The Notch pathway helps to pattern the tips of the Drosophila tracheal branches by selecting cell fates

The Drosophila tracheal system consists of a stereotyped network of epithelial tubes formed by several tracheal cell types. By the end of embryogenesis, when the general branching pattern is established, some specialised tracheal cells then mediate branch fusion while others extend fine terminal branches. Here evidence is presented that the Notch signalling pathway acts directly in the tracheal cells to distinguish individual fates within groups of equivalent cells. Notch helps to single out those tracheal cells that mediate branch fusion by blocking their neighbours from adopting the same fate. This function of Notch would require the restricted activation of the pathway in specific cells. In addition, and probably later, Notch also acts in the selection of those tracheal cells that extend the terminal branches. Both the localised expression and the mutant phenotypes of Delta, a known ligand for Notch, suggest that Delta may activate Notch to specify cell fates at the tips of the developing tracheal branches.     

Evidence for a novel Notch pathway required for muscle precursor selection in Drosophila.

The Notch pathway mediates cell fate choice in many species and developmental contexts. In the Drosophila mesoderm, phenotypic differences were observed when different components of the pathway were defective. To determine if these differences reflect variations in the signaling pathway or in the persistence of wild-type maternal products, we examined muscle precursors in embryos that lacked both maternally- and zygotically-derived gene products, called holonull embryos. Most holonull neurogenic embryos have the same number and arrangement of extra muscle precursors, but in Notch holonull embryos many additional cells also become muscle precursors. Thus Notch is active in cells where its known ligands and downstream effectors are not. These results indicate that Notch acts in two pathways to determine cell fates in mesoderm: the Delta-to-Notch-to-Suppressor of Hairless-to-Enhancer of split signaling pathway previously defined, and a second pathway that acts independently.     

Drosophila Notch Receptor Activity Suppresses Hairless Function during Adult External Sensory Organ Development

The neurogenic Notch locus of Drosophila encodes a receptor necessary for cell fate decisions within equivalence groups, such as proneural clusters. Specification of alternate fates within clusters results from inhibitory communication among cells having comparable neural fate potential. Genetically, Hairless (H) acts as an antagonist of most neurogenic genes and may insulate neural precursor cells from inhibition. H function is required for commitment to the bristle sensory organ precursor (SOP) cell fate and for daughter cell fates. Using Notch gain-of-function alleles and conditional expression of an activated Notch transgene, we show that enhanced signaling produces H-like loss-of-function phenotypes by suppressing bristle SOP cell specification or by causing an H-like transformation of sensillum daughter cell fates. Furthermore, adults carrying Notch gain of function and H alleles exhibit synergistic enhancement of mutant phenotypes. Over-expression of an H(+) transgene product suppressed virtually all phenotypes generated by Notch gain-of-function genotypes. Phenotypes resulting from over-expression of the H(+) transgene were blocked by the Notch gain-of-function products, indicating a balance between Notch and H activity. The results suggest that H insulates SOP cells from inhibition and indicate that H activity is suppressed by Notch signaling.     

A Genetic Analysis of Deltex and Its Interaction with the Notch Locus in Drosophila Melanogaster

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.     

Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways

The specification of polar, main-body and stalk follicle cells in the germarium of the Drosophila ovary plays a key role in the formation of the egg chamber and polarisation of its anterior-posterior axis. High levels of Notch pathway activation, resulting from a germline Delta ligand signal, induce polar cells. Here we show that low Notch activation levels, originating from Delta expressed in the polar follicle cells, are required for stalk formation. The metalloprotease Kuzbanian-like, which cleaves and inactivates Delta, reduces the level of Delta signaling between follicle cells, thereby limiting the size of the stalk. We find that Notch activation is required in a continuous fashion to maintain the polar and stalk cell fates. We further demonstrate that mutual antagonism between the Notch and JAK/STAT signaling pathways provides a crucial facet of follicle cell patterning. Notch signaling in polar and main-body follicle cells inhibits JAK/STAT signaling by preventing STAT nuclear translocation, thereby restricting the influence of this pathway to stalk cells. Conversely, signaling by JAK/STAT reduces Notch signaling in the stalk. Thus, variations in the levels of Notch pathway activation, coupled with a continuous balance between the Notch and JAK/STAT pathways, specify the identity of the different follicle cell types and help establish the polarity of the egg chamber.     

Differential requirements for the neurogenic gene almondex during Drosophila melanogaster development

During early development, the neurogenic genes of Drosophila melanogaster are involved in the control of cell fates in the neurectoderm; almondex (amx) belongs to this category of genes. We have identified the amx locus and rescued the amx embryonic neurogenic phenotype with a 1.5 kb DNA fragment. Using a small deficiency, we generated a new amx mutant background called amx(m), which is a null allele. Besides the characteristic neurogenic maternal effect caused by loss of amx, amx(m) flies display a new imaginal phenotype resembling loss of function of Notch. We describe amx-induced misregulation of the Notch pathway target E(spl) m7 in embryos and genetic interactions between amx and Notch pathway mutants in adult flies. These data show that wildtype amx acts as a novel positive regulator of the Notch pathway and is required at different levels during development.     

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.     

The little R cell that could

Drosophila eye development provides an excellent model system to study the role of inter-cellular signaling in the specification of unique cell fates. Behavioral screens by Benzer and his colleagues led to the identification of a gene, Sevenless, a receptor tyrosine kinase (RTK) receptor, required for the specification of the UV sensitive R7 cell. Genetic analysis further showed that the Ras/Raf/MAPK pathway function downstream of Sevenless in the specification of R7 fate. Signaling mediated by another RTK, EGFR and Notch have also been shown to function in either an antagonistic or a synergistic manner in the specification of cell fate during eye development. In some instances, these pathways are linked in a sequential manner by the regulation of the expression of Notch ligand, Delta by EGFR, while in others, these pathways function in a combinatorial fashion on enhancer elements to control target gene expression. In this review, we highlight the elegant genetic strategies used by several laboratories in early elucidation of the Sevenless pathway which helped link the RTK receptor to the Ras/Raf/MAPK cascade and discuss how EGFR and Notch signaling pathways are used in a reiterative manner and by combining in different modes, generate sufficient diversity required for the specification of unique cell fates.     

Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron

The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules) despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a gamma-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by a gamma-secretase inhibitor. These results establish distinct (non-redundant), instructive roles for Notch receptors in nephron segmentation.     

The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila

Drosophila sensory organ precursor (SOP) cells undergo several rounds of asymmetric cell division to generate the four different cell types that make up external sensory organs. Establishment of different fates among daughter cells of the SOP relies on differential regulation of the Notch pathway. Here, we identify the protein Lethal (2) giant discs (Lgd) as a critical regulator of Notch signaling in the SOP lineage. We show that lgd encodes a conserved C2 domain protein that binds to phospholipids present on early endosomes. When Lgd function is compromised, Notch and other transmembrane proteins accumulate in enlarged early endosomal compartments. These enlarged endosomes are positive for Rab5 and Hrs, a protein involved in trafficking into the degradative pathway. Our experiments suggest that Lgd is a critical regulator of endocytosis that is not present in yeast and acts in the degradative pathway after Hrs.     

Notch activity opposes ras-induced differentiation during the second mitotic wave of the developing Drosophila eye

Background  

EGF receptor acts through Ras and the MAPK cascade to trigger differentiation and maintain survival of most of cell types in the Drosophila retina. Cell types are specified sequentially by separate episodes of EGFR activity. All the cell types differentiate in G1 phase of the cell cycle. Before differentiating, many cells pass through the cell cycle in the "Second Mitotic Wave" in response to Notch activity, but no cell fates are specified during the Second Mitotic Wave. It is not known how fate specification is limited to G1-arrested cells.     

Spatially localized Kuzbanian required for specific activation of Notch during border cell migration

The transmembrane receptor Notch is used repeatedly during development for a variety of essential functions. During Drosophila oogenesis, Notch activity is required first to specify particular follicle cell fates, then to promote the differentiation of all follicle cell types, to promote border cell migration, and then to form dorsal appendages, raising the question as to how Notch activity is spatially and temporally regulated. Here we show the Notch activity pattern during oogenesis. Notch activation was found in many follicle cells at stage 6 but then at stage 9 was restricted to migrating border cells, despite uniform expression of Delta. Expression of Kuzbanian (KUZ), a metalloproteinase that can activate Notch as well as cleave other substrates, is enriched in border cells at stage 9; and dominant-negative KUZ caused a strong border cell migration defect, without affecting expression of markers of border cell fate or follicle cell differentiation. Constitutively active Notch rescued the migration defect due to dominant-negative KUZ, and conditional alleles of Delta and Notch also exhibited border cell migration defects. Expression of two different reporters of Notch activity was lost upon expression of dominant-negative KUZ. Taken together these results show that Notch activation and KUZ expression are restricted to border cells at stage 9 of oogenesis and are required for migration, but not differentiation, of these cells. This represents a previously unrecognized mechanism for achieving spatial restriction of Notch signaling.     

The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.     

Notch signaling in the regulation of tumor angiogenesis

The Notch signaling pathway is conserved in vertebrates and invertebrates and is involved in many developmental processes. Notch receptors and ligands are expressed on the cell surface enabling interactions between adjacent cells upon receptor-ligand binding. Notch signaling molecules have an important well-documented role in vascular development, differentiation, proliferation, apoptosis and tumorigenesis. Recently, several groups have identified the importance of Notch signaling in tumor angiogenesis. Notch activity increases specifically in tumor endothelium and in various tumors types and, in some studies, Notch signaling suppresses angiogenic processes. Because the Notch signaling pathway can mediate communication between various cell types in the tumor microenvironment, interactions between tumor cells and endothelial cells might promote angiogenesis, therefore targeting the Notch pathway might provide a novel strategy for anti-angiogenic therapies. Here, we discuss recent insights of Notch signaling in tumor angiogenesis.     

The combined action of two intercellular signaling pathways specifies three cell fates during vulval induction in C. elegans

Each of the six C. elegans vulval precursor cells (VPCs) has three potential fates (1 degree, 2 degrees, or 3 degrees). The fate of each VPC depends on two types of signals: a graded inductive signal that acts at a distance and a short-range lateral signal among the VPCs. We describe interactions among mutations that cause different misspecifications of VPC fates. Particular combinations of mutations cause all six VPCs to have a single fate independent of their positions. Our results suggest that specification of the three VPC fates is accomplished by two binary decisions, each effected by one of the two signaling pathways. The gene lin-12 acts in the lateral signaling pathway and specifies 2 degrees. The "vulvaless" and "multivulva" genes act in the inductive signaling pathway and specify 1 degree independently of lin-12 and 2 degrees via lin-12. We describe a model for the regulatory circuitry underlying VPC determination that includes a role for lin-12 in both autocrine and paracrine VPC signaling.