Extended Data Fig. 8: Genetic and chemical mutant rescue assays.
From: Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation
Related to Figs. 2, 3. a–d, Analysis of wild-type, Nrg1tm/tm, Tie2creRbpjfl/fl and Tie2creRbpjfl/flNrg1tm/tm mutant embryos showing histology (a), normalized ECM and trabecular area quantifications (b), immunofluorescence of N1ICD (c) and fold change in the number of N1ICD+ cells (d). e–g, Analysis of wild-type, Nrg1tm/tm, Tie2creNotch1fl/fl and Tie2creNotch1fl/flNrg1tm/tm mutant embryos showing histology (e), normalized ECM and trabecular area quantifications (f) and immunofluorescence of N1ICD (g). Note the persistent N1ICD staining in the Tie2creRbpjfl/flNrg1tm/tm mutant due to mosaic Cre-mediated deletion of Notch1. Here, N1ICD appears to nucleate touchdowns. Cre-mediated deletion was complete in Tie2creNotch1fl/flNrg1tm/tm mutants at E9.0, although mosaicism in Cre-recombination at an earlier time point may allow N1ICD restriction and nucleation of touchdowns. h, i, H&E of Nrg1tm/tm and wild-type embryos treated with 25 nM NRG1 or PBS as control for 24 h (h) with associated area quantifications (i). j–l, Immunofluorescence of N1ICD (j) and DLL4 (k) with quantification the number of N1ICD+ cells (l). m–q, Analysis of wild-type and Tie2creNotch1fl/fl embryos treated with 10 μM SU5416 or DMSO control for 24 h, showing histology (m), ECM and trabecular area quantifications (n) and immunofluorescence in SU5416- and control-treated wild-type embryos of N1ICD (o) and DLL4 (p), with fold change in the number of N1ICD+ cells (q). In H&E panels: trabecular myocardium (black arrows), ECM (white arrows). In all panels showing molecular expression: expression (arrows), reduced expression (arrowheads), increased expression (thick arrows). 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). a–f, j–l, o–q, n = 3 independent embryos were used; h, i, m, n, n = 5, except in h, i, n = 6 wild-type embryos treated with NRG1. 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 = 0.013; 2, P = 0.0003; 3, P = 0.012; 4, P = 0.009; 5, P = 0.009; 6, P = 0.029; 7, P = 0.033. d, 1, P = 0.024; 2, P = 0.03. f, 1, P = 0.039; 2, P = 0.023; 3, P = 0.034; 4, P = 0.012; 5, P = 0.009; 6, P = 0.045; 7, P = 0.034. i, 1, P = 0.0014; 2, P = 0.0006; 3, P = 0.03; 4, P = 0.0004; 5, P = 0.0046; 6, P = 0.022. l, 1, P = 0.02; 2, P = 0.0042. n, 1, P = 0.0025; 2, P = 0.024; 3, P = 0.016; 4, P = 0.0033; 5, P = 0.00015; 6, P = 0.0017. q, 1, P = 0.022. Scale bars, 50 μm (a, c, e, g, h, j, k, m, o, p).
