Figure 5

Tgif1 expression in Zic2 knockdown mice. (A) Total RNA was prepared from the head of E10.5 Zic2^+/+, Zic2^+/kd, and Zic2^kd/kd mouse embryos and subjected to RT-PCR analysis in the presence or absence of reverse transcriptase (RT⁺ or RT⁻, respectively). Specific primers were used for the detection of the indicated genes, and Zic2, Tgif1, Pai-1 (a direct target of Tgif1), Aldh1A2 (a downstream factor of Tgif1), Cyp26A1 (a downstream factor of Tgif1), and G3PDH (a housekeeping gene control). (B) Total protein extracts from E14.5 Zic2^+/+, Zic2^+/kd, and Zic2^kd/kd mouse head for western blotting analysis using the indicated antibodies. Tgif1 protein levels normalized to those of actin were 100% in Zic2^+/+, 74% in Zic2^+/kd, and 61% in Zic2^kd/kd in densitometric measurement of the blots. (C) In situ hybridization of E10.5 (a), whole-mount) and E11.5 (b–e), sections) Zic2^+/+ (left embryo in (a,b,d) and Zic2^kd/kd (right embryo in (a,c,e) mouse embryos. An antisense probe for Tgif1 was used in the experiment. Tgif1 expression was reduced in the diencephalon (asterisk in a,d’,d”) where the two genes are expressed in an overlapping manner at E9.5 (Fig. 4). The telencephalic hindbrain roof plate showed abnormal shape (#). Spina bifida-like anomaly existed in the caudal region (arrowheads), where also the Tgif1 expression was reduced.