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Fig. 1. Retinoid signaling is required for forebrain and FNP morphogenesis. (A) ALDH6, which synthesizes RA, is detected in ventral ectoderm (ec; arrow) of the presumptive FNP adjacent to the forebrain (f) of chick embryos at stage 10 (sagittal section, rostral towards right, dorsal on top). (B) RXR{gamma} is expressed in neural crest cells (arrows) that migrate out of the rostral neural tube of stage 10 embryos (dorsal view). (C) RARß is expressed in neural crest cells (arrows) that migrate between forebrain (f) neuroepithelium and overlying ectoderm (ec) of stage 10 embryos (sagittal section, rostral towards right, dorsal on top). Relative to cell density (blue nuclear stain), the neural crest shows higher expression levels (white dots) when compared with the neuroepithelium. (D) Ion exchange beads (asterisks) were soaked in RAR and RXR antagonists (100 µg/ml) and placed along the rostral margins of the forebrain (f) of stage 10 embryos. Midbrain (m), hindbrain (h). (E) DMSO control; (F) RAR/RXR antagonist-treated embryos at stage 36 showing the effects of disrupting retinoid signaling. Antagonist-treated embryos lack forebrain tissues, fail to form an FNP (fn), and have fused eyes (n=18, i.e. number of embryos examined with this phenotype). These results are further illustrated by sagittal histological sections of control embryos (G) and treated embryos (H). In treated embryos, the maxillary (mx) and mandibular (ma) processes are unaffected whereas the FNP (asterisk) and forebrain are absent. Eye (e), ear (ot), midbrain (m) and hindbrain (h). Two additional controls confirm that these defects are due to disruptions in retinoid signaling specifically. First, we treated embryos at stage 10 with beads soaked citral (I), which is an inhibitor of RA biosynthesis. These embryos also lack forebrain tissues and an FNP (asterisk) while the maxillary (mx) and mandibular (ma) processes are unaffected (n=22). Second, we used beads soaked concomitantly in all-trans RA and RAR/RXR antagonists (K). (J) Control embryos treated with RA alone have severe forebrain and FNP hypoplasia (asterisk; n=7), whereas embryos exposed to RA and RAR/RXR antagonists simultaneously (K) are relatively normal with a slightly shortened FNP (n=10). Scale bars: 100 µm in A; 200µm in B,D; 30 µm in C; 1 mm in E-H; 3 mm in I; 2 mm in J,K.