Extended Data Fig. 6: Analysis of ECM synthesis and degradation markers, and proliferation, in Nrg1tm/tm and Tie2creNotch1fl/fl mutants.
From: Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation
Related to Fig. 2. a–k, m, o–q, Analysis of ECM synthesis (a–h) and degradation (i–k) markers, BRG1 (m), Notch pathway (o–p) and VEGFA pathway (q) in left ventricle of Nrg1tm/tm mutants at E9.0 (a–q (second left)) and Tie2creNotch1fl/fl mutants at E8.5 (a–q (right)) relative to wild-type embryos (a–q (left, second from the right)) showing immunofluorescence analysis of HABP (a), CD44 (b), fibronectin (c), perlecan (d), aggrecan (e), laminin (f), collagen type I (g), collagen type IV (h), BRG1 (m), DLL4 (o) and VEGFR2 (q); ISH for Mmp2 (i), Adamts5 (j), Hyal2 (k) and Efnb2 (p); and qPCR (l). n, Percentage of BRG1 positive cells compared to the total number of cells in the left ventricle endocardium and myocardium of Nrg1tm/tm(n (left)) and Tie2creNotch1fl/fl (n (right)) mutants. r–u, Proliferation analysis by Ki-67 immunofluorescence of Nrg1tm/tm mutants at E9.0 (r (second left)) and E9.5–E10.0 (t (second left)); Tie2creNotch1fl/fl mutants at E8.5 (r (right)) and E9.5 (t (right)), relative to wild-type embryos (r, t, (left, second from the right); and the related quantifications of total myocardium proliferation and the proportion of trabecular cardiomyocytes compared to the total cardiomyocytes (s, u). Gene/protein expression panels: expression (arrow), reduced expression (arrowhead). In all immunofluorescence: markers associated with endocardium or ECM (red); markers associated with myocardium (orange); myocardium is stained for SMA (green dulled mask); nuclei (DAPI, blue). a–u, n = 3 independent embryos per genotype. l, n = 3 biological replicates each evaluated for three experimental replicates. Quantitative data are shown as mean ± s.e.m. Two-sided Student’s t-tests (without corrections for multiple comparisons) were used. Significant comparisons are indicated by numbers as follows. l, Left, 1, P = 0.0066; 2, P = 0.0038; 3, P = 0.017, 4, P = 0.035; 5, P = 0.036; right, 1, P = 0.0039; 2, P = 0.039; 3, P = 0.00053; 4, P = 0.0074; 5, P = 0.022. n, 1, P = 0.006. s, Left, 1, P = 0.029; right, 1, P = 0.012. u, Left, 1, P = 0.045; 2, P = 0.017; right, 1, P = 0.04; 2, P = 0.0031. Scale bars, 55 μm (a–k, m, o–r) and 40 μm (t).
