Fig. 1

miRNAs Predicted to Bind to Right-Handed EMT-TFs 3′ UTRs Are Asymmetrically Expressed in the Same Time Window than prrx1a in Zebrafish Embryos and Are Able to Downregulate Reporter Expression (A) Strategy to obtain vertebrate-conserved miRNAs able to regulate right-handed pathway EMT-TFs. A diagram of the species used is shown, with each EMT-TFs and the developmental time point where their asymmetric expression is evident by in situ hybridization. (L) Left; (R) Right; N = number of miRNAs selected (see also Figure S1A). prrx1a expression at the 20-somite stage, showing L/R asymmetric levels. The diagram below exemplifies the measurement of area and width of the ISH signal used in this manuscript. NC, Neural Crest positive signal was omitted for area calculation; a, resulting left and right areas measured; w, width of the signal indicated by arrows used for left and right LPM. (B) Luciferase reporter assay shows interaction of miR-34a, miR-92a, miR-125a, and miR-184 with zebrafish prrx1a 3′ UTR. miR-135b is not able to downregulate reporter expression. Data represent mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ns = not significant, two-sided Student’s t test WT seed versus seed mut, n = 4 independent experiments. (C) Four pre-miRNAs show higher expression on the left half at the 20-somite stage. Zebrafish embryos were collected and dissected right and left halves were pooled for total RNA isolation. qPCR for the indicated pre-miRNAs was performed and relative amounts were normalized to the left halves. Data represent mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, two-sided Student’s t test left versus right halves, n = 4 independent pools of 8 halves per pool. (D) In situ hybridization for mature miRNAs in 20-somite stage zebrafish embryos show higher levels of three miRNAs in the left LPM (LLPM) compared to the right LPM (RLPM). Zebrafish embryos were collected at 20-somite stage and cryosections of 10 μm were subjected to ISH using LNA double-DIG specific probes. Left (L) and right (R) sides are indicated on the picture. White box insets are magnified for comparison of LLPM versus RLPM. The asymmetric expression for miR-184 is apparent in the somitic mesoderm (som, yellow arrowhead) but not in the LPM (white box insets, read arrowheads). Detection of nuclei with DAPI (right panels) allows the localization of the different territories, LPM, som and neural tube. Scale bar: 250 μm. (E) pre-miRNAs asymmetric expression is transient. We collected 6 independent pools (8 embryo halves per pool), performed qPCR for each developmental stage (12- or 20-somite stages and 24 h post-fertilization), and calculated the R/L ratio of the normalized (to left halves) levels for prrx1a, pre-miR-34a, pre-miR-92a, and pre-miR-125a. We used spw levels as an internal control (Hirokawa et al., 2006). A value of 1 (dotted line) indicates bilaterally symmetric expression. Note that prrx1a and pre-miRNAs R/L ratio deviates from 1 only at 20-somite stage. Data represent mean of R/L ratio ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-sided Student’s t test left versus right halves, n = 6 independent pools of 8 halves/pool. (F) Zebrafish embryos were injected with synthetic miRNAs or with Cas9 alone (CRISPR-Ctrl) or with specific sgRNAs targeting miRNA sequences. Embryos collected at 20-somite stage were subjected to ISH for spw expression and its laterality quantified. Only miR-125a knockout affects the normal spw expression pattern. Scale bar: 250 μm. See also Figure S1.