The Journal of Neuroscience, October 22, 2014 • 34(43):14403–14419 • 14403 Development/Plasticity/Repair Repressing Notch Signaling and Expressing TNF␣ Are Sufficient to Mimic Retinal Regeneration by Inducing Mu¨ller Glial Proliferation to Generate Committed Progenitor Cells Clay Conner, Kristin M. Ackerman, Manuela Lahne, Joshua S. Hobgood, and David R. Hyde Department of Biological Sciences and the Center for Zebrafish Research, Galvin Life Sciences Building, University of Notre Dame, Notre Dame, Indiana 46556 Retinal damage in teleosts, unlike mammals, induces robust Mu¨ller glia-mediated regeneration of lost neurons. We examined whether Notch signaling regulates Mu¨ller glia proliferation in the adult zebrafish retina and demonstrated that Notch signaling maintains Mu¨ller glia in a quiescent state in the undamaged retina. Repressing Notch signaling, through injection of the ␥-secretase inhibitor RO4929097, stimulates a subset of Mu¨ller glia to reenter the cell cycle without retinal damage. This RO4929097-induced Mu¨ller glia proliferation is mediated by repressing Notch signaling because inducible expression of the Notch Intracellular Domain (NICD) can reverse the effect. ThisRO4929097-inducedproliferationrequiresAscl1aexpressionandJak1-mediatedStat3phosphorylation/activation,analogoustothe light-damagedretina.Moreover,coinjectingRO4929097andTNF␣,apreviouslyidentifieddamagesignal,inducedthemajorityofMu¨ller glia to reenter the cell cycle and produced proliferating neuronal progenitor cells that committed to a neuronal lineage in the undamaged retina. ThisdemonstratesthatrepressingNotchsignalingandactivatingTNF␣signalingaresufficienttoinduceMu¨llergliaproliferationthatgenerates neuronal progenitor cells that differentiate into retinal neurons, mimicking the responses observed in the regenerating retina. Key words: Ascl1; Mu¨ller glia; Notch signaling; quiescence; retinal regeneration; Stat3 Introduction mechanisms that will induce a regenerative response in mammals The damaged zebrafish retina undergoes a robust regeneration to restore vision. response that involves Mu¨ller glia dedifferentiation, cell cycle re- Notch signaling is an evolutionarily conserved pathway that entry, and production of neuronal progenitor cells that migrate regulates various steps during vertebrate development and is in- to the correct retinal layer to regenerate lost neurons (Vihtelic volved in regenerating the zebrafish nervous system (Bernardos and Hyde, 2000; Fimbel et al., 2007; Yurco and Cameron, 2007; et al., 2005; Raymond et al., 2006; Chapouton et al., 2010; Dias et Montgomery et al., 2010; Ramachandran et al., 2010, 2011; Nel- al., 2012; Wan et al., 2012). Canonical Notch signaling occurs son et al., 2012; Wan et al., 2012). In the damaged mammalian when a ligand binds a Notch receptor, leading to its cleavage to retina, however, Mu¨ller glia undergo reactive gliosis without re- release the Notch intracellular domain (NICD), which relocates generating retinal neurons (Ridet et al., 1997; Di Giovanni et al., to the nucleus and activates transcription of downstream Notch 2005; Bringmann et al., 2006; Va´zquez-Chona et al., 2011; de target genes (Groot and Vooijs, 2012). The Notch receptor, its Melo et al., 2012; Liu et al., 2013). Elucidating the signaling path- ligands, and downstream targets are differentially regulated in the ways that induce zebrafish retinal regeneration may reveal why damaged zebrafish retina (Raymond et al., 2006; Wan et al., the damaged mammalian retina fails to regenerate and provide 2012). Functionally, Notch signaling limits the number of prolif- erating Mu¨ller glia in the puncture-damaged zebrafish retina through a feedback loop that includes Ascl1a (Wan et al., 2012), Received Feb. 4, 2014; revised Aug. 22, 2014; accepted Sept. 14, 2014. an important regulator of Mu¨ller glia dedifferentiation and pro- Author contributions: C.C., K.M.A., M.L., J.S.H., and D.R.H. designed research; C.C., K.M.A., M.L., and J.S.H. per- liferation (Yurco and Cameron, 2007; Fausett et al., 2008; Ram- formed research; C.C., K.M.A., M.L., J.S.H., and D.R.H. analyzed data; C.C., K.M.A., M.L., J.S.H., and D.R.H. wrote the paper. achandran et al., 2010; Nelson et al., 2012). However, inhibiting This work was supported by National Eye Institute of National Institutes of Health Grant R01-EY018417 to D.R.H. Notch signaling by DAPT, a ␥-secretase inhibitor, did not induce and the Center for Zebrafish Research, University of Notre Dame. We thank Freimann Life Sciences technicians for Mu¨ller glia proliferation in undamaged retinas (Wan et al., 2012). their care and husbandry of the zebrafish; the Zebrafish International Resource Center (supported by National In contrast, two studies showed that high levels of Notch signal- Institutes of Health Grant P40-RR12546) for providing the her6 cDNA clones; Rebecca Wingert (University of Notre Dame) for the gift of the Tg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra) transgenic line; and members of the D.R.H. ing maintain both endocrine progenitor cells and brain neural laboratory for thoughtful discussions. stem cells in a quiescent state, whereas reducing Notch signaling The authors declare no competing financial interests. drives both cell populations to reenter the cell cycle (Chapouton Correspondence should be addressed to Dr. David R. Hyde, Department of Biological Sciences, 027 Galvin Life et al., 2010; Ninov et al., 2012). Sciences Building, University of Notre Dame, Notre Dame, IN 46556. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.0498-14.2014 Here, we investigated the role of Notch signaling in maintain- Copyright © 2014 the authors 0270-6474/14/3414403-17$15.00/0 ing Mu¨ller glia quiescence in the undamaged retina and its pos- 14404 • J. Neurosci., October 22, 2014 • 34(43):14403–14419 Conner et al. • Notch Signaling Regulates Mu¨ller Glial Quiescence sible interaction with TNF␣, which is produced by dying dark incubator at ϳ33°C, which corresponds to the temperature that the photoreceptors and is necessary to induce Mu¨ller glia prolifera- constant light-treated fish are maintained and allows for the initiation of tion (Nelson et al., 2013). Repressing Notch signaling in undam- Mu¨ller glia and neuronal progenitor cell proliferation in a relatively short aged retinas stimulates a subset of Mu¨ller glia to express the time frame. dedifferentiation markers, Ascl1a and Stat3, reenter the cell cycle, Heatshock-induced Notch activation. Adult double-transgenic Tg(hsp70l: Gal4);Tg(UAS:myc-notch1a-intra) fish (generous gift from R. Wingert, and produce neuronal progenitor cells. Interestingly, in the un- ␣ University of Notre Dame) were confirmed to have both transgenes by damaged Notch-repressed retina, applying TNF induces the PCR amplification of transgene-specific fragments in genomic DNA. A majority of Mu¨ller glia to reenter the cell cycle to produce pro- UAS:notch1a-specific 450 bp fragment was amplified between the genitors that commit to the neuronal lineage. These data indicate primers 5Ј-CATCGCGTCTCAGCCTCAC-3Ј and 5Ј-CGGAATCGT that the combination of TNF␣ and repression of Notch signaling TTATTGGTGTCG-3Ј, and a hsp70l:Gal4-specific 950 bp fragment was is sufficient to induce Mu¨ller glia and neuronal progenitor cell amplified with 5Ј-CGGGCATTTACTTTATGTTGC-3Ј and 5Ј-CATCA proliferation and subsequent differentiation of these progenitors TTAGCGTCGGTGAG-3Ј as published previously (Scheer and Campos- into different neuronal subtypes in the undamaged zebrafish Ortega, 1999; Scheer et al., 2001, 2002). For heat shock-induced Notch retina. activation, Tg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra) fish and albino adult zebrafish were placed in a warm water bath at 28°C, the water temperature was elevated 1°C every 3–4 min up to 38.2°C, and main- Materials and Methods tained for 1 h. The fish were then slowly cooled and maintained at 31°C Fish lines and maintenance. Male and female zebrafish (Danio rerio) used for 4 h before treatment with either RO4929097 or DMSO. Tg(hsp70l: in this study were 6–12 months old and maintained at the Freimann Life Gal4);Tg(UAS:myc-notch1a-intra) fish and albino fish were heat shocked Science Center at the University of Notre Dame as previously described three times (every 12 h) before the first injection and every 12 h during (Vihtelic and Hyde, 2000). All animal care and protocols were approved the previously described 3 d injection paradigm. by the University of Notre Dame Animal Care and Use Committee and TNF␣ purification and injection. The pQE30 plasmid containing re- are in compliance with the ARVO statement for the use of animals in combinant zebrafish TNF␣ cDNA was a generous gift from the Drapeau vision research. lab (Knogler et al., 2010). The plasmid was transfected into M15 cells Light treatment protocol and ␥-secretase inhibitor injections. Adult al- (QIAGEN), and recombinant TNF␣ protein was purified using the bino Tg(gfap:EGFP)nt11 transgenic zebrafish were dark-adapted for 14 d QIAExpressionist kit (QIAGEN). The purified TNF␣ was diluted to a and then immediately placed in constant light (18,000 lux) for 24 h, working concentration of 0.5 mg/ml with sterile 1 ϫ PBS and protease during which time maximal photoreceptor cell death occurs (Nelson et inhibitor mixture (tablets, Roche). The control lysate (CL) was an Esch- al., 2013). After light treatment, the fish were anesthetized in 1:1000 erichia coli protein lysate that was obtained from a bacterial culture that dilution of 2-phenoxyethanol (Sigma). A small incision was made in the lacked the pQE30-tnf␣ recombinant plasmid but was induced and puri- cornea adjacent to the iris of each eye with a sapphire blade scalpel fied in the same manner as TNF␣. We injected either 10% DMSO or 1 (World Precision Instruments) and ϳ1 ␮l of 1% DMSO (as a vehicle mM RO4929097 intraperitoneally and injected 0.5–1.0 ␮l of either the control), 250 ␮M Compound E (Santa Cruz Biotechnology), or 100 ␮M CL or the recombinant TNF␣ intravitreally as described above every RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] 12hfor4d. azepin-7-yl)-NЈ-(2,2,3,3,3-pentafluoro-propyl)-malonamide; Selleck- 5-Ethynyl-2Ј-deoxyuridine (EdU) and BrdU labeling and detection.