Figure 3. lin-4 and its targets lin-14 and lin-28 are expressed reciprocally during larval development.

(A) In an integrated lin-4::GFP reporter strain, GFP is up-regulated in the HSNs during larval development. The y-axis depicts raw average pixel intensity values, and n = 10 for each time point. Note that the change in GFP expression levels between L1 and Adult stages exceeded the dynamic range of the camera, and at the set exposure time, only pixel intensity values for L1, L2, and L3 were always within the linear range. Some images acquired for late L4 and young adult stages were oversaturated, and thus the changes in pixel intensity after L3 are likely to be under-representations of the true fold increases in GFP expression. EL1–4: Early Larval Stage 1–4. LL1–4: Late Larval Stage 1–4. YAd: Young Adult. (B) GFP expression from an integrated lin-14::GFP reporter was down-regulated in the HSNs by the L2 stage, while in lin-4(lf) mutants, GFP expression was maintained. **p-values≤0.01 (0.005 for L2 and 0.003 for L3). *p-value = 0.015. (C) A lin-28::GFP reporter strain (Line 10-2) was down-regulated in the HSNs during L2 and L3 stages. When it was crossed into lin-4(lf), GFP expression persisted throughout larval development. *p-value = 0.012. ***p-values<0.001 (2.5×10⁻⁵ for L3 and 6.3×10⁻⁷ for L4). For B and C, n≥10 for each time point. Note that for each strain, pixel intensity values for L2, L3, and L4 were normalized to average L1 values and do not represent absolute fluorescence intensities. In addition, the lin-14::GFP strain expressed GFP more weakly than the lin-28 reporter during the L1, and thus a much smaller decrease in relative pixel intensity was required for complete down-regulation of the lin-14::GFP transgene. p-values for differences in relative pixel intensity were obtained using the two-sample t-test. In (A–C), all error bars represent standard error of the mean (S.E.M.). (D) Representative images of HSNs from L1 (top) and L4 (bottom) animals bearing lin-4, lin-14, or lin-28 GFP reporters in wild-type or lin-4(lf) backgrounds as noted. Arrowhead: HSN. Scale bars represent 5 µm, and anterior is to the left and ventral is down.