Role of Chd7 in Developing Zebrafish Figure 3. Knockdown of chd7 affects eye and heart development. (A–B) Representative images of eye phenotypes induced by injection of chd7-MO in zebrafish embryos (B) compared with control embryos (A) at 72 hpf. Lateral view with anterior to right. Chd7 morphants had a reduced ocular size. Chd7-MO-injected zebrafish exhibited cardiac anomalies such as dysmorphic heart and tube-like heart shape (D) compared with control embryos (C). Insets in (C) and (D) represent an illustration of the heart phenotype in control or chd7 morphant embryos, respectively. (E–F) Bar graphs illustrating the prevalence of eye (E) and heart (F) 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. ***,0.001, *,0.01. doi:10.1371/journal.pone.0031650.g003 injected with 2 ng/nl of chd7-MO had several skeletal anomalies but exhibited no scoliotic curves (n = 6, 242 fish; Fig. 5 E,F). Notably, chd7-MO-injected (2 ng/nl) fish had smaller, irregular vertebral segments (n = 6; Fig. 5 F), wider intervertebral disc space (n = 6; Fig. 5 F) and smaller neural and hemal spines (Arrows; n = 6; Fig. 5 F). The pedicles of the neural spines were in some cases missing or fused. Mineralization can be assessed by the intensity and localization of the alizarin red S stain [21] .We observed a marked decrease in the alizarin red S staining of the vertebral segments of chd7-MO-injected fish (n = 6, 242 fish; Fig. 5 D–F) compared to control-MO ,suggesting a reduction in vertebral mineralization in chd7 morphants . These data suggest chd7 could play a critical role in vertebral development. Chd7 is required for proper somite polarity and segmental vascularization Morpholino effects on protein abundance typically last for ,7 days [22] and surprisingly, we observed post-embryonic vertebral defects in chd7-MO zebrafish. However, the skeletal malformation observed in chd7-MO injected fish (in Fig. 5) resembles the PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31650