To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR‐dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc‐tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3′UTRs identified miR‐199a‐3p (“miR‐199”) as a candidate regulator of EFTFs, and miR‐199 was shown to regulate rax1 in vivo. Targeted overexpression of miR‐199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir‐199 and the rax1 3′UTR reversed the small eye phenotype. Although targeted knockdown of miR‐199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR‐199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR‐199 knockdown. Our results indicate that miR‐199 plays both positive and negative regulatory roles in eye development.

中文翻译：

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miR-199在非洲爪蟾眼睛发育中同时发挥积极和消极的调节作用

为了研究microRNA（miR）在早期眼睛发育中的功能，我们询问眼野转录因子（EFTF）是否是非洲爪蟾胚胎中miR依赖性调控的靶标。在rax1启动子的控制下，从表达myc标签的AGO的转基因胚胎中共免疫沉淀了Argonaute（AGO）的核糖核蛋白复合物，包括miR和靶向的mRNA 。神经末期与Ago共免疫沉淀的所有EFTF的mRNA。对EFTF 3'UTR中miR结合位点的计算预测表明，miR-199a-3p（“ miR-199”）是EFTF的候选调节物，并且miR-199被证明可调节rax1。体内。有针对性的miR-199过表达导致小眼睛，EFTF表达减少和细胞增殖减少。抑制mir-199与rax1 3'UTR之间的相互作用可逆转小眼表型。尽管有针对性的敲低miR-199可使眼球完整无损，但它减少了视杯的生长并破坏了眼球的形成。通过对Xenopus转录组内miR-199候选靶标的计算鉴定，鉴定出ptk7为候选调控子。有针对性的ptk7过表达导致异常的视杯形成以及眼睛发育的减少或丧失，从而概括了miR-199敲除后看到的眼表型范围。我们的结果表明，miR-199在眼睛发育中同时发挥积极和消极的调节作用。