RESEARCH ARTICLE Age-Dependent M€ uller Glia Neurogenic Competence in the Mouse Retina Kati L € offler, 1 Patrick Sch€ afer, 1,2 Manuela V€ olkner, 2 Tina Holdt, 2 and Mike O. Karl 1,2 The mechanisms limiting neuronal regeneration in mammals and their relationship with reactive gliosis are unknown. M€ uller glia (MG), common to all vertebrate retinas, readily regenerate neuron loss in some species, but normally not in mammals. However, experimental stimulation of limited mammalian retina regeneration has been reported. Here, we use a mouse retina organ culture approach to investigate the MG responses at different mouse ages. We found that MG undergo defined spatio-temporal changes upon stimulation. In EGF-stimulated juvenile postmitotic retinas, most MG upregulate cell-cycle reg- ulators (Mcm6, Pcna, Ki67, Ccnd1) within 48 h ex vivo; some also express the neurogenic factors Ascl1, Pax6, and Vsx2; up to 60% re-enter the cell cycle, some of which delaminate to divide mostly apically; and the majority cease to proliferate after stimulation. A subpopulation of MG progeny starts to express transcription factors (Ptf1a, Nr4a2) and neuronal (Calb1, Calb2, Rbfox3), but not glial, markers, indicating neurogenesis. BrdU-tracking, genetic lineage-tracing, and transgenic-reporter experiments suggest that MG reprogram to a neurogenic stage and proliferate; and that some MG progeny differentiate into neuronal-like cells, most likely amacrines, no photoreceptors; most others remain in a de-differentiated state. The mouse MG regeneration potential becomes restricted, dependent on the age of the animal, as observed by limited activation of the cell cycle and neurogenic factors. The stage-dependent analysis of mouse MG revealed similarities and differences when com- pared with MG-derived regeneration in fish and chicks. Therefore, the mouse retina ex vivo approach is a potential assay for understanding and overcoming the limitations of mammalian MG-derived neuronal regeneration. Postmitotic MG in mouse retina ex vivo can be stimulated to proliferate, express neurogenic factors, and generate progeny expressing neuronal or glial markers. This potential regenerative competence becomes limited with increasing mouse age. GLIA 2015;63:1809–1824 Key words: regeneration, stem cells, neuroglia, mice, reprogramming, gliosis Introduction N on-mammalian vertebrates, for example some fishes, are able to regenerate their retina. In fish, M€ uller glia (MG) reactivate upon retinal injury, reprogram to gain stem-cell competence, re-enter the cell cycle, and divide asymmetrically to self-renew and to generate expanding neurogenic progeni- tors, which differentiate into all types of neurons to restore retinal structure and visual function (Goldman, 2014; Gor- such and Hyde, 2014; Lenkowski and Raymond, 2014). Conversely, mammalian MG are quiescent and respond to retinal damage by reactive gliosis (Bringmann et al., 2009; Dyer and Cepko, 2000a), rarely with proliferation, and do not naturally regenerate neurons (Joly et al., 2011; Karl et al., 2008; Karl and Reh, 2010; Roesch et al., 2008). Still, a few studies have suggested a very limited amount of neurogenic potential upon experimental stimulation of mammalian MG in- and ex vivo (Del Debbio et al., 2010; Giannelli et al., 2011; Harada et al., 2011; Insua et al., 2008; Karl et al., 2008; Lawrence et al., 2007; Ooto et al., 2004; Osakada et al., 2007; Pollak et al., 2013; Reyes-Aguirre et al., 2013; Takeda et al., 2008; Wan et al., 2007, 2008; Wang et al., 2012; Zhao et al., 2014), often using approaches inspired by studies in fish and chicks (Qin et al., 2011; Wan et al., 2012, 2014). Importantly, the potential MG-derived neuronal cell types and numbers differ considerably within the rodent stud- ies, which rarely verified cell sources and fates using View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22846 Published online May 6, 2015 in Wiley Online Library (wileyonlinelibrary.com). Received Jan 23, 2015, Accepted for publication Apr 8, 2015. Address correspondence to Mike O. Karl. E-mail: mike.karl@crt-dresden.de or mike.karl@dzne.de From the 1 CRTD—Center for Regenerative Therapies Dresden, Technische Universit€ at Dresden, Dresden 01307, Germany; 2 German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden 01307, Germany Grant sponsors: This project was financed from basic institutional funding (DFG CRTD and DZNE Helmholtz) to M.O.K. and supported by MedDrive Grant by the TU Dresden UKD Medical Faculty, PhD fellowship by ProRetina Deutschland e.V. to K.L., PhD fellowship to P.S. by the Herbert Funke Stiftung and research award and project grant by the Novartis Pharma GmbH. Additional Supporting Information may be found in the online version of this article. VC 2015 Wiley Periodicals, Inc. 1809