UNVEILING THE MOLECULAR MECHANISMS THAT CONTROL THE AXONAL LATERALITY OF DI1 SPINAL INTERNEURONS

Spinal neurons that ascend ipsilaterally or contralaterally to the brain contribute to bilateral neuronal circuits and are essential for somatosensory processing and motor coordination. However, the molecular mechanisms that determine axonal laterality are still not fully understood. Dorsal interneurons 1 (dI1) neurons are generated early during spinal cord development and comprise two subtypes: ipsilateral (dI1i) and contralateral (dI1c). Using a tamoxifen-inducible reporter mouse line (Atoh1-CreER) that labels all dI1 neurons, we determined when dI1i and dI1c subtypes are specified. We found that dI1i neurons are specified from embryonic day 8 (E8) onwards, whereas dI1c neurons begin to differentiate from E10 onwards. Thus, although dI1i and dI1c belong to the same neuronal class and convey similar sensory information, they are specified at distinct developmental stages. Consistent with this timing, at E11 most dI1 neurons project contralaterally, while at E12 dI1 projections are mixed, with a predominant ipsilateral component. We leveraged this temporal difference to perform single-nucleus RNA sequencing (snRNA-seq) and define the transcriptomic profiles of ipsilateral and contralateral dI1 neurons. This analysis identified candidate genes to regulate dI1 axonal laterality in the early spinal cord. In addition, we identified different subpopulations within both ipsilateral and contralateral dI1 neurons. Ongoing work focuses on functionally validating these candidate genes to determine their roles in midline crossing decisions.