Figure 5. Axud1 acts as a Wnt effector during neural crest specification.

(A) Immunostaining of transverse sections of HH9 embryos revealed Wnt1 expression in the dorsal neural tube. DAPI staining was used to visualize cell nuclei. (B) In situ hybridization showed that Axud1 expression lies within the Wnt1 positive domain (dashed outline). (C-D) Whole mount dorsal views of embryo in which control morpholino was electroporated on the left (blue) and Wnt1 morpholino or β-Catenin morpholino was electroporated onto the right side of the same embryo. (F,G) The same embryos as above, showing effects on expression of Axud1. Inactivation of Wnt signaling results in downregulation (downward arrow) of Axud1 expression. (E,H) Conversely, over-expression of Wnt1 (Wnt1 O/E) results in elevation of Axud1 levels in the neural folds (upward arrow). (I, J) Whole mount views of an embryo with control morpholino on the left and Wnt1 morpholino on the right. Knockdown of Wnt1 resulted in downregulation of FoxD3 expression. (K, L) Whole mount views of an embryo with control morpholino on the left and Wnt1 morpholino plus Axud1 expression vector on the right, which was able to rescue FoxD3 expression to near-normal levels (compare J with L; see also Fig. S3). (M-N) Quantitation of embryos following loss of function and rescue assays. The purple bar in (M) denotes enhancement of Axud1 expression following Wtn1 overexpression, while the blue and light blue bars represent strong or mild downregulation following loss-of-function assays. Error bars in E and G represent standard deviation. HH: Hamburger and Hamilton developmental stages. β-CatMo: β-Catenin morpholino, Wnt1DN: Wnt1 dominant negative construct, Wnt1Mo: Wnt1 morpholino, Wnt1 O/E: Wnt1 overexpression.