Differentiation trajectories of the Hydra nervous system reveal transcriptional regulators of neuronal fate.

The small freshwater cnidarian polyp uses adult stem cells (interstitial stem cells) to continually replace neurons throughout its life. This feature, combined with the ability to image the entire nervous system (Badhiwala et al., 2021; Dupre & Yuste, 2017) and availability of gene knockdown techniques (Juliano, Reich, et al., 2014; Lohmann et al., 1999; Vogg et al., 2022), makes a tractable model for studying nervous system development and regeneration at the whole-organism level. In this study, we use single-cell RNA sequencing and trajectory inference to provide a comprehensive molecular description of the adult nervous system. This includes the most detailed transcriptional characterization of the adult nervous system to date. We identified eleven unique neuron subtypes together with the transcriptional changes that occur as the interstitial stem cells differentiate into each subtype. Towards the goal of building gene regulatory networks to describe neuron differentiation, we identified 48 transcription factors expressed specifically in the nervous system, including many that are conserved regulators of neurogenesis in bilaterians. We also performed ATAC-seq on sorted neurons to uncover previously unidentified putative regulatory regions near neuron-specific genes. Finally, we provide evidence to support the existence of transdifferentiation between mature neuron subtypes and we identify previously unknown transition states in these pathways. All together, we provide a comprehensive transcriptional description of an entire adult nervous system, including differentiation and transdifferentiation pathways, which provides a significant advance towards understanding mechanisms that underlie nervous system regeneration.