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.
Keywords: Xenopus; ptk7; rax; eye field; microRNA.
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