Fig. 6

Sox4 negatively regulates ihhb expression. (A) ihhb and shha qPCR of mRNA from control and sox4 morphant whole embryos at 8, 10 and 12 hpf, and heads at 18 and 24 hpf. Relative transcript abundance was normalized to level of atp5h or gapdh. Y axis represents the average ratio of normalized sox4 morphant to control expression (three biological replicates; Studentós t-test, P<0.01). (B–G) Ihhb expression in control and sox4 morphants at 12 and 24 hpf detected by WISH. Representative images are presented from 15–20 individuals analyzed. Lateral images in B–E focus on the midline; therefore the eye is not visible. Arrows in B and C mark the ventral forebrain at the level of the optic vesicle. (H) Western blot analysis of protein lysates from control or sox4 morphant heads at 24 hpf. Two biological replicates were performed (sets 1 and 2) (I) qPCR of mRNA from vehicle- and cyclopamine-treated sox4 morphants (Studentós t-test, P<0.01). (J) Quantification of the proportion of embryos with coloboma at 48 hpf in sox4 morphants co-injected with two different doses of either ihhb MO or shha MO (sox4 MO alone: 51.24±2.7%, n=209/404; sox4 MO+0.5 ng/embryo ihhb MO: 19.33±2.52%, n=14/75; sox4 MO+1.0 ng/embryo ihhb MO: 10.67±2.08%, n=9/91; sox4 MO+2.0 ng/embryo of shha MO: 41.84±5.62%, n=53/125; sox4 MO+3.0 ng/embryo of shha MO, 34.39±5.03%, n=79/229) Fisherós exact test, P<0.0001 for sox4 MO+ihhb MO; P=0.1676 for sox4 MO+2.0 ng/embryo of shha MO; P=0.0001 for sox4 MO+3.0 ng/embryo of shha MO. f, forebrain; tel, telencephalon; di, diencephalon, hy, hypothalamus; mhb, midbrain–hindbrain boundary; n, notochord.