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).

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).

GENE STRUCTURE

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).

date revised: 5 February 2000 

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