Extended Data Fig. 9: Effect of COUP-TF2 overexpression during coronary vessel development.
From: Single-cell analysis of early progenitor cells that build coronary arteries
a, Schematic of transgenes used to study Coup-tf2 overexpression in coronary cells. b, c, Recombination is not complete in the SV with tamoxifen at E9.5 and E10.5 as shown in whole-mount confocal images (b) and quantification (c). Control GFP is visualized by direct fluorescence, and COUP-TF2OE through immunostaining for the myc tag. For c, ApjCreER RosamTmG, n = 5 hearts. ApjCreER Coup-tf2OE, n = 6 hearts. d, Tamoxifen dosing at E11.5 and E12.5 fills capillaries with recombined cells, but still resulted in Coup-tf2OE cells being excluded from arteries (A). e, Induction of Coup-tf2OE throughout vasculature shows that overexpressing cells can exist in arteries. f, Quantification of ventricle coverage at E12.5. n = 4 control hearts, n = 7 COUP-TF2OE hearts. ns, P > 0.05. P = 0.8868. g, Whole-mount confocal images of control and Coup-tf2OE hearts at different stages of development. Coronary migration (dotted line) on the dorsal side of the ventricle (outlined with solid line) is similar in both genotypes. h, High magnification of E12.5 Coup-tf2OE heart shown in g highlights the positioning of transgenic cells at both the leading front and trailing cells. i, COUP-TF2OE cells can become part of the JAG-1-positive artery if induced after pre-artery specification with Cx40CreER. j, Mosaic experiment in which constitutive expression of the NOTCH intracellular domain (NICD) is induced at the same time as Coup-tf2OE. This manipulation creates a vasculature containing three different transgene combinations: 1. NICD; 2. COUP-TF2OE; or 3. NICD + COUP-TF2OE (arrowheads). Those containing just the NICD (category 1) are the only transgenic cells that contribute to arterial vessels. k, Quantification of the percentage of endothelial cells in capillaries and arteries (Art) with the three transgenic combinations. NICD-expressing cells preferred arteries whereas COUP-TF2OE cells avoid arteries, the latter of which was not rescued by NICD. n = 6 hearts. **P ≤ 0.01; ****P ≤ 0.0001. For NICD capillary versus artery, P = 0.0070. For COUP-TF2OE capillary versus artery, P = 7.49224 × 10−5. For COUP-TF2OE + NICD capillary versus artery, P = 8.07734 × 10−5. l, The CDK inhibitor flavopiridol increased arterial specification (Cx40) in an SV sprouting assay. n = 33 control explants, n = 38 treated explants. ***P ≤ 0.001. m, Immunostaining of endothelial sprouts (VE-cadherin+) migrating from SV or atria tissue explants with Cx40 showed the increase in this arterial marker (arrowheads) with flavopiridol treatment. Data shown as mean ± s.d. A two-tailed unpaired t-test was performed to determine P values. Scale bars, b, 20 μm; d, e, g–j, 100 μm; m, 25 μm.
