Chd7 is expressed in the zebrafish brain, eye, otic vesicle, and somites. A,B: Sagittal sections of 24 hours postfertilization (hpf) zebrafish embryos showing chd7 expression in (A) the brain and (B) the somites. Inset in (B) shows chd7 expression along somite borders (arrowheads) at high magnification. e, eye; hb, hindbrain; mb, midbrain; ov, otic vesicle; s, somite; y, yolk. Scale bar = 200 μm.

Chd7 morpholino (MO) injection results in a gradient of phenotypic defects in developing zebrafish. A: Representatives of the classes of chd7 morphant phenotypes at 48 hours postfertilization (hpf). The top embryo is a control MO injected embryo. The remaining embryos represent the mild, moderate, and severe classes of chd7 morphants. B: Graph showing the percentage of embryos in each morphant phenotype category upon injection with 6 ng/nl of chd7 translation blocking, chd7 splice blocking, standard control, or chd7 5 base pair mismatch control morpholino. C: Reverse transcriptase-polymerase chain reaction (RT-PCR) exhibiting the loss of proper splicing of chd7 mRNA in chd7 splice blocking morphants. The increased band size is an indicator of the retention of chd7 intron 2 following mRNA processing. D: A green fluorescent protein (GFP) mRNA injected embryo illustrating the even distribution of the injected material throughout the animal. C, control morpholino; SB, splice blocking morpholino; WT, wild-type.

Loss of Chd7 results in asymmetric expression of somitogenesis patterning genes. A,D,G,J,M: Expression of cdx1a, dlc, her7, mespa, and ripply1 in control morpholino injected zebrafish embryos is largely symmetric in the somites, presomitic mesoderm, and tail bud at the 13-somite stage of development. B,E,H,K,N: Knockdown of Chd7 by chd7 translation blocking morpholino injection results in a loss of symmetric expression of cdx1a, dlc, her7, mespa, and ripply1 in zebrafish at the 13 somite stage. C,F,I,L,O: Graphs represent the percentage of animals exhibiting asymmetric gene expression in chd7 morphants (dark gray bar) and control morphants (light gray bar). Scale bar = 200 μm. *equals significance at the α = 0.05 level, **equals significance at α = 0.01, ***equals significance at α > 0.001.

Specificity of the chd7 riboprobe. A,B: Whole-mount in situ hybridization (WISH) performed using (A) antisense and (B) sense riboprobes for chd7 confirms the specificity of the antisense riboprobe. Zebrafish are at the 18 somite stage of development. Lateral view, anterior to the top. Scale bar = 200 μm.

Expression of spaw is bilateral in chd7 morphants. A: Spaw expression is limited to the left lateral plate mesoderm of the developing zebrafish in most control morphants. B: Spaw exhibits bilateral expression in most chd7 morphants indicating widespread laterality defects. Arrows denote spaw expression pattern. Dorsal view, posterior to the bottom. Scale bar = 200 μm.

Some Wnt target genes are up-regulated upon knockdown of Chd7. Cdx4 (A) and ntla (B) are up-regulated in chd7 morphants compared with control embryos. Large arrowheads denote up-regulation in chd7 morphants compared with controls. Both control and chd7 morphant embryos are captured in one image for each gene for proper comparison purposes. Lateral view, anterior to the top. Scale bar = 200 μm.