Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila

Thomas L. Jacobsen 1 2 Keith Brennan 0 1 Alfonso Martinez Arias 0 1 Marc A. T. Muskavitch 1 2 0 Department of Zoology, University of Cambridge , Cambridge CB2 3E1 , UK 1 Key words: Delta , Notch, Serrate, cis-interaction, fringe, Drosophila, Neuron 2 Program in Genetics, Cell and Developmental Biology, Department of Biology, Indiana University , Bloomington, Indiana , USA SUMMARY We find that ectopic expression of Delta or Serrate in neurons within developing bristle organs is capable of nonautonomously inducing the transformation of the pretrichogen cell into a tormogen cell in a wide variety of developmental contexts. The frequencies at which Delta can induce these transformations are dependent on the level of ectopic Delta expression and the levels of endogenous Notch signalling pathway components. The pre-trichogen cell becomes more responsive to Delta- or Serrate-mediated transformation when the level of endogenous Delta is reduced and less responsive when the dosage of endogenous Delta is increased, supporting the hypothesis that Delta interferes autonomously with the ability of a cell to receive either signal. We also find that a dominant-negative form of Notch, ECN, is capable of Intercellular communication mediated by distinct signalling pathways is central to the proper specification and differentiation of a wide variety of cell and tissue types throughout metazoan development. One such intercellular signalling pathway, the Notch pathway, was first discovered in Drosophila, and is deployed during oogenesis, embryogenesis and metamorphosis in flies. The Notch pathway plays central roles in the partitioning of cell fates within equivalence groups throughout development (Muskavitch, 1994; Artavanis-Tsakonas et al., 1995). Notch signalling exhibits a high degree of conservation among vertebrates and invertebrates, in the individual genes that make up the signal transduction pathway and the developmental roles they play (Muskavitch, 1994; ArtavanisTsakonas et al., 1995; de la Pompa et al., 1997). In Drosophila melanogaster, many genes have been identified that play critical roles in Notch receptor signalling events. Components within the pathway include the ligands Delta and Serrate, Notch, and the downstream effectors Suppressor of Hairless [Su(H)] and the proteins encoded by the Enhancer of split-Complex [E(spl)-C]. The ligands Delta and Serrate bind to Notch molecules located on adjacent autonomously interfering with the ability of a cell to generate the Delta signal. When the region of Notch that mediates trans-interactions between Delta and the Notch extracellular domain is removed from ECN, the ability of Delta to signal is restored. Our findings imply that cellautonomous interactions between Delta and Notch can affect the ability of a cell to generate and to transduce a Delta-mediated signal. Finally, we present evidence that the Fringe protein can interfere with Delta- and Serratemediated signalling within developing bristle organs, in contrast to previous reports of the converse effects of Fringe on Delta signalling in the developing wing. cells, initiating a cascade of events within the signal-receiving cell. In many cases, transduction of the Notch-mediated signal to the nucleus of the cell depends on the transcriptional activator Su(H), which physically interacts with the intracellular domain of Notch (Fortini and ArtavanisTsakonas, 1994). In cell culture, and in some contexts in vivo, the Su(H) protein translocates from the cytoplasm to the nucleus subsequent to Notch activation (Fortini and Artavanis-Tsakonas, 1994; Gho et al., 1996). Among the targets of Su(H) transcriptional activation are the hairy-like helix-loop-helix proteins of the E(spl)-C, which act primarily as transcriptional repressors (Delidakis and ArtavanisTsakonas, 1992; Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995; Oellers et al., 1994). In general, the effects of loss-of-function or gain-of-function mutations in any of these loci parallel one another, i.e., lead to reductions or increases in overall Notch signalling activity (Muskavitch, 1994; Artavanis-Tsakonas et al., 1995). The products of the Hairless (H) and fringe (fng) loci modulate Notch signalling. The Hairless protein interacts physically with Su(H) and antagonizes transduction of the Notch signal, possibly by sequestering Su(H) in the cytoplasm (Bang and Posakony, 1992; Brou et al., 1994; Bailey and Posakony, 1995). The Fringe protein appears to act at the level of ligand-receptor binding, antagonizing the function of the Serrate ligand (Fleming et al., 1997; Panin et al., 1997) and possibly potentiating transmission of the Delta signal in the larval wing disc (Panin et al., 1997). The event that initiates Notch signalling is the binding of the receptor to one of its ligands. The two identified Notch ligands in Drosophila, Delta and Serrate, are transmembrane proteins with extracellular domains that share extensive sequence homology within their amino-terminal domains (Vassin et al., 1987; Kopczynski et al., 1988; Fleming et al., 1990; Thomas et al., 1991). Cultured Drosophila cells that express either Delta (Delta+ cells) or Serrate (Serrate+ cells) are able to aggregate specifically with cells that express Notch (Notch+ cells; Fehon et al., 1990). Delta+ cells also display the ability to homotypically aggregate, implying that Delta, unlike Serrate or Notch, is able to self-associate in trans (Fehon et al., 1990). The ability of Delta+ or Serrate+ cells to aggregate with Notch+ cells is dependent on the presence of Notch EGF-like repeats (ELRs) 11 and 12, which are necessary and sufficient for the binding of either ligand (Rebay et al., 1991). While the activation of the full-length Notch receptor in vivo depends on the presence of ligand and the integrity of the ELR array, a truncated form of Notch composed of the intracellular domain acts as a constitutively active receptor (Lieber et al., 1993; Rebay et al., 1993; Struhl et al., 1993). This implies that the function of the Notch extracellular domain may be to restrict the activity of the intracellular domain prior to ligand binding. In contrast, removal of the intracellular domain from Notch yields a dominant-negative form of the receptor that inhibits Notchmediated signalling in vivo (Rebay et al., 1993; Klein et al., 1997). We are only beginning to understand interactions between Notch and its ligands. The multimerization states of the ligands and the receptor prior to and subsequent to ligandreceptor binding are unknown. It is also unclear whether ligand and receptor molecules located on the surface of the same cell are able to interact with each other. While interactions between Delta molecules on opposing cell surfaces have been observed in cultured cells (Fehon et al., 1990), the in vivo significance of this interaction, e.g. whether Delta on the surface of a signal-receiving cell can inhibit receipt of a Delta signal from an adjacent cell, (...truncated)