(A) Relative numbers of embryos freshly-laid by wild-type and mutant animals. wyIs333 is the marker that double-labels the HSN presynaptic specializations and vulval muscles. *p<0.0001, Fisher's exact test, n = 62–303 embryos. (B) Percentage of different stages of embryos freshly-laid by wild-type and mutants animals. *p<0.0001, Fisher's exact test, n = 62–303 embryos. (C) and (D) Ratiometric Ca2+ imaging in the vulval muscles in behaving wild-type (C) and lin-12(wy750) (D) animals. GCaMP3 (top) and mCherry (middle) were co-expressed in the vulval muscles and the GCaMP3/mCherry fluorescence ratio (bottom) was used to record Ca2+ transients (arrowheads). Time points are shown from Video 1 (Wild type) and Video 2 (lin-12(wy750) mutant). Vertical lines (II) indicate the vulval muscles at rest, and arrows indicate vulval muscle twitches (small) and egg-laying (large) contractions. Anterior (A), posterior (P), left (L), right (R), dorsal (D), ventral (V). Scale bars are 10 μm. (E–G) traces of vulval muscle GCaMP3/mCherry ratio change (ΔR/R) from the same wild-type (E) and lin-12(wy750) mutant (F) animals shown above (horizontal bars) and in the apx-1(wy755) mutant (G). Also indicated are egg-laying events (*), single transients limited to the anterior or posterior (A or P) vulval muscles (|), double transients occurring simultaneously in both anterior and posterior (A + P) vulval muscles (^), or delayed double transient where anterior and posterior transients within a body bend are separated by a visually discernible interval (horizontal bracket). (H–J) Quantitations of vulval muscle Ca2+ signaling (6-min recording per animal, 11 or 12 animals per genotype; error bars indicate 95% confidence intervals). (H) lin-12(wy750) and apx-1(wy755) mutants have more frequent Ca2+ transients than does the wild type. *p<0.0001, one-way ANOVA, n = 215–488. (I) Fewer synchronous double (A + P) Ca2+ transients in lin-12(wy750) and apx-1(wy755) mutants. *p<0.0001, chi-squared test, n = 200–305. (J) Consecutive anterior and posterior Ca2+ transients are delayed in lin-12(wy750) and apx-1(wy755) mutants. *p<0.0001; one-way ANOVA, n = 123–190.