For deletion mutations obtained from the C. elegans gene knockout consortium (ok, gk mutations) or the Japan National Bioresource Project (tm mutations), we backcrossed the mutant at least two times to N2 Dinaciclib molecular weight wild-type. For selected “hit” genes,

we retested the mutant after a second round of outcrossing and found consistent effects on regrowth. Deletions were genotyped by PCR; primer sequences are available on request. Transgenes were generated by standard procedures (see Supplemental Experimental Procedures). We chose a set of 654 genes based on the following criteria: (1) recognizable C. elegans, human similarity, as assessed by “best BLAST score” in Wormbase; (2) viable mutant strain; (3) known structural or functional category (e.g., kinase, channel); (4) expression in neurons (Wormbase). Some genes were prioritized based on RNAi screens for synaptic function ( Sieburth et al., 2005) or axonal guidance ( Schmitz et al., 2007). A few genes were selected based on expression in touch neurons ( Zhang et al., 2002). We performed laser axotomy essentially as described (Wu et al., 2007). To immobilize worms for EBP-2::GFP imaging without anesthetics, we used 12.5% agarose pads and a suspension of 0.1 μm diameter polystyrene beads (Polysciences) under the coverslip (C. Fang-Yen, personal communication). For live imaging of EBP-2::GFP, we collected

200 frames of 114 msec exposure each every 230 msec using the spinning disk confocal and generated kymographs using Metamorph Calpain (Molecular Devices) from a 40 μm ROI on the PLM axon

proximal to the cut site. Selleckchem MK8776 To apply taxol to regrowing axons in vivo, we grew animals on NGM agar plates containing 5 μM taxol (Sigma) for 24 hr prior to axotomy. One hour before axotomy, we injected 2–5 nl of 50 μM taxol in M9 buffer into the body cavity using standard injection protocols, and then recovered the animals on taxol-containing plates for 30 min. We axotomized PLM using our standard protocol except with 50 μM taxol in solutions. Control animals were injected with M9 buffer and cultured without taxol. Animals injected with buffer or taxol were healthy and grew at normal rates. The distribution of total regrowth length of axons in wild-type and controls passed standard tests of normality. In preliminary analysis, we used the Student’s t test or the Mann-Whitney test. Among 650 such two-way comparisons, 33 are expected to be significant at the 0.05 level by chance. Most genes discussed here displayed effects significant at the 0.01 level (red bars in bar charts of regrowth); we also discuss some genes that gave repeatable results at the 0.05 level (orange bars). To compare regrowth between experiments with different control means, we normalized each experimental data point by dividing it by its control mean. To correct for multiple comparisons, we used two approaches.