Trigeminal and facial branchiomotor neurons have shared muscle targets in zebrafish larvae

The trigeminal and facial branchiomotor neurons are believed to have unique sets of peripheral muscle targets in the first and second branchial arches, respectively. However, their innervation patterns in zebrafish have only been precisely mapped up to 48 h postfertilization (hpf). In this study, we dissected the final peripheral destinations of the trigeminal and facial motor nerves in the ventral branchial musculature of first-feeding zebrafish larvae. Using a combination of transgenic lines, we found that around 56 hpf, the facial nerve elongates anteriorly to reach the intermandibularis group of muscles, which are classically defined to be innervated by the trigeminal nerve. Intriguingly, the facial nerve could project onto these muscles ipsilaterally and contralaterally. Furthermore, the trigeminal nerve also elongates posteriorly into the hyoid arch territory around 53 hpf to reach the interhyoideus muscles. Targeted ablation of facial neurons revealed that the trigeminal nerve extends further posteriorly to reach the hyohyoideus inferior muscles between 3 and 4 days post fertilization. Strikingly, both these hyoid arch muscles formed neuromuscular junctions even in the absence of facial motor neurons, signifying functional relevance of their trigeminal innervation. Overall, we show that all ventral branchial muscles, except the hyohyoideus superior, receive dual innervation, while the hyohyoideus superior muscle is solely innervated by the facial nerve. We propose that this conjoint innervation of the anteroventral branchial muscles might be essential for their coordinated functional role in respiration or feeding.