Fig. 3. Inhibition of retinoid signaling with RAR/RXR antagonists causes a loss of FGF8 and SHH in the forebrain and FNP. Whole-mount in situ hybridization 48 hours after bead implantation at stage 10 demonstrates that control embryos (A) express FGF8 in the rostromedial forebrain, which appears as a midline horseshoe-shape (arrow), and in the ectoderm covering the FNP (fn), as well as in the maxillary (mx) and mandibular (ma) processes. (C) Treated embryos lose FGF8 in the forebrain and FNP ectoderm (asterisk; n=32). Note normal expression of FGF8 in the maxillary and mandibular processes. (B) Control embryos express SHH in the ectoderm of the FNP (fn; arrow) and forebrain (f), whereas treated embryos (D) show a downregulation of SHH (asterisk; n=35). Even at this early stage, the facial midline of treated embryos is hypoplastic, causing the eyes (e) to approximate one another. The lack of a forebrain is evidenced by a depression in the upper face. (E,F) Embryos treated with citral, which is an inhibitor of RA biosynthesis, lack FGF8 in the forebrain and ectoderm covering the FNP (E, asterisk; n=4) and SHH in the ectoderm of the FNP (F, asterisk) and forebrain (n=5). This result demonstrates that inhibiting retinoid signaling either at the point of RA biosynthesis or at the level of the receptors has similar downstream molecular and morphological consequences in the forebrain and FNP. Scale bars: 200 µm.