Role of Chd7 in Developing Zebrafish Figure 8. Chd7 is important for cranial ganglia development and proper projection of facial nerves. Dorsal view (rostal towards the top) of confocal fluorescent composite images of hindbrain branchiomotor neurons in control-MO injected (A) and chd7-MO-injected (B) 48 hpf Isl1-GFP transgenic embryos. (C, D) Confocal fluorescent composite images showing anti-3A10 antibody-stained axons of 48 hpf embryos. The broken lines indicate the area of significant loss of VII nerves in chd7 morphants. nV, trigeminal motoneurons; Va, anterior trigeminal motoneurons; Vp, posterior trigeminal motoneurons; nVII, facial motoneurons; nX, Vagal motoneurons; V, trigeminal nerve; VII, facial nerve. Scale bar: 50 mm. doi:10.1371/journal.pone.0031650.g008 differentiation by 52 hpf and become functional at around 3 dpf [37,38,39]. A functional retina consists of a laminated neuronal tissue with the ganglion cell layer on the inside, inner nuclear layer in the middle, and the outer nuclear layer on the outside. In between these layers are the synaptic layers: the inner and outer plexiform layer [36,40]. Notably, the retinas in chd7 morphants fail to laminate, which suggests a novel function for Chd7 in retinal patterning. Furthermore, our findings are similar to disruptions in retinal lamination as observed in the young zebrafish mutant that carries a mutation in brg1, a gene that encodes for a subunit of the Swi/Snf of ATP-dependent chromatin remodeling complexes [37]. Moreover, retinal ganglion cells were reduced and the photo- receptor layer were absent in the chd7-MO-injected embryos. These data suggest that Chd7 is required for the development of retinal cells. In Drosophila, the chd7 ortholog kismet is essential for transcription of the pro-neural factor atonal (Atoh1) and regulation of retinal photoreceptor cell development [41]. Drosophila ato mutants produce no photoreceptors in the eye [42]. Chd7 has also been recently shown to control the expression of sox9 [17]. Zebrafish sox9 mutants reveal that Sox9 is required for retinal differentiation and it also helps with retinal organization and regulates the number of photoreceptor cells [43]. Thus, it is likely that that loss of function of Chd7 disrupts the gene network that is crucial for retinal cellular development and organization. Several ganglionic defects have been reported in CHARGE patients, including defects in the facial and vestibulo-acoustic ganglia [44,45]. Chd7 knockdown resulted in important pheno- types relevant to cranial ganglia development, branchiomotor development, and vagal motor neuron positioning. Anomaly in facial nerves has also been demonstrated in CHARGE syndrome [5]. Loss of function of Chd7 resulted in a severe loss of facial nerves in the morphants. Altogether, these results suggest that Chd7 and downstream targets are important for neuronal development and proper axonal projections. Neural crest cells are multipotent cell populations with the ability to migrate, leading to the formation of several key developmental structures including bones, cartilages, nerves, and connective tissues [46]. CNC, specifically, migrate into the pharyngeal arches where they play a role in the formation of facial bone, muscle, and cartilage [47]. In addition, CNC migrate into the optic vesicle and otic placode, where they play key roles in the development of muscular and skeletal elements in the eye and PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31650