ARTICLE Evidence that the C-Terminal Domain (CtD) Autoinhibits Neural Repression by Drosophila E(spl)M8 Bhaskar Kahali, Jee-Eun Kim, Umesh Karandikar, Clifton P. Bishop, and Ashok P. Bidwai* Department of Biology, West Virginia University, Morgantown, West Virginia Received 10 August 2009; Accepted 5 October 2009 Summary: Analysis of the retinal defects of a CK2 phos- phomimetic variant of E(spl)M8 (M8S 159 D) and the trun- cated protein M8* encoded by the E(spl)D allele, suggest that the nonphosphorylated CtD ‘‘autoinhibits’’ repres- sion. We have investigated this model by testing for inhi- bition (in ‘‘trans’’) by the CtD fragment in its nonphos- phorylated (M8-CtD) and phosphomimetic (M8SD-CtD) states. In N 1 flies, ectopic M8-CtD compromises lateral inhibition, i.e., elicits supernumerary bristles as with loss of N signaling. This antimorphic activity of M8-CtD strongly rescues the reduced eye and/or bristle loss phenotypes that are elicited by ectopic M8SD or wild type M8. Additionally, the severely reduced eye of N spl /Y; E(spl)D/1 flies is also rescued by M8-CtD. Rescue is specific to the time and place, the morphogenetic fur- row, where ‘‘founding’’ R8 photoreceptors are specified. In contrast, the phosphomimetic M8SD-CtD that is pre- dicted to be deficient for autoinhibition, exhibits signifi- cantly attenuated or negligible activity. These studies provide evidence that autoinhibition by the CtD regu- lates M8 activity in a phosphorylation-dependent man- ner. genesis 48:44–55, 2010. V V C 2009 Wiley-Liss, Inc. Key words: Notch; lateral inhibition; E(spl)D; N spl ; neurogenesis INTRODUCTION N signaling has been intensively studied during Drosoph- ila neurogenesis, a process leading to stereotyped pat- terning of the compound eye and bristles (Baonza and Freeman, 2001; Bray, 2006; Campos-Ortega, 1997). Neu- ral development initiates with the expression of atonal (ato) or the achaete-scute complex (ASC), which encode basic-helix-loop-helix (bHLH) transcription fac- tors, the proneural activators (Cubas et al., 1991; Heit- zler et al., 1996; Jarman et al., 1994, 1995; Modolell and Campuzano, 1998; Skeath and Carroll, 1991). ato is required for eye development and ASC for the bristle. During early neurogenesis, these activators are expressed in groups of cells called the proneural clusters [PNC’s (Calleja et al., 2002; Dambly-Chaudiere and Ver- voort, 1998; Frankfort and Mardon, 2002; Gibert and Simpson, 2003; Hsiung and Moses, 2002)]. However, only a fixed number of cells from each PNC are selected to adopt the neural fate, while the others are redirected to an alternative fate. This selection begins when one cell of a PNC gains an advantage by expressing the high- est level of Ato/ASC. This cell is destined to form the ‘‘founding’’ R8 photoreceptor or the bristle sensory organ precursor (SOP), and inhibits other PNC cells from adopting the (default) R8/SOP fate. This N-depend- ent process is termed lateral inhibition (Lehmann et al., 1983; Simpson, 1990). The future R8/SOP expresses Delta (Dl) at a higher level, and activates N in adjoining cells of the PNC. As a result, N is cleaved and its intracel- lular domain (N ICD ) then elicits transcription of the Enhancer of Split Complex [E(spl)C, (Bailey and Posak- ony, 1995; Lecourtois and Schweisguth, 1995; Schrons et al., 1992)]. In cells receiving this inhibitory N signal, E(spl) repressors complex with the corepressor Grou- cho (Gro) and antagonize Ato/ASC, thereby preventing them from adopting the R8/SOP fate. Given the importance of E(spl) for lateral inhibition, numerous studies have sought to define their modes of action. These studies have turned out to be complicated, in part, because the E(spl)C encodes seven bHLH pro- teins with similar functional domains, and mutations affecting each transcription unit have been unavailable. Earlier studies that overexpression of E(spl) proteins elicits generalized bristle loss (Giebel and Campos- Ortega, 1997; Nakao and Campos-Ortega, 1996) raised the possibility of functional redundancy (Cooper et al., 2000). A more recent model, termed ‘‘the protein tether’’ (Giagtzoglou et al., 2003), proposes that, in addi- tion to DNA-binding, E(spl) proteins mediate repression by directly interacting with enhancer-bound Ato/ASC. One prediction of this model is that repression should reflect E(spl) dosage. Studies in the eye have produced *Correspondence to: Ashok P. Bidwai, Department of Biology, West Virginia University, Morgantown, WV, USA. E-mail: abidwai@wvu.edu Contract grant sponsor: National Institutes of Health, Contract grant number: EY015718 Published online 15 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/dvg.20581 ' 2009 Wiley-Liss, Inc. genesis 48:44–55 (2010)