Fig. 2: Characterization of Calb1⁺ and Nfib⁺Grm5⁺ MNs.

a UMAP visualization of all clusters (left), top three markers enriched in Calb1⁺ and Nfib⁺Grm5⁺ clusters (middle), and cholinergic genes (right). b Immunostaining reveals ventromedial positioning of the Calb1⁺Mnx1⁺ cells in the E13.5 mouse spinal cord. A high-magnification image is shown in the yellow square. c Immunostaining at E15.5 and P7 reveals that Calb1⁺ cells exhibit cholinergic identity by co-expressing Chat. High-magnification images are shown in the yellow square. d Calb1⁺Mnx1⁺ cells localize in the brachial and upper thoracic segments. e RNAscope-based fluorescence ISH of Nfib⁺Grm5⁺ cluster markers show that Nfib⁺Grm5⁺ and Grm5⁺B3glct⁺ coexpressing cells are present mainly in the sacral segments. f Immunostaining of Mnx1 with Nfib or Grm5 and Foxp1 with Lhx3 in adjacent sacral spinal cord sections (E13.5). Foxp1 and Lhx3 label sacral PGC and MMC MNs, respectively. Note that the Nfib⁺ Grm5⁺ cells are separated from sacral PGC MNs and partially overlap MMC MNs. Dashed lines outline the spinal cord boundary. Scale bars represent 50 μm. All immunostaining and in situ hybridization experiments were repeated on n = 3 embryos.