Fig. 3

Fig. 3 Knock down of APPb protein alone causes aberrant convergent-extension movements. (A) Knock down of APP levels by APPb-MO. Embryos were uninjected (lane 1) or injected with control-MO (lane 2) or with 5 ng (lane 3) or 10 ng (lane 4) of APPb-MO at the 1–2 cell stage, and harvested 48 hpf. Total protein extract was prepared from deyolked embryos and analyzed by SDS-PAGE and Western blot using anti C-terminal antibody. There is a dramatic reduction in APP levels (top panel) in APPb-MO-injected embryos (lanes 3, 4) compared to the controls. The same membrane was stripped and reprobed with anti-tubulin antibody (bottom panel). (B) Aberrant development in APPb knock down embryos. Morphology of live embryos at 24 hpf. Lateral views, anterior to the left and dorsal to the top. Embryos were uninjected (1), or injected with control-MO (2), or APPb-MO at 10 ng (3). Compared to uninjected or control-MO-injected embryos, APPb-MO-injected embryos had a shorter body axis and a deformed tail. At high magnification, the tail region of APPb-MO-injected embryos (6) show undulating curvature (highlighted by dotted lines) and deformed somites (4, 5). Scale bars are represented on the top right on the left panel, and equal 0.5 mm. (C) The curly tail defect is more pronounced at 48 hpf. Lateral views, anterior to the left and dorsal to the top, of live embryos at 48 hpf. Compared with uninjected (1) or control-MO-injected embryos (2), APPb-MO-injected embryos exhibit severely reduced body length and a short, curly tail (3; shown with thin black lines from hind yolk to the tail tip in each embryo). At high magnification, a mass of disorganized cells could be visualized at the tail tip (black arrow in 6) These abnormal growths were sometimes seen in the APPb-MO-injected embryos, but never in control embryos. An even higher magnification of the notochordal region of the tail reveals severe undulations in APPb-MO-injected embryos (shown by arrows in 9), not present in uninjected (7) and control-MO-injected embryos (8). (D) Aberrant notochord development in APPb knock down embryos. Embryos were fixed 24 hpf and labeled with an RNA probe for sonic hedgehog (shh), a marker for notochordal and floor plate cells. Arrows show severely undulating notochord in APPb-MO-injected embryos (3) compared to control embryos (1, 2).