Enhancer of split

DEVELOPMENTAL BIOLOGY

Embryonic

E(spl) is transcribed in the late blastoderm [Image]. It is first concentrated in two strips, each 2-3 cells wide on either side of the embryo in the neurogenic ectoderm ventral midline anlage, immediately dorsal of the mesodermal anlage (precursor), extending along the entire anterioposterior axis.

Transcript is present in the entire ectoderm during germ band extention, with the exception of some regions in the procephalic lobe, stomodeum and proctodeum [Images]. Transcripts become restricted to the epidermis during the extended germ band stage. In stage 11, transcripts are detected in the entire mesoderm. Only slight differences in the distribution of transcripts are found when comparing E(spl) with the other members of the E(spl)-C (Knust, 1987). Although little attention is paid to these differences, they likely represent some independent regulation of the different genes of the E(spl)-C, but it has been difficult to pinpoint.

E(spl)-C expression in the head starts during stage 8 in the central protocerebral, followed slightly later by the central deuterocerebral domain. E(spl)-C genes remain expressed at a high level in the both domains for several hours after neuroblasts have delaminated from these regions. During stage 10, the expression progresses and ultimately covers most of the procephalic ectoderm (Younossi-Hartenstein, 1996).

In head midline structures, in particular the optic lobe and stomatogastric nervous system, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express l’sc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).

Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless [Su(H)] mediate transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex [E(spl)-C], encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. The E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and malpha are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide. The five genes described in this paper are arrayed between mbeta and m7, both coding for bHLH proteins. Two other bHLH genes, m3 and m5 are intermingled with the five. Bearded and M4 are 16% identical. Furthermore, in transcripts of both Brd and m4 there are three common regulatory sequence motifs within the 3' UTR. These are known as the 'Brd box', the 'GY box' and the 'K box'. As in m4, the sequence motif of the Brd box is found twice in the 3'-UTR of malpha mRNA at similar positions but without a GY box. None of the other four non-bHLH E(spl)-C genes contains either Brd or GY box. The K box appears to be more common. It is found twice in the 3'-UTR of malpha and once each in the 3' UTRs of m2 and m6 (Wurmbach, 1999).

malpha and m4 embyonic expression patterns are nearly indistinguishable, and appear very similar to those of E(spl)-C bHLH genes, particularly m5, m7 and m8. The expression patterns suggest that both genes are under the same regulatory control as are the E(spl) bHLH genes and thus, might serve a role in Notch mediated cell differentiation. Surprisingly, also m2 transcripts accumulate in a pattern reminiscent of the transcript distribution of E(spl) bHLH genes, although there are no structural similarites with either the bHLH or the m4/malpha genes. Therefore m2 might serve as a Notch target gene. Unlike the other E(spl)-C genes, the gene is expressed within neuronal cells in the embryo. m6 mRNA accumulates in the CNS, brain and PNS, and in imaginal tissues. m1 is expressed in the digestive tract. Su(H) is shown to be the transmitter of Notch signaling to malpha, m4 and m2. Thus there are three types of Notch responsive genes. The bHLH genes are represented by m8 and others. m4 and malpha share structural similarity with Bearded. These Bearded family proteins share a presumptive basic amphipatic alpha-helical domain but differ with regard to other conserved sequence elements. m2, coding for a novel protein, represents the third class of Notch responsive genes (Wurmbach, 1999).

Robustness and flexibility and the role of lateral inhibition in the neurogenic network

Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? A model is presented of the core neurogenic network in Drosophila. This model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, the model exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms these test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. Two new measures are introduced for quantifying network robustness that mimic recombination and allelic divergence and these were used to reveal the shape of the domain in the parameter space in which the model functions. Lateral inhibition yields robustness to changes in prepatterns and a reconciliation of two divergent sets of experimental results is suggested. Finally, it is shown that, for this model, robustness confers functional flexibility. It is concluded that the neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. The model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification (Meir, 2002).

The experimental literature includes both support for, and refutation of, an important role for lateral inhibition in neural determination. The results of this analysis can account for both sets of experiments. If the prepattern that initiates neuroblast selection is well tuned, the prepattern plus a constant level of inhibition could select the winner, absent lateral inhibition. But lateral inhibition buffers the patterning against perturbations in the initial prepatterning (e.g., due to genetic or environmental variation, or 'developmental noise'). Seugnet (1997) reported that, with only constant production of Dl, 80% of proneural clusters developed normally, but 20% produced an extra NB. These experiments are interpreted to say that the prepattern is well tuned in most proneural clusters, but in 20%, either a poorly tuned prepattern or noise causes errors in the absence of lateral inhibition. This is a testable idea. One could remove lateral inhibition as Seugnet did. It would then be predicted that the embryo would be much more sensitive to hyper- and hypo-morphs in prepatterning genes such as extramachrochaete and hairy. It would also be predicted that such embryos would be more sensitive to mutations in genes within the network itself, such as missing or extra copies of Dl or N. The latter prediction is made because those mutations should change the threshold to which the prepattern is tuned. In the absence of lateral inhibition, a prepattern that was well tuned to the former threshold could not also be well tuned to the new threshold (Meir, 2002).

From these results, it is deduced that E(spl) greatly reduces the percentage of random parameter sets that enable lateral inhibition. It is believed this is because E(spl) acts as a homeostat. As the expression levels of the proneural genes (ac and sc) rise, their products activate E(spl). E(spl) then downregulates the proneural genes. As with the thermostat in a house, this negative feedback loop tends to keep the proneural genes at an intermediate level rather than allowing them to switch to either a high or low state. Both E(spl) autoinhibition, and to a lesser extent cis-Dl inhibition of N activation, help overcome this homeostat. On the face of it, this seems a strange design. The ac/sc network itself is a bistable switch that tends to go in the direction it is pushed and remain there. The switch and homeostat mechanisms are exact opposites. Removing the homeostat (the reduced model) makes it easier to find parameter sets that pass various tests (which all involve throwing the switch). Why incorporate counteracting mechanisms in the same circuit (Meir, 2002)?

It is, of course, possible that this is simply a vestige of the network's evolutionary history, with no design rationale. But electrical engineering suggests one possible advantage. An op-amp is a famous circuit that amplifies the difference between two inputs. Good op-amps can amplify a voltage difference more than a million-fold. Usually, though, engineers add a negative feedback circuit (that is, a homeostat). This greatly attenuates the gain but makes the amplifier much more stable; noise generated internally inside the op-amp will not affect the output signal. Reducing function to gain stability is common in other electrical circuits as well. These electrical circuits do not make good direct analogies to genetic networks, but the concept of adding negative feedback to increase stability might still apply. Perhaps the E(spl) homeostat reduces the network's sensitivity to developmental noise such as stochastic changes in transcription or translation rates, in the prepattern, or in the concentrations of modulators such as Da and Emc (Meir, 2002).

A related design benefit might be that the E(spl) homeostat prevents the network from switching individual cells on or off before the prepattern has a chance to decree the winner. A simple bistable switch consisting of ac and sc alone could not help but be thrown in one direction or the other by noise (as apparently takes place in C. elegans anchor cell specification). Adding E(spl) leads to a new, neither-on-nor-off steady state, which could enable the proneural switch to procrastinate until some extrinsic cue forces the system to choose one or the other switched state (Meir, 2002).

Larval

Enhancer of split complex genes manifest distinct patterns of expression in the wing imaginal disc. m8 and m7 mRNAs are detected in clusters of cells that correspond to the locations where sensory organ precursors (SOPs) develop. In addition m8 is also detected in cells at the dorsal/ventral boundary throughout the third instar. The expression of mgamma and mdelta is at times associated with the same SOPs, and at other times, with different SOPs. However mdelta and mgamma mRNAs are only detected in a subset of proneural clusters. Like m8, mgamma is also present at high levels at the dorsal/ventral boundary in early stages. The domain of mß is the most distinctive. It is expressed in the wing blade associated with developing veins, and is also present at the dorsal/ventral boundary and wing margin, and is expressed in a complex pattern elsewhere in the disc, with no simple association with developing sensory organs (de Celis, 1996). In the eye disc, m8 and m7 are expressed spanning the morphogenetic furrow, whereas mgamma and mdelta are expressed just posterior to the furrow. mgamma, mdelta and mß are expressed in the more posterior portions of the disc, where the recruitment of undifferentiated cells into ommatidial units occurs; there is little expression of m8 and m7 in this region (de Celis, 1996).

Effects of Mutation or Deletion

Ectopic expression of m5 or E(spl), both members of the E(Spl)-C, before bristle precursor division results in loss of sensory bristles from all parts of the adult fly. Ectopic expression after bristle precursor division produces bristles with aberrant cuticular structures. Reducing E(spl) gene function using mitotic recombination de-represses the neural fate and produces supernumerary sensory bristle neurons. Thus E(spl) inhibits neural fate during the selection of neural precursors, and also plays a role in restricting the neuronal fate to one of the four progeny cells of the bristle precursor (Tata, 1995).

Comparison between the phenotypes produced by Notch, Suppressor of Hairless and Enhancer of split mutations in the wing and thorax indicate the Su(H) and Notch requirements are not indistinguishable, but that Enhancer of split activity is only essential for a subset of developmental processes involving Notch function. For example Enhancer of split function is required for the segregation of a single sensory organ precursor in in wing morphogenesis but not for the correct differention of the progeny from each sensory organ precursor, requiring Notch and Su(H). Likewise, the ectopic expression of Enhancer of split proteins does not reproduce all the consequences typical of ectopic Notch activation. For example, no ectopic acitvation of wingless occurs when Enhancer of split proteins are ectopically expressed. It is suggested that the Notch pathway bifurcates after the activation of Su(H) and that Enhancer of split activity is not required when the consequence of Notch function is the transcriptional activation of downstream genes. Transcriptional activation mediated by Su(H) and transcriptional repression mediated by Enhancer of split could provide greater diversity in the response of individual genes to Notch activity (de Celis, 1996)

In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most superficial cell from the promuscular cluster, thus suggesting a role for the overlying ectoderm during its segregation. . Since epidermal Wg and Hedgehog (Hh) signaling has been shown to influence muscle formation, the SBM-associated lb expression was examined in embryos carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM progenitors. The initial influence of these signals is no longer observed later in development. In addition to signals from the epidermis, the activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of the majority of SBM fibers. During promuscular cluster formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an unknown factor. The lack of neurogenic gene function, known to be involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).

Eye development in Drosophila involves the Notch signaling pathway at several consecutive steps. At first, Notch signaling is required for stable expression of the proneural gene atonal (ato), thereby maintaining the neural potential of the cells. Subsequently, in a process of lateral inhibition, Notch signaling is necessary to confine neural commitment to individual photoreceptor founder cells. Later on, the successive addition of cells to maturing ommatidia is under Notch control. In contrast to previous assumptions, the recessive Notch allele split (N^spl) specifically involves loss of the early proneural Notch activity in the eye, which is in agreement with bristle defects as well. As a result, fewer cells gain neural potential and fewer ommatidia are founded. N^spl alleles are characterized by a smaller number of ommatidia, which usually contain less than the normal set of photoreceptors. Enhancement of this phenotype by the dominant mutation Enhancer of split [E(spl)^D] happens within the remaining proneural cells (in which Ato expression has been abolished). In line with genetic data, this process occurs primarily at the protein level due to altered protein-Protein Interactions and Post-transcriptional Regulation between the aberrant E(spl)^D and proneural proteins. Indeeed, in a yeast two-hybrid assay, the mutant M8*, representing the E(spl)^D alteration, binds significantly more strongly to proneural proteins, especially Achaete and Atonal. The mutant M8* protein does not interfere with the establishment of high Ato levels within intermediate-group cells. In contrast, m8* transcripts accumulate to a much higher level due to increase of mRNA stability caused by the deletion. Heterodimerization of M8* with other E(spl) bHLH proteins is indistinguishable from that of the wild-type M8 protein. Therefore the N^spl mutation reduces the inductive, proneural activity of N, which is normally required to stabilize expression of the proneural gene atonal. As a consequence fewer intermediate groups arise: these groups serve as reservoirs for future R8 cells. E(spl)^D potentiates the deficits of N^spl because in the compromized background the already lowered Ato levels in most presumptive R8 cells now drop below the threshold required to maintain neuronal fate. N^spl is the first Notch mutation known to specifically affect Notch inductive processes during eye development (Nagel, 1999).

The organization and function of the Notch signaling pathway in Drosophila are best understood with respect to the role of this pathway in the process of selection of neural progenitor cells. However, there is evidence that, in addition to neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these progenitor cells is increased in neurogenic mutants. It has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Additional elements of the Notch signaling pathway participate in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells (Giebel, 1999).

To test whether the Notch signaling pathway is involved in myogenesis, the effects of expression of a constitutively active Notch protein (Notch^intra) were examined. A second chromosomal effector line with an UAS-Notch^intra construct was used. This construct led to complete blocking of neural development upon activation with daG32. Embryos carrying that construct driven by daG32 or by 24B-Gal4, respectively, do not express any of the muscle founder cell markers S59 and Kruppel in the mesoderm. Therefore, it is assumed that no muscle progenitor cells are specified in these animals. Confirmation of this assumption is provided by the observation that no muscle fibers differentiate in these embryos, as shown by means of the expression of a myosin heavy chain (MHC) reporter gene. In mutants where fusion of myoblasts is blocked, founder cells express corresponding founder cell markers, while the non-founder myoblasts remain as undifferentiated rounded cells, which express certain muscle specific genes like myosin. Since Notch^intra expressing mesodermal cells are rounded and many of them express the MHC reporter, it is assumed that the MHC expressing cells are non-founder myoblasts that have failed to undergo fusion due to the lack of muscle founder cells (Giebel, 1999).

Further evidence for a Notch pathway role in myogenesis was obtained by overexpressing UAS-E(spl) in the mesoderm. Following Gal4 mediated activation of UAS-E(spl), the number of S59 and Kruppel positive cells is strongly reduced. This correlates with a defect in the number of differentiated muscle cells, as shown by MHC reporter gene expression. Again these data fit well with the results obtained on the development of the neuroectoderm, in which Gal4 driven UAS-E(spl) expression leads to strong reduction of CNS and PNS structures (Giebel, 1999).

echinoid (ed) encodes an immunoglobulin domain-containing cell adhesion molecule that negatively regulates the Egfr signaling pathway during Drosophila photoreceptor development. A novel function of Ed is shown, i.e., the restriction of the number of notum bristles that arise from a proneural cluster. Thus, loss-of-function conditions for ed give rise to the development of extra macrochaetae near the extant ones and increase the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, loss-of-function of ed reduces the expression of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss-of-function conditions for ed and for different components of the N pathway show clear synergistic interactions manifested as strong neurogenic bristle phenotypes. It is concluded that Ed is not essential for, but it facilitates, N signaling. It is known that the N and Egfr pathways act antagonistically in bristle development. Consistently, it is found that Ed also antagonizes the bristle-promoting activity of the Egfr pathway, either by the enhancement of N signalling or, similar to the eye, by a more direct action on the Egfr pathway (Escudero, 2003).

On the mechanism underlying the divergent retinal and bristle defects of M8* (E(spl)D) in Drosophila

Multisite phosphorylation has been implicated in repression by E(spl)M8. It is proposed that these phosphorylations occur in the morphogenetic furrow (MF) to reverse an auto-inhibited state of M8, enabling repression of Atonal during R8 specification. These studies address the paradoxical behavior of M8*, the truncated protein encoded by E(spl)D. It is suggested that differences in N signaling in the bristle versus the eye underlie the antimorphic activity of M8* in N⁺ (ectopic bristles) and hypermorphic activity in N^spl (reduced eye). Ectopic M8* impairs eye development (in N^spl) only during establishment of the atonal feedback loop (anterior to the MF), but is ineffective after this time point. In contrast, a CK2 phosphomimetic M8 lacking Groucho (Gro) binding, M8SDDeltaGro, acts antimorphic in N⁺ and suppresses the eye/R8 and bristle defects of N^spl, as does reduced dosage of E(spl) or CK2. Multisite phosphorylation could serve as a checkpoint to enable a precise onset of repression, and this is bypassed in M8* (Kahali, 2009).

Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

Notch signaling mediates multiple developmental decisions in Drosophila. This study examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, these results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells (Wang, 2011).

This study find that Notch signaling plays an essential role in the maintenance and expansion of neuroepithelial cells in the optic lobe; it also inhibits medulla neuroblast formation. Clonal analyses of several pathway components indicate that this dual function bifurcates downstream of Su(H) with E(spl)-C only partly involved in the inhibition of medulla neuroblast formation but not the maintenance and expansion of neuroepithelial stem cells (Wang, 2011).

In the optic lobe, Notch signaling plays a role analogous to lateral inhibition during embryonic CNS development. However, the selection of neuroblasts in the OPC neuroepithelium is an all-or-none process rather than selecting individual neuroblasts from the neuroepithelium. Medulla neuroblasts are generated in a wave progressing in a medial to lateral direction in the OPC neuroepithelium with all cells at a particular position along the medial-lateral axis differentiating into neuroblasts. Interestingly, this wave of medulla neuroblast formation coincides with the down-regulation of both Delta and Notch expression in the medial cells in the OPC, which might reduce Notch signaling activity, thereby allowing medulla neuroblasts to form. What factors drive the recession of both Delta and Notch expression in the OPC neuroepithelium along the medial-lateral axis is not known. When Notch signaling is inactivated, neuroepithelial cells in the OPC change cell morphology and differentiate into medulla neuroblasts prematurely. The results indicate that Notch signaling actively controls neuroepithelial integrity, possibly by regulating the adherens junction (AJ), since in Notch pathway mutant mosaic clones in the OPC, the apical determinants PatJ, Crumbs and aPKC are cell autonomously reduced or lost and the mutant cells change to rounded or irregular morphology. Further experiments will be needed to determine how Notch signaling activity affects the maintenance of neuroepithelial integrity, particularly the stability of the adherens junction (Wang, 2011).

Is neuroblast formation also actively inhibited by Notch signaling or simply a default state of neurogenic epithelial cells? In the latter model, Notch signaling may only maintain neuroepithelial integrity and promote their expansion while medulla neuroblasts form when the neuroepithelial integrity is disrupted. The argument against this model is that changes in neuroepithelial integrity are not always accompanied with cell fate changes. In N, Dl or Su(H) mosaic clones located in the OPC neuroepithelium, it was found that in about 25% of the clones, the mutant cells changed morphology or lost apical marker expression but did not become neuroblasts (Dpn-negative), whereas in E(spl)-C mosaic clones, Dpn⁺ cells were prematurely induced, which indicate that the cells begin to differentiate into neuroblasts, but these cells still retained columnar epithelial cell morphology and apical marker expression. This suggests that the suppression of neuroblast formation by Notch signaling activity is separable from the maintenance of neuroepithelial integrity and that medulla neuroblast formation is actively suppressed by Notch signaling. A possible scenario is that activation of the Notch pathway turns on the E(spl)-C genes, which in turn suppress proneural gene expression in the optic lobe neuroepithelia. Indeed, at least one member in the E(spl)-C genes, E(spl)m8, appears to be activated in the neuroepithelial cells by the Notch pathway, as the E(spl)m8-lacZ reporter is expressed in a pattern similar to Delta and Notch expression in the OPC and IPC. E(spl)m8 protein and possibly additional members of the E(spl)-C may suppress the expression of proneural genes in the optic lobe. The proneural genes of the achaete-scute complex (as-c) comprise four members, achaete, scute, L'sc, and asense. achaete is not expressed in the optic lobe, but scute is expressed in both the neuroepithelial cells and neuroblasts in the OPC implying that scute expression in the neuroepithelial cells is not suppressed by Notch signaling activity. By contrast, asense is only expressed in the neuroblast and GMCs and L'sc is transiently detected in an advancing stripe of neuroepithelial cells of 1-2 cells wide that are just ahead of newly formed medulla neuroblasts. Thus, E(spl)-C proteins may suppress L'sc and/or ase expression, the release of this suppression may allow the neuroepithelial cells to begin to differentiate into medulla neuroblasts. It should be noted, however, that the removal of the E(spl)-C activity does not seem to be sufficient to allow full differentiation of neuroepithelial cells into medulla neuroblasts, suggesting that additional factors downstream of Notch signaling may be involved in the suppression of medulla neuroblast formation (Wang, 2011).

The phenotypes of Notch pathway mutants are reminiscent of those of JAK/STAT mutants. For example, inactivation of either pathway led to early depletion of the OPC neuroepithelium; either pathway inhibits neuroblast formation, and ectopic activation of either pathway promotes the growth of the OPC neuroepithelium. The remarkable phenotypic similarities in Notch and JAK signaling mutant brains suggest that these pathways may act in a linear relationship such that activation of one pathway is relayed to the second, perhaps by inducing the expression of a ligand. Alternatively, these pathways may act in parallel and converge onto some key downstream effectors or target genes. Further experiments will be needed to test whether Notch interacts with JAK/STAT and if it does, to find out where the interaction occurs during the development of the optic lobe (Wang, 2011).

The roles of Notch signaling in mammalian brain development have been studied intensely. Many Notch pathway components have been examined in knockout mice, which showed defects in brain development. Mice deficient for Notch1 or Cbf all display precocious neurogenesis during early stages of nervous system development. This has led to the view that the role of Notch signaling in the mouse brain is to maintain the progenitor state and inhibit neurogenesis. However, it is not clear from these studies whether the premature neurogenesis in Notch signaling mutant mice was caused by premature differentiation of neuroepithelial stem cells into neurons or by premature differentiation of neuroepithelial stem cells into progenitor cells, which then generated neurons. In fact, it has been proposed that Notch activation can promote the differentiation of neuroepithelial stem cells into radial glial cells, the progenitor cells that generate the majority of neurons in the cerebral cortex. This is based on the observation that ectopic Notch activation using activated forms of Notch1 and Notch3 (N^ICD) caused an increase in radial glial cells as compared to control. The radial glial cells resemble medulla neuroblasts in the Drosophila optic lobe in that they are both derived from neuroepithelial stem cells and undergo asymmetric division to self-renew and generate neurons, although morphologically radial glial cells are still polarized while medulla neuroblasts have lost epithelial characters and are rounded in shape. Based on the current results, it is suggested that Notch signaling maintains the pool of neuroepithelial stem cells and promotes their expansion in both Drosophila and mammals and that the precocious neurogenesis in Notch signaling mutant brains arise due to premature differentiation of the neuroepithelial stem cells into the progenitor cells (Wang, 2011).

However, ectopic Notch activation may indeed promote progenitor cell proliferation in the brain. Ectopic neuroblasts were observed in the medulla cortex when N^ACT was ectopically expressed by the neuroblast/GMC driver insc-Gal4, by ubiquitous expression using hs-Gal4, or when numb¹⁵ mosaic clones were induced at later larval stages when neuroblasts normally begin to form. Since the results have shown that the Notch pathway is not essential for medulla neuroblast formation or self-renewal, the ectopic neuroblasts are a novel phenotype solely induced by ectopic Notch signaling activity. This is consistent with Notch activation promoting ectopic neuroblast formation in the central brain and VNC without being required for neuroblast self-renewal in these regions of the CNS; and Notch has been shown to be an oncogene in mammals. Since the sizes of the ectopic neuroblasts were in the range of GMC or neurons, they may resemble the transit-amplifying (TA) neuroblasts that are found in the dorsal-medial region of the central brain. The origin of these ectopic neuroblasts in the medulla cortex is not clear, but it is unlikely that they are derived from differentiated medulla neurons as ectopic expression of N^ACT using elav-Gal4, which is active in medulla neurons, did not result in ectopic neuroblasts and by the fact that ectopic neuroblasts can be induced in numb¹⁵ mosaic clones, which could only arise from mitotically active cells that include neuroepithelial cells, medulla neuroblasts, and ganglion mother cells (GMCs), but not neurons. The ectopic neuroblasts could be generated by a transformation of GMCs into a neuroblast identity as suggested for ectopic neuroblasts in brat mutant central brains. Ectopic Notch signaling activity may even directly promote the expansion of neuroblasts after they have differentiated from the neuroepithelial cells in the OPC. In either case, ectopic Notch signaling activity may block the normal path of neuronal differentiation and lock the cells in a proliferative state. This is indeed what was observed in numb¹⁵ mosaic clones in which numerous ectopic neuroblasts were induced in the medulla cortex without generating medulla neurons. Perhaps ectopic Notch signaling activity may also promote the proliferation of neural progenitors in vertebrates, such as the radial glial cells in the mouse brain (Wang, 2011).

Notch inhibits yorkie activity in Drosophila wing discs

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. This study shows that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. This cross-talk between the Notch and SWH pathways is shown to be mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). It is proposed that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs (Djiane, 2014).

In order to investigate the possibility of cross talk between the Notch and Sav/Warts/Hippo (SWH) pathways, the expression pattern of ex-lacZ, a reporter of Yki activity, which reveals the places where SWH activity is lowest was compared with NRE-GFP, which gives a direct read out of Notch activity. In the wing pouch these reporters direct expression in patterns that are complementary. Thus, ex-lacZ expression is completely absent from the dorso-ventral boundary where Notch activity, reported by NRE-GFP, is at its highest. Conversely, in late stage discs, ex-lacZ expression is higher in pro-vein regions where Notch activity (NRE-GFP) is low. Because ex-lacZ gives a mirror image of SWH activity, these results suggest that both Notch and SWH pathways are active together in the D/V boundary and are largely inactive in the pro-veins (Djiane, 2014).

The consequences of modulating Notch activity on the expression of ex-lacZ was tested as an indicator of its effects on SWH pathway. Expression of Nicd, the constitutively active form of the Notch receptor, promoted a strong down-regulation of ex-lacZ in the wing pouch. This effect was stronger in the region surrounding the D/V boundary and weaker towards the periphery. Little down-regulation occurred outside the pouch. Conversely, when Notch activity was impaired, through RNAi mediated knock-down in randomly generated overexpression clones, ex-lacZ levels were up-regulated. This effect was also only evident within the wing-pouch. Notch activity is therefore necessary and sufficient for the inhibition of ex-lacZ in the wing pouch, suggesting that it contributes to the normal down-regulation of ex-lacZ at the D/V boundary (Djiane, 2014).

In the wing pouch, ex-lacZ expression requires Yki. Therefore, to mediate the observed inhibition of ex-lacZ expression, Notch could either exert its actions upstream of Yki, by activating the SWH pathway, or downstream of Yki, by inhibiting Yki's transcriptional activity. To determine which of these alternatives is correct, the consequences of Notch activity on ectopic Yki expression were assessed. When over-expressed in a stripe of cells along the A/P boundary, Yki was able to promote strong expression of ex-lacZ at the periphery of the wing pouch. Strikingly, the high levels of Yki were not able to force ex-lacZ expression at the D/V boundary where Notch activity is highest. These results suggest that the actions of Notch, ie ex-lacZ down-regulation, are epistatic to Yki. This was further verified when high levels of Yki were expressed together with high levels of Nicd. In this case, Nicd suppressed the ex-lacZ expression, demonstrating that it wins out over Yki in the wing pouch. However, at the periphery of the discs, Nicd was unable to modify the effects of Yki over-expression on ex-lacZ levels. Taken together these results suggest that Notch-mediated down-regulation of ex-lacZ occurs at the level or downstream of Yki (Djiane, 2014).

Since a major output of Notch pathway activity is the up-regulation of gene expression, whether any of the directly regulated Notch target-genes could be responsible for antagonizing Yki was assessed. Amongst the direct Notch targets identified in wing discs, several are predicted to encode transcriptional repressors. These include members of the HES family, E(spl)mβ, E(spl)m5, E(spl)m7, E(spl)m8 and Deadpan (dpn), as well as the homeodomain protein Cut. All of these proteins are normally expressed at high levels along the D/V boundary, in response to Notch activity, and hence are candidates to mediate the repression of ex-lacZ (Djiane, 2014).

Over-expression of E(spl)mβ, E(spl)m5 or E(spl)m7 repressors had no effect on ex-lacZ expression. In contrast, over-expression of either E(spl)m8 or of dpn resulted in a robust down-regulation of ex-lacZ. The effect differed slightly from that from Nicd expression, in that ex-lacZ expression was not completely abolished and low levels persisted throughout the wing pouch domain. These results indicate that a subset of the HES bHLH proteins have the capability to repress ex-lacZ, and hence are candidates to antagonize Yki. Previous experiments have demonstrated that the E(spl)bHLH genes and dpn have overlapping functions, especially at the D/V boundary. Therefore to determine whether these factors normally contribute to the repression of ex-lacZ, it was necessary to eliminate all of the E(spl)bHLH genes in combination with dpn. To achieve this a potent RNAi directed against dpn was expressed in MARCM clones that were homozygous mutant for a deficiency removing the entire E(spl) complex. No derepression of ex-lacZ was detectable in such clones, suggesting that none of the E(spl)bHLH/dpn genes can account for the repression of ex-lacZ at the D/V boundary or in the wing pouch. Therefore even though E(spl)m8 and dpn expression is sufficient for ex-lacZ repression, they do not appear to be essential in the context of the wing pouch (Djiane, 2014).

An alternative candidate was Cut, which encodes a transcriptional repressor and is expressed at the D/V boundary in response to Notch signaling. Similar to some of the HES genes, over-expression of Cut promoted a down-regulation of ex-lacZ. This was most clearly evident at early developmental stages because Cut induced a strong epithelial delamination at later stages, confounding the interpretation. However no up-regulation of ex-lacZ was detectable when Cut function was ablated, using RNAi, even though Cut levels where efficiently reduced. Thus, as with the HES genes, Cut is capable of inhibiting ex-lacZ expression but does not appear to be essential for the regulation of ex under normal conditions in the wing pouch (Djiane, 2014).

Recent studies have demonstrated that, in the absence of Yki, several SWH target genes are kept repressed by Sd, the DNA-binding partner of Yki. This so-called 'default repression' requires Tondu-domain-containing growth inhibitor (Tgi), an evolutionarily conserved tondu domain containing protein, which acts as a potent co-repressor with Sd. There is no evidence that Drosophila tgiM is a target of Notch in the wing disc, making it an unlikely candidate to mediate the inhibitory effects on Yki-mediated ex-lacZ expression. However, vg, which encodes another Sd binding-partner with a tondu domain, is directly regulated by Notch in the wing pouch. It was therefore hypothesized that Vg could mediate the effects of Notch on Yki function and ex-lacZ down-regulation (Djiane, 2014).

In agreement with the hypothesis, when Vg was over-expressed it strongly inhibited ex-lacZ expression in the pouch and promoted a modest overgrowth of the tissue. This overgrowth is somewhat puzzling since it appears that Yki activity, as monitored by ex-lacZ, is lowered in the presence of excess Vg. How over-expressed Vg triggers overgrowth remains poorly understood, but has been proposed to involve a cross-talk with the wg pathway. More recently, it has been shown that the expansion of the pouch region is achieved by Vg activating transiently and non-autonomously Yki in cells not expressing Vg. These cells are then recruited to become wing pouch cells and turn on vg expression. This model predicts a wave of Yki activation around Vg positive cells. Therefore, the overgrowth seen when Vg is over-expressed, could be due to a non-autonomous effect where more cells are recruited as pouch cells at the expense of more peripheral cells. Alternatively, Vg could promote proliferation of the pouch cells by an as yet unidentified mechanism, independent of Yki (Djiane, 2014).

Conversely to over-expressed Vg inhibiting ex-lacZ expression, lowering the levels of vg using RNA interference in the whole posterior compartment resulted in a significant up-regulation of ex-lacZ. Vg knock down has proven difficult to achieve in small populations of cells, due to their elimination from the wing pouch, probably by cell competition. Thus, unlike the other factors tested, Vg is required for the repression of ex-lacZ in the wing pouch. It was further shown that, co-expressing with NICD a vg RNAi transgene in the patched domain, suppresses the NICD mediated ex-lacZ repression in the wing pouch. Taken together, these results suggest that Vg mediates the repressive effects of Notch on expanded expression (Djiane, 2014).

If the involvement of Vg downstream of Notch is a general mechanism for cross-talk between Notch and Yki, other targets of the Sd-Yki complex should be inhibited by Notch in a similar manner to ex-lacZ. However, apart from expanded, all other known Yki targets in the wing pouch, such as thread/DIAP1, diminutive/myc, and Cyclin E are also direct Notch targets. Their final expression patterns are therefore a reflection of the balance between different transcriptional inputs, in particular Notch and Yki. A model predicts that Notch could have a dual effect on the expression of genes: a positive direct effect through the NICD/Su(H) complex when bound in their promoters, but also a negative effect through the induction of Vg, which prevents the positive effect of Yki on Sd bound promoters (Djiane, 2014).

In agreement with this model, thread/DIAP1 and diminutive/myc, two well established Yki targets in wing discs, which are normally refractory to Notch mediated activation in the centre of the pouch, become susceptible to Nicd when Vg or Sd levels are lowered through RNAi (Djiane, 2014).

Focusing on DIAP1, it was decided to separate the Notch and Yki direct inputs on transcription by isolating the Hippo pathway Responsive Elements (HREs) from any potential Notch Responsive Elements (NREs). IAP2B2C-lacZ is a previously described DIAP1-HRE driving lacZ reporter expression that does not contain any NRE, at least based on Su(H) ChIP data and bio-informatics prediction of Su(H) binding sites. The model predicts that this IAP2B2C-lacZ reporter should be inhibited by Vg. In control wing discs, it was confirmed that IAP2B2C-lacZ is expressed at uniform low levels with a slight increase at the periphery of the pouch, where Vg protein levels have been shown to fade. The D/V boundary expression of DIAP1 is not reported by IAP2B2C-lacZ confirming that the NRE is absent in this reporter (Djiane, 2014).

Vg levels were lowered using moderate RNAi knocked down in the whole posterior compartment using the hh-Gal4 driver. In this experimental set-up, the posterior compartment is smaller than normal, and vg knock-down induced a 12% up-regulation of IAP2B2C-lacZ expression when compared to IAP2B2C-lacZ levels in the anterior control compartment, demonstrating that Vg has a negative effect on this reporter activity (there was no difference in IAP2B2C-lacZ expression between the anterior and posterior compartment in the pouch region of control discs). It is noted that IAP2B2C-lacZ expression was up-regulated in a small stripe of cells in the anterior compartment just at the boundary with vg depleted cell. This region was excluded from the quantifications, but suggests that the IAP2B2C-lacZ reporter fragment could be sensitive to a non-autonomous input acting around the boundary of cells with different Vg levels (Djiane, 2014).

It appears therefore, that at least for the two Yki targets ex-lacZ and IAP2B2C-lacZ, Vg inhibits their expression in the wing pouch. Previous studies reported independent roles of Vg and Yki on the activation of their targets, and could appear to contradict this newly described inhibitory role of Vg on Yki targets. However, in these previous studies, it was demonstrated that Vg and Yki do not require each other to promote wing pouch cell survival and to activate their respective targets, which does not rule out any negative cross regulation, as shown in this report (Djiane, 2014).

This analysis brings therefore new evidence of the central role of Vg in the complex network regulating wing disc growth, adding a new level of complexity in its interaction with the SWH pathway effector Yki. Thus, Notch induced expression of Vg could give rise to an Sd-Vg repressive complex that prevents expression of Yki targets. In situations where SWH signaling is lowest, Yki levels may be sufficiently high to overcome this repression. This suggests that in the wing pouch, Notch and SWH would act co-operatively rather than antagonistically (Djiane, 2014).

Outside of the pouch, at the wing disc periphery, sd and vg expressions are not promoted by Notch activity. Furthermore, other binding partners for Yki, such as Homothorax are expressed there and might substitute for Sd to control the expression of Yki targets in a way similar to what has been described in the Drosophila eye. The differential expression of these transcription factors in the disc could explain why Notch only has an inhibitory effect on Yki targets in the wing pouch. Furthermore, it is also worth noting that Notch has very different effect outside of the pouch, where it promotes Yki stabilization non-autonomously via its regulation of ligands for the Jak/Stat pathway (Djiane, 2014).

In summary, the evidence demonstrates that Notch activity can inhibit Yki under circumstances where Yki acts together with Sd. It does so by promoting the expression of Vg, a co-factor for Sd, counteracting the effects of Yki. This cross talk potentially extends to mammalian systems as the active form of NOTCH1, NICD1 promotes the up-regulation of VGLL3 (a human homologue of vg) in MCF-10A breast cancer derived cells. Thus, similar mechanisms may also be important in mediating interactions between the NOTCH and SWH pathways in human diseases (Djiane, 2014).

Because the end-point of SWH pathway activity is to prevent Yki function, the inhibitory effects of Notch on Yki could provide an explanation for those cellular contexts where the two pathways act co-operatively, as at the D/V boundary in the wing discs. Similar co-operative effects have been noted in the Drosophila follicle cells. However, in this case it is the SWH activity that is involved in promoting the expression of Notch targets. In other contexts, such as the mouse intestine, accumulation of Yap1, the mouse Yki homolog, and therefore inhibition of the SWH promotes Notch activity. These examples demonstrate that the interactions between Notch and the SWH are highly dependent on cellular context. The results suggest that some of these differences may be explained by the nature of the target genes that are regulated and by which Yki co-operating transcription factors are present in the receiving cells (Djiane, 2014).

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