(a) Position of let-7 binding sites in the 3’-UTRs of neural crest genes assessed in the reporter assay. Red boxes correspond to regions in the UTRs that are complementary to the let-7 seed sequence, while the blue boxes correspond to let-7 compensatory sites in the UTR, which are complementary to a region of the miRNA other than the seed sequence. Direct let-7 targets (Pax7, FoxD3, Myc) have seed sequence complementarity and, in the case of FoxD3, a compensatory binding site. These are absent in the UTRs of Sox10, Zic1, and Sox8. (b–f) Representative scatter plots of Sox10, Pax7, Sox8, Zic1 and cMyc UTR-reporter assay, showing the mCherry/GFP intensity ratio of each cell analyzed from the control (gene-UTR) and let-7a mimic transfected (gene-UTR +let7 GOF) halves of the same embryo. Each dot represents a single cell, and the medians and the 99% confidence intervals are overlayed on the scatter plots. (g–j) Single cell measurement of FoxD3 protein and NC2 mCherry-PEST reporter construct fluorescence in migrating NC cells. Transverse section of HH12 embryos, showing FoxD3 protein immunostaining (g) and FoxD3-NC2 enhancer reporter construct (h) in NC cells. Dotted lines show the migrating NC cells. The farthest migrated cells expressing FoxD3-NC2 enhancer (lower dotted region on (h)) are not positively stained for FoxD3 immunostaining, indicating decreased protein in these cells (g) Boxplots quantifying the fluorescent intensity of FoxD3 protein (i) NC2-mCherry-PEST reporter construct (j) in single neural crest cells as a function of the distance of the cells from the DNT. ‘NEAR’, ‘MID’ and ‘FAR’ corresponds to cells within 0–200 a.u, 201–350 a.u and 350–600 a.u from the DNT. (k) Bilateral electroporations of control vs. targeted Cas9 expression vectors were used to disrupt individual let-7 sites in the UTRs of the neural crest genes. UTR- Un-Translated Region, DNT- Dorsal Neural Tube, nt: neural tube, nc: neural crest, a.u- Arbitrary Units (as measured using ImageJ).