Fig. 4: Reduced TBX5 dosage caused VSDs, perturbations to IVS boundary position and integrity, and abnormal IVS cell arrangement.

a, Mid-posterior optical sections from lightsheet microscopy of a control (Tbx5^CreERT2/+;Mef2cAHF-DreERT2;ROSA26^Ai6/Ai66) heart show Tbx5⁺ lineage (ZsGreen) and Tbx5⁺/Mef2cAHF⁺ lineage (tdTomato immunostaining) cells. Tbx5⁺/Mef2cAHF⁺ lineage cells in the IVS extended from the base (a(i)) to the apex (a(ii)) of the heart, where cells were highly organized. b–d, A spectrum of phenotypes of Tbx5^CreERT2/flox mutants (Tbx5^CreERT2/flox;Mef2cAHF-DreERT2;ROSA26^Ai6/Ai66) were observed, including intact IVS (b–b(ii)), membranous VSD (c–c(ii), asterisk) and AV septal defect (double asterisk in d(i)). b(i),b(ii),c(i),c(ii),d(i), A maldistribution and disorientation of Tbx5⁺/Mef2cAHF⁺ lineage cells were observed in Tbx5^CreERT2/flox mutants. a–d(i), Scale bars, 200 μm. e–h, Linear plot profiles at positions along the anterior–posterior and apical–middle–basal axis (dashed lines) of control (116 profiles from 4 samples) and Tbx5^CreERT2/flox mutant (87 profiles from 3 samples) hearts for Tbx5⁺/Mef2cAHF⁺ lineage cells showed a leftward shift of boundary positioning and broadening of the Tbx5⁺/Mef2cAHF⁺ lineage position, consistent with lineage mixing. The image in e is a repeat of the image in a. f, Cartoon depiction. g,Examples of linear profile plots are shown, and aggregated data are depicted and quantified (h). Statistics determined by two-sided unpaired F-test to compare variance (P = 2.621 × 10⁻¹¹) and two-sided unpaired t-test (P = 9.741 × 10⁻⁵) to compare means. i–s, Orientation scores for cells from each channel (tdTomato⁺ or ZsGreen⁺) were delineated for each genotype, and distributions were plotted as a function of angle. Tbx5^CreERT2/flox mutant hearts scored worse for orientation of Tbx5⁺/Mef2cAHF⁺ lineage (tdTomato⁺) cells in the dominant direction (range from −5° to 5°) and scored higher in the orientation orthogonal to the dominant direction (range from 85° to 95° and −85° to −95°), as determined in s 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 = 3.207 × 10⁻¹⁰, 2.2 × 10⁻¹⁶, 7.839 × 10⁻⁹, for left-to-right plots). i–r, Scale bars, 100 μm. t, Tbx5⁺/Mef2cAHF⁺ lineage (tdTomato⁺) cells showed worse directional coherency scoring in Tbx5^CreERT2/flox mutants (99 regions from 4 controls, 74 regions from 3 mutants) by two-sided Wilcoxon rank sum test with continuity correction (P = 0.003845). u–y, Cell morphometry at E14.5 by WGA staining of cell membranes in the muscular IVS (mIVS) (dashed outline) of controls (3 planes per sample, 2 samples) and Tbx5 mutants (3 planes per sample, 2 samples). Scale bars, 100 μm (u, v), 15 μm (u(i), v(i)). Images of u–v(i)) are repeated in Supplementary Data Fig. 3b,b(i),f,f(i). Quantification of cell diameter (w), area (x) and eccentricity (y). z, Nuclei density shown using DAPI staining of the IVS. Statistics were determined by two-sided unpaired t-tests (w: P = 0.0002955, x: P = 2.9 × 10⁻⁸, y: P = 0.0007001, z: P = 5.296 × 10⁻⁵).

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