Figure 2.

Molecular validation of the RNA-seq data. (A-B) Bar graphs of the relative expression levels of selected genes at 13 hpf in rx3 ^-/- and siblings. (A) Relative expression levels of the genes from the RNA-seq plotted as log2 transformed RPKM. *, q-value < 0.001 by DEGseq. (B) Relative expression levels of the same genes quantified by real-time PCR and plotted as Ct values relative to the least abundant gene. *, Student’s t test p-value < 0.05. (C) Dot-plot showing the correlation between the expression levels determined from the RNA-seq and real-time PCR analyses. (D) In situ hybridisation confirms the down-regulation of the homeodomain genes six7 and hmx1 at the 6–8 somite stage using DIG-labelled anti-sense probes to these genes. In the eye field hmx1 transcription show normal expression in wild-type phenotype siblings but are prominently down-regulated in rx3 ^-/- mutants. Down-regulation of six7 was also evident in the eye field, where siblings show normal expression and mutants show some residual expression in the ventral eye. Sense negative controls showing the expression patterns obtained with sense probes are also shown. Arrows indicate the eye field, hollow arrows for dorsal images and full arrows for lateral images.