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.     

Role of Cre-loxP-mediated conditional gene targeting in understanding the function of Notch receptors

The Notch family of cell surface receptors and their ligands constitute an evolutionarily conserved signaling pathway that is used by invertebrates and vertebrates to regulate a broad spectrum of cell specification events through local cell interactions. After ligand binding Notch receptor undergoes proteolytic processing ultimately liberating the cytoplasmic domain of the Notch receptor which translocates to the nucleus and activates target genes. In all animal models tested, mutations in Notch genes invariably resulted in developmental abnormalities. In mammals, Notch signaling controls key stages of lymphocyte differentiation as well as activation and several abnormalities in Notch pathway have been suggested to cause human leukemias. Cre-loxP mediated conditional gene targeting significantly contributed to our current understanding of the physiological roles of different Notch family members in hematopoietic compartment. This technique helped to overcome embryonic lethality of Notch mutants providing the opportunity to inactivate specific Notch gene in adulthood.     

Notch receptor-ligand binding and activation: insights from molecular studies

The Notch receptor is part of a core signalling pathway which is highly conserved in all metazoan species. It is required for various cell fate decisions at multiple stages of development and in the adult organism, with dysregulation of the pathway associated with genetic and acquired diseases including cancer. Although cellular and in vivo studies have provided considerable insight into the downstream consequences of Notch signalling, relatively little is known about the molecular basis of the receptor/ligand interaction and initial stages of activation. Recent advances in structure determination of the extracellular regions of human Notch-1 and one of its ligands Jagged-1 have given new insights into docking events occurring at the cell surface which may facilitate the development of new highly specific therapies. We review the structural data available for receptor and ligands and identify the challenges ahead.     

Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels.     

Structural conservation of Notch receptors and ligands

The Notch signalling pathway is an evolutionarily conserved cell-to-cell communication system utilized multiple times and in many tissues during development. The outcome of an interaction between Notch and its ligands is highly influenced by factors both extrinsic and intrinsic to Notch expressing cells, suggesting that Notch functions either directly or in parallel with other signalling systems to regulate cellular differentiation events. Protein domains common to all ligands and receptors of this system suggest conserved functional properties that likely relate to regulatory mechanisms for Notch signalling. Within this review, the known functional properties of these domains are analyzed with respect to their contributions to ligand/receptor interactions and Notch signalling.     

Regulation of Notch signalling by endocytosis and endosomal sorting

Cell-cell signalling is an essential process in the formation of multicellular organisms. Notch is the receptor of an evolutionarily conserved signalling pathway regulating numerous developmental decisions. Indeed, its misregulation is linked to multiple developmental and physiological disorders. Notch and its ligands are distributed widely throughout development, yet Notch activity is highly controlled and restricted in time and space. Recent advances have highlighted that endocytosis followed by endosomal sorting of both the Notch receptor and its ligands is an essential mechanism by which Notch-mediated signalling is developmentally controlled.     

Notch signalling: a simple pathway becomes complex

A small number of signalling pathways are used iteratively to regulate cell fates, cell proliferation and cell death in development. Notch is the receptor in one such pathway, and is unusual in that most of its ligands are also transmembrane proteins; therefore signalling is restricted to neighbouring cells. Although the intracellular transduction of the Notch signal is remarkably simple, with no secondary messengers, this pathway functions in an enormous diversity of developmental processes and its dysfunction is implicated in many cancers.     

HES6 promotes prostate cancer aggressiveness independently of Notch signalling

Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high‐grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer‐specific up‐regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up‐ and down‐regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor‐independent manner. Using a Notch‐sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordingly pharmacological Notch pathway blockade did not inhibit cancer cell growth or viability. In contrast to canonical Notch targets, HES6, a HES family member known to antagonize Notch signalling, was not regulated by Notch signalling, but relied instead on androgen levels, both in cultured cells and in human cancer tissues. When engineered into prostate cancer cells, reduced levels of HES6 resulted in reduced cancer cell invasion and clonogenic growth. By molecular profiling, we identified potential roles for HES6 in regulating hedgehog signalling, apoptosis and cell migration. Our results did not reveal any cell‐autonomous roles for canonical Notch signalling in prostate cancer. However, the results do implicate HES6 as a promoter of prostate cancer progression.     

Multiple levels of Notch signal regulation (review)

Notch is a vitally important signalling receptor controlling cell fate determination and pattern formation in numerous ways during development of both invertebrate and vertebrate species. An intriguing pathway for the Notch signal has emerged where, after ligand-dependent proteolysis, an intracellular fragment of the receptor itself translocates to the nucleus to regulate gene expression. The nuclear activity of the Notch intracellular domain is linked to complexes regulating chromatin organization through histone deacetylation and acetylation. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. Regulation occurs at multiple levels including patterns of ligand and receptor expression, Notch-ligand interactions, trafficking of the receptor and ligands, and covalent modifications including glycosylation, phosphorylation and ubiquitination. Several Notch regulatory proteins have conserved domains that link them to the ubiquitination pathway, and ubiquitination of the Notch intracellular domain has recently been linked to its degradation. Different proteolytically derived isoforms of Notch have also been identified that may be involved in alternative Notch-dependent signals or regulatory mechanisms, and differences between the four mammalian Notch homologues are beginning to be appreciated.     

Expression of Notch pathway genes in the embryonic mouse metanephros suggests a role in proximal tubule development

The interaction of neighboring cells via Notch signalling leads to cell fate determination, differentiation and patterning of highly organized tissues. Mice with targeted disruption of genes from the Notch signal transduction pathway display defects in the developing somites, neurogenic structures, blood vessels, heart and other organs. Recent studies have added requirements for Notch signalling during kidney, pancreas and thymus morphogenesis. Here, we describe the expression of all four receptors (Notch1-4), the five transmembrane ligands (Dll1, 3, 4, Jag1 and Jag2), intracellular effectors (the Hey genes) and extracellular modulators (Lfng, Mfng, Rfng) in the developing mouse metanephros. Our results point to a Lfng-dependent role for Notch signalling in the development of nephron segments, especially the proximal tubules.     

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.     

Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1^Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1^Dll4ki/+ mice, the Dll1^Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.     

Notch signalling is required for both dauer maintenance and recovery in C. elegans

The Notch signalling pathway is conserved among higher metazoans and is used repeatedly throughout development to specify distinct cell fates among populations of equipotent cells. Mounting evidence suggests that Notch signalling may also be crucial in neuronal function in postmitotic, differentiated neurons. Here, we demonstrate a novel role for the canonical Notch signalling pathway in postmitotic neurons during a specialised ;diapause-like' post-embryonic developmental stage in C. elegans called dauer. Our data suggest that cell signalling downstream of the developmental decision to enter dauer leads to the activation of Notch-responding genes in postmitotic neurons. Consistent with this, we demonstrate that glp-1, one of the two C. elegans Notch receptors, and its ligand lag-2 are expressed in neurons during the dauer stage, and both genes are required to maintain this stage in a daf-7/TGFbeta dauer constitutive background. Our genetic data also suggest that a second Notch receptor, lin-12, functions upstream of, or in parallel with, insulin-like signalling components in response to replete growth conditions to promote dauer recovery. Based on our findings, cues associated with the onset of dauer ultimately trigger a glp-1-dependent Notch signalling cascade in neurons to maintain this developmental state. Then, as growth conditions improve, activation of the LIN-12 Notch receptor cooperates with the insulin-like signalling pathway to signal recovery from the dauer stage.