Science—Analysis of neurogenesis and regeneration in Mexican salamanders using single-cell multiomics techniques

Editor—Summer Leaf

The Mexican salamander (Ambystoma mexicanum) is an amphibian that represents one of the closest living relatives to amniotic animals, i.
e.
, quadruped vertebrates, and is therefore suitable for comparative study
of brain cell types, neuronal connections, and function.
After removing the dorsal region, salamanders can also regenerate the telencephalic by activating neurogenesis [1], which is also present in life after embryonic stage [2].

Neurogenesis
can be found in all metazoans with nervous systems.
However, neurogenesis is almost non-existent
after brain injury.
In addition, the molecular relationship between neurogenesis observed in axolotls and mammals has not been explored
.
In addition, similarities and differences during homeostasis and during regenerative neurogenesis in the axolotl brain are also unclear
.

Recently, Professor Barbara Treutlein from the Department of Biosystems Science and Engineering of ETH Zurich and the research group of Professor Elly M.
Tanaka from the Institute of Molecular Pathology of the Vienna Biological Center jointly published a paper in Science The research paper "Single-cell analyses of axolotl telencephalon organization, neurogenesis, and regeneration" delves into the study of axolotls Organization, evolution and regeneration
of the nervous system of tetrapods.
The authors used a single-cell genomic approach to identify cell populations in the homeostasis and regeneration of the salamander telencephalan.
Glutamatergic neurons similar to amniotic neurons in the hippocampus, dorsal and lateral cortex, as well as conserved GABAergic neuronal groups, were identified; The transcriptional dynamics and gene regulation relationship of neurogenesis in specific brain regions after embryo were inferred, and the conserved differentiation characteristics were interpreted.
and after brain injury, ependymoglia cells activate the specific state
of the injury before rebuilding the lost neuronal population and axon connections.

First, the team mapped single-cell nuclear transcriptome sequencing (snRNA-seq) of the salamander telencephalon (Figure 1).
）
。 The authors dissected the telcephalic brain into three regions: medial, dorsal and lateral, and analyzed them using single-cell multi-omics techniques (snRNA-seq, snATAC-seq).
A total of 48,136 nuclei were identified, clustered into 95 neuronal and non-neuronal groups.
Apply immunofluorescence staining and RNA in situ hybridization strand (HCR) reactions to locate cell types in brain regions
.
The results showed that ependymal glial cells were mainly distributed in each ventricular area, while neuroblasts were relatively sparsely distributed.
GABAergic neurons are sparsely distributed along the ventricular, dorsal and lateral regions, but densely distributed in the striatum.
Glutamatergic neurons are mainly localized on the ventricular, dorsal, lateral and frontal lobes; Oligodendrocytes and microglia are scattered in all ventricular regions
.
The authors also analyzed the abundance of each cell group in the cortical region
.
Together, these data provide a global map of the axolotl terminal brain cell population and suggest neurogenic regional specificity
.

Fig.
1 Cell diversity of salamander telencephalon

(Source: Lust, Katharina, et al.
, Science, 2022).

Next, the authors studied the regional conservancy of glutamatergic neurons in axolotls (Figure 2).

Glutamatergic neurons of the amniotic telencephale exhibit a high degree of transcriptome diversity
.
The authors analyzed the differences and similarities between glutamatergic neuron species from the following three aspects: (1) differentially expressed genes and differentially expressed transcription factors shared between salamanders and amniotic animals; (2) integrate single-cell and mononuclear datasets of salamanders, turtles, and mouse telencephals to calculate correlations between cell groups; (3) Spatial transcriptome technology was applied to locate the location of
glutamatergic neuronal subsets.
It was found that neurons on the inner side of the axolotl cerebral cortex (glutamatergic neurons) had obvious transcriptomic similarities with other amniotic hippocampal neurons (glutamatergic neurons), but no obvious differences between these neuronal groups in the hippocampal CA1, CA3 and dentate gyrus in the brain region were observed
。 Further analysis of the results suggests that there is a strong correspondence between neurons with transcriptomic similarities to neurons in the olfactory cortex of amniotic animals and neuroprojections that play a role in olfactory processing
.

Fig.
2 Salamander telcerebral glutamatergic neurons

(Source: Lust, Katharina, et al.
, Science, 2022).

Next, the authors applied a similar analytical approach to study the conserved characteristics of GABAergic neurons in axiar animals (Figure 3).

In GABAergic neurons, the authors identified a total of 30 cell subsets
.
Through the conservative analysis of transcription factors, it is inferred that these subsets mainly originate from the lateral, caudal and medial ganglion bulges (referred to as LGE, CGE, MGE, respectively).

Similarities
between subpopulations were then calculated by integrating single-cell transcriptome data with turtles and mice.
It was found that of the 13 subsets of axolotl GABAergic neurons originating from LGE, 11 subpopulations were associated with turtle or mouse striatum and olfactory bulb GABAergic cells are related
.
Further analysis showed that the LGE-derived GABAergic neurons of the striatum and olfactory bulb had strong transcriptional similarities between the three amniotic animals of axolotl, turtle and mouse.
CGE and MGE-derived GABAergic cell populations are distributed in all regions, while LGE-derived GABAERGIC cell populations are mainly located in the striatum
。 Together, these data strongly suggest the source of GABAergic nerve cells (i.
e.
, cell migration and localization) of the salamander telencephal.

Fig.
3 Salamander telcerebral GABA-ergy neurons

(Source: Lust, Katharina, et al.
, Science, 2022).

Transcriptomic analysis of apenchymal glial cells and neuroblasts at the end of the salamander (Figure 4).

Salamander terminal ependymal cells are clustered into three cell types: quiescent, active, and pro-neuro ), they have pronounced subcellular features of brain regions; In the post-embryonic axolotl brain, resting and active ependymal glial cells continue to express cortical characteristic genes (pallial patterning genes) in addition to anterior neurotype ependymal glial cells ）
。 For transgenital cells, neuroblasts are identified into two classes based on cellular characteristic gene expression: Slc17a6/7 (VGLUT) class and Gad1/2 (GABA) class; VGLUT classes are enriched in the medial and dorsal brain regions, while GABA classes are mainly enriched in the lateral brain regions
.
The authors also investigated the association
of ependymal glial cells and neuroblasts of axolotls with neuronal stem cells and neuronal precursor cells in the subventricular zone of adult mice.
It was found that the salamander telencephalus contained neuroblasts expressing the neurotransmitter characteristics of downstream neurons.
Salamander neuroblasts are most similar to mouse neuronal precursor cells and neuroblasts, while salamander ependymal glial cells have transcriptional similarities to mouse ependymal cells as well as neural stem cells
.

Fig.
4 Ependymal glial cells of salamander telencephalida nucleate into nerve cells

(Source: Lust, Katharina, et al.
, Science, 2022).

Transcriptomic dynamics analysis of post-glutamatergic neurogenesis in axolotl embryos (Figure 5).

The authors used Cre-loxP-mediated tracing techniques to label ependymal glial cells to study their self-renewal properties and determine their renewal patterns during post-embryonic neurogenesis, as well as the neurogenesis of
glutamatergic neurons from activated ependymal glial cells to glutamatergic neurons in the differentiation stage.
RNA rate analysis and URD[3]-based trajectory analysis are used to explore cellular and molecular dynamics of
neurogenesis.
The authors observed that although all neurogenic trajectories are rooted in active ependymal glial cells, not all trajectories contain neuroblast intermediates; The authors also found many genes that specifically expressed changes along pseudo-temporal trajectories; But some genes
that were consistently expressed along pseudo-time trajectories were also found.
These data highlight the regulatory relationships
that form neuronal diversity in the salamander telcephalus.

Fig.
5 Gene regulatory program of postembryonic neurogenesis

(Source: Lust, Katharina, et al.
, Science, 2022).

Study of cellular and molecular dynamics during salamander telcerebral regeneration (Figure 6).

To do this, damage the dorsal region of the telencephale (excise a 1×1×1 mm area) at different time points and apply snRNA-seq with EdU labeling A combined research strategy for S-phase cells, i.
e.
, Div-seq analysis[5].

First, in the first week after injury, the injury site is open, and EdU⁺ ependymal glial cells are distributed in the medial and lateral adjacent areas
of the injury site.
In the 2nd week after injury, the damaged site begins to shut down
due to the accumulation of EdU+ cells.
At subsequent injury time points, EdU⁺ cells continue to accumulate at the site of regeneration (i.
e.
, the site of injury) until the tissue structure is basically rebuilt
.
Next, the authors analyzed transcriptome changes
in EdU+ cells during regeneration.
Through data integration and cluster analysis, major cell types were identified, including ependymal glial cells, neuroblasts, glutamatergic and GABAergic neurons
.
The proportion of each cell type differs throughout the regeneration process, suggesting that the damage caused neurogenesis
.
Specifically, in the first week after injury, EdU⁺ cells are mainly composed of active ependymal glial cells, and the second after injury Week and week 4, the highest number of neuroblasts is high
.
From week 6 after injury, most EdU⁺ cells were glutamatergic and GABAergic neurons
.
Finally, the authors injected neurobiotin into the domains of regenerated neurons (Satb1+, Rorb⁺) to determine whether afferent and efferent neural projections were also reconstructed
.
It was found that, at week 8 post-injury, similar to non-injured brains, cell bodies labeled positive by neurobiotin were distributed in the olfactory bulb, accessory olfactory bulb, and amygdala, indicating that neural signaling inputs in these regions were re-established in the regenerated telencephalus at week 8 post-injury
。

Fig.
6 Regeneration of axolotl end brain injury

(Source: Lust, Katharina, et al.
, Science, 2022).