The first deltex null mutant indicates tissue-specific deltex-dependent Notch signaling in Drosophila

Notch (N) is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and was proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. Here, we report the first null mutant allele of Drosophila dx. We found that dx was involved only in the subsets of N signaling, but was not essential for it in any developmental context. A strong genetic interaction between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. Our epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). We also uncovered a novel dx activity that suppressed N signaling downstream of N.     

Interactions between enhancer of rudimentary and Notch and deltex reveal a regulatory function of enhancer of rudimentary in the Notch signaling pathway in Drosophila melanogaster

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, N^nd-p, was isolated as a lethal in combination with an e(r) allele, e(r)^p2. Both mutants are viable as single mutants. N^nd-p is caused by a P-element insertion in the 5′ UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that N^nd-p is a hypomorphic allele of N. The three viable notchoid alleles, N^nd-p, N^nd-1 and N^nd-3, are lethal in combination with e(r)⁻ alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the N e(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dx e(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)⁻ mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.     

Interactions between enhancer of rudimentary and Notch and deltex reveal a regulatory function of enhancer of rudimentary in the Notch signaling pathway in Drosophila melanogaster

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication, and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, N^nd-p, was isolated as a lethal in combination with an e(r) allele, e(r)^p2. Both mutants are viable as single mutants. N^nd-p is caused by a P-element insertion in the 5' UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that N^nd-p is a hypomorphic allele of N. The three viable notchoid alleles, N^nd-p, N^nd-1, and N^nd-3, are lethal in combination with e(r)^- alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the Ne(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dxe(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)^- mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.     

Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc

Notch (N) activation at the dorsoventral (DV) boundary of the Drosophila eye is required for early eye primordium growth. Despite the apparent DV mirror symmetry, some mutations cause a preferential loss of the ventral domain, suggesting that the growth of individual domains is asymmetrically regulated. We show that the Lobe (L) gene is required non-autonomously for ventral growth but not dorsal growth, and that it mediates the proliferative effect of midline N signaling in a ventral-specific manner. L encodes a novel protein with a conserved domain. Loss of L suppresses the overproliferation phenotype of constitutive N activation in the ventral, but not in the dorsal eye, and gain of L rescues ventral tissue loss in N mutant background. Furthermore, L is necessary and sufficient for the ventral expression of a N ligand, Serrate (Ser), which affects ventral growth. Our data suggest that the control of ventral Ser expression by L represents a molecular mechanism that governs asymmetrical eye growth.     

Down-regulation of Notch target gene expression by Suppressor of deltex

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.     

The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase.

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.     

Notch signaling and Notch signaling modifiers

Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency.     

Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea.

The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of &Dgr; expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis.     

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.     

Drosophila Ndfip is a novel regulator of Notch signaling

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch(+/-) wing phenotype and knockdown of dNdfip enhanced the Notch(+/-) wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate(+/-) wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation.     

Regulation of Notch signaling and endocytosis by the Lgl neoplastic tumor suppressor

The evolutionarily conserved neoplastic tumor suppressor protein, Lethal (2) giant larvae (Lgl), plays roles in cell polarity and tissue growth via regulation of the Hippo pathway. In our recent study, we showed that in the developing Drosophila eye epithelium, depletion of Lgl leads to increased ligand-dependent Notch signaling. lgl mutant tissue also exhibits an accumulation of early endosomes, recycling endosomes, early-multivesicular body markers and acidic vesicles. We showed that elevated Notch signaling in lgl⁻ tissue can be rescued by feeding larvae the vesicle de-acidifying drug chloroquine, revealing that Lgl attenuates Notch signaling by limiting vesicle acidification. Strikingly, chloroquine also rescued the lgl⁻ overgrowth phenotype, suggesting that the Hippo pathway defects were also rescued. In this extraview, we provide additional data on the regulation of Notch signaling and endocytosis by Lgl, and discuss possible mechanisms by which Lgl depletion contributes to signaling pathway defects and tumorigenesis.     

Notch signaling controls lineage specification during Drosophila larval hematopoiesis

Drosophila larval hemocytes originate from a hematopoietic organ called lymph glands, which are composed of paired lobes located along the dorsal vessel. Two mature blood cell populations are found in the circulating hemolymph: the macrophage-like plasmatocytes, and the crystal cells that contain enzymes of the immune-related melanization process. A third class of cells, called lamellocytes, are normally absent in larvae but differentiate after infection by parasites too large to be phagocytosed. Here we present evidence that the Notch signaling pathway plays an instructive role in the differentiation of crystal cells. Loss-of-function mutations in Notch result in severely decreased crystal cell numbers, whereas overexpression of Notch provokes the differentiation of high numbers of these cells. We demonstrate that, in this process, Serrate, not Delta, is the Notch ligand. In addition, Notch function is necessary for lamellocyte proliferation upon parasitization, although Notch overexpression does not result in lamellocyte production. Finally, Notch does not appear to play a role in the differentiation of the plasmatocyte lineage. This study underlines the existence of parallels in the genetic control of hematopoiesis in Drosophila and in mammals.     

Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling.

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. We have isolated four new alleles of Su(dx) and mapped the gene between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from loss-of-function of Notch signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, was also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development, and a role was demonstrated for the gene between 20 and 30 hr after puparium formation. This corresponds to the period when the Notch protein is involved in refining the vein competent territories. Taken together, our data indicate a role for Su(dx) as a negative regulator of Notch function.     

Asymmetric cell division and Notch signaling specify dopaminergic neurons in Drosophila

In Drosophila, dopaminergic (DA) neurons can be found from mid embryonic stages of development till adulthood. Despite their functional involvement in learning and memory, not much is known about the developmental as well as molecular mechanisms involved in the events of DA neuronal specification, differentiation and maturation. In this report we demonstrate that most larval DA neurons are generated during embryonic development. Furthermore, we show that loss of function (l-o-f) mutations of genes of the apical complex proteins in the asymmetric cell division (ACD) machinery, such as inscuteable and bazooka result in supernumerary DA neurons, whereas l-o-f mutations of genes of the basal complex proteins such as numb result in loss or reduction of DA neurons. In addition, when Notch signaling is reduced or abolished, additional DA neurons are formed and conversely, when Notch signaling is activated, less DA neurons are generated. Our data demonstrate that both ACD and Notch signaling are crucial mechanisms for DA neuronal specification. We propose a model in which ACD results in differential Notch activation in direct siblings and in this context Notch acts as a repressor for DA neuronal specification in the sibling that receives active Notch signaling. Our study provides the first link of ACD and Notch signaling in the specification of a neurotransmitter phenotype in Drosophila. Given the high degree of conservation between Drosophila and vertebrate systems, this study could be of significance to mechanisms of DA neuronal differentiation not limited to flies.     

Endosomal entry regulates Notch receptor activation in Drosophila melanogaster

Signaling through the transmembrane receptor Notch is widely used throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we systematically assessed Notch localization, processing, and signaling in a comprehensive set of Drosophila melanogaster mutants that block access of cargo to different endocytic compartments. We find that gamma-secretase cleavage and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. In mutants that block endosomal entry, we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data show that endosomal access of the Notch receptor is critical to achieve physiological levels of signaling and further suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.     

The Snail repressor positions Notch signaling in the Drosophila embryo

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. We have investigated the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, Notch(IC), in transgenic embryos using the eve stripe2 enhancer. The Notch(IC) stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'na?ve' embryos that contain low uniform levels of Dorsal. We suggest that these dual activities of Snail, repression of Notch target genes and stimulation of Notch signaling, help define precise lines of sim expression within the neurogenic ectoderm.