Figure 4. mir-71 expression and the tir-1 3′ UTR are differentially regulated in AWC^ON and AWC^OFF neurons.

(A, B) Images of mir-71p::GFP. The AWC^OFF cell body is outlined by dashed lines, which was done when the GFP intensity was temporarily enhanced with the Photoshop levels tool. (A′, B′) Images of ceh-36p::myr-TagRFP and str-2p::2Xnls-TagRFP. AWC^ON was identified as str-2p::2Xnls-TagRFP positive and ceh-36p::myr-TagRFP positive (A′). AWC^OFF was identified as str-2p::2Xnls-TagRFP negative and ceh-36p::myr-TagRFP positive (B′). (A″) Merge of A and A′ images from the same cell. (B″) Merge of B and B′ images from the same cell. (C) Quantification of mir-71p::GFP expression in AWC^ON and AWC^OFF cells. (D, E) Images of odr-3p::GFP::tir-1 3′ UTR. (D′, E′) Images of odr-3p::2Xnls-TagRFP::unc-54 3′ UTR and str-2p::myr-mCherry. The AWC^ON cell was identified as str-2p::myr-mCherry positive and odr-3p::2XTagRFP positive (D′). The AWC^OFF cell was defined as str-2p::myr-mCherry negative and odr-3p::2Xnls-TagRFP positive (E′). (D″) Merge of D and D′ images from the same cell. (E″) Merge of E and E′ images from the same cell. (F) Quantification of normalized GFP expression in AWC^ON and AWC^OFF cells. Normalized GFP expression was determined by calibrating GFP intensity with 2Xnls-TagRFP intensity of the same cell. All constructs, except for odr-3p::GFP::tir-1 3′ UTR, contain the unc-54 3′ UTR. All images were taken from first stage larvae. The single focal plane with the brightest fluorescence in each AWC was selected from the acquired image stack and measured for fluorescence intensity. Each animal was categorized into one of three categories: AWC^ON = AWC^OFF, AWC^ON>AWC^OFF, and AWC^OFF>AWC^ON based on the comparison of GFP intensities between AWC^ON and AWC^OFF cells of the same animal. We did not observe any animals that fell into the “AWC^ON = AWC^OFF” category from our GFP intensity analysis. Total number of animals for each category was tabulated and analyzed as described [86]. p-values were calculated using X ² test. Error bars represent standard error of proportion. Arrows indicate the AWC cell bodies. Arrowheads represent myr-TagRFP or myr-mCherry signal. Scale bar, 2 µm.