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• W2950384292 abstract "•Redox signaling and lipid peroxidation are active during retinogenesis•Regulation of H2O2 levels is critical to promote RPCs differentiation•High levels of 9-HSA affect RPC differentiation by activating Notch and Wnt pathways•9-HSA proliferative effects on RPCs occur via the inhibition of the HDAC1 in vivo Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However, their regulation and mechanism of action remain poorly studied in vivo during normal development. Here, we reveal that the fine regulation of hydrogen peroxide (H2O2) levels by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H2O2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation. Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However, their regulation and mechanism of action remain poorly studied in vivo during normal development. Here, we reveal that the fine regulation of hydrogen peroxide (H2O2) levels by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H2O2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation. Adult stem cells and progenitor cells require metabolic plasticity in order to adapt to a quiescent or highly proliferative state, respectively. Stem cells preference for aerobic glycolysis rather than oxidative phosphorylation has been proposed to be due to the hypoxic environment in which they replicate and the relatively low energy requirement of their quiescent state (Bigarella et al., 2014Bigarella C.L. Liang R. Ghaffari S. Stem cells and the impact of ROS signaling.Development. 2014; 141: 4206-4218Crossref PubMed Scopus (142) Google Scholar, Norddahl et al., 2011Norddahl G.L. Pronk C.J. Wahlestedt M. Sten G. Nygren J.M. Ugale A. Sigvardsson M. Bryder D. Accumulating mitochondrial DNA mutations drive premature hematopoietic aging phenotypes distinct from physiological stem cell aging.Cell Stem Cell. 2011; 8: 499-510Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Yeo et al., 2013Yeo H. Lyssiotis C.A. Zhang Y. Ying H. Asara J.M. Cantley L.C. Paik J.H. FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells.EMBO J. 2013; 32: 2589-2602Crossref PubMed Scopus (46) Google Scholar). Already a decade ago, Prozorovski et al. have highlighted the role of redox signaling in the regulation of neural progenitor fate (Prozorovski et al., 2008Prozorovski T. Schulze-Topphoff U. Glumm R. Baumgart J. Schröter F. Ninnemann O. Siegert E. Bendix I. Brüstle O. Nitsch R. et al.Sirt1 contributes critically to the redox-dependent fate of neural progenitors.Nat. Cell Biol. 2008; 10: 385-394Crossref PubMed Scopus (309) Google Scholar). The redox pathway has been proposed to regulate neural stem cell self-renewal and neurogenesis via PI3K/Akt signaling, further highlighting the physiological need for reactive oxygen species (ROS) as cellular signaling molecules (Le Belle et al., 2011Le Belle J.E. Orozco N.M. Paucar A.A. Saxe J.P. Mottahedeh J. Pyle A.D. Wu H. Kornblum H.I. Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner.Cell Stem Cell. 2011; 8: 59-71Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Tumor cells can also highjack the redox pathway by modulating their endogenous levels of ROS and downstream products to allow tumor progression (Gorrini et al., 2013Gorrini C. Harris I.S. Mak T.W. Modulation of oxidative stress as an anticancer strategy.Nat. Rev. Drug Discov. 2013; 12: 931-947Crossref PubMed Scopus (1079) Google Scholar, Piskounova et al., 2015Piskounova E. Agathocleous M. Murphy M.M. Hu Z. Huddlestun S.E. Zhao Z. Leitch A.M. Johnson T.M. DeBerardinis R.J. Morrison S.J. Oxidative stress inhibits distant metastasis by human melanoma cells.Nature. 2015; 527: 186-191Crossref PubMed Scopus (300) Google Scholar, Zhou et al., 2014Zhou D. Shao L. Spitz D.R. Reactive oxygen species in normal and tumor stem cells.Adv. Cancer Res. 2014; 122: 1-67Crossref PubMed Scopus (93) Google Scholar). Overall these reports highlight the importance of ROS level regulation for diverse physiological functions. However, to date, the “metabolic master switch that determines the fate of neural progenitors” remain uncharacterized (Prozorovski et al., 2008Prozorovski T. Schulze-Topphoff U. Glumm R. Baumgart J. Schröter F. Ninnemann O. Siegert E. Bendix I. Brüstle O. Nitsch R. et al.Sirt1 contributes critically to the redox-dependent fate of neural progenitors.Nat. Cell Biol. 2008; 10: 385-394Crossref PubMed Scopus (309) Google Scholar). Hydrogen peroxide (H2O2) has recently emerged as the major redox metabolite that acts as an essential messenger for several fundamental processes such as inflammation, hypoxia response, regeneration and wound-healing, cell proliferation, stem cell self-renewal, tumorigenesis, aging, and diabetes (Holmström and Finkel, 2014Holmström K.M. Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling.Nat. Rev. Mol. Cell Biol. 2014; 15: 411-421Crossref PubMed Scopus (666) Google Scholar, Sies et al., 2017Sies H. Berndt C. Jones D.P. Oxidative stress.Annu. Rev. Biochem. 2017; 86: 715-748Crossref PubMed Scopus (141) Google Scholar, Sies, 2017Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.Redox Biol. 2017; 11: 613-619Crossref PubMed Scopus (225) Google Scholar). In fish and amphibians, cell proliferation following injury correlates with the increase in the amounts of H2O2 at the site of amputation (Niethammer et al., 2009Niethammer P. Grabher C. Look A.T. Mitchison T.J. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.Nature. 2009; 459: 996-999Crossref PubMed Scopus (796) Google Scholar, Love et al., 2013Love N.R. Chen Y. Ishibashi S. Kritsiligkou P. Lea R. Koh Y. Gallop J.L. Dorey K. Amaya E. Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration.Nat. Cell Biol. 2013; 15: 222-228Crossref PubMed Scopus (203) Google Scholar, Hameed et al., 2015Hameed L.S. Berg D.A. Belnoue L. Jensen L.D. Cao Y. Simon A. Environmental changes in oxygen tension reveal ROS-dependent neurogenesis and regeneration in the adult newt brain.eLife. 2015; 4: 1725Crossref Scopus (22) Google Scholar). During central nervous system (CNS) development, we and others have reported a physiological function for H2O2 in controlling axon growth and regeneration (Gauron et al., 2016Gauron C. Meda F. Dupont E. Albadri S. Quenech'Du N. Ipendey E. Volovitch M. Del Bene F. Joliot A. Rampon C. et al.Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.Dev. Biol. 2016; 414: 133-141Crossref PubMed Scopus (23) Google Scholar, Meda et al., 2016Meda F. Gauron C. Rampon C. Teillon J. Volovitch M. Vriz S. Nerves control redox levels in mature tissues through Schwann cells and hedgehog signaling.Antioxid. Redox Signal. 2016; 24: 299-311Crossref PubMed Scopus (19) Google Scholar, Hervera et al., 2018Hervera A. De Virgiliis F. Palmisano I. Zhou L. Tantardini E. Kong G. Hutson T. Danzi M.C. Perry R.B.-t. Santos C.X.C. et al.Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.Nat. Cell Biol. 2018; 20: 307-319Crossref PubMed Scopus (12) Google Scholar, Rieger and Sagasti, 2011Rieger S. Sagasti A. Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin.PLoS Biol. 2011; 9: e1000621Crossref PubMed Scopus (0) Google Scholar, Wilson et al., 2018Wilson C. Muñoz-Palma E. González-Billault C. From birth to death: a role for reactive oxygen species in neuronal development.Semin. Cell Dev. Biol. 2018; 80: 43-49Crossref PubMed Scopus (10) Google Scholar). In our previous study, we revealed a dynamic landscape of H2O2 levels during morphogenesis, regulated through degradation to ensure appropriate levels in vivo (Gauron et al., 2016Gauron C. Meda F. Dupont E. Albadri S. Quenech'Du N. Ipendey E. Volovitch M. Del Bene F. Joliot A. Rampon C. et al.Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.Dev. Biol. 2016; 414: 133-141Crossref PubMed Scopus (23) Google Scholar). In this study, we sought to determine the role of the redox pathway and lipid peroxidation products as its possible mediators during the development of the zebrafish retina, taking advantage of its well-defined architecture and cell organization. At 2 days postfertilization (dpf) during zebrafish retinogenesis, retinal progenitor cells (RPCs) start differentiating in a central to peripheral manner. By 3 dpf, the central retina is fully differentiated and a peripheral stem cell niche, known as the ciliary marginal zone (CMZ), forms to ensure tissue homeostasis throughout the lifelong growth of the fish. The CMZ is comprised of three main cell domains demarcated by the expression of different transcription factors: the rx2 domain, which contains retinal stem cells (RSCs) and RPCs, the ccnd1 (zebrafish equivalent to cyclinD1) domain where cycling progenitors are found, and an atoh7 domain (also called ath5), which contains cycling and committed RPCs (Agathocleous and Harris, 2009Agathocleous M. Harris W.A. From progenitors to differentiated cells in the vertebrate retina.Annu. Rev. Cell Dev. Biol. 2009; 25: 45-69Crossref PubMed Scopus (143) Google Scholar, Ohnuma and Harris, 2003Ohnuma S.-I. Harris W.A. Neurogenesis and the cell cycle.Neuron. 2003; 40: 199-208Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Reinhardt et al., 2015Reinhardt R. Centanin L. Tavhelidse T. Inoue D. Wittbrodt B. Concordet J.P. Martinez-Morales J.R. Wittbrodt J. Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.EMBO J. 2015; 34: 1572-1588Crossref PubMed Scopus (17) Google Scholar, Wan et al., 2016Wan Y. Almeida A.D. Rulands S. Chalour N. Muresan L. Wu Y. Simons B.D. He J. Harris W.A. The ciliary marginal zone of the zebrafish retina: clonal and time-lapse analysis of a continuously growing tissue.Development. 2016; 143: 1099-1107Crossref PubMed Google Scholar). This precise spatial and temporal organization makes the zebrafish retina an excellent model to assess the effects of ROS and downstream products on stem cell and progenitor cell population in vivo. A downstream product of ROS is the peroxidized lipid 9-hydroxystearic acid (9-HSA), which we found endogenously produced in human embryonic intestine cells and colon adenocarcinoma as a signaling messenger produced by lipid peroxidation. The amounts of 9-HSA in cancer cells inversely correlating with tumor growth, making it a candidate endogenous mediator of redox signaling in proliferative tissues (Bertucci et al., 2002Bertucci C. Hudaib M. Boga C. Calonghi N. Cappadone C. Masotti L. Gas chromatography/mass spectrometric assay of endogenous cellular lipid peroxidation products: quantitative analysis of 9- and 10-hydroxystearic acids.Rapid Commun. Mass Spectrom. 2002; 16: 859-864Crossref PubMed Scopus (10) Google Scholar, Calonghi et al., 2007Calonghi N. Pagnotta E. Parolin C. Molinari C. Boga C. Dal Piaz F. Brusa G.L. Santucci M.A. Masotti L. Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells.Biochim. Biophys. Acta. 2007; 1771: 139-146Crossref PubMed Scopus (7) Google Scholar, Cavalli et al., 1991Cavalli G. Casali E. Spisni A. Masotti L. Identification of the peroxidation product hydroxystearic acid in Lewis lung carcinoma cells.Biochem. Biophys. Res. Commun. 1991; 178: 1260-1265Crossref PubMed Scopus (7) Google Scholar, Gesmundo et al., 1994Gesmundo N. Casali E. Farruggia G. Spisni A. Masotti L. In vitro effects of hydroxystearic acid on the proliferation of HT29 and I407 cells.Biochem. Mol. Biol. Int. 1994; 33: 705-712PubMed Google Scholar, Piretti et al., 1987Piretti M.V. Pagliuca G. Vasina M. Proposal of an analytical method for the study of the oxidation products of membrane lipids.Anal. Biochem. 1987; 167: 358-361Crossref PubMed Scopus (16) Google Scholar, Piretti and Pagliuca, 1989Piretti M.V. Pagliuca G. Systematic isolation and identification of membrane lipid oxidation products.Free Radic. Biol. Med. 1989; 7: 219-221Crossref PubMed Scopus (10) Google Scholar). Here, we reveal the in vivo physiological role of the redox pathway and lipid peroxidation during retinal development and homeostatic maintenance of the mature retina. Our results bring to light the cascade of events that leads to the upregulation of proneuronal genes in RPCs, which are required for transition from proliferation to differentiation. ROS homeostasis, resulting from their production and degradation within a tissue, is tightly regulated as shown for H2O2 regulation during morphogenesis (Gauron et al., 2016Gauron C. Meda F. Dupont E. Albadri S. Quenech'Du N. Ipendey E. Volovitch M. Del Bene F. Joliot A. Rampon C. et al.Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.Dev. Biol. 2016; 414: 133-141Crossref PubMed Scopus (23) Google Scholar). To uncover the dynamic of H2O2 production during the development of the retina, we first used embryos transgenically expressing a ratiometric sensor (HyPer) that we previously established as a reporter for endogenous levels of H2O2 and the atoh7:gap-RFP marker labeling the first differentiated cells in the neuroepithelium (Tg(ubi:HyPer); Tg(atoh7:gap-RFP)) (Gauron et al., 2016Gauron C. Meda F. Dupont E. Albadri S. Quenech'Du N. Ipendey E. Volovitch M. Del Bene F. Joliot A. Rampon C. et al.Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.Dev. Biol. 2016; 414: 133-141Crossref PubMed Scopus (23) Google Scholar). We imaged zebrafish retinae at two specific developmental stages: at 24 h postfertilization (hpf) when the undifferentiated neuroepithelium is highly proliferative and at 32 hpf when differentiation has started in the central part of the tissue (Figures 1A and 1A’ ). We observed a strong correlation between cell proliferation and high levels of H2O2. At 24 hpf, high amounts of H2O2 could be observed throughout the proliferating epithelium. In contrast, at 32 hpf, the central part of the tissue was depleted of H2O2 where cells are differentiating demarcated by atoh7:gap-RFP expression (Figures 1A, 1A’, S1A, and S1A’) (Zolessi et al., 2006Zolessi F.R. Poggi L. Wilkinson C.J. Chien C.B. Harris W.A. Polarization and orientation of retinal ganglion cells in vivo.Neural Dev. 2006; 1: 2Crossref PubMed Google Scholar). Indeed, quantifying H2O2 levels across the tissue showed that H2O2 levels remain high at the periphery of the developing retina where retinal progenitors are still proliferating, while they are drastically decreased in the central part of the tissue where differentiation has been initiated (Figure 1B). These results reveal the dynamics of H2O2 production in relation to cell differentiation and proliferation in the retina and suggest a putative role of this pathway for the development of the tissue. To get an insight into the source of H2O2, we investigated the expression of the superoxide dismutase 2 (Sod2) in the retina at 3 days dpf. In the mitochondria, Sod2 acts as an antioxidant enzyme by converting superoxide anions, a by-product of the mitochondrial electron transport chain, into H2O2 and O2 (Candas and Li, 2014Candas D. Li J.J. MnSOD in oxidative stress response-potential regulation via mitochondrial protein influx.Antioxid. Redox Signal. 2014; 20: 1599-1617Crossref PubMed Scopus (0) Google Scholar). By double fluorescent in situ hybridization, we compared the expression of sod2 to the retinal homeobox gene rx2, which labels stem and early progenitor cells in the CMZ of the 3 dpf zebrafish retina (Figures 1C–1C”’) (Wan et al., 2016Wan Y. Almeida A.D. Rulands S. Chalour N. Muresan L. Wu Y. Simons B.D. He J. Harris W.A. The ciliary marginal zone of the zebrafish retina: clonal and time-lapse analysis of a continuously growing tissue.Development. 2016; 143: 1099-1107Crossref PubMed Google Scholar). We found that sod2 and rx2 co-label the CMZ, pointing toward this enzyme as a source of H2O2 in stem and progenitor cells. We next aimed to assess how H2O2 scavenging occurs in the zebrafish retina over time. We first evaluated the expression dynamics of catalase, which encodes an antioxidant enzyme catalase, which transforms H2O2 into water and oxygen. Catalase is at the end of the antioxidant pathway that regulates cellular oxidative state oscillations, providing protection against ROS-induced damages (Lynch and Fridovich, 1978Lynch R.E. Fridovich I. Permeation of the erythrocyte stroma by superoxide radical.J. Biol. Chem. 1978; 253: 4697-4699Abstract Full Text PDF PubMed Google Scholar, Rhee et al., 2005Rhee S.G. Chae H.Z. Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling.Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (981) Google Scholar, Sies, 1991Sies H. Role of reactive oxygen species in biological processes.Klin. Wochenschr. 1991; 69: 965-968Crossref PubMed Scopus (0) Google Scholar, Spitz et al., 2004Spitz D.R. Azzam E.I. Li J.J. Gius D. Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology.Cancer Metastasis Rev. 2004; 23: 311-322Crossref PubMed Scopus (369) Google Scholar). We previously reported that catalase expression occurs mainly in the brain where fine-tuning of H2O2 levels is achieved by degradation (Gauron et al., 2016Gauron C. Meda F. Dupont E. Albadri S. Quenech'Du N. Ipendey E. Volovitch M. Del Bene F. Joliot A. Rampon C. et al.Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.Dev. Biol. 2016; 414: 133-141Crossref PubMed Scopus (23) Google Scholar). To test for catalase expression in the retina, we performed double fluorescent in situ hybridization for catalase and the basic helix-loop-helix (bHLH) transcription factor atoh7, a marker for retinal neurogenesis initiation and later for cycling RPCs in the CMZ (Figures 1D–1D’’ and S1B–S1B’’) (Kay et al., 2005Kay J.N. Link B.A. Baier H. Staggered cell-intrinsic timing of ath5 expression underlies the wave of ganglion cell neurogenesis in the zebrafish retina.Development. 2005; 132: 2573-2585Crossref PubMed Scopus (0) Google Scholar). Atoh7 and catalase transcripts co-localized at 2 dpf in the central retina where they are transiently expressed (Figures S1B–S1B’’) and at 3 dpf in the CMZ (Figures 1D–1D′’). To assess if atoh7 directs catalase expression, we next monitored catalase expression in the atoh7−/− mutant, also known as lakritz (Figures S1C and S1D) (Kay et al., 2001Kay J.N. Finger-Baier K.C. Roeser T. Staub W. Baier H. Retinal ganglion cell genesis requires Lakritz, a zebrafish atonal homolog.Neuron. 2001; 30: 725-736Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Interestingly, the absence of Atoh7 in the retina did not affect catalase expression, which is still found in the CMZ (Figures S1C and S1D). Therefore, catalase is expressed in the neurogenic domain labeled by atoh7 but is independent of Atoh7 function. Together, these results indicate dynamic expression of catalase in the retina first in cycling RPCs prior to their last division during development and later during retinal growth. ROS including H2O2 can react with polyunsaturated fatty acids of the lipid membrane and induce lipid peroxidation. We thus analyzed the levels of in vivo lipid peroxidation in the retina at 3 dpf using the lipid peroxidation sensor BODIPY-C11, which converts irreversibly from red to green when oxidized (Drummen et al., 2002Drummen G.P.C. van Liebergen L.C.M. Op den Kamp J.A.F. Post J.A. C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology.Free Radic. Biol. Med. 2002; 33: 473-490Crossref PubMed Scopus (0) Google Scholar, Bapat et al., 2002Bapat S. Post J.A. Braam B. Goldschmeding R. Koomans H.A. Verkleij A.J. Joles J.A. Visualizing tubular lipid peroxidation in intact renal tissue in hypertensive rats.J. Am. Soc. Nephrol. 2002; 13: 2990-2996Crossref PubMed Scopus (0) Google Scholar, Sasaki et al., 2009Sasaki M. Ozawa Y. Kurihara T. Noda K. Imamura Y. Kobayashi S. Ishida S. Tsubota K. Neuroprotective effect of an antioxidant, lutein, during retinal inflammation.Invest. Ophthalmol. Vis. Sci. 2009; 50: 1433-1439Crossref PubMed Scopus (0) Google Scholar). We found oxidized BODIPY-C11 at the outermost part of the CMZ, where stem and early progenitor cells are located (Figures 1E–1E”). To further reveal the dynamics of lipid peroxidation in the retina, we analyzed the localization of 4-hydroxynonenal (4-HNE), a known end product of lipid peroxidation that acts as a secondary messenger and marker of oxidative stress (Barrera et al., 2008Barrera G. Pizzimenti S. Dianzani M.U. Lipid peroxidation: control of cell proliferation, cell differentiation and cell death.Mol. Aspects Med. 2008; 29: 1-8Crossref PubMed Scopus (0) Google Scholar, Barrera, 2012Barrera G. Oxidative stress and lipid peroxidation products in cancer progression and therapy.ISRN Oncol. 2012; 2012: 137289PubMed Google Scholar, Pizzimenti et al., 2010Pizzimenti S. Toaldo C. Pettazzoni P. Dianzani M.U. Barrera G. The ‘two-faced’ effects of reactive oxygen species and the lipid peroxidation product 4-hydroxynonenal in the hallmarks of cancer.Cancers. 2010; 2: 338-363Crossref PubMed Scopus (0) Google Scholar). We assessed the levels of 4-HNE in RPCs and differentiated neurons at 3 dpf by immunohistochemistry (Schneider et al., 2001Schneider C. Tallman K.A. Porter N.A. Brash A.R. Two distinct pathways of formation of 4-hydroxynonenal. Mechanisms of nonenzymatic transformation of the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals.J. Biol. Chem. 2001; 276: 20831-20838Crossref PubMed Scopus (248) Google Scholar). In line with our previous result, we detected 4-HNE enrichment in the CMZ, while little or no signal was detectable in the differentiated part of the 3 dpf retina (Figure 1F). Taken together, our results indicate that H2O2 production, scavenging, and lipid peroxidation are active processes occurring within cycling RPCs during retinogenesis and retinal growth at specific differentiation steps (Figure 1G). In search of a possible molecular mechanism that could link H2O2 homeostasis and lipid peroxidation to cell proliferation and differentiation in the retina, we focused on 9-HSA as a putative endogenous mediator of the redox pathway in the retina. 9-HSA has previously been identified as an endogenous by-product of lipid peroxidation in colon carcinoma cells where it acts as a growth inhibitor (Cavalli et al., 1991Cavalli G. Casali E. Spisni A. Masotti L. Identification of the peroxidation product hydroxystearic acid in Lewis lung carcinoma cells.Biochem. Biophys. Res. Commun. 1991; 178: 1260-1265Crossref PubMed Scopus (7) Google Scholar, Calonghi et al., 2007Calonghi N. Pagnotta E. Parolin C. Molinari C. Boga C. Dal Piaz F. Brusa G.L. Santucci M.A. Masotti L. Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells.Biochim. Biophys. Acta. 2007; 1771: 139-146Crossref PubMed Scopus (7) Google Scholar, Gesmundo et al., 1994Gesmundo N. Casali E. Farruggia G. Spisni A. Masotti L. In vitro effects of hydroxystearic acid on the proliferation of HT29 and I407 cells.Biochem. Mol. Biol. Int. 1994; 33: 705-712PubMed Google Scholar). Given its importance in human cancer cell models, we assessed whether 9-HSA is endogenously produced in vivo in zebrafish under physiological conditions. We thus prepared lipid extracts from zebrafish embryos at different developmental time points, and then used a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to test for the presence of 9-HSA. We optimized the reverse-phase chromatographic separation efficiency by simultaneously analyzing 9-HSA and its isomer 10-HSA for clear separation of the two molecules (Figure S2A). Plotting the fragment ion 253.2 peak area versus the corresponding concentration (ng/mL) produced a calibration curve with linear behavior of the response over two orders of concentration magnitude (y = 283,78x – 42295, r2 > 0.995). We then used these optimized conditions to test for 9-HSA in zebrafish embryos across developmental stages and detected endogenously produced 9-HSA from prior to 1 through 4 dpf (Figure S2B). Taken together, the biological role of 9-HSA in cell culture and its endogenous production by the lipid peroxidation machinery in zebrafish suggest an in vivo role for this molecule in zebrafish development. We next asked whether 9-HSA has a role in RPC differentiation in the retina. To do so, we injected synthetic 9-HSA (or DMSO vehicle control) into 1-cell stage zebrafish embryos, then analyzed retina development at 2 and 3 dpf (Figure 2). At 2 dpf, retinal ganglions cells (RGCs) have differentiated, and at 3 dpf the fish retina is fully differentiated and contains all neuronal and glial cell types (Hu and Easter, 1999Hu M. Easter S.S. Retinal neurogenesis: the formation of the initial central patch of postmitotic cells.Dev. Biol. 1999; 207: 309-321Crossref PubMed Scopus (270) Google Scholar). We assessed the development of different retinal cell classes by immunofluorescent detection of Zn5 to mark all RGCs at 2 dpf, Parvalbumin to mark amacrine cells, GS to mark Müller glia cells, and Zpr1 to mark photoreceptors at 3 dpf (Figures 2A–2D). Relative to DMSO control retinae, 9-HSA-treated embryos displayed a strong reduction in differentiation markers for all tested retinal cell types (Figures 2A’–2D’). In some cases, like for the Zpr1 photoreceptor marker, the most central part of the retina appeared to be more affected than the peripheral regions (Figure 2D’). This can be explained by the retinal differentiation process, which temporally propagates from the central retina to the periphery (Stenkamp, 2007Stenkamp D.L. Neurogenesis in the fish retina.Int. Rev. Cytol. 2007; 259: 173-224Crossref PubMed Scopus (79) Google Scholar). Therefore, cells in the distal part undergo neuronal differentiation at later developmental stages, when exogenous 9-HSA amounts may have started to decrease. These observed defects in differentiation in 9-HSA-treated retinae were not associated with any increase in cell death at 1, 2, or 3 dpf as assessed by TUNEL assays (Figures S2C–S2H). Together our results demonstrate that RPCs overexposed to 9-HSA during early development fail to appropriately differentiate without undergoing apoptosis. 9-HSA supplementation to the human colon cancer cell line HT29 strongly inhibits cell proliferation and shifts cellular differentiation toward a benign phenotype (Calonghi et al., 2005Calonghi N. Cappadone C. Pagnotta E. Boga C. Bertucci C. Fiori J. Tasco G. Casadio R. Masotti L. Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer.J. Lipid Res. 2005; 46: 1596-1603Crossref PubMed Scopus (25) Google Scholar, Parolin et al., 2012Parolin C. Calonghi N. Presta E. Boga C. Caruana P. Naldi M. Andrisano V. Masotti L. Sartor G. Mechanism and stereoselectivity of HDAC I inhibition by (R)-9-hydroxystearic acid in colon cancer.Biochim. Biophys. Acta. 2012; 1821: 1334-1340Crossref PubMed Scopus (0) Google Scholar). To evaluate the effect of 9-HSA on the proliferation of RPCs during retinogenesis and later on the stem cell niche of the retina, we assessed the proliferative state of retinae from embryos injected with 9-HSA. We first evaluated the mitotic index of retinae from 9-HSA-injected embryos at 24 hpf by immunohistochemistry of the M phase marker phosphorylated histone H3 (pH3) and observed no difference in the number of pH3-positive cells in the retinae of 9-HSA- and DMSO-injected controls (Figures S2I–S2K). This indicates that 9-HSA does not affect the proliferation of RPCs at this early stage (Figure S2K). Later at 2 dpf, the retinal neuroepithelium is mainly differentiated in the central part, and by 3 dpf RPCs cells are restricted to the CMZ domain while the central part of the retina is entirely occupied by post-mitotic neurons and glia. Thus, we next tested for an effect of 9-HSA on S phase entry using a 12 h BrDU incorporation assay on 2 dpf 9-HSA- and DMSO-injected embryos (Figures S2L and S2M). While BrDU could mainly only be detected at the periphery of the retina in the nascent 2.5 dpf CM" @default.
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• W2950384292 date "2019-07-01" @default.
• W2950384292 modified "2023-10-15" @default.
• W2950384292 title "Redox Signaling via Lipid Peroxidation Regulates Retinal Progenitor Cell Differentiation" @default.
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