|
Fig. 5
Morpholino knockdown of trc40 results in wrb-/--like phenotypes. (A) Lateral view of 5 dpf uninjected larvae (top) and trc40 MO1 (translation-blocking, ATG), and trc40 MO2 (splice-blocking, SB) injected larvae. (B) Western blots of 15 µg protein lysates collected from whole larvae at 3 and 5 days after injection with 3 ng trc40 MO1, or MO1 with human trc40-eGFP mRNA. Membranes were immunoblotted with antibodies to detect GFP (top), TRC40 (middle) and γ-tubulin (bottom). (C) Live 5 dpf wild-type and trc40 morphants (MO1) larvae stained with the vital styryl dye DASPEI. (D) Immunohistochemistry of 5 dpf cryosections of Tg(nyx:mYFP) and Tg(nyx:mYFP) + trc40 MO1 morphant larvae using antibodies to detect YFP (green) and red/green double cones (zpr1, red). White arrows indicate bipolar cell dendritic protrusions into cone pedicles. (E) Quantification of dendritic protrusions observed within cone pedicles from cryosections of 5 dpf larvae (n = 11 wild-type, 8 trc40 MO1). (F) Gain of OKR versus log contrast for noninjected wild-type larvae (closed circles), 3 ng trc40 MO1 morphants (open down triangles) and 10 ng trc40 MO2 morphants (filled upward triangles) all at 5 dpf. Significance values indicated for trc40 MO2 morphants only. (G) Gain of OKR versus log contrast for noninjected larvae (filled circles), or larvae injected with 3 ng trc40 MO1 (downward gray triangles), 3 ng trc40 MO1 + trc40-eGFP mRNA (downward green triangles) and human trc40-eGFP mRNA alone (filled green circles). Significant values noted for only for trc40 MO1 morphants. Significance levels are as follows: *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. Scale bar: 5 µm.