Role of Chd7 in Developing Zebrafish Figure 5. Chd7 deficiency affects vertebral mineralization. Lateral view of control-MO (A) and chd7-MO-injected (B) fish at 3 dpf. Chd7 morphants exhibited a slight curvature of the long body axis (B). A significant percentage of the chd7-MO-injected (2 ng/nl) fish had deformed body axis shape (C). Lateral views of chd7 morphants (lower panel) and control siblings (upper panel) at 8 dpf (D) and 14 dpf (E) showed a reduced bone mineralization. Vertebrae with reduced or no hemal or neural spines were also observed in the chd7-MO-injected fish (F; black arrow). h, hemal spine; n, neural spine. ***,0.001. doi:10.1371/journal.pone.0031650.g005 expression of the photoreceptor layer was not present in the chd7- MO–injected embryos (n = 8; Fig. 7 E, F). Similarly, cone photo- receptor staining with Zpr-1 antibody was absent in chd7 morphants (Figure S2). These findings suggest a novel role of Chd7 in retinal organization and the development of photoreceptors. Chd7 is essential for correct positioning of cranial motor neurons and proper projection of facial and trigeminal motor axons Previous studies have demonstrated a role for Chd7 in neuronal development [27,28,29] and patients with CHARGE syndrome often display several neurological disorders. We found that chd7 is expressed in the developing zebrafish hindbrain and loss of function of chd7 leads to flattening of the head. To determine if these molecules are required for proper neural development in this area, we examined the cranial motor neurons of chd7-MO-injected embryos using Isl1-GFP transgenic zebrafish embryos. These embryos express GFP under the control of a motor neuron-specific Isl1 promoter. Interestingly, we observed a disrupted organization of the cranial motor neurons in the chd7-MO-injected embryos (n = 9; Fig. 8 B) when compared to control-MO-injected embryos (n = 9; Fig. 8 A). Chd7-MO-injected embryos demonstrated a severe disorganization and clustering of both Va and Vp clusters of the trigeminal (nV) neurons (Fig. 8 B). In addition, loss of Chd7 resulted in a reduction of facial (nVII) branchiomotor neuron populations (n = 9; Fig. 8 B). Furthermore, the vagal (nX) motor neurons in chd7-MO-injected embryos exhibited aberrant medio- lateral positioning and cell-to-cell spacing (n = 9; Fig. 8 B). Chd7- MO-injected embryos also exhibited extremely low levels of staining of the peripherally extending axons from nV neurons when compared to control-MO-injected embryos (n = 9; Fig. 8 C, D). Furthermore, chd7-MO injected embryos exhibited severe loss of the facial sensory ganglion and the axonal extensions from nVII neurons (n = 9; Fig. 8 C, D). Chd7 plays a key role in cranial neural crest development CHARGE syndrome is thought to result from the abnormal development of the neural crest. Recently, CHD7 and PBAF have been shown to cooperate during embryonic development to promote proper neural crest specification and cell migration [17]. We sought to examine the relationship between Chd7 and CNC development in zebrafish using a Fli1-GFP transgenic zebrafish line. We injected Fli1-GFP embryos with chd7-MO at the single cell stage and then assayed for CNC defects between 34–36 hpf by counting the number of CNC segments, which should approxi- mate pharyngeal arch number, within each embryo. We observed CNC defects in over 90% of chd7-MO-injected embryos (63 fish, Fig. 9 D). About one third of these had a reduction in the number of CNC segments, whereas the remaining animals showed more severe phenotypes characterized by the gross disorganization or absence of obvious CNC populations (Fig. 9 B, C). Disorganized CNC segments tended to possess fewer cells and lacked sharp borders between adjacent segments (Fig. 9 C). The phenotype of control-MO-injected zebrafish embryos mimicked that of wild type animals including well-defined segments and appropriate segment numbers (71 fish, Fig. 9 A). These data lend support for the function of Chd7 in proper CNC development and patterning. Whether the defects reported here are due to aberrant CNC PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31650