Role of Chd7 in Developing Zebrafish Figure 4. Knockdown of chd7 affects otolith development. (A) The otic vesicle of control-MO-injected fish contains two otoliths, by contrast, larva treated with a morpholino against chd7 (B,C) had smaller otoliths (*) or only a single otolith (Arrow). Lateral views with anterior to the left. Ot, otoliths (D) Bar graphs illustrating the prevalence of otolith phenotypes in wild-type (white bar), chd7-MO (black bar), control-MO-injected embryos (grey bar) and mRNA rescue (dark grey bar). ***,0.001, *,0.01. doi:10.1371/journal.pone.0031650.g004 phenotype observed when somite segmentation is disrupted [23,24]. To determine whether the abnormality in segmentation of the vertebral axis was due to developmental somite malforma- tion, we analyzed the somite segmentation phenotype. To do so, we examined expression of the segment-polarity gene, ephrin B2a (efnb2a). In situ hybridization expression data for efnb2a revealed defects in the delineation of somite borders. To verify this defect in somite borders, a segment boundary marker, titin a (ttna), was used. Specifically, zebrafish chd7 morphant embryos showed a loss of distinct borders between individual somites at the 13 somite stage, as exhibited by efnb2a expression (146 fish;p = 0.008, t-test; Fig. 6 A–C) and ttna expression (112 fish; p = 0.006, t-test; Fig. 6 D–F). Stage matched control MO injected embryos exhibited distinct expression of efnb2a within each somite (149 fish, Fig. 6 A–B), and ttna expression marked clear boundaries between adjacent somites (143 fish, Fig. 6 D–E). Sectioning of efnb2a stained embryos revealed that this defect occurs throughout the somitic tissue in chd7 morphants (inset, Fig. 6 A–B). Therefore, chd7 appears to be important for proper segment boundary formation. These results also suggest that the skeletal malformation observed later on during development in chd7 morphants was likely caused by defective somite segmentation. Furthermore, since efnb2a is involved in arterial-venous differentiation [25] and the development of intersegmental vasculature is dependent on proper segmentation of the presomitic mesoderm (PSM), we utilized a fli1: GFP transgenic zebrafish line to test for defects in vascular development caused by Chd7 knockdown. With this resource, we observed aberrant vascular organization. After 48 hours, chd7 MO injected embryos displayed improper patterning of the intersegmental vasculature along the long axis of the body (56 fish; p = 0.001,t-test; Fig. 6 G–I). Since these vessels form at the boundaries between somites [26], this phenotype is likely due to a lack of intersomite specification in chd7 morphants, and is similar to defects observed in other mutants defined by aberrant PSM segmentation [25]. Chd7 function is required for retinal organization We next thought to further analyze the eye morphology of the chd7 morphants. To permit a detailed assessment of the chd7-MO small eye phenotype, histological analysis was performed on embryos at 72 hpf (Fig. 7). Histology revealed that retinal organization was severely disrupted in chd7-MO–injected embryos (120 fish, Fig. 7 B). Specifically, the retinas lacked the chara- cteristic laminated structure of both control-MO (142 fish, Fig. 7 A) and wild-type embryos at 72 hpf. Immunostaining further confirmed disruption of retina formation. Retinal ganglion cells appear to be reduced and disorganized in the chd7-MO–injected embryos based on staining for the retinal ganglion marker Zn-8 (n = 5; Fig. 7 C, D). Staining with 3A10, a neurofilament marker was abnormal in that the normally recognizable stripe of PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31650