Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo
2015; Springer Nature; Volume: 34; Issue: 11 Linguagem: Inglês
10.15252/embj.201490706
ISSN1460-2075
AutoresRobert Reinhardt, Lázaro Centanin, Tinatini Tavhelidse, Daigo Inoue, Beate Wittbrodt, Jean‐Paul Concordet, Juan Ramón Martı́nez-Morales, Joachim Wittbrodt,
Tópico(s)Developmental Biology and Gene Regulation
ResumoArticle23 April 2015Open Access Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo Robert Reinhardt Robert Reinhardt Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Lázaro Centanin Corresponding Author Lázaro Centanin Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Tinatini Tavhelidse Tinatini Tavhelidse Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Daigo Inoue Daigo Inoue Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Beate Wittbrodt Beate Wittbrodt Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Jean-Paul Concordet Jean-Paul Concordet Muséum National d'Histoire Naturelle, Paris, France Search for more papers by this author Juan Ramón Martinez-Morales Juan Ramón Martinez-Morales Centro Andaluz de Biología del Desarrollo (CSIC/UPO/JA), Sevilla, Spain Search for more papers by this author Joachim Wittbrodt Corresponding Author Joachim Wittbrodt Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Robert Reinhardt Robert Reinhardt Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Lázaro Centanin Corresponding Author Lázaro Centanin Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Tinatini Tavhelidse Tinatini Tavhelidse Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Daigo Inoue Daigo Inoue Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Beate Wittbrodt Beate Wittbrodt Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Jean-Paul Concordet Jean-Paul Concordet Muséum National d'Histoire Naturelle, Paris, France Search for more papers by this author Juan Ramón Martinez-Morales Juan Ramón Martinez-Morales Centro Andaluz de Biología del Desarrollo (CSIC/UPO/JA), Sevilla, Spain Search for more papers by this author Joachim Wittbrodt Corresponding Author Joachim Wittbrodt Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany Search for more papers by this author Author Information Robert Reinhardt1,4,‡, Lázaro Centanin 1,‡, Tinatini Tavhelidse1, Daigo Inoue1, Beate Wittbrodt1, Jean-Paul Concordet2, Juan Ramón Martinez-Morales3 and Joachim Wittbrodt 1 1Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany 2Muséum National d'Histoire Naturelle, Paris, France 3Centro Andaluz de Biología del Desarrollo (CSIC/UPO/JA), Sevilla, Spain 4Present address: Developmental Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6221 546253; E-mail: [email protected] *Corresponding author. Tel: +49 6221 546499; E-mail: [email protected] The EMBO Journal (2015)34:1572-1588https://doi.org/10.15252/embj.201490706 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. Synopsis This study establishes Rx2 as functional determinant of neuro-epithelial progenitor fate and uncovers the gene regulatory network that governs Rx2 expression. Rx2-positive stem cells can give rise either to neuroretina or to retinal pigmented epithelium. A transcriptional core network of Sox2, TLX, Her9, and Gli3 confines rx2 expression to the peripheral CMZ. Repression of Rx2 (by Gli2 or in Rx2 mutant clones) favors formation of retinal pigmented epithelium. Rx2 balances the fate decision of retinal stem cells towards retinal pigmented epithelium or neural retina. Introduction Post-embryonic neurogenesis relies on the activity of neural stem cells (NSCs) (Adolf et al, 2006; Zhao et al, 2008). The fish neural retina (NR) constitutes an ideal model to study embryonic and post-embryonic NSCs in their physiological environment. It consists of seven main cell types distributed in three nuclear layers, and all these cell types are added lifelong from retinal stem cells (RSCs) that reside in the peripheral ciliary marginal zone (CMZ) (Johns, 1977; Lamba et al, 2008; Centanin et al, 2011). The CMZ also contributes to the retinal pigmented epithelium (RPE), a monolayer of pigmented cells surrounding and synchronously growing with the NR (Johns, 1977; Amato et al, 2004a; Moshiri et al, 2004; Centanin et al, 2011). As an attractive model for life-long neurogenesis as well as growth of the RPE, the CMZ has been extensively studied in fish, frog, and chicken (Amato et al, 2004b; Raymond et al, 2006; Lamba et al, 2008). The lack of genetic tools to follow lineages in these species, however, was a long-lasting limitation to validate putative RSC-specific markers, which in turn prevented understanding the regulatory framework that generates and maintains this stem cell fate. In medaka, retinal multipotent NSCs were identified by their virtue to form arched continuous stripes (ArCoS) via cell transplantation at early embryonic stages (Centanin et al, 2011) and by inducible recombination in late embryonic and post-embryonic stages (Centanin et al, 2014). These experiments demonstrated that the maintenance of the NR and the RPE is achieved by independent RSCs located in the CMZ. How the function of NSCs is maintained in the CNS remains largely unknown. Two of the best-studied factors expressed by post-embryonic NSCs in other niches are Sox2 and Tlx, which have been shown to be crucial for the self-renewal and differentiation of NSCs (Monaghan et al, 1995; Graham et al, 2003; Shi et al, 2004). Furthermore, fate-mapping studies showed that sox2 (Suh et al, 2007) and tlx (Liu et al, 2008) are markers for multipotent stem cells in the mammalian brain. Identifying target genes regulated by NSC-determining TFs is crucial for the understanding of the molecular and signaling pathways underlying adult neurogenesis. Previously, Sox2 has been demonstrated to bind and regulate the gfap regulatory element (Cavallaro et al, 2008). Target genes regulated by Tlx include cell cycle regulators p21 and pten (Sun et al, 2007). The function of Tlx is context dependent; inhibition of target genes relies on the interaction with HDACs (Sun et al, 2007), while Tlx can also act as activator of transcription (Iwahara et al, 2009). Characterizing a NSC regulatory network crucially depends on the identification of a reliable molecular marker and the main cis-acting regulators controlling its proper expression pattern. Here, we followed a long-term lineage analysis to identify the transcription factor (TF) retina-specific homeobox gene-2 (rx2) as a bona fide marker for multipotent adult RSCs in the post-embryonic medaka CMZ. We show that individual rx2+ RSCs give rise to progeny contributing either to NR or to RPE. Next, we identified TFs acting upstream of rx2 in a trans-regulation assay using the rx2 proximal cis-regulatory element (pCRE) and characterize sox2, tlx, gli3, and her9 as transcriptional modulators of stem cell features in the post-embryonic fish retina. Clonal expression of sox2 or tlx induces RSC-specific characteristics, including ectopic rx2 expression in differentiated neurons of the central retina. Conversely, conditional clonal gain of Gli3 or Her9 function leads to rx2 repression and inhibits stem cell proliferation in the post-embryonic CMZ. We also identify and validate the cis-regulatory motifs within the proximal rx2 pCRE that activate expression in the peripheral most CMZ, and repress expression in the adjacent RPE. Additionally, we show that the rx2 expression levels resulting from this transcriptional network establish the balance between NR and RPE stem cells. Our in vitro and in vivo results provide evidence for the importance of direct TF-DNA binding for proper spatial rx2 expression in RSCs. Taken together, we present a regulatory framework of TFs that establish, expand, and restrict RSC features in the post-embryonic retina and demonstrate a crucial function of Rx2 in the definition of retinal stem cell types. Results Rx2 labels the most peripheral cells in the ciliary marginal zone of the medaka retina To specifically target RSCs in the CMZ, we followed a candidate gene approach and systematically searched for genes and their regulatory regions with expression confined to the CMZ. Both in amphibians and fish, the retina-specific homeobox gene-1 and retina-specific homeobox gene-2 (rx1 and rx2 respectively) are expressed in the peripheral CMZ at embryonic and post-embryonic stages (Locker et al, 2006; Raymond et al, 2006; Borday et al, 2012). In medaka, rx2 is first expressed in the undifferentiated retinal progenitor cells (RPCs) that form the optic vesicle (Loosli et al, 1999). At later stages, when the fish retina already contains all the main cell types and is functionally active, rx2 expression is confined to photoreceptors (cones and rods in the outer nuclear layer, ONL), to the Müller glia cells, and to the peripheral most part of the CMZ, as revealed by in situ hybridization and immunostaining (Fig 1A and B) (Sinn et al, 2014). Medaka transgenic lines in which 2.4 kb of the proximal rx2 pCRE control the expression of a reporter fluorescent protein (FP) (rx2::H2B-mRFP and rx2::Tub-GFP) (Martinez-Morales et al, 2009; Inoue & Wittbrodt, 2011) (Fig 1B and C; Supplementary Fig S1) exhibit the same expression pattern, indicating that the 2.4-kb rx2 pCRE contains the regulatory cues driving rx2 expression to those cell types. Figure 1. Rx2 functions as retinal stem cell (RSC) marker A. rx2 mRNA is strongly detected in the peripheral CMZ of juvenile medaka (black box). B. Expression analysis of boxed area in (A). Transgenic reporter (rx2::Tub-GFP in green) overlaps with Rx2 immunostaining (in red). C. Cross-section of a transgenic rx2::H2B-mRFP juvenile medaka. rx2+ cells are located in the most peripheral domain of the CMZ (bracket), limited centrally by Atoh7::mYFP. rx2+ cells in the central retina are Müller glia (arrowhead) and photoreceptors (arrow). D, E. Transplantation of rx2::H2B-mRFP, Wimbledon cells into wild-type medaka demonstrates that rx2+ cells represent the most peripheral cells in an ArCoS. Data information: Scale bars represent 25 μm in (B), 50 μm in (C), and 100 μm in (D). Download figure Download PowerPoint The post-embryonic medaka retina grows outwards, with the RSCs located in the most peripheral domain of the CMZ (Centanin et al, 2011). A close analysis at rx2::H2B-mRFP transgenic juveniles revealed that rx2+ cells locate to the peripheral most domain of the CMZ (Fig 1C), suggesting that Rx2 constitutes a marker for RSCs. On the central side of the CMZ, differentiating Atoh7+ cells demarcate the boundary between the central most domain of the CMZ and the differentiated, layered retina (Fig 1C) (Del Bene et al, 2007; Cerveny et al, 2010). As expected for stem cells, rx2+ cells in the post-embryonic CMZ barely incorporate the thymidine analog bromodeoxyuridine (BrdU) when applied in short pulses (Supplementary Fig S2A) and similarly, there is only partial overlap with proliferation markers like phospho-histone H3 (pHH3) and proliferating cell nuclear antigen (PCNA) (Supplementary Fig S2B and C). Most of the BrdU+, PCNA+, and/or pH3+ cells of the CMZ map to the progenitor domain, between the rx2+ cells and the Atoh7+ cells. In medaka, transplantation of EGFP+ cells from the Wimbledon+/− line (a transgenic line that expresses EGFP ubiquitously during the entire life of the fish) (Centanin et al, 2011) into an unlabeled blastula results in fish with mosaic retinae. In these chimeric fish, RSCs were addressed by their property to form ArCoSs, which are continuous clonal strings of EGFP+ cells consisting of differentiated neurons and glia at central positions, and undifferentiated cells in the peripheral domain. We reasoned that if rx2+ cells were indeed RSCs, all the ArCoSs should contain rx2+ cells located at the most peripheral position of the clone. We therefore transplanted Wimbledon+/−, rx2::H2B-mRFP donor cells into wild-type hosts and consistently found rx2+ cells at the peripheral tip in all resulting ArCoSs (Fig 1D and E). Thus, the expression domain of rx2+ and the relative position of rx2+ cells within an ArCoS match the expected location of RSCs and suggest that rx2+ is a RSC marker in the mature medaka retina. Rx2 is a molecular marker of adult RSCs The ultimate validation of rx2+ as a stem cell marker is to follow the lineage of an individual rx2+ cell in the medaka CMZ during several months of post-embryonic life. The Gaudí toolkit allows single-cell labeling and lineage analysis in medaka (Centanin et al, 2014), based on Cre/LoxP-mediated recombination. When fish bearing a ubiquitous Cre driver and a Gaudí reporter are induced for stochastic recombination, single RSCs generate induced ArCoSs (iArCoSs) of the same characteristics than the ArCoSs generated by transplantation (Centanin et al, 2014). To address if rx2 marks RSCs and if the progeny of an rx2+ cell in the CMZ forms an iArCoS, we generated a transgenic line expressing a tamoxifen-inducible Cre recombinase under the control of the well-characterized 2.4-kb rx2 pCRE (rx2::ERT2Cre). We induced Cre recombinase within the rx2 expression domain to trigger recombination in the ubiquitously expressed four-color reporter cassette (Gaudí2.1) (Centanin et al, 2014). Tamoxifen induction of rx2::ERT2Cre, Gaudí2.1 at 10 dpf resulted in the specific labeling of individual rx2+ cells, as shown by stochastic expression of FPs labeling single photoreceptors, Müller glia, and peripheral cells in the CMZ (Fig 2A). Figure 2. Individual rx2-positive cells are stem cells for the neural retina (NR) or the retinal pigmented epithelium (RPE) The stochastic expression of fluorescent proteins (FPs) allows single-cell labeling within the rx2+ expression domain. ArCoS generation by individual rx2+ cells defines rx2 as a marker for RSCs for the NR, and the RPE. rx2 labels individual RSCs for (left) the NR and (right) the RPE. All rx2+ cells forming ArCoSs in the NR are multipotent, generating all NR cell types. Data information: CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; MG, Müller glia; ONL, outer nuclear layer; PRC, photoreceptor cell. Scale bars represent 50 μm (A) and 100 μm (B, C). Download figure Download PowerPoint Long-term lineage experiments showed that rx2+ cells formed iArCoSs and thus indeed represent RSCs (n = 162 red-colored iArCoSs distributed over 7 retinae, ranging from 15 to 31 iArCoSs per retina, average 23.1 iArCoSs/retina) (Fig 2B). Lineage analyses indicate as well that every single rx2+ post-embryonic NR stem cell analyzed is multipotent, equivalent to ArCoS-forming RSCs in transplantation experiments. Each iArCoS contains the full repertoire of differentiated retinal cell types of the NR (n= 125 NR iArCoSs) (Fig 2C). Remarkably, rx2+ RSCs as a population give rise to iArCoSs in both the NR and the RPE (Fig 2B). An individual rx2+ RSC, however, can be a stem cell either for the NR or for the RPE (n = 25 NR iArCoSs and 136 RPE iArCoS distributed over 8 retinae, 98.5% of independent iArCoSs). Identification of Sox2, Tlx, Gli3, and Her9 as transcriptional regulators in control of retinal stemness To identify genes controlling RSC features, we followed a high-throughput trans-regulation screen (Souren et al, 2009) to systematically detect factors operating on the rx2 pCRE. The trans-regulation screen is based on two nested screens. In a first step, it employs a high-throughput luciferase assay based upon the co-expression of an rx2 pCRE reporter construct driving firefly luciferase together with individual full-length candidate TFs (Fig 3A). This cell culture-based assay allows transcriptome scale analyses and has been used reliably to identify so far unknown upstream regulators (Souren et al, 2009). We took advantage of the relatively short 2.4-kb proximal rx2 CRE sufficient to recapitulate the rx2 expression pattern and assayed more than one thousand individual full-length cDNA clones, which represented a large complement of all putative medaka TFs. We controlled for transfection efficiencies in a dual luciferase-based screen in cultured cells through co-transfection of a control plasmid encoding Renilla luciferase (Fig 3A). To exclude potential false positives, we performed a secondary, nested, whole-mount in situ screen to analyze the expression pattern of putative candidate TFs relative to rx2 by a semi-automated whole-mount in situ hybridization approach (Quiring et al, 2004). We eventually selected activating or repressing candidates based on their co-expression with or adjacent to rx2 in the juvenile CMZ. Figure 3. Transcriptional regulators of rx2 are expressed in the post-embryonic CMZ A. A luciferase-containing vector (pGL4.1-rx2) was co-transfected with individual cDNA clones (pSport6.1-cDNA) and an internal control (pRL-CMV) to measure the transcriptional response of the rx2 pCRE in BHK cells. B–E. Dual luciferase assays show dose-dependent activation and repression of rx2 cis-regulatory activity in BHK cells. All values were normalized against the luminescence recorded in cells transfected with pCS2+ and reporters only. Values indicate averages of four replicates. Error bars indicate standard deviation. F–Q. Confocal stacks of two-color WISHs with probes against sox2 (G, red), tlx (J, red), her9 (M red), gli3 (P, red), and rx2 (H, K, N, Q, green) on medaka hatchlings. Vibratome sections show the distinct expression patterns of sox2, tlx, her9, and gli3, which partially overlap with the expression of rx2 in the peripheral CMZ. Dashed line demarcates boundary between RPE and NR. Scale bar represents 25 μm. Download figure Download PowerPoint This nested screening pipeline delivered clear candidates from the more than one thousand TFs analyzed: sox2 was the top activator, while gli3 and her9 (a medaka Hes1 ortholog) showed the strongest repressive activities. tlx—not initially present in the full-length TF library—showed a strong activation of the rx2 pCRE (Fig 3C) and was assayed in a parallel candidate screen because of its role in mouse NSCs (Yu et al, 1994; Monaghan et al, 1995; Shi et al, 2004). To test whether Sox2, Tlx, Her9, and Gli3 regulate rx2 transcription in a concentration-dependent manner, we performed dual luciferase assays with increasing amounts of the respective TF cDNA. For Sox2 (Fig 3B), we observed the activation of relative luciferase activity in a dose-dependent manner. Likewise, for Tlx (Fig 3C) activation of transcription peaked with the highest cDNA concentration (160 ng), implicating tlx as an activator of rx2 expression. Conversely, stepwise increase of Her9 resulted in the gradual reduction of reporter expression (Fig 3D). Interestingly, Gli3-mediated repression of rx2 pCRE activity was strongest at the lowest Gli3 concentration (Fig 3E), while increasing cDNA amounts led to a gradual reduction of its repressive potential. Next, we addressed the expression patterns of sox2, tlx, gli3, and her9 with respect to their putative target gene rx2 in the juvenile CMZ by two-color fluorescent whole-mount in situ hybridization (WISH). All four regulators are expressed in nested domains that partially overlap with the rx2 expression domain in the CMZ. We detected transcripts of the pan-neural determinant sox2 throughout the CMZ overlapping with the Rx2 expression domain (Fig 3F–H). tlx and her9 were both expressed in the central CMZ where they partially overlapped with the rx2 expression domain (Fig 3I–N). gli3 transcripts were found in the peripheral CMZ overlapping with rx2 expression and were also found in the adjacent RPE (Fig 3O–Q). Out of all the rx2 regulators identified in the trans-regulation screen, gli3 was the only factor expressed in the peripheral RPE adjacent to the CMZ. Gli3 and Her9 antagonize stem cell features in vivo To test whether these candidate factors regulate rx2 expression in vivo, we employed a conditional clonal analysis in the post-embryonic retina. For this purpose, we adopted a hormone-inducible binary gene expression system, which consists of a TF (LexPR) that upon hormone induction will dimerize and bind to the corresponding promoter element (LexOP) to activate the expression of following genes of interest (Emelyanov & Parinov, 2008). We established transgenic lines expressing LexPR in the CMZ under the control of the rx2 pCRE (Fig 4A). Upon addition of mifepristone (RU-486), dimerization and LexOP-dependent transcription were initiated (Fig 4B). By limited exposure to the hormone, we triggered mosaic expression of gli3 or her9 (and the co-expression of a fluorescent reporter protein) within the very specific Rx2 domain in the CMZ and Müller glia cells as well as in mature photoreceptor cells. This system allows co-expressing fluorescent reporter proteins and the gene of interest with high efficiency (up to 90%). Consequently, the analyses based on the expression of the fluorescent reporter are conservative and always underestimate the effects of the gene of interest. Figure 4. Clonal gain of gli3 or her9 restricts rx2 expression and proliferation in the CMZ A. Transgenic hormone-inducible lines for overexpression of gli3, her9, and H2B-EGFP (control). B. Addition of RU-486 induces mosaic overexpression of transgenes in the Rx2 domain. C–K. The cross-sections of transgenic control embryos (rx2::LexPR LexOP::H2B-EGFP, C–E) were compared to retinae mis-expressing gli3 (rx2::LexPR LexOP::Gli3 LexOP::H2B-EGFP, F–H) and her9 (rx2::LexPR LexOP::Her9 LexOP::H2B-EGFP, I–K) in the rx2 domain 9 days post-fertilization (dpf). Sustained clonal gain of Gli3 (F–H) or Her9 (I–K) inhibited rx2 expression as indicated by loss of Rx2 protein (in red). Scale bars represent 10 μm. L–T. Gli3 (O–Q) or Her9 (R–T) gain-of-function clones frequently lacked PCNA protein compared to control cells (L–N). Dashed lines demarcate RSC domain in the CMZ. Dotted outlines highlight affected cells. Scale bars represent 10 μm. Download figure Download PowerPoint To assess the repressive potential of gli3 and her9, respectively, on the expression of rx2 in vivo, we targeted their clonal expression to the peripheral rx2+ CMZ. Gain-of-function clones were highlighted by the co-expression of nuclear FPs (Fig 4F and I). The expression of Rx2 protein was determined by antibody staining and analyzed automatically by a threshold-based segmentation algorithm. The repression of the pCRE of rx2 by Her9 and Gli3 in the CMZ in this experiment not only affects the endogenous Rx2 but also the ectopic expression of the repressors. Consequently, we consistently underestimated the repressive potential Her9 and Gli3 on rx2. Six days after the clonal induction of Gli3 in the peripheral CMZ (rx2::LexPR LexOP::Gli3 LexOP::H2B-EGFP), Rx2 protein was lost in 39.4% of the induced gli3+ cells (n = 13/33) in the CMZ (compare Fig 4C–E to F–H). Similarly, 20% of the induced her9+ cells (rx2::LexPR LexOP::Her9 LexOP::H2B-EGFP) (n = 7/35) had lost Rx2 expression (compare Fig 4C–E to I–K), whereas the GFP alone had no effect on the rx2 expression (n = 1/34) (Fig 4C–E). These findings are in support of the hypothesis of Her9 and Gli3, confining rx2 expression as repressors in the central and peripheral domain of the CMZ. Since both Gli3 and Her9 showed the ability to repress rx2 expression in vivo, we next addressed the impact of ectopic gli3/her9 expression on the proliferative capacity of RSCs in the CMZ. PCNA staining, which labels S-phase cells within the CMZ, was low only in very few cells in the CMZ of GFP control retinae (n = 3/38). Conversely, PCNA expression was affected in the gli3 and her9 gain-of-function clones at rates comparable to those observed for the repression of Rx2. PCNA was severely affected in gli3 gain-of-function clones (n = 39/106) (compare Fig 4L–N to O–Q) or her9 gain-of-function clones (n = 21/64) (compare Fig 4L–N to R–T). Taken together, these results indicate that ectopic clonal gli3 and her9 expression in the CMZ represses rx2 expression and impacts negatively on the proliferation of RSCs. Sox2 and Tlx promote rx2 expression Since our in vitro characterization and the overlapping expression pattern of sox2 and tlx with rx2 consistently argued for an activating function of sox2 and tlx, we tested the consequences of acute clonal activation of sox2 (cska::LexPR LexOP::sox2) and tlx (cska::LexPR LexOP::tlx) gain-of-function (Fig 5A). Gain-of-function clones were marked by the expression of red FPs (LexOP::cherry), encoded by co-injected reporter plasmids. In combination with the ubiquitous cska promoter (Grabher et al, 2003), this approach allowed clonal, mosaic expression throughout all three nuclear layers of the differentiated retina (Fig 5B). Figure 5. Expression of sox2 or tlx promotes stem cell features in terminally differentiated neurons A. Hormone-inducible expression plasmids for co-injection into transgenic rx2::Tub-GFP or wild-type embryos. B. Positive clones were traced by the expression of FPs (LexOP::Cherry), encoded by co-injected reporter plasmids. In combination with the ubiquitous cska promoter, this approach facilitated mosaic expression throughout all three nuclear layers in transgenic rx2::Tub-GFP embryos. Expression of the candidate factors was hormonally induced (4 dpf) when the majority of cells in the central retina (CR) had exited the cell cycle and already differentiated into the neuronal and glial cell types. C, D. Compared to control (C), combined expression of tlx and sox2 (D) triggered ectopic rx2 pCRE activation. E–N. In addition to ectopic rx2 expression, PCNA protein was detected upon sox2 or tlx expression at 7 dpf. Data information: White arrowheads point to representative co-expressing cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RSCs, retinal stem cells. Scale bar represents 10 μm. Download figure Download PowerPoint We first examined the consequences of Sox2 and Tlx co-expression on rx2 promoter activity in vivo. The transgenic reporter line expressed FP under the rx2 pCRE (rx2::Tub-GFP). The combined expression of sox2 and tlx resulted in strong rx2 reporter activation (n = 40/48) in all three nuclear layers (Fig 5C and D). Individual clonal mis-expression of sox2 (n = 32/48) (Supplementary Fig S3A–D) or tlx (n = 142/173) (Supplementary Fig S3F–I) also resulted in ectopic rx2 reporter activation with high efficiency. To corroborate that sox2 and tlx activate endogenous rx2 expression in vivo, we combined WISH and immunohistochemistry in whole-mount preparations. Clones expressing sox2 (n = 53/62) (Supplementary Fig S3E–E‴) or tlx (n = 34/56) (Supplementary Fig S3J–J‴) efficiently triggered the ectopic expression of endogenous rx2 mRNA, which was never detected in controls. We next asked whether the clonal activation of sox2 or tlx was sufficient to trigger the ectopic induction of RSC features. While control clones in central retinal cell types never showed proliferating activity, the expression of Sox2 (n = 7/11) (Fig 5E–I) or Tlx (n = 3/23) (Fig 5J–N) resulted in the re-acquisition of proliferative features as indicated by PCNA staining. PCNA+ clones were observed in the INL and ONL, indicating that de-differentiation and re-initiation of proliferation were not restricted to one particular type of retinal neurons. Together, these results revealed that both sox2 and tlx induce the endogenous expression of rx2 in vivo, and re-activate the proliferative potential of post-mitotic cells. Sox2 and Gli3 regulate rx2 expression through direct protein–DNA interaction with the rx2 pCRE Our analysis showed
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