Ascl1a/Dkk/Β-Catenin Signaling Pathway Is Necessary and Glycogen Synthase Kinase-3Β Inhibition Is Sufﬁcient for Zebraﬁsh Retina Regeneration

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Ascl1a/Dkk/β-catenin signaling pathway is necessary and glycogen synthase kinase-3β inhibition is sufﬁcient for zebraﬁsh retina regeneration

Rajesh Ramachandran, Xiao-Feng Zhao, and Daniel Goldman1

Molecular and Behavioral Neuroscience Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109

Edited by David R. Hyde, University of Notre Dame, Notre Dame, IN, and accepted by the Editorial Board August 19, 2011 (received for review May 5, 2011) Key to successful retina regeneration in zebrafish are Müller glia necessary for proliferation of dedifferentiated MG in the injured (MG) that respond to retinal injury by dedifferentiating into a cy- retina and that glycogen synthase kinase-3β (GSK-3β) inhibition cling population of retinal progenitors. Although recent studies was sufficient to stimulate MG dedifferentiation into a pop- have identified several genes involved in retina regeneration, ulation of cycling multipotent progenitors in the uninjured ret- the signaling mechanisms underlying injury-dependent MG prolif- ina. Interestingly, Ascl1a knockdown limited the production of eration have remained elusive. Here we report that canonical Wnt neurons by progenitors in the GSK-3β inhibitor-treated retina. signaling controls the proliferation of MG-derived retinal progenitors. We found that injury-dependent induction of Ascl1a sup- Results pressed expression of the Wnt signaling inhibitor, Dkk, and Ascl1a-Dependent Suppression of dkk Gene Expression in Injured induced expression of the Wnt ligand, Wnt4a. Genetic and phar- Retina. Wnt signaling is a conserved pathway that affects many macological inhibition of Wnt signaling suppressed injury-depen- fundamental developmental processes (18). Deregulated Wnt dent proliferation of MG-derived progenitors. Remarkably, in the signaling often underlies cancer cell proliferation (19), and Wnt uninjured retina, glycogen synthase kinase-3β (GSK-3β) inhibition signaling may also participate in repair of the adult nervous was sufficient to stimulate MG dedifferentiation and the forma- system (20). Here we investigated whether Wnt signaling was

tion of multipotent retinal progenitors that were capable of dif- necessary for retina regeneration in zebrafish. We first asked CELL BIOLOGY ferentiating into all major retinal cell types. Importantly, Ascl1a whether any Wnt signaling components were regulated during expression was found to contribute to the multipotential character retina regeneration (Fig. 1A and Fig. S1B). For this analysis, of these progenitors. Our data suggest that Wnt signaling and RNA was purified from uninjured and injured retinas or from GSK-3β inhibition, in particular, are crucial for successful retina FACS-purified MG and MG-derived progenitors (Fig. S1A)at regeneration. 4 d post-retinal injury (dpi) as described (15). Interestingly, a number of genes encoding Wnt ligands (wnt2ba, wnt2bb, wnt4a, pyrvinium | XAV939 | transgenic zebrafish | heat shock | frizzled and wnt8b), Wnt receptors (fzd2, fzd3, fzd8a, fzd8b, and fzd9a), and a Wnt signaling antagonist (dkk1a) were induced in MG- ision loss is among the top disabilities afflicting the human derived progenitors, whereas others (wnt2, wnt3, dkk1b, and Vpopulation. Strategies for repairing the damaged or diseased dkk2) were suppressed. The expression and injury-dependent human retina have remained elusive. Unlike mammals, teleost regulation of Wnt signaling components in MG may suggest that fish such as zebrafish are able to regenerate a damaged retina they signal to each other after retinal injury. However, because that restores structure and function (1–3). Key to successful re- components of the Wnt signaling pathway are also expressed by generation are Müller glia (MG) that respond to retinal injury retinal neurons, they, too, may participate in the injury response. by generating multipotent progenitors that can regenerate all While analyzing the temporal expression pattern of Wnt major retinal cell types (4–8). Attempts to stimulate MG de- component genes, we observed a striking transient decline in dkk differentiation and retina regeneration in mammals have met expression throughout the retina from 6 to 15 h post-retinal in- with little success. In general, retinal injury stimulates a gliotic jury (hpi) (Fig. 1 B and C). This pan-retinal decline was unusual response where MG undergo morphological, biochemical, and in a model of focal retinal injury where all previously studied physiological changes (9), but rarely do these cells regenerate injury-responsive genes were confined to MG residing close to new neurons and glia, even when their proliferation is stimulated the injury site (4, 15, 16). Interestingly, Ascl1 expression is as- (10–14). sociated with DKK repression in human lung cancer (21), and in Mechanisms underlying retina regeneration in zebrafish are the injured retina ascl1a induction is correlated with dkk sup- just beginning to emerge, and it is anticipated that these mech- pression (Figs. 1B and 2A). Therefore, we investigated whether anisms may suggest novel strategies for stimulating retina re- the expression of these two genes was mutually exclusive. Indeed, generation in mammals. After retinal injury in zebrafish, genes in the uninjured retina, in situ hybridization showed that ascl1a associated with the formation of induced pluripotent stem cells was undetectable and dkk1b was readily apparent, whereas at 6 are activated in dedifferentiating MG (15). One of these genes, hpi the opposite was observed (Fig. 1C and Fig. S2A). At 4 dpi, lin-28, participates in an Ascl1a/Lin-28/let-7 microRNA signaling dkk1b was lacking from ascl1a+ progenitors but restored in pathway that contributes to MG dedifferentiation (15). Ascl1a neighboring cells (Fig. 1C and Fig. S2A). To further test the idea may also regulate the proliferation of dedifferentiated MG (16). In addition, injury-dependent induction of Pax6 appears to control the expansion of MG-derived progenitors, but not their Author contributions: R.R., X.-F.Z., and D.G. designed research, performed research, an- initial entry into the cell cycle (17). Although injury-dependent alyzed data, and wrote the paper. induction of Ascl1a and Pax6 are necessary for proliferation of The authors declare no conflict of interest. MG-derived progenitors, it is not clear how they are activated or This article is a PNAS Direct Submission. D.R.H. is a guest editor invited by the Editorial what signaling pathways underlie their effects. Board. Here we report that Ascl1a controls proliferation of dedif- 1To whom correspondence should be addressed. E-mail: [email protected]. fi ferentiated MG in the injured zebra sh retina via regulation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of a Wnt signaling pathway. We found that Wnt signaling was 1073/pnas.1107220108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1107220108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Fig. 1. Ascl1a inhibits the expression of dkk genes during retina regeneration. (A and B) Injury-dependent regulation of Wnt signaling component mRNAs. (C) Double in situ hybridization shows mutually exclusive ascl1a and dkk1b gene expression. (D) ascl1a and dkk1b expression in FACS-purified MG and non- MG from injured retinas. Values are relative to uninjured retina. *P < 0.009. (E and F) Ascl1a knockdown prevents injury-dependent dkk gene suppression. (F) Quantification of E by qPCR. Values are relative to uninjured retina. *P < 0.0001. (G) In situ hybridization showing Ascl1a knockdown relieves injury-dependent dkk suppression. Boxed region in low-magnification image is shown in higher magnification in the row below. Arrows point to ascl1a+/dkk1b+ cells. White dots identify autofluorescence in ONL. (H and I) Injection of zebrafish embryos with dkk1b:gfp-luciferase reporter and increasing amounts of ascl1a mRNA (H)orascl1a-targeting MO (I). *P < 0.005. (J) Lin-28 knockdown differentially affects injury-dependent dkk gene suppression. (K and L) Dkk1b overexpression inhibits cell proliferation at 4 dpi. *P < 0.003. (Scale bars, 10 μm.) ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

that ascl1a and dkk1b exhibit a mutually exclusive expression Because Ascl1a is a transcriptional activator, we suggest that it − pattern, we used FACS to isolate GFP+ MG and GFP retinal mediates dkk1b suppression via activation of an unidentified neurons (non-MG) from gfap:gfp transgenic fish retinas at 0 and transcriptional repressor. 8 hpi and GFP+ dedifferentiated MG from 1016 tuba1a:gfp We previously showed that Ascl1a regulates lin-28 expression transgenic fish retinas at 4 dpi (15). Quantitative PCR (qPCR) in the injured retina (15). Therefore, we tested whether Lin-28 showed that ascl1a was induced approximately sevenfold in non- mediated the effects of Ascl1a on dkk repression in the injured MG at 8 hpi, whereas dkk1b was suppressed (>90%) in this cell retina (Fig. 1J). Interestingly, Lin-28 knockdown completely re- population (Fig. 1D). Furthermore, at 4 dpi, ascl1a expression stored dkk1b and dkk2 expression, partially restored dkk3 ex- was suppressed in non-MG, but increased ∼170-fold in GFP+ pression, and had no effect on dkk4 repression. Therefore, MG-derived progenitors, whereas dkk1b was essentially elimi- Ascl1a uses both Lin-28-dependent and -independent mecha- nated from these cells (Fig. 1D). These data indicate a mutually nisms to regulate dkk gene expression. exclusive pattern of ascl1a and dkk gene expression. Because Dkk can antagonize Wnt signaling, its early decline The above data suggest that Ascl1a suppresses dkk gene ex- may be necessary to initiate Wnt signaling and retina regeneration. pression. To test this idea, we knocked down Ascl1a with pre- To directly test this idea, we took advantage of hs:Dkk1GFP viously validated ascl1a-targeting morpholino-modified antisense transgenic fish that harbor the zebrafish dkk1b–gfp fusion under oligonucleotides (MOs) (15, 16) and assayed dkk expression at control of the hsp70-4 promoter (22). These fish were used pre- 8 hpi (Fig. 1 E and F). Indeed, Ascl1a knockdown relieved injury- viously to demonstrate that canonical Wnt signaling mediates fin dependent dkk suppression. In situ hybridization assays also regeneration and that transgene activation in developing embryos showed that dkk1b gene expression returned after Ascl1a knock- phenocopies the effects of wnt8 loss of function (22). In addition, down (Fig. 1G). To further substantiate that Ascl1a can repress Dkk1GFP transgene overexpression blocked activation of the Wnt dkk1b gene expression, we coinjected zebrafish embryos with signaling reporter TOPdGFP (23). Therefore, the hs:Dkk1GFP a dkk1b:gfp–luciferase reporter and various amounts of either transgenic line is an excellent tool for studying the function of Wnt in vitro-transcribed ascl1a mRNA or ascl1a-targeting MO (Fig. 1 signaling. We confirmed that Dkk1b–GFP overexpression blocked H and I). In these experiments, Ascl1a overexpression decreased, fin regeneration and also verified heat shock-dependent Dkk1b– whereas Ascl1a knockdown increased dkk1b promoter activity. GFP expression in the retina (Fig. S3 A and B). Importantly,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1107220108 Ramachandran et al. Downloaded by guest on September 28, 2021 Fig. 2. Ascl1a regulates wnt4a expression via a Lin-28 independent pathway. (A) Time course of injury-dependent gene expression. (B and C) MO-mediated Ascl1a knockdown suppresses wnt4a gene induction at 2 dpi. *P < 0.0005. (D) wnt4a expression in MG and non-MG after retinal injury relative to uninjured control retina. *P < 0.0007. (E) ascl1a and wnt4a mRNAs are coexpressed in BrdU+ MG-derived progenitors at 4 dpi. (Scale bar, 10 μm.) (F) Lin-28 knockdown does not suppress injury-dependent wnt4a or fzd2 induction. Abbreviations are as in Fig. 1.

Dkk1b overexpression in the injured retina inhibited cell pro- To determine whether injury-induced β-catenin stabilization is liferation (Fig. 1 K and L and Fig. S3B). The retention of some necessary for retina regeneration, we stimulated β-catenin deg- progenitors beneath the injury site in Dkk1b-overexpressing fish radation in the injured retina of 1016 tuba1a:gfp fish by injecting may indicate a gradient of Wnt signaling emanating from the injury eyes with pyrvinium, a casein kinase 1-α activator (24), or site. In this case, Dkk1b levels may be insufficient to completely XAV939, a tankyrase inhibitor (25), along with a 3-h pulse of block Wnt signaling at the injury site. BrdU at 4 dpi. Pyrvinium and XAV939 dramatically reduced CELL BIOLOGY injury-dependent β-catenin accumulation, GFP transgene ex- Ascl1a-Dependent Induction of Wnt4a in Injured Retina. We next pression, and proliferation of MG-derived progenitors (Fig. 3 E investigated the temporal expression pattern of Wnt signaling and F and Fig. S5). These data are consistent with the idea that components wnt4a, wnt8b, and fzd2 after retinal injury and com- a canonical Wnt signaling pathway regulates proliferation of pared them to ascl1a expression. We focused on these genes be- MG-derived progenitors in the injured retina. cause their expression is undetectable in the uninjured retina but highly induced in MG-derived progenitors at 4 dpi (Fig. 1A). Like β-Catenin Stabilization Stimulates MG Dedifferentiation and Prolif- ascl1a, wnt4a and wnt8b were induced within 6 hpi, whereas fzd2 eration. We next investigated whether pharmacological stabili- was largely induced at ∼24 hpi (Fig. 2A). However, only wnt4a zation of β-catenin would enhance the regenerative response of appeared to be completely dependent on Ascl1a expression (Fig. 2 MG after retinal injury. For this analysis, we injected either LiCl, B and C), which is consistent with their coexpression in MG and GSK-3β inhibitor I, or vehicle (DMSO) into retinas of 1016 retinal neurons at 8 hpi (Figs. 1D and 2D) and their coenrichment tuba1a:gfp transgenic fish at the time of injury, and at 4 dpi fish in MG-derived progenitors at ∼4 dpi (Figs. 1D and 2 D and E and received a 3-h pulse of BrdU. GSK-3β inhibition resulted in Fig. S2B). Unlike injury-dependent dkk1b repression, wnt4a in- a large expansion in the number of GFP+/BrdU+ retinal production was independent of Lin-28 expression (Fig. 2F). genitors throughout the inner nuclear layer (Fig. S6A). Inspired by this finding, we introduced either the GSK-3β inhibitor or β-Catenin Stabilization Is Necessary for Proliferation of Dediffer- DMSO into the vitreous of an uninjured 1016 tuba1a:gfp fish entiated MG. Together, the above studies suggest a role for retina by injection through the front of the eye. Remarkably, the Wnt signaling in the injured retina. Because β-catenin stabiliza- GSK-3β inhibitor stimulated GFP transgene expression, β-cat- tion is a hallmark of canonical Wnt signaling, we assayed its enin accumulation, and BrdU incorporation in MG throughout expression in uninjured and injured retinas. We found that epi- the retina (Fig. 4A and Fig. S6B). TUNEL showed that the GSK- tope retrieval revealed nuclear β-catenin, whereas standard im- 3β inhibitor did not stimulate cell death (Fig. S6C). munohistochemical protocols showed cytoplasmic β-catenin To investigate whether the GSK-3β inhibitor actually stimu- (compare Fig. 3D Upper, which used standard protocol, with Fig. lated MG dedifferentiation similar to retinal injury or simply 3D Lower, which used epitope retrieval protocol). Because epi- forced MG into the cell cycle, we assayed for regeneration-as- tope retrieval hindered GFP detection, we used standard pro- sociated genes such as ascl1a, lin-28, pax6b, c-mycb, wnt4a, and tocols when anti-GFP and anti-β-catenin antibodies were used fzd2 (15–17). Indeed, the GSK-3β inhibitor stimulated a pattern on the same section. Retinal injury stimulated nuclear β-catenin of gene expression in the uninjured retina that was very similar to expression in MG near the injury site, and this induction was that after retinal injury (Fig. 4 B and C), and, at least for ascl1a, suppressed by Dkk1b overexpression in hs:Dkk1GFP transgenic this expression was confined to proliferating MG (Fig. S6D). fish (Fig. 3 A and B and Fig. S4A). Retinal injury in Wnt/β-cat- Thus, Wnt signaling feeds back to further activate genes asso- enin reporter TOPdGFP transgenic fish (23) stimulated GFP ciated with MG dedifferentiation and proliferation. expression in BrdU+/β-catenin+ progenitors located at the injury site (Fig. 3C and Fig. S4 F and G). In 1016 tuba1a:gfp transgenic Dedifferentiated MG in GSK-3β Inhibited Uninjured Retina Are fish (4), β-catenin was restricted to GFP+ progenitors (Fig. 3D) Multipotent. The above data suggest that GSK-3β inhibition in and coincided with the initiation of cell proliferation at ∼2 dpi the uninjured retina stimulates MG dedifferentiation and pro- (4) (Fig. S4 B and C). Quantification showed that 97% of the liferation. To investigate whether these cells are multipotent, we GFP+ and 95% of the BrdU+ cells colabeled with β-catenin at pulse-labeled them with BrdU 4 d post-GSK-3β inhibition and 4 dpi. We did not detect large changes in β-catenin (ctnnb1 or examined their fate 10–18 d later using retinal cell type-specific ctnnb2) mRNA after injury (Fig. S4E). antibodies. Remarkably, Zpr1+ cone photoreceptors, PKC+ bi-

Ramachandran et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 Fig. 3. β-Catenin accumulation in MG-derived progenitors is necessary for retina regeneration. (A and B) Injury-induced β-catenin expression at 4 dpi is blocked by Dkk1b overexpression in hs:Dkk1GFP fish. (C) Injury-dependent induction of GFP expression in the β-catenin reporter fish, TOPdGFP.(D) Accumulation of β-catenin in GFP+ MG-derived proliferating progenitors of 1016 tuba1a:gfp fish at 4 dpi. (Upper) Standard immunohistochemical protocol. (Lower) Epitope retrieval protocol. (E and F) Pyrvinium and XAV939 inhibit the generation of GFP+, β-catenin+, and BrdU+ MG-derived progenitors at 4 dpi in 1016 tuba1a:gfp fish. White dot identifies an area of autofluorescence that results from long exposures to detect fluorescent signals. *P < 0.001. (Scale bars, 10 μm.) Abbreviations are as in Fig. 1.

polar cells, GS+ MG, HuC/D+ amacrine, and Zn5+ differenti- Discussion ating ganglion cells were generated from these progenitors MG dedifferentiation and proliferation are key to retina re- (Fig. 4D). generation in zebrafish. We report here that activation of a ca- Our data suggest that Ascl1a activates a Wnt signaling path- nonical Wnt signaling pathway is necessary for proliferation of way that results in β-catenin stabilization and feeds back to fur- dedifferentiated MG. This conclusion is supported by the ther stimulate the expression of genes associated with MG observations that: (i) Stabilized β-catenin is first detected in MG dedifferentiation like ascl1a. Because Ascl1a was previously at ∼2 dpi when they begin proliferating; (ii) Dkk overexpression β shown to be necessary for MG dedifferentiation (15), we asked or -catenin destabilization suppressed injury-dependent MG β whether its induction in the GSK-3β–inhibited retina was nec- proliferation; and (iii) -catenin stabilization induced MG pro- essary for the generation of multipotent progenitors. To test this liferation in the uninjured retina. While investigating the temporal pattern of expression of Wnt idea, we intravitreally delivered GSK-3β inhibitor with a control signaling components to a focal retinal injury, we uncovered or ascl1a-targeting MO (Fig. S6E) and pulse labeled with BrdU a surprisingly robust pan-retinal suppression of dkk gene ex- at 4 dpi. Retinas were harvested 3 h and 6 d post-BrdU injection, pression that appeared to be dependent on Ascl1a induction. fl and immuno uorescence was used to quantify progenitor- These data suggest that signals reporting retinal injury are not + derived (BrdU ) retinal neurons and glia. Although Ascl1a confined to MG immediately surrounding the injury site, as knockdown almost completely suppressed MG proliferation in previously thought, but, rather, may rapidly diffuse throughout the injured retina (15), it only reduced MG proliferation by the retina. Although the nature of these signals remains un- ∼50% in the GSK-3β–inhibited retina (Fig. 4 E and F), sug- known, reactive oxygen species are potential candidates because gesting that Ascl1a may contribute to MG proliferation via they are rapidly induced by injury and can diffuse in tissues β-catenin–dependent and –independent mechanisms. and cross-membranes and regulate transcription factor activity Interestingly, GSK-3β inhibitor-induced progenitors (BrdU+), (26). Interestingly, a gradient of hydrogen peroxide was recently with and without Ascl1a knockdown, were not equivalent. First, shown to mediate wound detection in zebrafish (27). One progenitors with Ascl1a knockdown generated fewer neurons consequence of this retinal wide response to injury is the in- (40% less photoreceptor, 67% less bipolar, 43% less amacrine, duction of ascl1a gene expression, which leads to repression of and 100% less ganglion cells) and retained more MG charac- dkk gene expression. Interestingly, ascl1a induction and dkk repression is transient in retinal neurons, but maintained in MG- teristics (38% increase in BrdU+/GS+ MG) than those with derived progenitors. Although the mechanisms underlying this Ascl1a expression. Second, Ascl1a knockdown suppressed ex- regulation are not completely clear, it is likely that Ascl1a pression of a subset of genes associated with MG dedifferen- mediates dkk repression indirectly via the activation of a dkk tiation, such as lin-28, mycb, and wnt4a (Fig. 4G). These results transcriptional repressor. suggest that injury-dependent or GSK-3β inhibitor-dependent Although dkk suppression and wnt4a and 8b induction Ascl1a induction may not only stimulate MG dedifferentiation were observed at ∼6 hpi, β-catenin accumulation was not and proliferation but also influence their differentiation into detected until 2 dpi when MG-derived progenitors began pro- neurons and glia. liferating. This lag may reflect the need for induction of other

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1107220108 Ramachandran et al. Downloaded by guest on September 28, 2021 Fig. 4. β-Catenin stabilization stimulates MG dedifferentiation and the generation of multiple retinal cell types in the uninjured retina. (A) Intravitreal injection of GSK-3β inhibitor induces GFP, β-catenin, and cell proliferation in the uninjured retina of 1016 tuba1a:gfp transgenic ﬁsh. (B and C) Expression of regeneration- associated genes in uninjured retinas treated with the GSK-3β inhibitor. *P < 0.0001. (D) GSK-3β inhibitor-induced retinal progenitors proliferate and generate all major retinal cell types. (E and F) Effect of Ascl1a knockdown on MG proliferation in the GSK-3β inhibitor-treated retina. *P < 0.002. (G) Effect of Ascl1a CELL BIOLOGY knockdown on genes associated with MG dedifferentiation in the GSK-3β inhibitor-treated retina. [Scale bars, 10 μm(A and D); 20 μm(E).] Abbreviations are as in Fig. 1.

Wnt signaling components, such as Fzd2, that are induced contribute to the capacity of these progenitors to differentiate at later times during retina regeneration. Alternatively, low into neurons and glia. Indeed, we found that knocking down levels of Wnt signaling mediated by undetectable levels of Ascl1a expression in the GSK-3β inhibited retina dramatically β-catenin may participate in MG dedifferentiation at early reduced the production of new neurons from these progenitors. times after retinal injury, whereas at later times, higher levels of It is tempting to speculate that the poor regenerative capacity Wnt signaling may regulate events such as cell proliferation of mammals may be associated with their lack of expression of and differentiation. dedifferentiation-associated genes in proliferating MG. Consis- β We were most intrigued by the observation that GSK-3 in- tent with this idea, we note that NMDA-mediated retinal dam- hibition in the uninjured retina was sufficient to stimulate MG age combined with EGF-induced MG proliferation did not dedifferentiation into a cycling population of multipotent retinal stimulate retina regeneration or Ascl1 induction in the mouse progenitors. A similar result has been reported in rat retinal explants treated with GSK-3β inhibitors (11), suggesting a com- (13). In contrast, the postnatal chick retina does exhibit a limited mon mechanism underlying MG proliferation in zebrafish and amount of regeneration that is accompanied by a modest in- mammals. Interestingly, ciliary neurotrophic factor, FGF/insulin, duction of Ascl1 expression (35). In addition, overexpression of and glutamate have also been reported to stimulate MG pro- basic helix–loop–helix and homeobox genes in the injured rat liferation in fish, birds, and mammals, respectively (14, 28, 29). retina promoted the regeneration of cells with rod photorecep- Whether these factors stimulate a genetic program similar to that tor, amacrine, and horizontal cell phenotypes (36). Although the found in zebrafish MG-derived progenitors is not known; also full constellation of genes necessary for MG dedifferentiation unknown is whether Wnt signaling is necessary for these factors into a multipotent retinal progenitor is not yet defined, the to stimulate MG proliferation. zebrafish retina provides an ideal system for discovering these Wnt signaling in the adult regenerating zebrafish retina factors because of its robust regenerative response. appears to recapitulate development where Wnt signaling also The fact that Wnt signaling activation was sufficient to stim- regulates retinal progenitor proliferation (30). Interestingly, Wnt ulate MG proliferation in both rodent and zebrafish retinas signaling components are enriched in adult mouse MG (31, 32), suggests that this signaling cascade is a major control point for and this pathway appears to be involved in several mouse models MG dedifferentiation and proliferation. However, unlike in of retinal degeneration (33, 34). These studies suggest that, zebrafish, activation of Wnt signaling in mammals does not ap- although mammalian MG appear capable of activating the Wnt pear to be sufficient to stimulate robust retina regeneration. This signaling pathway, this pathway may need to collaborate with difference may result from incomplete activation of gene ex- other signal transduction cascades to stimulate MG dedifferentiation into multipotent progenitors. pression programs in mammals that underlie MG dediffer- The observation that early response genes (ascl1a, lin-28, entiation. It will be interesting to determine whether genes associated with retina regeneration in fish such as ascl1a, lin-28, c-mycb, pax6b, and wnt4a) associated with MG dedifferentiation were activated by GSK-3β inhibition in the uninjured retina and pax6 remain repressed in the injured mammalian retina suggests that stabilized β-catenin contributes to their expression exposed to GSK-3β inhibitors and whether overexpression of in MG-derived progenitors. These genes may be important for these genes or signaling pathways that activate these genes proliferating MG to function as multipotent stem cells and/or improves retina regeneration in mammals.

Ramachandran et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Methods BrdU Labeling, Immunofluorescence, and in Situ Hybridization. BrdU labeling, fl Plasmid Construction, RNA Isolation, PCR, and mRNA Synthesis. All primers immuno uorescence, and in situ hybridization were performed as described used in this study are listed in Table S1. A 4-kb zebrafish dkk1b pro- (4, 8, 15, 16). Double in situ hybridizations were performed according to moter was cloned to create dkk1b:gfp-luciferase. Total RNA was isolated manufacturer’s instructions (Perkin-Elmer). Detailed information is provided by using TRIzol (Invitrogen). PCRs were as described (15). Capped mRNAs in SI Methods. were synthesized by using the mMESSAGE mMACHINE kit (Ambion) according to manufacturer’s instructions. Detailed information is pro- ACKNOWLEDGMENTS. We thank D. Hyde, R. Moon, and R. Dorsky for vided in SI Methods. sharing transgenic fish; K. Cadigan for XAV939; J. Wan for oubain-treated retinal sections; M. Uhler for expression vectors and reagents; J. Beals for Animals, Heat Shock, Retinal injury, MO Delivery, and FACS. gfap:GFP, hs: help with confocal microscopy; the University of Michigan Flow Cytometry fi Core for cell sorting; P. Macpherson for statistics; and R. Karr and Dkk1GFP, TOPdGFP, and 1016 tuba1a:gfp sh and retinal lesions were de- fi fi T. Melendez for sh care. This work was supported by National Institutes scribed (4, 22, 23, 37). For heat shock, sh were transferred to preheated of Health Grant NEI R01 EY018132 (to D.G.), National Institutes of Health 36.5 °C water for 1 h every 12 h beginning 1 d before injury. MO delivery to National Institute of Child Health and Human Development Grant cells was facilitated by electroporation (16). MG and non-MG were purified T32HD007507 (to R.R.), and a University of Michigan Center for Organo- by FACS (15). Detailed information is provided in SI Methods. genesis grant (to X.-F.Z.).

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