Interactive Fly, Drosophila

Two products of the E(spl)-C, HLH-M5 and E(SPL), are capable of binding as homo-and heterodimers to the N-box, a sequence in the promoters of the Enhancer of split and achaete genes. This differs slightly from the E-box, the consensus binding site for other bHLH proteins. Both proteins were found to attenuate the transcriptional activation mediated by the proneural bHLH proteins lethal of scute and daughterless at the Enhancer of split promoter (Oellers, 1994).

Enhancer of split complex genes regulate Delta by acting through achaete-scute complex genes. Repression of Delta allows for the adoption of neural fate in adjacent cells (Heitzler, 1996).

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. What are the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo? Using the bicoid 3' UTR, localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic Dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo (Huang, 1997).

This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo. About half of the embryos carrying the Toll anteriorly expressed transgene exhibit a ventral gap in the endogenous ventral expression pattern of snail behind the ectopic anterior staining pattern. Although the identity of the repressor creating this gap is unknown, it is conceivable that members of the E(spl) complex encode putative snail repressors because previous studies have shown that the m7 and m8 genes are expressed in the lateral neuroectoderm of early embryos. Proteins coded for by these genes are known to repressors. These proteins might be regulated by the gene hierarchy responsible for D/V polarity (Huang, 1997).

To learn about the acquisition of neural fate by ectodermal cells, a very early sign of neural commitment in Drosophila has been analyzed, namely the specific accumulation of achaete-scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). An AS-C enhancer has been analyzed that directs expression specifically in SMCs. To delimit the sequences responsible for expression in SMCs, subfragments of a 3.7-kb fragment immediately upstream of scute were assayed for their ability to drive lacZ expression in wing discs. The necessary sequences are within a 356-bp fragment. This fragment specifically directed expression in SMCs. It also promotes expression in SMCs of other imaginal discs and of the embryonic PNS, but not in neuroectoderm neuroblasts. The SMC enhancer is shown to promote macrochaetae formation. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. The conserved sequences contain three E boxes: these are putative binding sites for bHLH proteins of the Achaete, Scute, and Daughterless type. The most proximal of the three is adjacent to an N box, a site that can be recognized by the E(spl)-C bHLH proteins. In addition, there are three copies of a motif reminiscent of a consensus binding site for the NF-kappaB family of transcription factors (named alpha1, alpha2, and alpha3), and three copies of a T-rich motif (termed beta1, beta2 and beta3) that does not fit with known protein-binding sequences. In spite of considerable effort, the NF-kappaB family member binding to the alpha motifs has not been identified (Culi, 1998).

In order to promote transcription, the SMC enhancer requires, besides proneural proteins, either additional activating factors or the removal of inhibitors. Activating factors might interact with the alpha and beta boxes necessary for efficient enhancer action. To identify the minimum number of different motifs sufficient to constitute an SMC-specific enhancer, the enhancer activity of a synthetic oligonucleotide containing two E1 boxes and one alpha2 box were examined. It promotes beta-galactosidase accumulation only in SMCs, although a weak one. A four tandem repeat of the same oligonucleotide drives much stronger lacZ activity and this also occurs exclusively in SMCs. In contrast, a four tandem repeat with E1 boxes, but without alpha2 boxes, drives strong expression in many cells of proneural clusters. A four tandem repeat of alpha2 boxes without E1 boxes fails to drive expression. Hence, both E and alpha boxes are sufficient, in the context of the minienhancer, to constitute an SMC-specific enhancer (Culi, 1998).

Promoters of other genes share similar enhancer motifs. The asense sequences that direct expression in SMCs contain several E boxes necessary for optimal expression in SMCs. The corresponding DNA from D. virilis was sequenced and compared with that of D. melanogaster. Similar to the sc SMC-enhancer, the stretches of D. virilis conserved DNA contain E boxes, one N box, two alpha boxes, and one beta box, supporting the relevance of these boxes for SMC enhancer function. Moreover, the neurogenic gene Bearded, which is expressed in proneural clusters and SMCs, contains in its regulatory region one E box, necessary for its expression, and one motif identical to the alpha2 box. An evolutionarily conserved alpha box is also found within the regulatory region of rough, a homeobox gene important for restricting photoreceptor R8 specification (Culi, 1998).

Notch signaling prevents more than one of a proneural cluster from becoming SMCs. When the N pathway is not operative, as for instance in Su(H) larvae or in larvae harboring a N^ts allele raised at a nonpermissive temperature, Ac and Sc proteins accumulate in many cells of proneural clusters at levels higher than in the wild type. The extra accumulation of Ac and Sc might be mediated by the cluster-specific enhancers, by the SMC enhancer (which under insufficient N signaling may promote expression in many cells of the proneural cluster as they become SMCs), or by both. To distinguish among these alternatives, an examination was carried out of the activity promoted by each type of enhancer, in both wild-type and in N^ts discs. N inactivation allows the SMC enhancer to drive expression in many cells of proneural clusters. Expression can occur in contiguous cells, indicating the failure of lateral inhibition. In contrast, N inactivation does not modify the activity of the enhancer that drives expression in the vein L3 and TSM (twin sensilla of the wing margin) proneural clusters, although the accumulation of Sc in these clusters is increased. Hence, the SMC enhancer is responsible for most of the increased levels of proneural protein that occur in proneural clusters under insufficient N function (Culi, 1998).

N signaling, triggered by Ac-Sc in the emitter cell, promotes in the receptor cell the accumulation of E(spl)-C proteins, the main effectors of this signal. E(spl)-C proteins are detectable in proneural cluster cells, except for the SMCs. This correlates with the SMCs being the cells that signal maximally and inhibit their neighbors from acquiring the neural fate, while the SMCs themselves are not inhibited. Ectopic accumulation of E(spl)-C protein prevents SMCs from emerging, as detected by a neuralized enhancer trap line and the consequent absence of SOs in the adult fly. Overexpression of UAS-E(spl)-m8 or UAS-E(spl)-m7 transgenes driven by da-GAL4 or the C-253 GAL4 lines block the activity of the SMC enhancer and the development of the corresponding SOs. In contrast, either of these overexpressions allowed normal accumulation of Ac and Sc in proneural clusters despite the high levels of ectopic E(spl)-m8 mRNA, which are severalfold higher than those in the wild type. However, overexpression with presumably stronger GAL4 drivers does interfere with ac-sc expression in proneural clusters. Taken together these results indicate that the function of the SMC enhancer is more sensitive to E(spl)-C inhibition than are the proneural cluster enhancers, and suggest that the SMC enhancer is the main target of lateral inhibition mediated by the N pathway (Culi, 1998).

Does E(spl)-m8 bind to the SMC enhancer, given the inhibition of SMC enhancer function by E(spl)-C? E(spl)-m8 binds to the N box and, unexpectedly, also protects a broad region of the enhancer (nucleotides 142-182), which does not contain sequences that fit the E(spl)-C consensus binding site. Binding to an enhancer with a mutated N box is weaker, and binding to an enhancer without the N box and the second E(spl)-m8-binding site is undetectable. Remarkably, the removal of one or both binding sites does not modify the SMC specificity of the enhancer, as might be expected if these binding sites mediated the repression of enhancer function in response to N signaling. E(spl)-m8 is unable to bind to the synthetic SMC-specific minienhancer. These results were extended to other E(spl)-C proteins by verifying that [similar to E(spl)-m8] E(spl)-m5 binds to an oligonucleotide with the E1-N sequence, but not to oligonucleotides containing only E2 or E3 boxes. Thus, it is concluded that the E(spl)-C proteins restrict enhancer function to SMCs by a mechanism that does not require direct interaction with enhancer DNA. Thus the Enhancer of split bHLH proteins block the proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-kappaB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment (Culi, 1998).

A common consequence of Notch signaling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. Each individual E(spl) gene was expressed using the GAL4-UAS system in order to analyse each gene's effect in a number of cell fate decisions taking place in the wing imaginal disk. A focus was placed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way: they antagonize neural precursor and vein fates. Yet the efficacy of this antagonism is quite distinct: E(spl)mbeta, which is normally expressed in intervein regions, has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. While E(spl)m8 is more effective in abolishing the notum microchaeta fate, E(spl)m7 is most active against wing margin bristles (Ligoxygakis, 1999).

During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. Two phases within this process have been discerned, based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes (Ligoxygakis, 1999).

How do E(spl) proteins, implicated in gene processing, come to activate cut expression? The present work suggests that cut expression requires Gro and partly also depends on E(spl)bHLH factors. One possibility is that E(spl) [at least E(spl)mgamma and E(spl)mdelta], like a number of other transcription factors, might have a dual function as either a transcriptional repressor or an activator, depending on context. Such an activation role has never been suggested before for either E(spl) or Gro. Alternatively, two models can be envisaged that reconcile a repressor activity of E(spl) proteins with their role in cut activation. In one, E(spl) can act by repressing a negative regulator of cut transcription. In the other, E(spl) can repress a negative regulator of Notch signaling. In the latter case, E(spl) expression would promote a positive feedback loop to enhance Notch signaling, thus increasing the signaling output from the severely compromised N^ts1 receptor at the restrictive temperature. The fact that no restoration is observed in the expression of two other Notch targets, namely wg and E(spl)m8-lacZ , argues against this hypothesis. A direct role of E(spl)mgamma and E(spl)mdelta in cut expression is favored, either as activators or as repressors of a repressor, but not as general positive regulators of Notch signaling (Ligoxygakis, 1999).

Ectopic expression of E(spl)mgamma/E(spl)mdelta is not sufficient for cut expression in a wild-type background. Rather, it appears that the ability of ectopic E(spl)mgamma/E(spl)mdelta to induce cut is spatially restricted to the normal domain of cut expression. Since activated Notch is sufficient to ectopically turn on cut, it follows that some other Notch-responsive event, other than E(spl) expression, must also contribute to cut expression. This is consistent with findings that early reduction of Notch activity abolishes cut expression despite concomitant ectopic expression of E(spl)mdelta. Molecular analysis has shown that cut expression requires the transcription factor Scalloped (Sd). sd is a candidate target gene of Vg, which in turn is initially activated by Notch independent of E(spl). It is possible that expression of vg and sd at the wing margin during early L3 could make these cells competent for cut expression. This would only be initiated later, when a second pulse of Notch signaling during mid-L3 activates (or relieves the repression of) cut via E(spl)mgamma or another Gro-interacting protein. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns (Ligoxygakis, 1999).

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).

E(spl) proteins are basic helix-loop-helix (bHLH) repressors, and most of them (m7, m8, mß, mdelta, and mgamma) are expressed in the posterior undifferentiated cells in eye discs. When E(spl) proteins (e.g., m7 and m8) are overproduced in eye discs, the yan enhancer activity is strongly reduced. Similarly, the level of Yan protein is also reduced. These results show that yan expression can be negatively regulated by E(spl) proteins. E(spl) proteins might act through an N box (5'-CACAAG-3') in the enhancer. Interestingly, mutations of the N box didn't cause upregulation of the reporter gene, but, instead, the reporter expression was abolished in all three transgenic lines. One explanation for this result is that the N box sequence might be shared by an activation element located in the region. Indeed, a Runt domain binding site (RBS) (5'-RACCRCA-3', R = purine) overlaps with the N box, which could mediate an effect by the Runt domain protein Lozenge (Lz), which has previously been shown to act as a transcriptional activator in the developing eye. Supporting this idea, the yan enhancer was completely inactivated in lz^r15 mutant eye discs. However, the level of Yan protein was not apparently affected by the lz mutation. This result suggests that Lz is not essential for the expression of the endogenous yan gene and that the loss of lz function could be compensated by other molecules so that yan expression is unaffected in lz mutants (Rohrbaugh, 2002) (Rohrbaugh, 2002).

Su(H)/CBF1 is a key component of the evolutionary conserved Notch signalling pathway. It is a transcription factor that acts as a repressor in the absence of the Notch signal. If Notch signalling is activated, it associates with the released intracellular domain of the Notch receptor and acts as an activator of transcription. During the development of the mechanosensory bristles of Drosophila, a selection process called lateral inhibition assures that only a few cells are selected out of a group to become sensory organ precursors (SOP). During this process, the SOP cell is thought to suppress the same fate in its surrounding neighbours via the activation of the Notch/Su(H) pathway in these cells. Although Su(H) is required to prevent the SOP fate during lateral inhibition, it is also required to promote the further development of the SOP once it is selected. Importantly, in this situation Su(H) appears to act independently of the Notch signalling pathway. Loss of Su(H) function leads to an arrest of SOP development because of the loss of sens expression in the SOP. These results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. This repressor activity is encoded by one or several genes of the E(spl)-complex. These results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl (Koelzer, 2003).

The removal of one copy of the E(spl)-C is already sufficient to relieve the block in SOP development in Su(H) mutants, indicating that the arrest is probably caused by the abnormal expression of one or more members of the complex. Although the complex encodes for several well-characterized repressors of neural development, it was not possible to pinpoint the repressor function to any particular gene. Many studies by various groups have studied the regulation of the genes of the E(spl)-C. From these studies, it is clear that only three genes of the complex are expressed in the cells of Su(H) mutant proneural clusters. All other members are either not expressed in the notal region of the wing imaginal disc or their expression is lost in the mutant cells. Previous studies have shown that both bearded-like proteins that are expressed in Su(H) mutant proneural clusters promote SOP development. Hence, it is unlikely that the abnormal expression of these genes causes the observed arrest in SOP development. Surprisingly, it was found that the strongest candidate, the bHLH repressor encoded by E(spl)m8, is also abnormally expressed in Psn mutants, where SOP development proceeds and the Su(H)/H-containing complex is intact. The observation is interesting, because it suggests that the activity of the whole Notch pathway is required to switch off the expression of E(spl)m8 in the SOP, but it also indicates that abnormal expression of the gene cannot be the reason for the arrest in SOP development in Su(H) mutant cells. Thus, the repressor activity might not be encoded by a specific member of the E(spl)-C (Koelzer, 2003).

One possibility is that the combination of the three abnormally expressed genes of the complex generates the repressing activity. An alternative is that Su(H) controls the expression of other genes that act in combination with the upregulated members of the complex to suppress SOP development. Another possibility is that more genes of the complex are de-repressed in Su(H) mutants at a level not detectable by the currently available methods. In this scenario, the weak expression of several bHLH-encoding genes will sum up to a level of repressor activity sufficient to stop SOP development. Using currently available techniques, it is very difficult to discriminate between these possibilities (Koelzer, 2003).

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