<p><b>A</b>. Fluorescent and transmitted light micrographs of late L1 larvae of the indicated genotypes, carrying SMAD::GFP (<i>cuIs5</i>) transgene. Bright fluorescence in the pharynx indicates DAF-7 activity. Animals were picked under transmitted light to provide an unbiased sample. Left panels correspond to boxed areas showing fluorescent pharynxes. <b>B</b>. Box-and-whisker plot of GFP intensity in animals of indicated genotypes (<i>n</i> = 1351, 770, and 118 animals, respectively; L1/L2 animals) carrying the SMAD::GFP transgene. Boxes show interquartile range, whiskers are 1-99<sup>th</sup> percentile. The outliers are shown as individual data points outside the whisker range. <b>C</b>. Development of <i>sa191</i> animals with bright <i>vs</i>. dim SMAD::GFP fluorescence. Decreased reporter activity correlates with stronger activation of dauer signaling (χ<sup>2</sup> = 38.81, df = 2). Animals were separated into dim and bright populations by eye using fluorescent stereo microscope. <b>D</b>. Over-expression of DAF-7 cDNA in the ASI neuron (ASI::DAF-7) (χ<sup>2</sup> = 81.71, df = 2) or expression of mCherry::DAF-7 fusion protein (χ<sup>2</sup> = 427.3, df = 2) rescues <i>sa191</i> dauer phenotype at 20°C. Since this is a non-integrated transgene, non-transgenic siblings (N-Tg sib.) were used as internal controls. 4xDel strain lacks four pro-growth IGF-like proteins, including DAF-28, INS-4 and INS-6. <b>E</b>. mCherry::DAF-7 fusion protein expressed from its cognate promoter rescues <i>daf-7(e1372)</i> loss-of-function mutation at 20°C. Non-transgenic siblings (N-Tg sib.) in the first generation only are also rescued. Data in B were analyzed by ANOVA followed by Bonferroni’s multiple comparison test, α = 0.05, ****<i>P</i><0.0001, ***<i>P</i><0.001. Data in C, D were analyzed by Chi-square test, α = 0.05, ****<i>P</i><0.0001.</p
