Figure 3. Knockdown of endogenous cellular chordin expression.

(A) Diagram of mir- shRNA^{chordin-3′UTR-1} and mir-shRNA^{chordin-3′UTR-2} against the 3′UTR of chordin gene, whose predicted secondary structure was shown at the bottom. Red brackets denoted the targeted regions. (B) Free-energy of the targeted regions and corresponding flanking sequence predicted with mFold software. (C) Phenotypes of chordin-deficient embryos. WISH analysis of chordin expression in the 6 hpf embryos injected with 200 pg of shRNA^EGFP-ORF (control), shRNA^{chordin-3′UTR-1} or shRNA^{chordin-3′UTR-2} mRNAs using a dig-labeled full-length 3′UTR of chordin as probe (upper panels). A significantly enlarged ICM (black arrowhead) with a partial loss of neural tissues (white arrow, 61/99) were observed in 24 hpf embryos injected with only shRNA^{chordin-3′UTR-1}, but not with shRNA^{chordin-3′UTR-2} and control (middle panels). A higher level of gata-1 expression was also detected in the shRNA^{chordin-3′UTR-1}-injected embryos, compared to control or shRNA^{chordin-3′UTR-2}-injetced embryos (bottom panels). (D) Morphology of embryos injected with chordin morpholino oligonucleotides and its 4-base pair mismatch control. Embryos at 6 hpf are dorsal view, and embryos at 24 hpf are lateral views with head to the left.