One-day adult hermaphrodites were anaesthetized for >20 minutes in a drop of 0.01% tetramisole (Sigma T1512) in M9 buffer on a 3% agar pad. Images of the PVD neuron in hermaphrodite C. elegans were analyzed by Nomarski optics and fluorescence microscopy using a Nikon eclipse Ti inverted microscope equipped with Yokogawa CSU-X1 spinning disk confocal microscope fitted with a 40 x oil Plan Fluor NA = 1.3 lens [22]. We used projections of z-stacked confocal images taken at ~0.5–0.6 μm intervals, digitally stored using iXon EMCCD camera (Andor). The acquisition of the images was done using MetaMorph software, utilizing 25% laser intensity at 488 nm, gain of 200 and 300 ms exposure for image acquisition. The multi-dimensional data (in the form of z-stacks) was projected onto a 2D plane using maximal intensity projection in Fiji/ImageJ software (NIH) to produce a 2D grayscale image.

For S7 Fig, L4s or young adult BP2117 hermaphrodites in which pmyo-2::GFP expression was not observed (and hence the hyEx372 extrachromosmal array was likely lost) were anesthetized and imaged as described above with the following exceptions: Apochromat 60x NA = 1.4 lens was used with 561 nm wavelength laser excitation (15–20% intensity, 100 ms imaging exposure time).

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