wnt11r Is Critical for Axonal Guidance and AChR Prepatterning
(A) The splice morpholino (SP-MO) targets the splice donor site of the wnt11r exon 3 (red arrow), and MO-induced aberrant splicing is shown in red. RT-PCR analyses of uninjected and wnt11r SP-MO injected embryos (arrows indicate the position of PCR primers).
(B) Quantification of wnt11r MOs injected embryos. TL-MO, translation initiation mopholino. Per embryo, 20 hemisegments were analyzed; n = hemisegments. Results are expressed as the mean of multiple injection experiments ± SEM, (*p < 0.001, t test).
(C–L) Wild-type, unplugged, and wnt11r MO injected embryos at 27 hpf (C–H) and at the 20 somite stage (I–L), stained for motor axons (znp-1, green) and AChR clusters (α-BTX, red). (E and F) In contrast to wild-type, unplugged embryos display characteristic stalling (arrowhead) and branches (arrows) at the choice point and lack all AChR clusters. (G and H) Injection of wnt11r MO causes unplugged-like axonal stalling (arrowhead), branching (arrow), and a strong reduction of AChR prepatterning (K and L). Note that the size and intensity of neural AChR clusters is reduced in wnt11r 27 hpf morphants (H).
(M–Q) Time-lapse images of Hb9-GFP-labeled wild-type (M and O) and wnt11r morphant CaP and VaP axons (N, P, and Q) as they exit from the spinal cord (M and N) and as they reach the somitic choice point (O–Q). Asterisks indicate the cell body of interneurons. (M and O) Wild-type CaP and VaP neurons extended one growth cone (arrow). Note the broad area (brackets) the two defasciculated wnt11r morphants CaP/VaP growth cones occupy (arrowheads in [N], [P], and [Q]), compared to wild-type (M and O).
Scale bars, 50 μm.