Knock-down of tbx5 genes causes cardiac looping defects. (a-c) Embryos injected with control MO or sub-optimal concentrations of tbx5a or tbx5b MOs. (d-d′′) tbx5a morphant phenotypes. (e-e′) tbx5b-morphant phenotypes. (f-h′′) Double knock-down of tbx5 genes (0.5 ng each MO (f-f′′), 1.5 ng each MO (g-g′′) and 3 ng each MO (h-h′′)). (i) Quantification of the degree of looping phenotypes: wt, complete; phenotype, incomplete looping s.o., sub-otimal. (j) Quantification of the looping orientation phenotypes. A χ² statistic has been calculated to assess significant differences between groups (**p < 0.001, *p < 0.05). Images are frontal views of 48 hpf embryos, and myl7 expression is used to highlight the developing heart.

tbx5 morphants exhibit cardiac jogging and lefty2 expression defects. (a-a′′) tbx5a morphant phenotypes. (b-b′′) tbx5b morphants. (c-e′′) Left, right and middle jog phenotypes obtained by co-injection of tbx5a and tbx5b MOs at 0.5 ng (c-c′′), 1.5 ng (d-d′′) or 3 ng (e-e′′) of each MO. (f) Control (ctrl) morphant. (g) Quantification of the phenotypes. (h-j) lefty2 expression in control (h) and double-morphant (i,j) 22-somite stage embryos. (k) Quantification of the phenotypes. A χ² test has been used to assess significant differences between groups (**p < 0.001). All images are dorsal views with anterior to the top, and myl7 expression is used to highlight the developing heart tube in a-f.

The hst mutation is a hypomorphic allele with regards to cardiac laterality. (a) tbx5a variants generated to perform the rescue experiments: a tbx5a full-length (tbx5a FL) version includes the whole N-terminal (N, black rectangle), T- (T, blue rectangle) and C-terminal (C, grey rectangle) domains; a heartstrings version (tbx5a hst) containing the whole N-terminal domain and T-domain and a truncated C-terminal domain; and a tbx5a severely truncated version (tbx5a trunc) that contains the whole N-terminal domain and a truncated T-domain. (b) Quantification of the rescue experiments (wt, left jog; phenotype, right and middle jog). A χ² statistic has been calculated to assess significant differences between groups (**p < 0.01, *p < 0.05). (c,d) Dorsal views with anterior towards the left of 26 hpf +/+ or +/hst (c) and hst mutant (d) embryos showing normal leftward jogging of the embryonic heart tube highlighted by myl7 expression.

tbx5 genes are required for dorsoventral retina organization. (a) Schematics of our quantification method. Expression of efn2a and ephB2 in control (ctrl) embryos (b-b′), tbx5a morphants (c-c′) and tbx5b morphants (d-d′). (e-g′) Expression of efn2a and ephB2 in embryos co-injected with different concentrations of both tbx5a and tbx5b MOs. (h-i) Quantification of the results obtained for the expression of efn2a (h) and ephB2 (i). (j-m) Retinal projections of 48 hpf ath5:GFP embryos injected with control, tbx5a, tbx5b or tbx5a and tbx5b MO. (n) Optic nerve diameter quantifications. Data are represented as the mean ± s.e. A Kruskal-Wallis test was used to determine statistical differences among experimental groups (*p < 0.05, **p < 0.001). D, dorsal; N, nasal; T, temporal; V, ventral.

tbx5b knock-down causes a delay in pectoral fin growth. (a,i,q) Pectoral fin morphology at 3 dpf. Dorsal views are shown with anterior to the top. (b-h) Expression of the developing pectoral fin markers in control (ctrl) MO-injected embryos. (c′-h′) Higher magnifications of (c-h). (j-p) Pectoral fin markers expression in tbx5b-morphant embryos. (k′-p′) Higher magnifications of (k-p). (r,s) tbx5a morphants. (t) Model for the differential requirements for the tbx5 genes during pectoral fin development. b-h, j-p,r,s are dorsal views with anterior to the left. c′-h′,k′-p′ are lateral views with anterior to the left.