Figure 5.

Differential control of NC factors by BMP signal attenuation. Shown is Northern blot analysis of RNA isolated from animal caps derived from embryos injected at the one-cell stage with a mixture of RNAs encoding Chd, Xwnt-3A, and AP2α. Uninjected (UI) embryo animal cap RNA and whole embryo (W) RNA are shown for comparison. Ethidium bromide staining of 18S ribosomal RNA is shown to confirm equal lane loading. All samples were cultured to stages 14 and 15. (A) Shallow gradient of Chd dosage ranging from 10 pg to 2.5 ng. AP2α up-regulation was elicited by as little as 10 pg of Chd RNA, whereas other NC markers (Sox9, Slug, and Xtwi) required more complete inhibition of BMP signaling (100–300 pg of Chd RNA). In all injected samples, the BMP-dependent homeobox gene Dlx3, and, to a lesser extent, Dlx5, was repressed, as were Sox2 and Otx2. (B) Steeper gradient of Chd dosage resulting in complete inhibition of BMP signaling. The results are similar to A, except the highest Chd dose (3 ng), which suppresses NC gene expression, restores the general neural plate marker Sox2 expression but not the anterior neural marker Otx2. The completeness of BMP signal inhibition can be judged by the reduction of Dlx5 transcripts to background levels. (C) Induction of NC gene expression in the absence of BMP signaling by AP2α. A very high Chd dose (4 ng) prevents BMP signaling, induces Sox2, and blocks NC induction as in B, whereas coinjection of 100 pg of AP2α RNA results in reactivation of NC markers and suppression of Sox2. The anterior neural plate marker Otx2 continues to be repressed under these conditions, presumably because of Wnt signaling.