DEV026906 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. dENotch1 activity and late expression of Notch pathway members. (A) Ectopic expression of dENotch1 detected with co-electroporated GFP (DMSO; green) leads to ectopic Nrarp expression in more medially ingressing mesoderm cells (DMSO; arrows), which are normally negative for Nrarp. DAPT treatment leads to a strong reduction of endogenous Nrarp expression and a failure of dENotch1 to induce Nrarp (right two panels). (B) Ectopic GFP expression does not affect L-fringe expression (top panels; right two panels show magnified views of the left two). Ectopic dENotch1 expression leads to a strong induction of ectopic L-fringe expression (bottom panels; right two panels show magnified views of the left two). (C) Expression patterns of indicated Notch pathway members at HH6-HH7 and HH10. The in situ was developed for prolonged period of time to reveal possible weak staining in the extraembryonic region, resulting in over-staining in embryonic regions.

• Supplemental Figure S2 -

  Fig. S2. Salt-and-pepper expression of Scl (Tal1) and dHAND in nascent ventral mesoderm cells. Embryo stages and section levels are indicated in left panels.

• Supplemental Figure S3 -

  Fig. S3. dENotch1-expressing cells are Scl negative and dHAND positive at HH5 and HH7. (A) Magnified whole-mount view of an HH5 embryo, showing that dENotch1-positive cells (brown) segregate from Scl-positive cells (blue). (B) Magnified whole-mount view of an HH5 embryo, showing that dENotch1 cells (brown) are located among dHand-positive cells (blue). (C) More prominent co-localization of dENotch1 cells (brown) and dHand-positive cells (blue) at HH7. (D) Magnified view of dHand and Scl double in situ hybridization shown in Fig. 3D.

• Supplemental Figure S4 -

  Fig. S4. Section views of HH5 embryos, showing that dENotch1-expressing cells are Scl negative and dHand positive. Arrows indicate co-positivity for the expression construct (anti-GFP or anti-Myc-tag in dENotch1) and in situ; arrowheads indicate single positivity for the expression construct. (A) Control GFP-expressing cells are observed in both Scl-positive and Scl-negative populations. (B) dENotch1-expressing cells are excluded from the Scl-positive population and are located in the dHand-positive population (D). (C) DAPT treatment results in the contribution of dENotch1-expressing cells to the Scl-positive population, similar to control. (E) Section views of HH5 embryos, showing that Scl-overexpressing cells are dHand negative. (F) Section views of HH5 embryos, showing that dHAND-overexpressing cells are Scl negative.

• Supplemental Figure S5 -

  Fig. S5. Scl acts downstream of Notch-mediated separation. When dENotch1 is co-introduced with Scl, SMC exclusive distribution (Fig. 1) is completed abolished, resulting primarily in blood contribution. (A-D) Stage HH10; (E,F) stage HH7. (A) Co-electroporation GFP is stained together with chicken ρ-globin. Ecdoterm-side view of the peripheral hemogenic region. Section is shown in C. (B) Co-electroporated GFP is stained together with dHand. Ecdoterm-side view of the peripheral hemogenic region. Section is shown in D. (E) dENotch1-expressing cells segregate from Scl-positive BC/EC progenitors. (F) Scl and dENotch1 co-expressing cells segregate from dHand-positive SMC progenitors, and are located in forming blood islands.

• Supplemental Figure S6 -

  Fig. S6. Changes in FGF pathway activity do not affect dHand expression and CA-β-Catenin does not induce BC/EC markers. (A) The FGF pathway is not involved in the early separation of SMCs and BC/ECs. Neither the electroporation of constitutive active FGFR2 nor SU5402 treatment affects dHand expression. (B) Ectopic CA-β-Catenin does not lead to ectopic expression of the BC/EC marker Lmo2, Vegfr2 or Ets1.

• Supplemental Figure S7 -

  Fig. S7. BMP pathway activity is revealed by anti-phopho-Smad1/5/8 staining. (A) An HH4 embryo stained by anti-phopho-Smad1/5/8 antibody. Right panels show sections indicated in left panel. No BMP activity is detected around the anterior primitive streak, reflecting the strong anti-BMP signals from the Hensen’s node. Sections of more-posterior primitive streak show phopho-Smad1/5/8 signals in most mesoderm cells. (B) Ectopic CA-ALK6, detected with co-electroporated GFP, leads to ectopic phopho-Smad1/5/8 signals (arrowheads in left panel inset; showing only the red fluorescent color for phospho-Smad) in ectoderm and mesoderm cells around the anterior primitive streak (right panel). Boxed area is magnified in D. (C) dENotch1 expression in the posterior primitive streak does not affect normal phopho-Smad1/5/8 staining. Boxed area is magnified in E.