VEGF signaling is plays a role in optic fissure fusion. (A) Whole mount Immunohistochemistry was used to visualize laminin (green) at 48 hpf after DMSO or DMH4 treatment. Central-proximal regions of the OF are displayed depicting fused, partially fused or unfused optic fissures. Scale bar = 50 μm. (B) Quantification of 48 hpf DMH4 treated embryos for fissure fusion (absence of laminin signal), partial fusion (partial retention of laminin) or failure to fuse (retention of laminin throughout the fissure). n = 41(12-48 hpf), 38(24-48 hpf), 38(28-48 hpf), 44(32-48 hpf), 40 (DMSO). ANOVA p < 0.0001. (C) 3D reconstructions of whole mount Tg[kdrl:mCherry] embryos treated with 100 μM DMH4 at various time points. Broken white line outlines the retina. Scale bar = 50 μm. (D) Quantification of the number of mCherry positive cells from 3D confocal stacks within the region of the OF after DMSO or DMH4 treatment. Individual embryo results are depicted. ANOVA p < 0.0001. (E) Fluorescent whole mount in situ hybridization of pax2a or tln1 probe at 32 hpf in DMSO or DMH4 treated embryos. OF expression is indicated with a yellow arrowhead. DMH4 treatment does not appear to alter pax2a expression but eliminates tln1 expression from the OF. Broken white lines outline the retinal lobes. Scale bar = 50 μm (F) qPCR results for tln1 expression in DMH4 treated embryos.
|
