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Fig. 1
Par3 is required for aortic lumen formation during zebrafish development. (a) Brightfield (left) and fluorescence (right) images of double transgenic Tg (kdrl:EGFP)s843;Tg (gata1:dsRed)sd2, control (top) and par3 MO (bottom) injected embryos at 48hpf. The par3 morphants exhibited pericardial (arrowhead) and brain (arrow) edema. In addition, they lacked circulation, and no erythrocytes could be observed in the trunk vasculature (arrowhead). Scale bar, 100 μm. (b) Transverse sections of 48hpf Tg(fli1a:EGFP)y1 control (top row) and par3 (mid row) morphants. Sections were stained with phalloidin for F-actin (red) and TO-PRO-3 iodide to visualize nuclei (blue). In control embryos patent lumens could be observed in both the dorsal aorta (red asterisk) and cardinal vein (blue asterisk). The par3 morphants showed narrow aortic lumens with present constrictions (arrowhead), while the vein was lumenized (white asterisk). As a comparison sections of amotL2a/b embryos were also included (bottom row). Note the similarity of the aortic phenotype. Scale bar, 10 μm. (c) Sagittal view of the DA (red bracket/ arrowhead) and PCV (blue bracket/ arrowhead) in control, par3 and amotL2 morphants. Both amotL2 and par3 morphants show DA constrictions (arrowheads) and reduced DA diameter. Scale bar, 50 μm. (d) Western blot analysis showing the knock-down efficiency of the par3 MO1. Alpha-tubulin was used to control for equal loading. (e) Quantification of the circulation defect in the par3 morphants. The phenotype could be partially rescued by co-injecting the morpholino with a human PAR3 mRNA. N(ctrl) = 147 embryos, N(par3 MO) = 154 embryos, N(par3 MO + hPAR3 mRNA) = 117 embryos. *** p ≤ 0.001. (f) Rescue experiment of the circulatory phenotype observed in the amotL2a/b MO zebrafish embryos. The circulation could be restored by co-injecting the morpholinos with a human AMOTL2 mRNA, but not with an AMOTL2 mRNA lacking the c-terminal PDZ-binding motif. N(ctrl) = 100 embryos, N(AmotL2 MO) = 111 embryos, N(AmotL2 MO + hAMOTL2 mRNA) = 141 embryos, N(AmotL2 MO + hAMOTL2 ΔPDZ mRNA) = 66 embryos, *** p ≤ 0.001.