Fig. 4

Chemical manipulation of zebrafish heart development by Bcl9–β-catenin inhibitor LH-2-40. (A,B) Treatment with 10 µM LH-2-40 from the two- to four-cell stages does not result in gastrulation defects (lateral views). (C–E) Bright-field images of 5-dpf DMSO controls (C) and representative embryos treated with 10 µM LH-2-40 from the four-cell stage on (D,E), as assessed for phenotype classes (quantified in F–H) (lateral views; anterior is to the left). Phenotypes in LH-2-40-treated embryos become visible at 5 dpf, comparable with phenotypes observed in bcl9^Δ29 mutants. LH-2-40-treated embryos show a variable phenotype expressivity, with mild to strong swim bladder inflation defects (arrowheads; D,E) linked with variable craniofacial defects (asterisks; D,E, cf. control in C). (F–J) Similar dose-dependent phenotype penetrance and expressivity are observed after Bcl9 inhibition at the four-cell, shield, and 18-somite (18 ss) stages, suggesting that the observed phenotypes result from perturbed Bcl9 function in craniofacial and heart development after somitogenesis. Mild phenotypes are characterized by mild craniofacial and swim bladder inflation defects. (D) Both phenotypes are more pronounced in the medium phenotype class. (E) Strong phenotypes are characterized by strong craniofacial defects and a complete failure to inflate the swim bladder. (I–P) Fluorescent and bright-field images of TCF:siamRed Bcl9-inhibited (K,L,O,P) and DMSO-treated (I,J,M,N) wild-type siblings at 3 and 5 dpf (lateral views; anterior is to the left). TCF reporter activity is severely reduced in the atrio–ventricular valve at 3 dpf (asterisks; I,K) and altered in the craniofacial cartilage (arrowheads; I,K). At 5 dpf, the TCF reporter is aberrantly patterned in the atrio–ventricular valve (asterisks; M,O) and fins (arrowheads; M,O). Bars: C–E,I–P, 200 µm; A,B, 500 µm.