Fig. 3

Salbutamol treatment of zMuSK MO-injected embryos improves motor axon pathfinding defects and AChR clustering. Lateral views of embryos with neuromuscular synapses labelled with antibodies against SV2 (green, presynaptic vesicles) and αBTX (red, postsynaptic AChRs). Scale bars = 10 µm. (A) zMuSK MO-injected embryos at 24 hpf demonstrate characteristic stalling of the outgrowing motor axon at the choice point on the horizontal midline (arrow). Following salbutamol treatment more axons cross the horizontal midline and extend to the periphery of the myotome (double arrow). (B) Quantification of the improvement of motor axon growth following salbutamol treatment. (**** indicates P < 0.0001, chi-square test, n = 240 treated and 240 untreated myotomal segments examined). (C) zMuSK embryos at 24 hpf display almost complete absence of AChR clustering. In salbutamol-treated zMuSK embryos, AChR clustering is restored. (D) Quantification of the improvement in AChR clustering following salbutamol treatment of zMuSK embryos. AChR clustering is significantly increased with the mean number of AChR clusters >20 μm² per myotome 1.16 in salbutamol treated embryos compared with 0.64 in untreated embryos (**** indicates P < 0.0001, Student’s t-test, n = 100 treated and 100 untreated myotomal segments examined). (E) zMuSK MO-injected embryos at 17 hpf. Untreated embryos lack elongated, diffuse prepatterned clusters on the myotomal surface prior to the arrival of the motor growth cone (boxed areas), but following salbutamol treatment some zMuSK embryos display prepatterned clusters. (F) Quantification of the effect of salbutamol on prepatterned AChR clustering in zMuSK embryos. Following salbutamol treatment, the number of prepatterned AChR clusters >3 μm² in zMuSK embryos increased, with a mean of 0.5 prepatterned clusters per myotome in untreated embryos, compared with 1.3 in salbutamol-treated embryos (* indicates P < 0.05, Student’s t-test, n = 30 untreated and 30 untreated myotomal segments examined).