Figure 3
Figure 3. Tie2-driven clonal analysis of early YS blood and endothelial precursors. (A-D) Tie2Cre-induced ROSA26R-EYFP detection by immunofluorescence in whole-mounts (A,C) and by immunohistochemistry in histologic sections (B,D) at E7 (A-B) and E8.5 (C-D). Boxed areas (B,D) are magnified in the panels to the right and show expression in endothelial (open arrowhead) and blood lineages (filled arrowhead). Scale bars, 100 µm. (E-G) In situ hybridization with a Cre probe at E6.5 (E) and E7.5 (G). The scheme in (F) depicts the epiblast fate map (modified from Kinder et al8). PS is highlighted in green in (E,G) and the blood-endothelium-heart forming region is outlined in red in (E). (H) Whole-mount E8.5 embryo showing Tie2CreERT2-induced labeling of cell clusters using the double reporter strategy (ROSA26R-LacZ and -EYFP). Scale bar, 500 µm. (I) Cross section of an E8.5 YS blood island showing a LacZ-labeled endothelial cell (open arrowhead) and EYFP-labeled hematopoietic cells (filled arrowhead). (J-K) Flat-mounted E8.5 YS showing general cell distribution of Tie2CreERT2-induced labeling and magnified details (J’-K’’). (K’’’) Detail of a BE cluster from a different embryo (LT26-E2). LacZ+ blood cells are marked with white arrowheads, LacZ+ endothelial cells with open arrowheads, and EYFP+ with black arrowheads. Scale bars, 20 µm. (L-M) Frequencies observed of B-, E-, and BE-clusters in the E8.5 YS after 4OHT induction at E6.5 (L) or E7.5 (M). (N) Single-cluster size distribution (dots) and average size (bars) of B-, E-, and BE-clusters in E8.5 YS after 4OHT induction at E6.5 and E7.5. Images in (A,C,E,G-H) were obtained with a Leica MZ-16 FA bionocular using a Plan Apo 1,0X objective and a Leica DFC310 FX camera. Images in (B,D,I-K’’) were obtained with a Nikon Eclipse 90i microscope using a Nikon DXM 1200c digital camera and 4X/0.2 NA objective (J) or 40X/0.95 NA objective, and Nomarski contrast (I-K’’). (See also supplemental Table 1.) a and arrowhead, anterior; b, blood; di, distal; e, endothelium; EE, extra-embryonic; h, heart; l, left; p and arrowhead, posterior; pr, proximal; r, right.

Tie2-driven clonal analysis of early YS blood and endothelial precursors. (A-D) Tie2Cre-induced ROSA26R-EYFP detection by immunofluorescence in whole-mounts (A,C) and by immunohistochemistry in histologic sections (B,D) at E7 (A-B) and E8.5 (C-D). Boxed areas (B,D) are magnified in the panels to the right and show expression in endothelial (open arrowhead) and blood lineages (filled arrowhead). Scale bars, 100 µm. (E-G) In situ hybridization with a Cre probe at E6.5 (E) and E7.5 (G). The scheme in (F) depicts the epiblast fate map (modified from Kinder et al8 ). PS is highlighted in green in (E,G) and the blood-endothelium-heart forming region is outlined in red in (E). (H) Whole-mount E8.5 embryo showing Tie2CreERT2-induced labeling of cell clusters using the double reporter strategy (ROSA26R-LacZ and -EYFP). Scale bar, 500 µm. (I) Cross section of an E8.5 YS blood island showing a LacZ-labeled endothelial cell (open arrowhead) and EYFP-labeled hematopoietic cells (filled arrowhead). (J-K) Flat-mounted E8.5 YS showing general cell distribution of Tie2CreERT2-induced labeling and magnified details (J’-K’’). (K’’’) Detail of a BE cluster from a different embryo (LT26-E2). LacZ+ blood cells are marked with white arrowheads, LacZ+ endothelial cells with open arrowheads, and EYFP+ with black arrowheads. Scale bars, 20 µm. (L-M) Frequencies observed of B-, E-, and BE-clusters in the E8.5 YS after 4OHT induction at E6.5 (L) or E7.5 (M). (N) Single-cluster size distribution (dots) and average size (bars) of B-, E-, and BE-clusters in E8.5 YS after 4OHT induction at E6.5 and E7.5. Images in (A,C,E,G-H) were obtained with a Leica MZ-16 FA bionocular using a Plan Apo 1,0X objective and a Leica DFC310 FX camera. Images in (B,D,I-K’’) were obtained with a Nikon Eclipse 90i microscope using a Nikon DXM 1200c digital camera and 4X/0.2 NA objective (J) or 40X/0.95 NA objective, and Nomarski contrast (I-K’’). (See also supplemental Table 1.) a and arrowhead, anterior; b, blood; di, distal; e, endothelium; EE, extra-embryonic; h, heart; l, left; p and arrowhead, posterior; pr, proximal; r, right.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal