Fig. 5. Neural induction is suppressed in FRL-1MO-injected embryos. (A,B) FRL-1MO or FRL-1-4misMO was injected into animal blastomeres of 8-cell-stage embryos, which were fixed at stage 30. (A) Embryo injected with 10 ng of FRL-1-4misMO. Transcripts of otx2 were detected in the pineal gland and in the eye. (B) Embryo injected with 10 ng of FRL-1MO, showing no expression of otx2, confirming the loss of anterior neural tissue. (C-F) FRL-1MO, FRL-1-4mis-MO or FRL-1MO-second was injected into animal blastomeres of 8-cell-stage embryos, which were fixed at stage 30 for whole-mount in situ immunohistology analysis using Neu-1. (C) Uninjected embryo. (D) Embryo injected with 15 ng of FRL-1MO. (E) Embryo injected with 40 ng of FRL-1-4misMO. (F) Embryo injected with 40 ng of FRL-1MO-second. (G.H) Histological analysis shows that the embryos injected with FRL-1MO had neural tissue defects without loss of mesodermal tissue, such as notochord and pronephric tube. nc; notochord, pt; pronephric tube, sc; spinal cord. FRL-1MO or FRL-1-4misMO was injected into animal blastomeres of 8-cell-stage embryos, which were fixed at stage 40. (G) Histological section of 10 ng of FRL-1MO-injected embryos. (H) Histological section of embryo injected with 10 ng of FRL-1-4misMO.

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