RA Induces cHB Cells, and RA and FGF, in Combination, Induce rSC Cells

<div><p>(A) Schematic drawing of a stage 4 embryo. Dotted line indicates the presumptive neural plate. Black box indicates caudal (C) neural plate explant isolated and cultured in vitro for 44 h. The red mark indicates the caudal margin of the explant labeled with DiI. Bars represent mean ± s.e.m. number of cells in Krox20<sup>+</sup><b><i>Hoxb4<sup>+</sup></i></b><i>/b8<sup>−</sup>/c9<sup>−</sup>, </i><b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup>/ </i></b><i>c9<sup>−</sup>, </i> and <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup>/c9<sup>+</sup></i></b> domains, respectively, as percentage of total cell number. </p> <p>(B–D) Each row represents consecutive sections from a single explant.</p> <p>(B) Control stage 4 C explants generated <b>Krox20<sup>+</sup></b> cells in the rostral, and <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup></i>/ <i>c9<sup>+</sup></i></b> cells were generated in the caudal region of the explant, adjacent to the DiI-labeled cells. A small domain of <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup>/ </i></b><i>c9<sup>−</sup></i> cells was generated in the medial region but no domain of cells expressing <i>Hoxb4</i> alone was generated ( <i>n</i> = 7 explants). </p> <p>(C) RA (10 nM) blocked the generation of Krox20<sup>+</sup> and induced <b><i>Hoxb4<sup>+</sup>/ </i></b><i>b8<sup>−</sup>/c9<sup>−</sup></i>cells in the rostral region. Adjacent to the DiI-labeled cells in the caudal region of the explant, <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup>/ </i></b><i>c9<sup>−</sup></i> cells, but no <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup>/c9<sup>+</sup></i></b> cells, were generated ( <i>n</i> = 6 explants). </p> <p>(D) RA (10 nM) and FGF4 (30 ng/ml), in combination, generated <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup></i></b><i>/c9<sup>−</sup></i> cells in both the rostral and caudal regions. No, or a few, <b><i>Hoxb4<sup>+</sup>/b8<sup>+</sup></i>/ <i>c9<sup>+</sup></i></b> cells appeared, and no Krox20<sup>+</sup> or <b><i>Hoxb4<sup>+</sup></i></b><i>/b8<sup>−</sup>/c9<sup>−</sup></i> cells were generated ( <i>n</i> = 5 explants). Scale bar represents 100 μm. </p></div>