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Fig. 2 Ocular defects induced by irx2a morpholinos. A: Schematic diagram showing the morpholino MO1 and morpholino MO2 targeted region in the irx2a pre-mRNA. MO1 blocked the translation of Irx2a by targeting the 52 UTR region of irx2a mRNA, whereas MO2 blocked the splicing of irx2a pre-mRNA by targeting the intron1-exon2 splice junction. B: RT-PCR analysis using irx1a- and irx2a-specific primers demonstrated a reduction of irx1a and irx2a transcripts in irx1a MO2- and irx2a MO2-injected embryos, respectively, at 48 and 96 hpf. Beta-actin was used as an internal control. irx2a transcript levels were reduced in irx1a MO2 morphants, suggesting that irx2a is a downstream target of irx1a. No difference in the abundance of irx1a transcripts was found in irx2a MO2 morphants, indicating that the irx2a MO2 was specific to irx2a. C–F: Embryos were raised in a bright environment until 96 hpf. As a result of visual impairment, irx2a MO2 morphants showed hyperpigmentation and smaller eye than the control morphants (E and F). The area of the eye was digitally measured (marked by the white dotted line in E) for each embryo.