Hif1α deficiency is associated with abnormal neurogenesis in vitro. (a) Relative gene expression of selected markers Nestin, Neurogenin 1, and Ascl1 (achaete-scute complex-like 1) involved in neurogenesis detected at the mRNA level during the differentiation of wild-type and HIF1α-deficient ESC by real-time quantitative PCR analysis in undifferentiated ESC (ESC) and in EBs differentiated for 10 days in adherent culture (5 + 10d). Data are presented as means ± SEM from at least 5 independent experiments (one-way ANOVA for each set of ESC and 5 + 10d; significance indicated only for dif.Hif1α+/+ versus dif.Hif1α−/−). (b, A) Protein levels of TUJ1 and BLBP (brain lipid-binding protein) detected by western blot in EBs differentiated for 10 days in adherent culture (5 + 10d). (b, B) Densitometric analysis was performed on the basis of 3 independent experiments (t-test for each marker separately, Hfi1α+/+ versus Hif1α−/−). (c, A) Illustrative picture of aggregate grown for 24 hrs (0d) in agarose microwell and (c, B) of 5-day-old EBs (5d) grown on an agar-coated plate. Scale bar: 200 μm. (c, C) Light microscope images illustrating the morphology of Hif1α+/+ and Hif1α−/− EBs after 10 days of differentiation in adherent culture (5 + 10d).