Fig. 6

Shha expression is upregulated in sox11 morphants.

(A) QPCR performed on mRNA from control and sox11 morphant heads at 24 hpf revealed a dramatic upregulation of shha expression, and a small but significant increase in ptc2 expression, in sox11 morphants compared to controls (n = 70 embryos per group, 3 independent biological replicates). Relative transcript abundance was normalized to gapdh levels. The Y-axis (log-scale) represents the mean ratio of sox11 morphant to control expression for three biological and three technical replicates. *p<0.01, Student′s t-test. (B) In situ hybridization with a shha probe on control (left) and sox11 morphant (right) embryos at 24 hpf revealed expanded shha expression in sox11 morphants throughout the brain and also in the notochord (inset). Numbers of embryos analyzed: n = 15 embryos per group, 3 independent repeats. (C) QPCR performed on mRNA from control and sox11 morphant heads at 8, 10 and 12 hpf demonstrated an upregulation of shha expression in sox11 morphants compared to controls. Relative transcript abundance was normalized to gapdh levels and is presented as the mean fold-change in expression relative to controls (n = 60 embryos per group, 3 independent biological repeats). **p<0.001, *p = 0.01, Student′s t-test. (D) Sox11 morphants (right) on the ptc2:EGFP background displayed elevated GFP expression in the midline at 12 hpf compared to control morphants (left). The bottom panels are an enlargement of the boxed area indicated in the top left panel. Number of embryos analyzed: control MO, n = 34; sox11 MO, n = 41, 3 independent biological replicates. (E) Co-knockdown of shha and sox11 reduced the proportion of embryos displaying abnormal lens and coloboma phenotypes at 24 hpf and 2 dpf, respectively. Number of embryos analyzed: 24 hpf control MO, n = 186; 2 dpf control MO, n = 165; 24 hpf sox11 MO, n = 199, 2 dpf sox11 MO, n = 182; 24 hpf shha MO, n = 249; 2 dpf shha MO, n = 231; 24 hpf sox11+ shha MO, n = 207; 2 dpf sox11+ shha MO, n = 190; 3 independent biological replicates. *p<0.0001, Student′s t-test. D, dorsal; V, ventral; A, anterior; P, posterior; hpf, hours post fertilization; dpf, days post fertilization; R, retina; di; diencephalon, tel, telencephalon; hy, hypothalamus; MO, morpholino.