C. elegans EMS mutagenesis screens are an important tool for identifying novel components of biological processes. Here, we report a screen to identify novel gain-of-function alleles promoting EGF pathway activity in vulval development. Simultaneous loss of the lin-15A and lin-15B loci confers a strong herniated multivulva (Muv) defect consistent with over-induction of 1° vulval fates, and lin-15(n765ts) animals are null for one locus and temperature sensitive mutant for the other (Huang et al., 1994). This hyper-induced vulval phenotype is due to de-repressed LIN-3/EGF expression in the hyp7 hypodermis surrounding the vulval precursor cells (VPCs), such that all VPCs receive an inappropriately high EGF signal (Cui et al., 2006). We reasoned that using temperature-sensitive lin-15(n765) mutant animals, we could titrate EGF activity to the level just barely insufficient for ectopic vulval induction. In this background an F1 screen should allow screening through tens of thousands of haploid genomes to uncover heterozygous activating mutations (gain-of-function alleles) sufficient to induce ectopic vulval formation.
To identify a non-inducing condition, we first assayed lin-15(ts) animals at a number of temperatures. We found that at 15°C, lin-15(ts) animals were not Muv, while Muv animals were observed at higher temperatures. We applied the standard 50 mM EMS mutagenesis protocol to late L4 lin-15(n765ts) animals, plated mutagenized P0 animals at 15°C, and screened F1 adult progeny for the Muv phenotype. We found an extremely high rate of positives, with 44/455 (9.7%) Muv F1 animals. Many putative mutants (19/44, 43.2%) segregated >5 Muv progeny, indicating that picked animals did not represent background noise, but rather were bona fide enhancer mutations that conferred a dominant Muv phenotype in this background at 15°C. Given this exceptionally high positive rate, we are probably identifying genes that are haploinsufficient for lin-3/EGF repression or EGFR pathway inhibition, which is similar to the sensitized Drosophila enhancer screen for haploinsufficiency in sevenless function, the E(sev) screen (Simon et al., 1991). Based on the estimated identifiable loss-of-function mutation rate of 5X10-4 for this EMS mutagenesis protocol, we estimate the gene target size of our screen to be roughly 84 genes.
Clearly this screen is unsuitable for our purposes. We speculate that we have identified a very large group of genes required for the transcriptional repression of lin-3/EGF, the synMuv genes, or another repressive function in vulval induction. Increased stringency should be achieved by using a lower temperature, thus making the screen less sensitive.
References
Cui M, Chen J, Myers TR, Hwang BJ, Sternberg PW, Greenwald I, Han M. (2006). SynMuv genes redundantly inhibit lin-3/EGF expression to prevent inappropriate vulval induction in C. elegans. Dev. Cell. 10, 667-72. 
Huang LS, Tzou P, Sternberg PW. (1994). The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development. Mol. Biol. Cell, 5, 395-412.
Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM. (1991) Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67, 701-716.
