Lejeune et al. BMC Genomics 2012, 13:91 http://www.biomedcentral.com/1471-2164/13/91 occurrence of neuronal cell loss, neuron dysfunction is indeed thought to be an early-stage component of the pathogenic process in HD [3,11]. However, neuronal cell dysfunction is not easily tractable in vivo. Interestingly, the genetic regulation of response to light touch by mechanosensory neurons in C. elegans has been the subject of detailed analysis [12]. By targeting polyQ expression to C. elegans touch cells, we previously gen- erated a model that recapitulates an early phase of expanded polyQ neurotoxicity, namely neuronal dys- function in the absence of cell death [13,14]. In these animals, exon 1 like htt fused to CFP was expressed in touch receptor neurons under the control of the promo- ter for the mec-3 gene (mec-3p) [14]. This promoter is active from the early stages of embryonic development through adulthood [15]. Young adults expressing normal polyQs (19Q) have a moderate loss of touch response with no or little change in touch cell morphology, whereas young adults expressing expanded polyQs (128Q) show a dramatic loss of touch response, a beha- vioral phenotype that is accompanied by a strong level of axonal dystrophy [14]. Axonal dystrophy is followed by late-onset axonal degeneration as observed in older adult animals (unpublished data). The loss of touch response in expanded-polyQ nematodes can be manipu- lated by genetic means, as exemplified by the protective effect of sirtuin overexpression and aggravating effect of loss-of-function in the sir-2.1/SIRT1–daf-16/FOXO cell survival pathway [14]. These animals thus provide an in vivo model in which to screen for modifiers of neuron dysfunction induced by expanded polyQs. Here, we used 128Q nematodes in a large-scale functional RNA inter- ference (RNAi) screen for modulation of response to touch. Additionally, we used 19Q nematodes to test a subset of the 128Q-confirmed hits for modulation of response to touch in 19Q nematodes. To this end, we generated polyQ strains with enhanced response of touch receptor neurons to double-standed RNA deliv- ered by feeding. We then undertook a semi-directed screening approach to test a total of 6034 genes includ- ing 5128 genes tested at random and 906 genes that belong to major signaling pathways (16-19). This led us to identify 662 genes that either reduce or aggravate the loss of response to light touch when knocked-down by RNAi in 128Q nematodes. Screening in 19Q nematodes suggested that greater than 90% of these genes specifi- cally modify 128Q cytotoxicity. In-depth data analysis emphasized a role for cell differentiation/survival, meta- bolic and neurodegenerative disease pathways in the modulation of expanded-polyQ cytotoxicity. Addition- ally, data analysis and comparisons to gene expression data from HD mice emphasized the potential value of this RNAi dataset for target discovery in HD. Page 2 of 14 Methods C. elegans strains Nematode strains were handled using standard methods [16]. Integrated polyQ strains used in this study were previously described [17,18]. Briefly, ID245 (igIs245[mec- 3p::htt57-19Q::CFP; lin-15(+); mec-7p::YFP]) and ID1 (igIs1[mec-3p::htt57-128Q::CFP; lin-15(+); mec-7p::YFP]) stably co-express the first 57 amino acids of human huntingtin (htt) fused to CFP under the control of the promoter of the mec-3 gene and YFP under the control of the promoter of the mec-7 gene. We previously reported that ID245 animals show a moderate loss of response to light touch at the tail whereas ID1 animals show a strong loss of response to light touch at the tail, the latter phenotype accompanied by axonal swelling [14]. The enhanced RNAi strain YY13 which carries the rrf-3(mg373) mutation [19] was provided by the Caenor- habditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). To construct polyQ strains with enhanced RNAi sensitivity in neurons, we crossed YY13 animals with either ID245 or ID1 animals to generate strain ID448 (igIs245[mec- 3p::htt57-19Q::CFP;lin-15(+);mec-7p::YFP];rrf-3(pk1426)) and strain ID447 (igIs1[mec-3p::htt57-128Q::CFP;lin-15 (+);mec-7p::YFP];rrf-3(pk1426)), respectively. The ID448 and ID447 strains were tested for sensitivity of touch receptor neurons to RNAi and subsequently used to screen the C. elegans ORF-RNAi library (Open Biosys- tems, AL) that includes clones from the C. elegans ORFeome library v1.1 [20] as detailed below. RNAi screen Bacterial clones from the C. elegans ORF-RNAi library were grown in 500 μl of LB culture medium supplemen- ted with ampicillin (100 μg/ml) and tetracyclin (12.5 μg/ ml), and they were induced by adding 10 μl of IPTG (12.5 mg/ml) 4 hours prior to incubation with nema- todes. Synchronized L1 larvae were obtained by hypo- chlorite extraction using standard methods [16] and they were grown at 20°C on Petri dishes until they reach the young adult stage. The day of incubation in 96-well plates (D0), two young adult animals were incu- bated per well. Each of the wells contained 33.6 μl of bacterial culture and 16.6 μl of M9 media supplemented with cholesterol, ampicillin, tetracyclin, fungizone and IPTG. Incubation was performed at 20°C during 4 days until the animals reached adulthood and produce late L4 larvae. To ensure appropriate feeding of the nema- todes in the well, 16.6 μl of bacterial cultures expressing RNAi clones were added at day 1. At day 4, animals were transferred to agar plates and allowed to recover for 15 minutes. RNAi clones producing lethality, larval arrest, egg-laying defect, developmental delay or any