NC expression of AP2α depends on Wnt signaling. Whole-mount in situ hybridizations with NC markers Sox9, Slug, and AP2α to embryos injected into one cell at the two-cell stage with 100 pg of plasmid DNA encoding Xenopus Xwnt-1 (Left) and Xwnt-3A (Center), or 1 ng RNA encoding GSK3β (Right), along with a β-galactosidase lineage tracer (red staining). In all cases the injected side is oriented to the right (red staining). Both Wnt treatments resulted in lateral and posterior expansion of NC territory, including expression of AP2α as well as the other NC markers. The reciprocal treatment with GSK3β, which interferes with downstream Wnt signaling (50), had the opposite effect: AP2α, Sox9 and Slug all were repressed. (Magnification: ×15.)

Ectopic expression of AP2α converts neural plate into NC. Whole-mount in situ hybridization of probes for Slug, Sox9 (NC), and Sox2 (neural plate) to embryos injected at the 16-cell stage with 100 pg of RNA encoding AP2α, along with β-galactosidase lineage tracer RNA (5-bromo-4-chloro-3-indolyl-β-d-galactoside, turquoise staining). Discrete domains of intense ectopic expression of both NC marker genes was observed (arrows) within the neural plate. Endogenous Slug and Sox9 expression is indicated by the arrowheads. Concomitant repression of neural plate identity also occurred, as indicated by the reduction in Sox2 expression (arrow). (Magnification: ×22.)

A model for NC induction. In the ectoderm, BMP signaling initiates and supports the expression of epidermal AP2α, at a basal level, and NC inhibitors. Because BMP signaling is attenuated by antagonists (Chd), this sequentially results in down-regulation of two NC inhibitors, the first of which may be the homeodomain gene Dlx3. With the first inhibitor removed, Wnt signaling results in up-regulation of AP2α, and, after silencing of the second inhibitor, induction of other factors such as Slug and Sox9. Slug reinforces the repression of the NC inhibitors, whereas Sox9 and AP2α positively regulate each other and Slug. All three factors are required for full induction and/or maintenance of NC. See Discussion for details.

Comparison of neurula stage expression of AP2α and other NC marker genes. (A) Whole-mount in situ hybridization with probes for AP2α, Slug, and Sox9 are shown in dorsal and lateral views. AP2α transcripts appear to extend more laterally than for the other two genes. This is shown more clearly in cross section in B, where the bracket indicates the Slug domain and the arrows point to the more extensive lateral AP2α. Note that under the conditions used here the epidermal expression of AP2α, which is considerably lower than for NC, is not apparent. The notochord is indicated by n. (Magnifications: A, ×7; B, ×42.)

Loss of function by MO antisense injections. (A) In vitro-coupled transcription/translation reactions with plasmid encoding the AP2α ORF in the presence of either control (Co-mo) or perfect-match AP2α MO (AP2-mo; 50 ng; see Materials and Methods for sequences). (B) Neurula stage embryos injected into one cell at the two-cell stage with 30 ng of AP2α MO or the control MO along with β-galactosidase mRNA as a lineage tracer and probed for expression of NC markers Slug and Sox9 and the neural plate marker Sox2. The injected sides are on the right in these dorsal views (reddish β-galactosidase staining). The AP2α MO reduces NC and slightly expands neural plate laterally. (C) AP2α-MO inhibits Slug expression in a dose-dependent manner (●). Values in parentheses indicate the number of embryos analyzed for each concentration of injected MO. Injection of 30 ng of a control MO had a minor effect on Slug expression (■). (D) Cross section of AP2α MO-injected embryo showing reduction in Slug. The injected side is to the right and the uninjected side is to the left (arrow). This also shows the reduced elevation of neural fold after AP2α MO injection. (Magnifications: B, ×7; D, ×42.)

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.