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First published online January 23, 2009
doi: 10.1242/10.1242/dev.026906

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Notch mediates Wnt and BMP signals in the early separation of smooth muscle progenitors and blood/endothelial common progenitors

RIKEN Center for Developmental Biology, Laboratory for Early Embryogenesis, Kobe, Hyogo 650-0047, Japan.

View larger version (112K): [in this window] [in a new window]   Fig. 1. Notch active cells are biased to become SMCs in chick. (A) Schematics of ventral mesoderm cell ingression from the posterior primitive streak at stage HH4 and their contribution to three major lineages at HH10. (B) Cells expressing dENotch1 become SMCs at HH10 (lower panels), compared with GFP-expressing cells, which contribute to all three lineages (upper panels). (Left) Whole-mount views (red, -globin; green, electroporated cells); (middle) sections showing the contribution of electroporated cells (red arrowheads, SMCs; yellow arrowheads, ECs; red arrows, BCs); (right) same sections as in middle panel co-stained with SMA. (C) A similar SMC contribution of dENotch1-expressing cells is seen in quail embryos. Red arrowheads, SMCs; yellow arrowheads, ECs; red arrows, BCs; green, QH1 co-staining. (Upper panel) Control GFP-expressing cells contribute to all three lineages; (lower panel) dENotch1-expressing cells have a predominant SMC contribution. Most SMCs can be clearly distinguished from QH1-positive ECs. (D) Magnified view of a region in the lower panel of C. SMCs that are closely associated with the vasculature can still be distinguished from ECs under high magnification.

View larger version (104K): [in this window] [in a new window]   Fig. 2. The Notch pathway is active during early extraembryonic mesoderm generation. (A) Expression of Delta1, Notch1, Lunatic-fringe (L-fringe) and Nrarp at HH3+. Lines indicate section levels shown in B. (B) Sections showing Delta1 and L-fringe expression at the posterior primitive streak level indicated in A. Salt-and-pepper positive staining for Delta1 (in both the epiblast and newly ingressed cells) and L-fringe (weakly in the epiblast cells and strongly in the newly ingressed cells) can be seen. (C) dENotch1 can induce Nrarp cell-autonomously, and both induced and endogenous Nrarp expression can be repressed by DAPT. (Top-left panel) DMSO treatment after dENotch1 electroporation; (bottom-left panel) DAPT treatment after dENotch1 electroporation. Right panels show magnified views near the posterior primitive streak of embryo shown on the left. (D) Inhibition of the Notch pathway by DAPT results in a specific reduction (right) of Notch pathway genes in the posterior primitive streak (left). The neural plate expression of Notch1, L-fringe and Hairy2, however, is not affected.

View larger version (89K): [in this window] [in a new window]   Fig. 3. Mutual antagonism of dHAND and Scl. (A) dHand expression from HH3 to HH10 in whole-mount views. Arrows indicate nascent extraembryonic mesoderm expressing dHand, which marks the SMC lineage. (B) Sections of an HH10 embryo, indicating dHand expression in both somatic (left and middle panels) and vascular (left and right panels) SMCs. (C) dHand-expressing cells are co-positive for SMA. (D) dHand and Scl are expressed in non-overlapping cells during early extraembryonic mesoderm generation (HH4-HH5). (E) dHAND-overexpressing cells are Scl negative, and become SMCs. Red arrowheads indicate regions magnified in right panels. (F)Scl-overexpressing cells are dHand negative, and become mainly BCs with minor EC contribution.

View larger version (67K): [in this window] [in a new window]   Fig. 4. Reduction of Notch activity results in a small increase in BC/EC contribution. (A) Control DMSO treatment from HH3 to HH10. Green indicates that GFP-expressing cells contribute to all three lineages. (B) DAPT treatment from HH3 to HH10 increases the contribution of labeled cells to the BC lineage. Although collapsed extraembryonic coelom makes the distinction of SMCs and ECs difficult, the relative contribution to BCs is assessed with the help of DAPI co-staining (top right). These BC lineage located cells are normal blood cells as revealed by RBC staining (bottom right). (C) Quantification of Scl-positive lineage contribution at HH5 for cells expressing either dENotch1 or DN-Su(H). Cell-autonomous Notch pathway activation by dENotch1 results in a strong bias (P<0.0001) against the Scl-positive, whereas inhibition by DN-Su(H) causes only a minor increase. (D) Quantification of the contribution of GFP-labeled cells in embryos treated with DAPT from HH3-HH10. DAPT causes a small increase in Scl-positive BC contribution (stained with RBC), in comparison with DMSO control.

View larger version (132K): [in this window] [in a new window]   Fig. 5. Roles of BMP and Wnt in SMC and BC/EC induction. Chick embryos were electroporated with expression constructs at HH3 and analyzed at HH4. Brown, cells expressing electroporated genes; blue, in situ staining with indicated genes. (A) Ectopic activation of the BMP pathway by CA-ALK6 leads to the ectopic expression of both Scl and dHand (red arrows). (B) Ectopic activation of the Wnt pathway by CA-β-Catenin results in ectopic dHand, but not Scl, expression. CA-β-Catenin overexpression in the endogenous Scl-expressing region results in its inhibition. Dotted line indicates the level of the section shown in the inset. (C) Ectopic Wnt pathway activation upregulates Notch pathway genes. (Top panels) Control GFP electroporation; (bottom panels) CA-β-Catenin electroporation. Insets indicate whole-embryo views. Yellow arrowhead in insets indicates magnified region. Red arrowheads indicate ectopic induction. (D) CA-β-Catenin induction of Notch pathway genes, L-fringe, shown here, is abolished by DAPT treatment (compare red arrowheads in top and bottom panels). Yellow arrowheads indicate the inhibition of endogenous L-fringe in the posterior primitive streak by DAPT.

View larger version (74K): [in this window] [in a new window]   Fig. 6. BMP-induced ectopic medial Scl expression is regulated by Notch activity. (A) Control GFP expression does not change Scl expression, which progressively weakens more medially. The presence of faint staining in part of the dorsal aorta is normal. (Left) Whole embryo view before anti-GFP staining, but after in situ analysis with Scl; (right) magnified view of the electroporated region after GFP staining of the embryo shown in the left panel. Arrow indicates the electroporated region with no Scl induction. Letter next to the arrow corresponds to the statistical analysis in E. (B) CA-ALK6 induces strong ectopic Scl expression medially (arrows in right panel), which is also prominent in the whole embryo view shown in the left panel. (C) CA-ALK6-induced ectopic Scl expression is abolished by dENotch1 co-expression. (D) DAPT treatment rescues induction by CA-ALK6. (E) Quantification of Scl ectopic induction by CA-ALK6. The dose effect of dENotch1 is indicated by the number reflecting the dENotch1 construct concentration (see also Materials and methods).

View larger version (40K): [in this window] [in a new window]   Fig. 7. A model for the separation of SMC, EC and BC lineages, and the role of Notch activity in separating SMC and BC/EC lineages in relationship with those of the BMP and Wnt pathways. (A) In this model, the main role of the BMP pathway is to ventralize mesoderm. Wnt pathway activation leads to SMC induction. The balance between SMCs and BC/ECs is mediated by the Notch pathway. After multipotential ventral mesoderm progenitors are segregated into dHAND-positive SMC progenitors and Scl-positive BC/EC progenitors, mutual inhibition of dHAND and Scl leads to fate reinforcement. BC/EC progenitors are further segregated into BCs and ECs, mediated by the FGF pathway. (B) A developmental view of how SMC, BC and EC lineages form between HH3 and HH10 in the chick embryo (colors as in A). Soon after the ingression of ventral mesoderm progenitors through the posterior part of the primitive streak (B1), cells are separated into Notch-activity-high and Notch-activity-low types (B2). This separation coordinates with the BMP- and Wnt-mediated induction of BC/EC and SMC progenitors to ensure the proper balance of these two lineages. BC/EC progenitors coalesce to form blood islands (B3). Blood island cells further differentiate into BCs and ECs, and SMC progenitors form both somatic and vascular SMCs (B4).

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