Notch signaling targets the Wingless responsiveness of a Ubx visceral mesoderm enhancer in Drosophila

BACKGROUND: Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. RESULTS: We report that Ubx(VM)B, a visceral mesoderm-specific enhancer of the Ubx gene of Drosophila, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf binding sites in the Ubx(VM)B enhancer. CONCLUSIONS: Our results show that, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on Ubx(VM)B. A possible target of this activity is dTcf. As dTcf has been shown to be capable of repressing Wingless targets, our results suggest that this repressive activity may be regulated by Notch. Finally, we suggest that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of dTcf and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with dTcf.     

A Highlights from MBoC Selection: Neuralized Promotes Basal to Apical Transcytosis of Delta in Epithelial Cells

Notch receptors mediate short-range signaling controlling many developmental decisions in metazoans. Activation of Notch requires the ubiquitin-dependent endocytosis of its ligand Delta. How ligand endocytosis in signal-sending cells regulates receptor activation in juxtaposed signal-receiving cells remains largely unknown. We show here that a pool of Delta localizes at the basolateral membrane of signal-sending sensory organ precursor cells in the dorsal thorax neuroepithelium of Drosophila and that Delta is endocytosed in a Neuralized-dependent manner from this basolateral membrane. This basolateral pool of Delta is segregated from Notch that accumulates apically. Using a compartimentalized antibody uptake assay, we show that murine Delta-like 1 is similarly internalized by mNeuralized2 from the basolateral membrane of polarized Madin-Darby canine kidney cells and that internalized ligands are transcytosed to the apical plasma membrane where mNotch1 accumulates. Thus, endocytosis of Delta by Neuralized relocalizes Delta from the basolateral to the apical membrane domain. We speculate that this Neuralized-dependent transcytosis regulates the signaling activity of Delta by relocalizing Delta from a membrane domain where it cannot interact with Notch to another membrane domain where it can bind and activate Notch.     

Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate.

BACKGROUND: The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS: Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS: Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.     

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. Here we have tested this proposal by analysing interactions between Delta- and Serrate-activated Notch signalling and Wingless signalling during wing development and patterning. We find that the 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 signalling but can also disrupt signalling through another pathway. One possibility is the Wingless signalling pathway as the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signalling. Furthermore, we observe that the outcome of the interactions between Notch and Wingless signalling differs when we activate Wingless signalling 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 signalling activated by Wingless leads to two effects, a reduction of Notch signalling and an activation of Armadillo.     

An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila.

BACKGROUND: The Drosophila Notch protein is a receptor that controls cell fate during embryonic development, particularly in lateral inhibition, a process that acts on groups of cells that share a particular developmental potential to restrict the number of cells that will adopt that cell fate. The process of lateral inhibition is implemented by the nuclear protein Suppressor of Hairless (Su(H)) and is triggered by the ligand Delta. Recent results have shown that the interaction between Delta and Notch triggers the cleavage of the intracellular domain of Notch which then translocates to the nucleus and binds to Su(H). RESULTS: We find that Notch plays a role in the patterning of the dorsal epidermis of the Drosophila embryo and that this function of Notch is independent of Su(H), requires Notch at the plasma membrane and targets the c-Jun N-terminal kinase (JNK) signalling pathway. Notch mutants show high levels of JNK activity and can rescue the effects of lowered JNK signalling resulting from mutations in the hemipterous and basket genes. Two regions of the intracellular domain of Notch are involved: the Cdc10/ankyrin repeats, which downregulate signalling through the JNK pathway, and a region carboxy-terminal to these repeats, which regulates this negative function. CONCLUSIONS: Our results reveal a novel signalling activity of Notch that does not require its cleavage and acts by modulating signalling through the JNK pathway. In the Drosophila embryo, this activity plays an important role in the morphogenetic movements that drive dorsal closure.     

Delta1 family members are involved in filopodial actin formation and neuronal cell migration independent of Notch signaling

Delta family proteins are transmembrane molecules that bind Notch receptors and activate downstream signaling events in neighboring cells. In addition to serving as Notch ligands, Notch-independent roles for Delta have been suggested but are not fully understood. Here, we demonstrate a previously unrecognized role for Delta in filopodial actin formation. Delta1 and Delta4, but not Delta3, exhibit filopodial protrusive activity, and this activity is independent of Notch signaling. The filopodial activity of Delta1 does not depend on the PDZ-binding domain at the C-terminus; however, the intracellular membrane-proximal region that is anchored to the plasma membrane plays an important role in filopodial activity. We further identified a Notch-independent role of DeltaD in neuronal cell migration in zebrafish. These findings suggest a possible functional link between Notch-independent filopodial activity of Delta and the control of cell motility.     

LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo

Activation of the Notch signaling pathway segregates the non-skeletogenic mesoderm (NSM) from the endomesoderm during sea urchin embryo development. Subsequently, Notch signaling helps specify the four subpopulations of NSM, and influences endoderm specification. To gain further insight into how the Notch signaling pathway is regulated during these cell specification events, we identified a sea urchin homologue of Numb (LvNumb). Previous work in other model systems showed that Numb functions as a Notch signaling pathway antagonist, possibly by mediating the endocytosis of other key Notch interacting proteins. In this study, we show that the vegetal endomesoderm expresses lvnumb during the blastula and gastrula stages, and that the protein is localized to the presumptive NSM. Injections of lvnumb mRNA and antisense morpholinos demonstrate that LvNumb is necessary for the specification of mesodermal cell types, including pigment cells, blastocoelar cells and muscle cells. Functional analysis of the N-terminal PTB domain and the C-terminal PRR domain of LvNumb shows that the PTB domain, but not the PRR domain, is sufficient to recapitulate the demonstrable function of full-length LvNumb. Experiments show that LvNumb requires an active Notch signal to function during NSM specification and that LvNumb functions in the cells responding to Delta and not in the cells presenting the Delta ligand. Furthermore, injection of mRNA encoding the intracellular domain of Notch rescues the LvNumb morpholino phenotype, suggesting that the constitutive intracellular Notch signal overcomes, or bypasses, the absence of Numb during NSM specification.     

Delta proteins and MAGI proteins: an interaction of Notch ligands with intracellular scaffolding molecules and its significance for zebrafish development

Delta proteins activate Notch through a binding reaction that depends on their extracellular domains; but the intracellular (C-terminal) domains of the Deltas also have significant functions. All classes of vertebrates possess a subset of Delta proteins with a conserved ATEV* motif at their C termini. These ATEV Deltas include Delta1 and Delta4 in mammals and DeltaD and DeltaC in the zebrafish. We show that these Deltas associate with the membrane-associated scaffolding proteins MAGI1, MAGI2 and MAGI3, through a direct interaction between the C termini of the Deltas and a specific PDZ domain (PDZ4) of the MAGIs. In cultured cells and in subsets of cells in the intact zebrafish embryo, DeltaD and MAGI1 are co-localized at the plasma membrane. The interaction and the co-localization can be abolished by injection of a morpholino that blocks the mRNA splicing reaction that gives DeltaD its terminal valine, on which the interaction depends. Embryos treated in this way appear normal with respect to some known functions of DeltaD as a Notch ligand, including the control of somite segmentation, neurogenesis, and hypochord formation. They do, however, show an anomalous distribution of Rohon-Beard neurons in the dorsal neural tube, suggesting that the Delta-MAGI interaction may play some part in the control of neuron migration.     

Notch-induced proteolysis and nuclear localization of the Delta ligand

The Delta protein is a single-pass transmembrane ligand for the Notch family of receptors. Delta binding to Notch invokes regulated intramembrane proteolysis and nuclear translocation of the Notch intracellular domain. Delta is proteolytically processed at two sites, Ala(581) and Ala(593) in the juxtamembrane and transmembrane domains, respectively (Mishra-Gorur, K., Rand, M. D., Perez-Villamil, B., and Artavanis-Tsakonas, S. (2002) J. Cell Biol. 159, 313-324). Controversy over the role of Delta processing in propagating Notch signals has stemmed from conflicting reports on the activity or inactivity of soluble extracellular domain products of Delta. We have examined Delta proteolysis in greater detail and report that Delta undergoes three proteolytic cleavages in the region of the juxtamembrane and transmembrane domains. Only one of these cleavages, analogous to cleavage at Ala(581), is dependent on the Kuzbanian ADAM metalloprotease. The two additional cleavages correspond to the previously described cleavage at Ala(593) and a novel unidentified site within or close to the transmembrane domain. Delta processing is up-regulated in co-cultures with Notch-expressing cells and is similarly induced by p-aminophenylmercuric acetate, a well documented activator of metalloproteases. Furthermore, expression of a truncated intracellular isoform of Delta shows prominent nuclear localization. Altogether, these data demonstrate a role for Notch in inducing Delta proteolysis and implicate a nuclear function for Delta, consistent with a model of bi-directional signaling through Notch-Delta interactions.     

Analysis of notch lacking the carboxyl terminus identified in Drosophila embryos

The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. We report here that certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch lacking the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor we designate NDeltaCterm.     

Endocytosis-independent mechanisms of Delta ligand proteolysis

Delta proteins function as cell surface ligands for Notch receptors in a highly conserved signal transduction mechanism. Delta activates Notch by "trans-endocytosis", whereby endocytosis of Delta that is in complex with Notch on a neighboring cell induces activating cleavages in Notch. Alternatively, proteolysis of Delta renders the ligand inactive by dissociating the extracellular and cytosolic domains. How proteolysis and trans-endocytosis cooperate in Delta function is not well understood. We now show that Drosophila Delta proteolysis occurs independent of and prior to endocytosis in neuroblasts and ganglion mother cells in vivo and cells in culture. Delta cleavage occurs at two novel sites that we identify in the juxtamembrane (JM) and transmembrane (TM) domains. In addition to the previously identified Kuzbanian ADAM protease, which acts on the JM domain, proteolysis in the TM domain is facilitated by a thiol-sensitive aspartyl protease that is distinct from Presenilin. Furthermore, cleavage in the TM domain is upregulated in the presence of Notch. Overall, Drosophila Delta proteolysis differs from the conventional regulated intramembrane proteolysis (RIP) mechanism by two criteria: (1) TM-domain processing of Delta is not sensitive to Presenilin, and (2) TM and JM domain cleavages occur independently of each other. Altogether, these data support a model whereby proteolysis can modulate Delta ligand activity independently of endocytosis.     

Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.