Differentiation trajectories of theHydranervous system reveal transcriptional regulators of neuronal fate

Abstract

The small freshwater cnidarian polyp Hydra vulgaris 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 Hydra 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 Hydra 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 Hydra neuron differentiation, we identified 48 transcription factors expressed specifically in the Hydra 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.

Relationship between neural activity and neuronal cell fate in regeneratingHydrarevealed by cell-type specific imaging

“…Generation of Tg(hym176c:tdTomato,tba1c:GCAMP7s) cj1-in transgenic strain Hydra transgenic line Tg(hym176c:tdTomato,tba1c:GCAMP7s) cj1-in was created by microinjecting a single plasmid containing two promoters and two transgenes. Nuclear tdTomato was driven by a 2022 bp section of the hym176c regulatory region, which should be specifically expressed in ec5 neurons found in the peduncle (Fig 1H,26), and GCaMP7s was driven by a 1901 bp section of the tba1c regulatory region, which was validated in Primack et al (2023) to be a pan-neuronal promoter. The plasmid injection solution was injected into Hydra vulgaris AEP 1-cell stage embryos using an Eppendorf FemtoJet 4x and Eppendorf InjectMan NI 2 microinjector (Eppendorf; Hamburg, Germany) under a Leica M165 C stereo microscope (Leica Microscopes, Inc; Buffalo Grove, Il).…”

“…The transcriptional state of all eleven Hydra neuron subtypes has been profiled using single cell RNA-sequencing [1,47]. Similar to findings in C. elegans [20], the Hydra neurons are best defined by unique combinatorial expression of specific genes, including transcription factors and neuropeptides [21,22].…”

“…To create this line we extracted the regulatory region of the hym176c gene, which is expressed specifically in ec5 neurons (Figure 1h) and used this to drive the expression of nuclear tdTomato. To be able to track ec5 expression in the context of the activity of the entire nervous system, we included a second expression cassette in the construct using GCaMP7s under the control of pan-neuronal promoter tba1c (identified in [47]). After obtaining a founding polyp with integration of the transgene in the interstitial lineage, which includes all neurons, we propagated the line through asexual reproduction, which gave us a continuous supply of this transgenic line for experimentation.…”

A new look at the architecture and dynamics of theHydranerve net

“…One transgenic strain uses the promoter of an alpha-tubulin gene driving expression of NeonGreen. This transgene has been demonstrated to be strongly expressed in all differentiated nerve cells in Hydra , and correspondingly the entire nervous system is brightly NeonGreen-positive (Primak et al 2023). Fixed polyps of this strain were stained with PNab and transgenic NeonGreen expressing nerve cells were scored for PNab staining (see Materials and Methods).…”

The internal metabolic state controls behavior inHydrathrough an interplay of enteric and central nervous system-like neuron populations

“…To approach the nature of the neuronal control of satiety in Hydra , we conducted a systematic screening of different neuronal populations by using previously established calcium imaging lines. In a first step, we took the advantage of a pan-neuronal Hydra line (alpha-tubulin promotor 15 ) and investigated neuronal activity in the body column ( Fig. 3A ).…”