Zebrafish pbx4, but not pbx3b, is required for early cardiac morphogenesis. (A) Alignment of human (Hs) and zebrafish (Dr) Pbx proteins in the region of the polyalanine tract. Numbers indicate amino acid positions. Partial PBC-A and PBC-B domains are underlined. The arrow marks the position of amino acid 136 in human PBX3. (B) Phylogenetic analysis of human (Hs), mouse (Mm) and zebrafish (Dr) Pbx genes. DmExd is the Drosophila Pbx gene ortholog extradenticle. (C) qRT-PCR analysis of Pbx gene expression in wild-type zebrafish embryos at four embryonic stages: eight-cell (∼1.25 hpf), 14 somites (s; ∼16 hpf), 24 hpf and 48 hpf. Levels of expression of each Pbx gene are shown relative to the expression of odc1. Error bars represent standard deviations for three technical replicates. (D) Schematic of zebrafish Pbx3b protein domains and inferred domains encoded by the CRISPR-Cas9-generated pbx3b^scm8 allele. (E) Images of live pbx3b^scm8/+ and pbx3b^scm8/scm8 larvae at 5 dpf. pbx3b^scm8/scm8 larvae show no obvious heart or other defects at least up to 7 dpf (n=15, pbx3b^scm8/scm8; n=14, pbx3b^scm8/+; n=7, pbx3b^+/+). Scale bar: 300 μm. (F) qRT-PCR analysis of Pbx gene expression in pbx3b^scm8/scm8 embryos relative to sibling pbx3b^+/+ embryos at 48 hpf. Levels of expression of each Pbx gene are normalized to the expression of eef1a1l1. Error bars represent standard deviations for four biological replicates. *P=0.0005, Student's t-test using Welch's correction for unequal standard deviations. (G) Western blot analysis of Pbx protein expression. pbx3^scm8/scm8 and pbx4^b557/b557 embryos were used to document identities of the proteins recognized by the anti-pan-Pbx antibody. The upper band is Pbx2, as previously described (Maves et al., 2007; Waskiewicz et al., 2002; G.H.F. and L.M., unpublished). Quantification of the middle band, normalized to Actin levels, shows that pbx3^scm8/scm8 embryos have 65% of wild-type levels, pbx4^b557/b557 embryos have 54% of wild-type levels and pbx3^scm8/scm8;pbx4^b557/b557 embryos have 10% of wild-type levels, demonstrating that the middle band consists of both Pbx3b and Pbx4. (H) Myocardial marker myl7 expression at 24 hpf appears normal in pbx3^+/+;pbx4^+/+ (n=11), pbx3^scm8/scm8;pbx4^+/+ (n=2) and pbx3^scm8/scm8;pbx4^b557/+ (n=15) embryos. pbx3^+/+;pbx4^b557/b557 (n=8) and pbx3^scm8/scm8;pbx4^b557/b557 (n=7) embryos show similarly disrupted early heart tube morphogenesis, as we previously described for pbx4^b557/b557 embryos (Kao et al., 2015). Dorsal views; anterior is up. Scale bar: 50 μm. (I) Expression of myocardial marker myl7 (red) and outflow tract marker elnb (green) (Miao et al., 2007) at 60 hpf appears normal in pbx3^+/+;pbx4^+/+ (n=8) and in pbx3^scm8/scm8;pbx4^+/+ (n=9) embryos. pbx3^+/+;pbx4^b557/b557 (n=11) and pbx3^scm8/scm8;pbx4^b557/b557 (n=10) embryos show variably disrupted myocardial and outflow tract morphogenesis, similar to what we previously described for pbx4^b557/b557 embryos (Kao et al., 2015). V, ventricle; A, atrium. Ventral views; anterior is up. Scale bar: 50 μm.

The pbx4 p.A131V variant enhances myocardial morphogenesis defects caused by loss of hand2. (A) Genetic crosses of zebrafish strains used to obtain embryos for the analyses of pbx4;hand2 mutant embryos. All adult breeder fish used in crosses 1-3 were ‘siblings’, derived from the same clutch from a group cross. (B-G) Myocardial marker myl7 expression at 24 hpf. Dorsal views; anterior is up. Animal numbers for phenotypic classes are provided in Table 2. (B) The heart tube appears normal in pbx4^+/+;hand2^+/+ embryos. (C) pbx4^b557/b557;hand2^+/+ embryos have a medial heart cone myl7 domain. (D) pbx4^+/+;hand2^s6/s6 embryos show a crescent-shaped myocardial fusion defect of the myl7 domains. (E) A similar phenotype is observed in pbx4^b557/+;hand2^s6/s6. (F,G) The myl7 fusion defect is more severe in pbx4^b557/b557;hand2^s6/s6 (F) and pbx4^scm14/b557;hand2^s6/s6 (G). (H) Quantitation of fusion defect of myl7 domains in different genetic combinations. myl7 distance measurements were made blind to embryo genotypes. The averages for the myl7 distances among the different genotypes were compared using one-way ANOVA, and P-values were corrected for multiple comparisons using Tukey's test. The boxes extend from the 25th to 75th percentiles, the whiskers are at the minimum and maximum, and the bar within the box represents the median. (I-N) Myocardial marker myl7 expression at 60 hpf. In I-J, ventral views; anterior is up. In K-N, anterior views; dorsal is up. Animal numbers for phenotypic classes are provided in Table 3. (I) The heart appears normal in pbx4^+/+;hand2^+/+ embryos. V, ventricle; A, atrium. (J) pbx4^b557/b557;hand2^+/+ embryos have dysmorphic hearts with bulges of the ventricular myocardium (arrow). (K) pbx4^+/+;hand2^s6/s6 embryos show an abnormally shaped, medial myocardium positioned more caudally between the eyes (asterisks). (L) A similar phenotype is observed in pbx4^b557/+;hand2^s6/s6. (M,N) More severe myl7 bilateral domain phenotypes are observed in pbx4^b557/b557;hand2^s6/s6 (M) and pbx4^scm14/b557;hand2^s6/s6 (N) embryos. Scale bars: 50 μm.