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• W2904212612 abstract "•Aryl hydrocarbon receptor (AhR) pathway is crucial for brain regeneration•High AhR levels promote conversion of ependymoglia to neurons during regeneration•Low AhR levels promote ependymoglial proliferation in the injured brain•AhR levels set the proper timing of restorative neurogenesis Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain. Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain. Regeneration in the mammalian CNS is largely limited (Dimou and Götz, 2014Dimou L. Götz M. Glial cells as progenitors and stem cells: new roles in the healthy and diseased brain.Physiol. Rev. 2014; 94: 709-737Crossref PubMed Scopus (161) Google Scholar) and restricted to either demyelinated axon repair (Dimou and Götz, 2014Dimou L. Götz M. Glial cells as progenitors and stem cells: new roles in the healthy and diseased brain.Physiol. Rev. 2014; 94: 709-737Crossref PubMed Scopus (161) Google Scholar) or, in very few cases, neuronal repair (Arvidsson et al., 2002Arvidsson A. Collin T. Kirik D. Kokaia Z. Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke.Nat. Med. 2002; 8: 963-970Crossref PubMed Scopus (2401) Google Scholar, Chen et al., 2004Chen J. Magavi S.S.P. Macklis J.D. Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice.Proc. Natl. Acad. Sci. USA. 2004; 101: 16357-16362Crossref PubMed Scopus (215) Google Scholar, Ernst et al., 2014Ernst A. Alkass K. Bernard S. Salehpour M. Perl S. Tisdale J. Possnert G. Druid H. Frisén J. Neurogenesis in the striatum of the adult human brain.Cell. 2014; 156: 1072-1083Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Neuronal replacement in mammals is limited to brain areas in close proximity to neurogenic zones (Brill et al., 2009Brill M.S. Ninkovic J. Winpenny E. Hodge R.D. Ozen I. Yang R. Lepier A. Gascón S. Erdelyi F. Szabo G. et al.Adult generation of glutamatergic olfactory bulb interneurons.Nat. Neurosci. 2009; 12: 1524-1533Crossref PubMed Scopus (261) Google Scholar). However, a large number of young neurons originating from the neurogenic zones fail to mature and integrate at the injury site and instead die (Arvidsson et al., 2002Arvidsson A. Collin T. Kirik D. Kokaia Z. Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke.Nat. Med. 2002; 8: 963-970Crossref PubMed Scopus (2401) Google Scholar, Brill et al., 2009Brill M.S. Ninkovic J. Winpenny E. Hodge R.D. Ozen I. Yang R. Lepier A. Gascón S. Erdelyi F. Szabo G. et al.Adult generation of glutamatergic olfactory bulb interneurons.Nat. Neurosci. 2009; 12: 1524-1533Crossref PubMed Scopus (261) Google Scholar). In contrast, the zebrafish CNS has the capacity to regenerate brain tissue after injury (Becker and Becker, 2015Becker C.G. Becker T. Neuronal regeneration from ependymo-radial glial cells: cook, little pot, cook!.Dev. Cell. 2015; 32: 516-527Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This regeneration also includes the replacement of lost neurons (restorative neurogenesis) (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar, Baumgart et al., 2012Baumgart E.V. Barbosa J.S. Bally-Cuif L. Götz M. Ninkovic J. Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.Glia. 2012; 60: 343-357Crossref PubMed Scopus (141) Google Scholar, Kishimoto et al., 2012Kishimoto N. Shimizu K. Sawamoto K. Neuronal regeneration in a zebrafish model of adult brain injury.Dis. Model. Mech. 2012; 5: 200-209Crossref PubMed Scopus (153) Google Scholar, Kroehne et al., 2011Kroehne V. Freudenreich D. Hans S. Kaslin J. Brand M. Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.Development. 2011; 138: 4831-4841Crossref PubMed Scopus (315) Google Scholar, Kyritsis et al., 2012Kyritsis N. Kizil C. Zocher S. Kroehne V. Kaslin J. Freudenreich D. Iltzsche A. Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain.Science. 2012; 338: 1353-1356Crossref PubMed Scopus (360) Google Scholar). Tremendous regeneration capacity coincides with the wide spread of ependymoglial cells producing different neuronal subtypes in the zebrafish brain throughout their lifetime (Kyritsis et al., 2012Kyritsis N. Kizil C. Zocher S. Kroehne V. Kaslin J. Freudenreich D. Iltzsche A. Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain.Science. 2012; 338: 1353-1356Crossref PubMed Scopus (360) Google Scholar). Notably, ependymoglial cells lining the ventricular surface in the adult zebrafish telencephalon generate new neurons that are recruited to the injury site (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar, Baumgart et al., 2012Baumgart E.V. Barbosa J.S. Bally-Cuif L. Götz M. Ninkovic J. Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.Glia. 2012; 60: 343-357Crossref PubMed Scopus (141) Google Scholar, Kishimoto et al., 2012Kishimoto N. Shimizu K. Sawamoto K. Neuronal regeneration in a zebrafish model of adult brain injury.Dis. Model. Mech. 2012; 5: 200-209Crossref PubMed Scopus (153) Google Scholar, Kyritsis et al., 2012Kyritsis N. Kizil C. Zocher S. Kroehne V. Kaslin J. Freudenreich D. Iltzsche A. Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain.Science. 2012; 338: 1353-1356Crossref PubMed Scopus (360) Google Scholar). Importantly, a considerable proportion of these additionally generated neurons fully differentiate into the appropriate neuronal subtypes and survive for more than 3 months (Baumgart et al., 2012Baumgart E.V. Barbosa J.S. Bally-Cuif L. Götz M. Ninkovic J. Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.Glia. 2012; 60: 343-357Crossref PubMed Scopus (141) Google Scholar, Kroehne et al., 2011Kroehne V. Freudenreich D. Hans S. Kaslin J. Brand M. Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.Development. 2011; 138: 4831-4841Crossref PubMed Scopus (315) Google Scholar). The activation of ependymoglial cells to produce additional neurons is preceded by the activation of microglial cells involved in the initial wound healing process. Importantly, the initial inflammation does not only remove cellular debris but also induces restorative neurogenesis (Kyritsis et al., 2012Kyritsis N. Kizil C. Zocher S. Kroehne V. Kaslin J. Freudenreich D. Iltzsche A. Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain.Science. 2012; 338: 1353-1356Crossref PubMed Scopus (360) Google Scholar), suggesting a biphasic regeneration process in the zebrafish brain. During the first phase, activated glial cells restrict the initial damage and clear cellular debris. The following second phase promotes the production of new neurons from the ependymoglia that are necessary for tissue restoration (Kyritsis et al., 2012Kyritsis N. Kizil C. Zocher S. Kroehne V. Kaslin J. Freudenreich D. Iltzsche A. Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain.Science. 2012; 338: 1353-1356Crossref PubMed Scopus (360) Google Scholar). This delay in restorative neurogenesis relative to the initial inflammatory phase therefore stands as a crucial mechanism to allow correct zebrafish brain regeneration. For this reason, understanding the specific molecular programs underlying the timely production of new neurons is critical to implement regeneration from endogenous glial cells in the mammalian brain. Despite their importance, these mechanisms, which are involved in the temporal control of restorative neurogenesis from ependymoglial cells, are not well understood. Therefore, we aim to identify them using both longitudinal analysis of injury-induced transcriptome changes in ependymoglial niches and the cell-type-specific manipulation of these pathways. To define the molecular pathways controlling restorative neurogenesis initiation in the dorsal neurogenic zone (VZ) of the zebrafish telencephalon (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar), we aimed to identify injury-induced changes in the transcriptome specific for the VZ. To this end, we compared changes in the transcriptome in laser-dissected tissue from 3 different telencephalic areas from intact and stab-wound injured brains (Barbosa et al., 2016Barbosa J.S. Di Giaimo R. Götz M. Ninkovic J. Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.Nat. Protoc. 2016; 11: 1360-1370Crossref PubMed Scopus (15) Google Scholar) using the Affymetrix Zebrafish Gene 1.x ST array (Figure 1A). We compared injury-induced changes in the VZ to changes in the brain parenchyma (DP), which is free of neuronal progenitors, to reveal a signature specific for this cell type. Moreover, a comparison with the medial neurogenic zone (MVZ) was performed to extract pathways specific for progenitors in the VZ engaged in the repair process. Non-supervised hierarchical clustering of injury-induced changes at 2 and 7 days after injury in these 3 different areas revealed several clusters of co-regulated genes. These clusters were either specific to the VZ (clusters 1 and 2), shared between neurogenic niches (clusters 3 and 4), or changed in all 3 analyzed areas (clusters 5 and 6; Figures S1A and S1B; Table S1). We reasoned that the molecular signature controlling the early response of the ependymoglia should be upregulated specifically in the dorsal neurogenic zone at 2 dpi, correlating with the first signs of ependymoglia reaction to injury (Baumgart et al., 2012Baumgart E.V. Barbosa J.S. Bally-Cuif L. Götz M. Ninkovic J. Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.Glia. 2012; 60: 343-357Crossref PubMed Scopus (141) Google Scholar), and should chase out at 7 dpi, as represented by cluster 1 (Figure S1A). We observed 192 genes specifically upregulated in the VZ at 2 dpi (Figure S1B). Ingenuity Pathway Analysis (IPA) of these genes revealed significant over-representation of several metabolic (green) and immune-system-related (blue) pathways (Figure 1B; Table S1). However, the most significantly regulated pathway was the aryl hydrocarbon receptor (AhR) signaling pathway (Figure 1B), placing it as our prime candidate for further analysis. We therefore analyzed the expression of AhR-signaling-regulated genes after injury and observed an upregulation of Irf9, Nfkbie, and Tgfb1 specifically at 2 dpi (Figure S1C), which was indicative of reduced AhR signaling. Taken together, our data revealed a specific inhibition of AhR signaling in the dorsal neurogenic zone at 2 dpi that then chases out at 7 dpi, suggesting its role in the initial control of restorative neurogenesis. To address the importance of AhR signaling levels in restorative neurogenesis, we either potentiated AhR signaling with a high-affinity AhR agonist, β-naphthoflavone (BNF; Berghard et al., 1992Berghard A. Gradin K. Toftgård R. The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes.Carcinogenesis. 1992; 13: 651-655Crossref PubMed Scopus (15) Google Scholar), or decreased it by morpholino-mediated knockdown. AhR is a ligand-dependent transcription factor that is restrained in the cytoplasm by a chaperone complex when inactive (Hestermann and Brown, 2003Hestermann E.V. Brown M. Agonist and chemopreventative ligands induce differential transcriptional cofactor recruitment by aryl hydrocarbon receptor.Mol. Cell. Biol. 2003; 23: 7920-7925Crossref PubMed Scopus (120) Google Scholar). Upon ligand binding, AhR translocates to the nucleus and activates the transcription of its downstream targets (Hestermann and Brown, 2003Hestermann E.V. Brown M. Agonist and chemopreventative ligands induce differential transcriptional cofactor recruitment by aryl hydrocarbon receptor.Mol. Cell. Biol. 2003; 23: 7920-7925Crossref PubMed Scopus (120) Google Scholar). BNF induces AhR translocation to the nucleus without a natural ligand and therefore activates AhR signaling (Soshilov and Denison, 2014Soshilov A.A. Denison M.S. Ligand promiscuity of aryl hydrocarbon receptor agonists and antagonists revealed by site-directed mutagenesis.Mol. Cell. Biol. 2014; 34: 1707-1719Crossref PubMed Scopus (93) Google Scholar). To minimize systemic effects, we injected 10 μg/g of body mass of BNF in the telencephalic ventricle using cerebroventricular microinjections (CVMIs) (Figure 1C). We first analyzed the efficiency of CVMI-administered BNF to activate AhR signaling based on the expression levels of cytochrome P450 1B1 oxidase (Cyp1b1), a transcriptional reporter for AhR signaling (Soshilov and Denison, 2014Soshilov A.A. Denison M.S. Ligand promiscuity of aryl hydrocarbon receptor agonists and antagonists revealed by site-directed mutagenesis.Mol. Cell. Biol. 2014; 34: 1707-1719Crossref PubMed Scopus (93) Google Scholar). Notably, we detected more than 2-fold higher levels of Cyp1b1 in the injured brains in BNF-treated animals compared to the vehicle treatment (Figure S1D). We then assessed the number of new neurons generated from ependymoglial cells (Figures 1C–1F). To follow both ependymoglia and their progeny, including newly generated neurons, we labeled ependymoglia by electroporation of a plasmid encoding for membrane-localized TdTomato (TdTomatomem) red fluorescent protein (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar) (Figure 1C), allowing the long-term tracing of the ependymoglial lineage (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar, Barbosa et al., 2016Barbosa J.S. Di Giaimo R. Götz M. Ninkovic J. Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.Nat. Protoc. 2016; 11: 1360-1370Crossref PubMed Scopus (15) Google Scholar). Elevated AhR signaling in the injured brain increased the number of newborn, HuC/D-positive neurons derived from the electroporated ependymoglial cells compared to the vehicle treatment (Figures 1D–1F). Conversely, the morpholino-mediated knockdown of aryl hydrocarbon receptor 2 (ahr2; Figures 1G and 1I), a major mediator of AhR signaling in zebrafish (Bello et al., 2004Bello S.M. Heideman W. Peterson R.E. 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish.Toxicol. Sci. 2004; 78: 258-266Crossref PubMed Scopus (67) Google Scholar), increased the ependymoglia proliferation compared to control morpholino (Figures 1H and 1I). Taken together, our data suggest a role for AhR signaling levels in controlling ependymoglial behavior after injury, with high levels of AhR signaling promoting neurogenesis from the zebrafish ependymoglia and low AhR signaling triggering their proliferation and/or self-renewal. To assess the importance of this regulation, we followed the fate of new neurons added after AhR potentiation using TdTomatomem-based fate mapping. We analyzed the fate of progeny at 5 (short-term tracing) and 14 days (long-term tracing) after injury (Figure 2A). As expected, the proportion of new TdTomatomem+ HuC/D+ neurons significantly increased after AhR potentiation compared to the vehicle-treated animals in short-term tracing. The proportion of TdTomatomem+ and HuC/D+ neurons increased further at 14 days compared to 5 days after vehicle treatment (Figures 2B–2D). Surprisingly, we observed that the number of TdTomatomem+, HuC/D+ neurons after AhR potentiation was significantly lower at 14 dpi (long-term tracing, Figure 2D) compared to 5 dpi (Figure 2D), suggesting impaired survival of additional neurons generated due to the inappropriate regulation of AhR signaling. To complement this analysis of precocious activation of AhR signaling, we interfered with the return of AhR signaling to basal levels after the initial decrease following injury and analyzed the proliferation of ependymoglia. To achieve the precise timing of interference with AhR signaling, we used a pharmacological approach and locally administered 6 μM AhR antagonist (AhR Antagonist II, SR1-CAS) using CVMI. Administration of the antagonist efficiently decreased AhR signaling, as measured by Cyp1b1 expression (Figure S2A). AhR levels were then kept low by the daily administration of the agonist starting at day 3 after injury, and the number of actively cycling, PCNA+ ependymoglia was determined at 7 dpi using intracellular fluorescence-activated cell sorting (FACS) (Figures 2E, 2F, and S2C), as previously described (Barbosa et al., 2016Barbosa J.S. Di Giaimo R. Götz M. Ninkovic J. Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.Nat. Protoc. 2016; 11: 1360-1370Crossref PubMed Scopus (15) Google Scholar). We chose to analyze samples at 7 dpi because this is the time point when the expression of Cyp1b1 is again increased to basal levels observed in the intact brain (Figure 4E). The number of proliferating ependymoglia significantly increased (Figures 2E and 2F) after antagonist treatment compared to the vehicle control. This increase correlates with our hypothesis that low AhR levels allow the proliferation of ependymoglia after injury. Our data, therefore, suggest a role of AhR in regulating the balance between the proliferation and differentiation of ependymoglia to ensure the proper timing of restorative neurogenesis.Figure 4AhR Signaling Is Regulated in the Population of Ependymoglia Undergoing Direct ConversionShow full caption(A) Micrograph with orthogonal projection and pixel intensity image depicting the surface of the ependymoglial cell layer in the Tg(gfap:GFP)mi2001 line. The signal is obtained using in vivo imaging. Note that two different states of ependymoglia cells can be distinguished based on the following levels of GFP expression: GFPhigh (arrow) and GFPlow (arrowhead).(B) Lineage trees of ependymoglia cells undergoing direct conversion in the injured brains after AhR agonist or vehicle treatment. Every line in the box represents a single ependymoglia cell followed during the 5-day-period of in vivo imaging.(C) FACS plots depicting the definition of the sorting gates (Brassy background) and sorting of gfap:GFPhigh and GFPlow ependymoglia in intact and injured telencephalons based on the contour plots.(D) Dot plot showing the real-time expression of glial genes in GFPhigh- and GFPlow-sorted cells from intact brains. Single dots represent individual animals indicating biological replicates. Each biological replicate is the mean of 4 technical replicates. Lines show mean ± SEM. ∗p ≤ 0.05 (unpaired t test).(E) Dot plot depicting the expression of Cyp1b1 in GFPlow ependymoglia-sorted cells from intact brains and after injury. Single dots represent individual animals indicating biological replicates. Each biological replicate is the mean of 4 technical replicates. Lines show mean ± SEM. ∗p ≤ 0.05 (repeated one-way ANOVA with Bonferroni post hoc test).(F) Gene set enrichment analysis (GSEA) plot depicting AhR pathway regulation at 1, 3, and 7 days after injury in the GFPlow ependymoglial subpopulation.(G) Micrographs with orthogonal projections depicting HuC/D and TdTomatomem double-positive cells negative for BrdU lacking radial morphology in AhR agonist- or vehicle-treated brains 5 days after injury. Images are presented as full z-projections of the confocal z stack.(H) Dot plot showing the percentage of neurons generated by direct conversion (double-positive HuC/D and TdTomatomem cells negative for BrdU) among all TdTomatomem-positive cells after treatment with AhR agonist or vehicle at 5 dpi (short term) and 14 dpi (long term). Single dots represent individual animals indicating biological replicates. Lines show mean ± SEM. ∗p ≤ 0.05 (Mann-Whitney test).DC, direct conversion; NES, normalized enrichment score.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Micrograph with orthogonal projection and pixel intensity image depicting the surface of the ependymoglial cell layer in the Tg(gfap:GFP)mi2001 line. The signal is obtained using in vivo imaging. Note that two different states of ependymoglia cells can be distinguished based on the following levels of GFP expression: GFPhigh (arrow) and GFPlow (arrowhead). (B) Lineage trees of ependymoglia cells undergoing direct conversion in the injured brains after AhR agonist or vehicle treatment. Every line in the box represents a single ependymoglia cell followed during the 5-day-period of in vivo imaging. (C) FACS plots depicting the definition of the sorting gates (Brassy background) and sorting of gfap:GFPhigh and GFPlow ependymoglia in intact and injured telencephalons based on the contour plots. (D) Dot plot showing the real-time expression of glial genes in GFPhigh- and GFPlow-sorted cells from intact brains. Single dots represent individual animals indicating biological replicates. Each biological replicate is the mean of 4 technical replicates. Lines show mean ± SEM. ∗p ≤ 0.05 (unpaired t test). (E) Dot plot depicting the expression of Cyp1b1 in GFPlow ependymoglia-sorted cells from intact brains and after injury. Single dots represent individual animals indicating biological replicates. Each biological replicate is the mean of 4 technical replicates. Lines show mean ± SEM. ∗p ≤ 0.05 (repeated one-way ANOVA with Bonferroni post hoc test). (F) Gene set enrichment analysis (GSEA) plot depicting AhR pathway regulation at 1, 3, and 7 days after injury in the GFPlow ependymoglial subpopulation. (G) Micrographs with orthogonal projections depicting HuC/D and TdTomatomem double-positive cells negative for BrdU lacking radial morphology in AhR agonist- or vehicle-treated brains 5 days after injury. Images are presented as full z-projections of the confocal z stack. (H) Dot plot showing the percentage of neurons generated by direct conversion (double-positive HuC/D and TdTomatomem cells negative for BrdU) among all TdTomatomem-positive cells after treatment with AhR agonist or vehicle at 5 dpi (short term) and 14 dpi (long term). Single dots represent individual animals indicating biological replicates. Lines show mean ± SEM. ∗p ≤ 0.05 (Mann-Whitney test). DC, direct conversion; NES, normalized enrichment score. Restorative neurogenesis in zebrafish is achieved by both an increase in the number of dividing neuronal progenitors (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar) and an increase in the direct conversion of ependymoglia into post-mitotic neurons (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar). To assess the direct conversion of ependymoglia after injury and AhR signaling activation, we sparsely labeled ependymoglia by the electroporation of TdTomatomem (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar) and followed individual labeled ependymoglia and their progeny for 5 days by live imaging (Figures 3A, 3B, S3A, and S3B). The identity of the followed cells was then assessed by post-imaging staining for neuronal marker HuC/D. This allowed the re-identification of previously imaged cells (Figures S3E–S3G). We electroporated plasmids in the Tg(gfap:GFP)mi2001 transgenic line expressing GFP in all ependymoglia (also retained in 25% of their progeny) to identify the ependymoglia identity by GFP expression and the radial morphology, as previously described (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar, Barbosa et al., 2016Barbosa J.S. Di Giaimo R. Götz M. Ninkovic J. Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.Nat. Protoc. 2016; 11: 1360-1370Crossref PubMed Scopus (15) Google Scholar). The direct conversion of ependymoglia into neurons was then characterized by the loss of radial morphology, migration out of the ependymoglial layer toward the brain parenchyma (Figures 3B and S3F), and the expression of the post-mitotic marker HuC/D prior to a change in radial morphology (Figure 3C; Figure S3G). In line with our previous observation (Barbosa et al., 2015Barbosa J.S. Sanchez-Gonzalez R. Di Giaimo R. Baumgart E.V. Theis F.J. Götz M. Ninkovic J. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.Science. 2015; 348: 789-793Crossref PubMed Scopus (127) Google Scholar), only a few ependymoglial cells divided (Figure 3D; Figures S3A and S3B), and the vast majority (85%) of labeled ependymoglia in vehicle-treated injured telencephalons remained quiescent. These ependymoglia cells did not change their identity within a 5-day imaging period (Figures 3D and S3A), whereas 14.2% of labeled cells directly converted (Figures 3B–3D). In contrast, after AhR signaling enhancement, 45% of labeled ependymoglial cells directly converted into post-mitotic neurons (Figure 3D). This increase in direct conversion was associated with the depletion of ependymoglia present in the dorsal neurogenic niche, as we observed a significant increase in the dorsal surface free of gfap:GFP-positive ependymoglia in BNF-treated brains compared to the vehicle treatment (Figures S3H and S3I; Videos S1 and S2). We did not observe any difference in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells at the dorsal telencephalon area between the BNF and vehicle treatment (Figures S3C and S3D). Therefore, we concluded that direct conversion causes ependymoglial depletion rather than cell death. Taken together, our data suggest that AhR signaling enhancement after injury leads to increased neurogenesis via the direct conversion and depletion of ependymoglia. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzF" @default.
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• W2904212612 date "2018-12-01" @default.
• W2904212612 modified "2023-10-18" @default.
• W2904212612 title "The Aryl Hydrocarbon Receptor Pathway Defines the Time Frame for Restorative Neurogenesis" @default.
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