Fig. 3. Specification of `area a' cells occurs after HH stage 4. (A) Left-hand panel: schematic of HH stage 4 embryo, showing `area a' (red) and Hensen's node (blue). Box indicates `area a' dissection. Right hand panel: whole-mount HH stage 4 embryo, showing Shh expression within Hensen's node (HN, white arrowhead) and anterior primitive streak. (B-E) Whole-mount views of HH stage 4 `area a' explants cultured for 40 hours and analysed for Shh, Hnf3b/Foxa2, Netrin1 and Sox2 expression. No expression of floor-plate markers is detected (B-D), but Sox2 expression (E) reveals that `area a' cells are specified as neural. (F-K) Midline neural explants isolated anterior to Hensen's node, in the region equivalent to `area a' and cultured in isolation for 24 hours. Explants taken from embryos at HH stage 4+ (F,G), HH stage 5 (H,I) and HH stage 6 (J,K) were processed for immunohistochemistry to determine expression of the floor-plate markers HNF3ß (F,H,J) and Shh (G,I,K). All panels show representative images of the results obtained and for each stage analysed show serial adjacent sections.