1. Volume 22, Issue 3, Pages 501-514 (March 2012)
Stalk Cell Phenotype Depends on Integration of Notch and Smad1/5 Signaling Cascades 
Iván M. Moya, Lieve Umans, Elke Maas, Paulo N.G. Pereira, Karen Beets, Annick Francis, Ward Sents, Elizabeth J. Robertson, Christine L. Mummery, Danny Huylebroeck, An Zwijsen 
Developmental Cell 
Volume 22, Issue 3, Pages (March 2012)
DOI: /j.devcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Developmental Cell 2012 22, 501-514DOI: (10.1016/j.devcel.2012.01.007)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Normal Vasculogenesis but Impaired Angiogenesis in dKOEC Embryos
(A) E13.5 control embryo (left) and embryos containing one functional allele of Smad1 or Smad5 in ECs.
(B–D) Embryos lacking the four alleles of Smad1 and Smad5 in endothelium. (B) Control and mutant E8.5 whole-mount and sectioned R26R reporter embryo stained with X-gal (blue). (C and D) Whole-mount and flat-mounted X-gal stained yolk sacs of control and mutant E9.5 R26R reporter embryos. In (D) magnified views are shown of the head and roof of the hindbrain (middle and right panels). cv, cardinal vein; da, dorsal aorta; s, somites. Scale bars: 250 μm (left panels) and 150 μm (D, middle and left panels).
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 2 Smad1/5 Signaling Regulates Angiogenesis
(A) Dorsal vascular plexus (anti-CD31/anti-Nrp1) in flat-mounted hindbrain roofs (E9.5). Asterisks and boxed areas (right panels) show sprouts quantified in (B).
(B and C) Quantification of sprouts (B) and dorsal anastomoses (C).
(D) Dorsal aortae (anti-CD31) from control and dKOEC E9.5 embryos. Arrowheads show ectopic sprouts.
(E and F) Quantification of proliferation (phospho H3-positive ECs) in the dorsal vascular plexus.
(G) Position of the Golgi apparatus (anti-GM130) in tip cells.
(H) Filopodia (anti-Nrp1) in endothelium (anti-CD31). Boxed region in (G and H) are magnified in right panels. tc, tip cell.
Quantifications are averages from several embryos (B, C, n ≥ 21; E, n ≥ 11). Scale bars: 200 μm (A, left panels), 20 μm (A, boxed area; G, left panels); 150 μm (D and F, left panels); 40 μm (H); 4 μm (G, boxed area), and 7 and 10 μm (H, boxed area, left and right, respectively).
See also Figures S2 and S3.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 3
Smad1/5 Signaling Regulates Stalk Cell Competence via Id Proteins
(A) Anti-p-Smad1/5/8 staining in endothelium of E9.5 control and dKOEC littermates. Nuclei of tip and stalk cells are highlighted by dashed lines.
(B) GFP localization (BRE:GFP) in early stalk cells (middle panels) and elongated sprouts (bottom).
(C) Id protein localization in wild-type yolk sac artery (top panels) and embryonic vasculature (middle panels). Absence of Id proteins in mutant sprouts (bottom panels). Yellow and white arrowheads indicate pan-Id/GFP positive or negative cells respectively. Asterisks represent red blood cells.
(D) Schematic representation of the dynamic p-Smad1/5/8 and Id/BRE:GFP localization in developmental angiogenic sprouts.
(E) Chimeric tube formation assay and quantification of Smad1/5KD (green, calcein) or WT (red, DiI-AC-LDL) leading “tip” cells.
(F) Chimeric tube formation assay and quantification of Id1OE/Id3OE (green, calcein) or WT (red, DiI-AC-LDL) leading “tip” cells. sc, stalk cell; tc, tip cell. Quantifications are averages from several sprout/tubular structures (E, n ≥ 50; F, n ≥ 49). Scale bars from top to bottom: 30 μm, 40 μm, 15 μm, and 30 μm in (A); 50 μm, 15 μm, 15 μm, and 15 μm in (B); 30 μm, 15 μm, 15 μm, and 30 μm in (C).
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 4 Cross-Signaling between Notch and Smad1/5 Pathways
(A) GFP localization in endothelium (anti-CD31) in E9.5 BRE:GFP dorsal explants exposed to different treatments. The arrows heads show ectopic intrasomitic vessels. Quantification of GFP-positive ECs from dorsal aorta.
(B–E) mRNA expression analysis of Id1-3 in embryo explants (B) and HUVECs (C). Hes1 and Hey1 expression in embryo explants (D) and HUVECs (E). Scale bars: 50 μm.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 5
Smad1/5-Mediated Regulation of Dll4/Notch Signaling in Endothelial Cells
(A and B) Aberrant Dll4 (A) and NICD (B) localization in dKOEC angiogenic sprouts. In (B), left and middle panels are identical. Boxed areas of presumptive tip cells are magnified at the right. Asterisks represent red blood cells.
(C and D) Effect of Smad1/5 siRNA (C) or Id1/3 overexpression in the regulation of tip- and stalk-cell-enriched transcripts in HUVECs.
(E and F) p-Smad1/5/8, Id1, and Hes1 proteins levels after Sm1/5 siRNA-mediated downregulation (E) and Id1/3 overexpression (F) in HUVECs. sc, stalk cell; tc, tip cell; tlc, tip-cell-like cells. Scale bars from left to right: 25 μm in (A); 30 μm in (B) left and middle panels and 5 μm in right panels.
See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

8. Figure 6 Interaction of Hes1 and Id Proteins in ECs
(A) Endogenous Hes1 and Id localization in HUVECs treated with Dll4, BMP6, DAPT, or Dorsomorphin.
(B) Hes1/Ids heteromers detected by in situ PLA using pan-Id and Hes1 antibodies.
(C) PLA signal quantification of (B).
(D and E) Reduced levels of Hes1/Ids heteromers upon Smad1/5 siRNA transfection.
(F and G) Dynamic regulation of Hes1/Ids and Hey1/Id3 heteromers in function of time. p values were calculated using two-tailed Student's t test (C and D, n ≥ 71; G, n ≥ 105). dorso, dorsomorphin hydrochloride.
Scale bars: 15 μm (A and F), 15 μm (B and D). See also Figure S7.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

9. Figure 7
Schematic Model of the Role of Smad1 and Smad5 in Dll4/Notch-Mediated Stalk Cell Behavior
(A) In wild-type embryos, the Dll4-rich leading tip cell (red) of the emerging sprout activates in adjacent cells higher levels of Notch signaling which directs them to acquire a stalk cell behavior (blue). Notch activation and target gene expression (Hey and Hes) downregulates the expression of VEGFR2, VEGFR3 and Dll4, and upregulates VEGFR1 resulting in stalk cell behavior. Downstream of Smad1/5 signaling, Id proteins release the negative autoregulatory loop of Hes1 in stalk cells. Conversely, increasing levels of the Notch target Hey1 will progressively inhibit Id steady-state levels, which will then indirectly cause attenuation of the Notch pathway by Hes1 downregulation.
(B) Absence of Id proteins in Smad1/5 deficient ECs will result in Hes1 downregulation. High levels of VEGFR2 and Dll4, and deficient Notch target gene expression in stalk cells turn them into a tip-cell-like cell phenotype (pink) and prevents the downregulation of Notch1 signaling (nuclear NICD accumulation) in the leading “tip” cells. The failure to balance tip versus stalk cell ratios and to transiently stabilize stalk cells, together with impaired tip cell polarity and directed migration, is likely to cause excessive sprouting and vessel coalescence in mutant embryos.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions