Fig. 4.

Fig. 4.

Myocardial Tnnt2a-eGFP degradation recapitulates the tnnt2a mutant phenotype. (A) Schematics of tnnt2a locus showing the eGFP insertion at the C-terminus of the protein in the tnnt2a^bns513 allele and a tol2-generated allele containing the zGRAD system with a TagBFP reporter separated by a P2A peptide under the control of the myocardial myl7 promoter; the zGRAD transgene is composed of the N-terminus of the F-box and WD repeat domain containing 11b (Nfbxw11b) protein and an anti-GFP nanobody (vhh). (B–D) Brightfield images of 48 hpf tnnt2a^{bns513/bns513} (B), myl7:zGRAD-P2A-TagBFP^+/- (C), and tnnt2a^{bns513/bns513}; myl7:zGRAD-P2A-TagBFP^+/− (D) embryos; arrowhead and asterisks indicate, respectively, the presence and absence of pericardial edema. (B'–D’ ) Brightfield images and kymographs of hearts from 48 hpf tnnt2a^{bns513/bns513} (B’ ), myl7:zGRAD-P2A-TagBFP^+/− (C’ ), and tnnt2a^{bns513/bns513}; myl7:zGRAD-P2A-TagBFP^+/− (D’ ) embryos; green lines outline the ventricle (V), blue lines outline the atrium (A), and vertical white lines indicate the reference axis of the kymographs. (E–G) Confocal image of hearts from 48 hpf tnnt2a^{bns513/bns513} (E), myl7:zGRAD-P2A-TagBFP^+/− (F), and tnnt2a^{bns513/bns513}; myl7:zGRAD-P2A-TagBFP^+/− (G) embryos stained for F-actin with Phalloidin; maximum z-projection; annotations correspond to the ventricle (V) and atrium (A). “Red hot” lookup table coloring (from Low to High) highlights the SD (B–D) or 3D variance (B’–D’). Diagrams indicate the anterior–posterior (A–P), dorsal–ventral (D–V), and left–right (L–R) axes.