Extended Data Fig. 7: Manipulation of ECM dynamics in RBEC assays and marker analysis of Tie2creNotch1GOF mutant embryos.
From: Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation
Related to Fig. 2. a–d, H&E analysis of wild-type and Tie2creNotch1fl/fl embryos treated with 20 turbidity reducing units (TRU) of hyaluronidase (HYAL) or PBS as control (a, b) or wild-type and Nrg1tm/tm embryos treated with 20 μM GM6001 or DMSO (c, d) in RBECs for 24 h, and accompanying ECM and trabecular area quantifications normalized to chamber perimeter (b, d). e–g, H&E of Tie2creNotchGOF embryos (e, f (right)) compared to wild-type embryos (e, f (left)) at E8.5 and E10.0 with morphological quantification of ECM and trabecular areas normalized to chamber perimeter (g). h–q, Molecular analysis of Tie2creNotchGOF embryos relative to wild-type embryos at E8.5 (h–l) and E9.5 (m–q): ISH of Adamts1 (h, m) and Has2 (l, p); immunofluorescence analysis of neoversican (i, n) and pERBB2 (k); RNA-scope of Nrg1 (o); qPCR analysis of the respective markers (j, q). Gene/protein expression panels: expression (arrow), reduced expression (arrowhead), increased expression (thick arrow). In all immunofluorescence images: markers associated with endocardium or ECM (red); markers associated with myocardium (orange); myocardium is stained for SMA (green dulled mask); nuclei (DAPI, blue). e–i, k–p, n = 3 independent embryos were used; a–d, n = 5, except in d, n = 6 wild-type embryos treated with GM6001. j, q, 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. b, 1, P = 1.99 × 10−6; 2, P = 0.025; 3, P = 9.28 × 10−6; 4, P = 0.00085; 5 = 2.83 × 10−5; 6, P = 0.00067; 7, P = 0.026; 8, P = 0.00014. d, 1, P = 0.024; 2, P = 0.0024; 3, P = 0.018; 4, P = 0.000021; 5, P = 0.016; 6, P = 0.022; 7, P = 0.028. g, Top, 1, P = 0.018; 2, P = 0.017; bottom, 1, P = 0.0071; 2, P = 0.03. j, 1, P = 0.03; 2, P = 0.041. q, 1, P = 0.034; 2, P = 0.00026; 3, P = 0.025; 4 = 0.038. Scale bars, 55 μm (a, c), 30 μm (e, h–l) and 40 μm (m–p).
