Background

The brain holds an astonishing cell type diversity of both neurons and glia across different regions. Understanding this diversity and the reprograming potential from one cell type to the other will be instrumental for the development of regenerative therapies after brain injury. The brain regional diversity arises during embryonic development when progenitors from different regions activate specific transcriptional programs. We know that neurons born in the same area at a similar time tend to be similar to each other, at least at the level of gene expression. There is evidence that this might be also the case for other brain cell types like astrocytes¹ . Given that neurons and glia often originate from the same progenitors², do they share transcriptomic profiles? If so, is this similarity brain region-specific? In their last preprint, Herrero-Navarro et al. explore these questions by comparing astrocytes and neurons from developing cortex and thalamus in mice. The authors show that neurons and astrocytes share region-specific transcriptomic and epigenetic signatures and are clonally related. They also perform astrocyte-to-neuron reprogramming and find that their new identity matches that of their close “neuronal siblings”.

Key findings

Neurons and astrocytes share region-specific transcriptomic profiles

The authors first isolated astrocytes from different regions of P7 mice brains using a GFAP::GFP reporter mice line. They dissected the cortex (primary somatosensory region, S1) and three nuclei of the thalamus (dorsolateral geniculate dLG, ventral posteromedial VPM and ventro-medial geniculate MGv nuclei), processed the samples in a fluorescence-activated cell sorter, which isolated GFP-GFAP-positive astrocytes and performed bulk RNA-sequencing for each of the regions (Fig.1). Differential expression analysis between thalamic versus cortical samples showed that genes enriched in thalamic astrocytes are quite similar to genes normally expressed in thalamic neurons (Gbx2, Tcfl2, Rora, Lef1), while genes enriched in cortical astrocytes are reminiscent to those expressed in cortical neurons (Tbr1, Neurod6, Citip2, Satbt2). Additionally, they performed RNA-seq in thalamic neurons (Gbx2::tomato mouse line) from the same three nucleus and they analysed a published dataset of cortical neurons (Figure 1), confirming the high degree of similarity between top-enriched genes of neurons and astrocytes of the thalamus (37%) and between neurons and astrocytes from the cortex (17%). Analysis of a recent single cell dataset in juvenile mouse containing both neurons and astrocytes from the cortex and thalamus³ confirmed their findings. Interestingly, they also show that astrocytes and neurons from the three different thalamic nuclei cluster separately in PCA plots, meaning that their transcriptional signatures are also nucleus specific.

Thalamic neurons and astrocytes are clonally related and show little dispersion across boundaries

The authors next explore the clonal origin of neurons and astrocytes in the thalamus by performing a series of in utero electroporation with a battery of plasmids encoding different fluorofores under the control of GFAP promoter, following transposase mediated integration (“StarTrack”⁴ for the specific labelling of astrocytes) or a ubiquitous promoter. They electroporated at E11.5 and analysed the distribution of the clones at P8. They observed that both astrocyte-only clones and mixed clones (containing both astrocytes and neurons originated from the same progenitor) tend to remain in the boundaries of a given nucleus. This means that the similarity observed in the transcriptional signatures of astrocytes and neurons of the same regions can be explained by their shared clonal origin and their little dispersion throughout development. Moreover, these experiments also show that the positional identity of neurons and astrocytes is acquired at early developmental stages and retained postnatally.

Astrocytes can be reprogrammed to become neurons, but not any neuron, they specifically form neuronal types that closely resemble their anatomical neighbours. And this ability is cell-autonomous.

The authors next tested the ability of astrocytes to be reprogrammed to neurons following a previously published strategy that involves Ngn2-Bcl2-mediated neuronal induction⁵. Taking advantage that retroviruses only transduce proliferating glia at the stage they performed the experiment (P3), they first injected the retroviral vector in cortex and thalamus and found that newly reprogrammed neurons express their corresponding cortical or thalamic molecular markers (Fig. 2).

To discard the effect of the environment, they next used in vitro cultures of astrocytes from cortex and thalamus. They infected the astrocytes with a retrovirus carrying a Ngn2 expression construct, obtaining the same outcome. Cortical astrocytes become cortical neurons while thalamic astrocytes become thalamic neurons. To further control for the environment effect, they repeated these experiments but this time co-culturing the astrocytes with astrocytes or neurons from the other tissue, still observing the same outcome (Fig. 3).

They even went further and tested this with astrocytes from different thalamic nuclei, finding that their reprogrammed neuronal identity reflects their nucleus of origin.

When trying to reprogram cortical astrocytes to thalamic neurons by using a thalamic fate determinant (Gbx2) in combination with Ngn2, they only caused a partial fate redirection, since only some thalamic signature genes were increased in these induced neurons.

These results indicate that, no matter what their environment is, astrocytes have an intrinsic tendency to form neurons of the same tissue. Finally, the authors find that epigenetic “primed” signatures inherited from progenitors could explain the differential induction potential of astrocytes born in different tissues, up to the level of different thalamic nuclei.

Why I find this preprint interesting

This preprint caught my eye because it deals with a very fundamental question in Developmental Biology (cell type specification, fate commitment and fate flexibility), and produces novel evidence with important biomedical implications. The authors’ assessment of fate flexibility of astrocytes is very elegant and proves they have the potential to become the neurons that surround them. This, I believe, makes them excellent targets for neuroregenerative therapies after brain injury.

Future directions and questions for the authors

• 1) In your clonal analysis using the ubiquitous promoter you find that mixed clones (giving rise to both neurons and astrocytes) tend to remain within a given thalamic nucleus territory while “only-neuron” _clones spread more broadly. Do you have any hypothesis to explain this different behaviour? Who might be these neurons that expand more broadly? Are they related with any known tangential migration?

   

  This is a very good question. Our data when looking only at the neurons reveals a slightly higher dispersion than in astrocytes. But it is important to mention that the number of clones containing only neurons is quite low. This result is, in any case, in agreement with previous studies (see Song-Hai Shi or Nakagawa’s work). Thalamic astrocytes seem to have a restricted allocation preference to a single nucleus, and we hypothesize that this might be mainly due to the timing of generation, and the position of the progenitor at the time of astrogenesis.

   

  2) Your study and others4 demonstrate that astrocyte reprogramming potential seems to be tissue-, cortical layer- and even thalamic nucleus- specific. This reinforce the idea of astrocytes as powerful candidates for local brain repair after injury (versus their classic view as the harmful cell type which form the glial scar) or in disease. Where is this field now? Where do you see it going next?

   

  We consider that this topic very promising and with lot of potential. Several groups have also made interesting discoveries that confirm that astrocytes are good candidate cells for brain repair, and our data is push even more into this direction by showing that astrocytes are molecularly “primed’’ to a region. Astrocytes have also the advantage to be resident cells that will not induce any immune response as it could occur with exogenous cell transplantation. Currently we consider that the field is moving towards finding less invasive reprogramming in vivo. The AAV delivery seems to be quite promising, and it is becoming the preferred option for targeting astrocytes in vivo. Finally, the next step of course will be to confirm the complete recovery of a long-range connection, which in fact has been recently shown in a few papers, and to confirm a functional recovery of a damaged circuit after reprogramming.

   

  3) Astrocytes do not spontaneously reprogram into neurons after brain damage in mammals (at least in the species that have been studied). But they do seem to hold the potential to do so. Do astroglial counterparts in more ancient animal groups have this ability? In other words, is this potentiality a remnant of an ability that was lost in modern vertebrates?

   

  Well, we are not experts in the properties or functions of astrocytes from an evolutionary point of view, but this is an interesting question. It is true that the nervous system in simpler animal models seems to be more plastic and with a higher capacity for neuronal regeneration, which is lost in superior vertebrates. We are not aware that astroglial cells could spontaneously be converted into neurons in response to damage. Perhaps, in more ancient animal, astrocytes cells remain in a more immature or less specialized state, making them easier to change their identity even spontaneously after a damage.

References

1. Hochstim, C., Deneen, B., Lukaszewicz, A., Zhou, Q. & Anderson, D. J. (2008). Identification of positionally distinct astrocyte subtypes whose identities are specified by a homeodomain code. Cell 133, 510–522
2. Rowitch, D. H. & Kriegstein, A. R. Developmental genetics of vertebrate glial cell specification (2010). Nature 468, 214–222.
3. Zeisel A, Hochgerner H, Lönnerberg P, et al. (2018). Molecular Architecture of the Mouse Nervous System. Cell 174, 999-1014.
4. García-Marqués, J. & López-Mascaraque, L. (2013) Clonal identity determines astrocyte cortical heterogeneity. Cortex 23, 1463–1472.
5. Gascón S, Murenu E, Masserdotti G, et al. (2016). Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming. Cell Stem Cell 18, 396–409.
6. Mattugini N, Bocchi R, Scheuss V, et al. (2019) Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex. Neuron 103, 1086–1095.

Tags: astrocytes, cortex, mouse, neurons, reprogramming, thalamus

doi: https://doi.org/10.1242/prelights.21264

Read preprint (2 votes)