Fig. 7. Forebrain and FNP defects can be ‘rescued’ by local application of either all-trans RA, or recombinant FGF2 and SHH proteins. Embryos were exposed to RAR/RXR antagonists at stage 10 and subsequently treated 8-10 hours later with either RA, or with FGF2 and SHH. Control embryos were treated at stage 10 with DMSO-soaked beads or RAR/RXR antagonist-soaked beads, which were removed after 8-10 hours. (A,B) By stage 19, DMSO-treated control embryos (A) exhibit normal SHH expression in the FNP ectoderm (fn), maxillary process (mx), and hyoid arch (hy), whereas RAR/RXR antagonist-treated embryos (B) lose SHH expression in the FNP ectoderm (asterisk), fail to develop a forebrain, and have eyes (e) that approximate one another. SHH expression remains unaffected in the maxillary process (mx) and hyoid (hy) arch. This control demonstrates that RAR/RXR antagonists elicit their effects between stage 10 and stage 12, as the RAR/RXR antagonist-soaked beads had been removed after 8-10 hours. Embryos exposed to RAR/RXR antagonists at stage 10 had their beads removed after 8-10 hours and then were treated with either RA, or with FGF2 and SHH. (C) By stage 19, expression of SHH is restored in the forebrain (f) and FNP (fn; arrow; n=15). SHH expression in the maxillary process (mx) and hyoid arch (hy) is unaffected. (D) DMSO-treated control embryos exhibit normal FGF8 expression in FNP ectoderm adjacent to the nasal pits (arrow) whereas RAR/RXR antagonist-treated embryos (E) lose this domain of FGF8 expression and show a collapse of the facial midline (asterisk). FGF8 expression remains unaffected in the maxillary (mx) and mandibular (ma) processes. (F) Embryos exposed to RAR/RXR antagonists at stage 10 had their beads removed after 8-10 hours and then were treated with either RA, or with FGF2 and SHH. Expression of FGF8 is restored adjacent to the nasal pits (arrow; n=11). FGF8 expression in the maxillary (mx) and mandibular processes (ma) is unaffected. By stage 36, embryos rescued with RA (G) or with FGF2 and SHH (H) have a well-developed FNP and forebrain tissues (n=27). Scale bars: 2 mm in A-F; 1 mm in G,H.