The axolotl is a type of Mexican salamander with astonishing regenerative capacity1. In our recent paper, we identified a signaling heterodimer that is formed directly after injury in the glial cells adjacent to the injury in axolotls. The c-Fos and JunB genes forming this heterodimer are not unique to animals with high regenerative capacity but they are present in humans too. In this paper I propose perspectives on molecular control of regeneration and future directions that need to be taken to advance our understanding of regeneration at a molecular level.
Diversity among species in mechanisms of regeneration
The ability to functionally regenerate is a phenomenon that has fascinated mankind for centuries. Aristotle (384–322 BC) made one of the first written observations of lizards regenerating their tails, yet centuries later the molecular blueprints for this fascinating skill set remain elusive2. Many animals and plants display various degrees of regenerative capacity and interestingly, there are many roads to the same endpoint even amongst highly related species3. Salamanders, for example, can functionally regenerate their limbs, which includes muscle fibers. The terrestrial newt does this by dedifferentiating its mature muscle to form mononucleate cells that act as progenitors for the new muscle, whereas the highly related aquatic Mexican salamander, the axolotl, utilizes its Pax7 positive satellite cells to regenerate its muscle fibers4. Why such closely related species use such different mechanisms to regenerate the same cell type is unknown, but it shows the importance of studying a wide range of research organisms to understand and eventually define the principles of regeneration.
Humans have very restricted regenerative capability but we can repair lesions in our muscle using endogenous satellite cells, similar to those found in highly regenerative species like axolotls, Parhyale and zebrafish. We can also repair lesions in our peripheral nervous systems; if our muscle is damaged, the nerves innervating that tissue are often also damaged and we can repair these types of injuries up to a certain size. However despite the ability to regenerate peripheral nerves, the scenario in the central nervous system is very different. After injury to the spinal cord in humans, surrounding cells, like glial cells, fibroblasts and immune cells, respond to the initial injury signal cues and migrate to the injury site. The injury activates a process termed reactive gliosis and those cells which migrate to the injury site start to express proteins that they normally don’t express and come together to form a glial scar. This glial scar has both positive and negative aspects—it is thought to prevent further injury but has also been shown to express proteins that inhibit axon regeneration5,6,7,8.
Similarities and differences in spinal cord regeneration
The spinal cord presents a similar diversity by which a specific animal will reach a functional end point. Lamprey, the most basal vertebrate that regenerates neurons, can regenerate after complete spinal cord transection and research from several laboratories has shown that its central nervous system consists of “good regenerators and bad regenerators”. However despite not all neurons having the capacity to regenerate, Lamprey can regain functional locomotive activity9,10,11,12,13,14. Work in zebrafish has shown a similar phenomenon, although exactly what percentage is regenerating is harder to define due to a larger number of neurons15,16. Axolotls are thought to, in fact, regrow all of their neurons after complete transection of their spinal cord17. This could be related to differences in how the severed neural tube regenerates. For example, in zebrafish, the glial fibrillar acidic protein (GFAP)-positive glial cells lining the central canal delaminate and stretch out to bridge the injury site18,19. Axolotls activate glial cell division and migration to fill in the missing portion of the neural tube, such that it is morphologically impossible to distinguish old from new tissue (Table 1)20. Lamprey and Xenopus appear to have quite different glial cells to other animals in that both animals seemingly lack a true GFAP in their genome. This may be why no true glial scar is formed in these animals after injury and hence a more permissive injury environment is presented to the severed neurons21,22,23. In Lamprey it would then suggest that intrinsic differences within neurons determine their regeneration capacity. In Xenopus the situation is more complicated. In the larval stages they can regenerate the spinal cord; glial cells adjacent to the injury upregulate the neural progenitor marker sox2, similar to axolotls and these cells migrate and divide to repair the injury. However after metamorphosis regenerative ability is lost24,25,26. This has been linked to the maturation of the immune system, which is known to play a role in reactive gliosis in humans (22–24).
Axolotl glial cell activation
In our recent paper we have begun to examine at a molecular level how axolotl glial cells are activated to divide and migrate in response to injury. We have identified a heterodimer consisting of c-Fos and JunB which is transiently activated and which functionally regulates the GFAP promoter preventing up-regulating GFAP expression. The c-Jun gene is present in axolotls and is repressed in response to injury by activation of miR-200a. Overexpression of c-Jun in the axolotl glial cells leads to upregulation of GFAP and other genes involved in reactive gliosis and ultimately blocks axon regeneration. In mammals, work from many groups have revealed that after spinal cord injury, glial cells show a prolonged upregulation of the canonical AP-1 transcription factor composed of the heterodimer c-Fos and c-Jun which instead activates the GFAP promoter27. This work illustrates that small changes in heterodimer formation can lead to substantially different outcomes probably due to very different signaling pathways activated downstream. Our research also brings up many interesting questions and potential avenues to follow. Recently we have tested whether changing the composition of the heterodimer can affect the mammalian GFAP promoter and indeed, preliminary in vitro experiments suggest that the non-canonical c-Fos:JunB heterodimer formed in axolotl after injury fail to induce high activation of the GFAP promoter. We have also begun to examine other species with high regenerative capacity to ask upregulation of the non-canonical AP-1 c-fos:JunB occurs. Initial data-mining of publicly available RNA sequencing data shows up-regulation of Fos and Jun family members in many regenerative species. However due to lack of high-level annotation of many genomes it is often unclear exactly which family member is activated in these data sets. Nevertheless, it is important to note that although Lamprey and Xenopus do not appear to have a GFAP gene, they activate Fos and Jun family members after injury23,28,29. The functional significance of this activation remains to be elucidated. It’s likely that injury-induced genes modulate several pathways to direct the response to injury in the spinal cord.
One of the future challenges is to determine, in axolotl, which are the important signaling pathways downstream of the GFAP promoter as well as identifying other potential pathways affected by the heterodimer. In our recent paper we carried out RNA sequencing on the axolotl spinal cord tissue 4 days post injury providing a wealth of data to be mined. As expected, markers of neuronal differentiation and synaptic signaling are downregulated after injury; however when miR-200a is inhibited we see mis-regulation of classical markers of neuronal differentiation and axon guidance. This could suggest that miR-200a may not just regulate c-Jun but also other target genes involved in regulating the differentiation state of cells after injury, and potentially some cells may revert to a more neural stem cell-like state in the axolotl after injury.
The role of the immune system
In addition, genes involved in the immune system are highly upregulated in regeneration. There has been renewed interest in the role of the immune system in pro-regenerative versus non-regenerative species in recent years. Earlier work in Xenopus limb regeneration suggested that the maturation of the immune system potentially was a large causative factor in the inability of the frog to regenerate after metamorphosis. However biology is rarely binary and work from many labs has shown that there is also a need for the immune system in regeneration. Ultimately it may all be about timing and duration of the immune stimulation. Recent work has very elegantly illustrated this aspect in two closely related species that have stark regenerative (Zebrafish) and non-regenerative (Medaka) responses to injury to the heart. In zebrafish recruitment of macrophages is necessary for activation of the regenerative program but it is not an absence of macrophages in Medaka that is responsible for its lack of regenerative ability, rather, an interesting difference in the timing and duration of immune cells recruited to the injury site30.
What pathways actually recruit immune cells to the injury site and control their dynamics is a big question that needs to be addressed. Data from many research organisms, especially zebrafish (because of the availability of transgenic lines for different immune cells) has provided interesting insights into the types of immune cells recruited to different types of injuries and their dynamics31. The next level is understanding their molecular control in detail. To date many signals like TGF-beta, Toll receptors and interleukins are know to be involved in recruiting immune cells. The AP-1 transcription factor has also been implicated. Interestingly JunB interacts with IL-1ß and with ATF3, genes with known roles in the immune system. This might suggest that in animals that lack a GFAP gene, Fos and Jun play roles in modulating the immune response in the spinal cord after injury.
The far-reaching goal of studying nervous system regeneration in species that can do it naturally is that one day this knowledge can inform strategies for therapies for patients with spinal cord injury and neurodegenerative diseases. It is clear from studying a small snapshot of organisms with functional regenerative capacity that there is no single pathway to regeneration. One question that scientists are often faced with is “how similar” are axolotl glial cells to human cells. To date we know from transcriptional profiling studies that glial cells from axolotl or zebrafish express many of the same genes as human cells. In the future it will be interesting to determine if the regulatory elements of these genes have evolved differently and whether they determine the initial response to injury in order to direct cells towards a certain pathway. Research from many groups using different organisms clearly shows that nature has evolved many different routes to regenerate functional spinal cords; however more in-depth knowledge is necessary to build blueprints for any one organism’s individual regenerative strategy.
Sabin, K. Z., Jiang, P., Gearhart, M. D., Stewart, R. & Echeverri, K. AP-1(cFos/JunB)/miR-200a regulate the pro-regenerative glial cell response during axolotl spinal cord regeneration. Commun. Biol. 2, 91 (2019).
Aristotle. Historia Animalium (Oxford, The Clarendon Press, 1910).
Sanchez Alvarado, A. & Tsonis, P. A. Bridging the regeneration gap: genetic insights from diverse animal models. Nat. Rev. Genet 11, 873–884 (2006).
Sandoval-Guzman, T. et al. Fundamental differences in dedifferentiation and stem cell recruitment during skeletal muscle regeneration in two salamander species. Cell Stem Cell 14, 174–187 (2014).
Fawcett, J. & Asher, R. The glial scar and central nervous system repair. Brain Res. Bull. 49, 377–391 (1999).
Göritz, C. et al. A pericyte origin of spinal cord scar tissue. Science (New York, NY) 333, 238–242 (2011).
Sandvig, A., Berry, M., Barrett, L. B., Butt, A. & Logan, A. Myelin-, reactive glia-, and scar-derived CNS axon growth inhibitors: expression, receptor signaling, and correlation with axon regeneration. Glia 46, 225–251 (2004).
Silver, J. & Miller, J. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5, 146–156 (2004).
Hanslik, K. L. et al. Regenerative capacity in the lamprey spinal cord is not altered after a repeated transection. PloS ONE 14, e0204193 (2019).
Oliphint, P. A. et al. Regenerated synapses in lamprey spinal cord are sparse and small even after functional recovery from injury. J. Comp. Neurol. 518, 2854–2872 (2010).
Smith, J. et al. Regeneration in the era of functional genomics and gene network analysis. Biol. Bull. 221, 18–34 (2011).
Selzer, M. E. Mechanisms of functional recovery and regeneration after spinal cord transection in larval sea lamprey. J. Physiol. 277, 395–408 (1978).
Wood, M. R. & Cohen, M. J. Synaptic regeneration in identified neurons of the lamprey spinal cords. Science (New York, NY) 206, 344–347 (1979).
Yin, H. S. & Selzer, M. E. Axonal regeneration in lamprey spinal cord. J. Neurosci. 3, 1135–1144 (1983).
Becker, T. & Becker, C. G. Axonal regeneration in zebrafish. Curr. Opin. Neurobiol. 27, 186–191 (2014).
Becker, T., Wullimann, M. F., Becker, C. G., Bernhardt, R. R. & Schachner, M. Axonal regrowth after spinal cord transection in adult zebrafish. J. Comp. Neurol. 377, 577–595 (1997).
Clarke, J. D., Alexander, R. & Holder, N. Regeneration of descending axons in the spinal cord of the axolotl. Neurosci. Lett. 89, 1–6 (1988).
Goldshmit, Y. et al. Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J. Neurosci. 32, 7477–7492 (2012).
Hui, S. P. et al. Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish. PloS ONE 9, e84212 (2014).
Sabin, K., Santos-Ferreira, T., Essig, J., Rudasill, S. & Echeverri, K. Dynamic membrane depolarization is an early regulator of ependymoglial cell response to spinal cord injury in axolotl. Dev. Biol. 408, 14–25 (2015).
Martinez-De Luna, R. I. et al. Muller glia reactivity follows retinal injury despite the absence of the glial fibrillary acidic protein gene in Xenopus. Dev. Biol. 426, 219–235 (2017).
Smith, J. J. et al. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nat. Genet. 45, 415–421 (2013). 421e411–412.
Herman, P. E. et al. Highly conserved molecular pathways, including Wnt signaling, promote functional recovery from spinal cord injury in lampreys. Sci. Rep. 8, 742 (2018).
Lee-Liu, D., Mendez-Olivos, E. E., Munoz, R. & Larrain, J. The African clawed frog Xenopus laevis: a model organism to study regeneration of the central nervous system. Neurosci. Lett. 652, 82–93 (2017).
Mendez-Olivos, E. E., Munoz, R. & Larrain, J. Spinal cord cells from pre-metamorphic stages differentiate into neurons and promote axon growth and regeneration after transplantation into the injured spinal cord of non-regenerative xenopus laevis froglets. Front. Cell. Neurosci. 11, 398 (2017).
Munoz, R. et al. Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells. Dev. Biol. 408, 229–243 (2015).
Gao, k. et al. Traumatic scratch injury in astrocytes triggers calcium influx to activate the JNK/c-Jun/AP-1 pathway and switch on GFAP expression. Glia 61, 2063–2077 (2013).
Lee-Liu, D. et al. Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages. Neural Dev. 9, 12 (2014).
Lee-Liu, D., Sun, L., Dovichi, N. J. & Larrain, J. Quantitative proteomics after spinal cord injury (SCI) in a regenerative and a nonregenerative stage in the frog xenopus laevis. Mol. Cell. Proteom.: MCP 17, 592–606 (2018).
Lai, S. L. et al. Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration. Elife 6, https://doi.org/10.7554/eLife.25605 (2017).
Caldwell, L. J. et al. Regeneration of dopaminergic neurons in adult zebrafish depends on immune system activation and differs for distinct populations. J. Neurosci. 39, 4694–4713 (2019).
The author declares no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Echeverri, K. The various routes to functional regeneration in the central nervous system. Commun Biol 3, 47 (2020). https://doi.org/10.1038/s42003-020-0773-z
Frontiers in Molecular Biosciences (2020)