BIOLOGICAL OVERVIEW

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 Dl^rev10 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 Dl^rev10;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 Dl^rev10;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 N^54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).

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

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

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).

Delta and Hairy establish a periodic prepattern that positions sensory bristles in Drosophila legs

In vertebrates and invertebrates, spatially defined proneural gene expression is an early and essential event in neuronal patterning. In this study, the mechanisms involved in establishing proneural gene expression were investigated in the primordia of a group of small mechanosensory bristles (microchaetae), which on the legs of the Drosophila adult are arranged in a series of longitudinal rows along the leg circumference. In prepupal legs, the proneural gene achaete (ac) is expressed in longitudinal stripes, which comprise the leg microchaete primordia. Periodic ac expression is partially established by the prepattern gene, hairy, which represses ac expression in four of eight interstripe domains. This study identifies Delta (Dl), which encodes a Notch (N) ligand, as a second leg prepattern gene. Hairy and Dl function concertedly and nonredundantly to define periodic ac expression. The regulation of periodic hairy expression was explored. In prior studies, it was found that expression of two hairy stripes along the D/V axis is induced in response to the Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) morphogens. This study shows that expression of two other hairy stripes along the orthogonal A/P axis is established through a distinct mechanism which involves uniform activation combined with repressive influences from Dpp and Wg. These findings allow formulation of a general model for generation of periodic pattern in the adult leg. This process involves broad and late activation of ac expression combined with refinement in response to a prepattern of repression, established by Hairy and Dl, which unfolds progressively during larval and early prepupal stages (Joshi, 2006).

Patterning of the leg imaginal disc along its circumference axis is controlled by the Hh, Dpp and Wg morphogens. This study sought to elucidate the molecular mechanisms through which these signals give rise to specific morphological features of the leg, the mechanosensory microchaetae. Patterning of leg mechanosensory microchaetae was shown to requires spatially defined expression of the proneural gene ac and its repressor Hairy. Expression of hairy in two pairs of longitudinal stripes, the D/V-hairy and A/P-hairy stripes, is directed by separate enhancers that are Hh-, Dpp- and Wg-responsive. The D/V-hairy and A/P-hairy stripes are differentially regulated by Dpp and Wg and distinct mechanisms are utilized to control hairy expression along the A/P and D/V axes. D/V-hairy expression is locally induced near the A/P compartment boundary by Hh signaling. In addition, Dpp and Wg positively influence expression of the dorsal and ventral components of the D/V-hairy stripes, respectively, by acting together with Hh to define the register of these stripes relative to the compartment boundary. In contrast, the A/P-hairy stripes, which are expressed orthogonal to the D/V-hairy stripes and A/P compartment boundary, are not activated via local induction. Rather, it appears that they are broadly activated along the leg circumference and repressed by Dpp dorsally and Wg ventrally to define their dorsal and ventral boundaries. This model for A/P-hairy regulation is supported by the observations that hairy is ectopically expressed in dorsal, but not ventral, clones lacking tkv or Mad function and that A/P-hairy expression is compromised by elevation of Dpp signaling. Furthermore, ventral, but not dorsal, clones lacking dsh function also ectopically express hairy and high-level Wg signaling results in loss of A/P-hairy expression (Joshi, 2006).

A potential caveat to this model for regulation of A/P-hairy expression is that conclusions were drawn from analysis of endogenous hairy expression rather than by examining expression directed by isolated A/P-hairy enhancer(s). Hence, it is possible that the ectopic hairy expression seen in tkv, Mad and dsh mutant clones is a result of expansion of D/V-hairy rather than A/P-hairy expression. However, several lines of evidence argue against this interpretation. First, through genetic and molecular analyses of D/V-hairy enhancer function, it has been demonstrated that Dpp and Wg positively regulate D/V-hairy expression, an observation that is inconsistent with the suggestion that D/V-hairy is ectopically expressed in clones unable to respond to Dpp or Wg signaling. Furthermore, in 3rd instar and early prepupal leg discs, stages at which the A/P-hairy stripes are not expressed, ectopic hairy expression is not observed in tkv mutant clones. Second, it was found that the D/V-hairy stripes can only be expressed in anterior compartment cells near the A/P boundary, which are the cells that receive and respond to Hh signal. Thus, it is unlikely that ectopic hairy expression observed in clones at distance from the compartment boundary, which receive little or no Hh signal, and in the posterior compartment, in which cells do not respond to Hh signal, corresponds to D/V-hairy expression. Finally, it was found that elevation of Dpp or Wg signaling specifically disrupts A/P-hairy but not D/V-hairy expression. Taken together, these findings are consistent with the conclusion that A/P-hairy rather than D/V-hairy is expressed in clones compromised in their response to Dpp and Wg signaling (Joshi, 2006).

This study identified Dl as a second prepattern gene that functions together with hairy to establish ac expression in the leg microchaete proneural fields. Several lines of evidence are are presented that support this conclusion. First, it was found that, beginning at 4 h APF, Dl expression is up-regulated in domains overlapping the microchaete proneural fields. This distribution of Dl is similar to that, in the notum, where Dl has been shown to regulate proneural ac expression. Second, it is shown that ac expression is expanded in legs with reduced Dl function. Third, it was found that elevated N signaling throughout the tarsus results in severely reduced ac expression. Finally, activation of N signaling was observed within the hairy-OFF interstripes (ac interstripes that do not express hairy), in agreement with the genetic requirement for Dl/N signaling in these domains. Based on these results, it is proposed that ac expression is activated broadly during mid-prepupal leg development but is confined to the microchaete proneural fields by a previously generated prepattern of repression, established by Hairy and Dl/N signaling. This hypothesis is supported by analysis of cis-regulatory elements that direct ac expression in the leg microchaete proneural fields (Joshi and Orenic, unpublished cited in Joshi, 2006). By generating rescue and reporter constructs, an enhancer has been identified that specifically controls expression of ac in the microchaete proneural fields. Unlike the hairy leg enhancers, no modular organization of the cis-regulatory elements that control expression of ac stripes in different regions of the leg is observed. Rather, preliminary analyses suggest that there is one enhancer consisting of an activation element that directs broad expression of ac along the leg circumference and two repression elements, which are N- or Hairy-responsive. This finding is consistent with genetic studies and the model for regulation of ac expression in the leg microchaete proneural fields (Joshi, 2006).

hairy and Dl function to repress ac expression in complementary domains. hairy encodes a transcriptional repressor which has been previously shown to directly repress ac expression in the wing by binding a specific site in the ac promoter. It is likely that Hairy acts through a similar site to repress ac expression in the leg. Dl represses ac expression via a different mechanism: presumably, cells of the microchaete proneural fields, which express high levels of Dl, signal to adjacent cells to activate N. This suggestion is supported by the observation that expression of two N-responsive reporters is specifically activated in cells corresponding to the hairy-OFF interstripes. One of the reporters used in this study, E(spl)mβ-CD2, and other similar reporters recapitulate endogenous E(spl)mβ-CD2 expression in wing and leg imaginal discs. E(spl)mβ is one of seven genes in the E(spl)-C that encode bHLH repressors related to Hairy. Hence, it appears that ac expression in the leg microchaete proneural fields may be established by a prepattern of periodically expressed bHLH repressors (Joshi, 2006).

N signaling is not activated within ac-expressing cells, even though these cells express high levels of Dl. This could be explained by a dominant-negative effect of Notch ligands on N signaling, which has been previously observed in the wing. In the wing, it has been shown that N signaling is not activated within cells expressing high levels of Dl and Ser but, rather, that these cells signal to adjacent cells to activate N signaling within the wing margin. Consistent with the hypothesis of a potential dominant-negative function for Dl in the leg microchaete proneural fields is the observation that over-expression of Dl along the leg circumference results in expansion of ac expression into the hairy-OFF interstripes, which would be expected if N signaling was disabled. Over-expression of N ligand expression has been shown to exert a similar effect in other tissues (Joshi, 2006).

A curious observation of this study is that, as suggested by genetic evidence and the expression of two N-responsive reporters, N signaling, with one exception, is not activated within the hairy-ON interstripes, even though each Hairy stripe is straddled on either side by a Dl stripe. This suggests either that Dl signals asymmetrically or that there is an asymmetric response to N signaling and raises questions regarding the underlying mechanism of asymmetric activation of N-target gene expression. A potential mechanism for asymmetric signaling by Dl is suggested by studies in the notum, in which it has been shown that the N receptor is distributed in a pattern complementary to Dl. If N levels were higher within the hairy-OFF vs. the hairy-ON interstripes in the leg, this could allow for preferential signaling within these domains. However, N expression was assayed in prepupal legs and it was found that N appears to be uniformly distributed along the leg circumference. Hence, either there is an asymmetric response to N or alternative mechanisms are responsible for establishing the directionality of Dl signaling in the leg, such as post-translational modification N signaling pathway components. For example, glycosylation of N by the Fringe glycosyltransferase influences its interactions with its ligands (Joshi, 2006).

Another intriguing finding is the overlap of N signaling with the V-Hairy stripe. This result was surprising because it would suggest redundancy between hairy and Dl/N signaling in this region. However, an absolute requirement was observed for hairy function in the ventral leg. An explanation for this puzzling finding is suggested by the specific loss of the V-Gbe+Su(H)m8-lacZ stripe in hairy mutant legs, which indicates that Dl/N signaling or responsiveness in the ventral leg is dependent on hairy function. The specific loss of N signaling in the ventral leg could be a result of the expansion of Dl expression in hairy mutant legs, which as explained earlier might have a dominant-negative effect on N signaling. This proposal is corroborated by the expansion of ac expression along the circumference of legs ectopically expressing Dl throughout the tarsus. The overlap of hairy and Dl/N signaling in the ventral leg raises questions regarding the function of Dl/N signaling in this domain. It was observed that V-hairy and Gbe+Su(H)m8-lacZ expression overlap only partially, suggesting that combined function of Dl and Hairy in the ventral leg could serve to establish a broader domain of repression in this region in comparison to other interstripe domains. This idea is supported by the morphology of the adult leg tarsus in which the spacing of bristles is most pronounced along the ventral midline. However, the function of N in the ventral leg is not as yet clear. It is plausible that there is a role for Dl/N signaling in the ventral leg that is unrelated to regulation of ac expression (Joshi, 2006).

The potential function of Dl as a regulator of proneural ac expression in the leg was suggested by studies in the notum, on which mechanosensory microchaetae are also organized in longitudinal rows. In the notum, Dl/Notch signaling, rather than Hairy, regulates periodic ac expression. The current studies suggest a distinct mechanism for leg microchaete patterning in which Hairy and Dl act together and nonredundantly to define periodic ac expression. In both the leg and notum, Dl signals to adjacent cells to repress ac expression. However, whereas in the notum Dl activates N signaling in cells on either side of each Dl/Ac stripe, in the leg, N signaling is activated (with one exception) only within the hairy-OFF interstripes. Although the pattern of mechanosensory bristles on the leg and notum is overtly similar, the bristle rows are more precisely aligned in the leg. The more organized pattern on the leg may be a consequence of the combined function of Hairy and Dl which might more precisely define the domains of proneural gene expression (Joshi, 2006).

Dl function is essential for proper patterning of ac expression and it is suggested that accurate positioning of the Dl stripes is necessary for activation of Notch signaling within appropriate domains. Hence, regulation of Dl expression is an important aspect of leg microchaete patterning. In legs lacking hairy function, Dl expression expands into four broad domains and ectopic hairy expression greatly reduces Dl expression, indicating that periodic expression of Dl is regulated in part by hairy. Concomitant with the expansion of Dl expression, there is loss of N signaling in the ventral leg, suggesting that hairy functions to create an apposition of cells expressing high levels of Dl to cells expressing low levels of Dl, which allows for activation of N signaling in the ventral leg. Regulation of Dl expression in proneural fields is not understood. A plausible hypothesis is that, like hairy, Dl expression is established in response to the morphogens that control pattern formation during leg development (Joshi, 2006).

This and previous studies suggest an outline of general genetic pathway for the regulation of ac expression in the leg microchaete proneural fields. This process involves broad and late activation, by an unknown factor, of ac expression along the leg circumference combined with refinement in response to a prepattern of repressors, which is established during larval and early prepupal stages. Hairy and Dl have been identified as the primary prepattern factors that regulate ac expression along the leg circumference. Position-specific expression of both hairy and Dl in longitudinal stripes is essential for proper ac expression. The longitudinal stripes of hairy are established in direct response to the Hh, Dpp and Wg signals, which globally pattern the leg, indicating that hairy acts as an interface between ac and these morphogens. Dl expression is regulated by Hairy, but its regulation is otherwise poorly understood. In addition to elucidating a pathway for establishment of periodic ac expression during leg development, these studies also provide insight into the mechanisms through which morphogens function to generate leg morphology (Joshi, 2006).

Periodic ac expression is established progressively. The first evidence of periodicity is expression of the longitudinal stripes of hairy expression. The D/V-hairy stripes are expressed first in the early 3^rd instar leg disc followed by the A/P-stripes between 3 and 4 h APF. Between 4 and 6 h APF, Dl expression within the mechanosensory microchaete primordia is established. Then, ac expression is activated uniformly along the leg circumference. By the time that ac expression is activated, the interstripe domains have been defined by the four Hairy stripes and Dl/N signaling (Joshi, 2006).

The delay of ac expression in the microchaete proneural fields until mid-prepupal stages is likely due to the requirement of ac function for formation of all leg sensory organs. Leg sensory bristles can be grouped into two broad categories based on their time of specification: one group includes the early-specified mechanosensory macrochaetae (large bristles) and chemosensory microchaetae, and the second group includes the more numerous late-specified mechanosensory microchaetae. During the 3rd instar and early prepupal stages, ac is expressed in small clusters of cells that define the primordia of early-specified bristles, while expression of ac in the mechanosensory microchaete primordia is activated later in the mid-prepupal stage. This late expression of ac is activated broadly along the leg circumference and is presumably delayed to allow for expression of the hairy and Dl stripes during earlier stages. Premature expression of this normally late ac expression would likely lead to disturbances in sensory organ patterning, suggesting that temporal control of ac expression is an important aspect of its regulation (Joshi, 2006).

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. Previous studies have shown that the NHR1 domain is required for Delta trafficking. This study shows that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, it was shown that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, evidence is provided that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization (Liu, 2012).

Neur is an E3-ubiquitin ligase that plays an essential role in Notch signaling by regulating the endocytosis of Notch ligands. It contains NHR domains, which are rare and conserved between vertebrates and invertebrates but not present in viruses, bacteria, fungi, or plants. In the Drosophila proteome, besides Neuralized, there are two other NHR-containing proteins, CG3894 and Bluestreak. In mammals, proteins containing NHR domains (also known as NEUZ) include the β-catenin regulator OzzE3 and lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Recent studies reported that the human homologue of Bluestreak serves to localize to the centrosome. Although the general role of the NHR domains is unclear, these domains tend to cluster, and most proteins contain two to six NHR domains. The significance of having more than one NHR domain in one protein is to yet be determined (Liu, 2012).

This study has investigated the role of the highly conserved NHR domains in Neur function. The NHR1 domain alone mediates the interaction between Neur and both Delta and Serrate. It was also shown that the NHR2 domain is required for Neur function, and while it is not required for the interaction with Notch ligands, it is involved in Dl internalization, a critical step in Notch activation. Moreover, the NHR domains play a role in Neur oligomerization, which in turn could contribute to Neur ubiquitination activity and ligand endocytosis (Liu, 2012).

The NHR1 domain mediates the interaction between Neur and the Notch ligands Delta and Serrate. Previous studies have shown that the NHR1 domain of Neur is both necessary and sufficient for the interaction with the Notch ligand Dl. Specifically, it was found that a point mutation in a highly conserved glycine residue within the NHR1 domain (G167E) abolishes the ability of Neur to bind Delta. Whether the NHR1 domain was also required to bind to Serrate, however, was unknown. In fact, in vitro studies in vertebrates suggested that the NHR2 domain in mouse Neuralized- like 1 (Neurl1) is sufficient to bind to Jagged1, the mouse orthologue of Serrate. In contrast, the current study found that the NHR2 domain is not required for Neur to bind Serrate in Drosophila and that the interaction is mediated entirely by the NHR1 domain. Other studies reported previously that the motifs on Dl and Ser that mediate the interaction with Neur are conserved. In comparisons of protein sequences, Jagged1 and Serrate share 40.7% similarity overall, while the overall similarity between Jagged1 and Dl is 33.8%. The NHR1 and NHR2 domains from Drosophila Neuralized have the same degree of amino acid similarity with the mouse Neurl1 NHR2 domain (33%). Since there is no clear correlation between protein sequence similarity and the ability of either the NHR1 or the NHR2 domain to interact with Notch ligands, whether NHR1 or NHR2 is important for interacting with ligands is likely to be species dependent. The Neur-Ser interaction was found to be abrogated by the G167E mutation in the NHR1 domain. Given that the NeurG167E mutant still retains ubiquitination activity, it is unlikely to affect overall protein folding. A previously reported structural analysis of the Drosophila NHR1 domain suggested that Gly167 resides in a hydrophobic core and that the Gly167 mutation presumably destabilizes the surrounding microenvironment. Therefore, the Gly167 mutation may result in spatial changes in the neighboring residues of the core, thus abolishing binding to ligands (Liu, 2012).

The G430E mutation reveals a distinct role for the NHR2 domain in the regulation of Neur activity and Delta trafficking. The data demonstrate that the NHR2 domain is required for Neur function in vivo. NeurG430E fails to rescue neur mutant embryos, while NeurNHR2 has some residual activity, which suggests that they affect different aspects of Neur function. The expression of NeurG430 in a heterozygous background, which does not have a neurogenic phenotype on its own, resulted in a significant increase in the percentage of neurogenic embryos, suggesting that NeurG430E has a negative effect on Neur function. In contrast, NeurNHR2 overexpression did not have any effect on heterozygous embryos, suggesting that it behaves as a loss-of-function allele. Despite the fact that the two NHR2 mutant proteins NeurG430E and NeurNHR2 behave differently, they both localize to the plasma membrane in the presence of Delta both in vitro and in vivo, and they are both capable of binding to the Notch ligands Delta and Serrate. However, both mutant proteins affect the extent of Dl internalization to various degrees. NeurG430E exhibits severely compromised ubiquitination activity and is no longer capable of inducing Delta internalization. NeurNHR2, on the other hand, retains ubiquitination activity but is much less efficient than WT Neur at directing Dl internalization in vivo or in Kc cells. The precise mechanism by which NeurNHR2 affects Delta endocytosis is unclear (Liu, 2012).

One possibility is that the NHR2 domain is required for Neur oligomerization. Neur was shown to form NHR domain-mediated oligomers by coimmunoprecipitation experiments. Therefore, the deletion of the NHR2 domain (NeurNHR2) may simply reduce the oligomerization potential of Neur, leading to a decrease in ligand endocytosis. In contrast, the point mutation (NeurG430) might disrupt the overall structure of the NHR2 domain, preventing oligomerization and resulting in a protein that has no ubiquitination activity and therefore can no longer internalize ligands. However, this model cannot fully explain the data, which show that NHR2 does not prevent Neur oligomerization and that Neur oligomerization still occurs in the absence of any NHR domains, suggesting that while the NHR domains may play a role in oligomerization, they are not necessary for this process. The data also show that although the G430E mutant loses ubiquitination activity, the double mutant containing the NHR1 deletion and the G430E mutation retains ubiquitination activity, which argues that G430E does not affect the overall folding of the NHR2 domain (Liu, 2012).

Another possibility is that the NHR1 and NHR2 domains initially form an intramolecular structure that is inactive and must be resolved for ubiquitination to promote ligand internalization. The G430E mutation may lock Neur into an intramolecular conformation through an NHR1-NHR2 interaction, such that this inactive form can still bind to Dl and Ser but cannot form oligomers and has no ubiquitination activity as a consequence of dysfunctional oligomerization. This model, in contrast to the former one, is supported by the data that demonstrate that the G430E mutant no longer forms oligomers and has no ubiquitination activity. Furthermore, when the NHR1 domain was removed from the G430E mutant (NeurNHR1G430E), the ubiquitination activity was restored, suggesting that the intramolecular loop can no longer form, whereas intermolecular interactions between NHR1 and NHR2 domains can occur. It may also explain the negative effect of NeurG430E on NeurWT: although NeurG430E has a reduced ability to bind WT Neur, it is still be able to sequester some portion of NeurWT into a nonfunctional intermolecular oligomer that can no longer ubiquitinate targets. In contrast, the deletion of the NHR2 domain would prevent intramolecular interactions but would be expected to have a reduced ability to form productive oligomers, leading to a defect in ligand internalization. Whether the NHR2 domain has additional roles in recruiting a protein(s) that promotes Notch ligand ubiquitination and endocytosis remains to be determined. Like Neuralized, other RING domain E3 ligases often function as oligomers, and they multimerize in different ways: some form heterodimers, such as Mdm2-MdmX, and some can form homo-oligomers, such as TRAF. The functional significance of RING E3 oligomerization is poorly defined. One previously proposed model is that the oligomerization of E3 ligases may functionally resemble the dimerization of receptor tyrosine kinases in such a way that autoubiquitination yields a mark that serves as a platform to assemble a signaling complex. Consistent with this idea, ubiquitination was seen in anti-Neur IPs when Dl was not present, consistent with the idea that Neur may be autoubiquitinated. Whether this autoubiquitination can initiate a cascade of further downstream ubiquitination events remains to be determined. It is possible that oligomerization is mediated via autoubiquitination and the interaction of ubiquitinated Neur with itself through a ubiquitin-binding motif (UIM). If so, then the NHR1-NHR2 interaction could also function to keep Neur in an inactive state by occluding the putative UIM. Such a model of Ubi-UIM complex formation was previously proposed for other endocytic proteins (Liu, 2012).

In summary, this study has shown that NHR domains are protein-protein interaction modules that are required for many aspects of Neur function. The NHR1 domain mediates the interaction between Neur and its targets Dl and Ser. Both NHR domains appear to regulate Neur activity by affecting its ability to form oligomers and/or interact with proteins required for the endocytosis of Notch ligands. Interestingly, NHR domains have been identified in several other proteins that are conserved between flies and humans. Whether Neur can form heterodimers with these other NHR-containing proteins and whether these heterodimers play a role in Notch signaling and other developmental processes remain to be determined (Liu, 2012).

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1

miR-1> is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. This study shows that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4. Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila. Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, it was found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart (Zhu, 2017).

Unexpectedly, overexpression of miR-1 in the anterior-posterior (AP) organizer of the wing disc results in a dose-dependent loss of L3 vein structures, consistent with de-repression of Notch or weakening of a regulatory mechanism that dampens the Notch signal. Using genetic techniques, it was determined that the loss of the distal aspect of L3 could be phenocopied by reducing the gene dose of Notch co-repressors or Nedd4; in the case of Nedd4, the regulation by miR-1 was direct. An expanded model is proposed in which miR-1 expression in the AP organizer has complex effects on Notch signaling owing to its regulation of ligand availability and receptor trafficking. As lower levels of miR-1 expression (18°C) caused wing-vein thickening and tortuosity, and higher levels (22°C) caused vein loss, Delta and Nedd4 may be differentially sensitive to miR-1 regulation, although these studies were not designed to address this issue. It is also possible that indirect effects, such as reductions in Nedd4-mediated ubiquitylation of positive effectors of the Notch receptor (e.g. Deltex) or perturbations in Delta-mediated cis-inhibition, contributed to the de-repression of Notch in the wing-based assay system (Zhu, 2017).

The findings in the mammalian heart indicate that the genetic and biochemical interaction between miR-1 and Nedd4l is physiologically relevant and may provide developmental or tissue-specific regulation of Nedd4l in the myocardium. It is speculated that the additional bands observed on western blots of heart lysates using an anti-Nedd4L antibody might result from post-translational modifications, because Nedd4L can autoregulate its stability through ubiquitylation of its HECT domain. Alternatively, they might represent heart-specific splice variants, because tissue-specific isoforms of Nedd4L have been found in the heart and the liver (Zhu, 2017).

Importantly, although miR-1-mediated reductions in Nedd4 activity caused wing-vein phenotypes induced by Notch, miR-1-mediated dysregulation of Nedd4L in the heart likely affects proteins outside the Notch pathway. Indeed, protein microarrays comparing human Nedd4 with human Nedd4L, suggest that Nedd4L (also known as Nedd4-2) preferentially targets ion channels, whereas Nedd4 targets are enriched for signaling pathways. Thus, in the heart, where miR-1 and murine Nedd4L are both expressed, their genetic and biochemical interaction might influence the excitability and connectivity of cardiomyocytes. Indeed, susceptibility to cardiac arrhythmias and sudden death in humans is associated with six genes that encode ion channels (SCN5A, KCNQ1, KCNH2, KCNE1, KCNE2 and RYR2). Murine Nedd4L regulates the cell-surface densities of the sodium channel, the voltage-gated type V alpha subunit (Scn5a), the potassium voltage-gated channel, KQT-like subfamily member 1 (Kcnq1) and the human Ether-a-go-go-related (KCNH2, previously hERG) channel. Furthermore, miR-1 directly regulates human KCNJ2, a channel that maintains cardiac resting potential. These findings suggest that the regulation of murine Nedd4l by miR-1 contributes to some of the electrophysiological abnormalities seen in miR-1 null mice. It would be interesting to determine whether Nedd4L is dysregulated in the heart after an infarction or under ischemic conditions, when miR-1 is upregulated and fatal cardiac dysrhythmias are common (Zhu, 2017).

Sensitized genetic backgrounds reveal differential roles for EGF repeat xylosyltransferases in Drosophila Notch signaling

In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, biochemical and genetic analysis was performed on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, it was found that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds (Pandey, 2018).

Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. This study reports that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions (Sood, 2022).

This study reports that Notch signaling regulates quiescence, entry and exit in Drosophila central brain (CB) NBs. Increasing Notch pathway activity induces CB NBs to exit cell cycle via a Dap-dependent mechanism. Dap, a cyclin-dependent kinase inhibitor and CIP/KIP family member, is a known Notch target gene as is Cyclin E, String (Cdc25) and E2F. Whether Notch regulates other cell cycle genes required for CB NB exit remains unknown. Once CB NBs stop dividing, Notch pathway activity becomes attenuated. Low to no Notch activity is required for CB NBs to exit quiescence in response to dietary nutrient cues. Thus, levels of CB NB Notch activity regulate both the entry and exit from quiescence. High Notch is required for entry, whereas low Notch is required for exit. Intestinal stem cells (ISCs) also experience a period of low to no Notch activity during mid-pupal stages and, if Notch is ectopically induced at this time, ISCs terminally differentiate into secretory enteroendocrine cells. Whether any of the quiescent CB NBs with ectopic Notch terminally differentiate remains unknown (Sood, 2022).

Although Notch is required for quiescence, most CB NBs do stop dividing, albeit late. This suggests that other genes or signaling pathways are required or that residual Notch activity is sufficient to induce CB NB quiescence. Unfortunately, Notch null mutants are embryonic lethal. In addition, this study has shown that Notch is sufficient to induce quiescence. Although quiescence occurs prematurely, it is still restricted to late embryonic stages. Restriction of Notch function (cell cycle exit) is likely due to CB NB intrinsic temporal programs. Temporal programs that likely vary across CB NB lineages, as is the case in the ventral nerve cord. Whether temporal programs regulate levels of Notch pathway activity or provide additional factors needed for CB NB cell cycle exit remains unanswered (Sood, 2022).

In mammals, Notch signaling is more complicated because of gene duplication. Yet, recently, Notch signaling has been shown to regulate neural stem cell quiescence. In Notch2 conditional knockout mice, more neural stem cells are actively dividing in the hippocampus and subventricular zone brain regions in adult animals compared with controls. This results in premature depletion of the neural stem pool and reduced neurogenesis in older mice. This is similar to what is reported in this study and, in the future, it will be interesting to determine which Notch ligands are required and whether neural stem cells in mammals use their newborn daughters for pathway activation (Sood, 2022).

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. This study found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets (Bhattacharya, 2017).

Differences were observed between how Notch signaling regulates the SMW compared to how Notch regulates photoreceptor differentiation (see Model of the cell cycle and cell fate specification in the morphogenetic furrow). The Notch pathway suppresses the specification of photoreceptor cells in a manner similar to regulation of neurogenesis by Notch in many other tissues. That is, activation of the transmembrane ligand Delta by the ubiquitin ligase Neuralized leads allows Delta to activate Notch, leading to release of the Notch intracellular domain, which then acts in a nuclear complex with Su(H) and Mastermind to induce the transcription of the E(spl)-C family of transcriptional repressors and prevent neural fate specification and differentiation. By contrast to the pathway regulating neural differentiation, cell cycle entry in the SMW occurred in the absence of the E(spl)-C. This suggested that a distinct transcriptional target of Su(H) is involved, but unusually, this Su(H) function did not seem to require the co-activator Mam. Although most studies find that Mam is required for transcriptional activation by Su(H), mutations in mam often have weaker phenotypes than other neurogenic genes. It is possible that Mam protein might exhibit exceptionally strong perdurance, but there is also in vitro evidence for mam-independent transcriptional activation by Su(H) (Bhattacharya, 2017).

SMW entry also occurred in the absence of neur. In the case of neurogenesis, cells lacking neur show a weaker neurogenic phenotype than cells lacking other components of the pathway, both in the embryonic CNS and in the eye, which is thought to reflect the activities of two other ubiquitin ligases, Mindbomb and Mindbomb2. No effect was found on the SMW of mutated mib1, but neither mib2 nor cells lacking two or more of these possibly redundant ubiquitin ligases were examined (Bhattacharya, 2017).

Eoles of ligands were examined in more detail. It has been reported that SMW entry depended on Delta, which is also the ligand necessary for regulation of neurogenesis in the eye as in other parts of the nervous system, but although their figures show an obvious reduction in cell cycle entry in the clones of cells lacking Dl, some cell cycle entry still seems to occur. Those experiments were repeated using cells lacking both Dl and the other known ligand Ser, with similar results, ie, cell cycle entry was obviously disrupted in clones lacking both Notch ligands, nevertheless some cells still entered the cell cycle. A potential complicating factor is that reducing Notch function typically results in excessive neurogenesis, raising the question of whether some Dl Ser mutant cells were prevented from entering the cell cycle by differentiation as photoreceptors. The excess neurogenesis of such clones was partially suppressed by expression of Argos or Ras-DN. Although there is clearly a considerable disruption of the SMW in the absence of N ligands, there is also clear evidence of cell cycle entry, occurring at approximately the same time as the normal SMW (Bhattacharya, 2017).

There are at least three possible explanations for these findings. First, it possible that another ligand besides Dl or Ser activates N in the SMW. Secondly, it has been reported that Dl can signal across several cell diameters using filopodia. During neurogenesis, rescue of Dl mutant clones appears to extend beyond cells immediately adjacent to wild type cells. Signaling across many cell diameters would be required to account for the cell cycle progression seen in Dl Ser mutant clones. Thirdly, Notch can be activated independently of ligands if delivered to certain cellular compartments. Recently it has been suggested that such ligand-independent activation is suppressed by cis-inactivation, in which ligands inhibit N signaling in the same cells, so that ligand-independent activation can be revealed in cells lacking Dl and Ser. Cis-inactivation is potentially an important contributor to the patterning of Notch-mediated lateral inhibition during neurogenesis, but direct evidence of such a role has not yet been obtained. If ligand-independent signaling is occurring in Dl Ser clones, some other pathway must be active near the SMW to explain why ligand-independent signaling cell cycle entry was limited to cells at the same stage posterior to the furrow as those that undergo the normal, ligand-dependent SMW (Bhattacharya, 2017).

The cell cycle targets of Notch are of some interest. Many Drosophila cell cycles depend on regulated transcription of Cyclin E. Cyclin E is clearly required for the SMW suggested that Cyclin E might not be limiting for SMW cell cycle entry, however, because Cyclin E was seen to accumulate more in the differentiating photoreceptor precursors that do not enter the SMW. Alternatively, the lack of Cyclin E accumulation in SMW cells might reflect Cyclin E protein instability in S phase, with accumulation only in differentiating cells that can't enter the cell cycle. A Cyclin E transcriptional reporter appears to accumulate in the same cells as the Cyclin E protein, however, which is not indicative of post-translational regulation (Bhattacharya, 2017).

It has been suggested that Cyclin A was part of the Notch dependent machinery promoting cell cycle entry in the SMW, although it was concluded that it could not be the only important Notch target. What was observed, however, was dependence of Cyclin A protein levels on Notch signaling that exactly paralleled the dependence of S-phase entry on Notch signaling in various genotypes. Like Cyclin B, Cyclin A protein is degraded by APC/Cyclosome activity during G1, so that Cyclin A protein cannot accumulate until the G1/S transition. Therefore these results are also expected if Cyclin A simply accumulates whenever the G1/S transition has occurred, and is not a direct target of Notch signaling (Bhattacharya, 2017).

In mammalian cells, c-Myc appears to be an important Notch target. Indeed, human Notch was first identified as a proto-oncogene that causes leukemia through c-Myc. c-Myc is an activator of the nucleolar protein fibrillarin, which transiently increases during the SMW. Although Dmyc is important for growth in Drosophila, so that clones of myc null mutant cells in the eye disc are small, they nevertheless enter the SMW at the normal time, indicating that myc is unlikely to be the critical Notch target in the SMW (Bhattacharya, 2017).

Emc is a transcriptional target of N signaling in the eye as in other tissues. Even though Emc protein levels seem to be less affected by N signaling than is emc RNA, it has been suggested that N regulates the eye cell cycle by upregulating emc, thereby inhibiting Da, which is proposed to inhibit cell cycle entry. Contrary to this model, previous studies have reported that Da is in fact required for the SMW. We investigated this potential role for emc directly, finding no evidence that emc was required for the SMW. These studies confirmed previous findings that da is required for S phase entry into the SMW, although not apparently for cell cycle activity anterior to the morphogenetic furrow (Bhattacharya, 2017).

The cell-autonomy of da requirement in the cell cycle had not been determined previously. da mutant cells sometimes enter the SMW at the posterior boundaries of da clones, where they were adjacent to wild type cells. This suggested that da was required for the production of a short-range non-autonomous signal that is required for SMW entry, and could be provided to da mutant cells that were near to wild type cells (Bhattacharya, 2017).

It is instructive to compare the effect of da on the cell cycle to that of ato. Ato is the heterodimer partner of Da that is required for R8 specification. Da is also required independently of ato to specify R2,R3,R4 and R5. Interestingly, this requirement in R2-5 seems to be incomplete, since some da mutant R2-5 cells do differentiate at reduced frequencies. Because of this requirement for da outside R8, R1-7 photoreceptor cells can differentiate near borders of ato mutant clones, but fewer photoreceptors are expected near the borders of da mutant clones, and mostly of the R1, R6 and R7 cell types. Proneural genes often promote Notch ligand expression, and accordingly ato is required for normal Dl expression during these stages of eye development. Reduced Dl expression is a plausible explanation for the absence of the SMW within ato clones, and the more extensive requirement for da in photoreceptor differentiation and Dl expression may explain why non-autonomous rescue of the SMW is more pronounced near the borders of ato clones than near the borders of da clones (Bhattacharya, 2017).

A model is present for SMW. Unpatterned proliferation of the eye imaginal disc is terminated ahead of the morphogenetic furrow by Hh and Dpp signaling. Hh and Dpp affect many aspects of eye development including expression of master regulators of retinal determination such as Eyeless, Teashirt, Dachshund, and Sine Oculis, as well as Homothorax. Cells posterior to the furrow can still respond to growth, because extra cell cycles are driven in unspecified cells by overexpression of CycD/cdk4, Inr, or myc, as well as by Cyclin E. It is possible that Notch regulates cellular growth in the SMW, since there appears to be a small but discernible increase in nucleolar size at this stage, and progenitor cells are not noticeably smaller after dividing, indicating that growth accompanies the SMW cell division. The SMW, however, does not depend on myc. It appears to depend on a transcriptional target of Su(H) outside of the E(spl)-C of bHLH genes and that can be transcribed in the absence of Mam. The possibility of a function of Su(H)/Nicd other than transcription cannot be ruled out. The SMW clearly depends on CycE, but CycE may not be the N-dependent gene required for SMW entry, because it is not obviously elevated in SMW cells. Contrary to a recent proposal, the SMW does not depend on the HLH gene emc and is not inhibited by the bHLH protein Da. Instead da is required for entry into the SMW. The positive requirement for da is cell-nonautonomous, as is the requirement for its heterodimer partner ato, and consistent with the role of ato and da in promoting Dl expression during retinal differentiation, which is likely to account for the cell cycle defect. Surprisingly, the requirement for Dl in the SMW cell cycle was not absolute, either because an unidentified ligand exists, long-range cell-nonautonomy occurs, or Dl (and Ser) may contribute to cis-inactivation of N, so that some ligand-independent Notch signaling could occur in the unphysiological circumstance that Notch ligands are completely absent (Bhattacharya, 2017).

Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions

This study reports that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, a mutant H3T3A was expressed to compromise asymmetric histone inheritance. Under this condition, an increase was observed in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, these results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells (Zion, 2023).

In multicellular organisms, asymmetric cell division (ACD) of adult stem cells serves as an important mechanism for tissue homeostasis and regeneration. Disruption of this precisely regulated cell division mode can result in the dysregulation of stem cells, leading to cancer or tissue degeneration. Epigenetic mechanisms enable different cell types within a multicellular organism to establish distinct cellular identities while carrying the identical genetic information. Canonical histone proteins H3, H4, H2A, and H2B are synthesized and incorporated into DNA during replication as an octamer structure, forming the fundamental unit of chromatin, the nucleosome. It is well known that nucleosomes and chromatin structure can affect cell fate decisions; however, it remains largely unclear how epigenetic information is retained or altered during cell divisions to produce cells with different identities in multicellular organisms. Previous studies have shown that H3 and H4 histones are asymmetrically inherited during ACD of the Drosophila male germline stem cells (GSCs), where preexisting (old) histones are retained in the self‐renewed stem cell, while newly synthesized (new) histones are enriched in the differentiating daughter cell. In contrast, old and new H2A and H2B are inherited more symmetrically during ACD of male GSCs. It is hypothesized that the old H3 and H4 histones retain an epigenetic memory that is inherited by the self‐renewed stem cell, while the newly synthesized histones lacking this information can be used to establish a new gene expression program in the differentiating cell. Complementary to this hypothesis, previous studies have reported differences in post‐translational modifications between preexisting and newly synthesized histones. Additionally, it has been shown that nucleosomal density displays differences between old and new histone‐enriched sister chromatids, with the old histone side having higher overall nucleosomes than the new histone side. Nucleosome density and position have profound impacts on many cellular processes by modulating DNA accessibility to different regulators, including pioneer factors, transcription factors, and cell cycle regulators. One functional readout of the inheritance of asymmetric chromatin statuses in GSC divisions is the asymmetric recruitment of the DNA replication component Cdc6, which allows asynchronous cell cycle progression in the resulting daughter cells. Furthermore, when asymmetric H3 segregation is disrupted, progenitor germline tumors and germ cell loss phenotypes are both detected, suggesting that this process is required for both stem cell maintenance and proper germline differentiation. Recently, a new study revealed that proper interactions between homologous chromosomes at a critical 'stemness' gene, stat92E locus, depend on asymmetric H3 inheritance, without which stat92E gene expression becomes misregulated. The findings of asymmetric histone inheritance in Drosophila male GSCs set a precedent in studying epigenetic inheritance modes in multicellular organisms. The question remains, however, of whether this phenomenon is germ cell‐specific or if it serves as a more general mechanism. It also remains unclear whether asymmetrically inherited histones aid in defining distinct cell fates at a single‐cell level. Addressing these questions will not only greatly enhance our current understanding of how epigenetic inheritance modes dictate cell fates, but it will also help establish new methods to identify bona fide stem cells and asymmetrically dividing cells in vivo (Zion, 2023).

To investigate the generality of asymmetric histone inheritance, this study use the Drosophila intestinal stem cells (ISCs) in the midgut as a model system. One feature of ISCs is that they can alternate between ACD, which produces a self‐renewed ISC and a differentiating daughter of either an enteroblast (EB) or a pre‐enteroendocrine (pre‐ee) cell, and symmetric cell division (SCD), which results in two self‐renewed ISCs. As the Notch (N) signaling pathway is critical for cellular differentiation in the ISC lineage, the expression of Delta can be used as an ISC‐enriched cellular marker. It is proposed that the ISC lineage is a great system to study histone inheritance due to its well‐characterized lineage, clearly distinguishable ISC‐specific mitosis, and abundant ISCs in vivo. Indeed, previous studies have shown asymmetric inheritance of old versus new centromere‐specific histone H3 variant CENP‐A (i.e., CID in Drosophila) in the ISCs. Using this system this study reports that asymmetric canonical histone inheritance applies to this somatic stem cell lineage during ACD of ISCs and that misregulation of this process leads to midgut hyperplasia with ISC‐like cells. Collectively, these results demonstrate that asymmetric histone inheritance could be a more general feature for asymmetric stem cell divisions. This study also offers insight into how histone inheritance could influence the establishment or maintenance of cell identities and how misinheritance could lead to diseases such as cancer (Zion, 2023).

To study histone distribution and inheritance patterns during ISC divisions, a dual‐color histone labeling and tracking system was optimized in the ISCs, similar to what has been previously used. In this study, the expression of labeled histones was driven by a cell type‐specific escargot‐Gal4 (esg‐Gal4) to turn on the UAS‐histone transgene in the ISC lineage. After a heat‐shock‐induced switch from eGFP to mCherry‐labeled histone expression, ISCs were allowed to undergo at least a complete round of DNA replication after an approximately 18‐h recovery time, shown by robust incorporation of new replication‐dependent canonical histones genome‐wide. This time frame was determined through a time course experiment, where the expression and incorporation of new H3‐mCherry began around 12 h after heat shock. By 24 h, robust signals of both H3‐eGFP and H3‐mCherry could be detected. However, by 36 h old H3‐eGFP signals became almost undetectable. This time lapse required for new H3 incorporation is consistent with the replication‐dependent incorporation mode for canonical histones. Contrastingly, the incorporation of new histone variant H3.3‐mCherry which is independent of DNA replication showed robust signals 12 h after heat shock. Quantification showed minimal flipped‐out without heat shock but efficient flipped‐out at 18 h after heat shock, which is the time point at which most imaging‐based analysis was performed. These results are consistent with previous data using GFP‐tagged H3 versus GFP‐tagged H3.3 that show distinct incorporation modes between DNA replication‐dependent incorporation of the canonical H3 and DNA replication‐independent incorporation of the H3.3 histone variant in Drosophila. The ISCs co‐labeled with both eGFP (old histones) and mCherry (new histones) signals entered the subsequent mitosis, when sister chromatids are condensed and segregated equally into the daughter cells. The mitotic ISCs can be labeled using a mitotically enriched H3S10ph mark (phosphorylation at Serine 10 of H3). Using this mitotic mark, esg driver‐labeled ISCs can be distinguished in this histone tracer assay. Old versus new histone distribution patterns were studied at different mitotic stages of ISCs (Zion, 2023).

A long‐standing biological question is how distinct cell fates are established, maintained, and changed by epigenetic mechanisms at the single‐cell level in multicellular organisms, where cells have identical genomes. In this study, using the Drosophila ISC lineage as a model system, the inheritance of different canonical histones H3, H4, and H2A was studied during the ISC divisions. In postmitotic pairs of cells with an asymmetric Delta expression pattern, asymmetric histone inheritance patterns are detected, where the Delta‐high cell inherits more old histones and the Delta‐low cell inherits more new histones. In contrast, histones are distributed more symmetrically between the postmitotic pair of cells with similar Delta expression. It was hypothesized that the observed differences in Delta expression patterns are indicative of distinct cell identities resulting from different cell division modes. An asymmetric Delta expression pattern, with one Delta‐high cell and one Delta‐low cell, may indicate that these two cells result from an asymmetric ISC division, where the Delta‐high cell will maintain an ISC identity while the Delta‐low cell will take on differentiation. In contrast, a symmetric Delta expression pattern could result from a symmetric ISC division, where both Delta‐expressing cells will maintain the ISC identity. Interestingly, the percentages of asymmetric versus symmetric Delta expression patterns in the postmitotic pairs (79.6% versus 20.4%) are largely consistent with the ratios of asymmetric versus symmetric ISC divisions as reported in other studies, as well as the H3 inheritance modes in the mitotic ISCs observed in this study (75.0% versus 25.0%). These findings link the asymmetric histone inheritance mode with the establishment of distinct cell identities after one cell division. Furthermore, this asymmetric inheritance mode is specific to H3 and to the less extent to H4, as old and new H2A histones are almost always inherited symmetrically. This molecular specificity could be explained by the incorporation of old H3 and H4 into chromatin as a (H3‐H4)2 tetramer, while old H2A and H2B are incorporated as (H2A‐H2B) dimers. Since H3 and H4 carry most of the post‐translational modifications that influence gene expression, the asymmetric inheritance of old versus new (H3‐H4)2 serves as an elegant mechanism for establishing distinct epigenomes in the daughter cells that arise from stem cell ACD, possibly leading to differential gene expression programs and potentially other distinct cellular features (see Model for old (green) and new (red) histone inheritance patterns and their potential roles in the ISC system (Zion, 2023).

The biological significance of the asymmetric histone inheritance is further exemplified when this pattern is disrupted by the H3^T3A histone mutant, where an increase was detected in Delta‐symmetric postmitotic pairs of cells as well as the overpopulated ISC‐like cells. Interestingly, based on intestinal morphology, hyperplasia was observed in the H3T3A mutant more often in the posterior midgut. Previous work in the field has identified the posterior midgut as a region that is more sensitive to tumor formation, acting as a 'tumor hotspot'. In the current experiments, the posterior midgut appears to be more sensitized to the H3T3A mutant, consistent with these findings. These data connect misregulated histone inheritance with potential changes in cell fate determination. Recently, large‐scale analysis identified histone mutations in 3.8% of human tumor samples, a ratio similar to the mutations of known cancer‐associated genes such as BRCA2 and NOTCH1. In particular, mutations at the Thr3 residue of H3 have been found in a variety of human tumor samples, including lung, breast, skin, bladder, and liver cancers. However, the molecular mechanisms underlying these 'oncohistones' are not fully understood. The current findings on the oncohistone H3T3A illuminate how this mutation could lead to the loss of proper epigenetic inheritance and the onset of tumor formation (Zion, 2023).

The asymmetric inheritance mode of histones was first reported during the ACD of male Drosophila GSCs, which opened a new avenue of research; however, many essential questions remained, such as whether this asymmetric histone inheritance mode is specific to germ cells, stem cells, and/or asymmetrically dividing cells. The results reported in this paper provide a solid basis to addressing these questions. Despite significant differences in the niche structure, signaling cascades for regulating stem cell activity, and cellular differentiation pathways between the ISC and male GSC lineages, several key features of asymmetric histone inheritance are common between these two stem cell systems. First, the cellular specificity in the GSC lineage has been demonstrated by asymmetric histone inheritance mode in asymmetrically dividing GSCs but not in symmetrically dividing spermatogonial cells. In the ISC lineage, this cellular specificity is manifested by ISCs displaying an asymmetric histone inheritance mode in Delta‐asymmetric postmitotic pairs but not for Delta‐symmetric postmitotic pairs. Second, this asymmetry has the molecular specificity for H3 and H4 histones in both systems, emphasizing the importance of these two canonical histones in carrying and passing on or resetting an 'epigenetic memory.' Finally, expression of the mutant histone H3T3A abolishes asymmetric histone inheritances in both systems, resulting in stem cell or progenitor cell hyperplasia. Therefore, these results demonstrate that the asymmetric histone inheritance mode is not specific to either germ cells or stem cells, but likely contingent on the asymmetric mode of mitosis with the mission to generate two distinct daughter cells (Zion, 2023).

Zdditionally, asymmetric histone inheritance could occur at a local level, likely at critical genes that regulate stem cell fate or proper cellular differentiation. Similar local asymmetry of histone inheritance was shown in induced asymmetrically dividing mouse embryonic stem cells, with the H3K27me3 as a key histone modification displaying distinct distribution at the differentially expressed genes between the two daughter cells\. It will be intriguing to explore in the future what histone modifications are associated with old versus new histones (Zion, 2023).

Furthermore, asymmetric sister centromere in recognizing sister chromatids during ACD has also been reported in Drosophila male GSCs and female GSCs. Asymmetric inheritance of old versus new centromere‐specific histone variant CENP‐A/CID has also been reported in the Drosophila ISCs. More studies in the future are needed to understand how global (i.e., in fly male GSCs and ISCs) and local (i.e., in fly female GSCs and mESCs) canonical histone asymmetries are established, which is likely by developmentally programmed DNA replication. Furthermore, how canonical histone asymmetries on sister chromatids are recognized and differentially inherited is likely due to the differential attachment of the mitotic spindle to asymmetric sister centromeres. Finally, the distinct distribution of epigenetic information between the two daughter cells derived from ACD probably prepares them for distinct cellular behaviors, which is crucial to development, tissue homeostasis, and regeneration of multicellular organisms (Zion, 2023).

It has been debated whether the two cells resulting from ISC division are intrinsically asymmetric, or only become asymmetric through the extrinsic signaling cues after ISC division. Previous studies demonstrate that intrinsic polarity mechanisms result in the asymmetric distribution and inheritance of Par proteins to the apical daughter cell during ACD of ISCs, in order to promote differentiation. Furthermore, differential Notch activities due to the polarized Par complex induce distinct cellular differentiation pathways. Recent work has also shown that the spindle orientation in ISCs is tightly linked with cell fate, where planar orientation gives rise to two ISCs and angular orientation generates the ISC/EB pair of daughter cells\. Through analyzing different mitotic stages of ISCs, separable old versus new H3 distribution is detectable in prophase and prometaphase ISCs regardless of the ISC division modes, indicating that this asymmetry is likely intrinsically established prior to mitosis. Interestingly, old versus new H3T3A signals are still separable in prophase and prometaphase ISCs, similar to that of wild‐type H3, suggesting that this mutant histone does not interfere with differential histone incorporation before mitosis. However, increased symmetric segregation patterns in anaphase and telophase H3T3A‐expressing ISCs indicate that sister chromatids differentially enriched with old versus new H3T3A signals cannot be properly recognized and segregated. Because flies have two major autosomes (2nd and 3rd chromosomes) in addition to the sex chromosomes, even the randomized segregation of sister chromatids could lead to an asymmetric pattern at a low percentage, as shown previously. However, even though rare asymmetric histone inheritance occurs with randomized segregation pattern, the daughter cell enriched with old histone is still the cell expressing a higher level of Delta, indicative of ISC identity. Together, these findings indicate that ISC cell fate is likely specified by the intrinsic epigenetic information and the polarized extrinsic cues possibly act to ensure their differential segregation pattern. In summary, given the unique features of the ISC system, such as the ability to precisely label each derivative cell in the entire lineage, the large number of ISCs in their endogenous niche, and the sensitivity of ISC activity to environmental changes such as nutrition as well as aging, it will become a new in vivo model system to study the fundamental principles of different histone inheritance modes and relevant biological consequences under physiological and pathological conditions (Zion, 2023).

Golgi-to-ER retrograde transport prevents premature differentiation of Drosophila type II neuroblasts via Notch-signal-sending daughter cells

Stem cells are heterogeneous to generate diverse differentiated cell types required for organogenesis; however, the underlying mechanisms that differently maintain these heterogeneous stem cells are not well understood. This study identified that Golgi-to-endoplasmic reticulum (ER) retrograde transport specifically maintains type II neuroblasts (NBs) through the Notch signaling. Intermediate neural progenitors (INPs), immediate daughter cells of type II NBs, provide Delta and function as the NB niche. The Delta used by INPs is mainly produced by NBs and asymmetrically distributed to INPs. Blocking retrograde transport leads to a decrease in INP number, which reduces Notch activity and results in the premature differentiation of type II NBs. Furthermore, the reduction of Delta could suppress tumor formation caused by type II NBs. These results highlight the crosstalk between Golgi-to-ER retrograde transport, Notch signaling, stem cell niche, and fusion as an essential step in maintaining the self-renewal of type II NB lineage (Zhang, 2024).

This study demonstrates that Golgi-to-ER retrograde transport (RT) is essential for the maintenance of type II NBs. In wild-type type II NBs, the Notch ligand Delta is distributed to the basal side and segregated to INPs during division. The Delta localized in NB-adjacent INPs activates Notch signaling in NBs. Blocking RT will disrupt biosynthesis and proliferation, leading to a decreased number of INPs, reducing the Delta protein, and consequently decrease the Notch activity. This disruption ultimately results in ectopic nuclear Pros expression and premature differentiation of type II NBs. This mechanism can be adopted to suppress the tumor development initiated from type II NBs (Zhang, 2024).

In the CNS, the Notch pathway is often involved in the maintenance of quiescence and self-renewal of NSCs. For instance, in the telencephalon of adult zebrafish, quiescent NSCs express the Notch3 to remain in a quiescent state. In the absence of Notch3, the ratio of activated NSC is significantly increased. However, once NSCs are activated, they still require Notch3 to maintain their stemness, and loss of Notch3 leads to a progressive decrease in the number of activated NSCs accompanied by an increase in neuron number. Similarly, in the subventricular zone of adult mammals, Notch signaling is highly active in quiescent NSCs and has a pivotal role in maintaining their quiescent state. Dysfunction in the Notch pathway will reactivate quiescent NSCs. In mammalian embryonic brain, Notch pathway is essential for the self-renewal of NSCs, as the loss of Notch leads to the premature differentiation of most NSCs into neuron and finally depletion. Likewise, in Drosophila type I NBs, loss of Notch delays entry into quiescence, while overexpression of Notch leads to an early entry into quiescence. In this study, it was found that reduced Notch activity induced by blocking RT selectively affects the self-renewal of type II NBs. Therefore, Notch pathway has different roles in two types of NBs, which is essential for entering quiescence in type I NBs and for maintaining the self-renewal in type II NBs. In conclusion, Notch plays critical roles in regulating quiescence and self-renewal of NSCs, which is determined by the cell state, developmental stage, or cell type of NSCs (Zhang, 2024).

The niche refers to the microenvironment, in which stem cells are located, and has a regulatory effect on stem cells. Generally, niche-forming cells are either cells independent of the stem cell lineage such as somatic cap cells or hub cells, which form the female or male Drosophila germline stem cell niche, respectively, or highly differentiated daughter cells of stem cells, like Paneth cells acting as the niche of mammalian intestinal stem cells. In the Drosophila CNS, previous studies have shown that highly differentiated glial cells form the NB niche, which regulates the quiescence, reactivation, survival, and proliferation of NBs. Therefore, the newly born daughter cells are not regarded as part of stem cell niche. However, recent studies have shown that the trans-amplifying cells generated by ISCs regulate the proliferation of ISCs through the Wnt pathway. In mammalian CNS, the intermediate progentior cells (IPCs) provide Notch ligands to activate Notch in NSCs. This study found that the newly generated INPs, as the NBs niche, provide Delta to trans-activate Notch and regulated the self-renewal of type II NBs. Together, these studies support the conception that the immediate daughter cells might function as the stem cell niche (Zhang, 2024).

The activation of the Notch pathway requires adjacent cells to provide ligands. This study has found that INPs provide Delta for maintaining the self-renewal of type II NBs, resembling the role of mammalian IPCs in producing Delta to activate NSCs in the CNS. However, the regulatory mechanisms of these two Notch activations are still different. In the Drosophila type II NB lineage, the NB expresses the highest level of Delta, which is distributed to INPs for activating Notch in type II NBs. Furthermore, knocking down Delta in type II NBs leads to a decrease in Notch activity, indicating that type II NBs produce Delta that is required for the activation of their own Notch signaling, resembling how Notch is activated to maintain the NSC quiescence in mammals. Therefore, tbis research deepens the understanding of the regulation of NB self-renewal through Notch signaling pathway (Zhang, 2024).

The Golgi-to-ER RT is a critical process in maintaining ER homeostasis and protein synthesis and secretion. Therefore, blocking this process is highly correlated with serious cellular defects and neurodegeneration diseases. For instance, in KDELR mutant mice, cells are sensitive to ERS, which finally develop into dilated cardiomyopathy. Protein misfolding or accumulation resulting from RT blocking is closely related to the development of many neurological diseases, such as Parkinson's disease and Alzheimer's disease. This study blocked RT in Drosophila type II NBs, which led to type II NB loss. However, type II NB loss was not due to ER stress (ERS) induction. Instead, blocking RT resulted in reduced biosynthesis and cell proliferation of type II NBs, eventually leading to attenuated Notch activity, and premature differentiation of type II NBs, which provides a new mechanism for future research on the consequences related to RT block. It is worth noting that several genes related to protein modification or epigenetics were discovered that were also specifically required for the maintenance of type II NBs in this screen, which possibly share the same mechanism of RT block, suggesting that regulating INP number may be a general mechanism on maintaining type II NBs (Zhang, 2024).

In this study it was observed that some retained type II NBs were labeled by mGFP (mCD8-GFP) after eliminating INPs, which is theoretically impossible to appear in the original type II NBs. It is speculated that the original type II NB is lost due to INP elimination, which creates room for an INP to occupy the NB position, receive Delta from other INPs, and eventually become the mGFP-labeled new NB. However, further evidence is required to confirm this hypothesis (Zhang, 2024).

This study revealed that Golgi-to-ER RT selectively maintains type II NBs by regulating Notch activity. Notch is highly expressed in various tumors, such as breast cancer, gliomas, malignant melanoma, and small cell lung cancer. The results demonstrated that blocking RT could effectively inhibit Notch-overexpressing tumors by reducing the number of INPs in Drosophila brain. Thus, targeting the cells that provide Notch ligands to tumor cells could potentially be a promising treatment to suppress tumors, but further research is needed in the future to fully explore this avenue (Zhang, 2024).

This study focused on Golgi-to-ER RT, which maintains Drosophila type II NBs by regulating Notch activity. During the division of type II NBs, Delta is asymmetrically distributed to INPs. In the future, it is necessary to explore the mechanisms that mediate Delta distribution. Besides, it was found that Golgi-to-ER RT maintains the self-renewal of Drosophila type II NBs. Therefore, using other types of mammal stem cells to explore the conservative regulatory mechanism of Golgi-to-ER RT in self-renewal is very meaningful (Zhang, 2024).

GENE STRUCTURE

There are two major transcripts and three minor ones. The 4.5 transcript is maternal (Kopczynski, 1989). The transcripts differ in the length of their 3'UTR. Minor transcripts consisting of introns of the Delta gene accumulate in the nucleus (Haenlin, 1990).

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

PROTEIN STRUCTURE

Amino Acids - 832

Structural Domains

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:  1 June 2024

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