Supplementary Figure 5: Extra-intestinal regulation of intestinal hrg-1.

(a) GFP expression in strain BW1946 (P_dbl−1::GFP). Arrowhead indicates head neurons. Arrow indicates tail neurons. ^∗ indicates body neuron. Scale bar = 100 μm (top panel); Scale bar = 20 μm (middle and lower panels). Images are representative of three independent experiments. (b) GFP quantification from IQ6011, IQ6311, and IQ6312 grown on OP50 supplemented with indicated heme concentrations. GFP fluorescence was quantified using COPAS BioSort. GFP is presented as relative expression on a scale from 1-100. Graph represents the mean and SEM of 50 worms per strain per heme concentration combined from three biological independent experiments. ^∗∗∗P < 0.001, ^∗∗P < 0.01, ^∗P < 0.05 compared to WT worms (two-way ANOVA). (c) GFP-DBL-1 expression in strain VC1478. GFP signal was detected in neurons and neuron support cells. “hn” indicates head neuron. “sc” indicates support cells. “cn” indicates ventral nerve cord neuron. Scale bar = 20 μm. Images are representative of three independent experiments. (d) wCherry expression in strain RW10745 (P_sma−9::HIS-24-wCherry). Arrow indicates hypodermal nuclei. Arrowhead and asterisks indicate intestinal and body neuron nuclei, respectively. Scale bar = 100 μm (top panel); Scale bar = 20 μm (middle and lower panels). Images are representative of three independent experiments. (e) GFP fluorescence quantified from IQ6011 expressing WT hrg-7 or hrg-7(tm6801) and fed dsRNA against vector, let-756, or snap-29. GFP was quantified using COPAS BioSort. GFP is presented as fold change compared to vector. Graph represents the mean and SEM of 1000 worms per treatment combined from four biological independent experiments. ^∗∗∗P < 0.001, ^∗∗P < 0.01, ^∗P < 0.05 (Dunnett’s test).