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).

Wiring between signaling pathways differs according to context, as exemplified by interactions between Notch and epidermal growth factor receptor (EGFR) pathways, which are cooperative in some contexts but antagonistic in others. To investigate mechanisms that underlie different modes of cross talk, this study has focused on argos, an EGFR pathway regulator in Drosophila melanogaster which is upregulated by Notch in adult muscle progenitors but is repressed in the wing. Results show that the alternate modes of cross talk depend on the engagement of enhancers with opposite regulatory logic, which are selected by context-determining factors. This is likely to be a general mechanism for enabling the wiring between these pathways to switch according to context (Housden, 2014).

Analysis of tissue-dependent responses to Notch demonstrates that, in argos, these are determined at the level of specific enhancers. These respond either to Su(H) or to the bHLH repressors downstream of Notch, giving rise to different consequences on argos expression and explaining how the logic of signaling pathway cross talk can be switched. Indeed, the different modes of argos regulation correlate with the relationship between Notch and EGFR pathways, with cooperative cross talk occurring in the adult muscle progenitors (AMPs), where the enhancer directly regulated by Su(H) is active, and antagonistic cross talk taking place in the wing pouch, where the repressive enhancer regulated by bHLH operates. Similar distinctive enhancers may also operate at different stages in development, where Notch first activates and then represses the expression of a gene via independent regulatory elements. In both cases it is likely that context-determining factors will alter the ability of specific enhancers to respond to distinct Notch inputs. These will then dictate how signaling pathways will act on the cognate gene, depending on which regulatory elements they make available (Housden, 2014).

Several observations, such as the inability of HLHmβ or HLHmβ-VP16 to alter expression of argos(p)-lacZ when expressed in the AMPs, suggest that, like Su(H), HLHmβ can occupy its binding sites only when the enhancer becomes accessible. Consistent with this possibility, another HLH family transcriptional repressor, Hairy, was shown to bind and repress only those enhancers that had been rendered accessible by prior binding of other factors. Alternatively, HLHmβ may still be capable of binding to its site in enhancer fragment argos2 but lacks the ability to mediate long-range repression, restricting its effects to transcription factors bound within the same vicinity, as observed for short-range repressors regulating even-skipped enhancers. Given that Hairy bHLH repressors can mediate long-range as well as short-range repression, this explanation seems unlikely. Furthermore, as studies of other bHLH factors, such as Myc, argue that they can only bind to chromatin in open conformations, the model in which enhancer accessibility is regulated seems the more probable explanation (Housden, 2014).

Thus, the context-dependent response of argos to Notch could be explained by a two-stage model. Key determining factors, such as Twist in the AMPs or Vvl in the wing pouch, would first regulate the accessibility of different enhancers in the argos intron. This would enable the second stage, which integrates the effects of Notch and EGFR. For example, in the wing pouch, multiple binding sites for the repressor Cic keep the gene repressed, except in regions where EGFR is active. Superimposed on this is the additional regulation from the E(spl)bHLH repressors, acting downstream of Notch to fine-tune the expression patterning within this active domain. Such a model is broadly consistent with two general principles proposed previously for gene regulation by signaling pathways. The first is the reliance on cooperation with context-determining transcription factors, fulfilled here by the requirements for Twist or Vvl. The second is the pivotal role played by repressors, which prevent enhancer activity in appropriate places, as seen here for Cic and E(spl)bHLH (Housden, 2014).

In Drosophila melanogaster the Enhancer of split-Complex [E(spl)-C] consists of seven highly related genes encoding basic helix-loop-helix (bHLH) repressors, intermingled with four genes that belong to the Bearded (Brd) family. Both gene classes are targets of the Notch signalling pathway. The Achaete-Scute-Complex [AS-C] comprises four genes encoding bHLH activators. Focussing on Diptera and the Hymenoptera Apis mellifera, the question arose how these complexes evolved with regard to gene number in the evolution of insects. In Drosophilids, both gene complexes are highly conserved, spanning roughly 40 million years of evolution. However, in species more diverged, like Anopheles or Apis , dramatic differences are found. Here, the E(spl)-C consists of one bHLH (mß) and one Brd family member (malpha) in a head to head arrangement. Interestingly in Apis but not in Anopheles, there are two more E(spl) bHLH like genes within 250 kb, which may reflect duplication events in the honeybee that occurred independently of those in Diptera. The AS-C may have arisen from a single sc/l'sc like gene which is well conserved in Apis and Anopheles and a second ase like gene that is highly diverged, however, located within 50 kb. Thus, E(spl)-C and AS-C presumably evolved by gene duplication to the current complex composition in Drosophilids in order to govern the accurate expression patterns typical for these highly evolved insects. The ancestral ur-complexes, however, consisted most likely of just two genes: (1) E(spl)-C contains one bHLH member of mß type and one Brd family member of malpha type, and (2) AS-C contains one sc/l'sc and a highly diverged ase like gene (Schlatter, 2005).

In total, 12 genes in D. melanogaster are known to encode Hairy/E(spl)-like proteins, i.e. bHLH proteins that also have the orange domain and a WRPW-type Gro-binding motif. Apart from the seven E(spl) bHLH proteins, these include Hairy, Deadpan, Side, Hey and Her. Moreover, there is similarity to Stich1/Sticky, which has a bHLH and an orange domain but not the typical Gro-binding motif. Since the number of E(spl) bHLH genes is not conserved in honeybee and mosquito, it was interesting to ask whether all the other genes were present. The Ensembl database was searched with the respective D. melanogaster protein sequences: orthologs were found of all genes except Her in both species. However, most of the predictions are incomplete. It is known from D. melanogaster that these genes contain introns, which complicates the search for potential coding sequences within genomic DNA. Thus, the protein sequence predictions are uncertain. With the sole exception of Dpn, all the proteins are better conserved between Drosophila and Anopheles than between Drosophila and Apis, confirming the evolutionary relationship. The best conserved proteins are Hey and Hairy. The Hey orthologs are 76% identical between Drosophila and Anopheles and 66% between Drosophila and Apis and the Hairy orthologs between 72% and 65%, respectively. Less conservation is found for Side, Dpn and Stich1 (62%/57% Side identity, 57%/59% Dpn identity and 60%/57% Stich1 identity), comparing fly with mosquito and honeybee, respectively. All proteins share the bHLH and orange domains. The WRPW motif of Hairy, Dpn and Side as well as the YRPW motif of Hey is present in the orthologs (Schlatter, 2005).

Extensive genome analyses in recent years revealed that there are not many examples of large gene complexes that are widely conserved. Prominent examples are the HOX (homeobox) complexes, which contain homeotic genes in Drosophila. HOX complexes are well conserved in metazoans despite some variations in gene number. HOX-genes encode regulatory proteins with specific individual functions and mutations affect different aspects of the body plan. Not surprisingly, it is almost only the homeodomain, which serves as sequence-specific DNA binding motif, that is conserved amongst different species. In contrast, similarity among bHLH proteins encoded by the E(spl)-C extends over the entire length, even within the same species, indicating rather recent duplication events. The D. melanogaster proteins M8/M5 and Mß/M3 are most similar with over 70% identity, whereas Mdelta is the most diverged. However, Mdelta still shares at least 50% identity with other E(spl) bHLH protein members. More interesting is the analysis of the overall similarity among these proteins. Here, any one of the proteins is compared with the other six and the result is averaged. Clearly, Mß (73%/64%, similarity/identity) closely followed by Mgamma (72%/63%) is most similar to all others, whereas Mdelta (66%/55%) shows the lowest values. One interpretation might be that the different bHLH genes evolved by duplication out of mß or mgamma. Remarkably, these two bHLH proteins in addition to M3 are the best conserved in the three Drosophila species. It is postulated that these are the most ancient proteins with the most general function and, therefore, the highest selection pressure. This hypothesis is supported by the finding that mß has the most general expression pattern from which the others can be derived by a decrease of gene activity. The conspicuous conservation of M3 might hint to an important function during egg development as this gene is also expressed maternally. The high degree of conservation of all E(spl) bHLH orthologous proteins in Drosophilids, which is clearly higher than the similarity within this protein family in D. melanogaster, indicates specific and non-redundant roles during development. Some of these functions have been identified in the past. It is conceivable that regulatory sequences were not duplicated or evolved more rapidly so that now highly dynamic expression patterns of these genes are found (Schlatter, 2005).

mß appears to be the ancestral bHLH gene of the E(spl)-C in Drosophilids based on its great similarity with all the other bHLH proteins. This assumption is strongly supported by the sequence conservation of the E(spl) bHLH proteins in A. gambiae and A. mellifera. The single E(spl) bHLH protein encoded by the mosquito genome has the highest identity to Mß. The genome of honeybee contains three prospective genes that encode proteins most highly related to E(spl) D.m.Mß and D.m.Mgamma. All three are clustered within a single sequence contig, albeit they span a large segment of about 250 kb, whereas the whole E(spl)-C in D. melanogaster comprises roughly 50 kb. Despite the fact that two of these genes possess introns just within the bHLH domain and at positions close to the ones found in the D. melanogaster genes dpn, hairy or Her , the amino acid sequence similarity classifies them clearly as E(spl) bHLH proteins. A comparison of Anopheles and Apis proteins reveals, that the presumptive Mß homologs have highest similarity (83%) and identity (76%), whereas the protein that classified as A.m.Mgamma is just 70% similar and 66% identical to A.g.Mß (Schlatter, 2005).

In Drosophilids, malpha is located close to mß and is transcribed in the opposite direction (head to head). This arrangement is likewise found in Anopheles and Apis. Notably, A.m.malphais next to A.m.mß, whereas the two Apis A.m.mgamma and A.m.mß' genes are much further apart. This arrangement is thought to be very ancient. In the beetle Tribolium, which on the tree of evolution is found even more deeply rooted (~300 Myr to Dipterans), two similar genes coding for Mß-like proteins (~65% and ~67% identity to D.m.Mß) are found and one is within ~18 kb of a gene coding for an Malpha-like protein (~52% identity to D.m.Malpha). It is postulated that the ur-complex consists of these two ancestral genes, malpha and mß. It is intriguing that they belong to the two different classes of Notch-responsive genes in the E(spl)-C, the bHLH and the Brd-class. In the fly, malpha and bHLH genes are similarly expressed. It is not unlikely that they share common regulatory elements that could explain their co-segregation in the process of evolution (Schlatter, 2005).

The Achaete-Scute complex (AS-C) is well conserved in D. virilis: all four genes, achaete (ac), lethal of scute (l'sc), scute (sc) and asense (ase) are found in the same order and orientation on the X-chromosome. As in D. melanogaster, the genes are without introns. All proteins share the typical bHLH motif of the AS-C proteins and this domain reveals the lowest evolutionary rate. However, compared with the bHLH proteins of the E(spl)-C the bHLH proteins of the AS-C evolve faster. The complex can be separated into two clusters that are distinguished by their rates of conservation. L'sc and Sc are well conserved with an identity between D. melanogaster and D. virilis of more than 75% ; in contrast, Ac and Ase are conserved with an identity of less than 69%. Note that the highest divergence that was found between these two species in the E(spl)-C was for M8 with still almost 81% identity (Schlatter, 2005).

Of the four AS-C gene members in D. melanogaster, ase stands out because it is much larger than the other three. In D. virilis, the size increase is even more striking: D.v.Ase is predicted to comprise 619 residues, whereas D.m.Ase is only 486 residues in length. This extension of more than 20% additional residues is caused by multiple insertions of repetitive sequences that code for poly-glutamine (Q), poly-alanine (A) and poly-asparagine (N) stretches. Like in D. melanogaster the unrelated gene pepsinogen-like (pcl) is located between l'sc and ase (Schlatter, 2005).

Genes related to achaete or scute have been identified in a large number of species, from hydra to mouse, and so these are also to be expected in the different insects. The AS-C was most intensely studied in various species of Schizophora flies, apart from Drosophila. The number of genes varies between one and four, however, is not strictly correlated with the position in the phylogenetic tree. For example, AS-C of Calliphora vicina contains three genes, whereas other dipteran flies like Drosophila contain four. Two genes are found in the branchiopod crustacean Triops longicaudatus. In Dipteran flies the expression patterns of the proneural genes are largely varied. This is regulated by positional information through the Iroquois Complex and pannier and in addition by a transcriptional feed-back loop involving AS-C proteins. Eventually, neural precursors are selected by the repressive activity of E(spl) bHLH proteins. In this way, location and number of the large bristles on the notum is precisely controlled. The mosquito is covered with rows of large sensory bristle, where number and position varies between individuals. This is in accordance with the fact that there is only one scute-like gene, A.g.ash that is expressed all over the presumptive notum in a modular pattern. Recently it was shown that the Anopheles A.g.ash gene can mimic the endogenous Drosophila genes and that overexpression leads to many ectopic bristles (Schlatter, 2005).

Although the bristle pattern on the notum of different Drosophilids varies slightly, bristle number and position is highly stereotyped. Therefore, it is not surprising to find the AS-C highly conserved within Drosophilids. Yet, the rate of change came unexpectedly and is quite remarkable outside of the bHLH domain. Compared to E(spl) bHLH proteins, those encoded by AS-C have a rather low degree of similarity, most notably Ac. In fact, the big flesh fly Calliphora vicina, which like Drosophila belongs to the Schizophora, is totally lacking the ac gene and is covered with bristles. In agreement, no ac was found in Anopheles or Apis, arguing for rapid evolution. The best conservation rate is found in Sc and L'sc suggesting high evolutionary pressure and maybe common ancestry. Not only the bHLH domain, but also two small stretches outside (aa 203; SPTPS in D. melanogaster L'sc) and also the C-terminus are of high similarity, the latter found identical in Calliphora. Presumably these protein domains are of functional importance. Indeed, the C-terminus acts as a transcriptional activation domain and is also used to recruit E(spl) bHLH proteins. Although the alignments of the respective genes of honeybee and mosquito to sc and l'sc are very similar, the tendency is toward a closer relationship to l'sc. However, it is proposed that this gene pair arose by duplication in the course of Drosophilid evolution, such that a common ancestor may be present in the other two species (Schlatter, 2005).

The rate of conservation is very limited for the Ase homologs. Decent conservation is found within the bHLH domain, and moreover, a further well-conserved box is present (NGxQYxRIPGTNTxQxL; x are differences between A. gambiae and D. melanogaster). This sequence is likewise detected in the Ase protein of C. vicina, which shares many more similarities with D.m.Ase. In Apis, there is no such conservation outside of the bHLH domain, which itself is highly diverged. The overall degree of conservation is so poor that further statements about the relationship are difficult. It is argued that this gene represents A.m.ase by its close proximity to A.m.ash, although other interpretations are similarly possible. An analysis of its expression pattern in honeybee may help to solve these questions (Schlatter, 2005).

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. This study found that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and genes of the Enhancer of split complex [E(spl)-C] were identified as the major targets of this repression. In addition, it was found that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. It is proposed that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation (Bardin, 2010).

Adult stem cells self-renew and, at the same time, give rise to progeny that eventually differentiate. This work provides evidence that one of the strategies used to maintain the identity of ISCs in Drosophila is to repress the expression of Notch target genes. Consistent with this finding, the loss of a general regulator of transcriptional repression, the Histone H2B ubiquitin protease Scrawny, gives a similar phenotype to Hairless (Buszczak, 2009). Additionally, several recent studies indicate that transcriptional repression of differentiation genes may be a central hallmark of stem cells in general (Bardin, 2010).

Drosophila larval neurogenesis is an excellent system for studying the balance between self-renewal and differentiation of a somatic stem cell (neuroblast). Neuroblasts (NBs) give rise to differentiated neurons and glia via intermediate precursors called GMCs or INPs. E(spl)mγ, E(spl)mβ, E(spl)m8 and Deadpan (Dpn), members of the basic helix-loop-helix-Orange protein family, are expressed in NBs but not in differentiated cells. Double mutation for the E(spl) complex and dpn severely affects the ability of NBs to self-renew, causing premature termination of proliferation. Single mutations produce only minor defects, which points to functional redundancy between E(spl) proteins and Dpn. Expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling from the GMC/INP daughter to the NB. When Notch is abnormally activated in NB progeny cells, overproliferation defects are seen. This depends on the abnormal induction of E(spl) genes. In fact E(spl) overexpression can partly mimic Notch-induced overproliferation. Therefore, E(spl) and Dpn act together to maintain the NB in a self-renewing state, a process in which they are assisted by Notch, which sustains expression of the E(spl) subset (Zacharioudaki, 2012).

This paper presents an analysis of the expression and function of two types of bHLH-O proteins expressed in Drosophila neuroblasts, Dpn and the E(spl) family. The main conclusions are that (1) these two types of factors have distinct expression modalities: E(spl)mγ and m8 are targets of Notch signalling, whereas Dpn is not; (2) these factors have redundant functions to maintain NBs in a self-renewing state in normal development, yet (3) in a pathological NB hyperproliferation context, Dpn and E(spl) have distinct functions (Zacharioudaki, 2012).

It was heretofore thought that embryonic NBs are cells that escape from Notch signalling, which is only perceived in the surrounding neuroectoderm. Only in the later post-embryonic period, were the NBs thought to respond to Notch. This study has shown that this happens much earlier, already in the embryonic NBs. Soon after the NB delaminates, a time when it sends, but does not receive, a Notch signal, it starts asymmetrically dividing. The results are consistent with the daughter GMCs sending a Delta signal back to their sister NBs, thereby initiating E(spl) expression. E(spl)mγ expression ceases when the NB enters quiescence, only to restart when proliferation resumes (Zacharioudaki, 2012).

Dpn, another bHLH-O protein, is also expressed in NBs, but much less dynamically. Its expression initiates upon NB delamination from the neuroepithelium and persists throughout its life. dpn does display some degree of dynamic expression, as it is rapidly turned off in the immature intermediate progenitors (iINPs), only to be reactivated upon maturation. Loss-of-function data clearly indicate that it is not a target of Notch in the NB, in contrast to E(spl). Paradoxically, dpn is induced upon Notch hyperactivation. This could be an indirect effect mediated through E(spl). Indeed, E(spl)mγ overexpression can induce ectopic dpn expression. Still, dpn does harbour a Notch-responsive enhancer that drives expression in larval NBs. This same region scored positively for Su(H) binding in a ChIP-chip approach in a cell line of mesodermal origin. How this enhancer contributes to the overall expression pattern of dpn will be a matter of future analysis (Zacharioudaki, 2012).

Despite their different expression modalities, Dpn and E(spl) have redundant functions in the larval NBs, as only double mutant clones show proliferation defects. These mutant NBs do not stop proliferating immediately, rather gradually terminate their cycling within a few days following homozygosing of the mutant alleles. It is proposed that Dpn/E(spl) keep the NB in an undifferentiated state and proliferation is a consequence of the ability of these cells to respond to mitogens. Upon Dpn/E(spl) loss, this state becomes unstable and prone to switch to a terminally differentiated state. This transition takes a few days, probably reflecting the time needed to accumulate pro-differentiation factors. A redundant role of Dpn/E(spl) in maintaining the undifferentiated state also during quiescence transpired from the genetic analysis of NB re-activation after embryogenesis. Whereas dpn^–/– NBs quite successfully re-entered the cell cycle, dpn^–/–;E(spl)^+/– NBs were unable to do so, despite trophic growth factor stimulation (Zacharioudaki, 2012).

E(spl) expression has been associated with the less differentiated of two alternative outcomes in other instances. For example, during NB formation, E(spl) genes are expressed in the undifferentiated embryonic neuroectoderm and not in the NBs. The same happens in the optic lobe neuroepithelium. This work has presented evidence for a similar role for Dpn/E(spl) in the NB. Excessive Dpn/E(spl) activity in GMCs/INPs can revert these partially differentiated cells back to a NB-like fate. For this reason, NB asymmetric divisions must ensure that Dpn and E(spl) are never expressed in the GMC or iINP. Regarding E(spl), it is proposed that this is ensured by the directionality of Notch signalling (GMC to NB). Dpn is also never seen to accumulate in the GMCs/iINPs, suggesting a repression mechanism at work in these cells, e.g., via Pros. These modes of transcriptional control are probably combined with active protein clearance by degradation (Zacharioudaki, 2012).

An anti-differentiation role has also been proposed for vertebrate homologues of Dpn/E(spl), the Hes proteins. Hes1, Hes5 and Hes3 are all expressed in proliferating neural stem cells of the embryonic CNS. Upon Hes knockout, neural stem cells prematurely differentiate resulting in a hypoplastic nervous system, with increasing severity as more Hes genes are lost. In an interesting analogy, only Hes1 and Hes5 are direct targets of Notch signalling. Another example where anti-differentiation during quiescence is mediated by high Hes1 expression are cultured fibroblasts and rhabdomyosarcoma cells. Similar to what was observed with Dpn/E(spl)-mutant embryos, a quiescence trigger, like serum depletion, can result in irreversible cell-cycle withdrawal, if Hes1 activity is compromised (Zacharioudaki, 2012).

The results have shed light on the paradox of why Notch loss of function has only minor effects in larval neurogenesis, whereas its hyperactivation causes significant overproliferation. Notch loss of function decreases E(spl) expression, leaving Dpn levels unaffected. Furthermore, Notch pathway disruption does not seem to directly affect NB proliferation, as Cyclin E expression is not eliminated. Therefore, Notch signalling from the GMC/iINP to the NB acts to ensure robustness in NB maintenance, in collaboration with Dpn (Zacharioudaki, 2012).

When Notch signalling is aberrantly activated in the GMCs/iINPs, both type I and type II lineages overproliferate, although the former do so with lower penetrance (fewer lineages) and expressivity (smaller clones). Yet, for both types of lineages, E(spl) genes are necessary to implement overproliferation. This is consistent with the hypothesis that ectopic E(spl)/Dpn activity in the GMCs/iINPs inhibits their differentiation and makes them competent to respond to mitogenic stimuli (Zacharioudaki, 2012).

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. This study demonstrates that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). Evidence is provided that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. The results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins (Li, 2017).

This study demonstrates that similar to the canonical Notch signaling, Dpn maintains the identity and self-renewal of type II NBs at least in part by inhibiting Erm expression. Loss of Dpn leads to the ectopic activation of erm in type II NBs and that removing Erm not only prevents the transformation of dpn mutant or Dpn knockdown type II NBs into type I-like NBs but also largely inhibits their premature termination of self-renewal. The results from gel-shift assays and reporter assays provide evidence to support that Dpn and E(spl) proteins suppress Erm expression by directly binding to at least two of the three putative bHLH-O binding sites in the erm enhancer (Li, 2017).

Although Dpn and canonical Notch signaling could function through a similar mechanism, these factors do not appear to be completely functionally redundant as previously suggested. First, during early 1st instar larval stages when type II NBs are still quiescent, the maintenance of type II NBs may mainly rely on Dpn in that Notch is not activated in quiescent type II NBs, as evidenced through the findings showing that the loss of Dpn at early 1st instar larval stages leads to ectopic Erm-mediated transformation and the premature loss of type II NBs. Second, after reactivation of type II NBs, both Dpn and Notch signaling are required to suppress the ectopic Erm expression in type II NBs because both the loss of Dpn and the components of the canonical Notch signaling pathways alone lead to ectopic Erm expression in type II NBs. However, the Notch signaling likely plays a dominant role in suppressing ectopic Erm expression and maintaining type II NBs. It has been previously shown that the loss of components of the canonical Notch pathway, including E(spl) proteins, leads to ectopic Erm expression and the transformation and premature loss of type II NBs, despite the presence of Dpn in the NBs, whereas the knockdown of Dpn after the reactivation of NBs only results in the weak ectopic activation of erm but not transformation or premature loss of type II NBs. Therefore, Dpn and Notch signaling may not be completely functionally redundant in suppressing the ectopic Erm expression or maintaining type II NBs, and their functions might be dependent on developmental stages. Furthermore, a recent study reported that Klu could also bind to the R9D11 enhancer to repress the expression of Erm. Thus, type II NBs likely utilize multiple mechanisms to ensure that erm will not be prematurely activated (Li, 2017).

Previous studies suggested that all E(spl) proteins share similar DNA sequences. However, results from the present study suggest that this similarity may not always be the case. Gel-shift assays show that only members of the E(spl) family, including E(spl)mγ, mβ, mδ, m3, and m7, can bind to the bHLH-O binding sites in the erm regulatory region, whereas the other two, E(spl)m5 and m8, cannot. The difference in their DNA binding specificity is consistent with differences in the amino acid sequences of their bHLH domains and their overexpression phenotypes in type II NB lineages. Therefore, although multiple E(spl) proteins have been shown to be expressed in larval NBs and at least two of them, E(spl)mγ and m8, are activated by Notch, these E(spl) proteins may bind to different DNA sequences and regulate the expression of different target genes, which may in turn determine their functional specificity (Li, 2017).

In contrast to the maintenance of type II NBs, the maturation of imINPs requires the activation of erm by PntP1 and shutdown of Dpn expression and Notch signaling. It has previously been shown that the loss of Erm or aberrant activation of dpn or Notch signaling in imINPs both lead to the dedifferentiation of imINPs and overproliferation of type II NBs. However, the functional relationship between the activation of erm and the absence of Dpn or Notch signaling in imINPs has never been established. This study demonstrates that the absence of Dpn and Notch signaling is essential for the activation of erm and subsequent Erm-mediated maturation of INPs. First, the results show that aberrant activation of dpn or Notch signaling inhibits the activation of erm in imINPs. Second, maintaining Erm expression in imINPs largely blocks the overproliferation of type II NBs resulting from the misexpression of E(spl) or Dpn proteins, suggesting that one main reason for the dedifferentiation of imINPs caused by Dpn or E(spl) overexpression is the suppression of Erm. However, the overproliferation of type II NBs resulting from the overexpression of Nintra or Numb knockdown can only be partially suppressed by concomitant Erm expression. Therefore, in addition to functioning through the canonical pathway to activate E(spl) expression, Notch may also act through noncanonical pathways, such as the mTORC2/Akt pathway, to regulate type II NB proliferation (Li, 2017).

How does Erm promotes INP maturation and prevents the dedifferentiation of imINP? It has previously been suggested that Erm prevents the dedifferentiation of INPs by activating pros expression and attenuating the response of INPs to self-renewing factors such as Dpn and E(spl) proteins. However, two pieces of evidence argue against this notion. First, the loss of Pros only induces the overproliferation of INPs but not the dedifferentiation of imINPs into type II NBs . Second, Erm is only expressed in imINPs, which do not express Dpn or E(spl) proteins. In the present study, evidence is provided demonstrating that Erm likely promotes INP maturation in part by inhibiting the expression and/or function of PntP1. These results show that the overproliferation of type II NBs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins, which leads to suppression of Erm expression, could be significantly inhibited by knocking down PntP1. These data strongly argue that the dedifferentiation of imINPs and generation of extra type II NBs resulting from the loss of Erm is in part due to de-repression of PntP1 expression and/or function in imINPs, which is consistent with the PntP1 function in specifying type II NBs and suppressing the activation of ase. Similar to other Ets family proteins that are commonly involved in tumorigenesis, PntP1 may also activate the expression of cell cycle regulators that promote nonproliferative imINPs to enter the cell cycle and initiate unrestricted tumorigenic overproliferation. However, PntP1 may not be the only target of Erm in imINPs. As shown in a recent study, in addition to PntP1, Erm also directly inhibits the expression of Grh-O in imINPs (Janssens, 2017). Therefore, Erm likely promotes the maturation of INPs by regulating the expression/function of multiple target genes (Li, 2017).

In conclusion, this study demonstrates here that similar to Notch signaling, Dpn maintains the identity and self-renewal of type II NBs in part by inhibiting Erm expression. Whereas in imINPs, the absence of Dpn and E(spl) proteins allows PntP1-mediated activation of erm, which in turn promotes INP maturation by inhibiting the expression and/or function of PntP1 and Grh-O in imINPs. Thus, the present study elucidates the mechanistic details of the maintenance of type II NBs and maturation of INPs (Li, 2017).