Fig. 11

Relationship between Foxe3 and Pitx3. Foxe3 and Pitx3 protein immunolocalization in 5 dpf larval frozen sections is shown in panels (A–E). Immediately adjacent sections were labeled with either Foxe3 or Pitx3 polyclonal antiserum (panels A and B, respectively; green signal). The two different antibodies detect their respective proteins in nearly identical patterns on these sections (arrows and arrowheads; nuclei were stained with propidium iodide [red signal]). The purified Foxe3 and Pitx3 antibodies were conjugated to either AlexaFluor-488 or -594, respectively and used to colabel lens sections (panels C–E). The individual signals corresponding to Foxe3 (green, panel C) or Pitx3 (red, panel D) overlap in the merged image (panel E). Scale bars = 50 μm. Abbreviations: GCL, ganglion cell layer. RT-PCR was used to assess foxe3 and pitx3 gene expression in the foxe3 or pitx3 morphants at 2, 6 and 12 hpf (panel F). Foxe3 and pitx3 transcripts are detected at all three time points in wild-type embryos. In addition, both foxe3 and pitx3 transcripts are detected in the foxe3 morphants. In contrast, foxe3 transcripts are not detected in the pitx3 morphants at 12 hpf. Control RT-PCRs lacking reverse transcriptase (no RT) failed to amplify any of the three targets.