Confirmation of the gmds gene as the twdrw685 locus by knock-down and rescue experiments. (A) The efficacy of the splice-blocking MO against gmds was confirmed by RT-PCR at 32 hpf. Total RNA of gmds morphants and 5-mis-pair control morphants was incubated with (RT+) or without (RT–) reverse transcriptase. Samples were amplified with primers in which the target region of the MO is covered. Therefore, a band is not detected if maturation of gmds mRNA is inhibited by the MO. ef1α; loading control. (B) Dorsal views of the gmds morphant and twdrw685 mutant embryos injected with GMDS mRNAs at 50 hpf (rostral towards the top). Wild-type embryos injected with the 5-mis-pair control MO (a), the gmds MO (b) and S-GMDS mRNA (e). The twdrw685 mutants injected with L-gmds mRNA (c,c′) and S-gmds mRNA (d). Completely rescued (c,d) and partially rescued (c′) mutants. Arrowheads indicate ectopic vagus motor neurons. (C) Time-lapse observation of the migration of vagus motor neuron progenitors in twdrw685 embryos injected with L-gmds mRNA. The broken line shows the outline of the hindbrain. Dorsal is towards the top.