Notch: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

Gene name - Notch

Synonyms - Abruptex (Ax), split (spl)

Cytological map position - 3C7

Function - receptor, lateral inhibition

Keywords - neurogenic, oncogene and tumor suppressor

Symbol - N

FlyBase ID:FBgn0004647

Genetic map position - 1-3.0

Classification - EGF family, ankyrin-repeat, SH3-domain

Cellular location - cell surface



NCBI links: Precomputed BLAST | Entrez Gene |
Recent literature
Michel, M., Aliee, M., Rudolf, K., Bialas, L., Julicher, F. and Dahmann, C. (2016). The selector gene apterous and Notch are required to locally increase mechanical cell bond tension at the Drosophila dorsoventral compartment boundary. PLoS One 11: e0161668. PubMed ID: 27552097
Summary:
In Drosophila, the wing imaginal disc is subdivided into a dorsal and a ventral compartment. Cells of the dorsal, but not ventral, compartment express the selector gene apterous. Apterous expression sets in motion a gene regulatory cascade that leads to the activation of Notch signaling in a few cell rows on either side of the dorsoventral compartment boundary. Both Notch and apterous mutant clones disturb the separation of dorsal and ventral cells. Maintenance of the straight shape of the dorsoventral boundary involves a local increase in mechanical tension at cell bonds along the boundary. The mechanisms by which cell bond tension is locally increased however remain unknown. This study used a combination of laser ablation of cell bonds, quantitative image analysis, and genetic mutants to show that Notch and Apterous are required to increase cell bond tension along the dorsoventral compartment boundary. Moreover, clonal expression of the Apterous target gene capricious results in cell separation and increased cell bond tension at the clone borders. Finally, using a vertex model to simulate tissue growth, an increase in cell bond tension at the borders of cell clones, but not throughout the cell clone, was found to lead to cell separation. It is concluded that Apterous and Notch maintain the characteristic straight shape of the dorsoventral compartment boundary by locally increasing cell bond tension.
Bi, P., Yue, F., Sato, Y., Wirbisky, S., Liu, W., Shan, T., Wen, Y., Zhou, D., Freeman, J. and Kuang, S. (2016). Stage-specific effects of Notch activation during skeletal myogenesis. Elife 5. PubMed ID: 27644105
Evolutionary Homolog Study
Skeletal myogenesis involves sequential activation, proliferation, self-renewal/differentiation and fusion of myogenic stem cells (satellite cells). Notch signaling is known to be essential for the maintenance of satellite cells, but its function in late-stage myogenesis, i.e. post-differentiation myocytes and post-fusion myotubes, is unknown. Using stage-specific Cre alleles, distinct roles were found of Notch1 in mononucleated myocytes and multinucleated myotubes. Specifically, constitutive Notch1 activation dedifferentiates myocytes into Pax7+ quiescent satellite cells, leading to severe defects in muscle growth and regeneration, and postnatal lethality. By contrast, myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles. Mechanistically, Notch1 activation in myotubes upregulates the expression of Notch ligands, which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity. These results highlight context-dependent effects of Notch activation during myogenesis, and demonstrate that Notch1 activity improves myotube's function as a stem cell niche.
Nemetschke, L. and Knust, E. (2016). Drosophila Crumbs prevents ectopic Notch activation in developing wings by inhibiting ligand-independent endocytosis. Development 143(23): 4543-4553. PubMed ID: 27899511
Summary:
Many signalling components are apically restricted in epithelial cells, and receptor localisation and abundance is key for morphogenesis and tissue homeostasis. Hence, controlling apicobasal epithelial polarity is crucial for proper signalling. Notch is a ubiquitously expressed, apically localised receptor, which performs a plethora of functions; therefore, its activity has to be tightly regulated. This study shows that Drosophila Crumbs, an evolutionarily conserved polarity determinant, prevents Notch endocytosis in developing wings through direct interaction between the two proteins. Notch endocytosis in the absence of Crumbs results in the activation of the ligand-independent, Deltex-dependent Notch signalling pathway, and does not require the ligands Delta and Serrate or γ-secretase activity. This function of Crumbs is not due to general defects in apicobasal polarity, as localisation of other apical proteins is unaffected. These data reveal a mechanism to explain how Crumbs directly controls localisation and trafficking of the potent Notch receptor, and adds yet another aspect of Crumbs regulation in Notch pathway activity. Furthermore, the data highlight a close link between the apical determinant Crumbs, receptor trafficking and tissue homeostasis.
Ling, X., Huang, Q., Xu, Y., Jin, Y., Feng, Y., Shi, W., Ye, X., Lin, Y., Hou, L. and Lin, X. (2017). The deubiquitinating enzyme Usp5 regulates Notch and RTK signaling during Drosophila eye development. FEBS Lett [Epub ahead of print]. PubMed ID: 28140449
Summary:
Usp5 belongs to the USP family of deubiquitinating enzymes (DUBs), which comprises the largest class of DUBs. Loss of Usp5 has been shown to impair development of photoreceptors in Drosophila eyes, but the detailed mechanism remained unclear. This study demonstrated that Usp5 regulates both Notch and receptor tyrosine kinase (RTK) signaling. Loss of Usp5 results in upregulation of Notch signaling and downregulation of RTK signaling, leading to impaired photoreceptor development. Moreover, genetic rescue experiments with Su(H) or Notch RNAi indicate that they mediate the regulation of RTK signaling by Usp5. This study provides mechanistic insight into how Usp5 regulates photoreceptor differentiation by Notch and RTK signaling in the Drosophila eye.
Corson, F., Couturier, L., Rouault, H., Mazouni, K. and Schweisguth, F. (2017). Self-organized Notch dynamics generate stereotyped sensory organ patterns in Drosophila. Science [Epub ahead of print]. PubMed ID: 28386027
Summary:
The emergence of spatial patterns in developing multicellular organisms relies on positional cues and cell-cell communication. Drosophila sensory organs have informed a paradigm where these operate in two distinct steps: prepattern factors drive localized proneural activity, then Notch-mediated lateral inhibition singles out neural precursors. This study shows that self-organization through Notch signaling also organizes the proneural stripes that resolve into rows of sensory bristles on the fly thorax. Patterning, initiated by a gradient of Delta ligand expression, progresses through inhibitory signaling between and within stripes. Thus Notch signaling can support self-organized tissue patterning as a prepattern is transduced by cell-cell interactions into a refined arrangement of cellular fates.
Lee, T. V., Pandey, A. and Jafar-Nejad, H. (2017). Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila. PLoS Genet 13(4): e1006723. PubMed ID: 28394891
Summary:
The Drosophila glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development. However, the molecular mechanisms underlying context-specificity of the shams phenotype is not known. It is hypothesized that Shams might affect Delta-mediated Notch signaling. This study found that altering the gene dosage of Delta affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites. Clonal analysis suggests that loss of shams promotes Delta-mediated Notch activation. Further, Notch trans-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of shams. In vivo, cell aggregation and ligand-receptor binding assays show that shams knock-down in Notch-expressing cells enhances the binding between Notch and trans-Delta without affecting the binding between Notch and trans-Serrate and cell surface levels of Notch. Removing one copy of endogenous ligands mimics the effects of loss shams on Notch trans-activation by ectopic Delta. This favors the notion that trans-activation of Notch by Delta overcomes the cis-inhibition of Notch by endogenous ligands upon loss of shams. Taken together, these data suggest that xylosylation selectively impedes the binding of Notch with trans-Delta without affecting its binding with cis-ligands and thereby assists in determining the balance of Notch receptor's response to cis-ligands vs. trans-Delta during Drosophila development.
He, L., Huang, J. and Perrimon, N. (2017). Development of an optimized synthetic Notch receptor as an in vivo cell-cell contact sensor. Proc Natl Acad Sci U S A 114(21): 5467-5472. PubMed ID: 28490499
Summary:
Detection and manipulation of direct cell-cell contact in complex tissues is a fundamental and challenging problem in many biological studies. This study reports an optimized Notch-based synthetic receptor (synNQ) useful to study direct cell-cell interactions in Drosophila. With the synNQ system, cells expressing a synthetic receptor, which contains Notch activation machinery and a downstream transcriptional activator, QF, are activated by a synthetic GFP ligand expressed by contacting neighbor cells. To avoid cis-inhibition, mutually exclusive expression of the synthetic ligand and receptor is achieved using the "flippase-out" system. Expression of the synthetic GFP ligand is controlled by the Gal4/UAS system for easy and broad applications. Using synNQ, cell-cell interactions within and between most fly tissues were successfully visualized, revealing previously undocumented cell-cell contacts. Importantly, in addition to detection of cells in contact with one another, synNQ allows for genetic manipulation in all cells in contact with a targeted cell population, which is demonstrated in the context of cell competition in developing wing disks. Altogether, the synNQ genetic system will enable a broad range of studies of cell contact in developmental biology.
Bhattacharya, A., Li, K., Quiquand, M., Rimesso, G. and Baker, N. E. (2017). The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins. Dev Biol [Epub ahead of print]. PubMed ID: 28919436
Summary:
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.
Prince, L. M. and Rand, M. D. (2017). Notch target gene E(spl)mdelta is a mediator of methylmercury-induced myotoxicity in Drosophila. Front Genet 8: 233. PubMed ID: 29379520
Summary:
Methylmercury (MeHg) is a ubiquitous environmental contaminant and neurotoxicant that has long been known to cause a variety of motor deficits. These motor deficits have primarily been attributed to MeHg targeting of developing neurons and induction of oxidative stress and calcium dysregulation. Studies in Drosophila have revealed that MeHg perturbs embryonic muscle formation and upregulates Notch target genes, reflected predominantly by expression of the downstream transcriptional repressor Enhancer of Split mdelta [E(spl)mdelta]. An E(spl)mdelta reporter gene shows expression primarily in the myogenic domain, and both MeHg exposure and genetic upregulation of E(spl)mdelta can disrupt embryonic muscle development. This study tested the hypothesis that developing muscle is targeted by MeHg via upregulation of E(spl)mdelta. Developmental MeHg exposure causes a decreased rate of eclosion that parallels gross disruption of indirect flight muscle (IFM) development. An increase in E(spl) expression across the pupal stages, with preferential E(spl)mdelta upregulation occurring at early (p5) stages, is also observed. E(spl)mdelta overexpression in myogenic lineages under the Mef2 promoter was seen to phenocopy eclosion and IFM effects of developmental MeHg exposure; whereas reduced expression of E(spl)mdelta shows rescue of eclosion and IFM morphology effects of MeHg exposure. No effects were seen on eclosion with E(spl)mdelta overexpression in neural and gut tissues. These data indicate that muscle development is a target for MeHg and that E(spl)mdelta is a muscle-specific mediator of this myotoxicity.
Kavaler, J., Duan, H., Aradhya, R., de Navas, L. F., Joseph, B., Shklyar, B. and Lai, E. C. (2017). miRNA suppression of a Notch repressor directs non-neuronal fate in Drosophila mechanosensory organs. J Cell Biol 217(2):571-583. PubMed ID: 29196461
Summary:
Although there is abundant evidence that individual microRNA (miRNA) loci repress large cohorts of targets, large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. This study identified a rare case of developmental cell specification that is highly dependent on miRNA control of an individual target. Binary cell fate choice in the Drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. The mir-279/996 defect phenocopies Notch loss of function during the sheath-neuron cell fate decision, suggesting the miRNAs facilitate Notch signaling. Consistent with this, mir-279/996 knockouts are strongly enhanced by Notch heterozygosity, and activated nuclear Notch is impaired in the miRNA mutant. Although Hairless (H) is the canonical nuclear Notch pathway inhibitor, and H heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-Notch signaling, H/+ does not rescue mir-279/996 mutants. Instead, Insensible (Insb), another neural nuclear Notch pathway inhibitor, was identified as a critical direct miR-279/996 target. Insb is posttranscriptionally restricted to neurons by these miRNAs, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. Thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving miRNA-mediated suppression of a Notch repressor to assign non-neuronal cell fate.
Kannan, R., Cox, E., Wang, L., Kuzina, I., Gu, Q. and Giniger, E. (2018). Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance. Development 145(2). PubMed ID: 29343637
Summary:
Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. This study shows that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. It furthers show that some Notch protein is tyrosine phosphorylated in Drosophila, that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, a model is proposed for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons.
Bellec, K., Gicquel, I. and Le Borgne, R. (2018). Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo. Development 145(13). PubMed ID: 29967125
Summary:
In Drosophila, the sensory organ precursor (SOP or pI cell) divides asymmetrically to give birth to daughter cells, the fates of which are governed by the differential activation of the Notch pathway. Proteolytic activation of Notch induced by ligand is based on the correct polarized sorting and localization of the Notch ligand Delta, the Notch receptor and its trafficking partner Sanpodo (Spdo). This study has identified Stratum (Strat), a presumptive guanine nucleotide exchange factor for Rab GTPases, as a regulator of Notch activation. Loss of Strat causes cell fate transformations associated with an accumulation of Notch, Delta and Spdo in the trans-Golgi network (TGN), and an apical accumulation of Spdo. The strat mutant phenotype is rescued by the catalytically active as well as the wild-type form of Rab8, suggesting a chaperone function for Strat rather than that of exchange factor. Strat is required to localize Rab8 at the TGN, and rab8 phenocopies strat. It is proposed that Strat and Rab8 act at the exit of the Golgi apparatus to regulate the sorting and the polarized distribution of Notch, Delta and Spdo.

BIOLOGICAL OVERVIEW

Notch is a surface receptor. It transmits signals received from outside the cell to the cell's interior. Notch ligands, such as Delta, Serrate and Scabrous interact with epidermal growth factor repeats contained in Notch's extracellular domain.

The intracellular domain of Notch binds Suppressor of Hairless, a multifunction transcription factor that acts as a signal transducing molecule shuttling between the cytoplasm and the nucleus. The intracellular domain of Notch might also have a nuclear function, as first suggested by Lieber, 1993. A nuclear function has been documented for the mammalian Notch homolog (Lu, 1996), and has now been documented for Drosophila as well (Struhl, 1998).

When Notch is bound by a ligand, a signal is passed across the cell membrane releasing the Suppressor of Hairless protein, freeing this protein to enter the nucleus and assume its role in activating transcription of Enhancer of split complex genes. E(spl)-C proteins act in turn to repress the adoption of neural and other differentiated states. Deltex, an intracellular docking protein, replaces Suppressor of Hairless as Su(H) leaves the site of interaction with the intracellular tail of Notch.

The Notch receptor is function is called neurogenic, but this confusing nomenclature refers to the phenotype established in the absence of functional Notch. Notch's function is to repress the adoption of differentiation by cells that carry the Notch protein. A look at the principle ligand of Notch (Delta) and its function makes the anti-neural function of Notch more easily understood.

Delta is not secreted, but is cell bound. The Delta-Notch interaction serves a cell adhesive function between ligand and receptor bearing cells. The receptor bearing cell is inhibited in assuming a differentitated state, while the ligand bearing cell is free to do so. During neurogenesis, this latter cell delaminates, that is, it migrates out of the epithelial cell layer in which it formerly resided, and assumes the differentiated state of a neuroblast in its new physical location within the developing nervous system. Thus Notch is involved in neurogenesis with respect to cells that bears the ligands for Notch: Delta, Serrate and Scabrous.

Lateral inhibition is a process whereby a single cell is fated to differentiate through the interaction of Notch-Delta, while other cells simultaneously retain their undifferentiated state. A state of competition is imposed upon a cluster of cells. Perhaps the single cell, seemingly selected at random, is the one with the highest density of ligand. However, very little is left to chance. Three other proteins are involved in fate determination of the selected cell. Inscuteable, Numb, Prospero assure a neural fate for the ligand bearing cell The selected cell proceeds along a neural differentiation pathway, synthesizing higher levels of the proneural proteins, Achaete and Scute.

Lateral inhibition is one of the major themes of development. The process of lateral inhibition and cell selection is repeated hundreds of times in Drosophila, with differentiation that takes place in nearly every kind of tissue. For example, Notch is required to limit the number of neural precursors, limit the number of muscle precursors, limit the growth of Malpighian tubules, and regulate the growth of the ovary. Notch also functions as receptor for both Serrate and Delta in organizing the dorsal-ventral boundary of the wing. One important target of Serrate and Notch in this context is wingless (Diaz-Benjumea, 1995).

Two extreme models can be envisioned for lateral inhibition. The first implicates the Notch pathway in the choice of a single precursor via a negative feedback loop. This process could be random in some cases. The second model postulates that the precursor is pre-determined by some mechanism other than Notch signaling, and that Notch signaling then serves only to mediate mutual, uniform repression of other cells and ensure development of a single precursor. Studies concerning the physical spacing of precursors for the microchaetes of the peripheral nervous system suggest the existence of a regulatory loop under transcriptional control between Notch and its ligand Delta. Activation of Notch leads to repression of the achaete-scute genes, which are themselves known to regulate transcription of Delta; this regulation may perhaps be direct (Seugnet, 1997a).

Neuroblast segregation was studied in embryos lacking both the maternal and the zygotic forms of either Notch or Delta. A seven-up-LacZ marker was used to follow neuralization of 5-2 and 7-4 neuroblast groups. In the absence of Notch signaling, the cells with an equivalence group do not enter the neural differentiation pathway simultaneously. Neuralization within a group is progressive with two or three cells segregating early and several more later. This suggests that neural potential is not evenly distributed among these cells. A requirement for transcriptional regulation of Notch and/or Delta during neuroblast segregation in embryos was tested by providing Notch and Delta ubiquitously at uniform levels. Neuroblast segregation occurs normally under conditions of uniform Notch expression, suggesting that transcriptional regulation of Notch is not necessary for many aspects of development of the larval CNS and PNS. In particular, it is dispensable both before and after neuroblast segregation, implying that it is not a necessary component of neuroblast segregation, per se. Under conditions of uniform Delta expression, a single neuroblast segregates from each proneural group in 80% of the cases; in the remaining 20%, more than one neuroblast segregates from a single group of cells. Thus transcriptional regulation of Delta is largely dispensable, with only a small percentage of multiple neurons segregating in each cluster. The possibility is discussed that segregation of single precursors in the central nervous system may rely on a heterogeneous distribution of neural potential between different cells of the proneural group. Genes such as achaete, scute, extramacrochaete, and wingless could be responsible for local differences in proneural activity. Notch signaling would enable all cells to mutually repress one another; only a cell with an elevated neural potential could overcome this repression (Seugnet, 1997a).

Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila

The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors. Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions. At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate. This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship. This proposal has been tested by analyzing interactions between Delta- and Serrate-activated Notch signaling and Wingless signaling during wing development and patterning. Cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra. This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signaling but can also disrupt signaling through another pathway. One possibility is the Wingless signaling pathway, since the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signaling. Furthermore, the outcome of the interactions between Notch and Wingless signaling differs when Wingless signaling is activated by expressing either Wingless itself or an activated form of the Armadillo. For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone. This result suggests that signaling activated by Wingless leads to two effects: a reduction of Notch signaling and an activation of Armadillo (Brennan, 1999a).

Expression of a dominant negative Notch molecule (Extracellular Notch or ECN) throughout the developing wing mimics the effects of loss of Notch function. However, Nintra cannot rescue the cell death caused by overexpressing ECN. Since Nintra provides constitutive signaling for Delta and Serrate during wing development and the effects of ECN are mediated by the sequestration of extracellular molecules that can interact with Notch, this suggests that the ECN molecule is sequestering extracellular molecules other than Delta and Serrate and attenuating signaling through another pathway. One candidate pathway is the Wingless signaling pathway, since the cell death caused by expressing the ECN can be rescued by activating Wingless signaling. Therefore, it is possible that the ECN molecule is sequestering the Wingless protein. The possibility that Wingless can bind the extracellular domain of Notch is supported by the results that are presented here, in particular, by two observations: first, that some of the deleterious effects of ECN can be suppressed by Wingless, but not Wingless signaling in the form of a constitutively active Armadillo molecule; and second, that this interaction requires specific EGF-like repeats of Notch, namely repeats 17-19 and 24-26 but not 10-12. Evidence for a physical interaction between Notch and Wingless has also been provided recently by Wesley (1999) who finds that the Wingless protein is enriched in a biopanning assay designed to identify proteins that interact with the extracellular domain of the Notch protein and that Wingless can be immunoprecipitated with Notch from embryo extracts and cultured cells. These experiments also show that the association of Wingless with Notch requires the integrity of a region of Notch centered around EGF-like repeats 24-26 (Wesley, 1999) which these experiments indicate are essential for the interactions that are described between Wingless and ECN during wing development and patterning (Brennan, 1999a).

High levels of Wingless throughout the developing wing induce widespread development of sensory organs, an observation that correlates with the requirement for Wingless in this process during normal development. However, it is consistently observed that an activated form of Armadillo has a much weaker effect than Wingless on neural development. However, the difference is unlikely to be due to a weak UASarm* insert used in these experiments since in other instances where only a Wingless signal is required, such as the induction of the wing primordium during the early events of wing development, overexpressing Arm* or Wingless has very similar effects. A possible insight into the differences that the expression of Wingless and Arm* has on neurogenesis comes from the experiments where these two proteins are coexpressed with the ECN molecule. In these experiments the phenotypes generated by expressing UASECN with UASwg or UASarm* are very similar; namely, disrupting Notch signaling by expressing the ECN protein makes UASarm* and UASwg functionally equivalent. This suggests that the difference between the phenotypes generated by expressing Wingless and Arm* on their own might arise from the ability of Wingless to inhibit Notch signaling, which Arm* is unable to do; attenuating Notch signaling blocks lateral inhibition, which leads to increased numbers of sense organs. Since Wingless can activate Armadillo, overexpression of Wingless can achieve both effects simultaneously (Brennan, 1999a).

When Arm* is coexpressed with ECN, the dominant negative molecule reduces Notch signaling, providing the function of Wingless that is missing in Arm* and thus making this molecule functionally equivalent to Wingless. These results raise the question of how Wingless signaling inhibits Notch signaling and where in the Wingless signaling pathway the cross-talk between the two pathways occurs. The inability of Arm* to inhibit Notch signaling indicates that the cross-talk must occur upstream of Armadillo. One possibility is that the inhibition occurs through Wingless interacting with the extracellular portion of Notch, preventing the Notch protein from interacting with its ligands. However, it is more likely to occur through the interaction of Dishevelled with the intracellular domain of the Notch protein, which has been shown previously to inhibit Notch signaling (Axelrod, 1996). In keeping with this, it has been found that overexpressing the Dishevelled protein can induce sense organ development as effectively as overexpressing Wingless; this suggests that Dishevelled can also disrupt Notch signaling as effectively as Wingless. Finally, it is possible that the interaction of Notch with both Dishevelled and Wingless is required to inhibit Delta signaling through Notch, since it has been shown previously that the ability to overexpress Dishevelled, which induces supernumerary sense organs, requires Wingless function (Axelrod, 1996). The interference of Wingless signaling with Notch signaling can also provide an explanation for the effects of ectopic expression of Wingless on the patterning of the veins and its sensitivity to the concentration of Delta. Overexpression of Wingless would reduce the availability of Notch for lateral inhibition by causing Dishevelled to sequester Notch into complexes that are unable to transduce the Delta signal. This would reduce the effectiveness of lateral inhibition signaling, an effect which would be exaggerated in situations of limiting signaling, as is observe in Dl heterozygotes or when Wingless is coexpressed with ECN (Brennan, 1999a).

The interaction of Wingless and Notch signaling that has been observed might also be important during normal neural development. Wingless and Delta have opposite effects during neurogenesis; Wingless promotes while Delta suppresses the development of sense organs. Various experiments suggest that during the segregation of neural precursors a reduction of Notch signaling in the precursors themselves is as important as the Delta-mediated activation of Notch signaling in the surrounding cells. It is possible that, like the activation of Notch by Delta, the suppression of Notch signaling is an active process mediated by the interaction of Wingless and Dishevelled with Notch. If this were the case, since both Delta and Wingless have spatially and temporally regulated patterns of gene expression, their interactions with Notch could contribute to the well-documented bias in the appearance of precursors from clusters of cells with neural potential. This competitive interaction could also account for the observed increases in Wingless signaling associated with reductions in Notch signaling during lateral inhibition (Brennan, 1999a).

Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, mutants were recovered that disrupt genes encoding serine palmitoyltransferase and Acetyl-CoA Carboxylase (ACC). Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EGFR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. This analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation (Sasamura, 2013).

The importance of lipid metabolism for the formation and maintenance of cell membranes is well established. Both serine palmitoyltransferase (SPT) and acetyl-CoA carboxylase (ACC) are critical enzymes that control different steps of lipid metabolism, and are highly conserved in diverse animal species. Genetic elimination of ACC1 or the SPT subunits Sptlc1 or Sptlc2 cause early embryonic lethality in mice, although the cellular basis for this lethality is unknown. In D. melanogaster, RNA-interfering disruption of ACC activity in the fat body results in reduced triglyceride storage and increased glycogen accumulation, and in oenocytes leads to loss of watertightness of the tracheal spiracles causing fluid entry into the respiratory system. This study demonstrates that D. melanogaster mutants lacking functional SPT or ACC exhibit endosomal trafficking defects, causing Notch, Wingless, EGFR, and Patched to accumulate abnormally in endosomes and lysosomes. These effects are accompanied by significant alterations in Notch and Wingless signaling, as revealed by changes in downstream target gene activation for both pathways. However, the mutants do not fully inactivate these developmental signaling pathways, and instead display phenotypes consistent with more complex, pleiotropic effects on Notch, Wingless, and potentially additional pathways in different tissues. These findings reinforce the importance of lipid metabolism for the maintenance of proper developmental signaling, a concept that has also emerged from studies demonstrating that: D. melanogaster mutants for phosphocholine cytidylyltransferase alter endosomal trafficking and signaling of Notch and EGFR; mutants for alpha-1,4-N-acetylgalactosaminyltransferase-1 affect endocytosis and activity of the Notch ligands Delta and Serrate; mutants for the ceramide synthase gene shlank disrupt Wingless endocytic trafficking and signaling, and mutants for the glycosphingolipid metabolism genes egghead and brainiac modify the extracellular gradient of the EGFR ligand Gurken (Sasamura, 2013).

Most strikingly, lace and ACC mutants also display prominent tissue overgrowth phenotypes. These tissue overgrowth effects are linked to changes in Notch and Wingless signaling outputs, and they involve gamma-secretase, Su(H), and Armadillo activities, suggesting that the overgrowth reflects an interplay of Wingless inactivation and Notch hyperactivation. Consistent with the findings, both Notch and Wingless regulate cell proliferation and imaginal disc size in D. melanogaster. Moreover, several observations indicate that Notch and Wingless are jointly regulated by endocytosis, with opposing effects on their respective downstream pathway activities, a dynamic process that might be especially sensitive to perturbations in membrane lipid constituents. Wingless itself exerts opposing effects on disc size that might depend on the particular developmental stage or disc territory. For example, hyperactivation of Wingless or inactivation of its negative regulators cause overproliferation, but Wingless activity can also constrain wing disc growth. Similar spatiotemporal effects might underlie the variability detected in studies with lace and ACC mutant clones, in which both tissue overgrowth and developmentally arrested discs were observed. Although no obvious changes were detected in downstream signaling for several other cell growth pathways that were examined, the trafficking abnormalities seen for other membrane proteins aside from Notch, Delta, and Wingless, as well as the incomplete suppression of the overgrowth phenotypes by blockage of Notch and Wingless signaling, suggest that other pathways might also be dysregulated in lace and ACC mutants, possibly contributing to the observed tissue overgrowth (Sasamura, 2013).

Wingless is modified by lipid addition, and lipoprotein vesicles have been suggested to control Wingless diffusion. In D. melanogaster embryos, endocytosis of Wingless limits its diffusion and ability to act as a long-range morphogen. Endocytosis can also affect Wingless signaling in receiving cells, where endocytosis both promotes signal downregulation and positively facilitates signaling. The apparently normal diffusion ranges for overaccumulated Wingless in lace and ACC mutant clones, yet reduced downstream target gene expression, is consistent with the idea that SPT and ACC act by promoting endocytic trafficking of Wingless in receiving cells rather than influencing the secretion and/or diffusion of Wingless from signal-sending cells (Sasamura, 2013).

The finding that lace and ACC mutant overgrowth phenotypes are also partially Notch-dependent is reminiscent of similar overproliferation phenotypes seen in certain D. melanogaster endocytic mutants, such as vps25, and tsg101. The overproliferation of disc tissue in these mutants is attributable to Notch hyperactivation, reflecting the fact that non-ligand-bound Notch receptors that are normally targeted for recycling or degradation are instead retained and signal from endosomes. Analogous effects are likely to contribute to the lace and ACC mutant overgrowth, where significant Notch overaccumulation was observed throughout the endosomal-lysosomal routing pathway. Some ectopic Notch signaling might emanate from the lysosomal compartment, which is enlarged and accumulates particularly high levels of Notch in lace and ACC mutant clones. Analysis of D. melanogaster HOPS and AP-3 mutants, which affect protein delivery to lysosomes, has identified a lysosomal pool of Notch that is able to signal in a ligand-independent, gamma-secretase-dependent manner (Sasamura, 2013).

How do SPT and ACC contribute to endosomal trafficking of Notch and other proteins? In the yeast SPT mutant lcb1, an early step of endocytosis is impaired due to defective actin attachment to endosomes, a phenotype that is suppressed by addition of sphingoid base. However, the trafficking abnormalities seen in lace and ACC mutants do not resemble those in the yeast lcb1 mutant, perhaps because endocytic vesicle fission is primarily dependent upon dynamin in D. melanogaster and mammals, instead of actin as in yeast. Nevertheless, the requirement for SPT and ACC in D. melanogaster endosomal compartments might reflect possible functions in endosome-cytoskeleton interactions. Another possibility is that the defective endosomal trafficking seen in lace and ACC mutants is caused by the inability to synthesize specific phospholipids needed for normal membrane homeostasis. Finally, lace and ACC might be important for the formation and/or function of lipid rafts, specialized membrane microdomains that have been implicated in both signaling and protein trafficking (Sasamura, 2013).

A remarkable feature of the lace and ACC mutant phenotypes that suggests an underlying defect in lipid biogenesis is the non-autonomous effect in mutant tissue clones, wherein nearby wildtype cells generate a secreted activity that diffuses several cell diameters into the mutant tissue and rescues the trafficking and signaling defects. One possibility is that these secreted activities are diffusible lipid biosynthetic products of SPT and ACC, which enter the mutant cells and serve as precursors for further biosynthetic steps that do not require SPT or ACC. An intriguing alternative is that the SPT and ACC enzymes are themselves secreted and taken up by the mutant cells. A precedent for this mechanism has recently been demonstrated for D. melanogaster ceramidase, a sphingolipid metabolic enzyme that is secreted extracellularly, delivered to photoreceptors, and internalized by endocytosis to regulate photoreceptor cell membrane turnover (Sasamura, 2013).

Recent work has highlighted the importance of lipid metabolism for oncogenic transformation, and ACC has been advanced as a promising target for cancer drug development. ACC is upregulated in some cancers, possibly as a result of high demands for lipid biosynthesis during rapid cell divisions. Sphingolipids and their derivatives are also thought to influence the balance of apoptosis and cell proliferation during tissue growth, and thus have also garnered attention as potential cancer therapy targets. The current findings regarding the requirements of SPT and ACC for proper trafficking and signaling of key developmental cell-surface signaling molecules, including Notch and Wingless, provide insights into how lipid metabolic enzymes might influence cell proliferation and tissue patterning in multicellular animals. Complex lipid biosynthesis is essential for the creation of the elaborate, interconnected, and highly specialized membrane compartments in which developmental pathways operate, and perturbations in lipid biosynthesis that are tolerated by the cell might nevertheless exert significant pleiotropic effects on developmental patterning, cell proliferation, and other cellular processes. Exploration of lipid metabolic enzymes as pharmacological targets must therefore take into account potentially unfavorable effects on critical signaling pathways controlling development and organogenesis (Sasamura, 2013).

A re-examination of the selection of the sensory organ precursor of the bristle sensilla of Drosophila melanogaster

The bristle sensillum of the imago of Drosophila is made of four cells that arise from a sensory organ precursor cell (SOP). This SOP is selected within proneural clusters (PNC) through a mechanism that involves Notch signalling. PNCs are defined through the expression domains of the proneural genes, whose activities enables cells to become SOPs. They encode tissue specific bHLH proteins that form functional heterodimers with the bHLH protein Daughterless (Da). In the prevailing lateral inhibition model for SOP selection, a transcriptional feedback loop that involves the Notch pathway amplifies small differences of proneural activity between cells of the PNC. As a result only one or two cells accumulate sufficient proneural activity to adopt the SOP fate. Most of the experiments that sustained the prevailing lateral inhibition model were performed a decade ago. This study re-examined the selection process using recently available reagents. The data suggest a different picture of SOP selection. They indicate that a band-like region of proneural activity exists. In this proneural band the activity of the Notch pathway is required in combination with Emc to define the PNCs. A sub-group in the PNCs was found from which a pre-selected SOP arises. The data indicate that most imaginal disc cells are able to adopt a proneural state from which they can progress to become SOPs. They further show that bristle formation can occur in the absence of the proneural genes if the function of emc is abolished. These results suggest that the tissue specific proneural proteins of Drosophila have a similar function as in the vertebrates, which is to determine the time of emergence and position of the SOP and to stabilise the proneural state (Troost, 2015).

This study has re-examined the development of the SOP of the MC (macrochaetae) using recently available reagents. Evidence was found that strongly suggests that the range of the Notch signal is restricted to the next cell: The elevated expression of Notch activity reporter Gbe+Su(H) around the SOP is observed only in adjacent cells. In addition, cells of PNCs that are not able to receive the Notch signal, but can send a strong signal to adjacent wildtype cells, cannot prevent a wildtype cell from adopting the SOP fate at a distance of two cell diameters away. Likewise, cells that are not able to send a signal cannot be prevented by wildtype SOPs from adopting the SOP fate more than one cell diameter away. These results suggest that the discovered filopodia of the SOP, which contact more remotely located cells do not extend the range of the inhibitory signal to these cells (Troost, 2015).

This study reveals the existence of a band of proneural activity. The PNCs are regions of elevated proneural activity in this band, rather than discrete clusters. In the band, the Notch pathway exerts an additional novel function, which defines the extent of the PNCs. In the absence of Notch function, most cells in the proneural band accumulate high levels of proneural activity that allows them to become SOPs. Thus, the pathway suppresses the proneural activity and the SOP fate in cells located between the PNCs in the proneural band. The short range of the Notch signal indicates that it is probably local mutual signalling among direct neighbours that generates the necessary Notch activity (mutual inhibition). The expression of Dl and Ser and the overall activity of Gbe+Su(H) (with exception of the halos) is unchanged in the absence of Ac and Sc. This suggests that the widespread activity of Notch in the notum that prevents most cells in the proneural band to become SOPs is not influenced by the proneural factors. It provides a baseline activity of Notch that suppresses the proneural activity in the band to prevent the formation of ectopic SOPs (Troost, 2015).

The presented results indicate that a subgroup within the PNCs exists, which is operationally defined via the requirement of the activity of Neur. The existence of a subgroup has previously been suggested on basis of experiments with a temperature sensitive allele of Notch. These data and the ones presented here, suggest that the cells of the subgroup require Notch activity that is stronger than the baseline activity to be inhibited from adopting the SOP fate. This increase in activity is generated by the nascent SOP through a Neur enhanced Dl signal: This study found that if only one cell in the subgroup is neur positive, it can prevent all other neur mutant members to adopt the SOP fate. Thus, initiating the expression of Neur first, is a critical step for a cell to adopt the SOP fate, since it allows a cell to strongly inhibit its neighbours. The inhibitory signal prevents the accumulation of sufficient proneural activity to also activate Neur in the neighbours. This inhibition is probably reflected in the observed halo of Gbe+Su(H) expression around SOPs. The findings are in good agreement with a previous study that showed that the level of Neur in a cell is a critical factor for the formation of the SOP of the microchaetae (mc) (Troost, 2015).

Loss of Notch activity results in expression of Neur and a dramatic increase in proneural activity in all cells of the PNC. Moreover, the nascent SOP, which contains the highest proneural activity, is the only cell that initiates Neur expression during normal development and expression of neur is abolished in ac sc mutant discs. These data indicate, that high proneural activity is required for the expression of Neur. Thus, the cell in the subgroup with the highest proneural activity is the cell that will express Neur first. The expression of Neur enables it to inhibit its neighbours from adopting the SOP fate by suppressing their proneural activity (Troost, 2015).

One generated through mutual signalling, which is not regulated by Ac and Sc and is sufficient to inhibit all cells in the proneural band outside the neur subgroup to become SOPs. This signalling requires the ubiquitously expressed Mib1 and antagonises the activity of Ac, Sc and Da. However, there is residual activity of Notch in mib1 mutants sufficient to prevent most cells from adopting the SOP fate. This residual activity is generated either independently of E3 ligases or by another unknown E3-ligase. In any case this component contributes to the baseline activity of the Notch pathway in addition to Mib1. The second activity on top of the baseline activity in the neur subgroup is generated by a Neur mediated strong signal from the nascent SOP. This signal suppresses the proneural activity of the other members of the neur subgroup. It is dependent on proneural activity, which initiates the expression of Neur. Thus, lateral inhibition is probably operating after the emerging SOP reaches a threshold of proneural activity. It serves to prevent the formation of supernumerary SOPs in the neur group and assures that other cells can generate the necessary SOP in case the selected one is lost (Troost, 2015).

How is the neur subgroup defined? It was found that the PNCs are small in their beginning, comprising the number of cells typical for the subgroup. These cells probably also constitute the small groups of SOPs observed in early third instar discs mutant for Psn. It is likely that E(spl)m8-SM expression defines this subgroup since this study shows that it is expressed in a small group of cells from which the SOP arises. This construct contains only one E box, the binding sites for Ac and Sc, and response to high proneural activity. It is therefore believed that the cells of the early PNC are the neur group and possess the highest proneural activity (Troost, 2015).

During normal development, a cell with more proneural activity is already recognisable at the early phase of the PNCs. This suggests the existence of a pre-selection mechanism that assures that one cell in the neur-subgroup is advanced in its development. Evidence for such a mechanism has been also previously found during rescue experiments studying the function of the proneural genes Ac and Sc. This study has obtained additional experimental evidence for this pre-selecting mechanism: In neur clones one of the cells is advanced in its development towards the SOP fate. Moreover, clonal analysis of kuz and Psn mutants revealed that wildtype cells at positions in the PNC where the SOP arises cannot be prevented from adopting the SOP fate, even if a mutant SOP that cannot be inhibited (e.g., kuz mutant), is its neighbour. The mutant cells can generate a strong inhibitory Notch signal. This indicates that the pre-selecting mechanism renders the wildtype SOP immune to the signal. The nature of this mechanism is not clear, nor whether it is always the same cell in a cluster that is selected (Troost, 2015).

Recent work demonstrated that in the eye disc a regulatory loop between Da and Emc assures correct expression of both factors and results in their complementary expression. Consequently, loss of emc function results in an increase of expression of Da. The consequences of this up-regulation for the proneural state of the mutant cells have not been investigated in detail. A published study focused on the eye imaginal disc and revealed that a few of the mutant cells in clones could adopt the neural fate. The neural cells do not express Runt, a marker expressed in the normal neural cells. Thus, the loss of emc does not result in the complete determination of the neural fate. The state of the vast majority of the cells in clones remained unknown. This study observed up-regulation of proneural activity in emc clones already in early third instar wing imaginal discs, indicating that it is an immediate reaction to the loss of emc function. Some of these cells progress to become SOPs. The increase in proneural activity was also observed in emc clones of the leg disc. Thus, the cells of imaginal discs must be permanently inhibited from adopting a proneural state through the activity of Emc. It has to be pointed out that this situation is remarkably similar to that in the early vertebrate embryo, where all cells of the blastula adopt the proneural state unless they are inhibited through BMP signalling. The cells of the neural plate maintain the proneural state due to the presence of BMP antagonists (Troost, 2015).

In the eye disc and during oogenesis expression of Emc is regulated by the Notch pathway. This study failed to find evidence that supports a regulatory relationship between Emc and the pathway in the notum during SOP development, since the loss of Psn function did not affect the expression of EMC. However, it has been previously shown that the expression of Emc along the dorso-ventral boundary in the wing primordium depends on the activity of the Notch pathway. This correlates well with the finding that this domain is independent of the activity of Da. However, the genetic network of the wing is significantly different from that in the notum. For example Notch signalling induces the expression of Wg along the D/V boundary. However, its expression in the proximal wing and in the notum is independent of the activity of the Notch pathway. This appears to be true also for the different domains of expression of Emc (Troost, 2015).

This study found that the function of ac and sc is dispensable for bristle development in the absence of emc function. How is the SOP fate initiated in these emc ac sc triple mutant cells? It is believed that the activity of Da is sufficient for SOP development in this situation for the following reasons: 1) Da is expressed ubiquitously and is required for the formation of all external sense organs. 2) Strong over-expression of Da induces bristle formation in cells that lack the whole AS-C. In contrast, over-expression of Sc fails to induce SOP formation in the absence of Da. 3) Da can form homodimers that bind to the same DNA target sequences as Ac/Da and Sc/Da heterodimers in bend-shift assays. 4) Loss of emc activity increases the activity of Da. This study show that this increase is independent of the activity of Ac and Sc. 5) The results show that Da regulates the expression of sca independently of Ac and Sc. 6) It has been shown that the mammalian homologue of Da, E2A, acts without its class II partners during B-cell development. Thus, it is likely that in the absence of function of emc, ac and sc, Da forms active homo-dimers that initiate the required neural program (Troost, 2015).

While it is clear that the activity of Ac and Sc is required during normal development, the formation of normal bristles in their absence after concomitant loss of emc function raises the question about their function. The data suggest that an important function is the neutralisation of Emc through formation of heterodimers with it or with Da. This releases Da from inactive heterodimers with Emc. The neutralisation of Emc by Ac and Sc, which are expressed in precise spatial and temporal regulated patterns, allows the differentiation of neural precursors at the correct position and time. The recent finding that a Sc variant without its transactivation domain is fully active fits well to this view of the function of Ac and Sc. Thus, through their intricate and dynamic expression, Ac and Sc and other tissue specific proneural factors determine when and where a neural precursor cell develops. In this view the function of the tissue-specific proneural genes of Drosophila, is similar to that in mammals where their orthologs also promote differentiation of neural precursors in a proneural field, the neural plate, at correct positions and time (Troost, 2015).

Based on the current results, a working model is suggested for the selection of the SOP of the MC: The differential expression of Emc defines a proneural band in the notum with changing proneural activity. The PNCs in this band are determined and positioned through the cluster-like expression of Ac and Sc, which increases the proneural activity at these positions. A baseline of activity of the Notch pathway generated by mutual inhibition prevents cells between the PNCs to accumulate high levels of proneural activity. In addition, it prevents cells located in the PNC, but outside the neur group, to accumulate high proneural activity required for adopting the SOP fate (Troost, 2015).

In the PNCs, expression of Ac and Sc neutralise Emc. Consequently, the proneural activity increases dramatically, since the released Da can form homodimers and/or heterodimers with Ac or Sc. The cells of the initial small PNCs later constitute the neur subgroup. The cells of this subgroup have the highest level of proneural activity and experience this activity also for the longest time. Within this subgroup a cell is pre-selected to become the SOP by a so far unidentified mechanism. Hence, it is the first to reach the threshold level of proneural activity required to initiate the expression of Neur. The expression of Neur enables it to efficiently inhibit the other cells of the subgroup through lateral inhibition. As a consequence these cells never accumulate sufficient proneural activity to activate Neur expression and to become a SOP. The strong signal also further activates the expression of Brd proteins that inhibit the activation of Neur, which might be accidentally activated weakly in one of the neighbours. This activation contributes to the precision of determination process. Thus, a combination of mutual and lateral inhibition mediated by the Notch pathway operates in the PNC during the determination of the SOP. Only the lateral inhibition component depends on proneural activity through transcriptional activation of expression of Neur (Troost, 2015).

The model differs from the lateral inhibition model in the following points: No feedback loop between expression of Dl and proneural activity and, hence, no differential Dl expression is required. Instead the future SOP is pre-selected and advanced in its development. Subgroups within a proneural band defined through its requirement of Neur exist. In this subgroup the activation of the expression of Neur is critical for SOP development since it enables a cell to potently inhibit its neighbours. The pre-selection mechanism favours a cell at the right position to initiate the expression of Neur before the others of the Neur group and therefore secures its development as SOP. Moreover, the existence of mutual signalling explains the inhibition of cells in the proneural band outside the subgroup without the necessity of signalling of Dl over longer distances (Troost, 2015).

Intra-lineage fate decisions involve activation of Notch receptors basal to the midbody in Drosophila sensory organ precursor cells

Notch receptors regulate cell fate decisions during embryogenesis and throughout adult life. In many cell lineages, binary fate decisions are mediated by directional Notch signaling between the two sister cells produced by cell division. How Notch signaling is restricted to sister cells after division to regulate intra-lineage decision is poorly understood. More generally, where ligand-dependent activation of Notch occurs at the cell surface is not known, as methods to detect receptor activation in vivo are lacking. In Drosophila pupae, Notch signals during cytokinesis to regulate the intra-lineage pIIa/pIIb decision in the sensory organ lineage. This study identified two pools of Notch along the pIIa-pIIb interface, apical and basal to the midbody. Analysis of the dynamics of Notch, Delta, and Neuralized distribution in living pupae suggests that ligand endocytosis and receptor activation occur basal to the midbody. Using selective photo-bleaching of GFP-tagged Notch and photo-tracking of photo-convertible Notch, this study showed that nuclear Notch is indeed produced by receptors located basal to the midbody. Thus, only a specific subset of receptors, located basal to the midbody, contributes to signaling in pIIa. This is the first in vivo characterization of the pool of Notch contributing to signaling. A simple mechanism of cell fate decision based on intra-lineage signaling is proposed: ligands and receptors localize during cytokinesis to the new cell-cell interface, thereby ensuring signaling between sister cells, hence intra-lineage fate decision (Trylinski, 2017).

Several methods are currently available to monitor in vivo the signaling activity of Notch by measuring the level and/or activity of NICD. By contrast, in vivo reporters for ligand-receptor interaction, conformational change of Notch in response to mechanical force, and S2 cleavage of Notch are lacking. Consequently, the subcellular location of Notch receptor activation in vivo and the relative contribution of the different pools of Notch to signaling remain unknown. Two complementary fluorescent-based approaches have been developed in this study to track where NICD comes from. Notch receptors present basal to the midbody along the pIIa-pIIb interface were shown to contribute to the accumulation of NICD, whereas receptors located apical to the midbody did not significantly contribute to NICD production. This study provides the first in vivo analysis of ligand-dependent Notch receptor activation at the cell surface. Moreover, the photo-bleaching and photo-conversion approaches used in this study should be broadly applicable in model organisms that can be genetically engineered and easily imaged (Trylinski, 2017).

Other sites of Notch activation had previously been proposed in pIIa. In one model, based on the specific requirements for Arp2/3 and WASp activities for both Notch signaling and actin organization, Dl at apical microvilli in pIIb would activate Notch located apically in pIIa. However, loss of Arp2/3 activity also disrupted cortical actin along the basal pIIa-pIIb interface, suggesting that regulation of the actin cytoskeleton at this location, rather than at microvilli, may be key for receptor activation. In a second model, Dl-Notch signaling was proposed to occur at the new apical pIIa-pIIb junction. This model was largely based on the detection of Notch at this location. The current study, however, indicated that this pool of Notch did not significantly contribute to the production of NICD in pIIa. In a third model, Notch activation was proposed to occur in specific Sara-positive endosomes in pIIa. Whereas the possible contribution of these endosomes to NICD production could not be directly addressed by photo-tracking, two lines of evidence suggest that their contribution can only be minor. First, live imaging of Notch failed to detect this pool indicating that this pool represents a minor fraction of Notch in pIIa. Second, symmetric partitioning of Sara endosomes did not affect the pIIa-pIIb decision, indicating that this proposed pool is not essential for fate asymmetry. Finally, the nature of the mechanical force acting on Notch at the limiting membrane of the Sara-positive endosomes remains to be addressed. In summary, all available data are fully consistent with the conclusion that receptor activation occurs mostly basal to the midbody (Trylinski, 2017).

Whereas these experiments identified the signaling pool of Notch along the pIIa-pIIb, they did not, however, address whether S3 cleavage takes place at the cell surface or intracellularly following endocytosis. Indeed, the photo-tracking approach used in this study did not inform whether the activation of Notch by Delta, i.e., s2 cleavage, is followed by S3 cleavage at the same location or whether S2-processed Notch is internalized to be further processed in signaling endosomes. It is noted, however, that the accumulation of lateral Notch observed in Psn mutant cells is consistent with S3 cleavage taking place, at least in part, at the cell surface (Trylinski, 2017).

This work also sheds new light on the general mechanism whereby Notch signaling is specifically restricted to sister cells within a lineage. In several tissues, including the gut, lung, and CNS, Notch regulates intra-lineage decisions between sister cells soon after mitosis. In this study it is proposed that Notch-mediated intra-lineage decisions are directly linked to division. Indeed, it is suggested that ligands and receptors localize to the lateral membranes that separate the two sister cells at cytokinesis so that Dl-Notch signaling is primarily restricted to sister cells. Thus, neighboring cells - belonging to other cell lineages - would not interfere with intra-lineage fate decisions. The current data indicating that Neur-dependent activation of Notch by Dl predominantly occurs along the pIIa-pIIb lateral interface, basal to the midbody during cytokinesis, fully support this model. Also, the observation that core components of the secretory machinery, e.g., Sec15, are specifically required for Notch signaling in the context of intra-lineage decisions is also consistent with this view. Thus, targeting both receptors and ligands along the newly formed interface during cytokinesis provides an elegant mechanism to restrict signaling between sister cells, thereby ensuring that intra-lineage signaling regulates intra-lineage fate decision. Because Notch generates fate diversity within neural lineages in both vertebrates and invertebrates, this mechanism of intra-lineage signaling may be conserved (Trylinski, 2017).


GENE STRUCTURE

cDNA clone length - 10.4kb There are other transcripts or splice variants lacking the first 5 exons. There appears to be a variant maternal transcript (Kidd, 1986).

Bases in 5' UTR - 799

Exons - nine

Bases in 3' UTR - 1262


PROTEIN STRUCTURE

Amino Acids - 2703

Structural Domains

There are two regions of high hydrophobicity, an N-terminal signal sequence and a transmembrane segment between residues 1745 and 1767. There is an epidermal growth factor domain repeated 36 times, each domain consisting of approximately 38 amino acids (Kidd, 1986).

The Drosophila Notch (N) gene encodes a conserved single-pass transmembrane receptor that transduces extracellular signals controlling cell fate. Evidence has been found that the intracellular domain of Notch gains access to the nucleus in response to ligand, possibly through a mechanism involving proteolytic cleavage and release from the remainder of the protein. These results suggest that signal transduction by Notch depends on the ability of the intracellular domain, particularly the portion containing the CDC10 repeats, to reach the nucleus and to participate in the transcriptional activation of downstream target genes (Struhl, 1998).

A sensitive approach was used to detect the physical presence of intracellular portions of Notch in the nucleus. The chimeric transcription factor Gal4-VP16 (GV) was inserted at various positions in otherwise wild-type Notch protein and the resulting N+-GV proteins expressed under heatshock control in embryos that also carry a UAS-lacZ transgene. The Gal4-VP16 protein contains the DNA-binding domain of the yeast Gal4 transcription factor coupled to the transcriptional activation domain of the viral VP15 protein. The UAS-lacZ gene contains copies of the UAS-binding site for Gal4 and is transcribed in response to Gal4 as well as the Gal4-VP16 protein. It was reasoned that expression of the UAS-lacZ gene would provide a sensitive assay for nuclear access of the inserted Gal4-VP16 domain and hence for events that lead to nuclear import of the Notch intracellular domain (Struhl, 1998).

Gal4-VP16 domains inserted in the intracellular domain of Notch do indeed have access to the nucleus, as judged by their ability to activate UAS-lacZ transcription. Access of a specific insertion positioned just C-terminal to the Notch transmembrane domain is ligand-dependent and correlates with Notch signal-transducing activity. A minimal fragment of the Notch intracellular domain was defined containing the CDC10 repeats that have intrinsic transducing activity. This intrinsinc transducing activity depends on its having access to the nucleus. Addition of sequences that permit or target this polypeptide to accumulate in the nucleus retain transducing activity, whereas sequences that target this polypeptide to extranuclear membranes block the activity. The presence of the VP16 activator domain renders the protein constitutively active, provided that it is inserted at a position that allows it to gain access to the nucleus irrespective of ligand. Adding repressor motifs from either Engrailed or Hairy blocks the signal-transducing activity of the resulting Notch proteins. It is suggested that the only limited step in the mechanism of signal transduction by Notch is the proposed ligand-dependent cleavage event that releases the intracellular domain from the membrane and allows it to enter into the nucleus. Notch signal transduction also appears to depend on several proteins that associate physically with the Notch intracellular domain, such as Su(H), Dishevelled, Deltex, and Numb, as well as other proteins such as Mastermind and Hairless (Struhl, 1998).


Notch continued: Evolutionary Homologs | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References
date revised: 5 February 2000 

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