Molecular address codes delineate circuit-specific motor neuron-muscle matchmaking in the developing tetrapod limb

An essential part of nervous systems development is the establishment and refinement of correct circuit architectures. In vertebrate neuromuscular circuits, this requires connecting the axons of motor neurons in the central nervous system to their corresponding muscle groups in the periphery. This process can roughly be sub-divided into two distinct phases: axon guidance towards their innervation territories, followed by the establishment of correct nerve-muscle connections. While the role of attractive and repulsive cues in axon guidance has been studied extensively, relatively little is known about the molecular interactions shaping motor neuron to muscle matchmaking that contribute to circuit specificity and refinement. Here, using the tetrapod limb as a model system, we focus on the maturation of three distinct neuromuscular circuits, targeting different proximal-distal and anterior-posterior territories of the developing chick forelimb, and sequence the transcriptomes of individually backfilled motor neurons as well as of the muscles they connect to. Comparative analyses across circuits suggest the presence of distinct molecular "address codes", based on individual signatures of matching profiles of secreted signaling factors and cell surface receptors. Furthermore, we probe for inherent transcriptional plasticity in this system, using an experimentally altered limb periphery that results in nerve miswiring, and sequence the corresponding motor neuron-muscle pairs. Lastly, to facilitate data exploration, we present an R Shiny app, to investigate circuit-specific ligand-receptor interaction profiles in both neuron-muscle and muscle-neuron directions, in control and experimentally altered limb configurations. Collectively, we present a resource to investigate the cellular and molecular basis for muscle-nerve matching during neuromuscular circuit refinement in the tetrapod limb, with implications for our understanding of vertebrate neuromuscular development and evolution, as well as regenerative approaches targeting re-innervation after injury or nerve damage.