Three-dimensional detailed redescription of the lamprey larval cranial muscles based on multiple imaging methods

Background The origin of the vertebrate jaw is one of the most long-standing and enigmatic topics in vertebrate evolution. To address this fundamental problem, lamprey larvae have long been the primary source of information on the cranial system of jawless vertebrates. However, their cranial anatomy remains contentious, largely because previous anatomical studies have relied primarily on conventional methodologies, leaving room for subjective identification. Here, we focused on cranial muscles whose morphology and innervation have been particularly controversial, performing high-resolution and objective imaging of the muscles and trigeminal nerve branches in three dimensions using various advanced techniques, including micro-computed tomography and light-sheet microscopy. Results Our detailed observations revealed several key characters that have previously been overlooked. First, an undescribed upper lip muscle is innervated by a sub-branch of the second major branch of the trigeminal nerve, which had long been thought to be purely sensory. Second, most lower lip muscles exhibit dual innervation by the second and third major branches of the trigeminal nerve. Last, the topographical arrangement of the lower lip muscle was elucidated in detail for the first time, suggesting that this muscle gives rise to the musculature of adult lingual apparatus through metamorphosis. Conclusions Our three-dimensional data revises previous views on the cranial morphology of lamprey larvae and provides a solid foundation for comparative morphological and developmental studies across extinct and extant vertebrates. In addition, the cutting-edge imaging methods employed here could be applied to metamorphic and adult lampreys, offering a promising approach for elucidating the morphogenesis of the adult head. Taken together, this study opens new avenues for evolutionary developmental and paleontological research on the early evolution of vertebrate heads.