Experimental procedures were performed in accordance with UK Animals (Scientific Procedures) Act, 1986 (PPL 70/7194). For whole-mount embryo in situ hybridization, embryos were dissected, fixed overnight in 4% paraformaldehyde in phosphate-buffered saline, and dehydrated through graded methanol solutions. Non-radioactive RNA in situ hybridization was performed as described33 before vibratome sectioning. RNA probes for Zic134 and En135 were digoxygenin labeled with the In Vitro Transcription kit (Roche Applied Science) followed by anti-digoxygenin-AP antibody (1:1,000) (Roche Applied Science) and NBT/BCIP (Sigma) staining to detect the hybridization signals.