miR-199 plays both positive and negative regulatory roles in Xenopus eye development

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.,© 2020 Wiley Periodicals, Inc.