Gene name - Delta
Cytological map position - 92A2
Function - ligand for Notch
Keywords - Notch pathway - neurogenic
Symbol - Dl
Genetic map position - 3-66.2
Classification - transmembrane ligand - EGF family
Cellular location - surface
Delta is one of the principle molecules in the Notch signaling pathway, and like the other proteins in this pathway is designated a neurogenic gene. As will be seen, Delta plays an especially important role in neurogenesis.
Both Delta, and its receptor Notch, are found expressed throughout the neuroectoderm. As a result of the gastrulation process, this layer of cells is positioned in the ventral part of the embryo (the ventral neuroectoderm). These cells are the precursors of neuroblasts, the stem cells from which the ventral nervous system (CNS) will arise. Cells destined to be neuroblasts delaminate from the neuroectoderm and migrate dorsally into the developing ventral nervous system. Delta plays a key role in the selection of cells that assume the neuroblast fate.
Through a process of selection known as lateral inhibition only a few cells in each segment become neuroblasts. It is a competitive process. A single cell, perhaps the one with the highest levels of Delta, sends a message to neighboring cells via the Notch receptor. This results in the inhibition of neuroblast development. The selected cell then delaminates and migrates dorsally. In this way, neuroblasts are born.
Because the Delta-Notch complex functions in cell adhesion (Fehon, 1990) this complex certainly must function in the delamination and migration process. The process of selection and inhibition is repeated many times, in many different aspects of development. The Delta-Notch pathway is highly conserved: Delta and Notch homologs are found throughout the metazoa. One of the major breakthroughs in developmental biology is an understanding of this mechanism, which has come about only in the last decade.
Expression of Delta has been studied during microchaeta (small bristle) development in Drosophila as well as in the regulatory relationships between the Delta-Notch signaling pathway and the proneural gene, achaete. The adult notum (the dorsal surface of thorax) is derived from the fusion of two heminota found at the anterior ends of the two wing/notal imaginal discs. Fusion takes place between 6 and 8 hours after puparium formation (APF). Within each notum, microchaeta sensory organ precursors (SOPs) arise within stripes of proneural cells arrayed from anterior to posterior. The stripe that develops in the center of the adult notum is designated stripe 1, and the stripe that contains the dorsocentral macrochaetae is designated stripe 5. Microchaeta stripes 1, 3, and 5 develop first, followed by stripes 2 and 4. The majority of microchaeta SOPs arise within proneural stripes between 10 and 12 hours APF. Delta is expressed in all microchaeta proneural cells and microchaeta SOPs, and is expressed dynamically in SOP progeny. Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that the initial Delta expression in the notum is not dependent on proneural gene function (Parks, 1997).
The Delta-Notch signaling pathway is required at two steps during proneural cluster formation and SOP specification. (1) Dl prevents specification of supernumerary SOPS. In this function Delta represses achaete expression between microchaeta proneural stripes. Thus Delta function is required to help define microchaeta proneural stripe boundaries. (2) Dl is required later within proneural stripes to repress achaete expression. This second function involves the choice between neural (neuronal and thecogen/glial) and non-neural (tormogen and trichogen support cells) cell fate in the two cells decendent from a single SOP. In the first stage of Delta function, the expression data indicate that Delta is transcribed and Delta protein is localized throughout the entire microchaeta proneural stripe. There are no asymmetries in Delta accumulation within proneural stripes at the transcriptional level; nor is there a decrease in Notch protein levels in nascent SOPs. This is in contrast to the predictions of lateral inhibition models that suggest a Delta-Notch feedback loop might result in higher Delta expression in the cell adopting the SOP fate and higher Notch expression in immediately surrounding cells. Within proneural stripes, therefore the expression data is most consistent with the idea of mutual inhibition, i. e., that microchaeta proneural cells within the entire equivalence group interact via Delta and Notch to inhibit adoption of the SOP fate (Parks, 1997).
After Achaete protein expression has, for the most part, resolved to cells adopting the SOP fate, there do appear to be asymmetries in subcellular Delta protein localization in the vicinity of SOPs. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is post-transcriptionally regulated, in part, by Delta-Notch signaling activity. Thus, Delta signaling is required for correct delineation of early proneural gene expression in developing nota. Later, within microchaeta proneural stripes, Delta-Notch signaling prohibits adoption of the SOP fate by repressing expression of the proneural gene achaete (Parks, 1997).
Not all Delta functions are involved with neurogenesis. During wing margin formation in the wing imaginal disc Delta is required in ventral cells at the dorsal/ventral boundary. Ectopic Delta induces wingless, vestigial and cut and causes adult wing tissue outgrowth in the dorsal compartment. Whereas Delta is required in ventral cells, Serrate, another ligand for Notch is required in dorsal cells, and Notch is required in both. Thus Delta and Serrate function as compartment-specific ligands for Notch in the wing disc, activating Notch, which in turn induces downstream genes required for wing formation (Doherty, 1996).
Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001).
Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001).
Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001).
Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001).
Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001).
These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001).
The simplest explanation of these results is that some signal or signals emanating from the cells posterior to, or within, the morphogenetic furrow are necessary for the specification of a neural competence zone ahead of the furrow. Within this zone, cells can respond to Delta-induced Notch activation by upregulating Atonal expression. A candidate for such a signal is the secreted protein Dpp. Dpp is expressed within the furrow in response to Hh signaling and has been proposed to define a 'pre-proneural' state in a zone anterior to the furrow. In order to analyse whether the function of Dpp is sufficient to generate the condition necessary for the neural activation by Notch signaling, clones that simultaneously express ectopic dpp and Delta were produced (Baonza, 2001).
Clones of cells that express dpp alone only induce neural differentiation along the margin of the eye discs; internal clones have no effect on neural induction. By contrast, clones that co-express Dl and dpp trigger neural differentiation everywhere ahead of the furrow. In all the clones studied, ectopic expression of Atonal and Elav was observed. The induction of neural differentiation occurs in all the cells surrounding the clone and not, as in Delta-expressing clones, only in the cells within the competence zone. In most of the clones analyzed, Atonal expression was found to be associated with an ectopic morphogenetic furrow induced by the clones. Thus, it is possible to observe clones with ectopic Atonal expression several cells away from the border of the clone and with Atonal expression restricted to isolated cells within the clone, reproducing the pattern of Atonal expression of the endogenous furrow. One interpretation of this result is that once Atonal is activated within and in the cells surrounding the clone, the normal cascade of ommatidial development is triggered, inducing an ectopic furrow that begins to move away from the clone (Baonza, 2001).
These observations lead to the conclusion that the expression of dpp is sufficient to enable all cells anterior to the furrow to activate neural differentiation in response to Notch. It is postulated that during normal development, Dpp primes the cells to become competent to differentiate neurally in response to Notch signaling, at a range of 12-15 cells anterior to the furrow (Baonza, 2001).
Loss of Dpp signaling during eye development causes furrow progression to slow down but not to stop: partial redundancy allows Hh signaling to induce neural differentiation in cells in which the Dpp signaling is blocked. Furthermore, clones of ectopic expression of Hh always induce neural differentiation and an ectopic furrow, even beyond the zone of Dpp-influenced cells, indicating that Hh is sufficient to trigger neural differentiation. The current model is that Dpp is important for furrow progression to occur efficiently and at a normal rate, but that it is not essential for neural differentiation to occur. This study shows that Dpp signaling has an important role in promoting the proneural function of Notch signaling by generating the 'pre-proneural' state ahead of the furrow. This does not, however, rule out the possibility that Hh signaling could also produce a similar effect. If the function of Dpp signaling can be rescued by Hh signaling, then it would be expected that the effects of ectopic activation of Notch signaling would be identical in a background where Dpp signaling is blocked (because in this case, Hh would replace Dpp function) (Baonza, 2001).
An examination was made of the effect of the ectopic expression of Delta when Dpp signaling is blocked, by inducing clones that co-express Delta and the negative Dpp signal regulator brinker. The use of brinker expression was evaluated as a way of inhibiting Dpp function in the eye by examining the phenotype of clones of brinker-expressing cells. brinker-expressing clones indeed mimic mad null clones in their ability to prevent the initiation of the morphogenetic furrow when they occur at the posterior margin of the disc (Baonza, 2001).
Double clones of brinker- and Delta-expressing cells only activate Atonal expression when they lie within four to five cells of the morphogenetic furrow. In addition, the position of the endogenous morphogenetic furrow is only slightly altered compared with control clones expressing Dl alone. Thus, the proneural action of ectopic Notch signaling anterior to the morphogenetic furrow is substantially reduced in cells in which Dpp signaling is inhibited. These results suggest that despite some partially rescuing short-range signal near the furrow (which is presumed to be Hh), Dpp signaling is required for the longer range ability of cells to initiate neural differentiation in response to Notch activation (Baonza, 2001).
The fact that the ectopic expression of Dpp does not reproduce the effects cause by the overexpression of Hh, indicates that additional Hh-dependent signals are needed to promote neural differentiation. The results suggest that Notch signaling could be one of these. According to this model blocking Notch and Dpp signaling would be sufficient to prevent neural differentiation, since it would block both Hh-induced intermediate signals. To analyze this possibility, double mutant clones of the strong Delta allele Dlrev10 and the medea allele med8 were induced. Medea is the Drosophila homologue of the mammalian MAD-related protein Smad4, and is required for transduction of the Dpp signal. Clones of med8 along the posterior eye margin cause similar phenotypes to mad minus clones, preventing the initiation of the morphogenetic furrow. Internal clones of med8 can reduce the expression of Atonal, especially the initial uniform expression. Occasionally (1/17), the expression of Atonal is totally removed in part of the clone. These phenotypes are similar to those described when Dpp signaling is blocked in mutant clones of the Dpp receptor thick vein (tkv). One phenotype of med8 clones could be found not accounted for by phenotypes caused by loss of other members of the pathway: in some clones (6/11) posterior to the morphogenetic furrow, Atonal is ectopically expressed, always in isolated cells. The basis for this phenotype is not understood, but it does not affect the region anterior to the furrow, which is under consideration here (Baonza, 2001).
Double Dlrev10;med8 mutant clones show a combination of the phenotypes observed in independent mutant clones of Delta and med. Thus, all internal clones analysed show Delta-like reduction of Atonal expression. In some of these clones there are regions where Atonal expression is totally lost, a phenotype observed in med clones. Also as in medea clones, posterior Dlrev10;Med8 clones are found that express Atonal ectopically. However, in this case, the Atonal expression is in clusters of cells, reflecting the fact that lateral inhibition is blocked in the absence of Delta (Baonza, 2001).
These results indicate that the initial expression of Atonal can be induced in the absence of Notch and Dpp signaling, implying that Hh signaling can, directly or via yet another intermediate, overcome the loss of function of both pathways (Baonza, 2001).
The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated (Baonza, 2001).
In mitotic clones of the Notch null allele N54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).
The most well characterized role of Notch signaling in R8 photoreceptor determination is mediating the process of lateral inhibition, which refines Atonal expression from a small group of cells to a single cell. However, an earlier and opposite role for Notch, this time promoting neural determination, has also been recognized, although how this 'proneural' function integrates with other pathways necessary for neural differentiation has been unclear. In this work, it has been shown that in normal eye development the proneural function of Notch signaling depends on prior Dpp signaling. Emc and Hairy, two negative regulators of Atonal expression, mediate the proneural function of Notch signaling in the eye. Thus, a model is proposed that links the upregulation of Atonal in the proneural groups with the downregulation of Hairy and Emc through the activation of Delta/Notch signaling (Baonza, 2001).
Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated.
Notch can only trigger Atonal upregulation in a zone extending 12-15 cells anterior to the furrow, and this zone is defined as the cells that receive the diffusible factor Dpp, whose source is in the furrow. Dpp acts to define a pre-proneural state that prepares cells for the imminent initiation of neural determination. This pre-proneural state is defined as the zone of cells that initiate Hairy and Atonal expression in response to Dpp signaling. A functional definition to this state can be added: all these cells are primed for neural differentiation because all can respond to Notch activation by upregulating Atonal levels (Baonza, 2001).
Simultaneous loss of Hairy and Emc activity leads to the precocious differentiation of photoreceptors in a competent region ahead of the morphogenetic furrow, a phenotype that resembles that caused by ectopic expression of Delta. In addition, ectopic Notch signaling downregulates Hairy and Emc ahead of the morphogenetic furrow, causing the accumulation of Atonal at high levels; conversely, loss of function of Notch signaling increased the levels of Hairy. It is concluded that Delta/Notch signaling regulates the expression of these negative regulators in the eye. Consistent with this proposal, Emc is also regulated by Notch in the developing wing disc (Baonza, 2001).
Although Notch signaling negatively regulates both Hairy and Emc, the ectopic expression of Delta does not affect both genes identically. Thus, whereas Hairy is removed both within the clone and in the neighboring cells, Emc is only downregulated autonomously within the clone. This distinction could be an artifact caused by the perdurance of ß-galactosidase. Alternatively, these differences may reflect a different requirement for Notch signaling in the regulation of both genes. Furthermore, the expression pattern of Hairy and Emc is different during the normal progression of the morphogenetic furrow. Hairy is precisely regulated, being expressed only in the cells anterior to the furrow, and is rapidly downregulated in the furrow. This precise regulation is crucial as shown by the ectopic expression of hairy. Emc has a much broader expression pattern in the eye disc, although it shows a similar upregulation followed by downregulation in the zone immediately anterior to the furrow (Baonza, 2001).
It is also worth pointing out that not only does the expression pattern of Emc and Hairy differ, but their exact mechanism of repression is also distinct. Hairy regulates bHLH proteins by a mechanism of direct DNA binding and transcriptional repression. Emc, however, forms complexes with bHLH proteins, preventing their DNA binding. Thus, Emc can antagonize the proneural function of Atonal by two distinct mechanisms: (1) Emc presumably binds to Atonal, rendering it incapable of activating its targets; (2) Emc controls the levels of Atonal. By analogy to its regulation of two other bHLH transcriptional regulators, Achaete and Scute, it is expected that Emc interferes with the autoregulatory upregulation of atonal expression. This positive autoregulation is an essential component of its accumulation in cells within the morphogenetic furrow. In conclusion, the proneural action of Notch signaling increases Atonal activity by two mechanisms: atonal is transcriptionally upregulated, and at the same time a repressive co-factor is removed. These concerted actions lead to the accumulation of active Atonal and thereby the initiation of neural differentiation (Baonza, 2001).
genomic DNA length - 25kb
cDNA clone length - 5.4 and 4.5 kb
Bases in 5' UTR -136
Exons - six
Bases in 3' UTR - 224
The protein sequence deduced from the cDNA sequence contains the following four elements: an N-terminal signal peptide required for transport across the membrane, an extracellular domain to residue 596, a transmembrane domain to residue 617, and an intracellular domain of 214 amino acids. There are five consensus asparagine-linked potential glycosylation sites. From residue 217 to 566 there are nine repeats of a 44 amino acid domain homologous to the vertebrate epidermal growth factor receptor. EGF repeats are found in Notch and in many other proteins including integrin and laminin (Kopczynski, 1988). The EGF domain has a major role in differentiation in many characterized developmental systems.
date revised: 24 November 2001
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