Mutations of both pku300 and fbn2b contributed to scote382 mutant phenotypes. The endocardium in the atrium, ventricle and CCV were labeled by the Tg(kdrl:EGFP) transgene. The endocardium was projected by red pseudocolor with Imaris software. (A-C) 2341 offspring from heterozygous scote382 mutant crosses produced 25.5% mutants that had defects in the atrial endocardium and CCV (B), 6.7% mutants that had only reduced atrial endocardium (C), and 67.8% siblings that were phenotypically normal (A), suggesting more than one gene mutated in the scote382 locus. Sequencing confirmed that the mutants (group II) with defects in the heart (endocardium and CCV) and tail (caudal vein) (supplementary material Fig. S3) were fbn2b-/- and pku300-/- (B), and the mutants (group I) with defects only in the heart (endocardium) were fbn2b+/- and pku300+/- (C). (D–F) 297 offspring from heterozygous scopku300 mutant crosses produced 25.5% mutants with defects in the atrial endocardium and CCV (E) and 74.6% normal siblings (D), which was supported by morpholino knockdown of pku300 with 9.6ng pku300MO (F). (G–I) A fbn2bMO (1.6ng) morphant showed reduced atrial endocardium but had normal CCV (I), compared with the control (H) and a pku300 morphant (G). a, atrium; v, ventricle. Scale bars: 50μm.