morphological differences from control morpholino injected or uninjected embryos under an Olympus SZX12 stereoscope at 24, 48 and 72 hpf. Isl1-GFP transgenic zebrafish were stage matched to 48 hpf and fixed in 4% paraformaldehyde for 4 h and then analyzed for differences in branchiomotor neuron development and migration from uninjected controls using a Zeiss AxioImager Z1 compound microscope. Images were photographed using a Zeiss LSM 510 confocal microscope at 488 nm under a 206 objective, and were compiled using Zeiss LSM Image Browser software. Fli1-GPF zebrafish were staged to 34–36 hpf and screened for CNC segmentation defects using an X-Cite UV light source with GFP filter mounted to a Zeiss M2 Bio Stereomicro- scope. Immunohistological procedures Zebrafish specimens were fixed in 4% paraformaldehyde and were either used for whole-mount immunostaining or embedded in paraffin. Transverse sections (1.5–3 mm) of paraffin-embedded specimens were deparaffinized in xylene and were rehydrated in a graded series of ethanol. Serial sections were collected from the central retina. For immunofluorescence microscopy, whole embryos and eye sections were washed several times in PBS and permeabilized for 30 min in 4% Triton-X 100 containing 2% bovine serum albumin (BSA) and 10% goat serum. Following permeabilization, tissues and retinal sections were incubated in the primary antibody 3A10 (Developmental Studies Hybridoma Bank; 1:500) for 48 h at 4uC on a shaker. Tissues and eye sections were washed several times in PBS over a 24 h period, and then incubated in the secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, Carlsbad, CA, 1:2000) for 4–6 h at room temperature. Animals were washed in PBS several times, de- yolked, cleared in 70% glycerol and mounted. Z-stack images were photographed using a Zeiss LSM 510 confocal microscope under a 206 objective, and were compiled using Zeiss LSM Image Browser software. For immunoperoxidase methods, slides were incubated with a blocking serum (Vectastain; Vector Laboratories, Burlingame, CA) for 45 minutes, after which they were blotted and then overlaid with the primary antibodies Chd7 (Santa Cruz Biotechnology, Santa Cruz, CA ; 1/600) and zn-8 (Developmental Studies Hybridoma Bank; 1/200) for 18 hours at 4uC. The slides were washed 3 times in PBS, pH 7.4, and incubated with secondary antibodies (anti- mouse (1:1000) or anti- rabbit (1:1000), Vectastain) for 1 hour at room temperature, followed by staining according to the avidin-biotin-peroxidase complex method (Vectastain ABC assay). Color was developed with 3,3-diamino- benzidine (Dako Diagnostics Inc., Mississauga, ON, Canada) containing hydrogen peroxide. Slides were counterstained with Harris modified hematoxylin (Fisher Scientific, Ottawa, ON, Canada). Eye sections were also stained with 1% Toluidine Blue. Images were captured using a Leica DMR microscope mounted with a Qimaging Retiga 1300 camera. Skeletal staining Juvenile zebrafish were fixed in 4% paraformaldehyde in PBS for 48 hr at 4uC and dehydrated to 100% ethanol over 2 days. Fish were then cleared in a 1% KOH solution containing approximately 4.5 ml of 3% H2O2 per 100 ml 1% KOH for 2 h, followed by 2 h in half this concentration (2.25 ml H2O2 per 100 ml 1% KOH). This latter step ensures a stepwise progression from H2O2 to water, which helps reduce tissue expansion. Zebrafish were then rinsed twice in distilled water, and transferred to a 30% saturated sodium tetraborate solution (Borax; Acros Role of Chd7 in Developing Zebrafish Organics) in water, overnight. Any remaining soft tissues were digested in a solution containing 1.0% trypsin and 2.0% Borax in water for 2–4 h. Digestion was deemed complete when the primary axis of the axial skeleton became visible. The fish were subsequently rinsed three times in distilled water to remove all residual trypsin, prior to staining for 12 h or more in a solution of 75% ethanol and Alizarin red S. Vertebral mineralization was assessed after Alizarin red S staining. Fish were examined using a Leica M205FA stereomicroscope. Digital images were collected with Leica DFC490 camera. Calcein staining was performed as described [70]. Briefly, fish were anesthetized in 0.6 mM MS-222 buffered to pH 7.0 and immersed in 0.2% calcein in 10% Hank’s solution buffered to pH 7.2 for 10 min followed by three 10 min washes in Hank’s solution. Vertebral mineralization was assessed after calcein staining using a Leica DMR microscope. Digital images were collected with a Qimaging Retiga 1300 camera. Sample number and statistical analysis The sample number (n) refers to the batch number and each batch number consisted of at least 40 fish. For each data set, we provided the batch number followed by the total fish number used. The sample number for wild-type fish consisted of 12 batches with a total of 510 fish used (n = 12,510 fish); 12 batches with a total of 436 fish for chd7 morphants (n = 12,436 fish); 12 batches with a total of 498 fish for control-MO injected embryos (n = 12, 498 fish) and 6 batches with a total of 249 mRNA rescue experiments (n = 6, 249 fish). Immunohistochemistry were performed on at least 6 batches for both control-MO and chd7- MO fish. Statistical analyses were performed and data were plotted in SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA). Significance was determined using paired Student’s t-tests, one-way anovas and Fisher’s least significant difference tests for normally distributed, equal variance data. Kruskal–Wallis anova and Dunn’s method of comparison were used for non-normal distributions. Supporting Information Figure S1 Chd7- MO injections are dosage dependent. As the concentration of the chd7-MO injection increased, the phenotypic defects became more severe. (A) Control-MO injected zebrafish showed no phenotypic defects 48 hpf and were comparable to wild type zebrafish at the same age. Embryos injected with 2 ng/nl (B), 4 ng/nl (C), or 6 ng/nl (D) chd7-MO showed increasing severity of developmental defects with increas- ing MO concentration. (TIF) Figure S2 Chd7 plays an essential role in photoreceptor development. The photoreceptor layer of control-MO (A) and chd7-MO-injected (B) embryos were stained with Zpr-1. Chd7 morphants lacked the photoreceptor layer. (TIF) Measurement of zebrafish size at different Table S1 ages. (TIF) Acknowledgments We are grateful to G. Laliberte´ and R. Milette for animal care. We would also like to thank Jeff Amack for the use of microinjection equipment and for Fli1-GFP transgenic fish. Thanks to Fiona Foley for help with the microinjections. The monoclonal antibodies 3A10 and zn-8 developed by Thomas M. Jessell and Jane Dodd were obtained from the Developmental PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e31650