Fig. 5

(A) Live imaging of MZnpc2^m/m and Mnpc2^+/m embryos. MZnpc2^m/m embryos had essentially normal gross morphology except the lack of otoliths at 22?hpf. Otic vesicles are shown in the insets (arrowheads). Older MZnpc2^m/m embryos manifested additional malformations, including abnormal head/brain development with reduced hindbrain, no circulating red blood cells, and a curved/twisted body axis at 30 and 48?hpf. All of the embryos from three separate crosses exhibited representing phenotypes as shown in each image (for 22?hpf: Mnpc2^+/m n=22, MZnpc2^m/m, n=18; for 30?hpf: Mnpc2^+/m n=28, MZnpc2^m/m n=33; for 48?hpf: Mnpc2^+/m n=24, MZnpc2^m/m n=21). Scale bars: 200??m (22?hpf); 500??m (30 and 48?hpf). (B) Filipin-positive puncta were found on the yolk surface of MZnpc2^m/m embryos at 24?hpf. This signal appears to be from the yolk syncytial layer. Mnpc2^+/m (n=25) and MZnpc2^m/m (n=21) embryos were examined from three experiments. Scale bar: 200??m. (C) Live imaging of otic vesicles at 30?hpf. Note that Mnpc2^+/m embryos (n=28) exhibited two otoliths in the oval-shaped otic vesicle whereas MZnpc2^m/m embryos (n=33) displayed a less-organized otic vesicle with no otoliths (arrowhead). Scale bar: 100??m. (D) Red blood cells stained with O-dianisidine at 30?hpf. Red blood cells accumulated in the posterior blood island (arrow) in the MZnpc2^m/m embryo (n=24), indicating that red blood cells were formed but not circulating in these embryos. This accumulation of RBCs was not observed in Mnpc2^+/m embryos (n=21). At 48?hpf, circulating blood cells were observed in caudal artery (red arrowhead) and vein (white arrowhead) of the Mnpc2^+/m embryos (n=18), whereas blood cells remained sequestered in the posterior blood island in MZnpc2^m/m embryos (asterisk) (n=23). Y, yolk stalk extension. Scale bars: 200??m (30?hpf); 100??m (48?hpf). All experiments were repeated three times.