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.     

Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts

Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.     

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.     

The Drosophila IgC2 domain protein Friend-of-Echinoid,a paralogue of Echinoid,limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway

The Notch signaling pathway is critical in cell fate specification throughout development. In the developing wing disc, single sensory organ precursors (SOPs) are selected from proneural clusters via a process of lateral inhibition mediated by the Notch signaling pathway. The epidermal growth factor receptor (EGFR) pathway has also been implicated in SOP formation. Here, we describe the Drosophila melanogaster gene friend of echinoid (fred), a paralogue of echinoid (ed), a gene recently identified as a negative regulator of the EGFR pathway. fred function was examined in transgenic flies by using inducible RNA interference (RNAi). Suppression of fred in developing wing discs results in specification of ectopic SOPs, additional microchaeta, and cell death. In eye-antennal discs, fred suppression causes a rough eye phenotype. These phenotypes are suppressed by overexpression of Notch, Suppressor of Hairless [Su(H)], and Enhancer of split m7. In contrast, overexpression of Hairless, a negative regulator of the Notch pathway, and decreased Su(H) activity enhance these phenotypes. Thus, fred acts in close concert with the Notch signaling pathway. Dosage-sensitive genetic interaction also suggests a close relationship between fred and ed.     

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.     

Drosophila deltex mediates suppressor of Hairless-independent and late-endosomal activation of Notch signaling

Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Here, we found that Dx was required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx required N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activated a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that was independent of the Delta (Dl)/Serrate (Ser) ligands- or Su(H). Dx caused N to be moved from the apical cell surface into the late-endosome, where it accumulated stably and co-localized with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibited the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)-independent N signaling.     

Notch signaling targets the Wingless responsiveness of a Ubx visceral mesoderm enhancer in Drosophila

BACKGROUND: Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. RESULTS: We report that Ubx(VM)B, a visceral mesoderm-specific enhancer of the Ubx gene of Drosophila, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf binding sites in the Ubx(VM)B enhancer. CONCLUSIONS: Our results show that, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on Ubx(VM)B. A possible target of this activity is dTcf. As dTcf has been shown to be capable of repressing Wingless targets, our results suggest that this repressive activity may be regulated by Notch. Finally, we suggest that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of dTcf and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with dTcf.     

Molecular separation of two signaling pathways for the receptor, Notch

Notch is required for many aspects of cell fate specification and morphogenesis during development, including neurogenesis and axon guidance. We here provide genetic and biochemical evidence that Notch directs axon growth and guidance in Drosophila via a “non-canonical”, i.e. non-Su(H)-mediated, signaling pathway, characterized by association with the adaptor protein, Disabled, and Trio, an accessory factor of the Abl tyrosine kinase. We find that forms of Notch lacking the binding sites for its canonical effector, Su(H), are nearly inactive for the cell fate function of the receptor, but largely or fully active in axon patterning. Conversely, deletion from Notch of the binding site for Disabled impairs its action in axon patterning without disturbing cell fate control. Finally, we show by co-immunoprecipitation that Notch protein is physically associated in vivo with both Disabled and Trio. Together, these data provide evidence for an alternate Notch signaling pathway that mediates a postmitotic, morphogenetic function of the receptor.