Targeted deletion of miR-430 in zebrafish. (A) Genetic deletion of zebrafish miR-430 cluster using TALENs. A 79.8 kb genomic fragment containing the zebrafish miR-430 was deleted using two pairs of TALENs. The TALEN binding sites were shown in colors and the inserted nucleotides were shown in lower case letters. MiR-430 isoforms were indicated by colored short lines. (B)In situ hybridization detection of pri-mir-430 expression in the WT and miR-430^–/– mutant embryos at 4 hpf. (C) Expression profiles of mature miR-430 isoforms in the WT and miR-430^–/– mutant embryos (12 hpf) by small RNA deep sequencing. The numbers of normalized reads were indicated. TPM, transcripts per kilobase million.

Morphological defects in the miR-430-deficient embryos. (A–O) The morphology of WT and miR-430-deficient embryos. The phenotypes are fully penetrant. Formation of the germ ring (black arrows) and shield (white arrows) were delayed in the miR-430^–/– mutants (A,B,F,G). The yolk was not enclosed by epiboly in the mutant at 9 hpf (C,H). The extension of the anterior-posterior body axis was reduced in the miR-430 mutants (D,E,I,J). The miR-430 mutants had no brain vesicles and lacked heart tissue and circulation (K,M). Tail blisters were observed in the mutant at 48 hpf (L,N). The miR-430 mimics but not the mismatched miR-430 with 2-nucleotide alterations could rescue the defects observed in the miR-430 mutants (O,P). The ratios of observed phenotypes were indicated.

Marker gene analysis. Marker gene expression of the WT and miR-430^–/– mutant was analyzed at shield stage (A), 75% epiboly (B), 6-somite (C,D), and 36 hpf (E). Marker genes of axis formation (A), germ layer formation (B), organ progenitor formation, (C) neuronal pattern (D), and heart formation (E) were analyzed in the miR-430^–/– mutants. The ratios of the observed phenotypes were indicated. FB, forebrain; MHB, midbrain hindbrain boundary; OV, otic vesicle; OP, optic placode; R3, rhombomere 3; R5, rhombomere 5.

Transcriptome analysis. Total mRNAs of embryos from WT, miR-430^–/– mutant and rescued groups were collected at shield stage for transcriptome sequencing. (A) Identification of miR-430-regulated genes. (B)MiR-430 binding site analysis. The ratios indicate the numbers of genes with canonical sites of the indicated miRNA in their 3′UTR/total number of analyzed genes. The miRNA binding sites were predicted by TargetScanFish 6.2. (C) GO analysis of the miR-430 regulated genes, performed by the DAVID system. (D) The expression of lft2 in the transcriptome of the WT, miR-430^–/– and rescued embryos. (E) Q-PCR analysis of lft2 mRNA expression levels in the WT, miR-430^–/– and rescued group. Data are expressed as mean values ± S.E.M (n = 4). (F) MO knockdown of lft2 partially rescued the miR-430-deficient phenotypes. The embryos were collected at 75% epiboly and 36 hpf for WISH analysis of marker gene expression.

miR-430 is required for the degradation of maternally provided transcripts and activation of zygotic gene expression. (A) Schematic representation of the expression profiles of miR-430-regulated genes during MZT (64/128-cell stages, oblong and 50 epiboly). The miR-430-regulated genes were grouped into three categories based on their expression profiles. The expression profiles of genes in the control set (B), up-regulated set (C), and down-regulated set (D) of the miR-430 mutants during MZT.

A reciprocal regulatory loop for miR-430 in the clearance of maternal transcript. (A) The transcripts of Nanog, Dicer1, Dgcr8, and AGOs were maternally provided. (B) The maternally provided transcripts Nanog, Dicer1, Dgcr8, and AGOs were significantly increased in the miR-430^–/– mutants and down-regulated in the rescued embryos at shield stage. (C) WISH detection of Dicer1 transcripts in the WT and miR-430^–/– mutants. The Dicer1 transcripts were maternally provided and degraded at shield stage in the WT but not miR-430-deficient embryos. (D) Repression of Dicer1 by miR-430 thorough miR-430 binding sites in their 3′UTR. Two miR-430 binding sites (GCACTT) were identified in the 3′UTR of Dicer1. GFP sensors with Dicer-3′UTR-WT and Dicer-3′UTR-Mut (two-nucleotide mutations in the miR-430 binding sites) were injected into embryos and fluorescence was examined at 6 hpf. (E) A proposed reciprocal regulatory loop between maternally provided transcripts and miR-430.

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