Hoescht stain

After enucleation, the lens and cornea were removed and each eye cup incubated in 4% PFA, incubated in a 30% sucrose solution before being frozen in OCT compound (VWR, Lutterworth, UK). Eyes were sectioned and dried before permeabilizing with 0.2% Triton-X 100. Slides were washed in PBS before blocking with 10% bovine serum albumin and 10% donkey serum. Slides were washed before primary antibody incubation (goat polyclonal, ABIN343052, AntibodiesOnline [Aachen Germany], used 1/200 in 1% bovine serum albumin, 1% serum) and secondary antibody incubation (donkey anti-goat 488, ab1050129 [Abcam, Cambridge, UK] used 1/400 in 1% bovine serum albumin and 1% serum). Hoescht stain was performed (Sigma-Aldrich, Gillingham, UK) (1/2,000 in PBS) before a final wash in PBS. ProLong Diamond antifade mounting medium (Life Technologies Ltd, Paisley, UK) was applied and slides were sealed before imaging.

In situ hybridization was carried out on whole-mount embryos according to Moustakas (2008) and on paraffin-embedded sections according to Albrecht (‘97) . Digoxigenin- or ³⁵S-labeled riboprobes were generated from linearized plasmids using T3 or T7 polymerase (Roche). For whole-mounts, mRNA expression was detected using alkaline phosphatase-coupled anti-digoxigenin antibody (Roche) and BM Purple (Roche). Turtle Bmp4 and Msx2 and chick Fgf8 probes were used with alligator embryos. Images of the radioactive in situ hybridization assays are the product of superimposing the pseudo-colored hybridization signal in Adobe Photoshop (Adobe, San Jose, CA) with a blue nuclear stain (Hoescht Stain, Sigma).