A method to investigate muscle target‐specific transcriptional signatures of single motor neurons

Abstract

Background: Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required. Results: To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in midgestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success. Conclusions:Our method provides a unique approach to study muscle targetspecific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

Distinct patterning responses of wing and leg neuromuscular systems to different preaxial polydactylies

“…However, these changes are likely to result in concomitant re-wirings and potential cell type or state re-specifications at higher levels of the central nervous system. It will thus be insightful to investigate how motor neurons connecting to extra muscles change their fate ( Berki et al, 2023 ), and what potential impact this might have at the level of the spinal cord, brainstem and upper motor circuits ( Mehring et al, 2019 ; Ruder and Arber, 2019 ).…”

Getting connected: Pathfinding and synaptogenesis in neural development, evolution, and disease

“…This reflects an outstanding need in the field, which is a detailed understanding of the molecular profiles that link specific classes of neurons with their unique targets. Toward this end, single‐cell sequencing techniques have been developed by Berki and colleagues 11 that employ dye tracing to mark and identify specific motor neuron groups based upon target innervation. This approach is bound to reveal the diversity of signaling pathways and combinatorial codes that facilitate the innervation of the vertebrate and invertebrate musculature in many species for which genetic tools are not readily available.…”