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A) Experimental strategy of E10.5 ventricular explants treated with recombinant Bmp2. B) Immunostaining for Erg (green) and Vegfr2 (blue) in E10.5 lineage labeled ventricular explants. Boxed regions show no ECs sprouting in explants treated with 2% FBS (vehicle) or 20ng/ml rBmp2. Combined Vegfa and Bmp2 treatment (rightmost panel) strongly induces endo-derived CVs sprouting (tdTomato+ cells, arrowheads). C) Quantification of total number of endo-derived CVs (tdTomato+ in sprouting ECs) from E10.5 ventricular explants shows increased endo-derived CVs sprouting in explants treated with combination of Vegfa and Bmp2. (rBmp2, n = 5 explants; Vegfa, n = 6 explants; combination, n = 6 explants) D) Experimental strategy of neonatal left coronary artery (lca) ligation on lineage traced hearts injected with cardiac specific <t>AAVs</t> expressing <t>GFP</t> <t>(AAV-</t> cTnT-GFP, vehicle) or Bmp2 (AAV-cTnT-GFP-p2A-Bmp2) in E and F . E) P18 tissue sections from BmxCreERT2;Rosa tdTomato injured hearts stained for tdTomato (red) and isolectin B4 (IB4, blue). Boxed regions show increased endo-derived CVs (arrowheads) in the left ventricles injected with AAV expressing Bmp2 injected hearts ( n = 9 hearts, lower panels) compared with vehicle-treated hearts (n=8, upper panels). F) Quantification of total number of tdTomato + CVs per vessel area. Scale bar=200 μM in B, and E (full view). Scale bar=50 μM in B, and E (boxed regions). Data are mean ± s.d. ***P ≤ 0.001, ****, P ≤ 0.0001, by Student’s t-test.
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A) Experimental strategy of E10.5 ventricular explants treated with recombinant Bmp2. B) Immunostaining for Erg (green) and Vegfr2 (blue) in E10.5 lineage labeled ventricular explants. Boxed regions show no ECs sprouting in explants treated with 2% FBS (vehicle) or 20ng/ml rBmp2. Combined Vegfa and Bmp2 treatment (rightmost panel) strongly induces endo-derived CVs sprouting (tdTomato+ cells, arrowheads). C) Quantification of total number of endo-derived CVs (tdTomato+ in sprouting ECs) from E10.5 ventricular explants shows increased endo-derived CVs sprouting in explants treated with combination of Vegfa and Bmp2. (rBmp2, n = 5 explants; Vegfa, n = 6 explants; combination, n = 6 explants) D) Experimental strategy of neonatal left coronary artery (lca) ligation on lineage traced hearts injected with cardiac specific AAVs expressing GFP (AAV- cTnT-GFP, vehicle) or Bmp2 (AAV-cTnT-GFP-p2A-Bmp2) in E and F . E) P18 tissue sections from BmxCreERT2;Rosa tdTomato injured hearts stained for tdTomato (red) and isolectin B4 (IB4, blue). Boxed regions show increased endo-derived CVs (arrowheads) in the left ventricles injected with AAV expressing Bmp2 injected hearts ( n = 9 hearts, lower panels) compared with vehicle-treated hearts (n=8, upper panels). F) Quantification of total number of tdTomato + CVs per vessel area. Scale bar=200 μM in B, and E (full view). Scale bar=50 μM in B, and E (boxed regions). Data are mean ± s.d. ***P ≤ 0.001, ****, P ≤ 0.0001, by Student’s t-test.

Journal: bioRxiv

Article Title: Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4

doi: 10.1101/2021.10.25.465773

Figure Lengend Snippet: A) Experimental strategy of E10.5 ventricular explants treated with recombinant Bmp2. B) Immunostaining for Erg (green) and Vegfr2 (blue) in E10.5 lineage labeled ventricular explants. Boxed regions show no ECs sprouting in explants treated with 2% FBS (vehicle) or 20ng/ml rBmp2. Combined Vegfa and Bmp2 treatment (rightmost panel) strongly induces endo-derived CVs sprouting (tdTomato+ cells, arrowheads). C) Quantification of total number of endo-derived CVs (tdTomato+ in sprouting ECs) from E10.5 ventricular explants shows increased endo-derived CVs sprouting in explants treated with combination of Vegfa and Bmp2. (rBmp2, n = 5 explants; Vegfa, n = 6 explants; combination, n = 6 explants) D) Experimental strategy of neonatal left coronary artery (lca) ligation on lineage traced hearts injected with cardiac specific AAVs expressing GFP (AAV- cTnT-GFP, vehicle) or Bmp2 (AAV-cTnT-GFP-p2A-Bmp2) in E and F . E) P18 tissue sections from BmxCreERT2;Rosa tdTomato injured hearts stained for tdTomato (red) and isolectin B4 (IB4, blue). Boxed regions show increased endo-derived CVs (arrowheads) in the left ventricles injected with AAV expressing Bmp2 injected hearts ( n = 9 hearts, lower panels) compared with vehicle-treated hearts (n=8, upper panels). F) Quantification of total number of tdTomato + CVs per vessel area. Scale bar=200 μM in B, and E (full view). Scale bar=50 μM in B, and E (boxed regions). Data are mean ± s.d. ***P ≤ 0.001, ****, P ≤ 0.0001, by Student’s t-test.

Article Snippet: AAVs particles were then purify from AAV-producing cells using AAVpro Purification Kit Maxi (Takara, Cat. #6666).

Techniques: Recombinant, Immunostaining, Labeling, Derivative Assay, Ligation, Injection, Expressing, Staining