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).