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Fig. 5. Functional knockdown of Oct-25 or Oct-60 expands the mesendodermal territory towards the upper equatorial region. (A) Immunoblotting using an Oct-25 antibody shows that Oct25MO specifically inhibits protein expression of Oct-25 in embryos. Lane 1, uninjected whole embryos at stage 10; lanes 2 and 3, embryos injected with 10 and 15 ng of ctrlMO; lanes 4 and 5, embryos injected with 10 and 15 ng of Oct25MO. Arrowhead indicates the band corresponding to Oct-25. (B) Immunoblotting using an antibody against the myc-tag shows that Oct60MOa1 specifically inhibits Oct-60MT protein expression. Lane 1, uninjected control animal caps at stage 10; lane 2, caps injected with 400 pg Oct-60MT RNA alone; lanes 3 and 4, caps co-injected with 400 pg Oct-60MT RNA and 20 or 40 ng of Oct60MOa1; lane 5, caps co-injected with Oct-60MT RNA and 40 ng of ctrlMO. (C) Posterior view of an embryo injected with ctrlMO showing normal development with fully closed blastopore at the end of gastrulation. (D–F) Embryos injected with Oct25MO (D), Oct60MOs (E), or a mixture of them (F) show similar defect in blastopore closure. (G) Whole mount in situ hybridization (vegetal view, lateral view, and tissue section) shows normal expression pattern for Xbra in embryos injected with ctrlMO. (H) In embryos injected with Oct25MO and Oct60MOs, the Xbra expression domain expands significantly towards the equatorial region. (I) Whole mount in situ hybridization (vegetal view, lateral view, and tissue section) shows normal expression pattern for Xsox17α in embryos injected with ctrlMO. (J) In embryos co-injected with Oct25MO and Oct60MOs, expression is enhanced and shifted towards the equatorial region. (K) Real-time RT-PCR shows significant upregulation of mesodermal genes and ectopic expression of endodermal genes in equatorial explants excised from embryos injected with Oct25MO and Oct60MOs. (L) Real-time RT-PCR shows downregulation of the neuroectodermal gene, Xsox2, in response to Oct-25 knockdown. |
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Fig. 1. Vegetally overexpressed Oct-25, Oct-60, Oct-91 or mOct-3/4 blocks mesendoderm formation. (A) An uninjected control embryo at stage 11.5 showing normal gastrulation. (B–E) Embryos injected with Oct-25 (B), Oct-60 (C), Oct-91 (D) and mOct-3/4 (E) show the same failure of blastopore formation and lack of gastrulation. (F) A control embryo at stage 10.5 shows normal expression of Xbra. (G–J) Embryos injected with Oct-25 (G), Oct-60 (H), Oct-91 (I) and mOct-3/4 (J) show nearly no or severely reduced expression of Xbra. (K) A control embryo at stage 10.5 shows normal expression of Xsox17α. (L–O) Embryos injected with Oct-25 (L), Oct-60 (M), Oct-91 (N) and mOct-3/4 (O) show nearly no or severely reduced expression of Xsox17α. All embryos above are shown in vegetal view. (P) Real-time RT-PCR shows that Oct-25, Oct-60 or mOct-3/4 injections at the vegetal pole significantly inhibit transcription of mesendodermal genes. |
