RESEARCH REPORT

Activation of Wnt signaling reduces ipsilaterally projecting retinalganglion cells in pigmented retinaLena Iwai-Takekoshi1,*, Revathi Balasubramanian2, Austen Sitko3, Rehnuma Khan1, Samuel Weinreb1,Kiera Robinson1 and Carol Mason1,2,3,4,‡

ABSTRACTIn mammalian albinism, disrupted melanogenesis in the retinalpigment epithelium (RPE) is associated with fewer retinal ganglioncells (RGCs) projecting ipsilaterally to the brain, resulting in numerousabnormalities in the retina and visual pathway, especially binocularvision. To further understand the molecular link between disruptedRPE and a reduced ipsilateral RGC projection in albinism, wecompared gene expression in the embryonic albino and pigmentedmouse RPE.We found that theWnt pathway, which directs peripheralretinal differentiation and, generally, cell proliferation, is dysregulatedin the albino RPE. Wnt2b expression is expanded in the albinoRPE compared with the pigmented RPE, and the expanded regionadjoins the site of ipsilateral RGC neurogenesis and settling.Pharmacological activation of Wnt signaling in pigmented mice bylithium (Li+) treatment in vivo reduces the number of Zic2-positiveRGCs, which are normally fated to project ipsilaterally, to numbersobserved in the albino retina. These results implicate Wnt signalingfrom the RPE to neural retina as a potential factor in the regulation ofipsilateral RGC production, and thus the albino phenotype.

KEY WORDS: Retinal pigment epithelium, Wnt2b, Zic2, Ciliarymargin zone

INTRODUCTIONRetinal ganglion cells (RGCs) are the output neurons from the eye tothe brain. During retinal development, RGCs are produced at theinterface of the retinal pigment epithelium (RPE) and neural retina,and are specified into two subtypes based on their laterality ofprojection – ipsilaterally or contralaterally. The proper proportion ofipsi- and contralateral RGC projections is crucial for binocularvision (Erskine and Herrera, 2014; Herrera et al., 2017; Petros et al.,2008). In mammalian albinism, a genetic disorder of melaninbiogenesis in both skin and the RPE, the number of ipsilateral RGCsis reduced (Herrera et al., 2017). When pigmentation is restoredin the RPE during RGC neurogenesis, the reduced ipsilateralprojection is rescued, suggesting that the pigmented RPE plays arole in controlling the number of ipsilateral RGCs (Cronin et al.,

2003). However, how pigment affects RGC specification andthus ipsi- versus contralateral RGC projection laterality even inpigmented retina is not understood.

In the developing albino mouse retina, ipsilateral RGCneurogenesis and subtype specification are disrupted (Bhansaliet al., 2014; Herrera et al., 2003; Rebsam et al., 2012). Duringthis period, albino RPE cells have irregular cell shape, fewermelanosomes and aberrant expression of junctional proteins(Iwai-Takekoshi et al., 2016). Because retinal neurons, includingRGCs, are produced at the interface of the RPE and the neural retina,perturbed RPE cell integrity may result in aberrant RPE-neuralretina communication and, in turn, altered ipsilateral RGCneurogenesis and subtype specification in albino mouse retina.

Here, to test the hypothesis that the RPE expresses extrinsicregulators influencing RGC neurogenesis, we investigated whetherpigmented and albino mouse RPE cells express different sets ofgenes and whether the genes that are altered could affect ipsilateralRGC production. We found that the embryonic albino RPEexpresses Wnt2b to a greater extent than pigmented RPE.Moreover, lithium treatment in vivo, known as an activator of Wntsignaling, led to a reduced number of RGCs that express Zic2, atranscription factor regulating the specification and guidance ofipsilateral RGCs (Herrera et al., 2003). These results suggestthat Wnt signaling is a potential molecular link between RPE andRGC generation and specification, especially for ipsilateralRGC production.

RESULTS AND DISCUSSIONThe pigmented and albino RPE are molecularly distinctTo unravel a role for the RPE in mechanisms of RGC specificationand to better understand the albino phenotype, we compared albinoand pigmented RPE by performing a microarray analysis on theRPE isolated from embryonic albino and pigmented retina (Fig. 1).We focused on the RPE at embryonic day (E) 13.5, when ipsilateralRGCs are produced and differentiate in the ventrotemporal (VT)retina (Dräger, 1985; Herrera et al., 2003). Pigmented and albinoRPE have distinct molecular features: 220 differentially expressedprobes were identified in the albino RPE, corresponding to 191different genes, of which 176 were upregulated and 15 weredownregulated (Table S1). For validation of the microarray, weanalyzed expression of select genes by RT-qPCR and in situhybridization (Fig. S1A,B). Gene profiling with Genespring(Table S1) did not identify secreted signaling genes, which wehypothesized would be released from the RPE to act in the retina. Toidentify signaling pathways that could be altered in albino RPEcompared with pigmented RPE, we applied Gene Set EnrichmentAnalysis (GSEA), which determines whether an a priori defined setof genes shows statistical significance between two biologicalphenotypes, in our case pigmented versus albino RPE (Fig. 1A,Tables S2-S4). In pigmented RPE, three gene sets involved inReceived 5 February 2018; Accepted 15 September 2018

1Department of PathologyandCell Biology, ColumbiaUniversity, Collegeof Physiciansand Surgeons, New York, NY 10027, USA. 2Department of Ophthalmology, ColumbiaUniversity, College of Physicians and Surgeons, New York, NY 10027, USA.3Department of Neuroscience, Columbia University, New York, NY 10027, USA.4Mortimer B. ZuckermanMind Brain Behavior Institute, Columbia University, New York,NY 10027, USA.*Present address: Division of Brain Function, National Institute of Genetics, Mishima,Shizuoka 411-8540, Japan.

‡Author for correspondence (cam4@columbia.edu)

R.B., 0000-0002-2209-0815; C.M., 0000-0001-6253-505X

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receptor and ligand binding were significant. The albino RPEtranscriptome was enriched in genes involved in cancer, signaling,promoter activity, cell differentiation, and cell cycle andproliferation. Gene sets associated with cytoskeleton and celljunction were enriched in albino compared with pigmented RPE,supporting our findings on disorganized RPE cell integrity in albinoretina (Iwai-Takekoshi et al., 2016). Gene sets associated withsignaling pathways and enriched in albino RPE included Hh, Notch,Bmp, Sfrp and Wnt (Fig. 1B. Table S3). Because GSEA focuses onsets of genes rather than individual genes, we searched the literatureto find candidate genes in the pathways that were recognized by theGSEA analysis (Bao and Cepko, 1997; Liu et al., 2003; Wang et al.,2016). Bmp4 (Fig. 2), Shh and Sfrp2 (Fig. S1) expression is similarin both genotypes. Notch2 expression is very faint in the pigmentedRPE but evident in albino RPE (Fig. S1). Compared with Notch2,which is expressed in the entire albino RPE, Wnt2b expression is ofinterest because it is expressed in the peripheral RPE, which isadjacent to where ipsilateral RGCs settle in the neural retina. Wethus focused on Wnt2b mRNA expression in the pigmented andalbino RPE (Fig. 1C). In pigmented retina, as melanin masks in situhybridization signals, we bleached melanin after the in situhybridization color reaction. At E13.5, Wnt2b is expressed in the

periphery of the ciliary margin zone (CMZ), as previously reported(Cho and Cepko, 2006; Kubo et al., 2003; Liu et al., 2003), both inalbino and pigmented retina. However, at E15.5, when expression ofZic2 is expressed in the majority of ipsilateral RGCs (Bhansali et al.,2014; Herrera et al., 2003), Wnt2b expression is expanded towardcentral retina in albino RPE compared with pigmented RPE(1.5-fold increase in albino RPE compared with pigmented RPE:Fig. 1D). RT-qPCR also indicated a trend of increased expression ofWnt2b in albino RPE (1.4-fold increase, Fig. S1D).

To ascertain whether this alteration of Wnt expression in albinoRPE at E15.5 is specific to Wnt2b, we examined other Wntsignaling components expressed in the peripheral retina. Wntinhibitory factor 1 (Wif1) (Ha et al., 2012) is enriched inpigmented RPE (Table S4), but a difference in intensity andpattern between genotypes was not apparent (Fig. 1E).Furthermore, expression of Fzd7, a Wnt receptor (Liu et al.,2003), was specifically enriched in the ventral CMZ, which isproposed as a neurogenic site for RGCs in embryonic retina(Marcucci et al., 2016). Although the spatial pattern and intensityof Fzd7 were similar in both pigmented and albino retina (Fig. 1F),the asymmetric Fzd7 expression implicates ventral retinal specificWnt signaling. These results indicate that the pigmented and

Fig. 1. Embryonic pigmented and albinoRPEhave differential gene expression. (A) GSEA for pigmented versus albinoRPE. Sets have a false discovery rate(FDR; q value)<0.05 and were hand curated into thematic categories. (B) GSEA for pigmented versus albino RPE focused on signaling pathways. Bars indicatethe number of gene sets with FDR<0.25. Parentheses indicate the number of gene sets with FDR<0.05. In pigmented RPE, no signaling gene set was detectedwith FDR<0.05 cut-off. (C) Wnt2b expression in peripheral RPE is expanded centrally in albino retina compared with pigmented RPE at E15.5 (arrowhead).(C1,C2) Ventrotemporal (VT) retina sections from a different embryo at E15.5 at higher magnification. d, dorsal; v, ventral. n=8 (E13.5), n=5 embryos (E15.5).(D)Quantification of line tracing of theWnt2b-positive area in the total perimeter of RPE. Four retinal sections (two rostral, one caudal and onewhere the optic nerveexits the eye) were analyzed. ON, optic nerve. Unpaired t-test (n=5 embryos). Bars represent mean. Error bars: ±s.e.m. *P<0.05; **P<0.01. (E) Wif1 expression inperipheral neural retina (arrowheads) at E13.5 (n=4 embryos) and E15.5 (n=3 embryos). (F) Fzd7 is enriched in the ventral peripheral retina compared with dorsalperipheral retina (arrowheads) at E13.5 and E15.5 in both genotypes (n=3 embryos). (F1,F2) Higher magnification of E15.5 retina. Scale bars: 100 µm.

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albino RPE differentially express Wnt2b, which is temporally andspatially correlated with ipsilateral RGC production.

Amarkerof neurogenesis, but not CMZmarker expression, isaltered in albino retinaPrevious studies have shown that a constitutively active Wnt modelin the mouse retina displayed increased ectopic expression of CMZmarkers such as Otx1 and Msx1 (Ha et al., 2012; Liu et al., 2007).We examined whether the expanded expression of Wnt2b in thealbino mouse RPE is associated with changes in patterning ofperipheral retinal structure. Otx1 and Bmp4 expression (Martinez-Morales et al., 2001; Zhao et al., 2002) is similar in pigmented andalbino CMZ at E13.5 (Fig. 2A). At E15.5, whenWnt2b is expandedin albino compared with pigmented retina (Fig. 1C,D), Otx1, Bmp4and Msx1 expression in the CMZ was again the same in bothgenotypes (Fig. 2B). Of note, Msx1 is expressed more intensely inventral than dorsal retina in both albino and pigmented retina, aspreviously described (Marcucci et al., 2016) (Fig. 2B).Next, we considered the possibility that Wnt2b expression in

the RPE overlying the CMZ may influence neurogenic potential ofthe CMZ. Connexin 43 (Cx43) interacts with regulators of cellproliferation, such as cyclin D1 (Swayne and Bennett, 2016).Intense Cx43 labeling is observed around progenitor cells in theCMZ of adult newt (Umino and Saito, 2002) and is associated withcell proliferation in albino rat retina (Tibber et al., 2007). We foundthat Cx43 is more highly expressed in albino peripheral retinacompared with pigmented retina at E13.5 (Fig. 2C). To determinewhether the Cx43 upregulation in albino retina reflects structuralchanges at the border of CMZ and neural retina, we examinedexpression of genes found in the neural progenitor layer anddifferentiated neural retina (Sox2 and Fzd5) (Wang et al., 2016).The spatial pattern of these genes was similar in albino andpigmented retina (Fig. 2D). The intense expression of Cx43 in the

albino CMZmay reflect alterations in the pace of RGC neurogenesis(Bhansali et al., 2014).

Because retinal area is unchanged in albino and pigmented miceat E15.5 (Bhansali et al., 2014), it is reasonable to assume thatexpansion of Wnt2b area in the albino RPE reflects anupregulation of Wnt activity. We assume that Wnt signaling hasdual (or multiple) functions in retinal development, depending onthe activation level: excess levels of Wnt activity lead tomacroscopic deficiencies in the structure of peripheral retinaand slight upregulation of Wnt signaling may affect onlydevelopment of RGC subtypes, such as ipsilateral RGCs versuscontralateral RGCs.

Wnt activation by lithium treatment reduces the number ofZic2-positive RGCs in pigmented retinaTo test whether Wnt signaling is involved in ipsilateral RGCneurogenesis, we activated Wnt signaling in vivo by treatingpregnant mice with lithium chloride (Lancaster et al., 2011; Liuet al., 2007) and quantifying the number of RGCs in the VT retina(Fig. 3A). We characterized ipsilateral RGCs by co-expression ofZic2 (Fig. 3B-E) and islet 1/2 (Isl1/2) (expressed in all RGCs). Asexpected from previous studies (e.g. Bhansali et al., 2014; Herreraet al., 2003; Marcucci et al., 2016), the number of ipsilateral RGCsis reduced by about half in control (NaCl-injected) albino retinacompared with control (NaCl-treated) pigmented retina (Fig. 3F). Incontrast, following lithium treatment, the number of ipsilateralRGCs in pigmented retina was reduced to numbers similar to thosein control albino retina (Fig. 3F). The number of total RGCs(expressing Isl1/2) (Fig. 3G) and the expression zone of Otx1(Fig. 3H) were also similar in lithium-treated and control retina,suggesting that peripheral retinal structure was not affected by thelithium treatment. Wnt activation in the lithium-treated pigmentedand albino retina was confirmed by increased expression of Wnt

Fig. 2. Expression of Cx43, but not of other genes in the CMZ, is enriched in embryonic albino retina. (A) Otx1 and Bmp4 are expressed similarlyin the CMZ albino and pigmented retina (n=3 embryos for Otx1, n=5 embryos for Bmp4). In situ hybridization following melanin bleaching. (B) Otx1, Bmp4and Msx1 are expressed similarly in the CMZ at E15.5 in both genotypes (n=3 embryos for Otx1 and Msx1, n=4 embryos for Bmp4). (C) Cx43 expression inthe CMZ is more intense in an expanded area in albino compared with pigmented retina (arrowhead) at E13.5 but this difference is less evident after E14.5(n=5 embryos for E13.5 and E15.5, n=3 embryos for E12.5 and E14.5). (D) The neural precursor cell markers Sox2 and Fzd5 are both expressed similarlyin pigmented and albino retina (n=3 embryos). Scale bars: 100 µm.

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target genes Axin2 and Lef1 beyond the levels observed in controlretina, as spreading of an existing domain (Fig. 3I,J). Lithiumactivates the Wnt pathway by inhibiting GSK3β activity, whichresults in stabilization of β-catenin and transcriptional activation oftarget genes. Therefore, the lithium response is more likely to beinduced in cells that are in a responsive state rather than in a broaderregion. These results suggest that Wnt activation is involved in theadjustment of the number of ipsilateral RGCs.Marccuci et al. (2016) proposed that, in the albino mouse retina,

diminished neurogenesis in the ventral CMZ correlates withreduced ipsilateral RGCs. To test the possibility that Wntactivation by lithium treatment affects mitosis in the ventral retinaand results in fewer ipsilateral RGCs, we compared the numberof PH3-positive M-phase nuclei in the CMZ and neural retina(Fig. 4A,B). A similar number of PH3-positive cells acrosstreatments and genotypes was observed in all regions of analysis,including dorsal/ventral CMZ and dorsal/ventral neural retina(Fig. 4C). These results suggest that, even with a reduction in thenumber of Zic2-positive cells in lithium-treated pigmented retina, atleast at the end point of lithium treatment, proliferation of retinalcells was not affected.How does Wnt signaling affect RGC output? Wnt/β-catenin

signaling functions as an extrinsic regulator of cell cycle controland cell fate specification in the central nervous system

(Megason and McMahon, 2002). Previous studies demonstratedthat increasing Wnt signaling induces progenitor cell markers inchick retina (Kubo et al., 2003) and delays neuronal differentiationin mouse cortex (Chenn and Walsh, 2002). Decreased Wnt activityis accompanied by reduced proliferation, but treatment with lithiumrescues this phenotype in mouse cerebellum (Lancaster et al., 2011).Moreover, gene profiling comparing E14 mouse retinal explantstreated with lithium to activate Wnt signaling to control retinalexplants revealed cell cycle inhibitors and genes involved inneuronal differentiation (Ha et al., 2012). These studies indicate thatactivation of Wnt signaling is likely associated with a slowing of thecell cycle and inhibition of differentiation.

The wave of ventrotemporal RGC production is delayed in thealbino retina compared with pigmented retina (Bhansali et al.,2014). This delay is linked to the altered expression of genes thatregulate ipsilateral and contralateral fate. Specifically, fewer Zic2-positive RGCs are born before E15.5 and more Zic2-negative RGCsafter E15.5 (Bhansali et al., 2014). The decrease in RGCs at E15.5in albino mice is specific to the Zic2-expressing region inventrotemporal retina, which is probably why retinal area isunchanged in albino and pigmented mice at E15.5. These resultssuggest that there is a brief time window of competence for Zic2expression to be fated as ipsilateral RGCs (∼E13) and Isl2 to befated as contralateral RGCs (∼E15) (Bhansali et al., 2014).

Fig. 3. Lithium treatment ofpigmentedmice leads to a reductionin the number of Zic2-positiveRGCs. (A) Timeline of LiCl or control(NaCl) injection into pregnantdams (arrowheads) and analysis.(B-E) There were fewer Zic2-positivecells (arrowheads) in lithium-treatedpigmented retina, similar to numbersin control (NaCl-treated) albino retina.(F) The number of Zic2 and Isl1/2double-positive cells decreased inlithium-treated pigmented VT retina,but not in lithium-treated albino retinaor control (NaCl-treated) pigmentedand albino retina at E15.5. (G) Thenumber of Isl1/2-positive cells (allRGCs) in lithium-treated and control(NaCl-injected) pigmented and albinoVT retina is unchanged at E15.5.(F,G) One-way ANOVA (n=7 embryosfrom five or six litters, two retinalsections/embryo). Bars representmean. Error bars: ±s.e.m. *P<0.05;**P<0.01; ***P<0.001. ns, notsignificantly different. (H) Ectopicexpression of Otx1 was not observedafter lithium treatment at E15.5; n=7embryos. (I,J) Wnt target genes, Axin2(I, in situ hybridization) and Lef1(J, immunohistochemistry) wereupregulated after lithium treatment inthe RPE (arrowheads in albino retina)and CMZ (arrows), but not in control(NaCl-treated) retina at E15.5. Melaninmasks immunohistochemistry andin situ hybridization signal in pigmentedRPE (melanin bleaching was notcompatible with Axin2 and Lef1staining). n=3 embryos. Scale bars:100 µm in B-E,I,J; 200 µm in H.

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Wnt activation by lithium treatment from E12.5 to E14.5 in thepresent study did not affect the number of differentiated RGCs(Isl1/2 positive) but led to fewer ipsilateral (Zic2-positive) RGCs atE15.5 (Fig. 4). Together with findings by Bhansali et al., the presentdata raise the possibility that activated Wnt signaling underliesdelayed neurogenesis/differentiation in the albino retina and inlithium-treated pigmented retina, affecting the competency ofretinal progenitor cells to express Zic2, and consequently leadingto a reduced number of ipsilateral RGCs.Wnt activation of lithium treatment reduced Zic2-positive

ipsilateral RGCs in pigmented but not in albino retina. Our resultmay indicate that ipsilateral RGCs affected by lithium treatment inpigmented mice are subsets of RGCs that might be absent in normalalbino retina. Subsets of ipsilateral RGCs have been discussed butnot characterized. Cyclin D2 knockout mice have 20% feweripsilateral RGCs than wild type (Marcucci et al., 2016), suggestingthat subsets of ipsilateral RGCs are produced by regulation ofcyclin D2.

The route from RPE to RGCsWe hypothesize that Wnt2b protein is released from peripheral RPEto the neural retina and signals to retinal precursor cells via Fzdreceptors, especially Fzd7, which is enriched in the ventral CMZ, orFzd5, which is enriched in ipsilateral RGCs (Wang et al., 2016).RT-qPCR analysis in isolated RPE at E15.5 did not show obviousupregulation of Wnt target genes in albino RPE (Fig. S1). Thiscould be explained by the co-expression of Wnt2b and Wif1.Whether Wnt and Wif1 interact with each other to play a role inRGC subtype specification is a pressing issue. The combination ofWnt2b and Wif1 expression in the peripheral RPE may represent atunable system by which the production of different cell types(ipsilateral and contralateral RGCs) can be controlled in a temporaland spatial manner. The VT retina produces ipsilateral RGCs in alimited time window then switches to produce contralateral RGCs.Wnt activation by lithium treatment, as performed in this study, is

not Wnt2b specific. Conditional inactivation of Wnt expression,for example, inhibition of ligand secretion by deletion of Wntlessin a RPE-specific manner, could directly test whether secretion ofWnt2b from RPE impacts the number of ipsilateral RGCs.Conditional inactivation of Wnt signaling in the presumptive RPE

induces abnormal development of the RPE (Fujimura et al., 2009).To maintain normal development of the RPE and analyze effects onRGC neurogenesis, manipulation ofWnt signaling could be specificto the period of RGC neurogenesis (E13-E15) and perturbed in theRPE itself. In summary, our past and present results support thehypothesis that cellular and molecular defects of the albino RPEduring RGC neurogenesis have an impact on communication withthe neural retina and the production of factors (potentially Wnts)that could affect ipsilateral RGC specification.

MATERIALS AND METHODSAnimalsMice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA;MGI: 1855985) and maintained in a timed-pregnancy breeding colony atColumbia University. Conditions and procedures were approved by theColumbia University Institutional Animal Care and Use Committeein protocols AC-AAAG9259 and AC-AAAG8702. Heterozygous mice(Tyr+/c-2J) were crossed with homozygous tyrosinase mutants (Tyrc-2J/c-2J)to generate litters containing homozygous Tyrc-2J/c-2J embryos (‘albino’)and heterozygous Tyr+/c-2J embryos (‘pigmented’). Pigmented littermateswere used as controls for albino embryos. Females were checked for vaginalplugs at approximately noon each day. Embryonic day (E) 0.5 corresponds tothe day when the vaginal plug was detected, with the assumption thatconception took place at approximately midnight.

Microarray analysisGene expression profiling was performed on E13.5 isolated RPE. To isolateRPE, the cornea, the lens and the neural retina were detached from eyecupand extraocular tissue was removed. The isolated RPE eyecup was treated inice-cold RNAprotect cell reagent (Qiagen) and processed for RNA isolationusing RNeasy Plus Mini Kit (Qiagen). For each biological replicate(pigmented and albino), RNAwas isolated from pooled RPE tissues of threeor four littermate embryos, generating more than 100 ng/µl for each sample.RNA quantity and quality were assessed using Agilent 2100 Bioanalyzer(Agilent). RNA samples (100 ng for each as input RNA) were labeled withbiotin by 3′ IVT Expression Kit (Affymetrix). The labeled RNA washybridized on Mouse Genome 430 2.0 Array chips (Affymetrix) andanalyzed using GeneSpringGx11 software (Agilent). Differentiallyexpressed genes were identified from three biological replicates byaverage expression level greater than 20 in at least one population, at least1.5-fold differential expression and corrected P-value less than 0.05 byBenjamini-Hochberg multiple testing correction. Gene set size filters

Fig. 4. Lithium treatment doesnot affect retinal proliferation, asmeasured by PH3 expression. (A) VTretinal sections at E15.5 labeled with PH3(green), Isl1/2 (magenta) and Hoechst(blue). (B) For cell quantification, thedomain of the ciliary marginal zone (CMZ)was delineated by the border of Isl1/2expression (magenta). Two sectors inthe CMZ domain and four sectors inthe neural retina (NR) were quantified.(C) The numbers of PH3-positive cellsin the ventrotemporal and dorsotemporalCMZ and neural retina. One-way ANOVA(n=8 embryos from six litters). Barsrepresent mean. Error bars: ±s.e.m.ns, not significantly different.Scale bar: 100 µm.

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(min=15, max=500) of Gene Set Enrichment Analysis (GSEA) resulted infiltering out 1898/10295 gene sets. The remaining 8397 gene sets were usedin the analysis. GSEA sets with FDR (q value)<0.05 were hand-curated intothematic categories to highlight transcriptional differences betweenpopulations. Analysis was carried out with GSEA software from theBroad Institute (Cambridge, MA, USA).

Quantitative RT-PCRcDNA from E13.5 RPE sheets (collected from 10-12 embryos from three orfour litters) was retrotranscribed from purified RNA using Superscript IIIReverse Transcriptase (Invitrogen). Quantitative PCR (qPCR) was performedusing aStratageneMX3000withSYBRGreenPCRKit (AppliedBiosystems)and a Takara Thermal Cycler Dice with SYBR Fast qPCR kit (KapaBiosystems). Changes in gene expression were quantified using the 2−ΔΔCT

method with normalization to hypoxanthine phosphoribosyltransferase(HPRT). qPCR-specific primers are listed in Table S5. A total of threebiological independent experiments were performed in triplicate (n=3).

Tissue preparationFor in situ hybridization, embryos were fixed by immersion with 4%paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4) at 4°Covernight. After fixation, tissue was washed with PBS, cryoprotected with30% sucrose in PBS for 24 to 72 h at 4°C and frozen in dry ice. Forimmunohistochemistry, embryos were fixed by immersion in 4% PFA inPBS for 1 to 1.5 h and cryoprotected in 10% sucrose in PBS. Consecutivecoronal sections (20 µm) were collected through the retina with a cryostat(Leica Biosystems) on Fisher frosted microscope slides.

Lithium treatment was performed as previously described by Lancasteret al. (2011). Briefly, we injected intraperitoneally equimolar lithiumchloride or sodium chloride (10 µl of a 600 mM stock solution in normalsaline per gram body weight) in timed pregnant dams every 24 h from E12.5until E14.5.

In situ hybridizationIn situ hybridization was performed as described previously (Kuwajimaet al., 2012) with specific antisense DIG-labeled riboprobes for Wnt2b(a gift from Dr T. Jessell, Columbia University, NY, USA), Fzd7, Sfrp2and Shh (gifts from Dr P. Bovolenta, Universidad Autónoma de Madrid,Spain), Otx1 and Msx1 (gifts from Dr X. Zhang, Columbia University,NY, USA and Dr V. Wallace, University of Toronto, Canada), Bmp4(a gift fromDr J. Dodd, Columbia University, NY, USA), Fzd5 (a gift fromDr G. Papaioannou, Columbia University, NY, USA), Notch2 (a gift fromDr G. Fishell, Harvard University, Boston, MA, USA), and Axin2 (a giftfrom Dr G. Oliver, Northwestern University, Evanston, Il, USA andDr F. Constantini, Columbia University, NY, USA). For Cx43 and Sox2(Wang et al., 2016) and Wif1, the unique sequence was amplified by PCRfrom E13.5 mouse RPE cDNA using primers generated from the mousesequence. In some cases, to reduce the concentration of melanin in pigmentedRPE, which masks the in situ hybridization signals, sections were bleachedafter color reaction of in situ hybridization as described previously (Foss et al.,1995; Iwai-Takekoshi et al., 2016; Orchard and Calonje, 1998). In brief,sections were incubated with 0.25% KMnO4 (Sigma 23851) in PBS at roomtemperature for 30 min, washed in PBS and then incubated in 1% oxalic acid(Sigma, O-0376) at room temperature for 1 min and washed again in PBS.Experiments were repeated at least three times in different animals fromdifferent litters and produced consistent results.

ImmunohistochemistryFor immunohistochemistry, slides were rinsed in PBS and antigen retrievalwas performed prior to blocking by incubating slides in 10 mM sodiumcitrate and 0.05% Tween-20 (pH 6.0) at 95°C for 20 min. Slides wereblocked in 10% normal goat serum and 0.2% triton in PBS for 30 min,then incubated in primary antibodies diluted in 1% normal goat serum and0.2% triton in PBS overnight at 4°C. Incubation with specific secondaryantibodies was performed for 2 h at room temperature. The followingprimary antibodies were used: rabbit anti-Zic2 (1:10,000; a gift fromDr S. Brown, University of Vermont, Burlington, VT, USA), mouseanti-Isl1/2 (1:100; a gift from Drs S. Morton, Columbia University, NY,

USA and T. Jessell), rabbit anti-PH3 (1:200, Millipore, #07-081), anti-Lef1(1:200, Cell Signaling, #2230S). Secondary antibodies used includeddonkey anti-rabbit Alexa488 and Alexa594, and donkey anti-mouse IgGAlexa488 and Alexa594 (1:500, Life Technologies). Hoechst 33258 (LifeTechnologies, #H3569) 1 µg/ml in PBSwas used to counterstain cell nuclei.

Quantification of gene expressionThe Wnt2b-positive region in the eye was analyzed from sections of retinaafter in situ hybridization at E15.5, imaged using a Zeiss AxioImager M2microscope. Four retinal sections were analyzed from each animal: twosections anterior to the section with optic nerve exit, the section with opticnerve exit and one posterior to the section with optic nerve exit. Contoursof RPE perimeter and theWnt2b-positive region in theRPEwere traced at 20×magnification using Neurolucida software (v11, MBF Biosciences; RRID:SCR_001775). The ratio of theWnt2b-positive perimeter over RPE perimeterwas calculated for each retinal section and averaged from five animals.

The expression domains of Axin2 and Lef1 in tissue from lithium-treatedanimals were analyzed in sections of E15.5 retina after in situ hybridizationand immunohistochemistry. Two sections per embryo and three embryosper condition were analyzed. The contours of the peripheral retina (a sectorof 300 µm long, as measured on the superficial aspect of the retina) and theAxin2-positive region in this region were traced at 10× magnification usingImageJ (NIH; RRID: SCR_003070). The ratio of the Axin2-positive areaover the peripheral retinal area was calculated for dorsal and ventral retina ofeach retinal section. Average mean intensity±s.e.m. of Axin2 expression:control (NaCl) pigmented, 25.06%±2.13; control (NaCl) albino,38.50%±5.70; lithium pigmented, 32.61%±4.15; lithium albino,52.89%±5.67. For Lef1 expression, the contours of the Lef1-positiveregion in the CMZ were traced at 20× magnification using ImageJ. Themean intensity of the traced region was measured from dorsal and ventralCMZ in each section. Average mean intensity±s.e.m. of Lef1 expression:control (NaCl) pigmented, 89.32±4.99; control (NaCl) albino, 75.41±7.83;lithium pigmented, 101.90±9.04; lithium albino, 122.38±7.75.

Quantification of cell number in retinal sectionsImmunostained sections were imaged using a Zeiss AxioImager M2microscope equipped with ApoTome, AxioCam MRm camera andNeurolucida software as previously described (Bhansali et al., 2014). Amax projection merged stack of eight images (2 µm steps) was acquired withthe ApoTome and 20× objective and used for analysis. After all images wererenamed for blinding to genotype and treatment, background intensity wasmeasured by ImageJ. We counted cells that met a threshold fluorescenceintensity of at least three times the intensity of background. After subtractionof background, Zic2- and Isl1/2-positive cell numbers were quantifiedwithin a sector of 240 µm long as measured in the superficial aspect of theretina by using Meta Imaging Series Metamorph, as previously described(Bhansali et al., 2014). To delineate the region of interest, four consecutivesectors of 60 µm were traced starting at the most peripheral Isl1/2-positiveRGCs in VT retina. Total cell counts from two consecutive sectionscaudal to the optic nerve were obtained from one animal and averagedfrom seven animals.

PH3 cell number was counted within a 200 µm long sector of the ciliarymarginal zone (CMZ) and a 400 µm long sector from the ciliarymargin towardcentral retina (see Fig. 4B). To delineate the region corresponding to the CMZ,two consecutive 100 µm long sectors were traced from the most peripheralIsl1/2 cells to the peripheral tip of the retina. To delineate the regionscorresponding to the neural retina,VT retinawas divided into four consecutivesectors of 60 µm eachwith the first sector traced starting at the most peripheralIsl1/2 RGCs. Total cell counts from four consecutive sections (two rostral tothe optic nerve, one at the optic nerve and one caudal to the optic nerve) wereobtained from one animal and averaged from counts from eight animals.

Statistical analysisTo determine statistical significance, all data were analyzed with GraphPadInStat version 3.1. After we confirmed the data met the assumptions of thetests, statistical significance was determined using an unpaired t-test andone-way ANOVA. A two-tailed P-value was used. Error bars represents.e.m. *P<0.05; **P<0.01; ***P<0.001. For RT-qPCR analysis, all

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measurements were shown as dot plots instead of determining statisticalsignificance because the total number of biological experiments is three.

AcknowledgementsWe thank Mika Melikyan for mouse breeding; John Peregrin, Sania Khalid andCorrine Quirk for technical support; and Jane Dodd and members of the Mason labfor discussions and comments on the manuscript. We also thank Dr Lorraine Clarkand members at Genomics Core at the Taub Institute, Columbia University MedicalCenter for assistance with the gene expression analysis based on AffymetrixGeneChips.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: L.I.-T., C.M.; Validation: L.I.-T.; Formal analysis: L.I.-T.;Investigation: L.I.-T., R.B., A.S., R.K., S.W., K.R.; Data curation: L.I.-T.; Writing -original draft: L.I.-T.; Writing - review & editing: L.I.-T., R.B., C.M.; Visualization:L.I.-T.; Supervision: C.M.; Project administration: L.I.-T., C.M.; Funding acquisition:L.I.-T., R.B., C.M.

FundingThis work was supported by the National Institutes of Health (R01 EY012736,EY015290 and R21 EY023714 to C.M., and P30EY019007 to M. E. Goldberg); theVision of Children (to C.M.); Fight for Sight, Uehara Memorial Foundation, DaiichiSankyo Foundation and Hayashi Memorial Foundation (to L.I.-T.); and by a KnightsTemplar Eye Foundation Career-Starter Research Grant (to R.B.). Deposited inPMC for release after 12 months.

Data availabilityMicroarray data have been deposited in Gene ExpressionOmnibus under accessionnumber GSE121467.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.163212.supplemental

ReferencesBao, Z.-Z. and Cepko, C. L. (1997). The expression and function of Notch pathwaygenes in the developing rat eye. J. Neurosci. 17, 1425-1434.

Bhansali, P., Rayport, I., Rebsam, A. and Mason, C. (2014). Delayedneurogenesis leads to altered specification of ventrotemporal retinal ganglioncells in albino mice. Neural Dev. 9, 11.

Chenn, A. andWalsh, C. A. (2002). Regulation of cerebral cortical size by control ofcell cycle exit in neural precursors. Science 297, 365-369.

Cho, S.-H. and Cepko, C. L. (2006). Wnt2b/beta-catenin-mediated canonicalWnt signaling determines the peripheral fates of the chick eye. Development133, 3167-3177.

Cronin, C. A., Ryan, A. B., Talley, E. M. and Scrable, H. (2003). Tyrosinaseexpression during neuroblast divisions affects later pathfinding by retinal ganglioncells. J. Neurosci. 23, 11692-11697.

Drager, U. C. (1985). Calcium binding in pigmented and albino eyes. Proc. Natl.Acad. Sci. USA 82, 6716-6720.

Erskine, L. and Herrera, E. (2014). Connecting the retina to the brain. ASN Neuro6, 1-26.

Foss, A. J., Alexander, R. A., Jefferies, L. W. and Lightman, S. (1995).Immunohistochemical techniques: the effect of melanin bleaching. Br. J. Biomed.Sci. 52, 22-25.

Fujimura, N., Taketo, M. M., Mori, M., Korinek, V. and Kozmik, Z. (2009).Spatial and temporal regulation of Wnt/beta-catenin signaling is essential fordevelopment of the retinal pigment epithelium. Dev. Biol. 334, 31-45.

Ha, A., Perez-Iratxeta, C., Liu, H., Mears, A. J. and Wallace, V. A. (2012).Identification of Wnt/beta-catenin modulated genes in the developing retina.Mol. Vis. 18, 645-656.

Herrera, E., Brown, L., Aruga, J., Rachel, R. A., Dolen, G., Mikoshiba, K.,Brown, S. and Mason, C. A. (2003). Zic2 patterns binocular vision by specifyingthe uncrossed retinal projection. Cell 114, 545-557.

Herrera, E., Erskine, L. and Morenilla-Palao, C. (2017). Guidance of retinal axonsin mammals. Semin. Cell Dev. Biol. 17, 30299.

Iwai-Takekoshi, L., Ramos, A., Schaler, A., Weinreb, S., Blazeski, R. andMason, C. (2016). Retinal pigment epithelial integrity is compromised in thedeveloping albino mouse retina. J. Comp. Neurol. 524, 3696-3716.

Kubo, F., Takeichi, M. and Nakagawa, S. (2003). Wnt2b controls retinal celldifferentiation at the ciliary marginal zone. Development 130, 587-598.

Kuwajima, T., Yoshida, Y., Takegahara, N., Petros, T. J., Kumanogoh, A.,Jessell, T. M., Sakurai, T. and Mason, C. (2012). Optic chiasm presentation ofSemaphorin6D in the context of Plexin-A1 and Nr-CAM promotes retinal axonmidline crossing. Neuron 74, 676-690.

Lancaster, M. A., Gopal, D. J., Kim, J., Saleem, S. N., Silhavy, J. L., Louie, C. M.,Thacker, B. E., Williams, Y., Zaki, M. S. and Gleeson, J. G. (2011). DefectiveWnt-dependent cerebellar midline fusion in a mouse model of Joubert syndrome.Nat. Med. 17, 726-731.

Liu, H., Mohamed, O., Dufort, D. and Wallace, V. A. (2003). Characterization ofWnt signaling components and activation of the Wnt canonical pathway in themurine retina. Dev. Dyn. 227, 323-334.

Liu, H., Xu, S., Wang, Y., Mazerolle, C., Thurig, S., Coles, B. L. K., Ren, J.-C.,Taketo, M. M., van der Kooy, D. and Wallace, V. A. (2007). Ciliary margintransdifferentiation from neural retina is controlled by canonical Wnt signaling.Dev. Biol. 308, 54-67.

Marcucci, F., Murcia-Belmonte, V., Wang, Q., Coca, Y., Ferreiro-Galve, S.,Kuwajima, T., Khalid, S., Ross, M. E., Mason, C. and Herrera, E. (2016).The ciliary margin zone of the mammalian retina generates retinal ganglion cells.Cell Rep. 17, 3153-3164.

Martinez-Morales, J. R., Signore, M., Acampora, D., Simeone, A. andBovolenta, P. (2001). Otx genes are required for tissue specification in thedeveloping eye. Development 128, 2019-2030.

Megason, S. G. and McMahon, A. P. (2002). A mitogen gradient of dorsal midlineWnts organizes growth in the CNS. Development 129, 2087-2098.

Orchard, G. E. and Calonje, E. (1998). The effect of melanin bleaching onimmunohistochemical staining in heavily pigmented melanocytic neoplasms.Am. J. Dermatopathol. 20, 357-361.

Petros, T. J., Rebsam, A. andMason, C. A. (2008). Retinal axon growth at the opticchiasm: to cross or not to cross. Annu. Rev. Neurosci. 31, 295-315.

Rebsam, A., Bhansali, P. and Mason, C. A. (2012). Eye-specific projections ofretinogeniculate axons are altered in albino mice. J. Neurosci. 32,4821-4826.

Swayne, L. A. and Bennett, S. A. L. (2016). Connexins and pannexins in neuronaldevelopment and adult neurogenesis. BMC Cell Biol. 17 Suppl. 1, 10.

Tibber, M. S., Becker, D. and Jeffery, G. (2007). Levels of transient gap junctionsbetween the retinal pigment epithelium and the neuroblastic retina are influencedby catecholamines and correlate with patterns of cell production. J. Comp. Neurol.503, 128-134.

Umino, Y. and Saito, T. (2002). Spatial and temporal patterns of distribution of thegap junctional protein connexin43 during retinal regeneration of adult newt.J. Comp. Neurol. 454, 255-262.

Wang, Q., Marcucci, F., Cerullo, I. and Mason, C. (2016). Ipsilateral andcontralateral retinal ganglion cells express distinct genes during decussation atthe optic chiasm. eNeuro 3, 1-28.

Zhao, S., Chen, Q., Hung, F. C. and Overbeek, P. A. (2002). BMP signaling isrequired for development of the ciliary body. Development 129, 4435-4442.

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