Curling down phenotypes in zebrafish embryos. (A–C) Overexpression of miR-214 results in ventrally curved embryos at 2 dpf, a phenotype that mimics the effect of injection of antisense morpholino oligonucleotides against dispatched homolog 2 (disp2^MO). A wild-type, uninjected embryo at 2 dpf is shown in (A) (UIC). (D) The 3′ UTR of disp2 contains three predicted MREs for miR-214.

Rescue of the curling down phenotype by miR-214. Single-cell zebrafish embryos, either uninjected (UIC; A) or injected with miR-214 RNA (B), antisense morpholino oligonucleotides against disp2 (disp2^MO; E), disp2 mRNA (D), or a combination of miR-214 and disp2 (C) were allowed to develop for 48 h before examination and quantitation (F) of the fraction exhibiting a curling down phenotype. The number of embryos exhibiting the curling down phenotype for miR-214 and disp2 co-injection compared to miR-214 injection alone was analyzed using Student's t-test (P < 0.01, n > 3).

Deletion analysis of disp2 MRE function. (A) As in Figure 2, GFP reporters were constructed that contain the indicated 3′ UTR sequences. (B and C) mRNAs derived from the reporters in (A) were injected into single-cell embryos in the presence or absence of co-injection of miR-214. Western blots of embryo lysates were performed with antibodies against GFP and the level of GFP was quantitated as above. Relative GFP levels are shown (± SEM) with asterisks representing significant differences between the control GFP levels and the indicated constructs as follows: P < 0.001 for constructs C and D1, P < 0.01 for constructs D2 and D3, P < 0.05 for construct D12 by Student's t-test, n > 3.

Genetic interaction between disp2 and miR-214. Whole-mount immunostaining of zebrafish embryos was performed using antibodies against the neural marker Prox1 (red) and the midbrain hindbrain boundary marker Engrailed (green). Embryos were positioned dorsal to the top, anterior to the left. Single-cell embryos were either uninjected (UIC; A) or injected with miR-214 (B), the combination of miR-214 and disp2 mRNA (C), disp2 mRNA (D) or disp2^MO (E). The relative number of Prox1 positive cells in the hindbrain compared to that in UIC was graphed in (F). Significant differences were observed between UIC and miR-214 injected embryos (P < 0.001), between UIC and disp2^MO injected embryos (P < 0.001) and between embryos injected with miR-214 alone and co-injected with miR-214 and disp2 mRNA (p < 0.05) by Student's t-test. In all cases, n > 3.

Combinatorial action of weak MREs. mRNAs encoding GFP reporters containing the complete disp2 3′ UTR sequence were injected into zebrafish embryos along with target protectors against the three disp2 MREs (TP1,2,3) in the presence or absence of exogenous miR-214. Single-cell embryos injected with all three target protectors were injected and examined for fluorescence at 1 dpf (A–D). Western blots of embryo lysates isolated from embryos injected either with all three target protectors or combinations thereof were performed with antibodies against GFP (E and G). Relative GFP levels were quantitated as above and values plotted (± SEM) with asterisks representing significant decreases between the GFP reporter alone and the indicated co-injections (F and H). Significance was analyzed using Student's t-test (P < 0.001 for construct C and miR-214 co-injection, P < 0.05 for construct C, miR-214, TP1 co-injection and construct C, miR-214, TP2,3 co-injection, n > 3).

The disp2 is targeted by miR-214. (A) GFP reporters were constructed that contain the indicated 3′ UTR sequences from disp2 (C, Δ3, Δ5), a synthetic 3′ UTR that contains two perfect pairing sites for miR-214 (2MRE), or the normal GFP 3′ UTR sequence (GFP). The predicted MREs for miR-214 are indicated by the colored rectangles. (B–K) mRNAs derived from the reporters in (A) were injected into single-cell embryos in the presence or absence of co-injection of miR-214. Fluorescence was examined at 1 dpf in living embryos. (L and M) Western blots of lysates from embryos injected as in (B–K) were performed with antibodies against GFP and the levels of GFP were quantitated as described in the Material and Methods section. Relative GFP levels (± SEM) were plotted with asterisks representing significant decreases between the control GFP construct and the indicated constructs. Significance was analyzed using Student's t-test (P < 0.001 for constructs 2MRE and C, P < 0.01 for construct Δ3; n > 3). (N) mRNAs encoding the complete disp2 3′ UTR fused to GFP were injected in the presence and absence of miR-20. Embryo lysates were prepared, and GFP levels were examined by western blot as above. (O–R) Single-cell embryos were injected as indicated in the presence or absence of antisense morpholino oligonucleotides against miR-214 (214^MO). Fluorescence was examined at 1 dpf. (S and T) Western blots of embryo lysates were performed and quantitated as above. Relative GFP levels (± SEM) were plotted with asterisks representing significant decreases between the GFP reporter alone and the indicated co-injections. Significance was analyzed using Student's t-test (P < 0.001, n > 3).