A switch in pdgfrb cell-derived ECM composition prevents inhibitory scarring and promotes axon regeneration in the zebrafish spinal cord

•Axon regeneration after spinal cord injury requires recruitment of pdgfrb+ cells•PDGFR signaling controls pdgfrb+ cell recruitment to the spinal lesion site•pdgfrb+ cells secrete axon growth-promoting ECM molecules (cthrc1a and col12a1a/b)•pdgfrb+ cells reduce synthesis of ECM molecules (lum and mfap2) that inhibit axon growth In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM. In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM. Spinal cord injury (SCI) severs axons that fail to regrow across the lesion site, leading to permanent functional deficits. Among the mechanisms that limit axon regeneration in the mammalian central nervous system (CNS) is the formation of fibrous (scar) tissue in the lesion core (Tran et al., 2018Tran A.P. Warren P.M. Silver J. The biology of regeneration failure and success after spinal cord injury.Physiol. Rev. 2018; 98: 881-917Crossref PubMed Scopus (239) Google Scholar). The scar forms due to the excessive deposition of extracellular matrix (ECM) by invading non-neural cells, most prominently platelet-derived growth factor receptor β (PDGFRβ)-positive fibroblasts of perivascular origin, and is considered a major barrier to regeneration (Dias et al., 2018Dias D.O. Kim H. Holl D. Werne Solnestam B. Lundeberg J. Carlén M. Göritz C. Frisén J. Reducing pericyte-derived scarring promotes recovery after spinal cord injury.Cell. 2018; 173: 153-165.e22Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar; Göritz et al., 2011Göritz C. Dias D.O. Tomilin N. Barbacid M. Shupliakov O. Frisén J. A pericyte origin of spinal cord scar tissue.Science. 2011; 333: 238-242Crossref PubMed Scopus (529) Google Scholar; Soderblom et al., 2013Soderblom C. Luo X. Blumenthal E. Bray E. Lyapichev K. Ramos J. Krishnan V. Lai-Hsu C. Park K.K. Tsoulfas P. Lee J.K. Perivascular fibroblasts form the fibrotic scar after contusive spinal cord injury.J. Neurosci. 2013; 33: 13882-13887Crossref PubMed Scopus (212) Google Scholar). Although different mechanistic concepts exist on how the lesion ECM interferes with axonal regrowth, surprisingly little is known about its regulation and composition (Bradbury and Burnside, 2019Bradbury E.J. Burnside E.R. Moving beyond the glial scar for spinal cord repair.Nat. Commun. 2019; 10: 3879Crossref PubMed Scopus (159) Google Scholar; Fawcett et al., 2012Fawcett J.W. Schwab M.E. Montani L. Brazda N. Müller H.W. Defeating inhibition of regeneration by scar and myelin components.Handb. Clin. Neurol. 2012; 109: 503-522Crossref PubMed Scopus (93) Google Scholar; Klapka and Müller, 2006Klapka N. Müller H.W. Collagen matrix in spinal cord injury.J. Neurotrauma. 2006; 23: 422-435Crossref PubMed Scopus (114) Google Scholar; Tran et al., 2018Tran A.P. Warren P.M. Silver J. The biology of regeneration failure and success after spinal cord injury.Physiol. Rev. 2018; 98: 881-917Crossref PubMed Scopus (239) Google Scholar). Attenuating perivascular cell-derived scarring or depriving the lesion site of ECM components improves permissiveness of the scar (Bradbury et al., 2002Bradbury E.J. Moon L.D. Popat R.J. King V.R. Bennett G.S. Patel P.N. Fawcett J.W. McMahon S.B. Chondroitinase ABC promotes functional recovery after spinal cord injury.Nature. 2002; 416: 636-640Crossref PubMed Scopus (1801) Google Scholar; Dias et al., 2018Dias D.O. Kim H. Holl D. Werne Solnestam B. Lundeberg J. Carlén M. Göritz C. Frisén J. Reducing pericyte-derived scarring promotes recovery after spinal cord injury.Cell. 2018; 173: 153-165.e22Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar; Klapka et al., 2005Klapka N. Hermanns S. Straten G. Masanneck C. Duis S. Hamers F.P. Müller D. Zuschratter W. Müller H.W. Suppression of fibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons, rescue of primary motoneurons in somatosensory cortex and significant functional recovery.Eur. J. Neurosci. 2005; 22: 3047-3058Crossref PubMed Scopus (129) Google Scholar; Ruschel et al., 2015Ruschel J. Hellal F. Flynn K.C. Dupraz S. Elliott D.A. Tedeschi A. Bates M. Sliwinski C. Brook G. Dobrindt K. et al.Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.Science. 2015; 348: 347-352Crossref PubMed Scopus (268) Google Scholar; Stichel et al., 1999Stichel C.C. Hermanns S. Luhmann H.J. Lausberg F. Niermann H. D'Urso D. Servos G. Hartwig H.G. Müller H.W. Inhibition of collagen IV deposition promotes regeneration of injured CNS axons.Eur. J. Neurosci. 1999; 11: 632-646Crossref PubMed Scopus (148) Google Scholar), thus providing a potential target for the development of therapeutic interventions following SCI. However, successful manipulation of the lesion ECM with the goal to enhance axon regeneration requires knowledge about potential growth-permissive, -promoting, or -inhibiting properties of specific ECM molecules, which remains limited. Unlike mammals, zebrafish exhibit an elevated regenerative capacity for the CNS and robustly recover locomotor function after SCI, both at larval and adult stages (Becker et al., 2004Becker C.G. Lieberoth B.C. Morellini F. Feldner J. Becker T. Schachner M. L1.1 is involved in spinal cord regeneration in adult zebrafish.J. Neurosci. 2004; 24: 7837-7842Crossref PubMed Scopus (129) Google Scholar; Mokalled et al., 2016Mokalled M.H. Patra C. Dickson A.L. Endo T. Stainier D.Y. Poss K.D. Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.Science. 2016; 354: 630-634Crossref PubMed Scopus (108) Google Scholar; Ohnmacht et al., 2016Ohnmacht J. Yang Y.J. Maurer G.W. Barreiro-Iglesias A. Tsarouchas T.M. Wehner D. Sieger D. Becker C.G. Becker T. Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish.Development. 2016; 143: 1464-1474Crossref PubMed Scopus (50) Google Scholar). Although the time course of regeneration differs (Figure 1A), restoration of swimming activity in both systems requires bridging of the non-neural lesion site by axonal connections (Becker et al., 2004Becker C.G. Lieberoth B.C. Morellini F. Feldner J. Becker T. Schachner M. L1.1 is involved in spinal cord regeneration in adult zebrafish.J. Neurosci. 2004; 24: 7837-7842Crossref PubMed Scopus (129) Google Scholar; Wehner et al., 2017Wehner D. Tsarouchas T.M. Michael A. Haase C. Weidinger G. Reimer M.M. Becker T. Becker C.G. Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish.Nat. Commun. 2017; 8: 126Crossref PubMed Scopus (64) Google Scholar). Similar to mammals, an injury to the zebrafish spinal cord leads to prominent ECM deposition in the lesion site. Yet, the zebrafish ECM is growth-conducive and necessary for regeneration. For example, collagen XII has been identified as a major constituent of the lesion matrix in zebrafish and its depletion impaired axonal regrowth and recovery of swimming function (Wehner et al., 2017Wehner D. Tsarouchas T.M. Michael A. Haase C. Weidinger G. Reimer M.M. Becker T. Becker C.G. Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish.Nat. Commun. 2017; 8: 126Crossref PubMed Scopus (64) Google Scholar). However, the cellular origin, identity, and regulation of ECM-producing cells, as well as the composition of the axon growth-promoting ECM, remain poorly defined. Here, we identify pdgfrb-expressing (pdgfrb+) myoseptal and perivascular cells as primary source of the axon growth-promoting ECM. We show that SCI triggers the recruitment of pdgfrb+ cells in a PDGFR signaling-dependent manner, which is essential for axons to bridge the lesion site. Mechanistically, we find that a switch in the composition of pdgfrb+ cell-secreted ECM is critical for axon regeneration and identify pro- and anti-regenerative ECM factors. Hence, we propose that after SCI, the lesion-specific composition of pdgfrb+ cell-derived ECM is a key determinant of regenerative success in zebrafish versus inhibitory scarring in mammals. To identify non-neural cells directing the regeneration response after SCI, we used a larval zebrafish transection model, which allows axonal regrowth and functional recovery within 2 days post-lesion (dpl) (Figures 1A and 1B; Wehner et al., 2017Wehner D. Tsarouchas T.M. Michael A. Haase C. Weidinger G. Reimer M.M. Becker T. Becker C.G. Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish.Nat. Commun. 2017; 8: 126Crossref PubMed Scopus (64) Google Scholar). We utilized the pdgfrb:GFP transgenic line that at 3 days post-fertilization (dpf) strongly labels blood vessel-associated mural (pericytes and smooth muscle cells) and floor plate cells (Figure 1C; Ando et al., 2016Ando K. Fukuhara S. Izumi N. Nakajima H. Fukui H. Kelsh R.N. Mochizuki N. Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish.Development. 2016; 143: 1328-1339Crossref PubMed Scopus (92) Google Scholar). However, GFP fluorescence intensity of the latter sharply decreased after 3 dpf (Figure 1D), indicating persistence of the GFP protein rather than transcriptional activity of the transgene. Additionally, a population of cells that was located between adjacent myotomes was labeled (Figures 1C and S1A). These myoseptal cells, previously described as tenocytes (Bricard et al., 2014Bricard Y. Rallière C. Lebret V. Lefevre F. Rescan P.Y. Early fish myoseptal cells: insights from the trout and relationships with amniote axial tenocytes.PLoS One. 2014; 9: e91876Crossref PubMed Scopus (16) Google Scholar), exhibited weaker GFP signal as compared to perivascular cells and were less abundant (36.2% ± 6.4% of total GFP+ cells; Figure S1B). We will, henceforth, refer to this heterogeneous population of myoseptal and perivascular cells as pdgfrb+ cells. To determine whether pdgfrb+ cells react to SCI, we imaged pdgfrb:GFP transgenics before and after lesion. This showed a 2.7-fold increase in the area coverage of pdgfrb+ cells in the lesion site at 1 dpl, the time point of active axonal regrowth (Figure 1A), as compared to unlesioned trunk (unlesioned: 28.3 ± 4.6 a.u., 1 dpl: 75.6 ± 8.6 a.u.; Figures 1D and S1C). Simultaneous visualization of flk1+ endothelial and pdgfrb+ cells showed that at 1 dpl, pdgfrb+ cells in the lesion center were not associated with blood vessels (Figure 1E). This observation is consistent with a scenario in which perivascular cells detach from blood vessels to populate the lesion site after SCI (Göritz et al., 2011Göritz C. Dias D.O. Tomilin N. Barbacid M. Shupliakov O. Frisén J. A pericyte origin of spinal cord scar tissue.Science. 2011; 333: 238-242Crossref PubMed Scopus (529) Google Scholar). We next analyzed the relationship of pdgfrb+ cells and regenerating axons at 1 dpl using transgenic reporter lines (Xla.Tubb:DsRED, elavl3:rasmKate2) or anti-acetylated tubulin immunohistochemistry (IHC) to label neurites. We frequently found axonal fascicles in close apposition to pdgfrb+ cells in the lesion site (23/26 axonal fascicles; Figures 1F, S1D, and S1E). Moreover, the distal end of 10 out of 11 axonal fascicles that had entered the lesion site, but not yet crossed it, was found in contact with pdgfrb+ cells, suggesting attraction of axons that grow toward the latter (Filous et al., 2014Filous A.R. Tran A. Howell C.J. Busch S.A. Evans T.A. Stallcup W.B. Kang S.H. Bergles D.E. Lee S.I. Levine J.M. Silver J. Entrapment via synaptic-like connections between NG2 proteoglycan+ cells and dystrophic axons in the lesion plays a role in regeneration failure after spinal cord injury.J. Neurosci. 2014; 34: 16369-16384Crossref PubMed Scopus (78) Google Scholar). Thus, pdgfrb+ cells are part of the lesion environment that regenerating axons encounter. To establish that the lesion site population of pdgfrb+ cells is derived from resident myoseptal and perivascular cells, we performed Cre-based genetic lineage tracing. We generated a transgenic line (pdgfrb:CreERT2) that enables the 4-hydroxytamoxifen (4-OH T)-inducible, non-leaky labeling of pdgfrb+ cells when used in combination with a Cre-responder line (hs:loxPDsREDloxPGFP) (Figures S1F–S1I). Repetitive imaging of the same animals showed that genetically labeled pdgfrb+ cells accumulated in the lesion site at 1 dpl and more pronounced at 2 dpl (Figure 1G). Importantly, genetically labeled pdgfrb+ cells accumulated in the lesion site in a similar manner when the 4-OH T withdrawal period between last treatment and first time point of analysis (1 dpl) was extended to several days. This excludes the possibility that cells that de novo upregulate pdgfrb expression after lesion were labeled due to 4-OH T retention (Figures S1J and S1K). Together, these data demonstrate that pdgfrb+ myoseptal and perivascular cells are recruited to the spinal lesion site. To gain further insight into the dynamics of the pdgfrb+ cell response after SCI and distinguish cells that proliferated from those recruited to the lesion site, we analyzed 5-ethynyl-2′-deoxyuridine (EdU) incorporation to assess their proliferation status. Although there was no difference in the number of pdgfrb+/EdU+ cells in the lesion site over baseline levels at 0.5 dpl (Figure S1L) and 1 dpl (Figure 1H), we observed a 2.8-fold increase in the number of pdgfrb+/EdU+ cells at 2 dpl (baseline: 16.7 ± 1.6 cells, lesion site: 47.3 ± 3.1 cells; Figure 1H). These data suggest that the rapid accumulation of pdgfrb+ cells in the lesion site during the early phase of regeneration is primarily due to cell recruitment, followed by cell proliferation. To examine whether the cellular response is conserved between larval and adult stages, we analyzed pdgfrb+ cell behavior after SCI in adult zebrafish. In unlesioned adults, the expression pattern of the pdgfrb:GFP reporter resembled that of larval zebrafish (Figures S1M and S1N; Ando et al., 2016Ando K. Fukuhara S. Izumi N. Nakajima H. Fukui H. Kelsh R.N. Mochizuki N. Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish.Development. 2016; 143: 1328-1339Crossref PubMed Scopus (92) Google Scholar). At 7 dpl, the onset of axonal regrowth (Figure 1A; Becker et al., 2005Becker T. Lieberoth B.C. Becker C.G. Schachner M. Differences in the regenerative response of neuronal cell populations and indications for plasticity in intraspinal neurons after spinal cord transection in adult zebrafish.Mol. Cell. Neurosci. 2005; 30: 265-278Crossref PubMed Scopus (48) Google Scholar), pdgfrb+ cells accumulated in the disorganized lesion center (Figure 1I). Importantly, pdgfrb+ cells that were genetically labeled before SCI in a stochastic manner using pdgfrb:CreERT2;hs:loxPDsREDloxPGFP transgenics, likewise appeared in the lesion site at 7 dpl (Figure 1J). In summary, these data show that SCI in zebrafish triggers recruitment of pdgfrb+ cells of myoseptal and perivascular origin to the lesion site. To test the role of pdgfrb+ cells in axon regeneration, we depleted them using pdgfrb:NTR-mCherry transgenics, which enable the nitroreductase (NTR)-mediated targeted ablation of pdgfrb+ cells upon exposure to the pro-drug metronidazole (MTZ). NTR-mCherry expression recapitulated that of GFP in pdgfrb:GFP;pdgfrb:NTR-mCherry transgenics, although less cells were labeled (Figure S2A). Treatment of pdgfrb:NTR-mCherry larvae with MTZ efficiently ablated transgene-expressing cells but did not overtly affect integrity of major blood vessels in the trunk (Figures 2A and S2B). Quantification of GFP+ cells in MTZ-treated pdgfrb:GFP;pdgfrb:NTR-mCherry transgenics showed a 54.3% reduction in the area coverage of pdgfrb+ cells in the lesion site at 1 dpl (DMSO: 52 ± 4.6 a.u., MTZ: 23.8 ± 2.5 a.u.; Figure 2B). Hence, NTR-mediated systemic depletion of pdgfrb+ cells results in a reduction of pdgfrb+ cells that are recruited to the lesion site. To assess whether axon regeneration is affected in pdgfrb+ cell-depleted animals, we analyzed axonal continuity across the lesion site ('axonal bridging'), shown to correlate with swimming function recovery, and thus, to robustly indicate anatomical repair (Wehner et al., 2017Wehner D. Tsarouchas T.M. Michael A. Haase C. Weidinger G. Reimer M.M. Becker T. Becker C.G. Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish.Nat. Commun. 2017; 8: 126Crossref PubMed Scopus (64) Google Scholar). We found that the proportion of animals with axonal bridge was reduced by 52.7% in MTZ-treated animals at 2 dpl (DMSO: 78.5% ± 4.1%, MTZ: 37.1% ± 5.1%; Figure 2C). Moreover, quantification of touch-evoked swimming at 2 dpl showed that MTZ-treated animals recovered to swim 34.2% of the distance covered by unlesioned MTZ-treated larvae (unlesionedMTZ: 54 ± 4.5 mm, 2 dplMTZ: 18.5 ± 2.5 mm), whereas the swimming distance covered by DMSO-treated lesioned animals was not significantly different as compared to their unlesioned controls (Figure 2C). Hence, depletion of pdgfrb+ cells inhibits axon regeneration and functional recovery. Importantly, quantification of the number of EdU+/elavl3+ neurons at 2 dpl showed that regenerative spinal neurogenesis (Ohnmacht et al., 2016Ohnmacht J. Yang Y.J. Maurer G.W. Barreiro-Iglesias A. Tsarouchas T.M. Wehner D. Sieger D. Becker C.G. Becker T. Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish.Development. 2016; 143: 1464-1474Crossref PubMed Scopus (50) Google Scholar) was not reduced in pdgfrb+ cell-depleted animals, indicating they were still capable of mounting a regeneration response (Figure S2C). To exclude systemic effects of NTR-mediated pdgfrb+ cell ablation on axon regeneration, we used an optogenetic approach. We generated Tet-responder (TetRE:memKillerRed) and Tet-activator (pdgfrb:TetA) transgenic lines to target a membrane-localized photosensitizer, memKillerRed, specifically to pdgfrb+ cells in a doxycycline (DOX)-inducible manner (Figures S2D and S2E) (Teh et al., 2010Teh C. Chudakov D.M. Poon K.L. Mamedov I.Z. Sek J.Y. Shidlovsky K. Lukyanov S. Korzh V. Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics.BMC Dev. Biol. 2010; 10: 110Crossref PubMed Scopus (74) Google Scholar; Wehner et al., 2015Wehner D. Jahn C. Weidinger G. Use of the TetON system to study molecular mechanisms of zebrafish regeneration.J. Vis. Exp. 2015; 100: e52756Google Scholar). Upon exposure to intense green light, KillerRed undergoes photobleaching and concomitantly generates reactive oxygen species, resulting in cell death (Bulina et al., 2006Bulina M.E. Chudakov D.M. Britanova O.V. Yanushevich Y.G. Staroverov D.B. Chepurnykh T.V. Merzlyak E.M. Shkrob M.A. Lukyanov S. Lukyanov K.A. A genetically encoded photosensitizer.Nat. Biotechnol. 2006; 24: 95-99Crossref PubMed Scopus (422) Google Scholar). We photobleached pdgfrb:TetA;TetRE:memKillerRed (short pdgfrb:memKillerRed) transgenics in a restricted region of the trunk prior to lesion (Figure 2D). Quantification of GFP+ cells in photobleached pdgfrb:memKillerRed;pdgfrb:GFP transgenics at 1 dpl showed a 68% reduction in the area coverage of pdgfrb+ cells in the lesion site (control: 115.7 ± 17.1 a.u., optoablated: 37.1 ± 8 a.u.; Figure 2E). Hence, pdgfrb:memKillerRed transgenics enable the spatiotemporally controlled depletion of pdgfrb+ cells. Importantly, we found that at 2 dpl, axonal bridging was reduced by 55.6% in optoablated animals (control: 92.4% ± 3%, optoablated: 41% ± 9.2%; Figure 2F), confirming results of NTR-mediated cell ablation. To corroborate our scoring results, we determined the thickness of the axonal bridge at 2 dpl in independent samples, shown to correlate with the recovery of touch-evoked swimming distance and to provide a sensitive readout for subtle changes in regeneration efficiency (Tsarouchas et al., 2018Tsarouchas T.M. Wehner D. Cavone L. Munir T. Keatinge M. Lambertus M. Underhill A. Barrett T. Kassapis E. Ogryzko N. et al.Dynamic control of proinflammatory cytokines IL-1beta and Tnf-alpha by macrophages in zebrafish spinal cord regeneration.Nat. Commun. 2018; 9: 4670Crossref PubMed Scopus (90) Google Scholar). Optogenetic depletion of pdgfrb+ cells reduced the average axonal bridge thickness by 62.5% (control: 30.4 ± 2.8 μm, optoablated: 11.4 ± 3.2 μm; Figure S2F). Together, these data show that pdgfrb+ cells are dispensable for regenerative neurogenesis but are required for axonal regrowth and functional recovery after SCI in larval zebrafish. To distinguish between the contribution of myoseptal and perivascular cells to axon regeneration, we reduced mural cell coverage of blood vessels by inhibiting developmental angiogenesis. We treated zebrafish embryos with a selective PDGFR inhibitor (PDGFR-i V), which resulted in largely normally developed larvae that lack major blood vessels in the trunk as previously described (Figure 2G; Wiens et al., 2010Wiens K.M. Lee H.L. Shimada H. Metcalf A.E. Chao M.Y. Lien C.L. Platelet-derived growth factor receptor beta is critical for zebrafish intersegmental vessel formation.PLoS One. 2010; 5: e11324Crossref PubMed Scopus (36) Google Scholar). Due to the absence of major blood vessels, we could mainly detect pdgfrb+ cells in myosepta with the exception of dorsal artery, posterior cardinal vein, and caudal vein, where mural cells where still present (Figure 2G). Thus, PDGFR-i V treatment during early development deprives the pdgfrb+ cell pool of perivascular cells, thereby providing a system to specifically assess their contribution to axon regeneration. In PDGFR-i V-treated animals, the area coverage of pdgfrb+ cells in the lesion site was reduced by 41.4% at 1 dpl (DMSO: 86.8 ± 8.7 a.u., PDGFR-i V: 50.8 ± 8.4 a.u.; Figure 2H). Given their lower abundance prior to lesion (0.57-fold, see Figure S1B), this suggests that the contribution of myoseptal cells to the pdgfrb+ cell population in the lesion site exceeds that of perivascular cells by >2-fold. Intriguingly, we found that the proportion of animals with axonal bridge at 2 dpl was reduced by 52% in PDGFR-i V-treated animals (DMSO: 84.9% ± 2.2%, PDGFR-i V: 41.1% ± 7.6%; Figure 2I). Moreover, in PDGFR-treated animals, the swimming distance was reduced by 44.7%, as compared to their unlesioned controls at 2 dpl. In contrast, swimming capacity of DMSO-treated lesioned animals was not significantly different, as compared to their unlesioned controls (unlesionedPDGFR-i V: 40.3 ± 2.4 mm, 2 dplPDGFR-i V: 22.3 ± 1.9 mm; Figure 2I). We conclude that perivascular cells are required for axon regeneration and functional recovery after SCI in zebrafish larvae. We next sought to identify signals governing the cellular response of pdgfrb+ cells after SCI. A candidate signal to control pdgfrb+ cell recruitment are platelet-derived growth factors (PDGFs). pdgfrb+ cells express pdgfrb (coding for PDGF receptor β; Pdgfrβ), and thus, are likely competent to respond to PDGF signals. We first analyzed the expression of the 6 zebrafish PDGF ligand-encoding genes by in situ hybridization (ISH). Transcripts of pdgfaa, pdgfab, pdgfba, pdgfc, and pdgfd but not pdgfbb were detectably upregulated in the lesion site at 20 h post-lesion (hpl) (Figures 3A and S3A). Hence, the zebrafish lesion site is a local source of PDGF ligands after SCI. To test the requirement of PDGFR signaling for pdgfrb+ cell recruitment, we inhibited it, using a selective PDGFR inhibitor (PDGFR-i IV; Ho et al., 2005Ho C.Y. Ludovici D.W. Maharoof U.S. Mei J. Sechler J.L. Tuman R.W. Strobel E.D. Andraka L. Yen H.K. Leo G. et al.(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamines: platelet-derived growth factor receptor tyrosine kinase inhibitors with broad antiproliferative activity against tumor cells.J. Med. Chem. 2005; 48: 8163-8173Crossref PubMed Scopus (31) Google Scholar). This reduced the area coverage of pdgfrb+ cells in the lesion site by 63.8% at 1 dpl (DMSO: 149.3 ± 14 a.u., PDGFR-i IV: 54.1 ± 10.1 a.u.; Figure 3B). Similarly, heat-induced overexpression of a dominant-negative pdgfrb (dnpdgfrb) in hs:dnpdgfrb transgenics reduced the area coverage of pdgfrb+ cells in the lesion site by 64.6% (wild type: 206.6 ± 29.8 a.u., hs:dnpdgfrb: 73.2 ± 24.1 a.u.; Figures S3B and S3C). In contrast, PDGFR-i IV treatment did not affect recruitment of pro-regenerative mpeg1+ macrophages (Tsarouchas et al., 2018Tsarouchas T.M. Wehner D. Cavone L. Munir T. Keatinge M. Lambertus M. Underhill A. Barrett T. Kassapis E. Ogryzko N. et al.Dynamic control of proinflammatory cytokines IL-1beta and Tnf-alpha by macrophages in zebrafish spinal cord regeneration.Nat. Commun. 2018; 9: 4670Crossref PubMed Scopus (90) Google Scholar) to the lesion site (Figure S3D). This supports that pdgfrb+ cell recruitment was specifically perturbed in PDGFR-i IV-treated animals. Strikingly, we found that upon PDGFR inhibition, axon regeneration and recovery of swimming function were impaired. The proportion of larvae with axonal bridge at 2 dpl was reduced by 75.9% after PDGFR-i IV treatment (DMSO: 77.2% ± 1.7%, PDGFR-i IV: 18.6% ± 2.3%; Figure 3C) and by 46.7% in hs:dnpdgfrb transgenics (wild type: 80.2% ± 1.8%, hs:dnpdgfrb: 42.8% ± 6.4%; Figure S3E). Moreover, in lesioned PDGFR-i IV-treated animals, the swimming distance was reduced by 59.6% at 2 dpl, as compared to unlesioned controls, whereas swimming capacity of DMSO-treated lesioned animals was not significantly different in comparison with their unlesioned controls (unlesionedPDGFR-i IV: 57 ± 4.5 mm, 2 dplPDGFR-i IV: 23 ± 2 mm; Figure 3C). In dnpdgfrb-expressing lesioned animals, the swimming distance was reduced by 26.8%, as compared to their unlesioned controls (unlesionedhs:dnpdgfrb: 33.2 ± 1.3 mm, 2 dplhs:dnpdgfrb: 24.3 ± 2 mm; Figure S3E). Thus, recruitment of pdgfrb+ cells to the lesion site coincides with axon regeneration and both processes are dependent on PDGFR signaling. To test the requirement of PDGFR signaling in pdgfrb+ cells for axon regeneration, we generated a Tet-responder line (TetRE:dnpdgfrb-mCherry) to selectively target dnpdgfrb expression to pdgfrb+ cells, when used in combination with pdgfrb:TetA transgenics (Figure S3F). Intriguingly, dnpdgfrb induction in pdgfrb+ cells was sufficient to reduce the average axonal bridge thickness by 38.3% at 2 dpl (pdgfrb:TetA: 32 ± 2 μm, pdgfrb:TetA;TetRE:dnpdgfrb: 19.7 ± 1.9 μm; Figure 3D). In contrast, neuron-specific targeting of dnpdgfrb did not induce a phenotype (Figures 3E and S3G). Thus, cell autonomous PDGFR signaling in pdgfrb+ cells is necessary for axon regeneration after SCI in larval zebrafish. Tenocytes and perivascular cells are a primary source of ECM during physiological and pathological processes (Dias et al., 2018Dias D.O. Kim H. Holl D. Werne Solnestam B. Lundeberg J. Carlén M. Göritz C. Frisén J. Reducing pericyte-derived scarring promotes recovery after spinal cord injury.Cell. 2018; 173: 153-165.e22Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar; Lin et al., 2008Lin S.L. Kisseleva T. Brenner D.A. Duffield J.S. Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am. J. Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar; Screen et al., 2015Screen H.R. Berk D.E. Kadler K.E. Ramirez F. Young M.F. Tendon functional extracellular matrix.J. Orthop. Res. 2015; 33: 793-799Crossref PubMed Scopus (109) Google Scholar). Hence, we hypothesized that pdgfrb+ cells facilitate axon regeneration in zebrafish through the secretion of a growth-promoting ECM. To this end, we transcriptionally profiled pdgfrb+ cells isolated from pdgfrb:GFP transgenics at 1 dpl and unlesioned controls by RNA-seq (Figure 4