guss, 2001).
Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).
These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).
Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).
Notch-dependent activation of wingless, cut and vestigial at the wing margin depends on the activity of Suppressor of Hairless. Su(H)-mutant cells lose expression of the vestigial early enhancer, of wingless and of cut in a cell autonomous manner. Clones of Su(H)-mutant cells cause loss of wing tissue and scalloping of the wing, but only in Notch mutant clones at the D/V boundary. vestigial expression at the D/V boundary does not depend on wingless, since misexpression of wild-type wg cDNA, which results in wing margin bristles, does not cause an expansion of vestigial expression. Likewise, wingless expression does not depend on an early function of vestigial. Both Notch and wingless cooperate to activate cut at the D/V boundary (Neumann, 1996).
The Notch receptor mediates cell interactions controlling the developmental fate of a broad spectrum of undifferentiated cells. By modulating Notch signaling in specific precursor cells during Drosophila imaginal disc development, it has been demonstrated that Notch activity can influence cell proliferation. The activation of the Notch receptor in the wing disc induces the expression of the wing margin patterning genes vestigial and wingless, and strong mitotic activity. However, the effect of Notch signaling on cell proliferation is not the simple consequence of the upregulation of either vestigial or wingless. On the contrary, Vestigial and Wingless display synergistic effects with Notch signaling, resulting in the stimulation of cell proliferation in imaginal discs (Go, 1998).
To explore the consequences of Notch signaling modulation during Drosophila development, the UAS-GAL4 system was used. Loss-of-function phenotypes were elicited through the expression of either a truncated, dominant negative form of the Notch receptor (d.n.N) lacking the intracellular domain, or the Hairless
(H) protein, a negative regulator of Notch signaling. Gain-of-function phenotypes were induced by expressing a constitutively activated form of the Notch receptor (act.N) (Go, 1998).
To examine the link between the H misexpression phenotypes and Su(H)-dependent Notch activity, transgenic animals were generated carrying a lacZ reporter construct driven by the fusion between multimerized Su(H)-binding sites and an E(spl)m gamma promoter, a known Su(H) target. This construct consists almost exclusively of engineered Su(H)-binding sites. In a cell culture based reporter, the expression from the reporter construct is induced by the simultaneous expression of Su(H) and act.N, while the expression of any one construct alone fails to induce transcription. Strong lacZ expression is detected in the posterior part of the eye disc of late third instar transgenic larva. This expression is effectively suppressed by misexpression of H using the GAL4 line T113 and results in small eye discs, indicating that H overexpression can suppress Su(H)-dependent Notch signaling in vivo. The size of the eye is significantly affected and, in extreme cases, the eye is missing. In addition to small eyes, small wings and halteres are observed as well as more typical Notch loss-of-function phenotypes, such as extra thoracic bristles. The 'small eye' phenotype induced by H expression is not associated with severe eye roughness. This 'small eye' phenotype, together with the wing and haltere abnormalities, is reminiscent of Serrate loss-of-function mutations. To further explore the possibility that the observed eye phenotype reflects Ser-dependent Notch signaling, the genetic interactions were examined with Beaded of Goldschmidt (BdG), a dominant negative mutation of Ser known to affect wing margin development. In combination with BdG, strong synergistic effects are observed displaying phenotypes characteristic of Ser, such as small eyes, wings and halteres. Therefore, H misexpression can mimic Ser loss-of-function mutations, raising the possibility that Ser/Notch signaling may control eye morphogenesis (Go, 1998).
To further investigate the role Notch signaling plays in morphogenesis, the H and d.n.N transgenes were expressed at the d/v compartment boundary of the wing disc using the vestigial-GAL4 driver. Misexpression of either H or d.n.N results in similar phenotypes, which range from wing margin notches to rudimentary wings. The effect of H misexpression can be suppressed by expressing act.N and vice versa. For example, the lethality associated with misexpression of act.N is suppressed by simultaneous expression of H. Conversely, the phenotypes elicited by H misexpression are largely suppressed by act.N. This mutual suppression is observed with other GAL4 lines as well. Given that the actions of act.N and H seem to be manifested through Su(H), it is likely that the mutual suppression of act.N and H is also mediated by Su(H). It is noteworthy that, even though both H and d.n.N act as antagonists of Notch signaling and the phenotypes associated with their expression are similar, their interactions with act.N are different. While act.N is an effective suppressor of the phenotypes induced by H misexpression, it fails to suppress the effects of d.n.N (Go, 1998).
The relationship between Notch signaling and the expression of vg and wg was examined, since the induction of both genes is considered to be essential for wing morphogenesis. When either d.n.N or H is misexpressed along the anterior/posterior (a/p) boundary using the ptc-GAL4 line, expression from the vg d/v boundary enhancer, as well as the wg enhancer, is effectively repressed near the intersection between the a/p and d/v boundaries. In contrast, the vg quadrant enhancer, which is normally silent at the intersection between a/p and d/v boundaries, is induced by the identical constructs. Essentially the opposite effect is observed when act.N is misexpressed, demonstrating that Notch signaling has opposite effects on two distinct enhancers of vg (Go, 1998).
The wing phenotypes elicited by misexpression of act.N are similar to those induced by Abruptex (Ax) mutations, which are Notch gain-of-function alleles associated with point mutations in the extracellular domain of the protein. A heteroallelic Ax combination results in the activation of Notch signaling and the expression of Notch downstream genes is induced. For instance, ectopic wg expression is found around the d/v boundary. Induction of the vg d/v boundary enhancer and repression of the vg quadrant enhancer around the d/v boundary are found, similar to the effect of expression of act.N. Activation of Notch signaling around the d/v boundary of the wing disc through either misexpression of act.N or the Ax mutations results in a substantial enlargement of the disc. BrdU incorporation experiments indicate that these phenotypes are associated with an elevated mitotic activity. BrdU incorporation is stimulated and is particularly obvious in the peripheral region of the wing pouch, suggesting that the periphery Is more responsive than other regions. Misexpression of act.N in other parts of the wing disc also results in the stimulation of mitotic activity. When act.N is expressed in the wing pouch, the disc grows in such a way that the characteristic folded structures of the wing pouch are pushed to the periphery. Conversely, the same structures are 'pushed' toward the d/v boundary when act.N is expressed in the periphery. When act.N is misexpressed in a discrete pattern in the periphery using the GAL4 line 766, a regional correspondence is observed between Notch signaling activation and high mitotic activity, demonstrating a local effect of Notch activity on cell proliferation in the periphery. However, as is particularly evident when the Notch receptor is activated around the d/v boundary, the region of Notch signaling activation does not coincide with the region of the highest mitotic activity. It is therefore concluded that the effect of Notch signaling on cell proliferation must be indirect (Go, 1998).
The effect of Notch activity on cell proliferation is not the simple consequence of vg induction Since vg is a direct target of Su(H)-dependent Notch signaling, it is possible that the mitogenic effect of Notch is mediated by the upregulation of vg. In this case, misexpression of Vg would be expected to result in phenotypes similar to those elicited by act.N. Misexpression of act.N in the dorsal side of the wing pouch, using the GAL4 line A9, induces expression from the vg d/v boundary enhancer as well as the wg enhancer The dorsal side of the wing pouch region appears enlarged. In contrast, when Vg is misexpressed in the same region, the dorsal side of the wing pouch becomes much smaller than the ventral side, while wg expression in the periphery of the dorsal side was suppressed. The loss of dorsal wing pouch induced by Vg misexpression is significantly rescued by expressing Wg simultaneously. This is consistent with the notion that the observed phenotype caused by misexpression of Vg is due to the repression of wg, whose expression in the wing pouch is more uniform at earlier stages. Misexpression of Wg alone in the dorsal side, unlike the misexpression of act.N, does not have a significant effect on cell proliferation in the wing pouch. These results indicate that the effect of act.N expression on mitosis is separable from vg induction. In addition, they indicate that Vg is capable of repressing wg expression in the wing pouch, but not at the d/v boundary (Go, 1998).
Misexpression of Vg compared to act.N has opposite effects in the wing disc. Thus, Vg misexpression in the wing disc induces wg downregulation and small discs. In contrast, misexpression of Vg in the eye discs upregulates wg and results in a clear enlargement of the discs, demonstrating that Vg can either repress or induce wg expression in a context-dependent manner. The observed context-dependent effect of Vg on wg expression raises the possibility that Notch signaling may be capable of modulating the way Vg affects wg expression. This is of particular interest in view of the possibility that Vg does not suppress wg expression at the d/v boundary because of the existing high level of Notch signaling activity. In fact, the simultaneous expression of act.N and Vg reveals a striking synergistic effect on cell proliferation. The most notable effects are in the eye discs, where tissue expressing the two proteins shows striking overgrowth associated with strong wg induction. The other discs are also clearly affected, displaying cellular overgrowth, but the effects are far less dramatic than the eye discs. This overgrowth phenotype is also evident when act.N and Vg misexpression are driven by dpp-GAL4, even though the synergistic effect is less dramatic. In contrast, the effect of misexpression of Vg with dpp-GAL4 on wg induction and cell proliferation in the eye discs is, in some cases, significantly suppressed by simultaneous expression of H. These experiments demonstrate that the proliferative potential of certain tissues can be modulated by the synergistic action of Notch with other genes. Moreover, they identify Notch signaling as an important factor in the way Vg affects wg expression and cell proliferation at the d/v boundary during wing morphogenesis (Go, 1998).
In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in imaginal
discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH)
transcriptional repressors, while 4 others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression
patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Strong
evidence is presented from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes are direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH
proteins Achaete and Scute. An extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such
dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. The very different wing imaginal disc expression patterns of
E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily conserved transcriptional enhancer elements that can confer these distinct
patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. The characteristic inactivity of the E(spl)mgamma enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity
of the N receptor. It is concluded that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their
transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in
which these modes of regulation are operative (Nellesen, 1999).
The Notch pathway plays a crucial and universal role
in the assignation of cell fates during development. In
Drosophila, Notch is a transmembrane protein that acts as
a receptor of two ligands, Serrate and Delta. The current
model of Notch signal transduction proposes that Notch is
activated upon binding its ligands and that this leads to the
cleavage and release of its intracellular domain (also called
Nintra). Nintra translocates to the nucleus where it forms
a dimeric transcription activator with the Su(H) protein. In
contrast with this activation model, experiments with the
vertebrate homolog of Su(H), CBF1, suggest that, in
vertebrates, Nintra converts CBF1 from a repressor into an
activator. The role of Su(H) in Notch
signaling during the development of the wing of
Drosophila has been assessed. The results show that, during this process,
Su(H) can activate the expression of some Notch target
genes and that it can do so without the activation of the
Notch pathway or the presence of Nintra. In contrast, the
activation of other Notch target genes requires both Su(H)
and Nintra, and, in the absence of Nintra, Su(H) acts as a
repressor. The Hairless protein interacts
with Notch signaling during wing development and
inhibits the activity of Su(H). These results suggest that, in
Drosophila, the activation of Su(H) by Notch involves the
release of Su(H) from an inhibitory complex, which
contains the Hairless protein. After its release Su(H) can
activate gene expression in the absence of Nintra (Klein, 2000).
Since Su(H) is expressed ubiquitously and continuously, the fact that Su(H) can promote transcription
without the presence of Nintra suggests that the activity of
Su(H) must be suppressed in the absence of Notch activation
during normal development. One possible mechanism for this
is the binding of an inhibitory factor. A candidate for this
function is the Hairless (H) protein, which has been shown to
interact with Su(H) and antagonize its DNA-binding activity. Furthermore, several reports show that H
antagonizes Notch signaling during adult PNS development. To test whether H is an
antagonist of Su(H) also during wing development, H mutant clones were induced in the wing pouch and it was asked whether
genes dependent only on Su(H) activity are expressed in these
clones. If H regulates the activity of Su(H), the removal of H
might lead to the activation of Su(H) and result in the
expression of its targets, e.g. the vestigial boundary enhancer (vgBE) and Serrate. Both are ectopically activated in H mutant clones.
The ectopic expression of the vgBE in the clones varies and is
strongest near the DV boundary. This graded
expression is possibly due to the requirement of a diffusible
factor coming from the DV boundary. One candidate for this
is Wg, which seems to be required for the proper expression
of the vgBE. The cells in the H mutant clones do not express
cut or wg, which are dependent on the presence of Nintra, suggesting that Notch is not activated in these clones (Klein, 2000).
The loss of H function seems to elicit Su(H)-dependent target
gene expression in the wing pouch, a region probably devoid
of Notch activity. This suggests that the inactivation of H is
sufficient to activate Su(H). To test further this conclusion, an examination was performed to see whether the activity of the vgBE is maintained in H
mutant wing pouches if Notch is concomitantly removed. For
this, Notch mutant clones were induced in H mutant wing discs. In H mutant wing pouches, weak ubiquitous
expression of the vgBE is observed throughout the whole area
of the wing, confirming the clonal analysis. vgBE is also active in several Notch mutant clones near
the DV and anteroposterior (AP) boundary, but the
activity is not maintained in all clones. One explanation for this
might be again the requirement of other so far unidentified
factors emanating from the two compartment boundaries. In
agreement with this, the vgBE enhancer has a late expression
domain along the AP boundary, suggesting an input from these
areas for its proper expression. However this domain is also dependent on Notch during normal
development. The removal of the Su(H)-binding site in the
enhancer leads to the loss of all expression domains in the wing
pouch, suggesting that Su(H) is required (Klein, 2000).
Therefore, the fact that the cells of several mutant clones do
express the vgBE suggest that the vgBE can be activated in the
complete absence of Notch activity and that the inactivation of
H is sufficient to activate Su(H). No activation of the vgBE was ever found
in Notch mutant clones induced in wild-type wing pouches, suggesting that during
wild-type development, the activity of Notch is
required to activate the vgBE. Hence, Notch
probably activates Su(H) through inactivation of H.
An examination was performed to see whether the degree of endogenous
Su(H) activation that results from the removal of H
is sufficient to elicit a biological effect. To assay this,
it was asked whether or not removal of H activity can
induce Su(H)-dependent development of the pouch in
wing discs in which Notch signaling is absent, such
as apterous and Presenilin mutant wing discs. Loss
of H function rescues the loss of wing development
of ap mutants: whereas ap mutants
have no wing pouch, ap;H double
mutants have large wing pouches with no margin
structures. The enlarged pouch of the
double mutant discs expresses spalt (sal) and the two
vg reporters, vgQE and vgBE, all of which are
expressed specifically in the wing pouch in a Notch/Su(H)-dependent manner and are not expressed in ap
mutants. In contrast, no wg expression
is induced in these double mutant discs,
suggesting that the observed rescue is likely to be due
to the activation of Su(H) in the double mutants. This
is strongly supported by the fact that Su(H);H double
mutants exhibit a small wing rudiment identical to
that of Su(H) mutants. Expression of UAS-vg by dpp-Gal4 in ap
mutant discs can recover the pouch-specific
expression domain of sal, suggesting that the activation of vg
expression by Su(H) is responsible for the recovered
sal expression in the ap;H double mutant wing discs.
Similar to overexpression of UAS-Su(H) in ap mutant
wing discs, the pouch in ap;H mutant discs develops
near the residual wg expression in the remaining
hinge. As
expected from the analysis of the wing discs, the
pharate adult ap;H double mutants have large wing
pouches, which are devoid of any margin like
structure such as innervated bristles (Klein, 2000).
The effects on wing development of removing H
in Psn mutants were examined. As in the case
of ap, loss of function of H effects a strong rescue
of the wing pouch in the Psn;H mutant discs in
comparison to the Psn mutant discs. However,
in this case, the morphology of the discs is more like wild type and, in contrast to ap;H mutant discs, the pouch
develops at its normal place. Closer
monitoring of double mutant discs reveals some expression of
wg and the vgBE along the DV boundary. This
suggests that, in contrast to the situation of ap mutants, in Psn
mutants, there is some activation of Notch and it seems that the
lack of H activity can enhance this residual signaling of Notch
at the DV boundary. This is remarkable considering that the
wing phenotype caused by the loss of Psn is stronger than that
caused by loss of Su(H) function.
Taken together, these results provide further evidence for a
positive transcriptional activity of Su(H). They further show
that H is an antagonist of Su(H) during early wing
development and that it suppresses the activity of Su(H) in the
absence of Notch signaling. The results also suggest that the
inactivation of H is sufficient to activate Su(H) and that the
activity of Notch is required to inactivate H during normal
development (Klein, 2000).
Overexpression of Su(H) leads to three
different responses: (1) activation, as is the case for vg, some
E(spl) genes, Dl and Ser; (2) inactivation, as shown for cut
and E(spl)m8; or (3) no effect, as is the case for wg. This
differential behavior is, at least in some cases, a consequence
of direct binding of Su(H) to the promoters: the vgBE as well
as the E(spl) genes contain Su(H)-binding sites to which Su(H)
binds; such sites are necessary for the
activation of these genes in vivo. Despite that, they react differently towards Su(H)
overexpression. Since E(spl)m8, which is suppressed by Su(H)
overexpression, can be activated by expression of Su(H)VP16
or Nintra, it is concluded that Nintra is required in addition to
Su(H) to activate E(spl)m8 expression. The results suggest that,
in this case, Nintra probably acts as an activation domain of a
dimeric transcription factor containing Su(H), as has been
proposed. From this, it
follows that Nintra might have two function during a Notch
signaling event: first it inactivates H, which leads to the release
of Su(H) and then, in some instances, it provides the
transactivation domain for free Su(H) to activate the expression
of target genes (Klein, 2000).
Flies carrying reporter lacZ constructs with up to 12 Su(H)-binding
sites do not display any activity in the wing disc. This suggests that Su(H) (even in association with
Nintra) is not sufficient to activate transcription and requires
other collaborating factors. It further suggests that, even in
promoters that can be activated by Su(H) in the absence of
Nintra, Su(H) probably interacts with other factors to promote
gene expression. This is confirmed by a study of the vgBE.
Although the Su(H)-binding site is absolutely necessary for its
activity, other sites are equally important. So far the factors that bind to these sites are not identified. The dependence of Su(H) on
these others factors is probably the reason for the differential
expression of Notch target genes in H and H/N mutant clones
that have been observed (Klein, 2000).
Recently it has been shown that Su(H) acts as a suppressor
of single minded transcription during the formation of the midline cells
in the embryonic central nervous system of Drosophila. This observation provides the first
evidence that Su(H), like its mammalian counterpart CBF1,
can act as a suppressor of transcription. The inactivation of the
cut and E(spl)m8 expression in absence of Nintra suggests that
Su(H) can act as a suppressor of gene expression also during
adult development and provides further evidence for a
suppressing activity of Su(H). However, this
suppression is context dependent and not a general feature of
Su(H). This context dependency might also exist for CBF1,
since only the reaction of a small number of genes towards its
activity has been tested so far and it is possible that some target
genes can be activated by CBF1 in the absence of Nintra in a
similar way, as has been shown for Su(H). In summary,
these results suggest that the consequence of the binding of
Su(H) to a promoter is dependent on its local architecture and,
therefore, Su(H) can at the same time activate and suppress
gene expression, like many other transcription factors.
The removal of both the maternal and zygotic expression
of H during embryogenesis seems to be of no consequence
for the embryo. Since
the overactivation of Notch/Su(H) signaling during
embryogenesis has deleterious consequences, this observation
contradicts the conclusion that H is required to inactivate
Su(H). However, the context dependency and differential
reaction of the target genes observed during wing development
offer two explanations for this discrepancy, without having
to postulate an unknown factor, which can functionally replace
H. First, it is likely that the interacting factors, which are
required for gene expression in concert with Su(H), are
different during embryogenesis and this could modulate the
responsiveness of the target promoters. This conclusion is
supported by the observation that the genes of E(spl)C,
although probably all requiring Su(H) for their expression, are
all very similarly expressed in the embryo, but their expression
pattern in the wing imaginal disc is very different. Another explanation is that the target promoters of binding Su(H)
during embryogenesis might be all of the type that require
the additional activity of Nintra. Therefore they would stay
inactive even in the presence of free Su(H) until Notch is
activated (Klein, 2000).
Many cell fate decisions in higher animals are based on intercellular communication governed by the
Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of
Hairless [Su(H)] mediate transcription of target genes. In Drosophila, the majority of Notch target
genes known so far is located in the Enhancer of split complex [E(spl)-C], encoding small basic
helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. The E(spl)-C contains three additional Notch responsive, non-bHLH genes: E(spl) region transcript m4 (m4) and malpha are structurally
related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or
maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are
unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide. The five genes described in this paper are arrayed between mbeta and m7, both coding for bHLH proteins. Two other bHLH genes, m3 and m5 are intermingled with the five. Bearded and M4 are 16% identical. Furthermore, in transcripts of both Brd and m4 there are three common regulatory sequence motifs within the 3' UTR. These are known as the 'Brd box', the 'GY box' and the 'K box'. As in m4, the sequence motif of the Brd box is found twice in the 3'-UTR of malpha mRNA at similar positions but without a GY box. None of the other four non-bHLH E(spl)-C genes contains either Brd or GY box. The K box appears to be more common. It is found twice in the 3'-UTR of malpha and once each in the 3' UTRs of m2 and m6 (Wurmbach, 1999).
malpha and m4 embyonic expression patterns are nearly indistinguishable, and appear very similar to those of E(spl)-C bHLH genes, particularly m5, m7 and m8. The expression patterns suggest that both genes are under the same regulatory conrol as are the differ E(spl) bHLH genes and thus, might serve a role in Notch mediated cell differentiation. Surprisingly, also m2 transcripts accumulate in a pattern reminiscent of the transcript distribution of E(spl) bHLH genes, although there are no structural similarites with either the bHLH or the m4/malpha genes. Therefore m2 might serve as a Notch target gene. Unlike the other E(spl)-C genes, the gene is expressed within neuronal cells in the embryo. m6 mRNA accumulates in the CNS, brain and PNS, and in imaginal tissues. m1 is expressed in the digestive tract. Su(H) is shown to be the transmitter of Notch signaling to malpha, m4 and m2. Thus there are three types of Notch responsive genes. The bHLH genes are represented by m8 and others. m4 and malpha
share structural similarity with Bearded. These Bearded family proteins share a presumptive basic amphipatic alpha-helical domain but differ with regard to other conserved sequence elements. m2, coding for a novel protein, represents the third class of Notch responsive genes (Wurmbach, 1999).
In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in
imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic
helix-loop-helix (bHLH) transcriptional repressors, while four others encode members of the Bearded family of small proteins. A striking diversity is observed
in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not
been elucidated. Strong evidence is presented from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily
responsible. Certain E(spl)-C genes are known to be direct targets of transcriptional activation both by the N-signal-dependent activator
Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Extensive sequence analysis of the promoter-proximal upstream
regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. The very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily
conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity
[each having three Su(H) and two proneural protein binding sites]. As originally defined by its structure in m4, m8 and mgamma, the Su(H) paired site (SPS) configuration consists of two high-affinity (YGTGRGAAM; M denotes A or C) Su(H) binding sites in an inerted repeat arrangement, with 300 bp between the first G of the two sites. In addition, the 'Y' of the upstream site is T, while that of the downstream site is C. Finally, the sequence between the two Su(H) sites includes the hexamer GAAAGT or its complement ACTTTC. The SPS motifs of m4 and mgamma in Drosophila hydei are remarkably conserved. A 43-nt block in m4 SPS contains the entire SPS motif and shows only a single varient position. Also all four bHLH activator binding sites in the two genes are conserved. A putative bHLH repressor binding site defined by the N box consensus sequence CACNAG does not appear to be as conserved as sites defined by CACGYG that have been shown to bind these proteins with high affinity. Thus of four distinct CACGYG sites (one in mgamma and three in m4 ), two are conserved, while apparently only one of seven distinct N box sites (three in mgamma and four in m4) is conserved. Conserved transcription factor binding sites are often accompanied by strongly conserved flanking sequences. It is concluded that the distinctive expression patterns of
E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct
proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative (Nellesen, 1999).
The product of the Drosophila gene Serrate acts as a short-range signal during wing development to
induce the organizing center at the dorsal/ventral compartment boundary, from which growth and
patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the
dorsal compartment of the wing disc have recently been confined to a genomic fragment in the
5'-upstream region of the gene (from -8 to -18 kb). This fragment, termed the dorsal wing regulator or DWR, responds to various
positive and negative inputs required for the early Serrate expression. Activation and maintenance
of expression in the dorsal compartment of the wing discs of second and early third instar larvae
depend on apterous, as revealed by reporter gene expression in discs either lacking or ectopically
expressing apterous. The DWR is not activated by ectopic fringe expression in the ventral compartment, suggesting that the observed induction of Serrate protein by ectopic Fringe is mediated by a different enhancer, which is active at later stages during wing development. The lack of Suppressor of Hairless results in a precocious repression of reporter gene expression along the margin, suggesting that the DWR of Ser responds to the postulated feedback loop mediated by the Notch signaling cascade to maintain expression in cells adjacent to the dorsal wing margin (Bachmann, 1998b).
In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional
regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch
target gene activation (Nagel, 2001).
These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by Nact. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by Nact/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of Nact are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).
A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of Nact as co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer
(Nagel, 2001).
These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).
These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).
Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two
evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation, is a target of Notch signaling during Drosophila eye
development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan
expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification (Rohrbaugh, 2002).
To investigate how yan expression is regulated, DNA fragments comprising a 20-kb genomic sequence surrounding the first exon of yan were tested for regulatory potential in corresponding transgenic flies. Through this approach, a 122-bp eye-specific enhancer located approximately 3.5 kb upstream of the first exon was identified. In eight out of nine transgenic lines, this enhancer activated expression of a bacterial lacZ reporter gene within posterior undifferentiated cells of eye discs. This recapitulates the endogenous yan gene expression in eye discs with the exception of the MF region. Three putative Su(H) binding sites were found in the yan enhancer. When tested through an in vitro electrophoretic mobility shift assay (EMSA), the Su(H) protein was shown to specifically bind to these sequences. Further, the yan enhancer became inactive in most of the posterior undifferentiated retinal cells when the Su(H) function was removed. All together, these loss-of-function and DNA binding analyses support the notion that Su(H) is required to promote yan transcription and that yan is a target gene of Su(H) in the eye (Rohrbaugh, 2002).
A candidate factor that may be involved in this compensation and could cooperate with Nintra/Su(H) proteins might be a DNA binding protein capable of interacting with a 5'-GAAACC/A-3' sequence. Two direct repeats of a 5'-GAAACC-3' sequence (hexamer, HEX) were found between S1 and S2. The second half of the S2 site might be considered as a third HEX, since there is only one variant base (as 5'-GAAACA-3'). When clustered point mutations were introduced into the first and second HEX, expression of the reporter gene was abolished in all six transgenic lines. Therefore, the HEX element is essential for the yan enhancer activity. Expression analysis of the HEX repeats has provided further evidence supporting the finding that the hexamer is an activation element. The reporter gene expression can be detected over the entire eye disc of all six lines when a six-copy concatomer of a 22-bp sequence containing three HEX repeats is used; however, one copy of this 22-bp oligonucleotide is not sufficient to induce gene expression in eye discs. It is proposed that a putative HEX binding protein functions together with Su(H) and Nintra to activate the yan enhancer. The nature of the HEX binding factor remains to be investigated (Rohrbaugh, 2002).
An Ets domain binding site (EBS, 5'-GGAA/T-3') was found within the S2 site. Since Yan is an Ets domain protein and a transcriptional repressor, whether Yan could be involved in autoregulation was examined. When a constitutively activated Yan (YanAct) was overproduced in eye discs, the reporter gene expression was strongly reduced. Since Yan is capable of negatively regulating yan transcription, this autoinhibitory mechanism might be used to prevent overproduction of Yan in undifferentiated cells. DNA binding data suggests that Yan can be directly involved in this negative regulation. However, this Yan-mediated autoinhibitory feedback appears to play a minor role in regulating yan expression, because the yan enhancer activity is apparently not affected in yan mutant clones produced in eye discs (Rohrbaugh, 2002).
A role for RTK signaling in regulating yan transcription was investigated. When the RTK pathway is constitutively activated by torD-DER or Ras1V12, the yan enhancer activity is greatly reduced. Thus, RTK signaling appears to negatively regulate yan transcription, in addition to its effect on Yan protein stability. Evidence supports a view that the inhibitory effect of RTK/Ras1 signaling on yan expression is mediated through the pointed (pnt) gene. Taken together, the results demonstrated that Pnt negatively regulates yan expression, and it is likely that Pnt is directly involved in repressing yan transcription. Although a role for Pnt as a transcriptional repressor has not been extensively investigated, pnt has been shown to negatively regulate hid transcription in embryos. Interestingly, a P-DLS motif is present in the Pnt protein (amino acids 356–360 in PntP1), which might mediate interaction with the transcriptional corepressor dCtBP. At this point, the data does not exclude the possibility that Pnt might also activate expression of a repressor, which in turn switches off yan transcription (Rohrbaugh, 2002).
The nesting of an Ets binding site within the S2 site suggests a possible mechanism whereby the binding of Pnt could interfere with Su(H)'s DNA binding activity. Indeed, increasing the amount of Pnt effectively prevents Su(H) from DNA binding. Such competition provides a mechanism by which RTK/Pnt signaling directly antagonizes Notch-mediated lateral inhibition at the transcriptional level. Since Ets binding sites are nested in many Su(H) binding sites, competitive occupancy of the common sequence could be a general mechanism for regulating expression of genes targeted by both Notch and RTK pathways (Rohrbaugh, 2002).
It is proposed that spatially restricted yan expression in the developing eye is coordinated by actions of multiple regulatory factors that include Su(H) and Pnt. Consequently, the yan enhancer provides an interface for Notch and RTK signals to oppose one another. The DNA binding analysis and mutagenesis of yan Su(H) binding sites provide evidence that supports a cell-autonomous role of Notch and RTK signaling in the regulation of yan expression. Interestingly, Yan expression is reduced not only in Su(H)D47 clones but also in some Su(H)+ cells that surround the mutant clones in eye discs. This result implies that loss-of-Su(H) function might also cause a cell-nonautonomous effect on yan expression, possibly due to upregulation of RTK signaling in those Su(H)+ cells. This upregulation may occur via an increase of a diffusible activator of the RTK pathway due to the loss of Su(H). The model presented here illustrates a mechanism that should help explain how progenitor cells are maintained in an undifferentiated state by Notch-mediated inhibitory signals and how they can be effectively induced for cellular differentiation by RTK-mediated inductive signals (Rohrbaugh, 2002).
The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).
These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).
During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing.
Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms
directing gcm expression. In the adult bristle lineage, gcm expression is negatively regulated by Notch signaling.
The effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial
cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is
required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch
activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain
developmental contexts. Evidence is presented that POU-domain protein Nubbin/PDM-1 is one of the factors that provides such context (Umesono, 2002).
Notch signaling promotes glial fate during asymmetric division in the embryonic dbd lineage. Notch is specifically activated in the presumptive DBD support glia cell (DBDG) owing to the negative regulation by Numb in the sibling cell, and provides instructive information to induce gcm transcription and glial development. Expression of gcm occurs quickly after the artificial activation of Notch, even in cells that have initiated neuronal development. In gcm mutants, DBDG are transformed into neurons, although the activation of Notch, visualized by the Su(H)-reporter, is normal in the presumptive glia. Likewise, ectopic expression of gcm in presumptive dbd neurons causes neuron-to-glia transformation without affecting Notch activity. These findings suggest that gcm expression appears to be the sole target of Notch activation in establishing glial fate in the dbd lineage. Within the 3.5 kb region upstream of the gcm gene, two sequences have been identified that perfectly match the consensus core sites for Su(H). Thus, gcm could be a direct target of Su(H), downstream of the Notch signaling pathway (Umesono, 2002).
The expression of vestigial during wing development is regulated
through two enhancers: the second intron or boundary
enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect
the patterns of expression directed by these regulatory regions:
vgBE produces a thin stripe over the
prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to
the vgBE and which fill in the developing wing blade. Both,
vgBE and vgQE, act as integrators of signaling systems that
drive wing development and, in this manner, these regulatory
regions determine the tempo and the mode of wing
development (Klein, 1999 and references).
The vgBE is activated first during the
second instar. Its expression pattern is
very similar to that of the Vestigial protein
at this stage suggesting that, in these early
stages, the vgBE is responsible for the
complete pattern of vg expression. Deletion analysis of the
enhancer reveals two regions essential for
its activity: a binding site for Suppressor
of Hairless and sequences contained in the
first 80 base pairs of the enhancer.
In an attempt to map the nature and
timing of the inputs into this enhancer, the activity of the wild-type vgBE and of
deletions of the two essential regions have been compared during wing development.
At the end of the second instar, lacZ reporter
expression from the wild-type vgBE
outlines a horseshoe over the ventral
region of the wing disc, with weak
expression in the ventral anterior region
where it overlaps with the expression of
wingless. lacZ expression
increases in this region at the beginning of the third instar. This increase does not occur in Suppressor of Hairless [Su(H)] mutants or in wg mutants.
The activity of the enhancer deleted for the Su(H)-binding
site is different from the wild type. The activity of this enhancer
is never initiated over the ventral region of the disc, where the
wing primordium is established and remains absent during later
stages. This result demonstrates that the activity of
Notch is required not only for the maintenance, but also for the
initiation of the expression of vg through the vgBE.
The activity of the enhancer deleted from 0-80 is similar to
that of the wild-type enhancer early on, but it never
acquires the high levels of activity in the anterior ventral region.
As the wing blade develops, a line of faint activity can be
detected over the DV boundary, but it fades quickly
and, by the end of the third instar, in most discs there is no
activity over the developing wing blade. This enhancer
still shows some activity in the flanking notal regions of the wing
disc in wild-type and Su(H) mutant discs. This
indicates the existence of additional inputs in the regulation of
the vgBE in the notal region. These results suggest that the cells in which the expression of the vgBE is upregulated at the end of the second instar represent the anlage of the wing and require Notch/Su(H) signaling. These cells are located at the DV interface, on the domain of wg expression and overlap the expression of nubbin. The suggestion that these cells represent the primordium of the wing pouch can explain why a deletion of the vgBE results in the abolition of the development of the wing pouch; in such a mutant, the anlage would never be defined (Klein, 1999).
Notch signaling is also required for the initial activity of the Quadrant Enhancer (vgQE)
The activity of the vgQE can be detected first at the beginning of the third instar, several hours after the upregulation of the vgBE, when it closely outlines the realm of the growing wing. This enhancer is only expressed in the growing wing blade and thus provides a
unique and most specific marker for wing blade tissue. A variety of experiments have shown that the vgQE receives a negative input from Notch signaling and a positive one from
Dpp. The presence of an E(spl)-binding
site in the sequence of the vgQE has led to the suggestion
that this suppression by Notch is mediated by the E(spl)
protein. However, no strong suppression of the
activity of the vgQE is found if E(spl)-m8 is ectopically
expressed, suggesting that the
effect of Notch requires other mediators. Although the vgQE is suppressed in the domain
of Notch activity, Notch signaling plays a non-autonomous
role in its activation. For example, the vgQE is
never active in Serrate (Ser) mutants in which wing
development initiates normally but is aborted very early. Ectopic expression of Delta
rescues the wing
pouch and leads to the activation of the vgQE.
Interestingly, this activity arises in regions devoid of Notch
signaling. This result suggests that
Notch signaling influences the activity of the vgQE in two
ways: it represses the activity of the vgQE autonomously but
it is also required for its activity in a non-autonomous way (Klein, 1999).
Wingless is shown to acts synergistically with Vestigial to
promote the activity of the vgQE.
It is clear that the activity of the vgBE is required for the
activation of the vgQE, but little is known about how this interaction
takes place. The observation that vgQE is activated in Ser
mutant discs in which vg is expressed ectopically
suggests that the activation of the vgQE is mediated by Notch
signaling through the activity of Vestigial on the vgQE. However, in this experiment, expression of wg is also
restored and this raises the possibility that activation
of the vgQE is mediated through the presumed organizing
activity of Wingless. This is probably not the case since ectopic
expression of wg alone does not lead to the activation of
the vgQE in Ser mutants. The inability of Wingless
to activate the vgQE in this experiment is not due to a general
insensitivity of the cells to Wingless signaling, since ectopic wg
is always capable of inducing hinge fate ectopically.
These results clearly demonstrate a requirement for vg in the
activation of the vgQE. However, clonal analysis has shown
that vg acts cell autonomously and therefore,
in the wild type, the non-autonomous effects of Notch on the
vgQE must be mediated by another, diffusible molecule(s),
which is under control of Notch signaling. A number of
studies suggest that Wingless has an influence on the
expression of vg in the wing pouch and that its expression at
the wing margin is under control of Notch signaling. Therefore, it is
possible that Wingless is mediating the non-autonomous effect
of Notch on the vgQE. It might be that Wingless acts by acting
on the vgQE to elevate and maintain the levels of vg expression
that had been induced by Vestigial through the vgBE. In
agreement with this proposal, it is found that the activity of the
vgQE is elevated in response to the ectopic expression of wg and that expression of a dominant negative Wingless molecule
suppresses the activity of the vgQE and reduces the size of the
wing pouch.
Altogether these results suggest that the upregulation of vg
expression in response to wg is mediated by the vgQE. This
conclusion is supported by the existence of several putative
TCF-1 binding sites in the vgQE. However,
the effects of Wingless are always restricted to the normal
domain of vg expression, in agreement
with the results presented above that Wingless alone is not
sufficient to initiate ectopic expression of vg through the vgQE.
These effects are likely to be mediated by Vestigial itself. The role of Wingless on this regulation is
to maintain and modulate the levels of activity of the vgQE.
Consistent with the conclusion that Wingless enhances the
effects of Vestigial, coexpression of Wingless and
Vestigial, which leads to the ectopic induction
of pouch and hinge fate in the notal regions, triggers a widespread and stable
expression of the vgQE throughout the wing disc (Klein, 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, 2000).
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, 2000).
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, 2000).
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, 2000).
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
scute10-1 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, 2000).
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, 2000).
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, 2000).
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, 2000).
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, 2000).
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, 2000).
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, 2000).
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) bind