DEV025916 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Components of Notch and Wnt signaling pathways are expressed in developing blast colonies. Expression patterns of Notch and Wnt signaling components in developing blast colonies. Six individual blast colonies were analyzed at each time point, as described in the legend of Fig. 1. Each lane represents an individual colony.

• Supplemental Figure S2 -

  Fig. S2. Serum free methylcellulose cultures support efficient blast colonies development. (A) Comparison of blast colony development in serum-containing and M10 serum-free methylcellulose media. Cultures were established with 2×10⁴ Flk1+ cells isolated from day 3.25 EBs and colonies were scored after 4 days of culture. (B) Comparison of the hematopoietic potential of blast colonies generated in serum-containing and M10 hemangioblast conditions. Flk1⁺ cells isolated from day 3.25 serum-stimulated EBs were plated at concentrations of 5×10⁴/ml into either serum-containing or serum-free M10 hemangioblast cultures. Four days later, 500 blast colonies were harvested from each group, the cells dissociated, and replated into hematopoietic methylcellulose cultures. Hematopoietic colonies were scored 5-8 days later. Error bars indicate standard deviation of triplicate plates. (C) Gene expression patterns of the blast colonies generated in serum-free conditions. Six individual colonies grown in M10 conditions were isolated at different time points and analyzed for the expression of genes indicative of mesoderm, hematopoietic and vascular development. Each lane represents an individual colony. Pos, positive control using a mixture of cDNAs from day 3 and day 9 EBs.

• Supplemental Figure S3 -

  Fig. S3. Numb isoform PRR^-s is preferentially expressed in 24 hour blast colonies. Twenty-four-hour-old blast colonies were harvested and analyzed by RT-PCR for the expression of numb isoform RPP^-S and RPP^-L. The upper band represents RPP^-L and the lower band RPP^-S. Details of the materials and methods can be provided on request.

• Supplemental Figure S4 -

  Fig. S4. Dox-induced expression of stabilized β-catenin, Notch1-IC and Numb protein in Flk1+ cells. Flk1⁺ cells isolated from serum-induced day 2.75 EB generated from either the sβ-cat, Notch1-IC or Numb ES cell lines were reaggregated for 24 hours in the presence or absence of Dox (2 µg/ml). Following this induction step, the aggregates were analyzed for the presence of β-catenin, Notch1-IC or Numb protein by western blotting. Arrow indicates the band of the induced PRR^-S isoform of NUMB (MW∼65 kDa).

• Supplemental Figure S5 -

  Fig. S5. Activation of RBP-Jκ-dependent Notch signaling following induction of Notch1C. RBP-Jκ luciferase reporter assays were performed as described previously in a modified manner (Schroeder et al., 2003). Details of the materials and methods can be provided on request. Shown is the RBP-Jκ-dependent luciferase activity normalized to renila luciferase activity. Error bars indicate the standard deviation of triplicate electroporations.

• Supplemental Figure S6 -

  Fig. S6. Morphology of the compact (core) colonies from DKK1-treated cultures compared to blast colonies. Four typical blast colonies from the DKK1-untreated group (−DKK1, upper panel) and four typical core colonies from the DKK1-treated group (+DKK1, lower panel) following 3 days of culture.

• Supplemental Figure S7 -

  Fig. S7. Hematopoietic and vascular potential of core colonies and blast colonies. Colonies generated the absence (−DKK1) or presence of DKK1 (+DKK1) were plated on a thin layer of Matrigel in liquid expansion assay. Shown are two cultures representative of each group (-DKK1, left panel; +DKK1, right panel).

• Supplemental Figure S8 -

  Fig. S8. Morphology and hematopoietic potential of Notch1-IC induced core colonies. (A) Morphology of 4-day-old colonies generated in the presence or absence of doxycyclin (−Dox or +Dox). Dox was added for the first 24 hours of culture. After this induction stage, the developing colonies were harvested, washed and replated into fresh M10 media without Dox. (B) Primitive erythroid and definitive hematopoietic potential of the colonies generated in the presence (+Dox) or absence (−Dox) of doxycyclin. Two-thousand colonies of each group (−Dox or +Dox) were picked, pooled, dissociated and replated into methylcellulose hematopoietic media. The resultant primitive erythroid and definitive colonies were scored as aforementioned. Error bars represent standard deviation of the mean number of colonies from triplicate plates. (***P<0.001).

• Supplemental Figure S9 -

  Fig. S9. Changes in Hey1 expression following Dox induction of Notch1-IC or Numb. Flk1+ cells isolated from day 2.75 EBs were aggregated in the presence or absence of Dox for the indicated periods of time and then harvested and analyzed by qPCR for the expression of Hey1. Average expression normalized to Actb is shown.

• Supplemental Figure S10 -

  Fig. S10. Sfrp1 and Wnt5a inhibit primitive erythroid development from Flk1+ cells. Flk1⁺ cells isolated from day 2.75 Notch1-IC EBs were allowed to reaggregate in serum-free media with VEGF in the presence or absence of SFRP1 (500 ng/ml) or Wnt5a (150 ng/ml). Following 2 days of culture, the aggregates were harvested and the cells plated (5×10⁴/ml) into hematopoietic methylcellulose cultures. Colonies were scored as in the legend to Fig. 4. Total colony numbers per treatment were determined by taking into account the total cell number post-reaggregation. Error bars represent standard deviation of the mean number of colonies from three independent experiments. (**P<0.01).

• Supplemental Figure S11 -

  Fig. S11. Expression of Numb, Notch1-IC and activated β-catenin in E7.5 embryos (20× magnification) and negative controls for different staged embryos. Confocal images of whole-mount immunostaining for (A) negative controls (embryo stained with rabbit IgG and Cy3-conjugated secondary antibody). (B) Numb, Notch1-IC and activated β-catenin in E7.5 mouse embryos and yolk sacs (acquired with 20× lens). Yellow arrows show the yolk sac; white arrowheads indicate the posterior primitive streak region in neural plate stage embryos, and white arrows indicate the embryo proper. Broken lines indicate the YS region in somite-stage embryos. Scale bars: 50µm. Red indicates Numb, Notch1-IC or activated β-catenin. Blue indicates DAPI.