tbx1 expression and heart looping defects in tbx1^-/- embryos.

(A) cmlc2 ISH in tbx1^-/- mutants and WT siblings at specification (14 somites), fusion (21 somites), linear heart tube (24 hpf), jogging (30–36 hpf) stages. (B) By 48 hpf the heart has finished looping in WT forming an acute angle between the atrial and ventricular axes (green lines), while in tbx1^-/- mutants the axes are nearly parallel. (C) Comparison of atrio-ventricular axis angles between wild-type (left, green) and tbx1^-/- mutants (right, red). N indicates the total number of embryos represented in the plot. (D) 5 micron sections of the hearts from 72 hpf embryos, showing wider ventricle and atrium and thinner heart walls in tbx1^-/- mutant as compared to WT sibling. Arrows indicate the AVC; a, atrium; v, ventricle. Scale bar: 50 μm.

Cell shape defects in tbx1^-/- mutants.

(A, B) Confocal projections of hearts from Tg(cmlc2:EGFP) embryos at 48 hpf stained with Alcama antibody (red) to demarcate cell boundaries. Cells in the outer and inner chambers are outlined in white and yellow respectively. The insets in E and F show a magnified view of the outer curvature cell outlined in white. The plot in C shows that cells in the outer chamber of tbx1^-/- mutants are rounder (circularity tending towards 1) as compared to WT. (D) Cell shape is unchanged in the inner chamber of tbx1^-/- mutants. Each bar in plots C and D represents data collected from 70 cells in 7 different embryos. Arrows point to the AVC; v, ventricle; a, atrium. Scale bars: 25 μm.

Regional differentiation defects in tbx1^-/- mutants.

48 hpf embryos stained for region-specific markers (A–H) and their schematic representation (I–L). Red lines indicate myocardium, grey lines indicate endocardium and blue lines indicate gene expression. bmp4 (OFT, AVC and IFT), notch1b (AVC endocardium) and versican (AVC myocardium) expression is down-regulated in tbx1^-/- mutants (E–G) as compared to WT siblings (A–C). The insets in C and G show versican expression in the ear as a control for ISH. anf expression is localized to the outer parts of the chamber myocardium in WT (D), but is broadly expressed in the ventricle and atrium in tbx1^-/- mutants (H). Arrows point to the AVC in all panels; v, ventricle; a, atrium. The plots in M–P show the penetrance of the phenotype as percentage embryos with absent/mis-localized expression of the respective gene. N depicts the total number of embryos represented in the plot. Scale bars: 50 μm.

Bulbous arteriosus defects in tbx1^-/- embryos.

Dissected hearts from 72 hpf embryos showing the BA region (A, D) outlined in white, with the bidirectional arrowheads in black and red showing the length and width of BA, respectively. (B, E) The corresponding images showing staining for Alcama antibody (red) and DAF-2DA (green). (C, F) tbx1^-/- mutants at the same stage have absent eln2 expression. Black arrows indicate the BA. (G) Quantification of BA width in WT and tbx1-/- mutants, while (H) demonstrates that BA length is reduced in tbx1^-/- mutants; * p-value = 0.0045. N = 13 for all measurements. Scale bars: 25 μm.

Heart looping and regional differentiation defects in wnt11r^-/- mutants.

(A) cmlc2 ISH showing the looping defect in wnt11r^-/- mutants. (B) Atrio-ventricular axis angle in wild-type (left, green) and wnt11r^-/- mutants (right, blue). N indicates the total number of embryos from 3 experiments. (C) Defective regional differentiation of heart in wnt11r^-/- mutants, similar to those seen in tbx1^-/- mutants. Scale bars: 50 μm.

wnt11r and tbx1 cooperate to regulate heart looping and differentiation.

(A-C) ISH showing wnt11r expression in the fusing heart fields (21 somites, A), linear heart tube (24 hpf, B) and fully looped heart (48 hpf, C) in WT embryos. (D, E) Dissected hearts from WT (D) versus tbx1^-/- mutant (E) embryos stained for wnt11r expression at 48 hpf. The heart boundaries are indicated by dotted lines. Arrows point to the AVC. Scale bars: 50 µm. (F, G) Quantification of penetrance of looping and regional differentiation defects in double heterozygotes as compared to single heterozygotes of tbx1 and wnt11r. WT is shown as negative control, and tbx1^-/- and wnt11r^-/- mutants as positive control for looping and regional differentiation defects. N depicts the total number of embryos from 3 experiments.

tbx1 and wnt11r regulate Alcama levels, and alcama regulates heart looping and differentiation.

(A, C) ISH analysis on 48 hpf embryos showing that wnt11r^-/- mutants have unaffected tbx1 expression. (B, D, E, F) Antibody staining showing that wnt11r^-/- (D) and tbx1^-/- (F) mutants have down-regulated Alcama expression when compared to WT siblings (B, E). cmlc2 ISH on 48 hpf embryos showing the various looping phenotypes observed in alcama morphants (H) and proper looping in an uninjected larva (G). notch1b is expressed in the AVC endocardium in an uninjected larva at 48 hpf (I), but is down-regulated in alcama morphants (J). Scale bars: 50 µm.

tbx1 expression and heart shape defect of tbx1^-/- 1arvae. ISH for tbx1 in WT embryos showing expression in the fusing heart fields (21 somites, A), linear heart tube (24 hpf, B) and the looped heart (48 hpf, C). A and B are dorsal views and C is a head-on view. Arrowheads in A and B point towards pharyngeal pouches and the cardiac cells are outlined in A–C. (D) Schematic of head-on view of 48 hpf heart. Arrow indicates the AVC; a, atrium; v, ventricle. Dotted red lines indicate the widths at the ends and at the widest part of the ventricle and atrium. The longitudinal axes (shown in green) were drawn by joining the midpoints m and n in ventricle and midpoints p and q in atrium. mo and pr were measured for length of ventricle and atrium, respectively (see Materials for details). (E) Measurement of lengths of the ventricle and atrium in WT and tbx1^-/- mutants. (F, G) Measurement of the widths of the ventricles at 48 hpf and atriums at 72 hpf in tbx1^-/- mutants and WT siblings in the expanded and contracted states. WT siblings are represented in green and tbx1^-/- mutants in pink. N indicates the total number of embryos represented in the plot. Scale bars: 50 μm.

Proliferation defects in tbx1^-/- mutants. (A, B) Confocal projections of hearts from cmlc2:gfp (all heart cells are green) embryos at 33 hpf stained with phospho-histone 3 antibody (proliferating cells are red). The insets in A and B show a magnified view of a co-labeled cell (yellow). (C, D) Plots showing the total (C) and proliferating (D) number of cells in WT and tbx1^-/- mutants. N is the total number of embryos. Arrows point to the AVC; v, ventricle; a, atrium. Scale bar: 25 μm.

Chambers are specified correctly in tbx1^-/- mutants. ISH analysis at 48 hpf for vmhc (A, C) and amhc (B, D) shows that the chambers are correctly specified in tbx1^-/-mutants. The dotted lines indicate the heart boundary. Scale bars: 50 μm.

tbx1^-/- embryos have defects in heart performance. (A) Measurement of ventricular contractility as shortening fraction [(width at diastole – width at systole)/width at diastole] shows no difference between WT and tbx1^-/- embryos. (B) Determination of atrium contractility between WT and tbx1^-/- embryos. The stroke volume (ventricular volume [diastole - systole]) plotted in C is unaffected in tbx1^-/- embryos, but the heart rate is decreased, shown in D. N = 19 in all experiments. WT siblings are represented in green and tbx1^-/- mutants in pink. p-values are reported under each measurement.

Candidate gene analysis for tbx1^-/- mutants. Whole mount lateral views (A, B, F, G) and head-on views (C–E, H–J) of 48 hpf embryos stained for known genes that affect heart looping. Expression of tbx2a (A, F), tbx3b (B, G), tbx5 (C, H) and tbx20 (D, I) was unaffected between WT siblings and tbx1^-/- mutants. (E, J) Dissected hearts from 48 hpf embryos show down-regulated expression of wnt11r in the mutant (J) versus WT (E). The heart boundary is shown by the red dotted line. Arrows point to the AVC. Scale bars: 50 7mu;m.

Unaffected BA in wnt11r ^-/- mutants. (A, B) Lateral and ventral views of 72 hpf larva showing normal eln2 and DAF-2DA staining respectively in wnt11r^-/- mutants. Scale bars: 50 μm.