Salamanders are tetrapod models to study brain organization and regeneration; however, the identity and evolutionary conservation of brain cell types are largely unknown. We delineated the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-cell genomic profiling. We identified glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved γ-aminobutyric acid–releasing (GABAergic) neuron classes. We inferred transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific neurogenesis and unraveled conserved differentiation signatures. After brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield insights into the organization, evolution, and regeneration of a tetrapod nervous system.
Salamander brains share some, but not all, structures with the mammalian brain. They also have greater capacity to regenerate in response to damage. Three groups now come together with single-cell transcriptomics analyses that set the salamander brain in evolutionary context (see the Perspective by Faltine-Gonzalez and Kebschull). By comparing salamander brains with those of lizard, turtle, and mouse, Woych et al . track the evolutionary innovations that gave rise to the mammalian six-layered neocortex, which salamanders do not have. Lust et al . take a close look at why the axolotl brain is so much more capable of regeneration than is the mammalian brain. Finally, Wei et al . compare the developmental and regenerative processes in the axolotl brain. —PJH
Molecular analysis of axolotl brain cells sheds light on evolutionary conservation, neural differentiation, and regeneration.
Salamanders, such as the axolotl ( Ambystoma mexicanum ), play a role in the study of tetrapod-conserved traits. Cell-type diversity in salamander brains and their relation to other vertebrate brains has until now been studied mainly histologically. Axolotl brains grow during postembryonic life, with new neurons generated by proliferating ependymoglia cells. Axolotl brains also regenerate after injury; however, it is still unclear how stem cells regenerate the brain and whether neuronal connections are appropriately recovered.
Single-cell and single-nucleus genomic profiling of the telencephalon has revealed diversity and evolutionary relationships of cell types and brain regions among several amniotes, including reptiles, birds, and mammals. These methods have also revealed molecular trajectories underlying developmental and adult neurogenesis. We molecularly characterized axolotl telencephalon cell types, neurogenesis, and evolutionary conservation. We applied single-nucleus genomic profiling to the axolotl telencephalon in steady state and during regeneration to investigate its cell-type diversity and molecular dynamics of homeostatic neurogenesis. We compared molecular profiles to understand injury-specific features of regenerative neurogenesis.
We determined the cellular diversity of the axolotl telencephalon using single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq), as well as spatial transcriptomics. We identified regionally distributed neuron, ependymoglia, and neuroblast populations and determined their conservation with amniotes by using comparative analyses. We found that the axolotl telencephalon contains glutamatergic neurons with transcriptional similarities to neurons of the turtle and mouse hippocampus, dorsal cortex, and olfactory cortex. Olfactory cortex–like neurons also show conserved neuronal input projections from the olfactory bulb. Axolotl γ-aminobutyric acid–releasing (GABAergic) inhibitory neurons show signatures of different subregions of the ganglionic eminence and resemble turtle and mouse GABAergic inhibitory neurons. We used trajectory inference to construct differentiation trajectories of homeostatic neurogenesis and found that ependymoglia largely progress through distinct intermediate neuroblast types and use specific gene regulatory networks to form distinct glutamatergic neuron types. We tracked cycling cells upon injury of the telencephalon and found an injury-specific ependymoglia transcriptional state characterized by up-regulation of wound healing and cell migration genes at the beginning of regenerative neurogenesis. Neurogenesis after injury progresses similarly to homeostatic neurogenesis and results in reestablishment of lost neurons and input projections from the olfactory bulb.
Our findings indicate that cell types and gene expression patterns associated with mammalian telencephalon regions are also evident in the amphibian brain. The evolutionary history of cell types clarified the larger divergence in glutamatergic compared with GABAergic neurons that we observed the axolotl, as was also seen in reptiles. We conclude that in the postembryonic axolotl, telencephalon neurogenesis progresses through diverse neuroblast progenitors, which are associated with specific neuron types and dependent on shared as well as specific regulatory programs. We found implementation of these same programs in regenerative neurogenesis, which indicates that brain injury activates neurogenesis through existing pathways after inducing an injury-specific ependymoglia state. Regenerated neurons reestablish their previous connections to distant brain regions, suggesting potential functional recovery. Our insight into how the axolotl brain regenerates may inform studies of brain regeneration in other organisms.