Extended Data Fig. 4: Additional samples of Tbx5 mutant hearts and supplementary quantification of distribution and cell arrangement.
From: A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects
a–h, Volume renderings from lightsheet microscopy of anterior and posterior surfaces, as well as anterior and mid-anterior 4-chamber optical sections of Tbx5CreERT2/+ control (Tbx5CreERT2/+;Mef2cAHF-DreERT2;ROSA26Ai6/Ai66) and Tbx5CreERT2/flox mutant (Tbx5CreERT2/flox;Mef2cAHF-DreERT2;ROSA26Ai6/Ai66) hearts at E14.5 display Tbx5+ lineage (ZsGreen) and Tbx5+/Mef2cAHF+ lineage (tdTomato immunostaining) cells. (a-h) scale bars: 200 microns. i–k, Quantification of the anatomical location of tdTomato+ and ZsGreen+ cells in Tbx5CreERT2/flox mutants (n = 3) and Tbx5CreERT2/+ controls (n = 4), by region at E14.5 as outlined in (i). Over 1200 3-dimensional regions of interest were drawn blinded, and then integrated fluorescence signal was assessed. j, Although no regional differences in the intensities of ZsGreen+ were detected, tdTomato+ displayed higher integrated fluorescence in the left ventricle (LV) and less signal in atrioventricular complex (AVCo) complex region by two-sided unpaired T-tests (AVCo: p = 0.01933, interventricular septum (IVS): p = 0.3679, left atrium (LA): p = 0.2829, LV: p = 0.0001574, right atrium (RA): p = 0.1678, right ventricle (RV): p = 0.05063). k, Length-normalized widths of LV, muscular IVS (mIVS), RV and total heart at E14.5 were not significantly different between control (n = 4) and Tbx5 mutants (n = 3) based on regions of interest in (i), by two-sided unpaired T-tests (p = 0.9654, 0.1277, 0.3797, 0.5631 for left-to-right plots). Median, first and third quartiles are shown in box plots. Further details are in Source data. l–u, Tbx5CreERT2/flox mutant hearts at E14.5 scored worse for orientation scores of Tbx5+ lineage (ZsGreen+) cells in the dominant direction (range from -5 to 5 degrees) and scored higher in the orientation orthogonal to the dominant direction (range from 85 to 95 degrees and -85 to -95 degrees), as determined (t) by two-sided Watson U2 test across all orientations (p < 0.001) or by two-sided Wilcoxon rank sum test with continuity correction for selected orientations (p-values < 2.2e-16 for left and middle plots, p-value = 0.0006863 for right plots). u, Tbx5+ lineage (ZsGreen+) cells demonstrated worse directional coherency scoring in Tbx5CreERT2/flox mutants by two-sided Wilcoxon rank sum test with continuity correction (p = 0.03316). (l-s) scale bars are 100 microns.
