Hemogenic endothelium markers in E8.5 YS endothelium and associated hematopoietic clusters. (A) CD41 expression in the YS endothelium and an associated hematopoietic cluster. (B) 3D surface rendering of the image in (A). Solid arrowheads indicate the CD41⁺ hematopoietic cluster and open arrowheads indicate CD41⁺ endothelial cells. (C-E) Confocal optical sections of the specimen in (A), showing symmetric Lyve1 and asymmetric CD41 signals (arrowheads). (F) Frequency of CD41^hi cells in the Lyve1⁺ endothelial cell population (left) and the frequency of Lyve1⁺ endothelial cell association with a CD41⁺ hematopoietic cluster (right). (G-N) Confocal microscopy images of CD41⁺Runx1⁺ hematopoietic clusters in close contact with the endothelium, and associated (G-J) or nonassociated (K-N) with a Lyve1⁺Runx1⁺ endothelial cell. Solid arrowheads indicate CD41⁺Runx1⁺ hematopoietic clusters and open arrowheads indicate CD41⁺Runx1⁺ endothelial cells. (O-R) Images showing different degrees of integration of Lyve1⁺CD41⁺Runx1⁺ cells in the YS endothelium. Flat cells highly integrated in the endothelium show low Runx1 and low CD41 (O-O’’), whereas increasingly round cells show progressively higher levels of Runx1 and CD41 (P-P’’,Q-Q’’), and completely round cells show high CD41 and Runx1 (R-R’’) and residual Lyve1 staining (small arrowheads in R,R’). Fluorescence from EYFP, Cy5, Cy3, and DAPI was obtained with a Leica SP5 confocal microscope using a 40X/1.25 NA objective. All 3D reconstructions in (A-B) were done with the Imaris software package (see also supplemental Movie 3).