Fig. 6. Ectopic application of BMP2 to forebrain explants. (A-D) Expression of Msx1 in cultured cephalic explants. Protein-soaked beads (represented by asterisks) were placed between rostral neural folds of five- to seven-somite wild-type cephalic explants in type 1 explants (A-C) and on the midline in type 2 explants (D). (A) BMP2 induces strong Msx1 expression in the ANR (arrow) after 6 hours. Msx1 is induced at large distances from the BMP2 source. (B,C) Msx1 induction is transient and is diminished after 9 (B) and 12 (C) hours. (D) BMP2 induces Msx1 adjacent to the caudal but not the rostral half of the bead (arrowhead). However, Msx1 is induced in the ANR (arrow). (E,F) Expression of Fgf8 and Shh (two-probe in situ hybridization) in type 1 (E) and type 2 (F) explants. (E) BMP2 suppresses the expression of Fgf8 in the ANR (arrow) and Shh in the rostral ventral neural midline (arrowhead) in both explant types, whereas Fgf8 in the isthmic organizer (io) is not affected. BSA beads had no effect in any explants. (G,H) Hypersensitivity of Chrd;Nog mutant explants to BMP signaling. BMP2-soaked beads were placed adjacent to both rostral and lateral neural folds in type 1+ explants. (G) At a concentration of 10 µg/ml, Msx1 was induced adjacent to both beads in wild-type explants (left), but induction was more robust in Chrd^-/-;Nog^+/- explants (right). (H) At a concentration of 0.1 µg/ml, Msx1 was not induced in wild-type explants (left), but was induced in Chrd^-/-;Nog^+/- explants (right). anr, anterior neural ridge; rvnm, rostral ventral neural midline.