Progressive changes in the replication timing of developmental regulator genes upon mesodermal differentiation of ES cells. (A) Isolation of cells at sequential stages of ES cell induction to hematopoietic progenitors. Bry-GFP ES cells were differentiated in embryoid bodies (EB), harvested at different times and samples isolated by FACS sorting on the basis of GFP and Flk1 expression.5 (B) Top panel: GFP (Bra/T) and Flk1 expression by ES cells (day 0) and in dissociated embryoid bodies (2.5, 3.75 days of differentiation). Bottom panel: Western blot analysis of Oct4 and Sox2 levels in sequential 3-fold dilutions of whole cell extracts from ES and FACS-sorted cell populations a, b, c, and d. Detection with tubulin antibody is shown as a loading control. (C) Equal numbers of undifferentiated ES cells and FACS-sorted differentiated cell populations were replated in ES cell medium containing LIF for 10 days on feeder layers and colonies scored after staining with methylene blue. Data are mean ± SD from 2 experiments carried out in triplicate. *Significantly different numbers of colonies compared with ES cells (P < .05, Student t test). (D) Replication timing of control loci (αGlobin, X141), ES- (Rex1 and Sox2), mesodermal- (Bra/T, Flk1, Ikaros, and Myf5), and neural-specific genes (Math1, Sox1, Nkx2-2, and Nkx2-9) in undifferentiated ES cells (black), and ES-derived GFP−/Flk1− (epiblast, gray), GFP+/Flk1− (mesoderm, green), and GFP+/Flk1+ (hemangioblast, red) samples. The nonexpressed nonbivalent Myf5 locus replicates late throughout, whereas the replication profile of Ikaros, a bivalent locus, is variable (according to developmental stage) but remains early. For each locus, the abundance of newly replicated DNA was measured by quantitative real-time polymerase chain reaction (PCR) in 6 cell-cycle fractions: G1, S1 to S4, and G2/M as described in supplemental Figure 2. Values are the amount of newly synthesized DNA, calculated as a percentage of total (sum of all fractions). Data are mean ± SD from 2 or 3 experiments.
