Figure 2.
Characterization of the over-expression microinjection consequences of the seven variants as assessed by in situ hybridization. A 2pg dose of SHH mRNA co-injected with 200pg of each distinctSMO mRNA into one-cell stage embryos. (A ) Representative lateral view of the craniofacial region phenotype at 48hpf; cyclopia at 24hpf as an uncommon severe phenotype is shown in the upper right panel. Arrows indicate less pigmentation of the retina as a consequence of co-injection. (B ) The biological response by over-expression analysis was measured by visual scoring of their phenotypes at 24hpf. An abnormal phenotype was observed for all variants including WTSMO . There is no statistically significant difference between each variant mRNA with respect to the percentage of embryos with an abnormal phenotype. (C, D ) In situ hybridization of embryos with pax6a as eye developmental makers at 19hpf and ventral view of the expression pattern. Suppression of pax6aexpression in the eye region is observed (C_typeII) compared withgfp injection (C_typeI) and quantified (D). (E ,F ) In situ hybridization of embryos with neurod4 as a forebrain and neural developmental maker at 24hpf. The dotted line indicates the edge of the eye and lens area. Mild reduction ofneurod4 expression in eye region is observed (E_type II) compared with gfp injection (E_typeI). (G and H )In situ hybridization of embryos with pax2a and ventral view of expression pattern. Enhancement of pax2a expression in the eye region is observed (G_typeII) compared with gfpinjection (G_typeI) and quantified (F). Error bars represent means with SD. **P < 0.01 (Student’s t-test).