DEV022475 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Table S3 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Arterial-venous connections in early embryos. (A,B) Whole-mount CD31 staining of E8.5 embryos at 9 ss (A) and 11 ss (B). Arrowheads indicate direct connections between DA and anterior CV. Scale bars: 100 mm.

• Supplemental Figure S2 -

  Fig. S2. Notch4 gain-of-function mutation causes vascular remodeling defects at E9.5. (A-D) The Tie2-tTA driver is specific for ECs, as shown by lacZ staining of the yolk sac (A,C, whole mount) and section (B,D) of E9.5 (A,B) and E12.5 (C,D) embryos carrying the reporter TRE-lacZ. ECs in the vitelline artery (arrows) and capillaries are stained for lacZ at E9.5 (A), and the staining is stronger at E12.5 (C). In embryo proper, a few ECs in the DA (da, arrows) and anterior CV (acv, arrowheads) are stained (B,D). (E) int3 mRNA expression, as detected by RT-PCR with int3-specific primers in pooled embryos and yolk sacs, begins at E8.5 (8-9 ss) and is restricted to embryos carrying the two transgenes. (F-H) Vascular defects of yolk sacs and embryos at E9.5. (F) Yolk sac arterial vessels (arrows). (G) Venous vessels in yolk sac (arrowheads). (H) Gross images of embryos. Note Tie2-tTA;TRE-int3 embryos displayed severe hemorrhage and enlarged pericardial sacs (H). Their yolk sacs lacked well-formed major vessels, indicating remodeling defects (F,G). At this stage, these Tie2-tTA;TRE-int3 embryos, expressing int3 in a temporally controlled manner, exhibited similar gross abnormalities and lethality to that seen in flk-int3 embryos, expressing int3 without temporal control (Uyttendaele et al., 2001). At E9.0 the phenotype was less severe, but still detectable (not shown). At e8.5 (9ss), mutant embryos and yolk sacs exhibited no detectable vascular defects (not shown). However, all Tie2-tTA;TRE-int3 embryos died between e10.5-11.5 (not shown). Black arrow, internal carotid arteries; red arrows, hemorrhage in the mutant; blue arrow, pericardiac sac (pcs). Scale bars: 100 mm in A-D; 800 mm in F-H.

• Supplemental Figure S3 -

  Fig. S3. Deletion of Notch1 causes reduction of DA and abolishes Dll4. (A,B) CD31 staining of the trunk region of E8.75 (15 ss) embryos. The DA (arrows) is atretic in the Notch1^−/− embryo (B) compared with the control (A). Anterior is leftwards and dorsal is upwards. (C,D) Cross-sections of E8.75 (15 ss) embryos stained by in situ hybridization for Dll4 show diminished expression in the Notch1-deficient mutant (D). Scale bars: 200 mm.