Ilaria Rebay

Specific EGF Repeats of Notch Mediate Interactions with Delta and Serrate: Implications for Notch as a Multifunctional Receptor

The neurogenic loci Notch and Delta, which both encode EGF-homologous transmembrane proteins, appear to function together in mediating cell-cell communication and have been shown to interact at the cell surface in vitro. To examine the role of the EGF repeats in this interaction, we performed an extensive deletion mutagenesis of the extracellular domain of Notch. We find that of the 36 EGF repeats of Notch, only two, 11 and 12, are both necessary and sufficient to mediate interactions with Delta. Furthermore, this Delta binding ability is conserved in the corresponding two repeats from the Xenopus Notch homolog. We report a novel molecular interaction between Notch and Serrate, another EGF-homologous transmembrane protein containing a region of striking similarity to Delta, and show that the same two EGF repeats of Notch also constitute a Serrate binding domain. These results suggest that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development.

Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila

Genetic analyses have raised the possibility of interactions between the gene products of the neurogenic loci Notch and Delta, each of which encodes a transmembrane protein with EGF homology. To examine the possibility of intermolecular association between the products of these two genes, we studied the effects of their expression on aggregation in Drosophila S2 cells. We find that Notch-expressing cells form mixed aggregates specifically with cells that express Delta and that this process is calcium dependent. In addition, we show that Notch and Delta can associate within the membrane of a single cell, and further, that they form detergent-soluble intermolecular complexes. Our analyses suggest that Notch and Delta proteins interact at the cell surface via their extracellular domains.

The activities of two Ets-related transcription factors required for drosophila eye development are modulated by the Ras/MAPK pathway

We show that the activities of two Ets-related transcription factors required for normal eye development in Drosophila, pointed and yan, are regulated by the Ras1/MAPK pathway. The pointed gene codes for two related proteins, and we show that one form is a constitutive activator of transcription, while the activity of the other form is stimulated by the Ras1/MAPK pathway. Mutation of the single consensus MAPK phosphorylation site in the second form abrogates this responsiveness. yan is a negative regulator of photoreceptor determination, and genetic data suggest that it acts as an antagonist of Ras1. We demonstrate that yan can repress transcription and that this repression activity is negatively regulated by the Ras1/MAPK signal, most likely through direct phosphorylation of yan by MAPK.

Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor

The Notch gene of Drosophila plays an important role in cell fate specification throughout development. To investigate the functions of specific structural domains of the Notch protein in vivo, a series of deletion mutants have been ectopically expressed under the hsp70 heat shock promoter. Two classes of dominant phenotypes are observed, one suggestive of Notch loss-of-function mutations and the other of Notch gain-of-function mutations. Dominant activated phenotypes result from overexpression of a protein lacking most extracellular sequences, while dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results support the notion that Notch functions as a receptor whose extracellular domain mediates ligand binding, resulting in the transmission of developmental signals by the cytoplasmic domain. Finally, the phenotypes observed suggest that the cdc 10/ankyrin repeat region within the intracellular domain plays an essential role in the postulated signal transduction events.

Yan functions as a general inhibitor of differentiation and is negatively regulated by activation of the Ras1/MAPK pathway

Drosophila yan has been postulated to act as an antagonist of the proneural signal mediated by the sevenless/Ras1/MAPK pathway. We have mutagenized the eight MAPK phosphorylation consensus sites of yan and examined the effects of overexpressing the mutant protein in transgenic flies and transfected S2 cultured cells. Our results suggest that phosphorylation by MAPK affects the stability and subcellular localization of yan, resulting in rapid down-regulation of yan activity. Furthermore, MAPK-mediated down-regulation of yan function appears to be critical for the proper differentiation of both neuronal and nonneuronal tissues throughout development, suggesting that yan is an essential component of a general timing mechanism controlling the competence of a cell to respond to inductive signals.

The transcription factor Eyes absent is a protein tyrosine phosphatase

Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.

Signaling circuitries in development: insights from the retinal determination gene network

Context-specific integration of information received from the Notch, Transforming growth factor β, Wingless/Wnt, Hedgehog and Epidermal growth factor receptor signaling pathways sets the stage for deployment of the retinal determination gene network (RDGN), a group of transcription factors that collectively directs the formation of the eye and other tissues. Recent investigations have revealed how these transcription factors are regulated by their interactions with each other and with effectors of the above signaling pathways. Further study of the RDGN may provide insights into how common cues can generate context-specific responses, a key aspect of developmental regulation that remains poorly understood.

Signal integration during development: insights from the Drosophila eye.

The Drosophila eye is a highly ordered epithelial tissue composed of ∼750 subunits called ommatidia arranged in a reiterated hexagonal pattern. At higher resolution, observation of the constituent photoreceptors, cone cells, and pigment cells of the eye reveals a highly ordered mosaic of amazing regularity. This relatively simple organization belies the repeated requirement for spatially and temporally coordinated inputs from the Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp), JAK‐STAT, Notch, and receptor tyrosine kinase (RTK) signaling pathways. This review will discuss how signaling inputs from the Notch and RTK pathways, superimposed on the developmental history of a cell, facilitate context‐specific and appropriate cell fate specification decisions in the developing fly eye. Lessons learned from investigating the combinatorial signal integration strategies underlying Drosophila eye development will likely reveal cell–cell communication paradigms relevant to many aspects of invertebrate and mammalian development. Developmental Dynamics 229:162–175, 2004.

The Spinocerebellar Ataxia 8 Noncoding RNA Causes Neurodegeneration and Associates with Staufen in Drosophila

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscle-blind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease.

Signal Integration During Development: Mechanisms of EGFR and Notch Pathway Function and Cross-Talk

ABSTRACT Metazoan development relies on a highly regulated network of interactions between conserved signal transduction pathways to coordinate all aspects of cell fate specification, differentiation, and growth. In this review, we discuss the intricate interplay between the epidermal growth factor receptor (EGFR; Drosophila EGFR/DER) and the Notch signaling pathways as a paradigm for signal integration during development. First, we describe the current state of understanding of the molecular architecture of the EGFR and Notch signaling pathways that has resulted from synergistic studies in vertebrate, invertebrate, and cultured cell model systems. Then, focusing specifically on the Drosophila eye, we discuss how cooperative, sequential, and antagonistic relationships between these pathways mediate the spatially and temporally regulated processes that generate this sensory organ. The common themes underlying the coordination of the EGFR and Notch pathways appear to be broadly conserved and should, therefore, be directly applicable to elucidating mechanisms of information integration and signaling specificity in vertebrate systems.