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The ETS transcription factor Etv1 mediates FGF signaling to initiate proneural gene expression during Xenopus laevis retinal development Minde Willardsen 1, David A. Hutcheson 1, Kathryn B. Moore, Monica L. Vetter

*

Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

A R T I C L E I N F O

A B S T R A C T

Article history:

Fibroblast growth factor signaling plays a significant role in the developing eye, regulating

Received 19 August 2013

both patterning and neurogenesis. Members of the Pea3/Etv4-subfamily of ETS-domain

Received in revised form

transcription factors (Etv1, Etv4, and Etv5) are transcriptional activators that are down-

27 September 2013

stream targets of FGF/MAPK signaling, but whether they are required for eye development

Accepted 25 October 2013

is unknown. We show that in the developing Xenopus laevis retina, etv1 is transiently

Available online 9 November 2013

expressed at the onset of retinal neurogenesis. We found that etv1 is not required for eye specification, but is required for the expression of atonal-related proneural bHLH transcrip-

Keywords: FGF Retina development Neurogenesis Etv1

tion factors, and is also required for retinal neuron differentiation. Using transgenic reporters we show that the distal atoh7 enhancer, which is required for the initiation of atoh7 expression in the Xenopus retina, is responsive to both FGF signaling and etv1 expression. Thus, we conclude that Etv1 acts downstream of FGF signaling to regulate the initiation of neurogenesis in the Xenopus retina. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

ETS

1.

Introduction

The initiation of retinal neurogenesis is a tightly controlled process in which multiple extrinsic signaling pathways act on a pool of retinal progenitor cells to regulate the onset of differentiation (Bassett and Wallace, 2012; Sinn and Wittbrodt, 2013). Together with intrinsic competence factors, these signaling pathways play a key role in controlling both differentiation and cell fate choice decisions. In particular, fibroblast growth factors (FGFs) are essential for neural patterning and development, and play significant roles in the developing eye. FGFs and their receptors are expressed in the optic vesicle in multiple species (Friesel and Brown, 1992; McFarlane

et al., 1995; Riou et al., 1996; Shiozaki et al., 1995; Song and Slack, 1994; Tannahill et al., 1992) and are involved in regulating patterning, proliferation, differentiation and survival in the developing retina (Cai et al., 2013; Guillemot and Cepko, 1992; Hochmann et al., 2012; Park and Hollenberg, 1989; Qin et al., 2011; Sievers et al., 1987; Zhao et al., 2001). For example, FGF signaling is necessary and sufficient to specify the neural retina domain in the developing optic vesicle (Fuhrmann, 2010). Gain of function studies in multiple species show that activating the FGF/MAPK signaling pathway can convert retinal pigment epithelium (RPE) to fully differentiated neural retina (Fuhrmann, 2010; Spence et al., 2007; Vergara and Del Rio-Tsonis, 2009; Yoshii et al., 2007; Zhao et al., 2001), while

* Corresponding author. Address: Department of Neurobiology and Anatomy, University of Utah, Rm. 517 Wintrobe, 20 North 1900 East, Salt Lake City, UT 84132, USA. Tel.: +1 801 581 4984; fax: +1 801 581 4233. E-mail address: [email protected] (M.L. Vetter). 1 Both authors contributed equally to this paper. 0925-4773/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mod.2013.10.003

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blocking FGF/MAPK signaling in the distal optic vesicle promotes RPE formation at the expense of neural retina (Cai et al., 2010). It has been shown in zebrafish and chick that FGF signaling is both necessary and sufficient to initiate retinal neurogenesis through a localized signaling center originating in the optic stalk (Martinez-Morales et al., 2005). FGF signaling has also been implicated in the spread of differentiation within the retina in chick (McCabe et al., 1999), and the regulation of retinal cell fate decisions in Xenopus (McFarlane et al., 1998). At the onset of neurogenesis, FGF signaling acts to trigger retinal ganglion cell differentiation, and has been shown to regulate the expression of the proneural basic helix-loop-helix (bHLH) factor Atoh7 in several species (Cai et al., 2010; Martinez-Morales et al., 2005; McCabe et al., 2006; Willardsen et al., 2009). Transgenic analysis in Xenopus has shown that FGF signaling is transduced through a 3.3 kb cis-regulatory atoh7 enhancer (Willardsen et al., 2009), suggesting positive regulation of atoh7 expression by intrinsic effectors of FGF signaling. However, no effectors linking FGF signaling to atoh7 expression have been identified. Etv4 (Pea3), Etv5 (Erm), and Etv1 (Er81), members of the Pea3/Etv4-subfamily of ETS-domain transcription factors, are transcriptional activators that are downstream targets of FGF/MAPK signaling, and are involved in a wide range of developmental processes (de Launoit et al., 2006; Graves and Petersen, 1998; Maroulakou and Bowe, 2000; Nakayama et al., 2008; Remy and Baltzinger, 2000). Etv4 is present in the developing chick retina and its expression is dependent upon FGF signaling (McCabe et al., 2006). In zebrafish, the initiation and propagation of shh expression in the eye is regulated by FGF signaling, and mediated by Etv4 and Etv5 which act through a specific enhancer in the shh gene (Vinothkumar et al., 2008). Thus, this gene family can mediate FGF signaling activity in the developing retina. In Xenopus laevis, Etv1 is the only family member expressed in the retina (Chen et al., 1999; Munchberg and Steinbeisser, 1999). Within the ETS domain, Xenopus Etv1 is entirely conserved with mouse and human and shows a high degree of conservation outside the ETS domain (Munchberg and Steinbeisser, 1999). In animal cap explants, etv1 expression is activated by FGF signaling, and endogenous expression in the marginal zone is blocked by a dominant-negative FGF receptor (XFD), thus etv1 expression is FGF-responsive in Xenopus (Chen et al., 1999). When etv1 is overexpressed by injecting of mRNA into two dorsal blastomeres of a 4-cell Xenopus embryo, the eye fails to form normally (Chen et al., 1999). However, whether this is due to early defects in tissue patterning or more specific disruption of eye or retina development is unclear. Thus the specific role of etv1 in retinal development is unknown. In this study, we sought to determine the relationship of etv1 to the process of retinal neurogenesis. Specifically, we performed a detailed retinal expression analysis and found that while etv1 is poised to affect the initial stages of retinogenesis, its expression is quickly downregulated. Loss of function analysis showed that etv1 is not required for retinal progenitor specification, but is required for the expression of atonal-related proneural bHLH transcription factors, including atoh7. Furthermore, inhibition of etv1 expression inhibited retinal neuron differentiation, consistent with Etv1

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acting as an effector of FGF signaling in retinal progenitors. Using transgenic reporters we show that the distal atoh7 enhancer, which is required for the initiation of atoh7 expression (Willardsen et al., 2009), is responsive to both FGF signaling and etv1 expression. Thus, etv1 participates in the initiation of neurogenesis in the retina by regulating proneural gene expression, and for atoh7 acts through a conserved enhancer that controls the initiation of atoh7 expression.

2.

Results

2.1. Etv1 is expressed in the retina prior to retinal neurogenesis FGF signaling plays a clear role in regulating retinal neurogenesis (Martinez-Morales et al., 2005; McCabe et al., 2006; McFarlane et al., 1998; Willardsen et al., 2009), and it has been proposed that the Pea3/Etv4-subfamily of ETS-domain transcription factors may mediate FGF signaling in the developing retina (McCabe et al., 2006; Willardsen et al., 2009). To explore this further we investigated the expression of etv1, which is the only family member expressed in the developing Xenopus retina (Chen et al., 1999), to determine whether its expression was correlated with the onset of retinal neurogenesis. Whole mount in situ hybridizations showed robust etv1 expression in the developing eye (stages 20, 22; Fig. 1A and B), and expression persisted in the eye at the onset of neurogenesis (stages 24, 26; Fig. 1C and D). At stage 24, when neurogenesis begins, in situ hybridization on sections through the optic vesicle showed weak expression in the surface ectoderm, as well as in the optic cup neuroepithelium, with highest expression in the ventral retina and optic stalk, consistent with previous reports of a midline source of FGF signal (Fig. 1F; (Martinez-Morales et al., 2005)). At stage 28, etv1 is still expressed throughout the retinal neuroepithelium and overlying surface ectoderm (Fig. 1G), but by stage 29/30 it is absent from the neural retina and optic stalk but strongly expressed in the developing lens (Fig. 1E and H). At later stages when retinal neurogenesis is complete, photoreceptors in the outer nuclear layer express etv1 (Fig. 1I). However, elsewhere in the retina etv1 does not appear to be expressed, including in the ciliary marginal zone (CMZ), which is a zone of continued proliferation and differentiation at the peripheral margins of the retina (Fig. 1I), although we cannot rule out very low levels of expression. The transient expression of etv1 in the optic vesicle, as well as its apparent absence from the CMZ suggests that its function is tied to the initiation of retinal neurogenesis rather than a more general involvement in retinal progenitor differentiation.

2.2. Etv1 is required for atonal class proneural gene expression Since FGF signaling is required to initiate retinal neurogenesis and proneural gene expression, we sought to determine whether etv1 was also required for expression of genes involved in retinal neuron development. First, we injected an antisense translation-blocking morpholino specific for etv1 into a dorsal animal blastomere at the 8-cell stage, which will target a majority of retinal neurons on the injected side

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(Moody, 1987a). In a second approach, we generated transgenic Xenopus embryos and expressed a dominant negative form of Etv1 lacking the C-terminal activation domain (Bosc and Janknecht, 2002), under the control of the human fzd5 enhancer, which directs transgene expression to the Xenopus optic vesicle beginning at stage 18–19 (Willardsen et al., 2009). Embryos were collected for whole mount in situ hybridization analysis at roughly stage 28. In both assays, early retinal progenitor genes vsx1 (not shown), rax, pax6, and sox2 were unaffected (Fig. 2A–I), indicating that etv1 is not required to confer eye identify or neural competence. Ascl3, a proneural bHLH gene of the achaete-scute class, which is expressed in early retinoblasts prior to the onset of neurogenesis (Kanekar et al., 1997), was not affected (Fig. 2J–L). Expression of ascl1 was also unaffected (data not shown). However in both etv1-MO injected embryos and dnEtv1 transgenic embryos, expression of the atonal related bHLH genes

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neurod4 (not shown), atoh7, neurod1, and neurog2 was reduced or lost as compared to controls (Fig. 2M–U). To control for the specificity of the etv1-MO, either inverse (etv1-INV-MO) or missense (etv1-MIS-MO) morpholinos were injected. These control morpholinos had only minor effects on retinal gene expression, with 10–20% of injected eyes showing weak variability in the expression of pax6, sox2, rax, and atoh7 (not shown). Importantly, neither of the two control morpholinos caused a loss of atoh7 expression as was seen in etv1-MO injected retinas. While rescue experiments were not possible for the etv1 MO due to effects with etv1 RNA injection (as discussed later), the etv1-MO phenotype was identical to that of the dnEtv1 transgenic embryos, confirming the specificity of the phenotype. Thus, similar to what was observed with block of FGF signaling (MartinezMorales et al., 2005; Willardsen et al., 2009), etv1 is specifically required for the expression of atonal-related proneural genes,

Fig. 1 – Expression of etv1 in the developing Xenopus laevis eye. Etv1 is detected by whole mount in situ hybridization during optic vesicle evagination (A, anterior view – bracket; inset, lateral view – arrow), in the pre-neurogenic optic vesicle (B, anterior view – bracket; inset, lateral view – arrow), and during the initial stages of retinal neurogenesis, (C and D, lateral view). By stage 29–30, etv1 is only expressed in the developing lens vesicle (E, lateral view – arrowhead). In situ hybridization on sections shows etv1 is most highly expressed in the ventral stalk and ventral optic vesicle at stage 24, with weaker expression in the neuroepithelium of the central to dorsal optic vesicle, and overlying surface ectoderm (F). At stage 28, etv1 is expressed throughout the retinal neuroepithelium and overlying surface ectoderm (G). At stage 29–30, the developing lens vesicle is strongly labeled, while there is no detectable expression of etv1 in the developing neural retina (H). At the end of embryonic retinal neurogenesis, etv1 is expressed in the photoreceptors of the outer nuclear layer but is absent from the rest of the central retina and also from progenitors in the ciliary marginal zone (I). Abbreviations: ov, optic vesicle; e, eye; lv, lens vesicle; se, surface ectoderm; ne, neuroepithelium; os, optic stalk; nr, neural retina; cmz, ciliary marginal zone; onl, outer nuclear layer.

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Fig. 2 – Loss of Etv1 function prevents proneural gene expression. Etv1 function was blocked by injection of etv1 MO at the 8cell stage (B,E,H,K,N,Q,T) or by transgenic expression of dnEtv1 using the eye-specific enhancer for human fzd5 (C,F,I,L,O,R,U). In situ hybridization on stage 28 embryos showed that expression of early retinal markers were unaffected relative to the uninjected side, including rax (A and B, n = 78/82 unaffected; C, n = 32/32), pax6 (D and E, n = 59/65; F, n = 15/15), sox2 (G and H, n = 51/58; I, n = 28/28), and the pre-neurogenesis bHLH gene ascl3 (J and K n = 37/37; L, n = 32/32). Expression of proneural bHLH genes required for retinal neurogenesis was reduced compared to the uninjected side: atoh7 (M and N, n = 70/124 reduced; O, n = 56/98), neurog2 (P and Q, n = 39/61; R, n = 20/34), and neurod1 (S and T, n = 53/103; U, n = 18/28). Arrows indicate the eye.

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but not retinal progenitor genes, consistent with Etv1 functioning as a mediator of FGF signaling during retinal development.

2.3. FGF signaling and etv1 are required to drive expression of the atoh7 distal enhancer Since atoh7 expression is dependent upon both FGF signaling and etv1 expression, we sought to determine whether Etv1 is acting through previously defined atoh7 regulatory sequences. We have shown that a GFP transgene consisting of 3.3 kb atoh7 upstream regulatory sequence recapitulates the pattern of endogenous atoh7 expression in transgenic Xenopus embryos, and that its expression is blocked by dominant negative FGF receptor (XFD) (Willardsen et al., 2009). Within this 3.3 kb region, a 252 bp extended distal enhancer is sufficient to accurately control transgene expression (Willardsen et al., 2009). To test whether this extended distal enhancer depends upon either etv1 expression or FGF signaling, we created transgenic embryos expressing GFP under the control of the atoh7 252 bp extended distal enhancer. Then, at the 8cell stage, a dorsal animal blastomere fated to give rise predominantly to one retina was injected with mRNA encoding mCherry either alone or together with XFD mRNA or etv1 MO. mCherry was used as a tracer to distinguish the targeted retina from the contralateral control retina. Injection of mRNA for mCherry alone did not affect expression of the GFP transgene (Fig. 3A and B), however retinas expressing

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either XFD or targeted with etv1 MO showed reduced or absent GFP transgene expression on the injected side (Fig. 3D and F). Thus the 252 bp distal enhancer is responsive to both FGF signaling and etv1 expression during Xenopus eye development. Within the 252 bp enhancer, a minimal 152 bp distal enhancer is highly conserved across Xenopus, zebrafish, mouse and human, and drives premature but spatially correct expression of the GFP transgene (Willardsen et al., 2009). We tested whether this smaller distal enhancer is also dependent upon FGF signaling and etv1 expression. Transgenic embryos were generated using a transgene consisting of multimerized 152 bp distal enhancers driving GFP, then injected at the 8 cell stage with mRNA encoding mCherry either alone or together with XFD mRNA or etv1 MO. Expression of GFP from the 152 bp distal enhancer was not affected by mCherry expression, but GFP expression was reduced or absent with expression of XFD (Fig. 3J), or injection of etv1 MO (Fig. 3L). Thus, we conclude that this highly conserved 152 bp distal enhancer is also responsive to both FGF signaling and etv1 expression activity. ETS domain factors bind to a core GGAA/T site and Etv1 was shown to bind preferentially to a core of RSMGGAWRY in vitro (Brown and McKnight, 1992). We searched the distal enhancer for potential binding sites and found three ETS core sites, but only one close match to the consensus Etv1 binding site. This site, GGCGGATTCA matches a consensus Etv1 binding site in all positions but the T immediately downstream of

Fig. 3 – Activity of the atoh7 distal enhancer is dependent upon both FGF signaling and etv1 expression. GFP transgene expression from either an extended 252 bp distal atoh7 enhancer (A–F) or a multimerized version of the 152 bp distal atoh7 enhancer (G–L) was unaffected by injection of mRNA for mCherry at the 8 cell stage (A and B, n = 47/49 unaffected; G and H, n = 22/23 unaffected). Coinjection of mRNA for XFD reduced or blocked expression of both transgenes on the mCherrypositive injected side relative to the uninjected side (C and D, n = 22/47 reduced; I and J, n = 16/28). Coinjection of etv1 MO had a similar effect, blocking transgene expression on the injected side (E and F, n = 37/55 reduced; K and L, n = 16/28). The eye domain is highlighted with a dashed circle.

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the core GGAT ETS-binding site (core underlined, mismatch italicized). To determine if Etv1 directly binds to the distal enhancer through this sequence and activates expression of atoh7, we mutated the core ETS-domain in the 152 bp distal enhancer by altering nucleotides to eliminate Etv1 binding (GTCT; (Brown and McKnight, 1992)), and assayed for transgene expression. We found that the transgene still promoted GFP expression in the retina (data not shown). Thus, although Etv1 regulates transgene expression through the distal enhancer, this putative Etv1 binding site is not required.

the proportion of Mueller glia relative to GFP mRNA alone (Fig. 4), consistent with the weak effects we observed with this morpholino on retinal gene expression (data not shown). Thus, etv1 is required for retinal neuron differentiation, and in its absence, retinal progenitors preferentially give rise to Mueller glia, consistent with a role for etv1 as a mediator of FGF signaling in retinal progenitors.

2.4. Loss of Etv1 blocks neurogenesis and promotes glial cell fate

In addition to FGF signaling, other factors are required to initiate proneural gene expression. In the Xenopus and mouse retina, it has been demonstrated that Pax6 directly regulates atoh7 expression (Riesenberg et al., 2009; Willardsen et al., 2009), but Pax6 also acts at multiple steps in the retinal development pathway, serving as a key upstream regulator of eye development (Shaham et al., 2012). Interestingly, expression of etv1 in the mouse cortex is dependent upon Pax6 (Tuoc and Stoykova, 2008). To determine whether this is the case during Xenopus eye development, we selectively blocked Pax6 and assayed for etv1 expression. mRNA encoding dominant-negative Pax6 (dnPax6) was injected into a dorsal blastomere at the 8-cell stage, embryos were grown to stage 24, and in situ hybridization was performed to assay the effect on etv1 expression. Blocking the Pax6 pathway did not cause a loss of retinal etv1 expression (Fig. S1 A and B). Another signaling pathway that is critical for retinal neurogenesis in Xenopus is the canonical Wnt/b-catenin pathway (Van Raay et al., 2005). The Fzd5 receptor activates this pathway to regulate expression of sox2, and thus proneural gene expression. To determine whether etv1 expression during retinal development is dependent upon this pathway, we injected fzd5 MO at the 8-cell stage and again observed no effect on etv1 expression in the developing eye (Fig. S1 C and D). As a control, we confirmed that chemical block (SU5402) of FGF signaling results in a loss of etv1 expression (Fig. S1 E and F; (Willardsen et al., 2009)). Thus, we conclude that in the Xenopus retina, etv1 acts in the context of FGF signaling and represents a positive neurogenic mechanism independent of the Wnt/Sox2 and Pax6 regulatory pathways.

Since FGF signaling is required for retinal neuron differentiation (McFarlane et al., 1998), we asked whether etv1 is also required for acquisition of retinal neuron fates. We injected etv1-MO + GFP mRNA or GFP mRNA alone into a dorsal animal blastomere at the 32-cell stage that is fated to give rise to roughly 8% of cells in the retina (Moody, 1987b). Retinal cell fate analysis was performed at stage 41/42, a timepoint when neurogenesis in the central retina is complete. Individual GFP-labeled cells were counted and scored for cell type based upon morphology and laminar position, as previously described (Moore et al., 2002). Blocking Etv1 caused a significant increase in non-neural Mueller glia as compared to GFP mRNA alone (Fig. 4). The increase in Mueller glia was coupled to a decrease in the percentage of some retinal neurons, specifically retinal ganglion cells and bipolar cells, with a modest effect on amacrine cell numbers. These findings are consistent with a block on proneural gene expression, as we have reported in other studies (van Raay et al., 2005; Aldiri et al., 2013). Injection of control etv1 INV MO had a weak effect on

2.5. Etv1 expression is independent of the Pax6 and Wnt retinal specification pathways

2.6. Ectopic etv1 is not sufficient to prematurely initiate proneural gene expression

Fig. 4 – Inhibiting Etv1 function in retinal progenitors biases cells to adopt the Mueller glial cell fate. Injection of etv1 MO caused a significant increase in the proportion of cells that become Mueller glia as compared to GFP alone, with a corresponding decrease in retinal neurogenesis, which was significant for retinal ganglion cells, amacrine cells and bipolar cells (N = 9 embryos, 3111 cells for GFP alone; N = 11 embryos, 1272 cells for etv1 MO). A control inverse morpholino (etv1 INV MO) had a weak effect (N = 8 embryos, 1447 cells). The percent representation of each cell type is a weighted average, and error bars represent SEM. #p < 0.05 and *p < 0.01, by Student’s t test. RGC, retinal ganglion cell; HC, horizontal cell; AM, amacrine cell, BP, bipolar cell; PR, photoreceptor cell; MG, Mueller glial cell.

Among the factors known to be required for atoh7 expression, none are sufficient alone to initiate atoh7 expression, but in the chick sclerotome, Pea3/Etv4-family ETS factors are both necessary and sufficient to activate expression of the bHLH gene scleraxis (Brent and Tabin, 2004). To determine whether etv1 is sufficient to activate atoh7 expression prior to its normal time of onset, we injected etv1 mRNA at the 8-cell stage. In situ hybridization on stage 23 embryos showed that expression of rax and pax6 remains normal in the presence of ectopic etv1, suggesting the eye is patterned normally and that retinal progenitors are present (Fig. 5A–F). Overexpression of etv1 did not induce premature expression of atoh7 or neurod1, indicating that etv1 is not sufficient to initiate retinal neurogenesis (Fig. 5G and J). Later, at stage 28, expression of both atoh7 and neurod1 was reduced (Fig. 5H and K) as

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Fig. 5 – Etv1 overexpression does not trigger early retinal neurogenesis. Early retinal patterning genes rax (A–C) and pax6 (D–F) were unaffected by 8-cell injection of mRNA for etv1. The proneural bHLH gene atoh7 (G, n = 49) and neurod1 (J, n = 76) were not expressed prematurely at stage22/23, indicating that etv1 was insufficient to initiate their expression. At stage 28, expression of both atoh7 (H and I) and neurod1 (K and L) was reduced on the injected side compared to the uninjected side (n = 24/48 for atoh7; n = 20/34 for neurod1). Arrows indicate the eye.

compared to the uninjected side (Fig. 5I and L), suggesting that maintaining ectopic etv1 expression interferes with normal proneural gene expression and thus retinogenesis.

3.

Discussion

3.1.

Etv1 regulates Xenopus retinal neurogenesis

FGF signaling triggers the initiation of neurogenesis in the vertebrate retina in multiple species, and several studies have shown atoh7 expression specifically depends upon FGF signaling (Martinez-Morales et al., 2005; McCabe et al., 2006; Willardsen et al., 2009). In this study, we have shown that the gene encoding the ETS transcription factor Etv1 is transiently expressed in the Xenopus retina at the onset of retinal neurogenesis, and functions as a downstream effector of FGF signaling to initiate proneural gene expression and regulate retinal neurogenesis. We further show that its expression is required for the activity of a conserved distal enhancer that regulates atoh7 expression. Our findings support a role for

etv1 in triggering the onset of retinal neuron differentiation by regulating proneural gene expression.

3.2. ETS transcription factor expression during eye development In Xenopus, etv1 is the only Pea3/Etv4-family ETS gene expressed in the developing eye (Chen et al., 1999). In chick, while etv1 is expressed in the retina, it appears that etv4 is most responsive to FGF signaling (McCabe et al., 2006). In zebrafish, both etv4 and etv5 are expressed throughout the central retina early in differentiation (36 hpf) (Vinothkumar et al., 2008), but interestingly, the expression of etv5 is maintained in all 3 nuclear layers into adulthood (Hochmann et al., 2012). Currently, no expression data exists for Pea3/Etv4-family ETS factors during murine retinal development. While there are species differences in the spatio-temporal expression patterns of these genes, in all species examined Pea3/ Etv4-family members are expressed in the retina early and in the photoreceptor layer during adult stages.

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We found that etv1 expression in the Xenopus optic vesicle precedes the onset of retinal neurogenesis, but is rapidly downregulated in the retinal neuroepithelium shortly after differentiation begins, and is absent during the time when most progenitors undergo differentiation. Further, unlike other retinal transcription factors with documented roles in embryonic retina development in Xenopus (Harris and Perron, 1998; Locker et al., 2009; Perron et al., 1998), etv1 does not appear to be expressed in the ciliary marginal zone (CMZ). This strongly suggests that etv1 is coupled with the initiation but not progression of embryonic neurogenesis in Xenopus, and is not associated with ongoing neurogenesis in the CMZ. Similarly, in the mouse retina conditional deletion of the protein tyrosine phosphatase Shp2, which is required for FGF-Ras signaling and etv5 expression, showed a requirement for initiation but not progression or maintenance of proneural gene expression and retinal neurogenesis (Cai et al., 2010). However, there is evidence that FGF signaling can regulate the progression of retinal neurogenesis in other species. In chick retina explants FGF signaling was shown to be required for progression of the wave of differentiation across the retina (McCabe et al., 2006). Furthermore, in zebrafish the initiation of shh expression and retinal neurogenesis is triggered by FGF3/8 while the propagation of shh expression in the eye is regulated by FGF19, and mediated by etv4 and etv5 (Vinothkumar et al., 2008). Thus, there may be species differences in the role of FGF signaling in regulating the initiation versus progression of retinal neurogenesis. In Xenopus, etv1 may trigger the expression of other factor(s) that act in a more sustained way to regulate retinal neurogenesis. Alternatively, mediators of FGF signaling other than etv1 may be involved in propagation of neurogenesis during Xenopus eye development and during ongoing neurogenesis in the CMZ. At stage 24, when retinal neurogenesis is initiated, expression was highest in the ventral retina and optic stalk region, consistent with a role for FGF signaling in ventral patterning in multiple species (Cai et al., 2013; Lupo et al., 2005; Take-uchi et al., 2003). In Xenopus, retinoic acid and FGFs cooperate with Shh to regulate ventral patterning of the eye (Lupo et al., 2005). In addition, FGF signaling regulates the expression of axon guidance cues in the developing optic tract (AtkinsonLeadbeater et al., 2009). In mouse, combined deletion of Fgfr1 and Fgfr2 results in ocular coloboma and optic nerve dysgenesis (Cai et al., 2013). In zebrafish, FGF signals also regulate early nasal-temporal patterning (Picker and Brand, 2005; Picker et al., 2009). Future studies will define whether ETS transcription factors function as downstream transcriptional mediators of FGF signaling in these patterning events. Although the expression of etv1 in the Xenopus retina is lost by mid-retinogenesis and remains absent in the CMZ, etv1 is strongly expressed in mature photoreceptors. Loss of FGF signaling has been associated with degeneration of photoreceptors in several species (Campochiaro et al., 1996; Fontaine et al., 1998; Green et al., 2001; Hochmann et al., 2012; Kinkl et al., 2001; Qin et al., 2011; Siffroi-Fernandez et al., 2008), thus Pea3/Etv4-group ETS factors may play a conserved role in maintenance of differentiated photoreceptors. In addition, the strong expression of etv1 in the developing lens is also consistent with the well-defined role for FGF signaling during lens development (Robinson, 2006).

3.3.

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Regulation of proneural gene expression

FGF signaling in Xenopus, zebrafish, chick and mouse is required to regulate both atoh7 expression and retinal neurogenesis (Cai et al., 2010; Martinez-Morales et al., 2005; McCabe et al., 2006; Willardsen et al., 2009). Pea3/Etv4-family ETS factors have been implicated as downstream responders to FGF signaling in the retina (Cai et al., 2010; McCabe et al., 2006; Willardsen et al., 2009), but not previously shown to be required for proneural gene expression or retinal development. Our data support the conclusion that vertebrate retinogenesis shares a conserved mechanism whereby FGF signaling via Pea3/Etv4-subfamily factors initiate proneural gene expression. We found that etv1 is required for the expression not only of atoh7, but also of neurod1 and neurog2, suggesting a general role in regulating expression of atonalrelated proneural factors. We found that both etv1 expression and FGF signaling were required for activity of the conserved minimal 152 bp atoh7 enhancer in the Xenopus retina, which we have proposed is an enhancer required to initiate atoh7 expression (Willardsen et al., 2009). However, previously we found using transgenic zebrafish that expression of GFP under the control of this 152 bp enhancer was not blocked by an inhibitor of FGF signaling SU5402, even though endogenous atoh7 expression was inhibited (Willardsen et al., 2009). It is possible that the enhancer responds to FGF signaling differently in Xenopus and zebrafish. Alternatively, there may be technical reasons that the inhibitor did not block enhancer activity in zebrafish. Here we clearly establish that FGF signaling and etv1 are required for activity of this minimal 152 bp enhancer during Xenopus eye development. While we identified a candidate ETS binding site, it was not required for enhancer activity. It is possible that additional more divergent ETS sites in the distal enhancer are involved. It is also possible that regulation of this distal enhancer by FGF signaling is indirect. For example, this pathway can control the expression of eye regulatory factors such as Lhx2 and Lhx9 (Atkinson-Leadbeater et al., 2009; Pottin et al., 2011). However, whether there are candidate intermediary factors remains to be defined.

3.4.

Regulation of retinal cell fate by etv1

We find that blocking etv1 has distinct effects on retinal cell fate, causing an increase in differentiation of Mueller glia at the expense of retinal neuron differentiation. This is consistent with a loss of proneural gene expression, and is similar to effects on retinal cell fate seen when proneural gene expression is reduced in other ways (Aldiri et al., 2013; Moore et al., 2002; Van Raay et al., 2005). The increase in Mueller glia is also consistent with cell fate changes observed by blocking FGF signaling in retinal progenitors by expression of XFD, a dominant negative FGF receptor (McFarlane et al., 1998). Interestingly, overexpression of FGF2 has the opposite effect, biasing retinal progenitors towards retinal ganglion cell fates and decreasing Mueller glia (Patel and McFarlane, 2000). Overall we conclude that Etv1 and other Pea3/Etv4 family effectors of FGF signaling represent a broad and conserved signaling pathway that is proneurogenic in the developing retina.

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In other tissues, ETS factors have been shown to be both necessary and sufficient for expression of some downstream targets such as the bHLH gene scleraxis (Brent and Tabin, 2004). While we have shown that etv1 is necessary for the expression of proneural bHLH factors in the retina, it is not sufficient to drive their premature expression in the optic vesicle, suggesting that other factors or signals are required to coordinate appropriate onset of proneural gene expression and differentiation. Surprisingly, overexpression of etv1 prevented proneural gene expression without altering expression of retinal progenitor genes, demonstrating that dynamic and transient expression of this factor is critical for normal eye development. Similarly, we previously found that transient activation of Wnt/b-catenin signaling is required for neurogenesis during Xenopus eye development, with both inhibition or activation of the pathway resulting in a block in neurogenesis (Agathocleous et al., 2009). Lupo et al. (2005) showed that overexpression of inducible FGFR in Xenopus altered patterning of the eye, inducing upregulation of ventral genes such as vax2 and pax2, and downregulation of pax6. Since we saw no effect on expression of pax6 when etv1 was overexpressed, it may be that etv1 is not sufficient to mediate the full effects of FGF signaling on ventral patterning of the eye. It is also possible that there are subtle effects on optic vesicle patterning with etv1 overexpression that alter the ability of proneural genes to be expressed.

3.5. Etv1 is expressed competence pathways

independent

of

key

retinal

Pax6 and Wnt/b-catenin signaling represent two additional conserved pathways that are necessary but not sufficient for the initiation of retinal neurogenesis. We show that the FGF effector etv1 is still expressed when these pathways are blocked, indicating that FGF/Etv1 represents an additional mechanism controlling retinogenesis, independent of these other retinal pathways. While etv1 and pax6 do not regulate each other’s expression, they are both required for activity of the distal atoh7 enhancer (Willardsen et al., 2009), which regulates the initial expression of atoh7 in the retina. We propose that the distal enhancer is a node at which multiple signals that control the initiation of atoh7 expression converge. Interestingly, transgenes driven by this minimal 152 bp enhancer are expressed beginning at stage 22, several hours before endogenous atoh7 expression begins, suggesting that all the necessary components and pathways required for initiation of atoh7 and retinogenesis may be present in the retina by stage 22 (Willardsen et al., 2009). Further exploration of the interactions of FGF/Etv1 signaling with other retinal specification factors will continue to shed light on early retinal development.

4.

Experimental procedures

4.1.

Molecular cloning and mutagenesis

The distal 152 bp enhancer 5 0 of atoh7 (previously called X5dis3), the multimerized 152 bp distal enhancer construct (previously called X5dis3mult), and the 252 bp extended distal

1 3 1 ( 2 0 1 4 ) 5 7 –6 7

65

atoh7 enhancer (previously called X5dis3 + 100bp5 0 ), were previously reported (Willardsen et al., 2009). The distal 152 bp enhancer was used for targeted mutagenesis of the core candidate ETS binding site (GGAT changed to GTCT). Dominant negative Etv1 (dnEtv1) was created by deleting the C-terminal activation domain of Etv1, as in (Bosc and Janknecht, 2002), and cloned into the pG1 vector under the control of the human fzd5 promoter that is specific to progenitors in the developing neural retina (Willardsen et al., 2009).

4.2.

Morpholinos

The following translation blocking morpholinos (Gene Tools LLC; Philomath, OR) were used in the 8 cell injections: etv1 MO: 5 0 -CTTGCTGGTCATAAAATCCATCCAT-3 0 (5 ng) etv1 MIS MO (6 base pair mismatch of etv1 MO – base changes underlined): 5 0 -CTTCCTCGTGATAAATGTCATCCAT-3 0 (5 ng) etv1 INV MO (inverse sequence of etv1 MO): 5 0 -TACCTACCTAAAATACTGGTCGTTC-3 0 (5 ng) fzd5 MO (30 ng) was previously described (Van Raay et al., 2005).

4.3.

Retinal cell fate analysis

etv1 MO (1 ng) was injected in one dorsal animal blastomere of 32 cell stage embryos along with 300 pg of GFP mRNA (Huang and Moody, 1993). 400 pg of GFP mRNA alone, or with control etv1 INV MO (1 ng) were injected as controls. GFP positive cells in stage 41/42 retinal sections were scored for cell type based on cell position and morphology as previously described (Moore et al., 2002).

4.4.

X. laevis transgenic procedure

Transgenic X. laevis embryos were generated as previously described (Hutcheson and Vetter, 2002; Kroll and Amaya, 1996). To detect GFP transgene expression, whole mount antibody staining was performed on all embryos using a polyclonal anti-GFP antibody (Torrey Pines) at 1:500 and an Alexa Fluor 488-conjugated secondary (Molecular Probes) at 1:1000 unless otherwise noted. 8 cell stage mRNA injections into transgenic embryos were performed as described in Willardsen et al. (2009).

4.5.

In situ hybridization and mRNA injection

Embryos were processed for in situ hybridization (ISH) as previously described (Kanekar et al., 1997). ISH probes were: rax (Mathers et al., 1997), pax6 (Hirsch and Harris, 1997), sox2 (Mizuseki et al., 1998), atoh7 (Kanekar et al., 1997), neurog2 (Ma et al., 1996), neurod1 (Lee et al., 1995), etv1 (Chen et al., 1999), and ascl3 (Turner and Weintraub, 1994). mRNA injections were done into a dorsal animal blastomere at the 8-cell stage in the following amounts: 600 pg dnPax6 (Chow et al., 1999), 500 pg XFD (dnFGFR, (Amaya et al., 1991)), 250 pg etv1. 500 pg mCherryCAAX mRNA (a gift from Kristin Kwan) or 800 pg eGFP mRNA were used to trace lineages of injected blastomeres.

66

MECHANISMS OF DEVELOPMENT

Acknowledgments We are grateful for technical support from Joy Corley. We thank Heithem El-Hodiri for sharing expertise and for helpful discussions. This work was supported by NIH Grant EY012274 to MLV.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.mod.2013.10.003.

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