RNA expression profiling in zebrafish embryos and larvae. A, B) lin28b expression in WT embryos at 1dpf (A) and 2dpf (B). The embryos show lin28b expression restricted to specific organ primordia such as the fin bud (FB, arrowhead) and NPZ = neuronal proliferation zone (arrow). C) 7d-WT larvae brain labeled with lin28b anti-sense probe. The brain contains a defined network of lin28b expressing cells, including projections to hypothalamus (Hy, arrow). D & E, comparisons of lin28b expression in 3d mRNA and MO injected embryos, showing strong and weak expression in the midbrain-hindbrain boundary at NPZ (arrows), and in the eyes (arrowheads). F & G, gnrh3 expression at 3dpf in WT and MO embryos. Although some of the fish showed clearly smaller head and eye size, the gnrh3 expression remained comparable in all fish. H & I, kiss2 expression in WT and MO embryos. In line with the qPCR results, the MO injected fish showed a reduction in the intensity of kiss2 staining. Scale bars = 100 μm. J) Changes in RNA expression for lin28b, notch1a, gnrh3 and kiss2 in lin28b MO or synthetic mRNA injected vs. WT and Rescue fish (combined injection of lin28b MO and mRNA) during the first five days of development. Over the five-day period, the MO injected embryos showed reduced and mRNA injected embryos increased expression of lin28b compared to WT (P = 0.00039 and P = 0.0042). No significant differences were observed in the expression of notch1a and gnrh3, however kiss2 mRNA levels were significantly reduced in the MO group (P = 0.033, Welch ANOVA with Games-Howell post-hoc test, error bars = SEM). N = 12–20. K) Representative images of 3-day old zebrafish, including fish injected with lin28b-blocking morpholino and synthetic lin28b mRNA. In all injected groups, most fish receiving injections as embryos showed no gross phenotypic abnormalities. Control injections shown here included phenol red and H20 only. Scale bars = 1 mm.
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