Muscle Staining, and Fluorescence/DIC Microscopy

To examine the muscle cytoskeleton, animals were collected and placed on poly-L-lysine coated slides and fixed with methanol and acetone for 5 min each at −20°C. Fixed worms were treated with rhodamine-conjugated phalloidin (0.2 U/ml, Sigma Chem. Co.) for 2 to 3 h at room temperature. Washed samples were mounted on the Nikon TE2000-U epifluorescence microscopes. Images were captured using a CoolSnap ES (Roper Scientific, Tucson, AZ, United States) and analyzed with Metavue software (version 7.5, Molecular Devices, Downingtown, PA, United States) or NIS Elements (version 5.02, Nikon Instruments, Melville, NY, United States). For DIC or fluorescence live samples, animals were fed with 2% NaN₃ or 5 mM levamisole solution on agarose pads and mounted on the Nikon TE2000-U or Eclipse Ni-U microscope. Samples were examined and analyzed for defects using a 20X or 40X Plan Fluor objective lens. Images were obtained using Coolsnap ES or DYNO monochrome camera (Photometrics, Tucson, AZ, United States) and analyzed using Metavue (version 7.5, Molecular Devices, Downingtown, PA, United States) or NIS Elements (version 5.02, Nikon Instruments, Melville, NY, United States) software.

For DNA staining, animals were washed off the plate and fixed with methanol in −20°C. The next day fixed worms were treated with ethanol series from 95–30%. After the final alcohol treatment, isolated animals were treated with 0.1 μg/ml of DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride, Sigma-Aldrich, St. Louis, MO, United States) in M9 buffer for more than 4 h with rotation. After three washes, samples were observed under the microscope.

Animals were stained with MH25, anti-PAT-3 antibodies (1:250 dilution, purchased from DSHB, Iowa City, IA, United States) (Francis and Waterston, 1991), and goat anti-mouse Cy3-conjugated antibody as secondary antibodies (1:500 dilution, Jackson Laboratory, Bar Harbor, ME, United States). Briefly, animals were placed on poly-L-lysine coated slides (Sigma-Aldrich, St. Louis, MO, United States) and fixed with ice-cold 100% methanol for 5–10 min. Fixed animals were blocked with 5% goat sera and treated with primary and secondary antibodies. Fluorescence images were obtained by using an Olympus FLUOVIEW FV1000 confocal microscope equipped with a PlanApo 60X oil-immersion objective lens and processed using its accompanying FLUOVIEW (version 4.2, Olympus Corporation, Center Valley, PA, United States) software.

Statistical analysis in Tables 1–3 and Supplementary Table S1 were performed with JMP Pro (version 14.0.0., SAS Institute, Cary, NC, United States). The 95% confidence interval for comparison was constructed using a chi-squared likelihood ratio test (Table 2 and Supplementary Table S1) (McHugh, 2013). For multiple comparison analysis, ANOVA analysis and the Tukey-Kramer HSD post hoc multiple comparison was used in Tables 1, ​,33 (Tukey, 1949).

Male percentage and mating success phenotypes of transgenic rescues, him-4 (e1267), N2, βpat-3(+) and double mutants.

Male gonad migration and abnormal tail phenotype of transgenic rescues, him-4 (e1267), N2, βpat-3(+) and double mutants.

Inviable zygote phenotype of transgenic rescues, him-4 (e1267), N2, βpat-3(+) and double mutants.