A single cell approach to study transcriptional regulation of cortico-pontine connectivity during mouse brainstem development

The projection neurons of the pontine nuclei (PN) constitute the primary mossy fiber input to the cerebellum, transmitting signals from the cerebral cortex. The connectivity pattern between cortical areas and PN and the complex organization of ponto-cerebellar connectivity have been extensively investigated. At the cellular level, an internal-external lamellar organization of cortical axon fields may topographically match an inside-out organization of PN neurons based on their birthdate (Altman and Bayer, 1987; Leergard et al.,1995). Moreover, our laboratory recently discovered an intrinsic organization of PN neurons according to their rostrocaudal origin in the precerebellar rhombic lip, which is topographically maintained through migration and nucleogenesis (Di Meglio et al., 2013; Maheshwari et al., 2020). However, little is still known about the establishment of the molecular diversity in PN neuron subpopulations during development, underlying such a complex cellular organization and cortico-pontine-cerebellar circuit connectivity. In this work, we used bulk and single-cell transcriptome profiling of PN neurons to evaluate molecular changes over development and in the context of lack of cortical inputs. We identified transcriptome developmental heterogeneity at the single-cell level in PN neurons, consistent with postulated differences in the degree of cell maturation and molecular identity due to distinct PN neuron birthdates. Moreover, in a mouse model lacking cortical input to PN neurons, we identified transcriptional changes at an early postnatal stage, suggesting a critical role of cortical innervation during a restricted time window in the maturation of sub-cortical nuclei. This study provides insights not only into the identification of intrinsic and extrinsic factors sequentially establishing neuronal identities but also information about regulators of the formation of complex circuitry during development.