In conclusion, we have examined the role of zebrafish Chd7. We provide evidence that zebrafish highly express chd7 in the retina, brain and somite boundaries. Loss of function of Chd7 resulted in several morphological defects similar to those observed in patients with CHARGE syndrome. We then performed a detailed analysis of uncharacterized defects of CHARGE syn- drome and show that the presence of Chd7 is crucial for proper neural, retinal and vertebral development in zebrafish. These data provide new insights on the role of Chd7 and the mechanistic link between defects in the Chd7 gene and the organs and systems dysfunction associated with CHARGE syndrome. Furthermore, on the basis of the overlap in clinical features between zebrafish chd7 morphants and CHARGE syndrome, we suggest zebrafish can be a valuable in vivo tool to further understand the pathophysiological mechanisms underlying the abnormalities associated with CHARGE syndrome. Materials and Methods Animals Wild-type AB, Isl1-GFP WIK transgenic and Fli1-GFP transgenic zebrafish (Danio rerio) embryos were raised at 28.5uC, and collected and staged using standard methods [64]. Wild-type AB stains fish and Fli1-GFP transgenic zebrafish were purchased from the Zebrafish International Resource Center (ZIRC; University of Oregon, Eugene, OR). Isl1-GFP WIK transgenic line was kindly provided by Dr. Hitoshi Okamoto (RIKEN Brain Science Institute, Wako, Japan) [65]. Embryos and larvae were anaesthetized in 0.02% tricaine (MS-222; Sigma Chemical, St. Louis, MO) in phosphate-buffered saline (PBS) prior to all procedures. Ethics Statement All protocols were carried out in compliance with the guidelines stipulated by the Canadian Council for Animal Care (CCAC), the CHU Sainte-Justine Research Center, as well as the Institutional Animal Care and Use Committee (IACUC) at Syracuse University and at the University of Montreal. This study was approved by the CHU Sainte-Justine Research Center ,University of Montreal (ZF- 09-60/Category B) and the Syracuse University (IACUC # 07- 010) ethics committees. Design and synthesis of DIG-labeled RNA probes for chd7, efnb2a and ttna Total RNA was isolated from blastula stage zebrafish embryos using the RNeasy Mini Kit (Quiagen, Valencia, CA). Reverse transcription was performed to make cDNA using the QuantiTect Reverse Transcription Kit (Quiagen, Valencia, CA). Chd7 fragments were amplified by PCR using 59-GCTATT- GACCGCTTCTCTCG-39 and 59-TGCTCCTTTACGCAG- GAGAT-39 primers (resulting in a 362 bp amplicon from about halfway through the cDNA sequence) and cloned into the pGEM- T Easy vector (Promega, Madison, WI) following the manufac- turer’s protocol. Zebrafish efnb2a [66] and ttna [67] plasmids were kind gifts from Dr. Steven Wilson and Dr. Andrew Oates respectively. To synthesize the antisense probe, the previously constructed plasmid was linearized using the restriction enzyme SpeI (New England BioLabs, Inc., Ipswich, MA) for 2 hours at 37uC. Linearization was confirmed by gel electrophoresis using a 1% agarose gel. Linearized plasmid was incubated with T7 RNA Polymerase (Roche Diagnostics, Indianapolis, IN) in the presence of digoxigenin (DIG) label (Roche Diagnostics, Indianapolis, IN) and RNase Inhibitor (Roche Diagnostics, Indianapolis, IN) for 2 hours at 37uC. To synthesize the sense strand, the restriction Role of Chd7 in Developing Zebrafish enzyme ApaI (New England Biolabs, Inc., Ipswich, MA) and SP6 RNA Polymerase (Roche Diagnostics, Indianapolis, IN) were used. Plasmid DNA was eliminated with DNase (Roche Diagnostics, Indianapolis, IN) by incubation at 37uC for 20 minutes. DIG- labeled RNA was precipitated in 0.2 M EDTA, 4 M LiCl, and 100% ethanol overnight at 220uC, and then resuspended in DEPC water. Agarose gel electrophoresis was used to confirm the presence of a purified probe and any unused probe was stored at 220uC. Whole mount in situ hybridization analysis Whole mount in situ hybridization was performed on staged zebrafish embryos using both sense and antisense chd7, efnb2a and ttna riboprobes. Methods for in situ hydridization analysis followed [68,69]. Briefly, staged embryos were fixed overnight in 4% paraformaldehyde, and then dehydrated in methanol. When ready to use, embryos were rehydrated in phosphate buffered saline with 0.1% Tween-20 (PBSt). Embryos were permeabilized by protein- ase K digestion and then hybridized with the riboprobes overnight at 70uC. The next day, embryos were put through graded solutions of 75%, 50%, and 25% prehybridized solution in 26 saline-sodium citrate (SSC) followed by a wash in 0.26 SSC for 30 minutes at 68uC. They were then placed in blocking solution for several hours, and incubated in a-DIG antibody overnight. Finally, embryos were washed again and incubated in staining solution in the dark until sufficient staining appeared on the embryos. Embryos were dehydrated in methanol to facilitate clearing of background staining and then rehydrated in PBSt. Embryos were stored in glycerol. Embryos in glycerol were visualized using a Zeiss M2 Bio Stereomicroscope, with motorized focus drive and X-Cite UV light source with GFP filter. Images were captured using a Zeiss Axiocam digital camera connected to an Antec PC and processed with Adobe Photoshop 7.0. Generation of zebrafish chd7 morpholino and mRNA rescue experiments To eliminate Chd7 function, two types of antisense morpholino oligonucleotide were used to disrupt the translation of chd7 transcripts. The translation-blocking morpholino, 59-TGCAGC- CAAGCTTAGAAGCAGGAC-39 and the splice blocking, 59- TTATTTTCTGGCACTAACCATGTCC-39 were synthesized by Gene Tools (Philomath, OR). The morpholino was injected into single-cell stage zebrafish embryos at doses of 2 ng/embryo, 4 ng/embryo and 6 ng/embryo. To validate the function of the chd7 splice blocking morpholino, RT-PCR was used to check for improper splicing using the following primer set: forward- 59- AGGTGGACTCCGAAGGAAAC-39, reverse- 59-CCGTCAT- CACCACATTTGAG-39. Amplified cDNA was visualized using gel electrophoresis. To confirm the specificity of the chd7 morpholinos, a mismatch morpholino (lower case) was used: TGgAcCCAAcCTTAcAAc- CAGGAC (Gene Tools. OR). Furthermore, rescue experiments were performed. Full-length wild type zebrafish chd7 gene was subcloned into pcs2+ vector and capped mRNA was synthesized using the Sp6 promoter and the mMessage mMachine Kit (Ambion). Three hundred picograms of the synthetic mRNA was then injected into embryos at the one-cell stage. Rescued embryos were visualized and counted using the Olympus SZX12 stereoscope. Injected and uninjected embryos were then incubated in embryo media at 28.5uC for 24 h, after which they were assessed for viability. Wild type, Isl1-GFP WIK and Fli1-GFP transgenic zebrafish embryos injected with chd7 morpholino were assessed for PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31650