A: Nucleotide sequences of the designed arl6ip-morpholino (MO). The arl6ip-MO1 targets the coding region of arl6ip mRNA, and arl6ip-MO2 targets the 5′-untranslated region. The arl6ip-control-MO served as a negative control MO, which was an inverted MO sequence of arl6ip-MO1. Square box, Arl6ip translation start site. B: Western blot analysis of total proteins extracted from the wild-type (WT) and the arl6ip-MO1-injected (MO) embryos, with β-Tubulin used as a control. The endogenous Arl6ip (arrow) from the arl6ip-morphants was completely absent. C-N: Phenotypic defects in the arl6ip-MO1-injected embryos were observed at the stages indicated. They were microphthalmia (small eyes; dotted blue arrow), pericardial edema (dotted green arrow), flat head, deformed trunk (dotted black arrow in H′-J′), pigment pattern disorder and pectoral fin defect (dotted red arrow). Black brackets indicate the extended yolk length by which we identified the different defect grades (M,N). Scale bar = 100 μm in M,N. oc, optic cup (solid blue arrow); h, heart (solid green arrow); f, fin (solid red arrow) were indicated in wild-type embryos. Scale bar = 10 μm in C′-E′,H′-J′,O-Q. The defective arl6ip-morphants (Q) were partially rescued to develop the wild-type-like phenotype (P) after co-injecting the synthetic arl6ip-mRNA with arl6ip-MO2. The white brackets indicate the eye dimensions. The histological sections were examined for the wild-type embryos and the arl6ip-morphants at 96 hpf after hematoxylin-eosin staining (R,S). Compared to the tissue morphology of wild-type embryos, arl6ip-morphants displayed many abnormalities of tissue development, including retina, tectum, myelencephalon, 3rd-7th pharyngeal arches, pneumatic duct, swim bladder, and gut.
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