Role of Chd7 in Developing Zebrafish Figure 9. Knockdown of chd7 results in defects in CNC development. Fli1-GFP transgenic embryos were used for MO injections to determine whether Chd7 depletion had an effect on CNC development. Embryos were injected at the 1 cell stage and examined for effects at 34–36 hpf. (A) Control-MO injected zebrafish embryos did not exhibit defects in CNC segment shape or number. Alternatively, the majority of chd7-MO-injected embryos exhibited aberrant CNC development including an improper number of CNC segments (B), as well as severely disorganized (C) or missing CNC populations. (D) Based on the t-test comparing means, there was a significant difference in CNC defects between chd7-MO and control- MO-injected zebrafish embryos. More than half of the chd7-MO-injected fish had deformed or missing CNC segments and about one third developed an improper number of segments. ** = 0.01; ***,0.001; e, eye; mb, midbrain; pa1–5, pharyngeal arches 1–5. doi:10.1371/journal.pone.0031650.g009 the inner ear, respectively [48,49]. Some key developmental defects in CHARGE patients can be traced to aberrant CNC development, including coloboma of the eye, external ear malformations, inner ear defects and a spectrum of facial defects [6,50,51]. Our chd7 morphant zebrafish embryos displayed obvious defects in CNC migration, based on observations of pharyngeal arch development and number. We did not fully characterize CNC activity in the optic nerve or otic placode of our chd7 morphants, however, we did observe and quantify significant defects in eye and otic vesicle development. CHD7 is essential for the formation of multipotent migratory neural crest in humans and Xenopus [17] and recently, CHD7 was found to play a role in proper neurogenesis of the inner ear in mice [27]. Our results support a role for Chd7 in proper CNC migration and differentiation, and substantiate the use of chd7 morphant zebrafish as an in vivo animal model for CHARGE Syndrome. The chd7 –MO-injected embryos displayed a very specific defect in otolith and semicircular canal formation and they also exhibited a circling swimming behavior consistent with vestibular dysfunc- tion. Such inner ear malformations are very similar to those reported in CHARGE syndrome patients [52,53,54,55]. In addition, CHARGE patients have been reported to have vestibular problems and hyperactivity [56], and this may be due to inner ear defects. CHD7 is necessary for proliferation of inner ear neuroblasts and inner ear morphogenesis by the maintenance of Fgf10, Otx2 and Ngn1 expression [27]. Fgf signaling and Otx genes have also been shown to be important for inner ear development in zebrafish [57,58,59,60]. Thus, our zebrafish model for CHARGE syndrome can be useful to further explore the mechanisms underlying inner ear anomalies in CHARGE pathogenesis. For instance, it will be interesting to investigate whether loss of function of chd7 in zebrafish results in a downregulation of Fgf10 and Otx2 and whether overexpression of one or more of these genes could rescue chd7 –MO phenotypes. Heart defects have been reported to be associated with CHARGE syndrome [2,3,5,61,62]. Here we report that the hearts of chd7-MO-injected embryos are developmentally impaired and have signs of severe pericardial edema. Heart anomalies described in CHARGE syndrome patients include ventricular and atrial septal defects and conotruncal defects [61,63]. In this study, we did not fully characterize the observed heart defects in the chd7 morphants. The atrial and ventricular chambers in these fish can be distinguished, but these compartments were dysmorphic and more tube-like than the well-defined and tightly looped chambers of wild-type fish. Future studies using chd7 zebrafish morphants could help to identify components in the Chd7 regulatory network that are essential for heart development and further our understanding of one of the major anomalies reported in CHARGE patients. PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31650