DEV020131 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. c-myc^-/- embryos at 8.25 dpc display an abnormal placental morphology. The arrowhead indicates ectoplacental trophoblasts. Ch, chorion; Epc, ectoplacental cone. Scale bar: 200µm.

• Supplemental Figure S2 -

  Fig. S2. Quantitative morphometric analysis of the vasculature in Tie2-Cre;c-myc^flox/- mutant yolk sacs. Whole-mount yolk sacs were stained with anti-CD31 antibodies (details can be provided on request). Capillary images were taken by focusing on the capillary bed between the vitelline artery branches in the embryonic hemisphere of the embryonic yolk sac, which was predictably uniform in size. Capillary diameter and intercapillary space measurements were made with the ruler and pencil tools of Slidebook 4.0 (Intelligent Imaging Innovations, Denver, CO). Vessel diameters were measured from edge to edge at a point in the vessel located equidistant from its adjacent branches. Five mutant and five littermate control yolk sacs were analyzed, and 50 capillary diameters and intercapillary spaces were measured for each sample. (A-C) The area between capillaries (A), width of capillaries (B) and diameter of vitelline arteries (C) in control (white bars) and Tie2-Cre;c-myc^flox/- mutant (black bars). *P<0.01 by Student’s t-test.

• Supplemental Figure S3 -

  Fig. S3. c-Myc staining in primary cultures of embryonic ECs. (A) ECs were isolated from embryos at 10.5 dpc and stained with anti-c-Myc (red), anti-CD31 (green) antibodies and DAPI (blue). Hollow arrowheads indicate EC nucleus. Scale bar: 20 µm. (B) ECs were isolated from embryos at 10.5 dpc and stained with anti-N-Myc (red), anti-CD31 (green) antibodies and DAPI. Orange dots are anti-CD31 magnetic beads used for isolating ECs. Scale bars: 20µm. Hollow arrowheads indicate EC nucleus.

• Supplemental Table S4 -

  Fig. S4. Vav-iCre is activated in fetal liver cells at 11.5 dpc. (A-D) Vav-iCre-mediated gene excision occurs primarily during definitive hematopoiesis at 11.5 dpc in the fetal liver. Black arrows indicate lacZ+ cells in the fetal liver of lacZ-stained whole-mount embryos carrying Vav-iCre and Rosa26R reporter alleles at 11.5dpc (A) and circulatory lacZ+ cells in the yolk sac (B). Paraffin cross-sections (5 µm) of whole-mount lacZ-stained embryo showing the lacZ+ cells in fetal liver (C) and in the heart (D). The endocardium is lacZ negative (white arrowheads). Scale bars: 100 µm in B-D. (E-H) Vav-iCre;c-myc^flox/− embryos exhibit hemorrhaging at 11.5 dpc (E,G). Scale bars: 2 mm. (I,J) The Vav-iCre;c-myc^flox/- fetal liver at 11.5dpc (I). The mutant fetal liver is pale and smaller than that of control. Scale bar: 200 µm. (K) The percentage of YFP+ cells in each hematopoietic lineage from a single fetal liver of Vav-iCre;RosaYFP reporter embryos. Dots indicate the percentage of YFP+ cells under the specific cell marker from a single fetal liver. In total, 10 fetal livers from 11.5 dpc embryos were used in this assay.

• Supplemental Figure S5 -

  Fig. S5. Tie1-Cre mediated gene excision occurs preferentially in ECs over hematopoietic cells. (A) Whole-mount image of the head region of lacZ stained 10.5 dpc embryos with Tie1-Cre and Rosa26R alleles. (B) Tie1-Cre positive endocardial cells are evident in cross section. Black arrowheads, non-endothelial lacZ+ cells; white arrowheads, endothelial cells. Scale bar: 100 µm. FL, fetal liver; AH, atrium of heart; Da, dorsal aorta. C, cross-section of embryonic dorsal aorta. Tie1-Cre positive endothelial cells are shown by anti-β-Gal antibody staining (Red) and anti-CD31 staining (Green). Scale bar: 50µm.

• Supplemental Figure S6 -

  Fig. S6. Vav-iCre activity in fetal liver HCs at 11.5 dpc from Tie2-Cre, Vav-iCre, Tie1-Cre embryos with RosaYFP reporter allele. (A) Fetal liver cells were isolated from embryos bred with Tie2-Cre;RosaYFPR, Vav-iCre;RosaYFPR and Tie1-Cre;RosaYFPR. Cre activity was analyzed as the percentage of YFP+ cells in each hematopoietic lineage (Ter119+, CD11b+ and B220+).