BMP-regulated Dpysl2 and Kdm6b play an important role in early neural differentiation. (A) Dpysl2 expression is increased during neural differentiation, and BMP4 impairs this increase at the early stage (day 1, 3). R1 cells were cultured in neural differentiation medium in the presence (+) or absence (−) of 10 ng/mL BMP4 for 5 d or treated with BMP4 for 4 h before harvest (4 h). Dpysl2 expression was determined by qRT-PCR. (B) Dpysl2 knockdown reduces Sox1 expression. Sox1 expression was determined in the Dpysl2 shRNA D1- or D2-expressing cells that were subjected to neural differentiation. (C) Dpysl2 knockdown reduces nestin expression. Nestin expression was determined by immunofluorescence in the shRNA D1- or D2-expressing cells at day 5 of neural differentiation. (D) SMAD represses the Dpysl2 promoter activity. (Left) Schematic map of the Dpysl2 promoter-driven luciferase reporters. TSS, transcriptional start sites; SBE, SMAD-binding element (for both SMAD1/5 and SMAD4) that is predicted from ChIP-chip analysis (Supplemental Tables S1, S2). Reporter plasmid (0.1 μg), Renilla plasmid (10 ng), SMAD1 and SMAD4 (0, 0.05 μg, or 0.1 μg, each) were cotransfected into HEK293T cells. At 36 h post-transfection, cells were harvested for luciferase assay. The experiment was performed in triplicate, and the data are presented as mean ± SEM of three independent experiments after normalization to Renilla activity. (E) DPYSL2 increases Sox1 and nestin expression during early neural differentiation. R1 ES cells were transfected with pEGFP-Dpysl2 or pEGFP-C3 empty vector respectively and then replated for neural differentiation. RNA was extracted at day 3 for qRT-PCR analysis. (F) Kdm6b expression is reduced by BMP4 in the early stages of neural differentiation. (G) Kdm6b knockdown reduces Sox1 expression. Sox1 expression was determined in the shRNA K1- or K2-expressing cells that were subjected to neural differentiation similarly as in B. (H) Kdm6b knockdown decreases nestin expression. Nestin expression was determined by immunofluorescence in shRNA K1- or K2-expressing cells at day 5 of neural differentiation.
