Figure 1. Hypothalamic-like differentiation of the embryonic stem cell line EB5.

A, Pluripotency factors rapidly decreased in differentiating EB5 cells over 4 days following withdrawal of leukemia inhibitory factor (-Lif) as evidenced by qRT-PCR analysis. B, Markers for neuroectoderm and rostral hypothalamic development conversely increased during EB5 cell differentiation. C, qRT-PCR analysis of markers for dorsal-ventral hypothalamic patterning and PVN neurons demonstrated strong increases following 25 days of differentiation. D, PVN markers (Brn2 and Arnt2) further increased following treatment with the sonic hedgehog (Shh) antagonist cyclopamine as evidenced by qRT-PCR. Application of the Shh agonist purmorphamine blocked this increase. Bars represent standard deviations from the mean (sem) from at least 3 independent experiments.