The fate of Wt1-derived cells, marked by ®-gal expression in Wt1^GFPCre/+; Rosa26^fsLz hearts (a-c,e), or RFP expression in Wt1^GFPCre/+;Z/Red hearts (d,f,g-j), was analyzed by immunohistochemistry (a-f) and live cell imaging (g-j). a. Overview of contribution of Wt1-derived cells to myocardium. b-d. Cryosections of E15.5 heart stained for genetic lineage tracers (β-gal or RFP) and cardiomyocyte-specific markers Tnnt2 and Actn1. e-f. Co-expression of genetic lineage tracers with cardiomyocyte markers in dissociated E15.5 heart culture. g-j. Live cell imaging of Wt1-derived cells, identified by RFP fluorescence. g. Spontaneous contraction of an RFP⁺ cells. Transmission line scan along the paths indicated by dashed lines. Arrowheads indicate contraction of an RFP⁺ cell (g1). A non-contractile cell is shown in g2. h. Fluo-4 AM calcium imaging of a cluster of beating cells, one of which was RFP⁺. Calcium oscillations in the RFP⁺ cell, measured by line scan along the white line, showed calcium sparks (black arrowhead, h1) preceding calcium waves (red arrowhead). i. Calcium oscillations in the RFP⁺ cell was synchronized with adjacent cardiomyocytes, indicating electrical coupling. Plots correspond to scans within colored boxes shown in h. j. Caffeine augmentation of the amplitude of calcium oscillations in an RFP⁺ cell. Blue staining, a-f, DAPI. Scale bars: a, 50 ⌠m, others 10 μm.

a-c. The Nkx2-5 fate map was determined in Nkx2-5^IRES-Cre; Rosa26^fsLz (a-b) or Nkx2-5^Cre; Gata4^flap (c) embryos. Nkx2-5-driven Cre activated lineage tracer expression in PE (asterisk). d. Nkx2-5 fate, marked by expression of alkaline phosphatase (AP, red membrane staining) compared to Wt1 immunohistochemistry (green nuclear staining). Wt1 was expressed in Nkx2-5-derived cells in PE (white arrowhead). e. Immunohistochemistry comparing Nkx2-5 (red) and Wt1 expression (green). Yellow arrowhead indicates anterior heart field, which did not express Wt1. f.Nkx2-5^gfp ES cells were differentiated in embryoid body (EB) culture for the indicated number of days. The cells were then dissociated, and GFP⁺ cells were isolated by flow cytometry. Wt1 expression in GFP⁺ cells was measured by quantitative reverse transcription PCR. Bars indicate mean ± sd. n=3. Scale bars: 50 ⌠m. A, atrium . RV, right ventricle. LV, left ventricle. OFT, outflow tract.

a. At E9.5, Wt1 was expressed in proepicardium (PE) and in scattered pericardial cells over the surface of the heart (arrowheads, a2). b-c. At E15.5, Wt1 expression was confined to the epicardium, as determined by in situ hybridization (ISH, b) and immunohistochemistry. d-f. Co-expression of Wt1 and GFPCre expression in Wt1^GFPCre/+ E9.5 embryos. Wt1 and GFPCre were co-expressed in PE and urogenital ridge (UR). g. Wt1-driven GFPCre was confined to epicardium (arrowheads), and not detected within myocardium, at E9.5-E15.5 (representative images for E10.5, E12.5, and E14.5 are shown). Arrows indicate autofluorescent red blood cells. All images except b show immunohistochemical staining. A, atrium. V, ventricle. LV and RV, right and left ventricle. Lu, lung. OFT, outflow tract. Scale bars: 50 ⌠m.

a-h. Temporal restriction of Cre labeling with Wt1^CreERT2. Wt1^CreERT2 did not recombine the fate mapping reporter Rosa26^fsLz in the absence of tamoxifen (b). Tamoxifen treatment at E10.5 and E11.5 induced Wt1^CreERT2 labeling of cardiomyocytes, as determined in both tissue sections (c) and in dissociated heart culture (g-h). Also labeled were SMCs adjacent to endothelial tubes (d-e). i-k. E11.5 Wt1^GFPCre/+ heart cells actively expressing Wt1, as determined by GFP fluorescence, differentiated into cardiomyocytes. Wt1⁺ epicardial cells were enriched in early digestion fractions compared to late digestion fractions. GFP⁺ cells from early fractions were plated either with mitotically inactivated feeders (j) or without feeders (k). l-m. CMFDA dye, selectively incorporated into E11.5 epicardium, was found in CMs at 48 hours. Brief incubation of E11.5 heart explants with the dye CMFDA resulted in selective labeling of epicardium (0 hours culture). After 48 hours in explant culture, dye labeled cells were present in the myocardial wall. A subset of dye-labeled cells co-expressed the CM markers Nkx2-5, cardiac troponin I (Tnni3), and Actn1. Arrows indicate co-expression. Scale bars: 10 ⌠m.