Echinoid facilitates Notch pathway signalling during Drosophila neurogenesis through functional interaction with Delta

The Notch intercellular signalling pathway is important throughout development, and its components are modulated by a variety of cellular and molecular mechanisms. Ligand and receptor trafficking are tightly controlled, although context-specific regulation of this is incompletely understood. We show that during sense organ precursor specification in Drosophila, the cell adhesion molecule Echinoid colocalises extensively with the Notch ligand, Delta, at the cell membrane and in early endosomes. Echinoid facilitates efficient Notch pathway signalling. Cultured cell experiments suggest that Echinoid is associated with the cis-endocytosis of Delta, and is therefore linked to the signalling events that have been shown to require such Delta trafficking. Consistent with this, overexpression of Echinoid protein causes a reduction in Delta level at the membrane and in endosomes. In vivo and cell culture studies suggest that homophilic interaction of Echinoid on adjacent cells is necessary for its function.     

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.     

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.     

A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila

The anterior-posterior axis of Drosophila becomes polarized early in oogenesis, when the oocyte moves to the posterior of the germline cyst because it preferentially adheres to posterior follicle cells. The source of this asymmetry is unclear, however, since anterior and posterior follicle cells are equivalent until midoogenesis, when Gurken signaling from the oocyte induces posterior fate. Here, we show that asymmetry arises because each cyst polarizes the next cyst through a series of posterior to anterior inductions. Delta signaling from the older cyst induces the anterior polar follicle cells, the anterior polar cells signal through the JAK/STAT pathway to induce the formation of the stalk between adjacent cysts, and the stalk polarizes the younger anterior cyst by inducing the shape change and preferential adhesion that position the oocyte at the posterior. The anterior-posterior axis is therefore established by a relay mechanism, which propagates polarity from one cyst to the next.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Notch signalling coordinates tissue growth and wing fate specification in Drosophila

During the development of a given organ, tissue growth and fate specification are simultaneously controlled by the activity of a discrete number of signalling molecules. Here, we report that these two processes are extraordinarily coordinated in the Drosophila wing primordium, which extensively proliferates during larval development to give rise to the dorsal thoracic body wall and the adult wing. The developmental decision between wing and body wall is defined by the opposing activities of two secreted signalling molecules, Wingless and the EGF receptor ligand Vein. Notch signalling is involved in the determination of a variety of cell fates, including growth and cell survival. We present evidence that growth of the wing primordium mediated by the activity of Notch is required for wing fate specification. Our data indicate that tissue size modulates the activity range of the signalling molecules Wingless and Vein. These results highlight a crucial role of Notch in linking proliferation and fate specification in the developing wing primordium.     

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.     

Notch responds differently to Delta and Wingless in cultured Drosophila cells

Notch, a cell surface receptor, is required for producing different types of cells during development of Drosophila melanogaster. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to ligand Wingless. The means by which Notch initiates these different intracellular activities was examined in this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta resulted in accumulation of approximately 120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch did not accumulate in cells treated with Wingless, but the approximately 350-kDa full-length Notch molecules accumulated. These results indicate that N responds differently to binding by Delta and Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane.     

Planar cell polarity controls directional Notch signaling in the Drosophila leg

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. Here, we show that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor.     

Notch signalling in Drosophila: three ways to use a pathway

Cell-cell interactions mediated by Notch are critical at multiple stages of development. Our current understanding of the Notch signalling pathway suggests a comparatively simple transduction mechanism. However, this core pathway can be deployed in three different types of developmental process: lateral inhibition, lineage decisions and boundary formation. These illustrate how the activity of the pathway can be modulated both at the cell surface, through availability and effectiveness of ligand interactions, and inside the cell, through effects on the transduction pathway and the responsiveness of target genes.     

Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

Joint morphology in the insect leg: evolutionary history inferred from Notch loss-of-function phenotypes in Drosophila

Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. We showed previously that strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). Here, we show that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement.     

Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints.     

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.