Role of Chd7 in Developing Zebrafish Figure 6. Chd7 knockdown leads to somite boundary and segmental vasculature defects. Efnb2a expression in control morphant (A) and chd7 morphant (B) zebrafish embryos at the 13-somite stage of development. Top is anterior. Insets in (A) and (B) represent coronal sections through control or chd7 morphant embryos, respectively, at the 13 somite stage. Posterior views with dorsal towards the top. Expression of ttna, a segment boundary marker reiterates the defects in segment boundary formation in chd7 morphants (E) compared to controls (D). Dorsal views with anterior to the top. Segmental vasculature patterning is defective (arrowheads) in chd7 morphants (H) compared to control morphants (G) as visualized using fli1: GFP transgenic zebrafish at 48 hpf. Sagittal views with anterior to the right. Graphs show percentages of animals exhibiting efnb2a expression defects (C), ttna defects (F), or segmental vasculature patterning defects (I). The dark gray bar represents chd7 morphants and the light gray bar represents control morphants. Scale bar: 200 mm. ***,0.001, **,0.01. doi:10.1371/journal.pone.0031650.g006 migration or improper pharyngeal arch patterning remains to be investigated. Discussion The present results show a novel and essential role of Chd7 in retinal organization, axial patterning and bone development and mineralization. Furthermore, knockdown of zebrafish chd7 results in abnormalities similar to those described in CHARGE Synd- rome, as we observed inner ear, heart, eye, cranial ganglia, neural crest and skeletal defects in chd7 morphants. Collectively, our findings demonstrate that Chd7 function is essential for proper neural and cranial neural crest development. In addition, to our knowledge, this is the first study to report the requirement of chd7 in retinal and vertebral development. Chd7 has been implicated in spinal deformities [20] and scoliosis is one of the minor clinical features in some CHARGE patients [11]. Here, loss of function of Chd7 resulted in the curvature of the body axis, irregular vertebral segments and smaller neural and hemal spines. The chd7 morphants did not exhibit any skeletal defects reminiscent of a scoliotic adult phenotype, however, suggesting that either scoliosis in CHARGE patients is secondary to the effects of attenuated Chd7 levels, or due to primary effects that occur later in development. Our findings also showed that zebrafish embryos deficient in Chd7 mineralize vertebrae more slowly than control siblings, indicating a critical role for Chd7 in promoting miner- alization during normal development. Delays in vertebral miner- alization may result from a general retardation in growth and development; however, we did not detect any distinguishable differences in length between the chd7 morphants and controls at all developmental stages examined (Table S1). Interestingly, a similar delay of mineralization in zebrafish has been reported after knockdown of collagen XXVII [30]. Furthermore, in the absence of BMP signaling, zebrafish bone mineralization was delayed [31]. Chd7 has been shown to regulate BMP signaling [27,32]. Thus, it is possible that disruption of Chd7 affected the transcription of genes that are crucial for bone mineralization. We also show that a loss of somite border identity during somitogenesis, as exhibited by efnb2a and ttna expression, is linked to a loss of organized segmental vasculature following chd7 MO injection and skeletal defects. We hypothesize that early defects in efnb2a expression lead to improper segmental patterning later in development, affecting structures such as segmental vasculature and bone mineralization. The link between the process of somitogenesis and the defects in border specification will require further investigation. For example, since efnb2a expression persists after somite epithelialization, the irregular PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31650