(A–G′) Confocal images of PA2 sections of control (A–C‴), Numb/Numbl DKO (D), and chimeric (E–G′) embryos, immunostained with PH3, RFP, Isl1 antibodies. Asterisks indicate PH3+ RFP+ Isl1+ (triple positive) cells located in outer layers of RFP+ Isl1+ cell population (outlined, A and E). Boxed areas in (A), (B), and (E) are shown in higher magnification in (B), (C–C‴), and (F–G′), respectively. No PH3+ cells are found in RFP+ cells (asterisks, F–G′). RFP+ cells were outlined in white (F–G′). (H and I) Percentage of donor-derived Isl1+ Nkx2.5− cells in PA2 is shown in (H) and number of PH3+ RFP+ Isl1+ cells per 12-micron PA2 section is shown (I). Data are mean ± SD; n = 10; *p<0.05. (J and K) Relative percentage of EdU+ cells in ES cell-derived Mesp1+ progenitor, Isl1+ Nkx2.5− CPCs or Nkx2.5+ CPCs transfected with control or Numb/Numbl DKO siRNA (J) or Isl1+ Nkx2.5− CPCs transfected with control (CTV) or Numb overexpression construct (CTV-Numb) (K). Data are mean ± SD; n = 3; *p<0.05. The Mesp1+, Isl1+ Nkx2.5−, or Nkx2.5+ cells were FACS-purified from day 4 Mesp1Cre; Ai9, day 5 Isl1Cre; Ai9, or day 6 Nkx2.5GFP ES cells, respectively. Dapi (blue) was used to counterstain the nuclei. p values were determined using the paired Student t test. Scale bars, 10 μm (B, C, F, G). 50 μm (A, D, E). fe, foregut endoderm; pa, pharyngeal arch ec, endocardial layer; mc, myocardial layer; pc, pericardial layer.