Zebrafish Dnd1 (drDnd1) Counteracts Inhibition of Nanos by miR-430
(A) One-cell-stage zebrafish embryos were injected with Dnd1 morpholino or control morpholino. RNA was extracted and subjected to quantitative RT-PCR analysis to compare endogenous levels of nanos1 and vasa to odc, and nanos to vasa.
(B) One-cell-stage zebrafish embryos were coinjected with DsRed-nos1-3′UTR and gfp-vasa-3′UTR together with dead end morpholino or control morpholino.
(C) One-cell-stage zebrafish embryos were coinjected with RNA containing the venus open-reading frame fused to the wild-type nanos1 3′UTR (3′nos1wt), RNA containing the cfp open-reading frame fused to the miR-430-binding site mutated nanos1 3′UTR (cfp-3′nos1mut1) and vasa-dsRed (for labeling the germinal granule for easier identification of germ cells) together with dead end morpholino or control morpholino. Error bars depict the standard error of the mean (SEM); the p value was calculated using t test.

URRs Are Required for Dnd1 to Efficiently Repress miR-430-Mediated Nanos Inhibition
(A) RNA containing the DsRed open-reading frame fused to the wild-type nanos1 3′UTR (3′nos1wt) was coinjected into one-cell-stage zebrafish embryos together with RNA containing the gfp open-reading frame fused to different versions of the nanos1 3′UTR. The different nanos1 3′UTRs that were used are shown above; mutations are marked in red. The ratio between the signal intensity provided by GFP whose open-reading frame was fused to either one of the nanos UTRs was divided by that originating from DsRed that was fused to the wild-type nanos RNA UTR. Representative single cells are shown in the right panels.
(B) An experimental setting similar to that described in (A) was used to examine the function of nanos UTR containing a combination of mutations in the miR-430 and putative Dnd-binding sites. The different nanos1 3′UTRs that were used are shown above; mutations are marked in red. The ratio between the signal intensity provided dividing the signal intensity of GFP by that of DsRed whose open-reading frame was fused to the wild-type nanos RNA UTR. Representative single cells are shown in the right panels. Error bars depict the standard error of the mean (SEM); p value was calculated using Ttest.

Zebrafish DND1 (drDND1) counteracts inhibition of TDRD7 by miR-430 through binding to adjacent URRs.
(A) One-cell-stage zebrafish embryos were injected with dead end morpholino or control morpholino. RNA was extracted and subjected to Q- RTPCR analysis to compare endogenous levels of TDRD7 and Vasa to Odc, and TDRD7 to Vasa.
(B) One-cell-stage zebrafish embryos were co-injected with DsRed-TDRD7-3′UTR (3′TDRD7) and gfp-vasa-3′UTR (3′vasa) together with dead end morpholino or control morpholino.
(C) One-cell-stage zebrafish embryos were co-injected with RNA containing the yfp open reading frame fused to the wild-type TDRD7 3′ UTR (3′TDRD7wt), RNA containing the cfp open reading frame fused to the miR-430 binding site mutated TDRD7 3′ UTR (cfp- 3′TDRD7mut1) and vasa-dsRed (for labelling the germinal granule for easier identification of germ cells) together with dead end morpholino or control morpholino.
(D) RNA containing the DsRed open reading frame fused to the wild-type nanos-1 3′ UTR (3′nos1wt) was co-injected into one-cell-stage zebrafish embryos together with RNA containing the gfp open reading frame fused to different versions of the TDRD7-1 3′ UTR. The different TDRD7 3′ UTR that were used were: wild-type TDRD7 3′UTR (3′TDRD7wt) or TDRD7 3′UTR where the putative DND1 interaction sequence was mutated (3′TDRD7mut2). Error bars depict the standard error of the mean (SEM), p-value was calculated using Ttest.