Inhibiting angiogenesis disrupts optic fissure fusion mechanics. (A) Fluorescent whole mount in situ hybridization of vegfaa, vegfab and vegfc at 32 hpf in WT and pax2a−/− embryos. Loss of pax2a results in decreased vegfaa, ab and c expression. DAPI is depicted in blue. Scale bar = 100 μm. (B) qPCR results for vegfaa, vegfab and vegfc expression from heads of pax2a+/+ and pax2a−/− embryos at 32 hpf. (C) 3D confocal images of Tg[kdrl:mCherry] (red) embryos treated with 100 μM DMH4 between 24-72 hpf. DNA was stained with DAPI (blue). Scale bar = 50 μm. (D) 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 = 22 (DMSO), 52 (100 μM), 22 (50 μM), 24 (25 μM) ANOVA p < 0.0001. (E) Quantification of laminin signal intensity within the central-proximal region of the OF, normalized to regions of laminin staining juxtaposed to the lens. Relative pixel intensities are displayed. ANOVA p < 0.0001. (F) Quantification of F-actin signal intensity (phalloidin staining) within the central-proximal region of the OF, normalized to regions of F-actin signal within the lobe of the retina. Relative pixel intensities are displayed. ANOVA p < 0.0001.
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