The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye

Notch signaling is one of the most important pathways in development and homeostasis, and is altered in multiple pathologies. Study of Drosophila eye development shows that Notch signaling depends on the HLH protein Extramacrochaetae. Null mutant clones show that extramacrochaetae is required for multiple aspects of eye development that depend on Notch signaling, including morphogenetic furrow progression, differentiation of R4, R7 and cone cell types, and rotation of ommatidial clusters. Detailed analysis of R7 and cone cell specification reveals that extramacrochaetae acts cell autonomously and epistatically to Notch, and is required for normal expression of bHLH genes encoded by the E(spl)-C which are effectors of most Notch signaling. A model is proposed in which Extramacrochaetae acts in parallel to or as a feed-forward regulator of the E(spl)-Complex to promote Notch signaling in particular cellular contexts.     

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.     

Non-cell autonomous control of apoptosis by ligand-independent Hedgehog signaling in Drosophila

Hedgehog (Hh) signaling is important for development and homeostasis in vertebrates and invertebrates. Ligand-independent, deregulated Hh signaling caused by loss of negative regulators such as Patched causes excessive cell proliferation, leading to overgrowth in Drosophila and tumors in humans, including basal-cell carcinoma and medulloblastoma. We show that in Drosophila deregulated Hh signaling also promotes cell survival by increasing the resistance to apoptosis. Surprisingly, cells with deregulated Hh activity do not protect themselves from apoptosis; instead, they promote cell survival of neighboring wild-type cells. This non-cell autonomous effect is mediated by Hh-induced Notch signaling, which elevates the protein levels of Drosophila inhibitor of apoptosis protein-1 (Diap-1), conferring resistance to apoptosis. In summary, we demonstrate that deregulated Hh signaling not only promotes proliferation but also cell survival of neighboring cells. This non-cell autonomous control of apoptosis highlights an underappreciated function of deregulated Hh signaling, which may help to generate a supportive micro-environment for tumor development.     

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.     

Negative Regulation of Notch Signaling by Xylose

The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.     

Drosophila liquid facets-Related encodes Golgi epsin and is an essential gene required for cell proliferation, growth, and patterning

Epsin and epsin-Related (epsinR) are multi-modular proteins that stimulate clathrin-coated vesicle formation. Epsin promotes endocytosis at the plasma membrane, and epsinR functions at the Golgi and early endosomes for trans-Golgi network/endosome vesicle trafficking. In Drosophila, endocytic epsin is known as Liquid facets, and it is essential specifically for Notch signaling. Here, by generating and analyzing loss-of-function mutants in the liquid facets-Related (lqfR) gene of Drosophila, we investigated the function of Golgi epsin in a multicellular context. We found that LqfR is indeed a Golgi protein, and that like liquid facets, lqfR is essential for Drosophila viability. In addition, primarily by analyzing mutant eye discs, we found that lqfR is required for cell proliferation, insulin-independent cell growth, and cell patterning, consistent with a role in one or several signaling pathways. Epsins in all organisms share an ENTH (epsin N-terminal homology) domain, which binds phosphoinositides enriched at the plasma membrane or the Golgi membrane. The epsinR ENTH domain is also the recognition element for particular cargos. By generating wild-type and mutant lqfR transgenes, we found that all apparent LqfR functions are independent of its ENTH domain. These results suggest that LqfR transports specific cargo critical to one or more signaling pathways, and lays the foundation for identifying those proteins.     

Regulation of EGFR and Notch signaling by distinct isoforms of D-cbl during Drosophila development

Cells receive and interpret extracellular signals to regulate cellular responses such as proliferation, cell survival and differentiation. However, proper inactivation of these signals is critical for appropriate homeostasis. Cbl proteins are E3-ubiquitin ligases that restrict receptor tyrosine kinase (RTK) signaling, most notably EGFR (Epidermal Growth Factor Receptor), via the endocytic pathway. Consistently, many mutant phenotypes of Drosophila cbl (D-cbl) are due to inappropriate activation of EGFR signaling. However, not all D-cbl phenotypes can be explained by increased EGFR activity. Here, we report that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. The long isoform, D-CblL, regulates the EGFR. We found that the short isoform, D-CblS, preferentially restricts Notch signaling. Specifically, our data imply that D-CblS controls the activity of the Notch ligand Delta. Taken together, these data suggest that D-Cbl controls the EGFR and Notch/Delta signaling pathways through production of two alternatively spliced isoforms during development in Drosophila.     

Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.     

TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.     

The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub

Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. Previous studies have shown that its central region is essential for function even though its three domains are poorly conserved and intrinsically disordered. Using these disordered domains as affinity reagents, we have now identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to a number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. We conclude that the Gro central domains mediate multiple interactions required for repression, thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro.     

The tiny Hairless protein from <Emphasis Type="Italic">Apis mellifera</Emphasis>: a potent antagonist of Notch signaling in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

The Notch signaling pathway is fundamental to the regulation of many cell fate decisions in eumetazoans. Not surprisingly, members of this pathway are highly conserved even between vertebrates and invertebrates. There is one notable exception, Hairless, which acts as a general Notch antagonist in Drosophila. Hairless silences Notch target genes by assembling a repressor complex together with Suppressor of Hairless [Su(H)] and the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). Now with the availability of genomic databases, presumptive Hairless homologues are predicted, however only in insect species. To further our understanding of Hairless structure and function, we have cloned the Hairless gene from Apis mellifera (A.m.H) and characterized its functional conservation in Drosophila.     

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. We previously showed that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell–GSC junction. Here, we further dissect the molecular and cellular mechanisms underlying these processes. We show that insulin and Notch ligands directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. We also report that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, we also find that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell–GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function.     

In Vivo Analysis of the Notch Receptor S1 Cleavage

A ligand-independent cleavage (S1) in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.