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. 2014 Jun 6;5:4054. doi: 10.1038/ncomms5054

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Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

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In Vav1-Cre;R26R-tdTomato embryonic hearts, tdTomato+ (Vav1+) haematopoietic stem cells (HSCs) contribute to the developing epicardium from E12.5 to E16.5. At E14.5 (a,b) and E16.5 (c,d), tdTomato+ (Tom) cells reside in the epicardium and underlying myocardium (white inset boxes in a and c are highlighted at higher magnification in b and d, respectively; white arrowheads in b and d highlight tdTomato+ cells specifically in the epicardium). At E14.5, further immunostaining for combined α-CD45+ and tdTomato (e) revealed that all CD45+ cells residing within the developing epicardium were Vav1-tdTomato+ (f–h; white arrowheads highlight double-positive cells), whereas Vav1-tdTomato+ cells negative for CD45+ were localized within the underlying myocardium (f–h). The CD45+ HCs were distinct from the Wt1+ contribution as indicated by CD45+ immunostaining in conjunction with lineage tracing in WtCreERT2;R26R-tdTomato developing hearts at E14.5 (i–l; white inset boxes in i and k are highlighted at higher magnification in j and l, respectively; white arrowheads in j and l highlight CD45+ cells specifically in the epicardium). The number of CD45+ cells that contributes to the epicardium increases from E10.5, coincident with formation of the epicardium, through to E16.5 with a significant increase between E12.5 and 14.5 (mean % CD45+ cells ± s.e.m.; P≤0.001; n=6 hearts; m). In Vav1-tdTomato-labelled embryos, tdTomato+ cells do not contribute to the proepicardial organ (peo; indicated in blue in n; rest of heart indicated in green via Nkx2.5Cre;R26R-EYFP) at E9.5 (o; peo is demarcated by Wt1 immunostaining) and CD45+ cells do not localize to the peo (p), suggesting tdTomato/CD45+ HCs that contribute to the forming epicardium arise from an alternate source, previously described as the haemagenic endothelium and/or fetal liver (Supplementary Fig. 6). ep, epicardium; lv, left ventricle; my, myocardium; peo, proepicardial organ; rv, right ventricle. Scale bars, 200 μm (a,i); 20 μm (b,d,f–h); 400 μm (c,e); 50 μm (j,l); 100 μm (i,o,p); 600 μm (n). All statistics are calculated by Students t-test; *P≤0.001.