One of the most remarkable features of vertebrate morphology is the complexity of the head that harbours a “big” brain, many sensory organs, and an assemblage of bones, cartilages and muscles organized in a much-elaborated way, and its evolution remains an unanswered question. Concerning muscles, their developmental origins are diverse, (some derive from the somites (tongue), others from the paraxial unsegmented pharyngeal mesoderm, whereas the extraocular muscles develop from the prechordal plate). Vertebrates belong to the chordate phylum, with the tunicates and the cephalochordates. Both tunicates and cephalochordates (i. e. amphioxus) are filter feeders and share a pharynx adapted to this mode of nutrition, little sensory organs and reduced anterior centralized nervous system, as well as relatively simple oropharyngeal/atrial muscles. It has been proposed that vertebrates acquired their complex head during evolution as an adaptation to a predatory life style and Partner1 proposed a scenario for the evolution of the head muscles of vertebrates from a chordate ancestor with a mesoderm organization similar to that of cephalochordates. However, how anterior mesoderm derived muscles and associated motoneurons (MNs) co-evolved is still a major question. Thus, the aim of this project is to provide clues about the evolution of the motoneurons associated with anterior mesoderm derived muscles. In other words: are the motoneurons contacting non-myomeric mesodermal anterior muscles of the three chordate clades homologous? To achieve this, we will use cutting-edge techniques (scRNA-seq/ATAC-seq, transgenesis, retrograde labelling, etc.) applied to three chordate models (amphioxus, ascidian and mouse) to provide the information needed for an exhaustive comparison of anterior muscle/neuron systems, in order to define the co-evolution scenario of these structures in relation to the appearance of the complex vertebrate head.