Spinal cord regeneration - the origins of progenitor cells for functional rebuilding.

The spinal cord is one of the most important structures for all vertebrate animals as it connects almost all parts of the body to the brain. Injury to the mammalian spinal cord has devastating consequences, resulting in paralysis with little to no hope of recovery. In contrast, other vertebrate animals have been known for centuries to be capable of functionally regenerating large lesions in the spinal cord. Here, we will review the current knowledge of spinal cord regeneration and recent work in different proregenerative animals that has begun to shed light on the cellular and molecular mechanisms these animals use to direct cells to rebuild a complex, functional spinal cord.

Regulation of stem cell identity by miR-200a during spinal cord regeneration.

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo cell tracking allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3'UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.

Spinal Cord Transection In Xenopus laevis Tadpoles.

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.

Non-canonical Hedgehog signaling regulates spinal cord and muscle regeneration in Xenopus laevis larvae.

Inducing regeneration in injured spinal cord represents one of modern medicine's greatest challenges. Research from a variety of model organisms indicates that Hedgehog (Hh) signaling may be a useful target to drive regeneration. However, the mechanisms of Hh signaling-mediated tissue regeneration remain unclear. Here, we examined Hh signaling during post-amputation tail regeneration in Xenopus laevis larvae. We found that while Smoothened (Smo) activity is essential for proper spinal cord and skeletal muscle regeneration, transcriptional activity of the canonical Hh effector Gli is repressed immediately following amputation, and inhibition of Gli1/2 expression or transcriptional activity has minimal effects on regeneration. In contrast, we demonstrate that protein kinase A is necessary for regeneration of both muscle and spinal cord, in concert with and independent of Smo, respectively, and that its downstream effector CREB is activated in spinal cord following amputation in a Smo-dependent manner. Our findings indicate that non-canonical mechanisms of Hh signaling are necessary for spinal cord and muscle regeneration.

CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1ß rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

A latent lineage potential in resident neural stem cells enables spinal cord repair.

Injuries to the central nervous system (CNS) are inefficiently repaired. Resident neural stem cells manifest a limited contribution to cell replacement. We have uncovered a latent potential in neural stem cells to replace large numbers of lost oligodendrocytes in the injured mouse spinal cord. Integrating multimodal single-cell analysis, we found that neural stem cells are in a permissive chromatin state that enables the unfolding of a normally latent gene expression program for oligodendrogenesis after injury. Ectopic expression of the transcription factor OLIG2 unveiled abundant stem cell-derived oligodendrogenesis, which followed the natural progression of oligodendrocyte differentiation, contributed to axon remyelination, and stimulated functional recovery of axon conduction. Recruitment of resident stem cells may thus serve as an alternative to cell transplantation after CNS injury.

AP-1cFos/JunB/miR-200a regulate the pro-regenerative glial cell response during axolotl spinal cord regeneration.

Salamanders have the remarkable ability to functionally regenerate after spinal cord transection. In response to injury, GFAP+ glial cells in the axolotl spinal cord proliferate and migrate to replace the missing neural tube and create a permissive environment for axon regeneration. Molecular pathways that regulate the pro-regenerative axolotl glial cell response are poorly understood. Here we show axolotl glial cells up-regulate AP-1cFos/JunB after injury, which promotes a pro-regenerative glial cell response. Injury induced upregulation of miR-200a in glial cells supresses c-Jun expression in these cells. Inhibition of miR-200a during regeneration causes defects in axonal regrowth and transcriptomic analysis revealed that miR-200a inhibition leads to differential regulation of genes involved with reactive gliosis, the glial scar, extracellular matrix remodeling and axon guidance. This work identifies a unique role for miR-200a in inhibiting reactive gliosis in axolotl glial cells during spinal cord regeneration.

Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons.

After injury to the mature central nervous system (CNS), myelin-derived inhibitory ligands bind to the Nogo-66 tripartite receptor complex expressed on axonal growth cones, comprised of LINGO-1 and p75NTR/TROY and induce growth cone collapse through the RhoA pathway. We have also shown that amphoterin-induced gene and open reading frame-3 (AMIGO3) substitutes for LINGO-1 and can signal axon growth cone collapse. Here, we investigated the regeneration of dorsal root ganglion neuron (DRGN) axons/neurites after treatment with a short hairpin RNA (sh) AMIGO3 plasmid delivered with a non-viral in vivo-jetPEI vector, and the pro-survival/axogenic neurotrophin (NT) 3 in vitro and in vivo. A bicistronic plasmid, containing both shAMIGO3 and NT3 knocked down >75% of AMIGO3 mRNA in cultured DRGN and significantly overexpressed NT3 production. In vivo, intra-DRG injection of in vivo-jetPEI plasmids containing shAMIGO3/gfp and shAMIGO3/nt3 both knocked down AMIGO3 expression in DRGN and, in combination with NT3 overexpression, promoted DC axon regeneration, recovery of conduction of compound action potentials across the lesion site and improvements in sensory and locomotor function. These findings demonstrate that in vivo-jetPEI is a potential non-viral, translatable DRGN delivery vehicle in vivo and that suppression of AMIGO3 disinhibits the growth of axotomised DRGN enabling NT3 to stimulate the regeneration of their DC axons and enhances functional recovery.

Xenopus laevis as a Model Organism for the Study of Spinal Cord Formation, Development, Function and Regeneration.

The spinal cord is the first central nervous system structure to develop during vertebrate embryogenesis, underscoring its importance to the organism. Because of its early formation, accessibility to the developing spinal cord in amniotes is challenging, often invasive and the experimental approaches amenable to model systems like mammals are limited. In contrast, amphibians, in general and the African-clawed frog Xenopus laevis, in particular, offer model systems in which the formation of the spinal cord, the differentiation of spinal neurons and glia and the establishment of spinal neuron and neuromuscular synapses can be easily investigated with minimal perturbations to the whole organism. The significant advances on gene editing and microscopy along with the recent completion of the Xenopus laevis genome sequencing have reinvigorated the use of this classic model species to elucidate the mechanisms of spinal cord formation, development, function and regeneration.

Immunization with neural derived peptides plus scar removal induces a permissive microenvironment, and improves locomotor recovery after chronic spinal cord injury.

BACKGROUND: Immunization with neural derived peptides (INDP) as well as scar removal-separately-have shown to induce morphological and functional improvement after spinal cord injury (SCI). In the present study, we compared the effect of INDP alone versus INDP with scar removal on motor recovery, regeneration-associated and cytokine gene expression, and axonal regeneration after chronic SCI. Scar removal was conducted through a single incision with a double-bladed scalpel along the stump, and scar renewal was halted by adding α,α'-dipyridyl. RESULTS: During the chronic injury stage, two experiments were undertaken. The first experiment was aimed at testing the therapeutic effect of INDP combined with scar removal. Sixty days after therapeutic intervention, the expression of genes encoding for TNFα, IFNγ, IL4, TGFß, BDNF, IGF1, and GAP43 was evaluated at the site of injury. Tyrosine hydroxylase and 5-hydroxytryptamine positive fibers were also studied. Locomotor evaluations showed a significant recovery in the group treated with scar removal + INDP. Moreover; this group presented a significant increase in IL4, TGFß, BDNF, IGF1, and GAP43 expression, but a decrease of TNFα and IFNγ. Also, the spinal cord of animals receiving both treatments presented a significant increase of serotonergic and catecholaminergic fibers as compared to other the groups. The second experiment compared the results of the combined approach versus INDP alone. Rats receiving INDP likewise showed improved motor recovery, although on a lesser scale than those who received the combined treatment. An increase in inflammation and regeneration-associated gene expression, as well as in the percentage of serotonergic and catecholaminergic fibers was observed in INDP-treated rats to a lesser degree than those in the combined therapy group. CONCLUSIONS: These findings suggest that INDP, both alone and in combination with scar removal, could modify the non-permissive microenvironment prevailing at the chronic phase of SCI, providing the opportunity of improving motor recovery.

Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration. We performed a genome-wide profiling screen for secreted factors that are up-regulated during zebrafish spinal cord regeneration. We found that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupted spinal cord repair, and transgenic ctgfa overexpression or local delivery of human CTGF recombinant protein accelerated bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration.

miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair.

UNLABELLED: Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers including inflammation. microRNA (miR)-155-5p is a small, noncoding RNA that negatively regulates mRNA translation. In macrophages, miR-155-5p is induced by inflammatory stimuli and elicits a response that could be toxic after SCI. miR-155 may also independently alter expression of genes that regulate axon growth in neurons. Here, we hypothesized that miR-155 deletion would simultaneously improve axon growth and reduce neuroinflammation after SCI by acting on both neurons and macrophages. New data show that miR-155 deletion attenuates inflammatory signaling in macrophages, reduces macrophage-mediated neuron toxicity, and increases macrophage-elicited axon growth by ∼40% relative to control conditions. In addition, miR-155 deletion increases spontaneous axon growth from neurons; adult miR-155 KO dorsal root ganglion (DRG) neurons extend 44% longer neurites than WT neurons. In vivo, miR-155 deletion augments conditioning lesion-induced intraneuronal expression of SPRR1A, a regeneration-associated gene; ∼50% more injured KO DRG neurons expressed SPRR1A versus WT neurons. After dorsal column SCI, miR-155 KO mouse spinal cord has reduced neuroinflammation and increased peripheral conditioning-lesion-enhanced axon regeneration beyond the epicenter. Finally, in a model of spinal contusion injury, miR-155 deletion improves locomotor function at postinjury times corresponding with the arrival and maximal appearance of activated intraspinal macrophages. In miR-155 KO mice, improved locomotor function is associated with smaller contusion lesions and decreased accumulation of inflammatory macrophages. Collectively, these data indicate that miR-155 is a novel therapeutic target capable of simultaneously overcoming neuron-intrinsic and neuron-extrinsic barriers to repair after SCI. SIGNIFICANCE STATEMENT: Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers, including inflammation. Here, new data show that deleting microRNA-155 (miR-155) affects both mechanisms and improves repair and functional recovery after SCI. Macrophages lacking miR-155 have altered inflammatory capacity, which enhances neuron survival and axon growth of cocultured neurons. In addition, independent of macrophages, adult miR-155 KO neurons show enhanced spontaneous axon growth. Using either spinal cord dorsal column crush or contusion injury models, miR-155 deletion improves indices of repair and recovery. Therefore, miR-155 has a dual role in regulating spinal cord repair and may be a novel therapeutic target for SCI and other CNS pathologies.

Further insight into molecular mechanism underlying thoracic spinal cord injury using bioinformatics methods.

The present study aimed to explore the molecular mechanisms underlying the development of thoracic spinal cord injury (SCI). The gene expression profile of GSE20907, which included 12 thoracic non­injured spinal cord control samples and 12 thoracic transected spinal cord samples at different stages of SCI, was obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified using the limma package in R/Bioconductor. DEG­associated pathways were analyzed using the Kyoto encyclopedia of genes and genomes database. A protein­protein interaction (PPI) network was constructed and transcription factors (TFs) were predicted using cytoscape. Compared with the control samples, there were 1,942, 396, 188 and 396 DEGs identified at day 3 (d3), week 1 (wk1), wk2 and month 1 (m1), respectively. Cluster analysis indicated that the DEGs at m1 were similar to those in the control group. Downregulated DEGs were enriched in nervous system disease pathways, such as Parkinson's disease. Upregulated DEGs were enriched in immune response­associated pathways, such as Fc γ R­mediated phagocytosis at early stages (d3 and wk1). Upregulated DEGs were enriched in pathways associated with cancer and pyrimidine metabolism at wk2 and m1, respectively. In the PPI network, nodes including RAC2, CD4, STAT3 and JUN were identified. Furthermore, ATF3, JUN and EGR1 were identified as TFs associated with SCI. In conclusion, the results of the present study showed that the number of DEGs decreased in a time­dependent manner following SCI. OLIG1, ATF3 and JUN may represent SCI regeneration­associated genes. Immune-associated inflammation was shown to be important in SCI, and SCI exhibits causal associations with other diseases, including cardiovascular disease and cancers. The present study provided novel insight into the molecular mechanisms of SCI regeneration, which may aid in the development of strategies to enhance recovery following SCI.

Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells.

Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2(/)3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2(/)3(+) cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2(/)3(+) neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism that is lost in non-regenerative froglets.

Sponge-mediated lentivirus delivery to acute and chronic spinal cord injuries.

The environment within the spinal cord after injury, which changes in the progression from the acute to chronic stages, limits the extent of regeneration. The delivery of inductive factors to promote regeneration following spinal cord injury has been promising, yet, few strategies are versatile to allow delivery during acute or chronic injury that would facilitate screening of candidate therapies. This report investigates the intrathecal delivery of lentiviruses for long-term expression of regenerative factors. Lentivirus-filled sponges were inserted into the intrathecal space surrounding the spinal cord, with transgene expression observed within multiple cell types that persists for 12 weeks for both intact and injured spinal cord, without any apparent damage to the spinal cord tissue. Sponges loaded with lentivirus encoding for Sonic hedgehog (Shh) were investigated for acute (delivered at 0 weeks) and chronic (at 4 weeks) injuries, and for multiple locations relative to the injury. In an acute model, sponges placed directly above the injury increased oligodendrocyte and decreased astrocyte presence. Sponges placed caudal to the injury had reduced impact on oligodendrocytes and astrocytes in the injury. In a chronic model, sponges increased oligodendrocyte and decreased astrocyte presence. Furthermore, the effect of Shh was shown to be mediated in part by reduction of Bmp signaling, monitored with an Msx2-sensitive reporter vector. The implantation of lentivirus-loaded biomaterials intrathecally provides the opportunity to induce the expression of a factor at a specified time without entering the spinal cord, and has the potential to promote gene delivery within the spinal cord, which can influence the extent of regeneration.

Decreased anti-regenerative effects after spinal cord injury in spry4-/- mice.

Previously, we have demonstrated a role for fibroblast growth factor (Fgf) in spinal cord regeneration in both zebrafish and mouse. We have shown that exogenous Fgf2 treatment attenuates astrocytic gliosis and induces glia cells to become progenitors that undergo neurogenesis as well as differentiating into bipolar astrocytes that support axonal regeneration (Goldshmit et al., 2012, 2014). One of the downstream signaling target genes of Fgf is spry4, which acts as a feedback inhibitor for Fgf signaling. In this study we examined the effects of increased endogenous Fgf signaling, in spry4-/- mice, on the early events that occur after spinal cord injury (SCI). We demonstrate that in spry4-/- mice inflammatory responses, such as tumor necrosis factor α (TNFα) secretion and macrophage/neutrophil invasion into the lesion site are reduced. In addition, astrocytic gliosis is attenuated and neuronal survival is increased. These results further support a pro-regenerative role of Fgf after SCI, and suggest that increased endogenous Fgf signaling after SCI may contribute to functional recovery and therefore presents this pathway as a target for new therapy development.

Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages.

BACKGROUND: Xenopus laevis has regenerative and non-regenerative stages. As a tadpole, it is fully capable of functional recovery after a spinal cord injury, while its juvenile form (froglet) loses this capability during metamorphosis. We envision that comparative studies between regenerative and non-regenerative stages in Xenopus could aid in understanding why spinal cord regeneration fails in human beings. RESULTS: To identify the mechanisms that allow the tadpole to regenerate and inhibit regeneration in the froglet, we obtained a transcriptome-wide profile of the response to spinal cord injury in Xenopus regenerative and non-regenerative stages. We found extensive transcriptome changes in regenerative tadpoles at 1 day after injury, while this was only observed by 6 days after injury in non-regenerative froglets. In addition, when comparing both stages, we found that they deployed a very different repertoire of transcripts, with more than 80% of them regulated in only one stage, including previously unannotated transcripts. This was supported by gene ontology enrichment analysis and validated by RT-qPCR, which showed that transcripts involved in metabolism, response to stress, cell cycle, development, immune response and inflammation, neurogenesis, and axonal regeneration were regulated differentially between regenerative and non-regenerative stages. CONCLUSIONS: We identified differences in the timing of the transcriptional response and in the inventory of regulated transcripts and biological processes activated in response to spinal cord injury when comparing regenerative and non-regenerative stages. These genes and biological processes provide an entry point to understand why regeneration fails in mammals. Furthermore, our results introduce Xenopus laevis as a genetic model organism to study spinal cord regeneration.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

BACKGROUND: Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. RESULTS: In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. CONCLUSION: As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish.

BACKGROUND: Among the vertebrates, teleost and urodele amphibians are capable of regenerating their central nervous system. We have used zebrafish as a model to study spinal cord injury and regeneration. Relatively little is known about the molecular mechanisms underlying spinal cord regeneration and information based on high density oligonucleotide microarray was not available. We have used a high density microarray to profile the temporal transcriptome dynamics during the entire phenomenon. RESULTS: A total of 3842 genes expressed differentially with significant fold changes during spinal cord regeneration. Cluster analysis revealed event specific dynamic expression of genes related to inflammation, cell death, cell migration, cell proliferation, neurogenesis, neural patterning and axonal regrowth. Spatio-temporal analysis of stat3 expression suggested its possible function in controlling inflammation and cell proliferation. Genes involved in neurogenesis and their dorso-ventral patterning (sox2 and dbx2) are differentially expressed. Injury induced cell proliferation is controlled by many cell cycle regulators and some are commonly expressed in regenerating fin, heart and retina. Expression pattern of certain pathway genes are identified for the first time during regeneration of spinal cord. Several genes involved in PNS regeneration in mammals like stat3, socs3, atf3, mmp9 and sox11 are upregulated in zebrafish SCI thus creating PNS like environment after injury. CONCLUSION: Our study provides a comprehensive genetic blue print of diverse cellular response(s) during regeneration of zebrafish spinal cord. The data highlights the importance of different event specific gene expression that could be better understood and manipulated further to induce successful regeneration in mammals.