A sequencing-based screening method identifies regulators of EGFR signaling from nonviable mutants in Caenorhabditis...

Suppressor screens can identify genetic modifiers of biochemical pathways but generally require that the suppressed mutant be viable and fertile. We developed a screening method that obviated this requirement and enabled the identification of mutations that partially suppressed the early developmental arrest and lethality caused by loss of the epidermal growth factor (EGF) receptor ortholog LET-23 in Caenorhabditis elegans. We chemically mutagenized animals carrying the loss-of-function allele let-23(sy15), recovered let-23(sy15) homozygotes that escaped early developmental arrest but were nevertheless inviable, and sequenced their genomes. Testing of candidate causal mutations identified 11 genes that, when mutated, mitigated the early lethality caused by loss of EGF signaling. These included genes encoding homologs of the small guanosine triphosphatase (GTPase) Ras (let-60), which is a downstream effector of LET-23, and of regulators of the small GTPase Rho, including the homolog of the phosphotyrosine-binding protein TENSIN (tns-1). We also recovered suppressing mutations in genes encoding nuclear proteins that protect against DNA damage, including the homolog of MutS homolog 4 (him-14). Genetic experiments were consistent with the repression of Rho activity or the activation of the DNA damage response compensating for the loss of EGF signaling. This sequencing-based, whole-animal screening method may be adapted to other organisms to enable the identification of mutations for which the phenotype does not allow the recovery of viable animals.