An integrated transcriptomic approach for identifying enhancers in limb motor pools.

Motor neurons (MNs), which control body movement, consist of distinct motor pools, that target specific muscles. The precise delineation of these motor pools necessitates an understanding of their specialized gene expression profiles. By integrating single-cell transcriptomics and epigenomics, using scATAC-seq, scRNA-seq and histone modification datasets, we identified cis-regulatory elements (CREs) specific to five MN-lineage clusters, which exhibit distinct gene ontology terms, transcription factor (TF) binding motifs, and disease-associated traits across them. Notably, LIM-homeodomain (LIM-HD) TF motifs and their footprints were differentially enriched in corresponding individual motor column CREs, including a combined Isl1 and Lhx3 motif consistent with their known role in MN identity specification. Within the genomic locus of Etv4, a gene essential for limb motor pools, we identified three enhancer candidates among CREs enriched in LMC neurons. Functional assays demonstrated that these enhancers exhibited robust and specific activity in ETV4-expressing motor pools when electroporated into chicken neural tubes. This study demonstrates that an integrated in silico analysis of epigenetic and transcriptomic data at the single-cell level can predict putative CREs governing the identity of distinct MN subgroups, providing insights into motor pool diversification and gene regulation.