Transgene‐mediated RNA interference defines a novel role for notch in chemosensory startle behavior

The Notch signal transduction system functions in the developmental differentiation of a wide variety of tissues in every metazoan thus far examined (Artavanis-Tsakonas et al., 1999). In addition, recent evidence has shown that Notch function is important even after cell fate decisions are made. Examples include control of neurite arborization (Redmond and Ghosh, 2001) and postdevelopmental maintenance of flight and lifespan (Presente et al., 2001; Vilkki and Portin, 1987). Since the parallel discovery that the presenilin genes encode modifiers of Notch signal (Levitan and Greenwald, 1995) and are the most common target gene mutation in familial Alzheimer’s disease (AD mutation database, http://molgen-www. uia.ac.be), there has been much debate as to the potential of Notch signal dysfunction in Alzheimer’s disease pathology. While many mutations in presenilin or its homologs demonstrate gain-of-function properties with respect to increased deposition of the amyloidsenile plaques (Haass and Baumeister, 1998), recent work has shown that these mutations act as partial loss-of-function with regard to Notch signaling (Song et al., 1999) and Notch function in neuronal growth in nematodes (Wittenburg et al., 2000). Some groups have begun to address the effects of presenilin loss-of-function in the fly and the mouse (Feng et al., 2001; Ye et al., 1999; Yu et al., 2001), but because of the ever-increasing number of gene products affected by presenilin (Marambaud et al., 2002; Ni et al., 2001; Van Uden et al., 1999) it is difficult to assign behavioral phenotypes to a specific pathway. Our goal is to develop a reagent that, in combination with existing resources in the Drosophila community, will enable us to selectively inhibit Notch in the adult and allow us to determine if Notch dysfunction plays a role in neurodegeneration. Much of our current understanding of the biology of Notch signaling has been derived from classic loss-offunction studies involving Drosophila in which the Notch locus has been mutated. These studies have been aided by the ability to conditionally inhibit Notch functions using temperature-sensitive alleles (Shellenbarger and Mohler, 1975) or to express dominant-negative transgenes in a temporally and/or spatially specific manner (Lieber et al., 1993; Rebay et al., 1993). However, both approaches have serious limitations. For example, it is not clear how Notch signaling is inhibited at the nonpermissive temperature. This is true even for the widely used N allele, and there is ample evidence to indicate that at the elevated temperatures these alleles do not act as true genetic nulls (Shellenbarger and Mohler, 1975). Thus, even very small amounts of functional Notch protein can be transcriptionally active in these mutants and this residual Notch function may mask important phenotypes. Similarly, the dominant-negative activities of many engineered Notch constructs have shown themselves to be initially perplexing (Sun and Artavanis-Tsakonas, 1997). For example, a simple hypothesis is that a transgene encoding only the Notch extracellular domain (N) would compete with endogenous Notch for the ligands Delta and Serrate. However, this is complicated by the discovery that other ligands such as Wnt/Wingless can bind to the extracellular domain of Notch (Wesley, 1999). How Wingless (Wg) may signal through Notch is unknown, but it has been shown that Wg and Delta produce differing effects on Notch processing (Wesley and Saez, 2000). Thus, it is unclear whether the N truncation interferes with Wg/Notch signaling. In addition, it is possible that high expression of this construct may bind the diffusible Wg ligand and prevent it from reaching other physiological Wg targets such as cells expressing the Frizzled receptor. Therefore, one must consider the caveat that any activity resulting from the overexpression of this truncation construct may be unrelated to endogenous Notch signaling. Finally, it is often difficult or impossible to produce enough dominantnegative protein to completely overcome endogenous Notch signaling, and as more protein is produced there is always the complication of nonspecific toxicity. Thus, as our understanding about the molecular mechanisms involved in Notch signaling increases, many questions are raised about the specificity of the dominant negative strategy for intervention.