Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib) gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the α1,4-N-acetylgalactosaminyltransferase-1 (α4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of α4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that α4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by α4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL–protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.

Activation of the Raf kinase by GTP-bound Ras is a poorly understood step in receptor tyrosine kinase signaling pathways. One such pathway, the epidermal growth factor receptor (EGFR) pathway, is critical for cell differentiation, survival, and cell cycle regulation in many systems, including the Drosophila eye. We have identified a mutation in a novel gene, aveugle, based on its requirement for normal photoreceptor differentiation. The phenotypes of aveugle mutant cells in the eye and wing imaginal discs resemble those caused by reduction of EGFR pathway function. We show that aveugle is required between ras and raf for EGFR signaling in the eye and for mitogen-activated protein kinase phosphorylation in cell culture. aveugle encodes a small protein with a sterile ␣ motif (SAM) domain that can physically interact with the scaffold protein connector enhancer of Ksr (Cnk). We propose that Aveugle acts together with Cnk to promote Raf activation, perhaps by recruiting an activating kinase. Many receptor tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), signal through the Ras/ mitogen-activated protein kinase (MAPK) pathway (Nishida and Gotoh 1993). These receptors have important developmental functions and are also misregulated in a variety of cancers (Holbro and Hynes 2004), making it critical to understand their signaling mechanism. Genetic screens in Drosophila and Caenorhabditis elegans, coupled with biochemical analysis in cultured cells, have allowed the identification of numerous Ras/MAPK pathway components (Rubin et al. 1997;Moghal and Sternberg 2003).The Drosophila eye is a particularly useful system for genetic analysis of the EGFR pathway, which has welldefined functions in photoreceptor development (Freeman 1997;Halfar et al. 2001;Yang and Baker 2003). As the morphogen Hedgehog (Hh) drives progression of the morphogenetic furrow across the eye disc, it induces the expression of the transcription factor Atonal, which specifies the first photoreceptor to differentiate in each cluster, R8 (Jarman et al. 1995;Dominguez 1999). Once the R8 photoreceptor has been specified, it sequentially recruits additional photoreceptors, cone cells, and pigment cells from the surrounding pool of undifferentiated cells. The signal for this recruitment is the EGFR ligand Spitz (Spi), which is secreted by R8 and subsequently by other photoreceptors as they differentiate (Freeman 1996). Production of the downstream feedback inhibitor Argos (Aos), which binds to Spi and blocks its binding to the receptor (Klein et al. 2004), restricts the response to Spi to a small number of cells, allowing the stepwise recruitment of ommatidial cell types (Freeman 1997). A second RTK, Sevenless, also contributes to R7 differentiation (Freeman 1996). The precursors of the R2, R5, R3, and R4 photoreceptors remain arrested in the G1 phase of the cell cycle after leaving the morphogenetic furrow. This arrest also requires EGFR signaling, but occurs at a lower threshold tha...