Transgenic lines were scored as egg-laying defective if young adults (24 h after late L4) had, on average, greater than 30 eggs in utero. To score the developmental stages of newly laid eggs, young adults were transferred to fresh nematode growth medium plates with bacteria, five worms per plate, for 1 h at room temperature and then removed. Operated worms were assayed individually, and a single operated worm was assayed two and four times.
Eggs on the plate were examined under a high-power dissecting microscope and categorized as described in Figure 2.
Distributions of the developmental stages of eggs laid by worms of different genotypes were analyzed with the Wilcoxon Mann-Whitney rank-sum test, a nonparametric test of statistical significance, as implemented in the Coin package of the R statistical analysis program49 . The results of statistical tests of significance can be found in Supplementary Table 1. Egg-laying defects of many strains were also independently quantified by measuring the number of retained eggs in staged adults as previously described19 (link), and we observed similar interactions between receptor- and ligand-encoding genes, receptor- and G protein-encoding genes, and genes in the egl-6 signaling pathway and acetylcholine biosynthesis genes (data not shown).
Laser microsurgeries were performed on L2-stage larvae as described50 (link). Cell identifications were made on the basis of nuclear position and cell morphology. A gcy-33∷gfp transgene was used in some experiments to identify BAG neurons (see Supplementary Methods).