Fig. 3

Impairment of Igf2bp1 perturbs retinotectal projections.

(a) The e3i3 MO targeted to the e3i3 splice junction in Igf2bp1 pre-mRNA. (b) Left gel: forward primer targeted to exon 1 in the 5′UTR (Fprimer) and a reverse primer targeted to exon 3 (Rprimer1) yielded no detectable product as expected with exon 3 deletion. Right gel: Fprimer and a reverse primer targeted to exon 4 (Rprimer2) yielded a PCR product that was TOPO-TA cloned and sequenced (c) to verify exon 3 deletion (blank lanes were cropped out of gel image), which results in a severely truncated protein (d). (e-n) Transmitted light images of whole 3 dpf control (e,f) and e3i3 MO-injected morphants (g-i) and head regions (j-n) with eyes (yellow outline) and hearts (blue arrowheads). (o-s) 3D projections made from confocal z-stacks taken with a 30x silicone immersion lens on a confocal microscope, of Tg(isl2b:mCherryCAAX)^zc23 3 dpf embryos stained with α-DsRed (red) and counterstained with TO-PRO-3 (blue), with one example each for uninjected (o), or injected with negative control MO (p), e3i3 MO (mild (q), moderate (r), severe (s)), reconstructed from confocal z-stacks with FluoRender software, rotated to give lateral (top panels), coronal (middle panels) and dorsal (bottom panels) views. Scale bar is 100 µm.