Figure 4

LHX9α/β protein expression is specific to the epicardium of the heart. (A-C) Immunofluorescence analysis of LHX9α/β expression in frozen sections of E11.5 heart. (A) LHX9 (red; A and C) does not co-localize with cardiac muscle marker TNNT2 (cardiac troponin T) (green; B-C). LHX9-positive cells are localized to the outmost cardiac layer (arrows). Scale bar 50 μM. (D-L). LHX9α/β-positive cells (red; D, G, J, F, I, L) are localized to the outermost cardiac layer (arrows) in E11.5 (D-F) and E12.5 control (G-I) and Fog2 mutant (J-L) cardiac sections. The endothelial marker ENG (endoglin) (green; E-F; H-I; K-L) labels the epicardial cells already committed to the endothelial lineage beginning at E12.5 (arrowheads). Scale Bar: 50 μM. Control sections required longer time exposures to detect the weaker LHX9 staining in the wild-type hearts.