Multiple alignment of nucleotide sequences of miR-92a and miR-92b, and their target sites in gata5 of Japanese flounder. (A) The cDNA sequence of gata5 is shown and its coding sequence is represented in upper-case letters and the flanking non-coding sequences in lower-case letters; the amino acids are exhibited below the coding sequence region, and the asterisk marks the stop codon. MREs in the 3′ UTR are underlined. (B) The seed regions of miR-92a and miR-92b are underlined, and identical bases in different species are shown by dashes (–). pol: Paralichthys olivaceus; ola: Oryzias latipes; dre: Danio rerio; ccr: Cyprinus carpio; bbe: Branchiostoma belcheri; xtr: Xenopus tropicalis; mmu: Mus musculus; hsa: Homo sapiens. (C) The mCherry reporter fused to the 3′ UTR of gata5. Base pairing between miR-92 and the two MREs is shown.

gata5 is a target gene of miR-92. The expression of mCherry was observed as red fluorescence at 12 h after injection of one-cell zygotes of zebrafish. Sample numbers for each group were about 100 zygotes. (A–H) Embryos were injected with reporters containing either the full-length gata5 3′ UTR (full) or a construct in which MRE(s) were deleted (D1: MRE 1 deleted; D2: MRE 2 deleted; D12: MRE 1 and MRE 2 deleted). (A–D) Reporter plasmids were injected alone. (E–H) Reporter plasmids were co-injected with miR-92. (I) The proportion of fluorescent embryos in (A–D). (J) The proportion of fluorescent embryos in (E–H). Fluorescence-positive and -negative embryos are depicted in red and blue, respectively.

Effects on larval phenotype after overexpression and inhibition of expression of miR-92. Representative tailbud-stage embryos of untreated (A), miR-92 overexpression (B, C) and miR-92 inhibition (D–F) groups. miR-92-overexpressing embryos (B, C) showed an intumescent body with sinuous spine compared to untreated embryos (A), while inhibition of miR-92 (D–F) resulted in an intumescent body and abnormal tail. Sample numbers for each group were about 30 zygotes.

Relative gene expression at the 50% epiboly and tailbud stages after overexpression and inhibition expression of miR-92. (A, B) Relative expression of gata5 in the miR-92 group (A) and LNA-92 group (B). (C, D) Relative expression of sox17 in the miR-92 group (C) and LNA-92 group (D). (E, F) Relative expression of ntl in the miR-92 group (E) and LNA-92 group (F). All data are shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. Sample numbers for each group were about 30 zygotes.

Tailbud ISH analysis of ntl after overexpression and inhibition of miR-92. About 25 zygotes were treated for each group, and representative embryos are shown. (A) Untreated group. (B) LNA-92 injection group. (C) TE buffer injection group. (D) miR-92 injection group. The red arrow marks the region of ntl expression.

Expression profiles of miR-92a and miR-92b after treatment of larvae with TU. The time was calculated from larval hatching of the untreated group. 20 dph: pre-metamorphosis (metamorphosis of the larva has not begun); 22 dph: early metamorphosis (the right eye of the larva begins to move); 27 dph: mid-metamorphosis (the right eye of the larva has moved to the top of the head); 35 dph: the larva is settled. All data are shown as mean ± SEM (n = 3). **P < 0.01. Sample numbers for each group were about 500 zygotes.