Molecular Profiling defines evolutionarily conserved transcription factor signatures of major vestibulospinal neuron groups

Vestibulospinal neurons are organized into discrete groups projecting from brainstem to spinal cord, enabling vertebrates to maintain proper balance and posture. The two largest groups are the lateral vestibulospinal tract (LVST) group and the contralateral medial vestibulospinal tract (cMVST) group, with different projection lateralities and functional roles. In search of a molecular basis for these differences, we performed RNA sequencing on LVST and cMVST neurons from mouse and chicken embryos followed by immunohistofluorescence validation. Focusing on transcription factor (TF)-encoding genes, we identified TF signatures that uniquely distinguish the LVST from the cMVST group and further parse different rhombomere-derived portions comprising the cMVST. Immunohistofluorescence assessment of the CNS from spinal cord to cortex demonstrated that these TF signatures are restricted to the respective vestibulospinal groups and some neurons in their immediate vicinity. Collectively, these results link the combinatorial expression of TFs to developmental and functional subdivisions within the vestibulospinal system. Transcriptome profiles of small pools of retrogradely labeled, acutely dissociated, manually sorted vestibulospinal neurons from mouse and chicken embryos with 3-6 biological replicates per sample condition (projection target). RNAseq libraries prepared from medially adjacent tissue served as a control for both mouse and chicken samples (4 biological replicates), and for the mouse an additional control group was comprised of unlabeled, acutely dissociated and manually sorted cells from caudally adjacent tissue (4 biological replicates).