extra macrochaetae

Transcriptional Regulation

Tamou, coded for by polychaetoid, a Drosophila cell-cell junction-associated protein, is homologous to mammalian ZO-1, a member of the membrane-associated guanylate kinase homolog family. It is suggested that TAM is in an emc activating pathway. Mutation in tam gives a phenotype resembling mutation in emc. Mammalian ZO-1 can bind directly to the cytoskeletal element alpha-spectrin, and also binds occludin, an integral membrane protein at the tight junction. It is proposed that ZO-1 plays a role in the structural linkages between the tight junction and cytoskeletal networks. ZO-1 also colocalizes with cadherins in nonepithelial cells lacking tight junctions. Mutation in tam reduces the transcription of emc and causes enlargement of proneural clusters resulting in emergence of supernumerary precursor cells, and consequently in extra mechanosensory organs (Takahisa, 1996).

Irregular facets (If) is a dominant mutation of Drosophila that results in small eyes with fused ommatidia. Previous results showed that the gene Krüppel (Kr), which is best known for its early segmentation function, is expressed ectopically in If mutant eye discs. However, it was not known whether ectopic Kr activity is either the cause or the result of the If mutation. This study shows that If is a gain-of-function allele of Kr. The If mutation was used in a genetic screen to identify dominant enhancers and suppressors of Kr activity on the third chromosome. Of 30 identified Kr-interacting loci, two were cloned, and whether they also represent components of a natural Kr-dependent developmental pathway of the embryo was tested. The two genes, eyelid (eld) and extramacrochaetae (emc), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique Kr-expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the Kr gain-of-function mutation If provides a tool to identify genes that are active during eye development and that a number of them function also in the control of Kr-dependent developmental processes (Carrera, 1998).

Kr expression defines the Malpighian tubule anlage at late blastoderm stage and becomes restricted to a ring of cells at the midgut/hindgut boundary from where Kr-expressing Malpighian tubule precursors evert. Previous studies have shown that the specification of Malpighian tubule fate and the segregation of the cells depend on Kr expression in the Malpighian tubule anlage. In Kr-deficient embryos, the respective cells become part of the hindgut epithelium (Carrera, 1998).

Once the tubules evert, Kr expression becomes restricted to a single cell, termed the "tip mother cell". The singling-out process of this cell from an equivalence group of Malpighian tubule precursors involves the activated Notch pathway, which restricts the proneural bHLH proteins encoded by the achaete-scute-complex (ASC) genes to the tip mother cell. In this cell, the ASC proteins act in concert with bHLH protein encoded by daughterless (da) to maintain Kr expression. The tip mother cell divides once, and the daughters give rise to the tip cell, which controls proliferation during tubule elongation and differentiates neuronal characteristics, and an excretory cell, termed "satellite cell". The satellite cell loses Kr expression in a Notch-dependent manner, whereas Kr expression is maintained in the tip cell until the end of embryogenesis (Carrera, 1998).

emc expression accompanies Malpighian tubule development in a manner similar to Kr expression. However, once the tip cell is formed, the patterns of expression become complementary, meaning that emc expression continues in all cells of the elongating Malpighian tubules except in the tip cell. To test whether the complementary patterns of Kr and emc expression reflect a regulatory effect of emc on Kr, as indicated during eye development in the If mutant, Kr expression was examined in the Malpighian tubules of emc mutant embryos. Multiple Kr-expressing cells are seen in emc mutant Malpighian tubules. This finding is consistent with the previous finding that emc mutant embryos develop multiple tip cells and that each of them continues to express achaete. Virtually the same observations have been made with Notch mutants, and Notch acts toward restricting the activity of the proneural bHLH proteins, which are required to maintain Kr expression first in the tip mother cell and subsequently in the tip cell. However, although the activated Notch pathway acts through transcriptional repression of the ASC genes, emc protein antagonizes proneural bHLH activities by sequestering the proteins as heterodimers that are incapable of binding to DNA. The results are therefore consistent with the proposal that emc functions in the control of Kr expression by antagonizing proneural bHLH activities that are required to maintain Kr expression in the tip mother cell (Carrera, 1998).

The Eld protein shows a nuclear localization, consistent with its suspected function as a transcription factor. It appears to act in multiple signaling pathways because it antagonizes wingless activity, suppresses Ras1 activity in the eye, and blocks Notch-dependent neuronal differentiation. During Malpighian tubule development, eld is expressed in a restriced area of the everting precursors that corresponds to the equivalence group of cells expressing the proneural genes (Carrera, 1998).

eld mutant embryos exert a distinct phenotype during Malpighian tubule development that is linked to Kr activity. Whereas the anlage and the four tubules evert normally, each tubule develops two instead of the normal one tip cell. Tip cell development is under the control of Kr activity, so it was next asked whether and when Kr expression is altered in eld mutant embryos. In correspondence with the mutant phenotype, the initial expression of Kr, including its restriction to the tip mother cell, appears to be normal. However, once the tip mother cell has undergone division, two instead of only one of the daughter cells maintain Kr expression. This indicates that eld activity is necessary to prevent Kr expression in the sibling of the tip cell and allows for its differentiation into a satellite cell. Thus, although emc is necessary for the restriction of Kr to the tip mother cell, eld functions specifically at the subsequent step during Malpighian tubule development where an alternative and Kr-dependent cell fate decision is taken between the daughters of the tip mother cell (Carrera, 1998).

Notch signaling is required first for the selection of the tip mother cell and subsequently for the distinction between its daughters to either develop a tip cell or a satellite cell. Consistently, in Notch mutant embryos, all cells of the proneural equivalence group develop first into tip mother cells; these cells divide and subsequently develop into the multiple tip cells that continue Kr expression. In contrast, only two tip cells were found in eld mutants. This finding implies that, if eld acts in a Notch-dependent manner and/or mediates Notch signaling, its activity is required only for the second of the two Notch-dependent differentiation steps during Malpighian tubule development. Thus, eld participates as an optional component in the Notch-signaling pathway and is needed to prevent, directly or indirectly, the maintenance of Kr expression in the satellite cell that would otherwise develop into a second tip cell (Carrera, 1998).

The results of this study demonstrate that gene activities that were identified via an artificial experimental situation, namely the ectopic expression of Kr in the developing eye disc, can lead to the identification of integral components of a Kr-dependent developmental pathway during embryogenesis. In the eye imaginal disc, emc suppresses Kr activity whereas eld has an opposite effect, but both act during embryonic Malpighian tubule development as negative regulators of Kr. No explanation is available for this phenomenon. It could mean, in negative terms, that the Kr misexpression screen turned up dosage-sensitive genes affecting cell fate that were several steps downstream from Kr activity and thus have no direct interaction with Kr. Thus, each gene identified in the modifier screen represents a candidate gene that needs to be evaluated critically through additional criteria as outlined here for eld and emc. The additional screening is essential to distinguish between direct Kr interactors and genes that mediate different read-outs of the Kr pathway in cells that have a different organ or tissue competence. However, in view of the fragmentary information concerning the spatial and temporal control of postblastodermal Kr expression and in view of the fact that the few Kr target genes of Kr were identified by molecular approaches, this experimental strategy to assess components of a Kr-dependent regulatory circuitry seems a valid one (Carrera, 1998).

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001).

Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001).

Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001).

Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001).

Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001).

These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001).

The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated (Baonza, 2001).

In mitotic clones of the Notch null allele N^54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).

The most well characterized role of Notch signaling in R8 photoreceptor determination is mediating the process of lateral inhibition, which refines Atonal expression from a small group of cells to a single cell. However, an earlier and opposite role for Notch, this time promoting neural determination, has also been recognized, although how this 'proneural' function integrates with other pathways necessary for neural differentiation has been unclear. In this work, it has been shown that in normal eye development the proneural function of Notch signaling depends on prior Dpp signaling. Emc and Hairy, two negative regulators of Atonal expression, mediate the proneural function of Notch signaling in the eye. Thus, a model is proposed that links the upregulation of Atonal in the proneural groups with the downregulation of Hairy and Emc through the activation of Delta/Notch signaling (Baonza, 2001).

Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated (Baonza, 2001).

Notch can only trigger Atonal upregulation in a zone extending 12-15 cells anterior to the furrow, and this zone is defined as the cells that receive the diffusible factor Dpp, whose source is in the furrow. Dpp acts to define a ‘pre-proneural’ state that prepares cells for the imminent initiation of neural determination. This pre-proneural state is defined as the zone of cells that initiate Hairy and Atonal expression in response to Dpp signaling. A functional definition to this state can be added: all these cells are primed for neural differentiation because all can respond to Notch activation by upregulating Atonal levels (Baonza, 2001).

Simultaneous loss of Hairy and Emc activity leads to the precocious differentiation of photoreceptors in a competent region ahead of the morphogenetic furrow, a phenotype that resembles that caused by ectopic expression of Delta. In addition, ectopic Notch signaling downregulates Hairy and Emc ahead of the morphogenetic furrow, causing the accumulation of Atonal at high levels; conversely, loss of function of Notch signaling increased the levels of Hairy. It is concluded that Delta/Notch signaling regulates the expression of these negative regulators in the eye. Consistent with this proposal, Emc is also regulated by Notch in the developing wing disc (Baonza, 2001).

Although Notch signaling negatively regulates both Hairy and Emc, the ectopic expression of Delta does not affect both genes identically. Thus, whereas Hairy is removed both within the clone and in the neighboring cells, Emc is only downregulated autonomously within the clone. This distinction could be an artifact caused by the perdurance of ß-galactosidase. Alternatively, these differences may reflect a different requirement for Notch signaling in the regulation of both genes. Furthermore, the expression pattern of Hairy and Emc is different during the normal progression of the morphogenetic furrow. Hairy is precisely regulated, being expressed only in the cells anterior to the furrow, and is rapidly downregulated in the furrow. This precise regulation is crucial as shown by the ectopic expression of hairy. Emc has a much broader expression pattern in the eye disc, although it shows a similar upregulation followed by downregulation in the zone immediately anterior to the furrow (Baonza, 2001).

It is also worth pointing out that not only does the expression pattern of Emc and Hairy differ, but their exact mechanism of repression is also distinct. Hairy regulates bHLH proteins by a mechanism of direct DNA binding and transcriptional repression. Emc, however, forms complexes with bHLH proteins, preventing their DNA binding. Thus, Emc can antagonize the proneural function of Atonal by two distinct mechanisms: (1) Emc presumably binds to Atonal, rendering it incapable of activating its targets; (2) Emc controls the levels of Atonal. By analogy to its regulation of two other bHLH transcriptional regulators, Achaete and Scute, it is expected that Emc interferes with the autoregulatory upregulation of atonal expression. This positive autoregulation is an essential component of its accumulation in cells within the morphogenetic furrow. In conclusion, the proneural action of Notch signaling increases Atonal activity by two mechanisms: atonal is transcriptionally upregulated, and at the same time a repressive co-factor is removed. These concerted actions lead to the accumulation of active Atonal and thereby the initiation of neural differentiation (Baonza, 2001).

Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).

The expressions of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) protein lacking a basic motif, and Notch (N), were examined because both genes have been shown to be involved in the control of cell proliferation in the wing. In third instar larvae, emc is expressed at a low level throughout the wing disc and at a higher level in two stripes of cells corresponding to the prospective A margin and the AP organizer. Unmodified at the A margin, emc expression is completely lost from the AP organizer cells in either col¹ or col¹/kn¹ mutant discs, showing that Col is required for emc transcription in the L3-L4 intervein primordium. Levels of N protein are high in intervein regions and low in presumptive vein territories in late third instar. In col¹ mutants, N is downregulated in the L3m provein domain. col requirement for emc and N upregulation in the AP organizer cells is consistent with the reduced cell number in the central region of col¹ mutant discs (Crozatier, 2002).

Targets of Activity

Several genes encoding transcription factors of the helix-loop-helix (HLH) family (such as Daughterless (DA), Sisterless-b (SIS-B), Deadpan (DPN) and EMC) regulate Sex lethal. DA/SIS-B heterodimers bind several sites on the SXL early promoter with different affinities and consequently tune the level of active transcription from this promoter. Repression by the DPN product of DA/SIS-B dependent activation of Sex-lethal results from specific binding of DPN protein to a unique site within the promoter. This contrasts with the mode of EMC repression, which inhibits the formation of the DA/SIS-B heterodimers (Hoshijima, 1995).

One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. This work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. It is proposed that Notch signaling targets two distinct 'Repressors of twist' - the proteins encoded by the Enhancer of split complex [E(spl)C] and the HLH gene extra machrochaetae (emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, this work suggests a conserved mechanism for Notch in early mesodermal patterning (Tapanes-Castillo, 2004).

Analysis of Notch mutant embryos revealed that Notch signaling is essential for Twist regulation at mesodermal subdivision. However, comparison of Notch and Su(H) mutant embryos indicated that Notch regulates Twist differently from Su(H). At stage 10, uniform high Twist expression was maintained in N^null mutants; by contrast, Su(H)^null mutants have a wild-type-like Twist pattern. Furthermore, while constitutive activation of Notch represses Twist expression at stage 10, constitutive expression of a transactivating form of Su(H) [Su(H)-VP16] increases Twist expression. Despite these differences, double mutant analysis and rescue experiments demonstrate that Notch requires Su(H) to repress Twist. Moreover, further rescue experiments show that Notch signaling acts as a transcriptional switch, which alleviates Su(H)-mediated repression and promotes transcription. In addition, genetics, combined with promoter analysis, suggest that Notch and Su(H) have multiple inputs into twist. Notch/Su(H) signaling both directly activates twist and indirectly represses twist expression by activating proteins that repress Twist. Finally, the data indicate that Notch targets two distinct 'Repressors of twist' - E(spl)-C genes and Emc. It is proposed that Notch signaling activates expression of E(spl)-C genes, which then act directly on the twist promoter to repress transcription. Since removing groucho enhances the phenotype of the E(spl)-C mutant embryos, it is suggested that the corepressor, Groucho, acts with E(spl)-C proteins and the Hairless/Su(H) repressive complex to mediate direct repression of twist. The second 'Repressor of twist', Emc, mediates repression of Twist in an alternative fashion. It is hypothesized Emc activity inhibits dimerization of Da with itself or another bHLH protein. This, in turn, prevents Da from binding DNA and activating twist transcription. Since Emc is expressed in the embryo prior to stage 10, it is likely that the transition from uniform high Twist expression to a modulated Twist pattern involves Emc inhibition of Da activity at stage 9. In conclusion, this work uncovers how Notch signaling impacts a network of mesodermal genes, and specifically Twist expression. Given that Notch signaling directs cell fate decisions in many Drosophila embryonic and adult tissues and that Notch regulates Twist in adult flight muscles, these data may suggest a more universal mode of Notch regulation (Tapanes-Castillo, 2004).

The distinct mesodermal phenotypes of Notch and Su(H) mutants can be explained by Notch acting as a transcriptional switch. This aspect of Notch signaling has been described in other systems, and the early Drosophila mesoderm appears no different in this regard. However, these data suggest that there is more to the phenotypes; that is, additional layers of Notch regulation in the transcriptional control of twist (Tapanes-Castillo, 2004).

Genetic experiments, as well as promoter analysis, raised the hypothesis that Notch signaling regulates twist directly, as well as indirectly by activating expression of a 'repressor of twist.' This indirect repression of twist concurs with the role of Notch in activating E(spl) transcriptional repressors. Moreover, a mechanism involving direct and indirect regulation is consistent with Su(H) mutant phenotypes. In Su(H)^null embryos, neither twist nor repressor of twist (for example, emc) are repressed. The de-repression of both genes at the same time results in Twist expression appearing 'wild-type-like'. When a constitutively activating form of Su(H) is expressed, both twist and repressor of twist are activated. In these embryos, high Twist domains are expanded, but uniform high Twist expression is not observed because repressor of twist is expressed (Tapanes-Castillo, 2004).

However, simple direct and indirect regulation [through emc and E(spl)-C genes] by Notch still does not fully explain the phenotypes of Notch mutants. Both twist and repressor of twist should be repressed in N^null embryos because Su(H) will remain in its repressor state. While the N^null phenotype was consistent with repressor of twist being repressed, twist was still strongly expressed. Additionally, constitutive Notch activation should cause both twist and repressor of twist to be expressed. Consequently, N^intra was expected to cause a phenotype similar to that caused by Su(H)-VP16. Contrary to these predictions, panmesodermal expression of N^intra represses Twist, consistent with only repressor of twist being strongly expressed. Taken together, these results suggested that at stage 10, the twist promoter is less receptive to Notch/Su(H) activation than to Notch/Su(H) repression. As a result, constitutive activation of Notch represses twist, while loss of Notch activates twist ectopically (Tapanes-Castillo, 2004).

While Notch signaling has the ability to activate twist, Notch/Su(H) signaling ultimately leads to repression of twist at stage 10. This predominance of repression can be explained in two ways: (1) direct Notch activation of the twist promoter is overpowered by Notch activated repressors of twist; and (2) a repressor of twist gene, such as E(spl), is more responsive to Notch/Su(H) activation than twist. These ideas are discussed below in light of the results (Tapanes-Castillo, 2004).

The first model proposes that while Notch signaling might directly promote both twist and repressor of twist activation, repressors of twist might suppress an increase in twist transcription. The data suggest that Notch regulates multiple repressors of twist, including E(spl)-C genes and Emc. On the twist promoter, these multiple repressors could overwhelm Su(H) activation. Hence, twist would be transcriptionally repressed rather than activated. In Su(H)-VP16 embryos, the constitutive activating ability of Su(H) on the twist promoter might inhibit some of this repression. Consequently, Twist is ectopically expressed at high levels (Tapanes-Castillo, 2004).

The data are also consistent with the second model, which proposes that twist and a repressor of twist gene, such as E(spl), respond differently to Notch activation. The reason for this differential response is provided by the concept of Notch instructive and permissive genes. Transcription of Notch instructive genes requires the intracellular domain of Notch (N^icd) first to alleviate Su(H)-mediated repression and then to serve as a coactivator for Su(H). Transcription of Notch permissive target genes requires N^icd solely to de-repress Su(H); Su(H) bound to other coactivators and/or other transcriptional activators is necessary for permissive gene activation. Since panmesodermal expression of N^intra does not activate twist, it is concluded that simple de-repression of Su(H) is insufficient to activate twist expression and that other factors are required. Hence, Notch acts permissively on the twist promoter. By contrast, panmesodermal expression of N^intra is sufficient to activate a repressor of twist, resulting in the strong Twist repression. Since E(spl)-C genes have been categorized as Notch instructive target genes, it is suggested that E(spl)-C genes are the Notch instructive repressor of twist genes in this system. Although Notch can upregulate Emc expression, the inability to see a change in Emc expression in N^null and Su(H)^null mutants suggests Emc is not a Notch instructive target gene. Thus, based on all of this work, the instructive and permissive target gene regulation model is currently favored (Tapanes-Castillo, 2004).

Post-transcriptional regulation

In common with several transcription units of the E(spl)-C, including E(spl)m4, Bearded contains two novel heptanucleotide sequence motifs in its 3' untranslated region (UTR), suggesting that all these genes are subject to a previously un-recognized mode of post-transcriptional regulation. These sequence motifs are called the Brd box (AGCTTTA) and the GY box (GTCTTCC). Like known sequence elements that function in post-transcriptional regulation, both of these motifs are found in a single orientation and specifically in the UTRs of the genes that include them. Many mRNAs are translationally inactive until they undergo additional cytoplasmic polyadenylation, a process controlled by cytoplasmic polyadenylation elements (CPEs). Polyadenylation is implicated in Brd box function. Negative regulation by the Brd box motif affects steady-state levels of both RNA and protein. This result indicates that Brd boxes have an additional role in regulating translation, beyond the effect attributable to transcript level differences. Thus, the Brd 3' UTR confers negative regulatory activity in vivo. This activity is spatially and temporally general, in that most or all cells are able to respond to Brd boxes. This suggests that some genes expressed outside of proneural clusters may be regulated by these motifs as well. Three other genes that encode negative regulators of PNS development also contain these sequences in their 3' UTRs. In particular, kuzbanian (kuz) and extramacrochaetae (emc) each include single Brd boxes, while hairy (h) contains a GY box. emc also includes four copies of a GY box-related sequence (GTTTTCC) in its 3' UTR, which may be relevant for its regulation. kuz has functions in SOP selection and lateral inhibition, so its expression certainly includes proneural clusters. However, emc and h are expressed in spatial patterns that are largely complementary to proneural clusters in the leg and wing imaginal discs, and are thus possible examples of genes regulated by the Brd box (and possibly the GY box) in territories outside the clusters. Interestingly, the Emc and H proteins, as members of the HLH family, are structurally related to the E(spl)-C bHLH proteins. In contrast, kuz encodes a metalloprotease/disintegrin protein of the ADAM family (Lai, 1997 and references).

The 3' untranslated regions (3' UTRs) of Bearded, hairy, and many genes of the E(spl)-C contain a novel class of sequence motif, the GY box (GYB, GUCUUCC); extra macrochaetae contains the variant sequence GUUUUCC. The 3' UTRs of three proneural genes include a second type of sequence element, the proneural box (PB, AAUGGAAGACAAU). The full 13 nt PB is found once each in ac, l'sc, and ato, along with a second, variant version in both l'sc and ato. The presence of these motifs in such distantly related paralogs as hairy and certain bHLH genes of the E(spl)-C (for the GYB), and ato and two genes of the AS-C (for the PB), indicates that both classes of sequence element are subject to strong selection. Furthermore, both the PB and the GYB are conserved in the orthologs of ac and E(spl)m4 from the distantly related Drosophilids D. virilis and D. hydei, respectively, though these 3' UTRs are otherwise quite divergent from their D. melanogaster counterparts. These findings strongly suggest functional roles for both of these sequence elements (Lai, 1998).

Intriguingly, the central 7 nt of the PB and the GYB are exactly complementary, and are often located within extensive regions of RNA:RNA duplex predicted to form between PB- and GYB-containing 3' UTRs. Indeed, using in vitro assays, RNA duplex formation has been observed between the ato/Brd and ato/m4 3' UTR pairs that is PB- and GYB-dependent. It is noteworthy that the predicted duplex interactions involving the GYB of Brd are significantly stronger than those involving the GYBs of the other transcripts. For example, Brd and ato are perfectly complementary over 18 contiguous nucleotides. This difference in the degree of PB:GYB-associated complementarity is likely to have functional consequences (Lai, 1998).

In C. elegans, small antisense RNAs encoded by lin-4 mediate translational repression of lin-14 and lin-28 transcripts by binding to complementary sequences in their 3' UTRs. In Drosophila, PB- and GYB-bearing transcripts may likewise participate in a regulatory mechanism mediated by RNA:RNA duplexes, but with the feature that both partners are mRNAs that also direct the synthesis of functionally interacting proteins. The opportunity to form such duplexes clearly exists, since transcripts from proneural genes and their regulators very frequently accumulate in coincident or overlapping patterns. Moreover, while 7 nt is the minimum length of complementarity between any PB and any GYB, the longest possible uninterrupted duplex between a given GYB-bearing transcript and a given proneural partner is almost always considerably longer (8-12 nt). It is worth noting that in a lin-4/lin-14 duplex that has been shown to be sufficient for proper regulation in vivo, the longest region of uninterrupted complementarity is only 7 nt (Lai, 1998 and references therein).

The formation of the postulated RNA duplexes may serve to regulate proneural gene function, consistent with the known roles of hairy, emc, and the bHLH genes of the E(spl)-C. This might explain occasional C-to-U transitions in the GYB sequence (in emc and D. hydei m4); these variants retain complementarity with the PB due to G:U base-pairing. It is equally plausible that GYB-containing transcripts are regulated by duplex formation. A third very interesting possibility is that RNA:RNA duplexes formed between PB- and GYB-containing transcripts function to initiate a downstream regulatory activity affecting as-yet-unknown targets. Ample precedent exists establishing the trans-regulatory potency of double-stranded RNA. In any case, the apparent capacity of transcripts from the proneural genes and their regulators to form duplexes in their 3' UTRs suggests further complexity in the already complex regulatory interactions that control Drosophila neurogenesis (Lai, 1998).

Protein Interactions

daughterless and three genes of the achaete-scute complex act positively in the delaminating of the sensory organ precursors of cell fate. Both emc and hairy act as negative regulators. EMC, but not Hairy, antagonizes DNA binding of da/achaete-scute heterodimers (Van Doren, 1991).

EMC protein forms heterodimers with proteins of Achaete, Scute, Lethal of scute and Daughterless, and inhibits transcription activation by these proteins (Cabrera, 1994). EMC is also thought to inhibit other bHLH transcription factors as well (Cubas, 1994).

Posttranscriptional Regulation

The EMC homolog in humans, (ID) has sequences in its 3' UTR that are bound by the human homolog of ELAV, a Drosophila RNA binding protein. The 3'UTR of EMC RNA has sequence homology to regions in ID that are bound by the human homolog of ELAV (King, 1994).

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