© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Echeverri, K.  The various routes to functional regeneration in the central nervous system. Communications Biology, 3(1), (2020): 47, doi:10.1038/s42003-020-0773-z.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

Echeverri, Karen

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COMMENTThe various routes to functionalregeneration in the central nervoussystemKaren Echeverri1*The axolotl is a type of Mexican salamander with astonishing regenerativecapacity1. In our recent paper, we identiﬁed a signaling heterodimer that isformed directly after injury in the glial cells adjacent to the injury in axolotls. Thec-Fos and JunB genes forming this heterodimer are not unique to animals withhigh regenerative capacity but they are present in humans too. In this paper Ipropose perspectives on molecular control of regeneration and future directionsthat need to be taken to advance our understanding of regeneration at amolecular level.Diversity among species in mechanisms of regenerationThe ability to functionally regenerate is a phenomenon that has fascinated mankind for cen-turies. Aristotle (384–322 BC) made one of the ﬁrst written observations of lizards regeneratingtheir 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, thereare many roads to the same endpoint even amongst highly related species3. Salamanders, forexample, can functionally regenerate their limbs, which includes muscle ﬁbers. The terrestrialnewt does this by dedifferentiating its mature muscle to form mononucleate cells that act asprogenitors for the new muscle, whereas the highly related aquatic Mexican salamander, theaxolotl, utilizes its Pax7 positive satellite cells to regenerate its muscle ﬁbers4. Why such closelyrelated species use such different mechanisms to regenerate the same cell type is unknown, but itshows the importance of studying a wide range of research organisms to understand andeventually deﬁne the principles of regeneration.Humans have very restricted regenerative capability but we can repair lesions in our muscleusing 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 ourmuscle is damaged, the nerves innervating that tissue are often also damaged and we can repairthese types of injuries up to a certain size. However despite the ability to regenerate peripheralnerves, the scenario in the central nervous system is very different. After injury to the spinal cordhttps://doi.org/10.1038/s42003-020-0773-z OPEN1 Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA, USA. *email: kecheverri@mbl.eduCOMMUNICATIONS BIOLOGY |            (2020) 3:47 | https://doi.org/10.1038/s42003-020-0773-z | www.nature.com/commsbio 11234567890():,;in humans, surrounding cells, like glial cells, ﬁbroblasts andimmune cells, respond to the initial injury signal cues and migrateto the injury site. The injury activates a process termed reactivegliosis and those cells which migrate to the injury site start toexpress proteins that they normally don’t express and cometogether to form a glial scar. This glial scar has both positive andnegative aspects—it is thought to prevent further injury but hasalso been shown to express proteins that inhibit axonregeneration5–8.Similarities and differences in spinal cord regenerationThe spinal cord presents a similar diversity by which a speciﬁcanimal will reach a functional end point. Lamprey, the most basalvertebrate that regenerates neurons, can regenerate after completespinal cord transection and research from several laboratories hasshown that its central nervous system consists of “good regen-erators and bad regenerators”. However despite not all neuronshaving the capacity to regenerate, Lamprey can regain functionallocomotive activity9–14. Work in zebraﬁsh has shown a similarphenomenon, although exactly what percentage is regenerating isharder to deﬁne due to a larger number of neurons15,16. Axolotlsare thought to, in fact, regrow all of their neurons after completetransection of their spinal cord17. This could be related to dif-ferences in how the severed neural tube regenerates. For example,in zebraﬁsh, the glial ﬁbrillar acidic protein (GFAP)-positive glialcells lining the central canal delaminate and stretch out to bridgethe injury site18,19. Axolotls activate glial cell division andmigration to ﬁll in the missing portion of the neural tube, suchthat it is morphologically impossible to distinguish old from newtissue (Table 1)20. Lamprey and Xenopus appear to have quitedifferent glial cells to other animals in that both animals seeminglylack a true GFAP in their genome. This may be why no true glialscar is formed in these animals after injury and hence a morepermissive injury environment is presented to the severedneurons21–23. In Lamprey it would then suggest that intrinsicdifferences within neurons determine their regeneration capacity.In Xenopus the situation is more complicated. In the larval stagesthey can regenerate the spinal cord; glial cells adjacent to the injuryupregulate the neural progenitor marker sox2, similar to axolotlsand these cells migrate and divide to repair the injury. Howeverafter metamorphosis regenerative ability is lost24–26. This has beenlinked to the maturation of the immune system, which is known toplay a role in reactive gliosis in humans (22–24).Axolotl glial cell activationIn our recent paper we have begun to examine at a molecularlevel how axolotl glial cells are activated to divide and migrate inresponse to injury. We have identiﬁed a heterodimer consisting ofc-Fos and JunB which is transiently activated and which func-tionally regulates the GFAP promoter preventing up-regulatingGFAP expression. The c-Jun gene is present in axolotls and isrepressed in response to injury by activation of miR-200a.Overexpression of c-Jun in the axolotl glial cells leads toupregulation of GFAP and other genes involved in reactive gliosisand ultimately blocks axon regeneration. In mammals, work frommany groups have revealed that after spinal cord injury, glial cellsshow a prolonged upregulation of the canonical AP-1 transcrip-tion factor composed of the heterodimer c-Fos and c-Jun whichinstead activates the GFAP promoter27. This work illustrates thatsmall changes in heterodimer formation can lead to substantiallydifferent outcomes probably due to very different signalingpathways activated downstream. Our research also brings upmany interesting questions and potential avenues to follow.Recently we have tested whether changing the composition of theheterodimer can affect the mammalian GFAP promoter andindeed, preliminary in vitro experiments suggest that the non-canonical c-Fos:JunB heterodimer formed in axolotl after injuryfail to induce high activation of the GFAP promoter. We havealso begun to examine other species with high regenerativecapacity to ask upregulation of the non-canonical AP-1 c-fos:JunBoccurs. Initial data-mining of publicly available RNA sequencingdata shows up-regulation of Fos and Jun family members inmany regenerative species. However due to lack of high-levelannotation of many genomes it is often unclear exactly whichfamily member is activated in these data sets. Nevertheless, it isimportant to note that although Lamprey and Xenopus do notappear to have a GFAP gene, they activate Fos and Jun familymembers after injury23,28,29. The functional signiﬁcance of thisactivation remains to be elucidated. It’s likely that injury-inducedgenes modulate several pathways to direct the response to injuryin the spinal cord.One of the future challenges is to determine, in axolotl, which arethe important signaling pathways downstream of the GFAP pro-moter as well as identifying other potential pathways affected by theheterodimer. In our recent paper we carried out RNA sequencingon the axolotl spinal cord tissue 4 days post injury providing awealth of data to be mined. As expected, markers of neuronaldifferentiation and synaptic signaling are downregulated afterinjury; however when miR-200a is inhibited we see mis-regulationof classical markers of neuronal differentiation and axon guidance.This could suggest that miR-200a may not just regulate c-Jun butalso other target genes involved in regulating the differentiationstate of cells after injury, and potentially some cells may revert to amore neural stem cell-like state in the axolotl after injury.The role of the immune systemIn addition, genes involved in the immune system are highlyupregulated in regeneration. There has been renewed interest inthe role of the immune system in pro-regenerative versus non-regenerative species in recent years. Earlier work in Xenopus limbregeneration suggested that the maturation of the immune systempotentially was a large causative factor in the inability of the frogto regenerate after metamorphosis. However biology is rarelybinary and work from many labs has shown that there is also aneed for the immune system in regeneration. Ultimately it may allbe about timing and duration of the immune stimulation. RecentTable 1 A summary of the current state of data regarding glial cells and composition of the AP-1 factor activated in them afterspinal cord injury.Species Functional CNS Regeneration GFAP+Glial cells AP-1 components activated after injury ReferencesHumans No Yes c-Fos, c-Jun 5,7,8,27Axolotls Yes Yes c-Fos, JunB 1Zebraﬁsh Yes Yes Fos, Jun 19Xenopus Larvae only No Fos, Jun 28,29Lamprey Yes No Fos, Jun 23In many species the exact Fos or Jun family members are not speciﬁcally identiﬁed in transcriptional proﬁling dataCOMMENT COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-0773-z2 COMMUNICATIONS BIOLOGY |            (2020) 3:47 | https://doi.org/10.1038/s42003-020-0773-z | www.nature.com/commsbiowork has very elegantly illustrated this aspect in two closelyrelated species that have stark regenerative (Zebraﬁsh) and non-regenerative (Medaka) responses to injury to the heart. In zeb-raﬁsh recruitment of macrophages is necessary for activation ofthe regenerative program but it is not an absence of macrophagesin Medaka that is responsible for its lack of regenerative ability,rather, an interesting difference in the timing and duration ofimmune cells recruited to the injury site30.What pathways actually recruit immune cells to the injury siteand control their dynamics is a big question that needs to beaddressed. Data from many research organisms, especially zeb-raﬁsh (because of the availability of transgenic lines for differentimmune cells) has provided interesting insights into the types ofimmune cells recruited to different types of injuries and theirdynamics31. The next level is understanding their molecularcontrol in detail. To date many signals like TGF-beta, Tollreceptors and interleukins are know to be involved in recruitingimmune cells. The AP-1 transcription factor has also beenimplicated. Interestingly JunB interacts with IL-1ß and with ATF3,genes with known roles in the immune system. This might suggestthat in animals that lack a GFAP gene, Fos and Jun play roles inmodulating the immune response in the spinal cord after injury.Future outlookThe far-reaching goal of studying nervous system regeneration inspecies that can do it naturally is that one day this knowledge caninform strategies for therapies for patients with spinal cord injuryand neurodegenerative diseases. It is clear from studying a smallsnapshot of organisms with functional regenerative capacity thatthere is no single pathway to regeneration. One question thatscientists are often faced with is “how similar” are axolotl glialcells to human cells. To date we know from transcriptionalproﬁling studies that glial cells from axolotl or zebraﬁsh expressmany of the same genes as human cells. In the future it will beinteresting to determine if the regulatory elements of these geneshave evolved differently and whether they determine the initialresponse to injury in order to direct cells towards a certainpathway. Research from many groups using different organismsclearly shows that nature has evolved many different routes toregenerate functional spinal cords; however more in-depthknowledge is necessary to build blueprints for any one organ-ism’s individual regenerative strategy.Received: 7 January 2020; Accepted: 14 January 2020;References1. 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 duringaxolotl spinal cord regeneration. Commun. Biol. 2, 91 (2019).2. Aristotle. Historia Animalium (Oxford, The Clarendon Press, 1910).3. Sanchez Alvarado, A. & Tsonis, P. A. Bridging the regeneration gap: geneticinsights from diverse animal models. Nat. Rev. Genet 11, 873–884 (2006).4. Sandoval-Guzman, T. et al. Fundamental differences in dedifferentiation andstem cell recruitment during skeletal muscle regeneration in two salamanderspecies. Cell Stem Cell 14, 174–187 (2014).5. Fawcett, J. & Asher, R. The glial scar and central nervous system repair. BrainRes. Bull. 49, 377–391 (1999).6. Göritz, C. et al. A pericyte origin of spinal cord scar tissue. Science (New York,NY) 333, 238–242 (2011).7. Sandvig, A., Berry, M., Barrett, L. B., Butt, A. & Logan, A. Myelin-, reactiveglia-, and scar-derived CNS axon growth inhibitors: expression, receptorsignaling, and correlation with axon regeneration. Glia 46, 225–251 (2004).8. Silver, J. & Miller, J. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5,146–156 (2004).9. Hanslik, K. L. et al. Regenerative capacity in the lamprey spinal cord is notaltered after a repeated transection. PloS ONE 14, e0204193 (2019).10. Oliphint, P. A. et al. Regenerated synapses in lamprey spinal cord are sparseand small even after functional recovery from injury. J. Comp. Neurol. 518,2854–2872 (2010).11. Smith, J. et al. Regeneration in the era of functional genomics and genenetwork analysis. Biol. Bull. 221, 18–34 (2011).12. Selzer, M. E. Mechanisms of functional recovery and regeneration after spinalcord transection in larval sea lamprey. J. Physiol. 277, 395–408 (1978).13. Wood, M. R. & Cohen, M. J. Synaptic regeneration in identiﬁed neurons of thelamprey spinal cords. Science (New York, NY) 206, 344–347 (1979).14. Yin, H. S. & Selzer, M. E. Axonal regeneration in lamprey spinal cord. J.Neurosci. 3, 1135–1144 (1983).15. Becker, T. & Becker, C. G. Axonal regeneration in zebraﬁsh. Curr. Opin.Neurobiol. 27, 186–191 (2014).16. 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).17. Clarke, J. D., Alexander, R. & Holder, N. Regeneration of descending axons inthe spinal cord of the axolotl. Neurosci. Lett. 89, 1–6 (1988).18. Goldshmit, Y. et al. Fgf-dependent glial cell bridges facilitate spinal cordregeneration in zebraﬁsh. J. Neurosci. 32, 7477–7492 (2012).19. Hui, S. P. et al. Genome wide expression proﬁling during spinal cordregeneration identiﬁes comprehensive cellular responses in zebraﬁsh. PloSONE 9, e84212 (2014).20. Sabin, K., Santos-Ferreira, T., Essig, J., Rudasill, S. & Echeverri, K. Dynamicmembrane depolarization is an early regulator of ependymoglial cell responseto spinal cord injury in axolotl. Dev. Biol. 408, 14–25 (2015).21. Martinez-De Luna, R. I. et al. Muller glia reactivity follows retinal injurydespite the absence of the glial ﬁbrillary acidic protein gene in Xenopus. Dev.Biol. 426, 219–235 (2017).22. Smith, J. J. et al. Sequencing of the sea lamprey (Petromyzon marinus) genomeprovides insights into vertebrate evolution. Nat. 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J.Neurosci. 39, 4694–4713 (2019).Author contributionsK.E. wrote the manuscript.Competing interestsThe author declares no competing interests.Additional informationCorrespondence and requests for materials should be addressed to K.E.Reprints and permission information is available at http://www.nature.com/reprintsPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional afﬁliations.COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-0773-z COMMENTCOMMUNICATIONS BIOLOGY |            (2020) 3:47 | https://doi.org/10.1038/s42003-020-0773-z | www.nature.com/commsbio 3Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directly fromthe copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2020COMMENT COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-0773-z4 COMMUNICATIONS BIOLOGY |            (2020) 3:47 | https://doi.org/10.1038/s42003-020-0773-z | www.nature.com/commsbio

The various routes to functional regeneration in the central nervous system

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The various routes to functional regeneration in the central nervous system

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