Figure 4.

Abnormal erythroid and megakaryocyte differentiation in Tie2-Cre;N1ICD YS. (a) YS cells centrifuged on 96-well plates, revealing the low hemoglobin content of Tie2-Cre;N1ICD YS, indicating defective erythroid differentiation. (b) Representative dot plots of double-stained WT and electronically gated EGFP⁺ cells for the erythroid marker TER119 and the transferrin receptor CD71. Boxed areas define CD71⁺⁺TER119⁻ (R1) and CD71⁺⁺/TER119⁺ (R2) cells. The graph in the right shows the relative percentage of R1 and R2 cell populations (mean±S.E.M.; N=6) in WT and electronically gated EGFP⁺ and EGFP⁻ cells. (c) Microphotographs of May Grümwald-Giemsa staining in WT and FACS-purified EGFP⁺ Tie2-Cre;N1ICD cells. (d) Representative dot plots of WT and electronically gated EGFP⁺ cells for TER119 and c-Kit. Numbers are relative percentages of the TER119⁺c-Kit⁺, cells boxed (mean±S.E.M.; N=6). The right graph shows the quantification of numbers of TER119⁺c-Kit⁺ cells/YS calculated as in Figure 3j (mean±S.E.M.; N=6) for WT and electronically gated EGFP⁺ and EGFP⁻ cells. (e) The graph represents the mean fluorescence intensity (MFI) data obtained from E9.5 WT and electronically gated EGFP⁺ and EGFP⁻ cells from Tie2-Cre;N1ICD mice stained for TER119, as mean±S.E.M. (N=4). Staining of blood cells at E11.5 is shown as a control. (f) Quantifications of embryo-derived primitive (βH1) and definitive (β1) hemoglobins are shown in the bar graph. WT and FACS-purified EGFP⁺ and EGFP⁻ Tie2-Cre;N1ICD cells were submitted to RNA extraction, cDNA was synthesized and RT-qPCR was performed as indicated in Materials and Methods. The Bio-Rad CFX Manager software was used to calculate the C_T of each reaction, and the relative amount of specific cDNA in each sample was determined by the ΔC_T method. Results are displayed as the relative expression of each transcript over Gα gene expression (mean±S.E.M.; N=4). (g) Representative FACS analysis of WT and Tie2-Cre;N1ICD mice stained for TER119, CD45/Mac1, CD41 and c-Kit. TER119⁻CD45/Mac1⁻ cells were gated (left dot plot) and analyzed for CD41 and c-Kit, as indicated in the plots from WT and electronically gated EGFP⁺ cells. The boxes identify the c-Kit⁺CD41⁺ erythro-myeloid progenitor (EMP) population; numbers are percentages (mean±S.E.M.; N=4). The right graph shows the EMP quantification/YS calculated as in Figure 3j (mean±S.E.M.; N=4) for WT and electronically gated EGFP⁺ and EGFP⁻ cells. (h) Representative FACS analysis of WT and electronically gated EGFP⁺ cells stained for CD9 and CD41. Boxed areas define the double-positive CD9⁺⁺CD41⁺⁺ megakaryocytic population and the CD9⁺CD41⁺ cell population. The graphs in the right show the relative percentages and absolute numbers of both cell populations in WT and electronically gated EGFP⁺ and EGFP⁻ Tie2-Cre;N1ICD YS (N=5). (i) Left panel, Representative graph of the relative percentage of megakaryocyte progenitors (MK-CFUs) present in FACS-purified CD41⁺ cells from WT versus EGFP⁺ populations (N=3). Right panel, Relative percentages of MK-CFUs expressing acetylthiocholiniodide (AcH) in WT versus EGFP⁺ cells (N=3). (j) Microphotographs showing cultures of CD9⁺⁺CD41⁺⁺ FACS-purified cells from E9.5 WT and EGFP⁺ Tie2-Cre;N1ICD YS cells at 48 h of culture. *P<0.05, **P<0.01, ***P<0.001