Fig. 5. Wnt1 is required for ectopic induction of mDA neurons by Fgf8 and Shh. (A) In situ hybridization with Th probe (dark blue) on E8.0-8.5 wild-type (Wnt1^+/+ and ^+/-; n=20/28) and Wnt1^-/- (n=8/9) mouse anterior neural plate explants 6 days after implantation of Fgf8b-coated beads (arrowheads) close to the ventral midline of the presumptive forebrain. No induction of Th-positive cells was seen after implantation of BSA-coated (control) beads (data not shown; n=8/9 for wild-type and n=4/5 for mutant explants). (B) In situ hybridization of wild-type E8.0-8.5 mouse anterior neural plate explants 24 hours after implantation of Fgf8b-coated beads with Foxg1b (light blue) and Shh (red) to confirm the forebrain and ventral identity of the tissue where the bead was implanted (arrowhead). No Wnt1 expression (dark blue) was detected in the tissue surrounding the bead located outside the endogenous Wnt1 domain (arrowhead). However, expression of Wnt1 was maintained at a distance from the bead when compared with the contralateral control side of the explant (broken line coincides with the ventral midline). Repression of endogenous Wnt1 around another Fgf8b-coated bead located in the presumptive dorsal midbrain is apparent (arrow). (C) RT-PCR for Th, Pitx3, Nr4a2, Aldh1a1 and Wnt1 on total RNA from pooled wild-type and Wnt1^-/- explants cultured under the same conditions. Detection of ubiquitously expressed glyceraldehyde-3-phosphate dehydrogenase (Gapd) was used as control. Expression of Nr4a2 and Aldh1a1 is not restricted to mDA neurons and was therefore detected in wild-type as well as in Wnt1^-/- explants.