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Fig. 3 Specific Morpholinos Deplete the Embryo of miR-375 (A) Sequence alignment of the two miR-375 genes from zebrafish and design of morpholinos targeting the dre-miR-375–1 and dre-miR-375–2 precursors. (B) Northern blot analysis of the effect of morpholinos on the expression of miR-375 derived from injected pri-miRNA mRNAs for miR-375–1 and miR-375–2. MO-375–1 overlap loop and loop morpholinos target exclusively the pri-miR-375–1 construct, and MO-375–2 overlap loop and loop morpholinos target exclusively the pri-miR-375–2 construct. Co-injected miR-206 serves as a loading and injection control. Embryos were collected 8 h after injection. (C) Northern blot analysis of the effect of morpholinos on endogenous miR-375 expression at 24 hpf and 48 hpf. MiR-206 serves as loading control. (D) In situ hybridization for pri-miR-375–1 and pri-miR-375–2 on wild-type (WT) and morpholino-injected embryos. Arrowheads indicate the pituitary gland and the pancreatic islet. (E) Analysis of GFP expression in 24-h embryos injected with a miR-375 GFP sensor construct, a synthetic miR-375 duplex and MO miR-375. Red fluorescent protein (RFP) serves as an injection control. NIC, noninjected control.