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Figure 3. Overexpression of miR‐199 leads to defects in eye development. Unilateral (a–i) or bilateral (j) injections of miR‐199 were carried out to evaluate the effects of miR‐199 overexpression on eye phenotype and EFTF expression. (a–c) Embryo injected with miR‐199a‐3p in two left anterior dorsal blastomeres at the 16‐cell stage. Eye on injected side (a) is smaller than the eye on the uninjected side (b,c), and shows a coloboma. Injected sides are marked with asterisks. (d–h) In situ hybridizations of selected EFTFs (rax, optx2, tbx3, pax6), and otx2. With the exception of pax6 (h), all show restricted expression on the injected side. Scale bars approximately 200 μm. (i) Frequencies of small eye and coloboma phenotypes; n = 48 embryos from eight independent experiments. (j) Q‐RT‐PCR showing expression of EFTFs and otx2 in embryos following targeted overexpression of miR‐199 or MTT‐199. Embryos were injected at the 8‐cell stage as described and collected for RNA isolation and analysis at st. 18 (N = 3 independent experiments; *p < .05; unpaired Student's t test). EFTFs, eye field transcription factors; Q‐RT‐PCR, quantitative reverse‐transcriptase‐polymerase chain reaction |
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Figure 6. miR‐199 knockdown leads to defects in eye development. (a) Phenotypes of embryos injected with the LNA oligonucleotide inhibitor of miR‐199 (LNA‐199). An embryo injected with a control LNA sequence (ctrl LNA) is shown in (i); (ii)–(v) show the range of phenotypes resulting from LNA‐mediated knockdown of miR‐199. Scale bar, 250 μm. The percentages showing each phenotype are indicated. (N = 76 embryos across seven independent experiments). (b and c) Neural tubes isolated at st. 24 from embryos injected unilaterally with either the control (b) or the miR‐199 (c) LNA oligonucleotide. The injected side is marked with an asterisk. Scale bar, 250 μm. (N = 4 independent experiments). (d) Quantitative RT‐PCR showing expression of EFTFs and otx2 in st. 18 embryos following targeted knockdown of miR‐199. Values shown represent the mean ± S.E.M. of the fold difference between embryos injected with LNA‐199 and the control LNA. (N = 3 independent experiments; *p > .05; **p > .01; unpaired Student's t test). (e–g) In situ hybridization to show expression of the EFTFs rax (e), optx2 (f), and pax6 (g) in embryos injected unilaterally with LNA‐199. The injected side is marked with an asterisk. Scale bars, 100 μm. EFTFs, eye field transcription factors; LNA, locked nucleic acid |
