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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 |
