chd7-MO exhibited phenotypes marked by curvature of the long axis of the body, a flattening of the head, abnormal tail fins, smaller eyes and heart defects (n = 12, A total of 498 fish used; Fig. 2, 3 A–C). These defects were observed in a dosage-dependent manner (Figure S1). As embryos were injected with increasing concentrations of chd7-MO, the resulting defects became more severe (Figure S1 C–D) To exclude the possibility that this phenotype may have resulted from non-specific or mistargeting effects of the MO, we also designed and injected zebrafish embryos with a chd7 splice- blocking morpholino (SBMO) and control 5-bp mismatch MO. We recently showed by RT-PCR analysis that the morpholino- targeted region exhibited abnormal splicing of chd7 in embryos injected with chd7 SBMO and normal splicing in uninjected and control-MO injected embryos [19]. Embryos injected with the chd7 SBMO exhibited morphological defects similar to those observed in the chd7-MO morphants (Fig, 2 D). On the other hand, all control MO-injected larvae developed normally (n = 12, 436 fish; Fig. 2 E and Fig. 3) and no apparent phenotype was observed when compared to untreated wild-type embryos (n = 12, 510 fish; Fig. 2 A). To further confirm that the lack of Chd7 underlies all the phenotypic alterations observed in chd7-MO zebrafish, a rescue experiment was performed. We found that expression of chd7 mRNA which had no complementary sequence to chd7-MO can correct all the developmental defects (n = 6, 249 fish; Fig. 2 E) and no apparent phenotype was observed when compared to control-MO fish (Fig. 2). Thus, the amount of newly- synthesized Chd7 coded by the rescuing mRNA in the initial stages was sufficient to prevent the developmental anomalies imparted by the chd7-MO. Altogether these findings were consistent with a reduction in Chd7 expression and they suggest that the phenotype observed with chd7-MO-injected embryos was a specific and due to a direct result of Chd7 protein depletion. We focused the analysis of the loss of function of Chd7 in embryos injected with 2 ng/nl of chd7-MO. At 48 hpf, we found that chd7-MO-injected (2 ng/nl) embryos exhibited a small eye phenotype (Fig. 3B,E) compared to control-MO (Fig. 3A,E) and wild-type fish (Fig. 3E). We observed that the hearts of chd7-MO- Role of Chd7 in Developing Zebrafish injected embryos were developmentally impaired and have signs of severe pericardial edema compared to controls and wild-type fish (Fig. 3 F). The atrial and ventricular chambers in these fish can be distinguished (Fig 3. D), but these compartments were dysmorphic and more tube-like (inset, Fig. 3 D) than the well-defined and tightly looped chambers of control-MO injected fish and wild-type fish (Fig. 3C). Injection of chd7 mRNA significantly rescued the morpholino eye and heart phenotypes (Fig. 3E–F). At 48 hpf, chd7-MO-injected (2 ng/nl) embryos also displayed a very specific defect in otolith formation (n = 6, 214 fish; Fig. 4B– D). We observed that 63% of the morphants exhibited otoliths that were asymmetric in size (135/214; p = 0.006, ANOVA; Fig. 4 B,D), and 15% possessed only one otolith (32/214; p = 0.008, ANOVA; Fig. 4 C–D). The enlarged or single remaining otolith was often shaped irregularly as opposed to the round and smooth otoliths in control-MO (n = 6, 249 fish; Fig. 4 C), wild-type embryos or mRNA rescue embryos (Fig. 4-D). In addition, the general size and morphology of the ear and the formation of semicircular canals were affected in morphants with a single otolith. Fish with defective otoliths also had a pronounced curvature of the body axis, usually remained on their sides at the bottom of their dish, and exhibited a circling swimming behavior upon a touch (tail tap) or acoustic (dish tap) stimulation (data not shown). Chd7 plays critical roles in primary axis development and vertebral mineralization Loss of function of Chd7 led to a pronounced curvature of the body axis in 25% of the morphants (n = 12; p,0.001, ANOVA; Fig. 5 A–C) compared to control-MO (n = 12; Fig. 5 A,C) and wild-type embryos (n = 12; Fig. 5 C). This phenotype was completed rescued by co-injection of chd7 mRNA (n = 6; Fig. 5 C). Recently, CHD7 gene polymorphisms were associated with susceptibility to idiopathic scoliosis in human populations [20]. We therefore sought to examine vertebral phenotypes at various developmental stages (Fig. 5 D–F). Chemical staining of the vertebrae at later stages revealed that the spine of zebrafish Figure 2. Chd7- MO injections and mRNA rescue experiments. Control-MO injected (E) chd7-MO+chd7-mRNA co-injected (F) zebrafish showed no phenotypic defects at 48 hpf and were comparable to wild type zebrafish (A) at the same age. Embryos injected with 2 ng/nl chd7-MO (B–C), or 2 ng/nl (D) chd7-SBMO showed several developmental defects. doi:10.1371/journal.pone.0031650.g002 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31650