Temporal profiling of photoreceptor lineage gene expression during murine retinal development

Gene Expression Patterns 23-24 (2017) 32e44

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Temporal profiling of photoreceptor lineage gene expression during murine retinal development Tooka Aavani a, b, Nobuhiko Tachibana a, c, Valerie Wallace d, Jeffrey Biernaskie e, Carol Schuurmans a, b, c, * a

Sunnybrook Research Institute, Biological Sciences Platform, Room S1-16, 2075 Bayview Ave, Toronto, ON, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Department of Biochemistry and Molecular Biology, Cumming School of Medicine, Alberta Children's Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, T2N 4N1, Canada d Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network and Department of Ophthalmology and Vision Sciences and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada e Department of Comparative Biology and Experimental Medicine, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 November 2016 Received in revised form 3 February 2017 Accepted 7 March 2017 Available online 10 March 2017

Rod and cone photoreceptors are photosensitive cells in the retina that convert light to electrical signals that are transmitted to visual processing centres in the brain. During development, cones and rods are generated from a common pool of multipotent retinal progenitor cells (RPCs) that also give rise to other retinal cell types. Cones and rods differentiate in two distinct waves, peaking in mid-embryogenesis and the early postnatal period, respectively. As RPCs transition from making cones to generating rods, there are changes in the expression profiles of genes involved in photoreceptor cell fate specification and differentiation. To better understand the temporal transition from cone to rod genesis, we assessed the timing of onset and offset of expression of a panel of 11 transcription factors and 7 non-transcription factors known to function in photoreceptor development, examining their expression between embryonic day (E) 12.5 and postnatal day (P) 60. Transcription factor expression in the photoreceptor layer was observed as early as E12.5, beginning with Crx, Otx2, Rorb, Neurod1 and Prdm1 expression, followed at E15.5 with the expression of Thrb, Neurog1, Sall3 and Rxrg expression, and at P0 by Nrl and Nr2e3 expression. Of the non-transcription factors, peanut agglutinin lectin staining and cone arrestin protein were observed as early as E15.5 in the developing outer nuclear layer, while transcripts for the cone opsins Opn1mw and Opn1sw and Recoverin protein were detected in photoreceptors by P0. In contrast, Opn1mw and Opn1sw protein were not observed in cones until P7, when rod-specific Gnat1 transcripts and rhodopsin protein were also detected. We have thus identified four transitory stages during murine retina photoreceptor differentiation marked by the period of onset of expression of new photoreceptor lineage genes. By characterizing these stages, we have clarified the dynamic nature of gene expression during the period when photoreceptor identities are progressively acquired during development. © 2017 Published by Elsevier B.V.

Keywords: Retinal progenitor cells Rod and cone photoreceptors Transcription factors Temporal sequence Mouse

1. Introduction The neural retina is a laminar structure that is comprised of one glial and six neuronal cell types that are organized into three cellular layers. The outer nuclear layer (ONL), which lies on the apical side of the retina, contains the cell bodies of cone and rod

* Corresponding author. Sunnybrook Research Institute, Biological Sciences Platform, Room S1-16, 2075 Bayview Ave, Toronto, ON, MRN 3M5, Canada. E-mail address: [email protected] (C. Schuurmans). http://dx.doi.org/10.1016/j.gep.2017.03.001 1567-133X/© 2017 Published by Elsevier B.V.

photoreceptors. The inner nuclear layer (INL), which is in the center of the retina, is populated by amacrine, horizontal and bipolar cell interneurons as well as Müller glia, which are the only glial cells generated in the retina. Finally, the ganglion cell layer (GCL), on the basal side of the retina, contains ganglion cells and displaced amacrine cells. In addition, the retinal pigment epithelium (RPE) is a non-neural epithelial cell layer that overlies the ONL. Eye formation begins with an evagination of the diencephalon at E8.5 in mice, leading to the development of bilateral optic pits that contact the head ectoderm to give rise to optic vesicles by E9.0 (Fuhrmann, 2010; Wawersik and Maas, 2000). Inductive signals

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from the optic vesicles cause the formation of lens placodes in the overlying ectoderm by E9.5. By E10.0, there is a coordinated invagination of the lens placode and the optic vesicle, resulting in the formation of a lens pit and double-layered optic cup (Fuhrmann, 2010; Wawersik and Maas, 2000). The inner layer of the optic cup adjacent to the lens gives rise to the neural retina, while the outer layer gives rise to the RPE. In the murine retina, cellular differentiation begins at E10 and lasts until P5-6 in the central retina and P11 in the periphery, followed by eye opening at P14 (Cepko, 1996; Young, 1985). Lineage tracing experiments using retroviral gene transfer, DNA or dye labelling, epitope tagging, or clonal density cultures demonstrated that early RPCs are multipotent, giving rise to all seven retinal cell types (Alexiades and Cepko, 1997; Fekete et al., 1994; Holt et al., 1988; Jensen and Raff, 1997; Moody et al., 2000; Turner and Cepko, 1987; Turner et al., 1990; Wetts and Fraser, 1988). Retinal cells are generated in a sequential albeit overlapping order that is conserved among vertebrates (Marquardt and Gruss, 2002; Wassle and Boycott, 1991). RGCs are the first cells to differentiate, followed closely and in an overlapping manner by horizontal cells, cone photoreceptors and amacrine cells, all of which are primarily generated during the embryonic period in mouse (Cepko, 1996, 1999; Young, 1985). Rod photoreceptors, bipolar cells and Müller glia cells are the next cell types to be generated, also in a highly overlapping fashion, peaking during the early postnatal period (Cepko, 1999; Cepko et al., 1996; Young, 1985). While lineage tracing experiments support the existence of multipotent RPCs, committed precursors that give rise to specific retinal cell types also exist (Pearson and Doe, 2004). RPCs give rise to committed photoreceptor precursors that progress to fully differentiated photoreceptors expressing genes involved in phototransduction and morphogenesis (Swaroop et al., 2010). Rod development occurs in an early and late phase, with some rod precursors generated as early as E14, but most generated in the late phase beginning after E19, peaking at P4 and extending to P10 (Cepko et al., 1996; Morrow et al., 1998; Rapaport et al., 2004). Notably, the onset of rhodopsin expression, a terminal differentiation marker of a rod fate, does not occur until 5.5e6.5 days after rod precursors exit the cell cycle (or 8.5e12.5 days for early-born rods) (Morrow et al., 1998). In contrast, cone precursors are generated as early as E11, peaking at E13-E16, and completing their genesis by P0 (Carter-Dawson and LaVail, 1979; Rapaport et al., 2004). Similar to rods, the expression of mature cone markers is delayed, with Opn1sw transcripts detected at around P0, several days after most cones have already exited the cell cycle (Swaroop et al., 2010). Several transcription factors have been identified that control photoreceptor fate specification and differentiation, but a comprehensive comparison of the onset and offset of their expression has not been reported. A temporal analysis of photoreceptor gene expression is warranted as current attempts to generate induced photoreceptors by cellular reprogramming requires a detailed understanding of normal lineage progression. We therefore characterized the temporal expression patterns of a panel of 11 transcription factors and 7 non-transcription factors previously implicated in the development of photoreceptor lineages. 2. Results and discussion 2.1. Onset of gene expression in photoreceptor lineages at earliest embryonic stage (E12.5) We set out to perform a temporal analysis of gene expression in murine photoreceptor lineages at seven different stages of retinal development (E12.5, E15.5, P0, P7, P14, P21, P60), encompassing the period of cone (E12.5-P0) and rod (E15.5-P14) genesis and

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photoreceptor differentiation, maturation and maintenance (P14P60). The eleven transcription factors we examined included members of the homeodomain (Cone-rod homeobox (Crx), Orthodenticle homeobox 2 (Otx2)), nuclear hormone receptor (RAR-related orphan receptor beta (Rorb), Thyroid hormone receptor beta (Thrb), Retinoid X receptor gamma (Rxrg), Nuclear receptor subfamily 2, group E, member 3 (Nr2e3)), basic-helix-loop-helix (bHLH) (Neurogenic differentiation 1 (Neurod1), Neurogenin 1 (Neurog1)), zinc finger (Spalt like transcription factor 3 (Sall3), PR domain containing 1, with ZNF domain (Prdm1)) and neural zipper (Neural retina leucine zipper gene (Nrl)) families. In addition, we examined seven nontranscription factor markers, including those labeling cones (Arrestin 3 (Arr3), Opsin 1 (cone pigments), medium-wave-sensitive (color blindness, deutan) (Opn1mw), Opsin 1 (cone pigments), shortwave-sensitive (color blindness, tritan) (Opn1sw), peanut agglutinin (PNA e a lectin), rods (Guanine nucleotide binding protein, alpha transducin 1 (Gnat1), Rhodopsin (Rho)) and both rods and cones (Recoverin (Rcvrn)). We began our analysis at E12.5, just after the first cone photoreceptor precursors have exited the cell cycle. RNA in situ hybridization on tissue sections revealed that at E12.5, transcripts for Crx (Fig. 1A,A0 ), Otx2 (Fig. 1H,H0 ), Rorb (Fig. 1O,O0 ), Neurod1 (Fig. 1V,V0 ) and Prdm1 (Fig. 1CC,CC0 ) were detected in the upper outer neuroblast layer (ONBL), which we have designated the presumptive ONL (pONL). In contrast, none of the other genes that we analysed were expressed at this time point (Figs. S1e3). Between E12.5 and P0, Crx expression was detected in the pONL and in scattered cells throughout the ONBL, where dividing RPCs are giving rise to committed precursors for different neuronal lineages (Fig. 1AeC,A0 -C0 ). By P7, when differentiation is complete in the central retina, and up to P60, Crx transcripts were primarily localized to the ONL, with an additional narrow band of Crxþ cells also detected in the upper INL, in a subset of bipolar cells (Fig. 1DeG,D0 -G0 ), as previously described (Samson et al., 2009). Crx is a transcriptional target of Otx2 and is required for both cone and rod development (Chen et al., 1997; Furukawa et al., 1997; Nishida et al., 2003). While photoreceptors are properly specified in Crx null mutants, these cells do not differentiate normally; outer segments do not form properly, synaptic terminals and phototransduction pathways are defective, mature markers are downregulated (e.g., Rho, Arr) and photoreceptor degeneration ensues (Chen et al., 1997; Furukawa et al., 1999; Livesey et al., 2000; Morrow et al., 2005). Otx2 transcripts were similarly detected in the pONL and in scattered cells throughout the ONBL as early as E12.5, peaking at P0 of development, with additional expression also detected in the developing inner neuroblast layer (INBL), where differentiating INL and GCL cells accumulate, and in the RPE (Fig. 1HeJ,H0 -J0 ). However, by P7, Otx2 was expressed in only a few scattered cells in the ONL and RPE, with predominant expression instead detected in bipolar cells in the INL, as previously shown (Koike et al., 2007), a pattern that was maintained between P14-P60 (Fig. 1KeN,K0-N0 ). Otx2 is thought to be the earliest acting transcription factor in the photoreceptor lineage, similar to its Drosophila ortholog, orthodentricle (otd) (Finkelstein and Boncinelli, 1994), which is also an ortholog of Crx (Plouhinec et al., 2003; Tahayato et al., 2003). Conditional retinal deletion of Otx2 results in a complete loss of both rod and cone photoreceptors, and a reduction in expression of several key genes involved in photoreceptor development and survival, including Crx, Nrl, Nr2e3 and Neurod1 (Nishida et al., 2003; Omori et al., 2011). In contrast, overexpression of Otx2 promotes ectopic photoreceptor cell genesis (Nishida et al., 2003). Otx2 specifies a photoreceptor fate in part by inducing the expression of Crx (Beby and Lamonerie, 2013; Chen et al., 1997; Furukawa et al., 1997; Hennig et al., 2008). Additional genes that function downstream of Otx2 are Vsx2 and Prdm1, with the loss of Prdm1 also leading to a

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Fig. 1. Temporal expression profiles of early onset transcription factors in the ONL (E12.5). Section RNA in situ hybridization to assess the expression of Crx (A-G,A0 -G0 ), Otx2 (HN,H0 -N0 ), Rorb (O-U,O0 -U0 ), Neurod1 (V-Z,AA,BB,V0 -Z0 ,AA0 ,BB0 ) and Prdm1 (CC-II,CC'-II0 ) in the retina at E12.5, E15.5, P0, P7, P14, P21 and P60. Higher magnification images of boxed areas are shown in A0 -Z0 and AA0 -II'. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer; onbl, outer neuroblastic layer; os, outer segment; p-onl, presumptive outer nuclear layer; re, retina; Scale bars, 250 mm.

loss of rod photoreceptors (Brzezinski et al., 2010). Rorb was also expressed in the developing pONL between E12.5 and P0, but it had much higher levels of expression in RPCs in the ONBL, and in differentiating cells in the INBL, as previously reported (Chow et al., 1998; Ji et al., 2014), compared to Crx and Otx2 (Fig. 1OeQ,O0 -Q0 ). However, by P7, Rorb expression had declined in the ONL and was instead predominant in the INL, with INL/GCL-

specific expression persisting between P14 and P60. This is in line with previous studies highlighting the expression of Rorb in bipolar cells in the INL (Lai et al., 2015) (Fig. 1ReU,R0 -U0 ). Rorb is required for rod differentiation, as mutant mice generate many fewer rods and instead overproduce S-cones, in part due to the loss of Nrl expression, which is an essential determinant of a rod fate (Jia et al., 2009; Swaroop et al., 2010). There are two isoforms of Rorb; Rorb1 is

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required to initiate Nrl expression as well as Rorb2 expression, with Rorb2 playing a role in maintaining Nrl expression at a threshold level required to specify a rod fate (Fu et al., 2014). Notably, S-cones are poorly differentiated in Rorb mutants, indicating that this transcription factor is also required for cone maturation, in part because it is required to initiate Opn1sw transcription in cooperation with Crx (Srinivas et al., 2006). Neurod1 transcripts were also detected in the developing pONL and in scattered ONBL and INBL cells from E12.5 to P0 (Fig. 1VeX,V0 X0 ). Neurod1 transcripts were also detected in the ONL and to a lesser extent, the INL at P7, but from P14, expression started to taper off and by P60, Neurod1 expression was no longer detectable (Fig. 1YeZ,Y0-Z0 ,AA-BB,AA0 eBB0 ). Neurod1 mutants develop agerelated rod photoreceptor degeneration, indicating a late role for this gene in photoreceptor cell survival (Morrow et al., 1999; Pennesi et al., 2003). However, more than one bHLH transcription factor is required for photoreceptor genesis, as it is only in triple mutants of members of the bHLH gene family (i.e., Ascl1;Math3;Neurod1 triple knock-outs) that photoreceptor differentiation is perturbed (Akagi et al., 2004). Finally, Prdm1 transcripts were also detected in the pONL beginning at E12.5, peaking in expression at P0, when transcripts were also detected throughout the ONBL and in the INBL (Fig. 1CCEE,CC0 -EE0 ). While Prdm1 transcripts were detected in some, albeit fewer, ONL cells at P7 and P14, Prdm1 expression was no longer detectable in the ONL at P21 and P60 (Fig. 1FF-II,FF0 eII0 ). Prdm1 acts downstream of Otx2 to promote a photoreceptor fate, with fewer rod and cone photoreceptors generated in Prdm1 mutants (Katoh et al., 2010). In summary, of the transcription factors expressed at the earliest stage of photoreceptor development that we analysed, Otx2 and Prdm1 are required for the differentiation of both rods and cones, Crx and Neurod1 are required for the survival of these two photoreceptor populations, and Rorb is essential to specify a rod fate, and for proper cone differentiation. There therefore does not appear to be a cone-specific signature at E12.5, even though cones are the only photoreceptors differentiating in this early temporal window. In this regard it is interesting that rods are derived from S-cone lineages during evolution, resulting in rods and S-cones sharing some gene expression profiles (Kim et al., 2016). 2.2. Onset of gene expression in photoreceptor lineages in midembryogenesis (E15.5) We next assessed gene expression by RNA in situ hybridization at E15.5, at the peak of cone photoreceptor differentiation, and at a time point when rod photoreceptors are just beginning to differentiate. At this stage, we saw the expression of four additional transcription factors in the pONL, namely Thrb (Fig. 2A,A0 ), Neurog1 (Fig. 2F,F0 ), Sall3 (Fig. 2K,K0 ) and Rxrg (Fig. 2P,P0 ). None of these four genes were expressed at E12.5 (Fig. S1), suggesting that their expression is initiated in the developing photoreceptor layer between E12.5 and E15.5. Thrb transcripts were detected in the pONL of E15.5 and P0 retinas, with lower levels of expression observed in the lower ONBL (Fig. 2AeB,A0 -B0 ). In contrast, Thrb expression was no longer detected in the retina between P7 and P60 (Fig. 2CeE,C0 -E0 , data not shown). Thrb plays a critical role in the selection of an M-cone cell fate, and is expressed in Nrl-negative photoreceptor precursors that are destined to become cones (Ng et al., 2001; Roberts et al., 2005; Xiao and Hendrickson, 2000). Within photoreceptor precursors, Thrb represses Opn1sw transcription and transactivates Opn1mw (Ng et al., 2001; Roberts et al., 2005; Xiao and Hendrickson, 2000). Notably, M- and S-cones have opposing gradients, and the loss of Thrb (and hence, M-cones), perturbs the normal distribution of S-

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cones, suggesting that these gradients develop in a co-dependent fashion (Roberts et al., 2005). A cone-specific cis-regulatory module has been identified in the upstream regulatory region of Thrb, which is regulated by Otx2 and Onecut1 (Emerson et al., 2013). Notably, Onecut1 is expressed in very early cones, and loss of this factor leads to precocious rod differentiation (Emerson et al., 2013). While we did not detect Neurog1 transcripts in the E12.5 retina (Fig. S1), it was expressed at elevated levels in E15.5 and P0 retinas, marking photoreceptor precursors in the pONL, RPCs in the ONBL, and differentiating cells in the INBL in a salt-and-pepper-like pattern characteristic of proneural genes (Fig. 2FeG,F0 -G0 ). By P7 and through to P60, Neurog1 expression was no longer detected in the retina (Fig. 2HeJ,H0 -J0 , data not shown). As a proneural gene, Neurog1 has been shown to play a critical role in cell specification and differentiation in different lineages in the CNS and PNS (Fode et al., 2000; Ma et al., 1999). In the retina, Neurog1 is required in chick for photoreceptor differentiation in vitro (Yan et al., 2009). Conversely, overexpression of Neurog1 promotes the generation of ectopic retinal cells, including photoreceptors (Li et al., 2010; Yan et al., 2009, 2015). Sall3 expression was expressed at E15.5 (Fig. 2K), and not at E12.5 (Fig. S1), with transcripts detected in a small number of presumptive photoreceptors scattered throughout the upper pONL and in RPCs in the ONBL at E15.5 and P0 (Fig. 2K,L,K0 ,L0 ). By P7, Sall3 expression was nearly lost in the ONL and instead was predominant in the upper INL (Fig. 2M,M0 ). The expression of Sall3 in horizontal cells in the INL is in line with previous reports (de Melo et al., 2011). At P14, P21 and P60, Sall3 expression was no longer detected in the retina (Fig. 2N-O,N0 -O0 , data not shown). The early expression of Sall3 in the ONL is consistent with its role in inducing S-cone specific gene expression (de Melo et al., 2011; Domingos et al., 2004; Mollereau et al., 2001; Sprecher et al., 2007). Finally, Rxrg expression was not detected at E12.5 (Fig. S1), but was expressed in a small number of cells in the developing photoreceptor layer at E15.5 and P0, in addition to being expressed in the INBL, where differentiating INL/GCL cells are accumulating (Fig. 2PeQ,P0 -Q0 ). However, by P7, Rxrg was only expressed in a few scattered cells in the ONL, and no expression was detected at P14, P21 and P60 (Fig. 2ReT,R0 -T0, data not shown). Rxrg acts together with Thrb to suppress Opn1sw expression, influencing the choice between M- and S-cone acquisition (Roberts et al., 2005). Of note, three of the four transcription factors that begin to be expressed between E12.5 and E15.5 play a role in the specification or differentiation of M- and S-cones (i.e., Thrb, Sall3, Rxrg), implicating this stage as a critical period for the decision by photoreceptors to acquire a particular cone fate. Neurog1 has been studied in less detail, so it may be that this proneural gene also plays a role in the differentiation of a particular subtype of cone photoreceptors. 2.3. Onset of transcription factor gene expression in photoreceptor lineages at P0 We next assessed gene expression by RNA in situ hybridization at P0, when cone genesis has ended (although these cells are still undergoing terminal differentiation) and at the peak of rod photoreceptor precursor production. At this stage, we saw the expression of two additional transcription factors in the ONL, namely Nrl and Nr2e3, which were not expressed at E15.5 (Fig. S2), indicating that they initiate their expression between E15.5 and P0. Nrl transcripts were detected throughout the pONL at P0, and in scattered cells in the ONBL, likely corresponding to newborn photoreceptor precursors (Fig. 3A,A0 ). By P7 and through to P60, Nrl transcripts were specifically detected throughout the ONL and not in other sites of the retina (Fig. 3BeE,B0 -E0 ). Consistent with this

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Fig. 2. Temporal expression profiles of mid-onset transcription factors in the ONL (E15.5). Section RNA in situ hybridization showing the expression of Thrb (A-E,A0 -E0 ), Neurog1 (F-J,F0 -J0 ), Sall3 (K-O,K0-O0 ) and Rxrg (P-T,P0 -T0 ) in the retina at E15.5, P0, P7, P14, and P21. Higher magnification images of boxed areas are shown in A0 -T'. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer; onbl, outer neuroblastic layer; os, outer segment; p-onl, presumptive outer nuclear layer; re, retina; Scale bars, 250 mm.

expression profile, Nrl is an important determinant of a rod cell fate, with Nrl mutants generating all cone-like photoreceptors (cods) instead of rods (Mears et al., 2001; Swaroop et al., 2010). An important transcriptional target of Nrl is Nr2e3, which acts with Nrl to induce rod genes and turn off cone genes and initiate rod gene transcription (Chen et al., 2005; Cheng et al., 2004, 2011; Haider et al., 2000). Notably, the conversion of rods to cone-like cells in Nrl mutants has allowed investigators to identify several new genes specifically expressed in cone lineages by performing transcriptional profiling in Nrl mutant retinas (Corbo et al., 2007). Nr2e3 transcripts were also detected in the pONL at P0, albeit at low levels (Fig. 3F,F0 ), and not at E15.5 (Fig. S2). By P7 and through

to P60, Nr2e3 transcripts were detected throughout the ONL and at even higher levels in the outer segments (Fig. 3GeJ,G0 -J0 ). Nr2e3 is a direct transcriptional target of Nrl and is also involved in suppressing the expression of cone photoreceptor markers and in transactivating rod photoreceptor markers in concert with Crx (Chen et al., 2005; Cheng et al., 2004, 2011; Haider et al., 2000; Peng et al., 2005). Thus, the two transcription factors that initiate their expression in photoreceptor lineages between E15.5 and P0 are both involved in specifying a rod fate and repressing the acquisition of a cone fate, consistent with the decline in cone genesis and peak of rod genesis occurring at this time.

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Fig. 3. Temporal expression profiles of late-onset transcription factors in the ONL (P0). Section RNA in situ hybridization showing the expression of Nrl (A-E,A0 -E0 ) and Nr2e3 (FJ,F0 -J0 ) in the retina at P0, P7, P14, P21 and P60. Higher magnification images of boxed areas are shown in A0 -J'. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer; onbl, outer neuroblastic layer; os, outer segment; p-onl, presumptive outer nuclear layer; re, retina; Scale bars, 250 mm.

2.4. Onset of expression of cone differentiation markers in midembryogenesis (E15.5)

2.5. Initiation of cone and rod differentiation marker expression at P0

We next assessed the expression of well-known differentiation markers for both cone (PNA, Arr3, Opn1sw, Opn1mw), rod (Rhodopsin, Gnat1) and rod and cone (Rcvrn) lineages so as to better understand photoreceptor lineage progression. At E15.5, when cone differentiation is at its peak and rod photoreceptors precursors are only beginning to be produced, only two cone markers were detected, namely PNA lectin staining (Fig. 4A,A0 ) and Arr3 protein (Fig. 4G,G0 ). The early detection of cone markers has been previously confirmed for PNA and Rxrg (Fei, 2003; Roberts et al., 2005). Neither PNA nor Arr3 were expressed at E12.5 (Fig. S1), indicating that the expression of these two cone differentiation markers initiates between E12.5 and E15.5. PNA lectin binding was detected in the outer segments of developing cone photoreceptors at E15.5, and this labeling was maintained at P0 and through to P60 (Fig. 4AeF,A0 -F0 ). PNA is a lectin that specifically binds to intracellular and extracellular matrix material in cone photoreceptors (Blanks and Johnson, 1984). Arr3 was similarly detected in cone outer segments at E15.5 and persisting through the postnatal period to P60 (Fig. 4GeL,G0 -L0 ). Arr3 plays an important role in phototransduction by binding to light-activated phosphorylated cone opsins (Nikonov et al., 2008). Mice that are deficient for cone arrestin display defects in contrast sensitivity and visual acuity, and over time, develop a cone dystrophy (Deming et al., 2015). Thus, sometime between E12.5 and E15.5, when transcription factors involved in the specification and differentiation of cone photoreceptors begin to be expressed, we also see the onset of detection of two early markers of a cone fate e PNA lectin labeling and Arr3 protein.

We next examined the expression of rod and cone differentiation markers at P0, when cone genesis has ended and rod photoreceptor precursor production is at its peak. At P0, we detected transcripts for Opn1mw (Fig. 5A,A0 ,A00 ) and Opn1sw (Fig. 5F,F0,F00 ), which label M- and S-cones, respectively, as well as Rcvrn protein (Fig. 5K,K0 ), which marks rods and cones, in the pONL. In contrast, none of these markers were expressed in the E15.5 retina, indicating that they turn on sometime between E15.5 and P0 (Fig. S2). Opn1mw transcripts were detected in the upper pONL, where inner and outer segments begin to develop, as early as P0 and persisting through to P60 (Fig. 5AeE). However, significantly fewer cone photoreceptors expressed Opn1mw in ventral domains of the retina (Fig. 5A0 -E0 ) compared to dorsal domains, indicative of a dorsal-bias in expression (Fig. 5A00 -E00 ), consistent with previous reports (Daniele et al., 2011). Opn1mw encodes a green sensitive opsin that has sensitivity to medium-long wavelength light, and Thrb, which is first expressed at E15.5 in the pONL, is known to be a direct transcriptional activator of Opn1mw (Nathans et al., 1986; Szel et al., 1993, 2000). Opn1sw transcripts were similarly detected in the upper pONL where inner and outer segments begin to develop, at P0 and through to P60 (Fig. 5FeJ). Opn1sw transcripts were distributed in an opposing gradient to Opn1mw, with higher expression in the ventral retina (Fig. 5F0 -J0 ) and lower expression in the dorsal retina (Fig. 5F00 -J00 ), as demonstrated previously (Daniele et al., 2011). Opn1sw encodes a blue-sensitive opsin that reacts to short wavelength light (Nathans et al., 1986; Szel et al., 1993, 2000). Interestingly, the deletion of Opn1sw does not result in the death of Scones, in part due to a compensatory increase in Opn1mw expression (Daniele et al., 2011). However, the distribution of M-cones is perturbed in the absence of Opn1sw (Daniele et al., 2011).

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Fig. 4. Temporal expression profiles of early-onset cone differentiation markers in the ONL (E15.5). Staining of E15.5, P0, P7, P14, P21 and P60 retinas with two cone markers, the lectin PNA (A-F,A0 -F0 ) and an antibody to cone Arr3 (G-L,G0 -L0 ). Higher magnification images of boxed areas are shown in A0 -L'. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer; onbl, outer neuroblastic layer; os, outer segment; p-onl, presumptive outer nuclear layer; re, retina; Scale bars, 250 mm.

Finally, we observed that Rcvrn protein was detected in the pONL and in developing inner and outer segments at P0 (Fig. 5K,K0 ), with expression widespread throughout the photoreceptor layer from P7-P60 (Fig. 5LeO,L0 -O0 ). Rcvrn is a calcium binding protein from the calmodulin family that regulates guanylate cyclase activity in rod and cone photoreceptors (Dizhoor et al., 1991; Polans et al., 1991, 1993; Sampath et al., 2005; Vinberg et al., 2015). Thus, at P0 we observe the expression of the cone opsins, which are late differentiation markers in M- and S-cone lineages. The timing of detection of Opn1mw and Opn1sw expression coincided with the onset of transcriptional inhibitors of a cone fate (i.e., Nrl, Nr2e3). However, it may be that cone opsin expression initiates a few days before these transcriptional repressors turn on, or these transcription factors may not be able to repress cone opsins at this ‘early’ developmental stage, or Nrl/Nr2e3 and Opn1sw/mw may be expressed in distinct populations of cells. Future studies would be required to distinguish between these possibilities. In contrast, the onset of Rcvrn expression at P0 correlates with the onset of Nr2e3 and Nrl expression, transcription factors that play an active role in initiating the transcription of rod differentiation markers. 2.6. Onset of expression of late rod and cone differentiation markers at P7 Finally, we examined the expression of rod and cone differentiation markers at P7, when cones are undergoing terminal differentiation and rods are still being generated. At this stage, we saw the onset of detection of Gnat1 transcripts, as well as Opn1mw, Opn1sw and Rhodopsin protein (Fig. 6), none of which were expressed at P0 (Fig. S3). Gnat1 transcripts were first detected in the ONL at P7, with apparent higher levels of expression at P14, and persistent expression until P60 (Fig. 6AeD,A0 -D0 ). Gnat1 encodes a transducin that localizes to the plasma membrane of photoreceptors, where it plays a critical role in phototransduction (Carrigan et al., 2015).

Gnat1 mutants display an absolute absence of electrical response from rod photoreceptors, whereas cone photoreceptors respond normally to light (Carrigan et al., 2015). Over time, retinal degeneration occurs in Gnat1 knock-out mice, resulting in blindness after 13 weeks postnatal (Calvert et al., 2000). By P7 we were also able to detect Opn1mw (Fig. 6EeE00 ) and Opn1sw (Fig. 6IeI00 ) protein, appearing one week after transcripts were detected for these two cone opsins. Similar to the distribution of Opn1mw transcripts, Opn1mw protein was detected in a low ventral to high dorsal gradient from P7 to P60 (Fig. 6EeH,E0 -H0 ,E00 H00 ). Conversely, Opn1sw was expressed in a high ventral to low dorsal gradient between P7 and P60 (6I-L,I0 -L0 ,I00 -L00 ). Notably, while we observed Opn1mw (Fig. 6E0 ,E00 ) and Opn1sw (Fig. 6L00 ) staining in the GCL, this is non-specific background staining as we do not see transcripts for these proteins in the GCL (Fig. 5). Finally, at P7 we also observed the initiation of Rhodopsin expression in the ONL and inner and outer segments, an expression pattern that persisted up to P60 (Fig. 6MeP,M0 -P0 ). Rhodopsin is a rod opsin, and interestingly, Rhodopsin null mice undergoing rod photoreceptor degeneration by 2.5 months postnatal, demonstrating that this visual pigment is required for photoreceptor survival (Humphries et al., 1997). Thus, between P0 and P7 we see the onset of expression of terminal differentiation markers for both rods and cones, several days after these cells have exited the cell cycle and begun to differentiate. As reported in previous studies, cell cycle exit is inferred by the expression of Nrl and Crx (Akimoto et al., 2006; Garelli et al., 2006; Hennig et al., 2008). The photoreceptor opsins are in particular late markers of photoreceptor lineages. 3. Summary Photoreceptor cell differentiation occurs over a wide temporal window. After exit from their final mitosis, committed cone and rod photoreceptor precursor mature over weeks or months, depending

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Fig. 5. Temporal expression profiles of mid-onset rod and cone differentiation markers in the ONL (P0). Section RNA in situ hybridization showing the expression of Opn1mw (A-E) and Opn1sw (F-J) at both low (A-E,F-J) and high (A0 -E0 ,F0 -J0 ,A00 -E00 ,F00 -J00 ) magnification of boxed areas in the ventral (A0 -E0 ,F0 -J0 ) and dorsal (A00 -E00 ,F00 -J00 ) retina at P0, P7, P14, P21 and P60. Section immunohistochemistry showing the expression of Rcvrn protein at low (K-O) and high (K0-O') magnification at P0, P7, P14, P21 and P60. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer; onbl, outer neuroblastic layer; os, outer segment; p-onl, presumptive outer nuclear layer; re, retina; Scale bars, 250 mm.

on the species. To better understand the stages of photoreceptor maturation, we characterized the temporal patterns of expression of 11 homeodomain, bHLH, nuclear hormone receptor, zinc finger and leucine zipper family transcription factors previously implicated in photoreceptor development, as well as 7 non-transcription factors involved in various stages of photoreceptor differentiation, in the murine retina. With this study we were able to identify four stages of photoreceptor development characterized by the onset of expression of key genes (Fig. 7). The first phase (E12.5), which was the earliest stage analysed in our study, was characterized by the expression of transcription factors essential in both rod and cone lineages (Otx2, Prdm1, Crx, Neurod1, Rorb), even though only cone

photoreceptors are differentiating at this time. There therefore does not appear to be a cone-specific early-gene expression signature, possibly because rods develop from the S cone lineage (Kim et al., 2016). In contrast, the second phase of photoreceptor development (E15.5) was characterized by the expression of several genes involved in cone specification and differentiation (i.e., Thrb, Sall3, Rxrg), and one factor important in photoreceptor lineages (Neurog1) for which a cone-specific function has not been ascribed. Corresponding to the onset of expression of cone-specific transcription factors, the first cone differentiation genes were also expressed at E15.5 in our study (PNA, cone arrestin). In the third phase of photoreceptor development (P0), when cone genesis has ended

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Fig. 6. Temporal expression profiles of late-onset rod and cone differentiation markers in the ONL (P7). Section RNA in situ hybridization showing the expression of Gnat1 at both low (A-D) and high (A0 -D0 ) magnification of boxed areas in the retina at P7, P14, P21 and P60. Section immunohistochemistry showing the expression of Opn1mw (E-H) and Opn1sw (I-L) protein at both low (E-H,I-L) and high (E0 -H0 ,I0 -L0 ,E00 -H00 ,I00 -L00 ) magnification in the ventral (E0 -H0 ,I0 -L0 ) and dorsal (E00 -H00 ,I00 -L00 ) retina at P7, P14, P21 and P60. Section immunohistochemistry showing the expression of rhodopsin at low (M-P) and high (M0 -P0 ) magnification at P7, P14, P21 and P60. gcl, ganglion cell layer; inl, inner nuclear layer; le, lens; onl, outer nuclear layer; onbl, outer neuroblastic layer; os, outer segment; p-onl, presumptive outer nuclear layer; re, retina; Scale bars, 250 mm.

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and rod photoreceptor genesis has reached its peak, the expression of two transcription factors involved in rod genesis was observed, namely Nrl and Nr2e3. At this stage, we also observed Rcvrn expression, which is a rod and cone differentiation marker (Sampath et al., 2005; Vinberg et al., 2015), as well as transcripts for the cone opsins Opn1sw and Opn1mw, indicating that cones are differentiating in this temporal window. Finally, at P7, the expression of additional differentiation markers was observed in both rods (Gnat1, Rhodopsin) and cones (Opn1sw and Opn1mw protein). By characterizing photoreceptor lineage progression in vivo, we will be better poised to examine lineage progression in cellular reprogramming studies. Throughout most of the CNS, including the retina, new neurons are not made in adulthood. Cell-based therapies must thus be devised to replace lost cells with healthy cells in order to restore tissue function. Amongst the major causes of vision loss are a group of retinal disorders associated with the degeneration or dysfunction of photoreceptors, including cone-rod dystrophies, retinitis pigmentosa, and age related macular degeneration. Researchers have taken advantage of the wealth of information on the molecular control of retinal cell fate specification (reviewed in (Wallace, 2011)) to induce embryonic stem (ES) and induced pluripotent stem (iPS) cells to differentiate into photoreceptor lineages (Ikeda et al., 2005; Lamba et al., 2006, 2009; Mellough et al., 2012; Meyer et al., 2011; Osakada et al., 2008; West et al., 2012). However, ES-and iPS-derived photoreceptors have a limited capacity to functionally integrate, and more worrisome, injection of these cells into the subretinal space can result in tumor formation (West et al., 2012), highlighting the safety issues associated with using pluripotent cells as a source of donor cells. A more thorough characterization of the developmental profile of photoreceptors differentiating in a dish will allow us to examine whether lineage progression is properly initiated by comparing to in vivo

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studies such as ours. Ultimately, by understanding the factors that are involved in photoreceptor cell fate specification, differentiation and maintenance, we will be better able to develop cell replacement therapies to treat blindness. 4. Experimental procedures 4.1. Animal maintenance and tissue preparation CD1 male and female mice were crossed and embryos were staged considering the morning a vaginal plug was detected as E0.5. Embryos were fixed in 4% paraformaldehyde (PFA)/diethylpyrocarbonate phosphate buffered saline (DEPC-PBS) at 4  C overnight. For later postnatal stages (P7-P60), animals were first perfused with 4%PFA/DEPC-PBS before eyes were dissected and immersed in fixative overnight. To maintain the orientation of the retina, eyes were kept in the head. After 24 h, eyes were rinsed with DEPC-PBS and transferred to 20% DEPC-sucrose/1X DEPC-PBS overnight at 4  C. Heads were then embedded in optimal cutting temperature (OCT) compound and 10 mm sections were cut. 4.2. RNA in situ hybridization Digoxygenin (Dig)-labeled RNA probes were made using T3 (Roche), T7 (Invitrogen) or SP6 (Biolabs) RNA polymerases and digRNA labeling mix according to the manufacturer's instructions (Roche). Templates and anti-sense riboprobes for the following cDNAs were generated: Crx (Image clone ID 4527863; RE HindIII, Pol T3), Otx2 (a gift from Gillemot lab; RE XbaI, Pol T3), Rorb (Image clone ID 5358124; RE SalI, Pol T3), Neurod1 (Image clone ID 4511370; RE KpnI, Pol T7), Prdm1 (Image clone ID 40048956; RE SpeI, Pol T7), Thrb (Image clone ID 40057420; RE XbaI, Pol T7),

Fig. 7. Summary of the temporal expression profiles of transcription factors and photoreceptor differentiation genes during retinal development. A summary of all the transcription factors as well as cone and rod markers has been shown in this figure. Dotted lines indicate the period when levels of gene expression decline. Yellow lines indicate expression of transcription factors, green lines are representative of cone photoreceptor markers and red lines indicate rod photoreceptor markers.

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Neurog1 (Image clone ID 30146192; RE NheI, Pol Sp6), Sall3 (Image clone ID 6833590; RE EcoRI, Pol T3), Rxrg (Image clone ID 5707723; RE EcoRI, Pol T3), Nrl (a gift from Corbo lab; RE KpnI, Pol T7), Nr2e3 (Image clone ID 4512088; RE SalI, Pol T7), Opn1mw (Image clone ID 4507311; RE BamHI, Pol T7), Opn1sw (Image clone ID 4511808; RE EcoRI, Pol T3) and Gnat1 (Image clone ID 6490797; RE SalI, Pol T7). RNA in situ hybridization was performed on 10 mm transverse sections of the retina collected on SuperFrost Plus slides (Thermo Scientific). 1 ml of dig-probe was diluted in 200 ml of hybridization buffer [50% formamide, 10% dextran sulphate, 1 mg/ml yeast tRNA, 1X Denhardt's, 1X salt (0.2M NaCl, 10 mM Tris-HCl, pH 7.5, 6.5 mM NaH2PO4$2H20, 5 mM Na2HPO4, 5 mM EDTA)]. Sections were hybridized with riboprobes in a humidified chamber overnight at 65  C. On the second day, sections were washed twice for 30 min each in 1X SSC, 50% formamide, 0.1% Tween 20 at 65  C. Sections were then washed twice for 30 min each in 1X MABT (100 mM Maleic acid, 150 mM NaCl, 0.1% Tween 20, pH 7.5) at room temperature. Sections were blocked in blocking solution (2% blocking reagent [Roche], 1X MABT, 10% normal horse serum) for 1 h at room temperature. Sections were then incubated overnight at room temperature in anti-dig alkaline phosphatase antibody (2 ml, Roche) diluted in 1 ml of 5XMABT and 1 ml of normal horse serum adjusted to 5 ml total with MilliQ water. On the third day, sections were washed with 1X MABT 5  20 min. Sections were then washed one time in NTMT (100 mM NaCl, 50 mM MgCl, 100 mM Tris pH 9.5, Tween 20) for 10 min at room temperature, followed by an additional 10 min wash with NTMT containing 5 mM levamisole. Finally, sections were stained in NTMT þ levamisole solution containing 0.33 mg/ml NBT (Roche) and 0.26 mg/ml BCIP (Roche) substrates. Staining was stopped in 2e4 h, or overnight if necessary. Staining was stopped by washing sections in water. Slides were then dried at room temperature and mounted using Permount SP15-100 Toluene Solution (Fisher Scientific). 4.3. Section immunohistochemistry 10 mm transverse sections of the retina were collected on SuperFrost Plus slides (Thermo Scientific). Sections were washed in PBS 1 PBS/0.1% Triton X (PBT). Sections were then blocked with blocking solution (10% normal horse serum/PBT) for 1 h at room temperature. Primary antibodies were diluted in blocking solution and incubated on the sections overnight at 4  C. The next day, sections were washed with PBT and then incubated with secondary antibodies diluted 1/500 in blocking solution at room temperature for 45 min. Sections were washed 3 times for 10 min each with PBT. Nuclei were stained in 4’,6-diamidino-2-phenylindole (DAPI- Santa Cruz Biotechnology) diluted in PBT (1:5000) for 5 min. Sections were washed with PBS once by 10 min and mounted using AquaPolymount (Polysciences). Primary antibodies used included: rabbit anti-Arr3 (1/200, Millipore, AB15282), rabbit anti-Opn1sw (1/ 500, Millipore AB5407), rabbit anti-Opn1mw (1/500, Millipore AB5405), mouse anti-Rho (1/500, Millipore, MAB5356), rabbit antiRcvrn (1/1500, Millipore AB5585) and rhodamine-conjugated Peanut Agglutinin (PNA) (1/1000, Vector RL1072). 4.4. Image processing Images were captured with a Leica DMRXA2 optical microscope, QImaging RETIGA 2000R or QImaging RETIGA EX digital cameras and Openlab5 software (Improvision). The images were processed using Adobe Photoshop software. Acknowledgements The authors would like to thank Rajiv Dixit and Natalia Klenin

for technical support. We would also like to acknowledge Brain Canada and the Weston Foundation for operating support to CS, JB and VW, and Canadian Institutes of Health Research (89994) and Lion's Sight Centre support to CS. NT was supported by a CIHR/ ACHRI Training Grant in Genetics, Child Health and Development. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.gep.2017.03.001. References Akagi, T., Inoue, T., Miyoshi, G., Bessho, Y., Takahashi, M., Lee, J.E., Guillemot, F., Kageyama, R., 2004. Requirement of multiple basic helix-loop-helix genes for retinal neuronal subtype specification. J. Biol. Chem. 279, 28492e28498. Akimoto, M., Cheng, H., Zhu, D., Brzezinski, J.A., Khanna, R., Filippova, E., Oh, E.C., Jing, Y., Linares, J.L., Brooks, M., Zareparsi, S., Mears, A.J., Hero, A., Glaser, T., Swaroop, A., 2006. 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