P2X7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

ABSTRACT In contrast to mammals, zebrafish undergo successful neural regeneration following spinal cord injury. Spinal cord ependymo-radial glia (ERG) undergo injury-induced proliferation and neuronal differentiation to replace damaged cells and restore motor function. However, the molecular cues driving these processes remain elusive. Here, we demonstrate that the evolutionarily conserved P2X7 receptors are widely distributed on neurons and ERG within the zebrafish spinal cord. At the protein level, the P2X7 receptor expressed in zebrafish is a truncated splice variant of the full-length variant found in mammals. The protein expression of this 50 kDa isoform was significantly downregulated at 7 days post-injury (dpi) but returned to basal levels at 14 dpi when compared to naïve controls. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi but did not affect neuronal differentiation at 14 dpi. Our findings suggest that unlike in mammals, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish and may also work to curb the proliferative response of ERG following injury.


INTRODUCTION
Spinal cord injury (SCI), characterized by irreversible sensory, motor, and autonomic dysfunction, is an ever-growing global health burden (Ding et al., 2022).Per year, an estimated 900,000 individuals across the world incur an SCI, significantly adding to the 20,600,000 global cases currently documented (Ding et al., 2022;Noonan et al., 2012).Although substantial progress has been made in elucidating the pathophysiology of this condition, treatment options remain extremely limited (Krueger et al., 2013;Lynch and Cahalan, 2017).This is due to the maladaptive cellular injury response within the central nervous system (CNS) that results in secondary cell death, neuroinflammation, reactive gliosis, and axonal degeneration.
In contrast to mammals, several non-mammalian vertebrate species, such as Osteichthyes, demonstrate a high degree of regenerative ability following injury to the CNS.Indeed, zebrafish (Danio rerio) undergo injury-induced neurogenesis following SCI and achieve functional recovery within 6-8 weeks (Becker et al., 1997;Hui et al., 2010;Reimer et al., 2008).This adaptive process is largely driven by ependymo-radial glia (ERG) lining the central canal of the spinal cord.Although the functions of ERG within the adult zebrafish spinal cord are not fully understood, in response to injury, these cells undergo a massive proliferative response and can differentiate into distinct neuronal types that are integrated into the spinal circuitry (Reimer et al., 2008).While a few signaling pathways including, but not limited to, dopamine, serotonin, and wingless-related integration site/β-catenin have been implicated in regulating this injury response, the molecular cues necessary for driving adult injury-induced neurogenesis have yet to be fully clarified (Barreiro-Iglesias et al., 2015;Briona et al., 2015;Reimer et al., 2013).Thus, identifying the signals employed by these species to restore neuron populations and locomotor function following injury may be integral to the development of neurorestorative treatments for mammals.
One evolutionarily conserved signaling pathway that has been extensively explored following SCI in mammals, but not in zebrafish, is the purinergic system (Stefanova and Scott, 2022).Nucleoside triphosphates and their metabolites regulate a plethora of cellular processes within the CNS (Burnstock and Ulrich, 2011;Gomez-Villafuertes, 2016;Oliveira et al., 2016;Ribeiro et al., 2016).These ligands have extensive receptor families, including metabotropic adenosine receptors, A 1 , A 2A , A 2B , A 3 ; ionotropic nucleotide receptors, P2X 1-7 ; and metabotropic nucleotide receptors, P2Y 1,2,4,6,11,12,13,14 (Burnstock, 2018).In the context of CNS regeneration, P2X 7 receptors are of particular interest.These receptors function as non-selective cation channels activated by high concentrations of extracellular adenosine 5′triphosphate (ATP).When ATP stimulation is sustained, as in the first few hours following mammalian SCI, certain splice variants of the P2X 7 receptor may form a reversible, but cytotoxic, channel pore (Andrejew et al., 2020;De Salis et al., 2022;Di Virgilio et al., 2018;Wang et al., 2004).Other P2X 7 splice variants have been shown to mediate proliferation and neuronal differentiation of embryonic and adult neural progenitor cells in mammals (Glaser et al., 2014;Leeson et al., 2019;Leeson et al., 2018;Tsao et al., 2013).Remarkably, while the P2X 7 gene is present across all Metazoa, its specific properties and functions appear to differ across vertebrates (Benzaquen et al., 2019;Liang et al., 2015;López-Castejón et al., 2007;Rump et al., 2020).Given the varied, yet significant role of this receptor following mammalian SCI, in this study we focused on the P2X 7 receptor.We aimed to uncover its potential role in injury-induced ERG cell proliferation and neuronal regeneration following SCI in adult zebrafish.
Here, we examined cellular expression of P2X 7 receptors within the naïve and injured spinal cord, quantified temporal changes in P2X 7 protein expression following injury, and assessed the functional significance of P2X 7 receptor activation on injury-induced neurogenesis.Our results demonstrated that a truncated P2X 7 variant was prominently expressed on neurons and ERG.Curiously, expression of this variant became significantly downregulated during the period of peak proliferation following SCI.Given that exogenous activation of P2X 7 was found to promote proliferation of ERG, downregulation of P2X 7 may act as the needed brake signal that reduces proliferation prior to differentiation of neural progenitors within the injured zebrafish spinal cord.

RESULTS
P2X 7 is expressed by ependymo-radial glia and neurons within the adult zebrafish spinal cord In addition to mediating various homeostatic functions, ERG are essential for neural regeneration following CNS injury in zebrafish (Briona and Dorsky, 2014).The cell bodies of ERG line the central canal of the spinal cord, while their processes extend out to the pial surface.Immunohistological analyses showed that P2X 7 colocalizes with GFAP, a marker for ERG (Becker and Becker, 2015;Lyons et al., 2003), in the thoracic spinal cord of naïve and injured (7 and 14 dpi) fish (Fig. 1).Within the naïve cord (Fig. 1A-C′), at 7 dpi (Fig. 1D-F′), and at 14 dpi (Fig. 1G-I′), colocalization of P2X 7 and GFAP + was detected in both the cell bodies and cellular processes of ERG.
We next examined expression of P2X 7 on proliferating cells within the zebrafish spinal cord.During spontaneous regeneration, ERG undergo a massive proliferative response at 7 dpi (Hui et al., 2015).We found that P2X 7 receptors are expressed by PCNA + cells in the naïve zebrafish spinal cord (Fig. 2A-C′) and at 7 dpi both rostral (Fig. 2D-F′) and caudal (Fig. 2G-I′) to the lesion.Curiously, not all PCNA + cells colocalized with P2X 7 .Furthermore, P2X 7 staining was found primarily around the periphery, consistent with expression on the cellular membrane of PCNA + cells (Fig. 2).
Lastly, we examined expression of P2X 7 on neurons within the zebrafish spinal cord using the pan-neuronal marker HuC/D which stains both mature and immature neurons (Ghosh and Hui, 2016;Hui et al., 2010).Following SCI in adult zebrafish, peak injury-induced neurogenesis occurs at 14 dpi (Reimer et al., 2008).We found that within the adult zebrafish spinal cord, P2X 7 receptors are expressed by HuC/D + cells in naïve fish (Fig. 3A-C′) and at 14 dpi both rostral (Fig. 3D-F′) and caudal (Fig. 3G-I′) to the lesion.P2X 7 demonstrated colocalization with small and large HuC/D + cells, typically corresponding to immature and mature neurons respectively (Fig. 3).

Differential expression of P2X 7 following spinal cord injury in adult zebrafish
In response to mammalian SCI, protein expression of the P2X 7 receptor is significantly upregulated by 3-7 dpi and remains elevated until at least 30 days (Fan et al., 2020;Gomez-Villafuertes et al., 2015;Kobayashi et al., 2011).To assess temporal changes in P2X 7 protein expression following zebrafish SCI, we performed quantitative protein assays (western blotting) on spinal cord tissue collected from naïve animals and from individuals that sustained a spinal cord transection during various time periods of recovery (Fig. 4A-B).Injured tissue was collected at 1 dpi during peak secondary cell death, at 7 dpi during peak ERG cell proliferation, and at 14 dpi during peak neurogenesis (Barreiro-Iglesias et al., 2015;Hui et al., 2015;Reimer et al., 2008;Tsarouchas et al., 2018) (Fig. 4C).
Since mammals and zebrafish have highly conserved genes encoding the 'ballast domain', otherwise known as the extended C-terminal tail of the P2X 7 receptor, we probed the zebrafish spinal cord with a P2X 7 antibody targeting the full-length receptor (McCarthy et al., 2019;Rump et al., 2020).This antibody typically detects the full-length 68-85 kDa mammalian P2X 7 receptor.We established antibody specificity for P2X 7 using mouse brain tissue as a positive control and pre-absorption with a blocking peptide as a negative control (Carvalho et al., 2021;Leeson et al., 2018) (Fig. 4D).In zebrafish samples, we identified immunoreactive bands in the 50 kDa region; meanwhile, those in the 68-85 kDa region were either completely absent or demonstrated limited staining in just a few individuals.The molecular weight of the protein detected in zebrafish corresponded to a recognized P2X 7 splice variant in mammals, namely P2X 7 B, that is reported to lack much of the C-terminus (Cheewatrakoolpong et al., 2005).Protein expression of the 50 kDa isoform was consistent with naïve levels at 1 dpi (Fig. 4E,F), became significantly downregulated within the injury site and caudal to the lesion at 7 dpi (Fig. 4G,H), and returned to basal levels of expression at 14 dpi (Fig. 4I,J) when compared to naïve controls.Given that ERG undergo a massive proliferative response at 7 dpi in adult zebrafish, we quantified proliferation by analyzing the number of PCNA + expressing cells within the injured spinal cord at this time point (Bologna-Molina et al., 2013;Hui et al., 2015).The use of PCNA as a marker for ERG proliferation has been validated by several research groups.Specifically, it has been demonstrated that staining with bromodeoxyuridine (BrdU); a marker for cells in the S-phase of the cell cycle, and PCNA; a marker for cells in the early G 1 and S-phases of the cell cycle, yield very similar results in the examination of ERG cell proliferation within zebrafish brain and spinal cord tissue (Grandel et al., 2006;Hui et al., 2015;Reimer et al., 2008).Consistent with previous work by Barreiro-Iglesias and colleagues (2015), we detected a significant increase in the number of PCNA + expressing cells around the central canal at 7 dpi when compared to naïve controls both rostral (naïve, n=5; saline, n=9; BzATP, n=8; JNJ, n=8; one-way ANOVA with Dunnett's post hoc test, P<0.0001) (Fig. 5J) and caudal (naïve, n=5; saline, n=10, P<0.0001; BzATP, n=9, P<0.0001; JNJ, n=6, P=0.0007) (Fig. 5K) to the lesion.Quantification of PCNA + cells around the central canal indicated that the number of proliferating cells decreased as distance from the lesion increased (Fig. 5I).Indeed, we noted that injurystimulated cell proliferation around the central canal occurred primarily within the first 400 μm rostral and caudal to the lesion with relatively little effect in the distal spinal cord (Fig. 5I).Thus, for each sample (n), we limited our counts to spinal sections within the first 400 μm rostral and caudal to the injury site and compared the values between groups.
To directly test the involvement of P2X 7 signaling in mediating ERG cell proliferation, we administered intraperitoneal injections of P2X 7 agonist BzATP or P2X 7 antagonist JNJ 47965567 at 3 and 6 dpi, and then quantified proliferation around the central canal at 7 dpi (Fig. 5B).Vehicle injections delivered in the same time intervals served as controls.We used 100 μM BzATP as this concentration specifically activates only P2X 7 and had previously been shown to activate these receptors in larval zebrafish in vivo (Chang et al., 2011).We used a JNJ concentration of 200 μM based on in vivo studies in murine species (Bhattacharya et al., 2013;Ly et al., 2020).To test the efficacy of this concentration of JNJ in zebrafish, we performed a concentration curve analysis to test three different concentrations of the antagonist, specifically 20 μM, 200 μM, and 2 mM.The respective means of summative PCNA + cell counts across the rostral spinal cord were 133.00±5.66, 191.75±70.65, and 159.50±54.45 (data not shown).All three treatment concentrations were well tolerated by adult zebrafish with no adverse effects.Given that there was no statistically significant difference between PCNA + cell counts among the three groups, we selected the concentration (200 μM) previously reported to be effective in vivo for all subsequent experiments.Fig. 5I.When comparing vehicle to BzATP treated fish, we found a significant increase in the number of PCNA + cells around the central canal both rostral (saline, n=9; BzATP, n=8; P=0.0306) (Fig. 5C,D,J) and caudal (saline, n=10; BzATP, n=9; P=0.0447) (Fig. 5F,G,K) to the lesion.Meanwhile, JNJ injections had no effect on proliferative events rostral (Fig. 5E,J) or caudal (Fig. 5H,K) to the lesion.
To assess the involvement of P2X 7 signaling in mediating neuronal regeneration, we administered intraperitoneal injections of P2X 7 agonist BzATP or P2X 7 antagonist JNJ 47965567 at 3, 6, 9, and 12 dpi, and then analyzed HuC/D + expression at 14 dpi (Fig. 6B).Vehicle injections delivered in the same time frames served as controls.Quantification of HuC/D + cells, which represents a compound value of both newly generated and pre-existing neurons, within the injured spinal cord indicated that the total number of neurons present decreased slightly as distance from the lesion increased (Fig. 6I).This effect was more pronounced rostral to the lesion.Again, for each sample (n), we limited our counts to spinal sections within the first 400 μm closest to the injury site (as above) and compared these values between treatment groups.When comparing vehicle to BzATP or JNJ treated fish, we found there were no significant differences in the total number of HuC/D + expressing cells both rostral (Fig. 6C-E,J) and caudal (Fig. 6F-H,K) to the lesion.

DISCUSSION
The P2X 7 receptor has been implicated in various cellular functions and disease pathologies, including CNS injury.However, its role in adaptive plasticity and modulating injury-induced ERG cell proliferation and neuronal regeneration in adult zebrafish is unknown.Here, we show that the P2X 7 receptor has widespread distribution throughout the adult zebrafish spinal cord.Remarkably, we failed to detect expression of the full-length P2X 7 receptor, suggesting that the predominant P2X 7 receptor isoform expressed in zebrafish differs from that expressed in murine species.Using pharmacological manipulation in vivo, we found that P2X 7 receptor activation is sufficient to induce proliferation of ERG but is not a necessary mediator of either proliferation or neurogenesis in injury-induced responses.
Within the mammalian CNS, P2X 7 receptors are expressed by immune cells, glia, and neural stem cells; however, expression by neurons appears more controversial with contradictory evidence supporting both possibilities (Fan et al., 2020;Genzen et al., 2009;Kaczmarek-Hajek et al., 2018;Khan et al., 2019;Leeson et al., 2018;Matute et al., 2007;Wang et al., 2009;Wang et al., 2004).While P2X 7 mRNA is also expressed throughout the brain and spinal cord of larval zebrafish, thorough cellular characterization within larval or adult zebrafish CNS tissue has not yet been done (Appelbaum et al., 2007;Chang et al., 2011).In our study, we observed widespread distribution of P2X 7 expression throughout the thoracic spinal cord of adult zebrafish with specific localization to ERG, proliferating cells around the central canal, and neurons.We found more consistent colocalization of P2X 7 with larger HuC/D + expressing neurons compared to smaller cells, suggestive of temporal changes in P2X 7 expression over the course of neuronal maturation.Indeed, other groups have found that P2X 7 expression changes as neural differentiation and maturation progresses in mouse embryonic stem cells and in human induced pluripotent stem cell-derived neurons (Francistiová et al., 2021;Glaser et al., 2014).These findings suggest that cellular expression of P2X 7 may share similarities between zebrafish and mammals.
The rather diverse portfolio of roles for P2X 7 receptors may be attributed to the existence of different splice variants that perform distinct functions (Adinolfi et al., 2018;Burnstock and Knight, 2018).Alternative splicing is highly varied across species and in terms of P2X 7 , there have been 13 splice variants identified in humans and five in mice.Of particular relevance to regeneration is the full-length protein; namely, human P2X 7 A and murine P2X 7 a, and the C-terminus truncated proteins; namely human P2X 7 B and murine P2X 7 b/P2X 7 c (Benzaquen et al., 2019;Cheewatrakoolpong et al., 2005;Masin et al., 2012;Sluyter, 2017).The full-length protein in humans and murine species retains its canonical role as an ion channel but is also capable of mediating cytotoxic cell death via pore formation (Cheewatrakoolpong et al., 2005;Di Virgilio et al., 2018;Leeson et al., 2018).Similarly, C-terminus truncated variants function as ion channels; however, unlike P2X 7 A/P2X 7 a, they demonstrate reduced channel activity and do not typically form cytotoxic pores (Cheewatrakoolpong et al., 2005;Karasawa et al., 2017;Masin et al., 2012).Prior to our study, different P2X 7 receptor splice variants in zebrafish had not yet been identified.We detected a truncated protein matching the molecular weight of human P2X 7 B.However, the human P2X 7 gene only shares 42% similarity with zebrafish when comparing genomic organization and chromosomal localization (Sluyter, 2017).Thus, the exact function of the zebrafish P2X 7 protein we identified is unlikely to be identical to that in mammals because other components of the P2X 7 receptor, like the transmembrane domains, do not share strong sequence homology between mammalian and fish species (Rump et al., 2020).These significant structural differences may account for why certain characteristic functions of mammalian P2X 7 receptor activation, such as interleukin-1 beta (IL-1β) secretion, are not shared by zebrafish P2X 7 receptors (López-Castejón et al., 2007).While confirmation of this potential isoform requires future analysis, our data suggests that zebrafish P2X 7 mRNA undergoes alternative splicing to generate distinct proteins that contribute to yet unknown functions of this receptor.
We hypothesize that the downregulation of P2X 7 at 7 dpi may serve as a brake on ERG cell proliferation.In support of this, we found that pharmacological activation of P2X 7 using BzATP increased total PCNA + expression around the central canal at 7 dpi, while pharmacological inhibition with JNJ-47965567 had no effect.In agreement, multiple lines of evidence suggest that in mammals, elevated P2X 7 receptor expression potentiates cellular proliferation in populations of highly proliferative stem or cancer cells (Francistiová et al., 2021;Giuliani et al., 2014;Pegoraro et al., 2020).Similarly, treatment of mouse embryonic stem cells with a P2X 7 agonist increased proliferation (Glaser et al., 2014).We propose that within the zebrafish spinal cord, P2X 7 receptor activation may serve as a potent stimulus for cellular proliferation, and under homeostatic conditions, P2X 7 may function to maintain basal rates of ERG proliferation.To prevent uncontrolled cell growth at 7 dpi, the period of peak ERG proliferation, zebrafish downregulate protein expression of the 50 kDa P2X 7 isoform.This downregulation does not adversely affect the rate of proliferation, suggesting that various other signaling pathways simultaneously promote ERG re-entry into the cell cycle (Barreiro-Iglesias et al., 2015;Briona et al., 2015;Reimer et al., 2009;Reimer et al., 2013).
In our study, we also detected basal levels of expression of the 50 kDa P2X 7 isoform during the period of peak injury-induced neurogenesis (14 dpi).In response to chronic P2X 7 receptor agonism or antagonism, we detected no statistically significant changes in the total number of HuC/D + cells, indicating that P2X 7 signaling likely does not play a principal role in neurogenesis Fig. 4. Temporal changes in P2X 7 protein expression following SCI in adult zebrafish.Schematic representations of naïve and injured spinal cord areas of analysis (A-B), as well as tissue collection timeline (C).Representative western blot and corresponding total protein in naïve zebrafish (Zf ) spinal cord tissue, mouse (Ms) hippocampal tissue as a positive control (+), and pre-absorption of naïve zebrafish spinal cord tissue with a blocking peptide as a negative control (-) (D).Representative western blot and corresponding total protein at 1 dpi (E), 7 dpi (G), and 14 dpi (I).Quantitative analysis of the 50 kDa isoform showed significant downregulation within the injury (In, n=10, one-way ANOVA with Dunnett's post hoc test, P=0.0079) and caudal to the lesion (Ca, n=9, P=0.0046) at 7 dpi when normalized to naïve tissue (n=9) (H).Protein expression of the 50 kDa isoform showed basal levels of expression at 1 dpi (F) and 14 dpi (J).Data presented as means±s.d.**, significant differences, P<0.01.Created with BioRender.com.
following SCI in adult zebrafish.Within the mammalian CNS, the role of P2X 7 in the regulation of neurogenesis is unclear.Some groups report that as neuronal differentiation progresses, P2X 7 expression decreases (Francistiová et al., 2021;Glaser et al., 2014).Along these lines, Glaser and colleagues (2014) found that treatment of mouse embryonic stem cells with a P2X 7 receptor agonist had no effect on neuronal differentiation, while treatment with a P2X 7 receptor antagonist increased neuronal differentiation.In stark contrast, others suggest that P2X 7 receptor agonism promotes neural differentiation of mouse embryonic neural progenitor cells (Tsao et al., 2013).Curiously, one group studying phenotype specification of mouse embryonal carcinoma cells found that P2X 7 receptor activation promotes gliogenesis (Yuahasi et al., 2012).Thus, it may be possible that in our experiments, pharmacological activation of P2X 7 pushed more ERG cells toward a glial cell fate.An alternative explanation for the observed effect of BzATP and JNJ 47965567 on zebrafish spinal cord regeneration may be due to an indirect effect on phagocytosis.Interestingly, in the absence of extracellular ATP, P2X 7 receptors on astrocytes or adult neural progenitor cells from rodent brains can facilitate phagocytosis in vitro (Leeson et al., 2018;Yamamoto et al., 2013).Phagocytosis is mediated by the closed (unbound) state of the P2X 7 receptor.Interestingly, certain non-competitive P2X 7 receptor antagonists like AZ10606120 block fluorescent dye uptake (via cytotoxic pore formation) without affecting P2X 7 mediated phagocytic ability of human monocytes (Ou et al., 2018).Given that JNJ-47965567, the non-competitive P2X 7 receptor antagonist used in this study, likely binds the same site, it seems feasible that P2X 7 receptor inhibition by this compound similarly did not inhibit endogenous phagocytic ability (Ly et al., 2020).This is important because CNS injuries and neurodegenerative diseases are typically associated with significant cellular degeneration and cell death, which create considerable cellular debris.In cases like these, and perhaps following SCI in adult zebrafish, treatment with P2X 7 receptor antagonists may dampen secondary cell death by inhibiting P2X 7 mediated cytotoxic cell death.Simultaneously, these antagonists may enhance debris clearance and subsequent regeneration by promoting P2X 7 mediated phagocytic activity.
In conclusion, our results suggest that, unlike in mammals, P2X 7 signaling does not play a maladaptive role following SCI in adult zebrafish.Using immunohistochemical techniques, we detected P2X 7 expression on glia and neurons.In vivo analysis of the effects of P2X 7 agonism or antagonism showed that while P2X 7 activation is sufficient to promote proliferation, it is not necessary for mediating proliferation or neurogenesis.Future studies examining the possible effects of P2X 7 on differentiation of other cell types within the spinal cord may offer some new insights into injuryinduced glial or immune cell regulation.In all, our findings suggest alternative roles of the purinergic system following CNS injury in teleost fish versus mammals.

Spinal cord lesion
Prior to any surgical procedure, individual fish were acclimated to individual housing within an environmental chamber maintained at 28°C in a 14-h light cycle and fed dry food twice daily for 48 h.As previously described (Becker et al., 1997), adult fish were anesthetized by immersion in pH neutralized 0.02% aminobenzoic acid ethyl methyl ester (MS222; Sigma-Aldrich, St. Louis, MO, USA) in sterile water for ∼1 min.The anesthetized fish were then placed on a pre-wet agar plate under a dissection microscope, and a small vertical incision was made at the level of the thoracic spinal cord.The muscle layers were then blunt dissected and carefully separated with forceps, the vertebral column was exposed, and the spinal cord underwent full transection with visual confirmation of completeness. 1 μl of Vetbond tissue adhesive gel was quickly placed on the external incision.Fish were transferred to a new tank with sterile water and recovered in an environmentally controlled chamber again maintained at 28°C and set to a 14-h light cycle.Fish were not fed for 24 h post anesthesia, but regular feeding twice daily commenced afterwards and continued until the end of the experiment.Fish were housed individually and monitored twice daily to assess physical appearance (eye condition, fin and skin condition, mucus production, spinal deformities, abdomen size), clinical signs (changes in food consumption, respiratory rate, posture in water), and behaviour (hypo or hyperactivity).Fish were euthanized if they displayed grossly abnormal physical appearance, clinical signs, or behaviour.Water was changed and assessed for quality daily.

Immunohistochemical procedures
To localize expression of select proteins, adult zebrafish spinal cord tissue was processed for histology.Zebrafish were euthanized by immersion overdose in pH neutralized 0.1% MS222 in sterile water for 5 min and immediately transcardially perfused with 200 µl saline followed by 3000 µl 4% paraformaldehyde (PFA; Sigma-Aldrich, 158127) dissolved in 0.01 M phosphate buffered saline (PBS).The vertebral column and spinal cord was then removed and placed in 4% PFA overnight before being transferred to a 20% sucrose solution (in 0.01 M PBS) for another 12-24 h.Following fixation and cryoprotection, the vertebral column was dissected to expose the injured spinal cord, the injury site was identified, and each rostral and caudal segment was isolated.From each spinal cord segment, a section of 550 µm from the injury site was embedded in Tissue Plus O.C.T. Compound Clear (ThermoFisher Scientific, Waltham, MA, USA, SGN4585) and stored in a 7×7 mm base bold (Ted Pella, Redding, CA, USA, 27147-1) that was flash frozen in liquid nitrogen and stored at −80°C.To prepare the sections for cryosectioning, the spinal cord blocks were warmed to −20°C and mounted on a sectioning stage.Frozen spinal cord sections (10 µm thick) were collected on gelatin coated slides using a Leica CM1900 cryostat.Gelatin coated slides were used to minimize detachment of sections from slides.They were pre-made by dipping frosted slides in a 40-50°C solution of 0.5% 300 bloom gelatin and 0.05% chromium potassium sulphate for 1 min.Once the liquid was drained, the slides were left to dry at 57°C for 3-4 h.

Protein assays
Protein samples were prepared from 2 mm sections of adult zebrafish thoracic spinal cord tissue isolated from the following: (1) thoracic spinal cord tissue from naïve fish, (2) tissue within the injury site, (3) tissue adjacent to the rostral side of the injury site, and (4) tissue adjacent to the caudal side of the injury site.Tissue samples from each location of the injured zebrafish spinal cord were collected at either 1, 7, or 14 dpi.
All samples were flash frozen in liquid nitrogen and stored at −80°C before being mechanically homogenized in 20 μL of 1× Brain Extraction Buffer (25 mM HEPES pH 7.3, 150 mM KCl, 8% glycerol, 0.1% NP-40, Roche ULTRA protease inhibitor, and Roche PhoSTOP phosphatase inhibitor) using a Teflon pestle for several minutes (Reynolds et al., 2021).The homogenate was chilled on ice for 15 min, prior to centrifugation at 10,000 rpm for 10 min at 4°C.The supernatant was removed and transferred to a new microtube.Samples were chilled on ice until used in the DC protein assay (BioRad, Mississauga, Ontario), which standardized protein content for western blotting.A standard curve consisting of serial dilutions of bovine serum albumin (BSA; Sigma-Aldrich, A-7906) was prepared.The standards and samples were prepared per manufacturer's instructions (Bio-Rad).Plates were gently shaken for 15 min prior to spectrophotometer (xMark Microplate Absorbance Spectrophotometer, Bio-Rad) readings taken at 750 nm.Samples were flash frozen in liquid nitrogen and returned to storage at −80°C.
Protein samples (10 μg) were loaded on precast polyacrylamide TGX Stain-Free, 4-12% gradient, SDS gels (BioRad) and were separated electrophoretically at 125 V for 45-50 min.Each loaded protein sample corresponded to tissue from an individual fish (n).Protein extraction from adult mouse brain tissue was loaded as a positive control, while protein extraction from adult naïve zebrafish whole brain tissue was loaded as a cross-gel control.Following electrophoresis, gels were activated by UV exposure for 45 s to permit the visualization of total protein loaded within each lane.Proteins were transferred onto polyvinylidenedifluoride (PVDF; BioRad) membranes using the TransBlot Turbo system using the settings 'turbo'>'1 mini-gel' (BioRad).Membranes were then imaged for total protein prior to incubation in 10 mM sodium citrate and 0.05% Tween-20 ( pH 6) overnight at 60°C.The next day, membranes were incubated in 5% BSA (Sigma-Aldrich) in 1× Tris-buffered saline solution with Tween-20 for 1 h and then gently rotated for 3 h at RT in primary antibody dilution.This included anti-P2X 7 (rabbit polyclonal; 1:500, Alomone Labs, Jerusalem, Israel, APR-004, RRID: AB_2040068).A negative control for the antibody was performed using a blocking peptide specific for the antibody binding site.This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).Membranes were pre-incubated with the blocking peptide overnight at 4°C prior to primary antibody exposure (as above).Membranes were then incubated in secondary antibody (donkey antirabbit horseradish peroxidase, 1:2500, Cytiva Lifescience, Marlborough, MA, USA, NA934VS, RRID: AB_772206) for 2 h at RT, developed in enhanced chemiluminescence (BioRad) substrate for 5 min, and imaged using a ChemiDoc system (BioRad).

Experimental design and statistical analysis
For qualitative assessment of P2X 7 colocalization with various cell types, sample sizes consisted of 3 fish per group.Meanwhile, for cell quantification experiments, the sample size was 3-10 fish per treatment group.For all immunohistochemical analysis, 4-6 sections encompassing either 550 µm naïve spinal cord or 550 µm rostral and 550 µm caudal to the lesion site were analyzed per fish (n).Quantification of cells detected in spinal cord transection images was completed using ImageJ with filters for background subtraction using settings for a rolling ball radius of 75 pixels.Manual counts of PCNA + cells within a 30 µm radius of the central canal were completed for each section of tissue.Automated counts for HuC/D + cells within the entire cross-section were completed for each section of tissue.HuC/D + and DAPI + channels were made into binary images that were then overlayed.The overlayed image was put through an automated cell counter and set to exclude any particles smaller than 20 pixels.All cell counts and data analysis were completed by an individual who was blinded to the experimental groups.
For quantitative protein analysis, the sample size ranged from 6-10 adult zebrafish for every time point.ImageLab 6.1 software (BioRad) was used to quantify the relative densitometry of bands of interest and total protein.Each protein of interest was normalized to the loading control and cross-gel control lanes to account for variation of membrane transfers.Results were expressed as fold change relative to the naïve control.Error bars are representative of standard deviation (s.d.).Statistical significance between groups was determined using one-way ANOVA and post-hoc Dunnett test for multiple comparisons.
P2X 7 agonism increases injury-induced ependymo-radial glial cell proliferation around the central canal
Centre at McMaster University.All experiments were approved by the McMaster Animal Research Ethics Board (Animal Utilization Protocol 19-08-22).