Figure 4. Rb maintains the osteoblastic fate commitment in normal osteoblasts and regulates fate choice during normal development in vivo.

a, Calvarial osteoblasts were prepared from e18.5 Rb^fl/fl or p53^fl/fl embryos and infected with Ad-GFP or Ad-Cre at P1. Five days later, the cells were induced with differentiation media and then assayed for osteogenesis and adipogenesis at 0, 14 and 25 days by staining with Alizarin Red and Oil-Red-O. A representative timepoint (25 days) is shown. b, qPCR was also used to assess osteogenic and adipogenic markers in the un-induced Rb^fl/fl (wt) versus Rb^fl/fl+Ad-Cre (Rb^−/−) osteoblasts. Bars represent the mean of three independent experiments (+/− SD). c, Alizarin Red (bone mineralization) and Alcian Blue (cartilage) staining of e15.5 skeletons (top panel), e18.5 calvaria (middle panel) and e18.5 limbs (botton panel) from Meox2-Cre;Rb^+/+ and Meox2-Cre;Rb^fl/fl littermate embryos. Arrows mark visible skeletal defects. qPCR was used to assess osteogenic (Runx2, Alp, and Bsp) and adipogenic (Ap2 and C/ebpα) markers in mRNA extracted from the calvarial bones of e18.5 Meox2-Cre;Rb^+/+ and Meox2-Cre;Rb^fl/fl embryos. Bars show the mean of three embryos arising in two independent crosses (+/− SD). d, Brown adipose tissue (BAT) was dissected from the backs of Meox2-Cre;Rb^fl/fl embryos (n=10) and their Meox2-Cre;Rb^+/+ littermate controls. All 10 showed a dramatic expansion of the brown fat compartment. A representative example is shown (upper two panels). Introduction of the LSL-LacZ reporter into this model, and LacZ staining confirmed equal, widespread expression of Cre in the control and Rb mutant BAT (third panel). H+E staining of BAT (bottom panel).