Random retrospective clonal analysis strategy. (A) Transgenic strategy with 2 independent reporters; ROSA26R-LacZ and ROSA26R-EYFP combined with the RERT transgene in which CreERT2 is expressed from the RNApol II gene. (B) Depiction of the intended strategy. After induction with low-dose 4OHT at E5.5, individual LacZ⁺ (blue) or EYFP⁺ (red) epiblast cells eventually migrate through the PS (E6.5) and colonize the extra-embryonic region (E7.5). (C) Examples of E6-6.5 embryos containing cells labeled with LacZ⁺ (X-Gal-stained) or GFP⁺ (antibody-detected) after 4OHT induction at E5.5. Scale bars, 500 µm. (D-E) E8.5 embryos retrieved after low dose 4OHT induction at E5.5 and containing either LacZ⁺ (D, arrowhead) or EYFP⁺ (E, arrowhead) cell clusters in the blood island region of the YS. Scale bars, 500 µm. (F-H) A flat-mounted E8.5 YS (F), its mask showing labeled cells (G), and the cell cluster segmentation revealed by color coding (H). (I-R) Histologic sections of the blood island region showing LacZ⁺ mesothelial (I-J), endothelial (K-L), and blood cells (M-N), and EYFP⁺ endothelial (O-P) and blood cells (Q-R). (J,L,N,P,R) Show co-localization with Lyve1⁺ immunostaining. Scale bars, 10 µm. Images in panels (C-E) were obtained with a Leica MZ-16 FA bionocular using a Plan Apo 1,0X objective and a Leica DFC310 FX camera. Images in (F) and (I-R) were obtained with a Nikon Eclipse 90i microscope using a Nikon DXM 1200c digital camera and 4X/0.2 NA objective (F) or 40X/0.95 NA objective and Nomarski contrast (I-R) (see also supplemental Figure 1). a and arrowhead, anterior; di, distal; IRES, internal ribosome entry site; l, left; neo, neomycin resistance gene; p and arrowhead, posterior; pA, polyadenylation site; PGK, phosphoglycerate kinase-1 promoter; pr, proximal; r, right; SA, splice acceptor.