Accepted Manuscript Research report Sox2 Expression in the Visual System of two Teleost Species Laura DeOliveira-Mello, Juan M. Lara, Rosario Arévalo, Almudena Velasco, Andreas F. Mack PII: DOI: Article Number: Reference: S0006-8993(19)30404-4 https://doi.org/10.1016/j.brainres.2019.146350 146350 BRES 146350 To appear in: Brain Research Received Date: Revised Date: Accepted Date: 22 February 2019 20 June 2019 23 July 2019 Please cite this article as: L. DeOliveira-Mello, J.M. Lara, R. Arévalo, A. Velasco, A.F. Mack, Sox2 Expression in the Visual System of two Teleost Species, Brain Research (2019), doi: https://doi.org/10.1016/j.brainres. 2019.146350 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Manuscript Number BRES-D-19-00251 Sox2 Expression in the Visual System of two Teleost Species Laura DeOliveira-Mello1, Juan M. Lara1, Rosario Arévalo1, Almudena Velasco1, and Andreas F. Mack2 1. Dept. Cell Biology and Pathology, IBSAL-Institute of Neurosciences of Castilla and León, University of Salamanca, Spain. 2. Institute of Clinical Anatomy and Cell Analytis, Eberhard-Karls Universität Tübingen, Oesterbergstr. 3, 72074 Tübingen, Germany. E-mail contacts: (laura.mello@usal.es), (rororo@usal.es), (mraa@usal.es), (malmu@usal.es), and (an.mack@uni-tuebingen.de) Corresponding author: Andreas F. Mack, PhD Institute of Anatomy University of Tübingen Österbergstrae 3, D-72074 Tübingen, Germany an.mack@uni-tuebingen.de Declarations of interest: none Abstract: The visual system of teleost fish shows growth and regeneration capacities during the entire animal's life. Thus, the visual system of adult fish serves as a model for studying neurogenesis in the vertebrate central nervous system (CNS). Our study focused on the expression pattern of Sox2 in the fish visual system. Sox2 is a transcription factor known for its function in keeping stem cell properties, and as a regulator of cell fate during development, especially in the visual system. We used two different fish species: Astatotilapia burtoni and Danio rerio. In the visual system of fish, we identified Sox2 positive cells in the stem cell niche in the peripheral retina, in Müller cells and amacrine cells in the differentiated retina, and glial cells in the optic nerve (ON). We did not observe hardly any Sox2 expression in the optic nerve head (ONH). In the ON, Sox2 positive glial cells were lining the fascicles of new axons. Taking together, the broad spectrum of Sox2 expression indicates that this protein has different functions in the CNS of adult vertebrates. The results suggest that Sox2 has functions associated with the pathway of new axons from the retina. To understand the variety of cell types and subtypes and their plasticity potential in the visual system of fish will be essential to comprehend the growing and regenerating CNS in adult vertebrates. Highlights: In the fish retina, Sox2 is mainly expressed in proliferative, amacrine and glial cells. Some, but not all glial cell types in the optic nerve of fish express Sox2. No Sox2 positive cells are found in the optic nerve head. In the optic nerve, some Sox2+ cells align with bundles of growing axons. Differences in expression suggest different functions for Sox2 in the adult CNS. Keywords: Sox2, visual system, continuous growth, zebrafish, cichlid fish, glial cells Acknowledgements: This study was supported by Fund for Red de Terapia Celular de Castilla y León. L. DeOliveira-Mello was supported by a grant from Banco de Santander. The authors would like to thank M.T. Sánchez, M. Martín-García, L. Macher, U. Mattheus, for their excellent technical assistance. 2 Abbreviations: ChaT: Choline acetyltranferase CNS: Central Nervous System CR: Calretinin DAPI: 4′,6-Diamidine-2′-Phenylindole Dihydrochloride DCX: Doublecortin DMSO: Dimethyl Sulfoxide GCL: Ganglion Cell Layer GFAP: Glial Fibrillary Acid Protein GS: Glutamine Synthetase INL: Inner Nuclear Layer IPL: Inner Plexiform Layer NFL: Nerve Fiber Layer ON: Optic Nerve ONH: Optic Nerve Head ONL: Outer Nuclear Layer OPL: Outer Plexiform Layer PBS: Phosphate Buffer Saline PCNA: Proliferating Cell Nuclear Antigen PGZ: Peripheral Germinal Zone PV: Parvalbumin 3 1. Introduction The visual system of vertebrates consists of the retina, optic nerve (ON), and the brain centers processing visual information. In contrast to mammals, the visual system of teleost fish shows continuous growth (Fig. 1) and spontaneous regeneration during the entire animal's life (Fernald, 1991; Stuermer et al., 1992; Parrilla et al., 2009; Bejarano-Escobar et al., 2014). In fish, retinal neurogenesis continues after embryonic development by the preservation of a peripheral germinal zone (PGZ) in the circumferential edge of retinal tissue (Easter and Nicola, 1996; Velasco et al., 2000). Neurogenesis in adult mammals, in contrast to fish, is a comparatively rare event and restricted to few brain locations; in addition, axons in the mammalian central nervous system (CNS) are very ineffective to re-establish damaged connections. Consequently, the retina and the ON of adult fish are common models to study neurogenesis and regeneration in the vertebrate CNS (Bastmeyer et al., 1991; Hitchcock et al., 1992; Ghosh and Hui, 2016; Bollaerts et al., 2017). Research in many laboratories has shown that all cellular and synaptic elements are added during adult growth of the retina, and are rebuilt during regeneration (Hitchcock et al., 1992; Otteson et al., 2001; Mack et al., 2003; Lenkowski and Raymond, 2014). Thus, the visual system of fish has been used as an experimental model to study neurogenesis, not only during development but also in life-long growth (Goldman et al., 2001; Arenzana et al., 2011; Fleisch et al., 2011). Several studies have demonstrated the participation of astrocytes and oligodendrocytes to support, guide, nourish, and myelinate the constantly added new axons (Blaugrund et al., 1993; Alvarez-Buylla et al., 2002; Alunni et al., 2010; Dimou and Gotz, 2014; Falk and Goetz, 2017; Garcia-Pradas et al., 2018). Therefore, our study focused on glial cells as main candidates involved in the processes of continuous growth of the fish visual system. Previous reports have demonstrated the presence of various glial precursors, that generate different subtypes of astrocyte and oligodendrocyte populations, not only in the retina but also in the ON and optic nerve head (ONH) thereby accommodating the increasing number of axons in the ON (Lillo et al., 1998, 2001; Arenzana et al., 2011; Parrilla et al., 2012; 2016). We were interested in the generation of glial cells in the visual system and hypothesized that they might express the stem cell marker protein Sox2 since it has 4 been implicated in retinal growing processes in teleosts (Graham et al., 2003; Ito et al., 2010; Reinhardt et al., 2015; Ghosh and Hui, 2016). Sox2 is a transcription factor known for its primary function in stem cell proliferation, which has also been established as a regulator of cell fate during development (Pevny and Placzek, 2005; Taranova et al., 2006; Surzenko et al., 2013; Bachleda et al., 2016). During visual system development, Sox2 is widely expressed throughout the presumptive neural retina and the lens pit (Kamachi et al., 2001; Yang et al., 2009). After development, Sox2 protein is no longer detected in cell nuclei; instead expression is also reported in the cytoplasm of mature cells (Hever et al., 2006). It is not yet clear why Sox2 expression is maintained in mature cells, but it is known that down-regulation of Sox2 expression during development affects the expression of other transcription factors of the Group B Sox family which comprises Sox1, Sox2, and Sox3 (Wegner and Stolt, 2005; Kamachi and Kondoh, 2013; She and Yang, 2015). Furthermore, the absence of Sox2 in embryonic development causes microphtalmia or anophtalmia (Hever et al., 2006; Verma and FitzPatrick, 2007; Schneider et al., 2009). Several reports suggest that Sox2 plays a role in adult tissue homeostasis and regeneration, not only in the CNS but also in other tissues like ciliated cells and/or club cells in the respiratory system (Kondoh et al., 2004; Sarkar and Hochedlinger, 2013). Previous studies have shown the presence of several cell types in the retina that express Sox2 protein, not only in fish but also in mammals (Hever et al., 2006; Fischer et al., 2010; Wu et al., 2017; Kautzman et al., 2018). Our goal was to identify cell types expressing Sox2 in the retina, ONH, and ON (preencephalic visual system) of teleost fish. We used two fish species, the zebrafish (Danio rerio), and the cichlid fish (Astatotilapia burtoni, formerly known as Haplochromis burtoni). D. rerio is an established teleost model organism (Lieschke and Currie, 2007; Gemberling et al., 2013; Ghosh and Hui, 2016), and A. burtoni is a model organism for behavior (Hofmann, 2003; Maruska and Fernald, 2018) and visual system growth adding substantial retinal tissue, and ON fibers throughout life (Fernald, 1991; Mack et al., 2003; see Fig. 1). We were primarily interested in Sox2 positive cells associated with growth processes, yet in addition to the expression of Sox2 in adult stem cells, we identified the Sox2 positive amacrine cell type, and several glial cells in the preencephalic fish visual system, one of them aligned with new axons in the ON. The study suggests multiple roles of Sox2 in the adult fish visual system. 5 2. Results We analyzed the expression pattern and the distribution of Sox2 protein in the adult pre-encephalic visual system (retina, ONH and ON) in two model fish species: Astatotilapia burtoni and Danio rerio. We observed a largely similar distribution pattern of Sox2 positive cells in both species with some differences detailed below. After labeling retinal sections with anti-Sox2 antibodies, we detected Sox2 expression in the PGZ of both species. In the retina we also found several Sox2 positive cells in the inner nuclear layer (INL), in the ganglion cell layer (GCL) and dispersed cells in the outer nuclear layer (ONL); in addition, some cells were Sox2 positive in the inner plexiform layer (IPL) and few cells in the nerve fiber layer (NFL). The ONH was largely devoid of Sox2 positive cells, in contrast, stainings of the ON revealed several Sox2 positive cells. Figure 1 shows the enormous size increase of the ON during growth and the presence of Sox2 positive cells in the ON in small and larger fish. Sox2 immunolabeling was always located exclusively in the nuclei of the cells. This general pattern indicated that Sox2 expression was not restricted to stem and/or proliferating cells. In order to identify and characterize the different types of Sox2 positive cells in the visual system, we performed double immunostainings with known markers for proliferative, glial, and amacrine cells. We also labeled axons from new cells generated in the PGZ to study their relationship with Sox2 positive cells in the retina, ONH, and ON. 2.1. Sox2 positive cells in the retina To find out to what extent Sox2 positive cells in the retina represent stem cells we double labeled sections with proliferating cell nuclear antigen (PCNA). In the peripheral growth zone (PGZ; Figure 2), all PCNA labeled cells were also Sox2 positive in both species (Fig. 2A, B). These PGZ cells were never labeled with any of the other cellspecific markers for glial cells [glutamine synthetase (GS) or glial fibrillary acid protein (GFAP)] nor for neurons [parvalbumin (PV), choline acetyltransferase (ChaT), calretinin (CR), and doublecortin (DCX); see table 1]. In the differentiated retina we observed Sox2 positive cells in the ONL, INL, and GCL in both species, in addition few Sox2 cells were also present in the IPL and NFL of the cichlid retina (Fig. 2 C). Staining for PCNA in the ONL, we found some but not all Sox2 positive cells double labeled (supplementary Fig. 1A), and vice versa, not all 6 PCNA immunoreactive cells were positive for Sox2 in the cichlid retina (Fig. 2C’’). Few double positive cells were found at the innermost NFL where it was recently shown that new axons are located in the cichlid fish NFL (Garcia-Pradas et al., 2018). In the zebrafish retina, PCNA positive cells in the ONL were never positive for Sox2 (Fig. 2B’’). However, the majority of Sox2 positive cells in the differentiated retina was negative for PCNA in both species. We analyzed the potential relationship of Sox2 positive cells with newly formed neurons and their axons by immunolabeling with DCX (in A. burtoni), and with neurolin (Zn8) (in D. rerio) which would possibly indicate a guidance function. Newly formed cells next to the PGZ and their extensions stained with DCX or Zn8 but were negative for Sox2 (Fig. 2D-D’’’ and suppl. Fig. 1C). Thus, the triple label revealed Sox2 positive cells in the PGZ (Fig. 2D), DCX positive cells in a transition zone (Fig. 2D’), and CR positive cells in the mature retina (Fig. 2D’’). In the NFL, the few Sox2 positive cells did not show any particular pattern or alignment with new DCX positive axons, but as mentioned above, some of the Sox2 cells positive for PCNA were located at the innermost NFL (Fig. 2C-C’’). Most Sox2 positive cells in the retina were located within the INL in two different sublayers and with different intensities of immunoreactivity. Sox2 cells in the outer INL sublayer showed elongated nuclei and were positive for GFAP (Fig. 3A’, C’) and GS (Fig. 3B’). This identified them as Müller cells and is shown in detail in Fig. 3A’’’, B’’’ and C’’’. Sox2 positive cells in the inner INL showed stronger immunofluorescence, had round nuclei and were at the location of amacrine cells. We therefore stained for the amacrine markers PV, CR and ChaT (Fig. 4 and supplementary Fig. 1B). Some Sox2 positive cells in the INL and GCL were double stained with PV (Fig. 4A-A’’, C-C’’), however, there were also cells positive for only one of the markers. In cichlid fish, most PV positive cells were also positive for Sox2 and located in the INL, whereas in zebrafish more double labeled cells were found in the GCL. For CR and ChaT immunostains we did not find any co-localization with Sox2 positive cells in either species studied (Figs. 4B’, D’; suppl. Fig. 1B). 2.2. Sox2 positive cells in the ONH We did not see hardly any Sox2 positive cells in the ONH in either species. The counterstaining with DAPI or DRAQ5TM showed numerous cell nuclei with different 7 morphologies in this portion of the visual system, but they were not positive for Sox2 (Fig. 5A, A’’, C). In the ONH we observed an intricate immunolabeling for DCX (Fig. 5A-A’’). The DCX labeled axons from newly formed, differentiating neurons (Fig 2D’) appeared crossed at the level of the ONH, the zone free of Sox2 positive cells (Fig. 5AA’’’). Sections of the ONH stained for DCX (new axons) CR (mature axons) and Sox2 (Fig. 5B-B’’), showed a complete lack of co-localization (Fig. 5D’’’). Few cells close to the ONH were positive for GFAP and Sox2 (Fig. 5C-C’’’) in an area that is still part of the NFL. Similar results were found for Zn8 in the zebrafish. Differentiating cells in the transition zone next to the PGZ, and their growing axons in the NFL were positive for Zn8 (and negative for Sox2; data not shown). 2.3. Sox2 positive cells in the ON Sox2 positive cells were distributed along the entire extension of the ON in both species (Figs. 1, 5D, and 6). The immunohistochemistry for DCX (Fig. 5A, B and D) in the ON enabled us to demonstrate the entrance zone of new and growing axons arriving from the peripheral retina. A high number of Sox2 positive cells arranged in parallel rows were located around the axons positive for DCX (Fig. 5D’’, D’’’). In contrast, the Sox2 positive cells in the region of older axons did not show such a specific distribution pattern. We used double immunohistochemistry with Sox2 and the same markers used in the retina (PCNA, GFAP, GS, PV, CR, ChaT, DCX and Zn8) to characterize the Sox2 positive cells in the ON. Only few PCNA positive cells (Fig. 6A’, B’) were positive for Sox2 (Fig. 6A’’). GFAP in the ON is highly variable depending on species (and possibly antibodies) and has not been shown to be constitutively expressed in the fish ON (Levine, 1989; Marcus and Easter, 1995; Koke et al., 2010). In the ON of A. burtoni we found Sox2 positive cells with and without staining for GFAP (Fig. 6D-D’’). In addition, we found Sox2 positive cells in the ON of D. rerio with immunostaining for GS (Fig. 6C-C’’’) but also cells with only one of the markers (Sox2 positive and GS negative, or Sox2 negative and GS positive). As expected, we did not find any staining for PV, CR or ChaT along the entire extension of the ON. 8 2.4. Quantification of Sox2 positive cells in the pre-encephalic visual system of fish We found Sox2 positive cells in all parts of pre-encephalic visual system, but apparently the cell density and distribution was not the same in the analyzed parts. We considered four different zones for the statistical analysis: the GCL of the retina, the ONH, the first 300 µm of ON (ON1) and the retino-distant ON beyond 300 µm (ON2; Fig. 7). Because our data did not have a homogeneous variance (Fig. 7A), we applied two non- parametric tests to determine the statistical differences between the four zones. The Kruskal-Wallis test resulted in a significant difference (p-value ≤ 0,01) in the Sox2 positive cell density in the four zones (Fig. 7A’). In order to detect what group showed the biggest differences we applied the Mann-Whitney-U test with a Bonferroni correction. The analyses showed that the Sox2 positive cell density had a significant difference in the analyzed areas, with the exception of the comparison between retina and the distant ON (Fig. 7A’). The density was highest in the ON1 region, and lowest in the ONH. 3. Discussion In this study, we performed an immunohistological characterization of Sox2 positive cells in the adult pre-encephalic visual system of two species of teleost fish – Astatotilapia burtoni (Acanthopterygii; Perciformes; Cichlidae) and Danio rerio (Ostariophysi; Cyprinifomes; Cyprinidae) – to identify the cell types expressing this transcription factor in adult, growing animals. We found that Sox2 is expressed in some but not all proliferating cells, in various glial cells and a subclass of neurons. These results are summarized and generalized for both fish species, in a schematic diagram depicting Sox2 positive cells with cell-specific labels and the analyzed structures of the visual system (Fig. 8). Most previous reports discussed the functions of Sox2 in the context of proliferation capacities and stem cell properties. In fact, Sox2 expression together with other transcription factors is used to re-program cells into stem cells (Yu et al., 2007; Park et al., 2008). Our study shows that Sox2 is present not only in stem cells but also in glial and differentiated neuronal cells. Thus, we suggest that the Sox2 protein has different functions in addition to keeping proliferative capacities in the fish visual system. 9 3.1. Sox2 positive in proliferative cells As mentioned above, the function of the Sox2 protein has been mainly attributed to proliferation capacities. Indeed, our study combining Sox2 staining with PCNA identified proliferating cells in different zones of the pre-encephalic visual system (PGZ, retina and ON). The co-localization of Sox2 and PCNA has been reported before in other zones of the CNS (Alunni et al., 2010; Ito et al., 2010; Sirbulescu et al., 2015), but this work reports for first time both markers in the pre-encephalic visual system of fish. As expected, all cells of the PGZ were double positive for both markers (Fig. 2AA’’, B-B’’). In the differentiated retina, all PCNA positive cells found in NFL were also positive for Sox2 (Fig. 2C-C’’), but most PCNA positive cells in the ONL were negative for Sox2 (Fig. 2B-B’’, C-C’’ and supplementary Fig. 1A). This is remarkable because (1) all Sox2 and PCNA positive cells in the NFL were located very close to new axons and were most likely glial cells recently described (Garcia-Pradas et al, 2018), and (2) the absence of Sox2 in PCNA positive cells in the ONL previously identified as rod precursors (Cid et al., 2010; Song et al., 2017), indicates that Sox2 is not necessary for cells to stay in the cell cycle for final photoreceptor differentiation. Similarly, although we detected reactivity for both Sox2 and PCNA in the ON, we did not find many cells double labeled. This result supports again the notion that Sox2 is not necessary for proliferation, and some non-proliferative cells keep Sox2 expression. Since all cell bodies in the ON are glial cells, we can conclude that Sox2 is not required for glial cell proliferation. Whether Sox2 is involved in maintaining proliferative and differentiation capacities in ON glial cells remains to be shown. 3.2. Sox2 in glial cells Previous studies reported the presence of Sox2 in retinal Müller cells (Hever et al., 2006; Surzenko et al., 2013; Bachleda et al., 2016; Lust and Wittbrodt, 2018). Our results confirmed these findings by the localization and typical cell shape, and by double staining of Sox2 with GFAP and GS markers. We detected that all outer sublayer of Sox2 positive cells in the INL were also positive for GFAP or GS. The expression of Sox2 by Müller cells is not surprising since these cells are regarded as potential stem cells in the fish retina especially during regeneration (Bernardos et al., 2007; Craig et al., 2010). 10 The use of GFAP antibodies to characterize Sox2 positive cells in the ON implicates some difficulties since GFAP is not constitutively expressed in the ON of all fish (Levine, 1989). Thus, we found many Sox2 and GFAP double labeled cells in the ON of A. burtoni, but not in zebrafish. However, in zebrafish we characterized Sox2 and GS double labeled cells in the ON, yet not all Sox2 cells were GS positive, nor all GS cells positive for Sox2. Since all ON components are axons or glial cells we can assume that glial cells can express Sox2 protein also in ON. These cells very likely are members of the astrocyte family but could also be glial precursor cells. Sox2 has been shown to be expressed in astrocytes in mouse retina playing a role in retinal vascular development (Kautzman et al. 2018). However, the fish species we used do not have any intraretinal blood vessels and therefore no intraretinal astrocytes. 3.3. Sox2 in neuronal cells Using antibodies for the calcium-binding proteins PV and CR to characterize Sox2 positive cells we found several different cell populations. Many Sox2 positive cells were positive for PV and none for CR (Fig. 4). These cells were identified as amacrine and displaced amacrine cells, according to the localization in the retina (Weruaga et al., 2000; Mack et al., 2004, Mack, 2007). To our knowledge, this is the first report showing Sox2 in PV expressing amacrine cells in the fish retina. Whereas some studies in mice inmunodetected ChaT in Sox2 positive amacrine cells (Whitney et al., 2014), in fish we did not co-localize any ChaT reactivity in Sox2 positive cells. This indicates that Sox2 plays different roles in the pre-encephalic visual system of different vertebrate species. DCX positive and Zn8 positive cells were negative for Sox2 protein but these markers for newly differentiating neurons and their axons allowed us to investigate the relationship of growing axons with Sox2 expressing cells (see 3.4). Obviously, the ON did not show any immunoreactivity for neuronal markers. 3.4. Role of Sox2 in the pre-encephalic visual system fish Our investigation and many previous studies (e.g. Ito et al., 2010; Maerz et al., 2010) provide evidence that Sox2 has an important function keeping stem cell characteristics also in the fish visual system. In the retina, our results show that Sox2 and PCNA markers co-localized, not only in PGZ but also in NFL, and in a few cases in the ON. 11 This function could also apply for Müller cells, which are known to have proliferative capacity in the growing and regenerating visual system of fish (Surzenko et al., 2013; Lust and Wittbrodt, 2018). In addition, the proliferative function of Sox2 is a likely explanation for the PCNA/Sox2 positive cells in the NFL which could be glial precursors to generate the recently described oligodendroglial-like cells (Garcia-Pradas et al., 2018) in order to myelinate new axons. This is supported by the recent report that Sox2 is a positive regulator for myelination in the developing and postnatal mouse brain (Zhang et al., 2018). Current bibliography does not provide much information about the expression of Sox2 in the ONH (Fischer et al., 2010; Tiwari et al., 2014). It is remarkable that the area where new axons re-order in the ONH labeled by DCX (Fernandez-Lopez et al., 2016; Garcia-Pradas et al., 2018), was free of Sox2 positive cells. This could have functional importance in the continuous growing process that occurs in teleost fish (see in 3.4). To confirm that the re-arranging DCX positive fibers in the ONH were not astrocyte processes similarly oriented in this zone (Parrilla et al., 2009; 2012), we performed double stains with DCX and GFAP. The results did not show any co-localization of DCX and GFAP fibers in the ONH (supplementary Fig. 1D). Previous work reports the presence of resident glial precursors in the ON (Lillo et al., 2002; Garcia and Koke, 2009; Parrilla et al., 2009), which provide new glial cells necessary to receive, guide, support, orient and myelinate the new axons. (Maggs and Scholes, 1986; Arenzana et al., 2011). For quantitative analysis we selected the sections of cichlid fish due to the larger size compared to zebrafish and the precise retinotopic and chronological organization of the ON (Scholes, 1991). The high density of Sox2 positive cells in the first portion of the ON could be a pool of potential proliferative cells (Parrilla et al., 2009) to support the continuous growth of the fish visual system. In addition, Sox2 positive cells show a parallel distribution around new axons entering and running at the new edge of the ON as a fascicle. The fascicle itself is negative for Sox2 (Fig. 5A’’’, C’’’). These Sox2 positive cells possibly indicate a neurochemical niche for the new axons, coming from the entire circumferential retinal periphery, to compact themselves in a fascicle to enter the ON (Maggs and Scholes, 1990; Scholes, 1991). The absence of Sox2 positive cells in the ONH, and the complete lack of co-localization with new axon markers suggest the existence of this particular neurochemical niche. In fact, the region of new axons in the ON of A. burtoni has previously been shown to be surrounded by a 12 particularly high expression of tight junction molecules (Mack and Wolburg, 2006). In the present study, we provide evidence that Sox2 cells have a close relationship with the pathway of new axons in the growing fish visual system. We hypothesize that this implies signaling to indicate to new axons the correct zone they need to cross in order to reach the ON entrance, pass into correct fascicle in the ON (Fig. 6), and form the chronological distribution of ON fibers. This view is also supported by the few Sox2 positive cells aligning with new axons in the NFL. Liu et al. (2014) have shown that Sox2 can work as a repressor in neuronal stem cells. Other studies discuss the role of Sox2 in the differentiation processes in development (Kondoh et al., 2004; Hever et al., 2006; Kautzman et al., 2018). The function of this transcription factor in differentiated cells remains unclear. However, this could also indicate that fish maintain a high plasticity of their mature cells, or at least of their progenitor cells. 3.5. Conclusions In summary, we found Sox2 positive cells co-labeled for either markers of cell proliferation, of the astroglial family, or of neuronal cells in retina and ON. We did not observe any Sox2 expression in the ONH, yet in the ON, Sox2-positive glial cells were lining the fascicles of new axons. Taking together, the differences of Sox2 expression indicate that this protein has several different functions in the CNS of adult vertebrates. Besides known functions in stem cell pluripotency, Sox2 cells could be involved in neurochemical signaling and/or a pool of potential proliferative cells, in possible combination with other regulatory factors, in the fish visual system. Our results suggest that this function is associated with the pathway navigation of the new axons from the retina. Understanding the variety of cell types and subtypes in the visual system of fish and their plastic capacities could be the key to comprehend the growing processes in the adult vertebrate CNS. 4. Experimental procedure 4.1. Animals For this study we used 7 adult specimens of Astatotilapia burtoni wild type measuring between 3-5 cm of standard length, and 10 specimens of AB (5) and 13 Tg(GFAP:EGFP) (5) strains Danio rerio measuring more than 1.5 cm. For the comparison of ON sizes we used material from previous studies (Garcia-Pradas et al., 2018). All fish were bred in our own colonies, cichlid fish (Astatotilapia burtoni) in the Institute of Clinical Anatomy und Cell Analysis (University of Tübingen, Germany) and zebrafish (Danio rerio) in the Institute of Neuroscience of Castilla y León (University of Salamanca, Spain). All animals were maintained in aquaria under a 12 h light/dark cycle without food restrictions at 28.5±1ºC. The animals were deeply anaesthetized with MS222 and sacrificed by decapitation. All procedures were performed in accordance with the guidelines of European Union Council Directive (2010/63/EU). Animal procedures were approved by local authorities (Regierungspräsidium Tübingen and Animal Ethical Committee of University of Salamanca) before experimentation in both institutions. 4.2. Tissue preparation To analyze the components of the pre-encephalic visual system (retina, ONH and ON) we dissected eyes, ONs and brain. The samples were fixed in 4% paraformaldehyde dissolved in phosphate buffer saline 0.1M pH 7.2 (PBS) overnight at 4ºC. After the fixation the tissues were rinsed in PBS and cryoprotected in 30% sucrose, overnight at 4ºC, embedded in OCT medium and sectioned on a cryostat at 14 µm thickness. 4.3. Immunohistochemistry We combined the immunohistochemical detection of Sox2 with different proteins to characterize the Sox2 positive cells. The specificity of the commercial antibodies used in this study has been previously reported (Table 1). For zebrafish experiments we also used the immunohistochemical technique in a Tg(GFAP:EGFP) transgenic line (Bernardos and Raymond, 2006). Sections were pre-incubated in PBS with 0.02% (v/v) Triton X-100 (Probus), 5% (v/v) normal donkey serum (Jackson) and 1% (v/v) dimethyl sulfoxide (DMSO; Sigma). For PCNA labeling, sections were treated with 2N HCl in PBS for 30 min before preincubation. After blocking, they were incubated with the primary antibodies (Table 1) diluted in PBS with 0.02% (v/v) Triton X, 1% (v/v) DMSO and 5% (v/v) normal donkey serum, overnight at 4ºC. 14 After washes with PBS, sections were incubated with fluorescent secondary antibodies for 90 minutes at room temperature in PBS with 0.02% (v/v) Triton X, 1% (v/v) DMSO and 5% (v/v) normal donkey serum (Table 1). Nuclei were labeled with either DAPI (Sigma-Aldrich) or DRAQ5 (Thermo Fisher Scientific). Negative controls without first or secondary antibodies were performed. Sections were mounted with Mowiol® or Fluoromont®. 4.4. Image acquisition Sections were examined on a LSM510 confocal microscope (Zeiss®, Oberkochen, Jena, Germany), or an Axio Observer Z1 apotome microscope (Zeiss®, Oberkochen, Jena, Germany) using laser excitations at 488, 543, and 633 nm in sequential scans with appropriated filter sets. The images were captured with the ZEN 2009®, ZEN 2011® and Leica Confocal Software®, coupled to each microscope respectively. Images were linearly adjusted for contrast, brightness, and uniform the color code, and the images plates were assembled with Adobe® Photoshop® CS6. 4.5. Statistical analysis We counted cells from random areas of 1000 µm2 in 25 sections, selected from at least 5 different adult cichlid fish. The DRAQ5 and Sox2 labeled cells were counted in the retina, ONH and two different zones of ON. The cell quantification was performed under 20x and 40x objectives. The statistical analysis was performed using the software SPSS® using the test of Levene and two non-parametric tests, Kruskal-Wallis and Mann-Whitney-U tests (applying the Bonferroni correction to multiple comparisons). 15 5. References Alunni, A., Hermel, J.-M., Heuze, A., Bourrat, F., Jamen, F., Joly, J.-S., 2010. Evidence for Neural Stem Cells in the Medaka Optic Tectum Proliferation Zones. Dev. 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Figures captions Figure 1: Size increase of the optic nerve (ON) in Astatotilapia burtoni shown in sections stained with antibodies for Sox2 from three different sized animals with standard lengths of 1 cm (A), 3 cm (B) and 11 cm (C). Note the same scale for the three lower images. In the small animal (A), the section goes through the head including both eyes, on the left retinal layers with Sox2 positive cell in the ganglion cell layer (GCL) and inner nuclear layer (INL) are shown. In A', the area of the ON is magnified to show ON entering the brain, B' is a higher power view of B with a different scale. There were many Sox2 positive cells in the center of the ON and also at the ON edge where new axons grow towards the brain (arrows), even in the large fish (C). The enormous difference in eye and optic nerve sizes enable us to study growth and neurogenesis in an adult vertebrate system. Figure 2: Sox2 in proliferating cells of the retina A. burtoni (A, C, D) and D. rerio (B). In the PGZ, Sox2 (A and B) and PCNA (A’ and B’) immunoreactivities are localized in the same cells (white arrows in A’’’ and B’’’; red arrows indicate PCNA positive cells; green arrows indicate Sox2 positive cells). In the differentiated retina, Sox2 positive cells are found in the ONL, INL, IPL, GCL and NFL (C), but very few of these cells are also positive for PCNA (C’, C’’) cells with only one of the labels are shown for Sox2 or PCNA. D depicts the maturation zones in the peripheral retina (D) where Sox2 positive cells are in the PGZ (D, D’’’; green arrows), new neurons labeled with DCX (D’, D’’’; yellow arrows) in the transition zone, and CR positive neurons (D’’, D’’’; red arrows) in the differentiated retina. No co-localization of the labels is found (D’’’). Cells were counterstained with DRAQ5 (blue). Scale bars = 20 µm. Figure 3: Sox2 in Müller cells: Several Sox2 positive cells (green arrows) (A, B and C) in the INL are also positive (white arrows) for GFAP (A’-A’’ and C’-C’’) or GS (B’B’’). In both species (A and B: A. burtoni; C: D. rerio) the Sox2 immunoreactivity appears in the nuclei and GFAP and GS in the cytoplasm (details in A’’’, B’’’ and C’’’; white arrows indicate double labels). Cells were counterstained with DRAQ5 (blue). Scale bars: 20 µm. Figure 4: Co-localization (white arrows) of Sox2 (green arrows) with PV (red arrows) in the INL (A, C). Some cells have both markers (white arrows). Apparently, the A. burtoni (A’) retina shows a higher density of PV positive cells than D. rerio (C’) where most double labeled cells are found in the GCL. No co-localization for Sox2 and CR (red arrows) are observed in retina of either species (A. burtoni B-B’’ and D. rerio D-D’’). Cells were counterstained with DRAQ5 (blue). Scale bars: 20 µm. Figure 5: Characterization of Sox2 positive cells in the ONH and ON of A. burtoni. The DRAQ5 nuclear label indicates many cells in the ONH but none of them is positive for Sox2 (A, A’’). Combined immunolabeling for new axons with DCX (A’, B’) does not show any co-localization with Sox2 (A’’’, green and yellow arrows), nor with CR positive fibers (B’’, (B’’’; green, yellow and red arrows). Some Sox2 positive cells are found close of the ONH (C), but a detailed analysis shows that these cells are part of the NFL. These Sox2 positive cells (C’) are also positive for GFAP (C’’, C’’’; white arrows). The new axons re-arrange in the ONH (A’) to fasciculate into a chronological organization in the ON (D) where new fibers from the circumferential retinal periphery 23 run at one edge of the ON (yellow arrows in D’’ and D’’’). In contrast to the ONH, Sox2 positive cells occur in a relatively high density at the beginning of the ON (green arrows in A’’’) especially surrounding the fascicle of new axons. Sox2 positive cells do not directly co-localize with the axon markers (yellow arrows) but surround fiber bundles (green arrows in A’’ and D’’). Cells were counterstained with DRAQ5 (blue). Scale bars: 20 µm. Figure 6: Sox2 characterization in the ON. Sox2 (green arrows) and PCNA (red arrows) positive cells in the ON of A. burtoni (A-A’’’) and D. rerio (B-B’’’). A few Sox2 and PCNA double positive cells (A’’ white arrows) in the first portion of ON, close to ONH, are observed but most proliferating cells in the ON are not positive for Sox2. Double immunolabeling of Sox2 (green arrows) and GS (red arrows) in D. rerio (C), shows some cells with both markers (white arrows in C’’, C’’’). In the ON of A. burtoni (D), (but not in D. rerio) GFAP staining was present and some Sox2 positive cells are double labeled (white arrows; single label green arrows). Cells were counterstained with DRAQ5 (blue). Scale bars: 20 µm. Figure 7: Quantification of Sox2 positive cells from selected regions as indicated reveals a highly significant difference between the variance of groups (p-value ≤ 0,01). Thus, the analysis was performed by non-parametric tests (A), which shows a highly significant difference (p-value ≤ 0,01) between the medians of the analyzed regions, with the exception of the retina vs. ON2 comparison (A’). Figure 8: Schematic summary of the general Sox2 expression pattern in the visual system of fish. TABLE 1: List of antibodies used Primary Antibody Source Anti-Choline Chemicon acetyltransferase (ChaT) Anti-Calretinin (CR) Swant Anti-Doublecortin Santa Cruz (DCX) Biotechnology Anti-Glial fibrilar acid Sigma Aldrich protein (GFAP) Anti-GFAP (zrf1) ZIRC Anti-Glutamine Millipore Synthetase (GS) Anti-Proliferating cell Santa Cruz nuclear antigen (PCNA) Biotechnology Anti-Parvalbumin (PV) Sigma Anti-Sox2 Abcam Anti-Neurolin DSHB Secondary Antibody Fluorescence Anti-Rabbit A546 Anti-Mouse A555 Anti-Goat A488 Anti-Rabbit A488 Catalog number Host Dilution AB144P Mouse 1:100 6b3 Mouse 1:1000 sc-8066 Goat 1:100 G6171 Mouse 1:300 zrf1 Mouse 1:400 mab302 Mouse 1:500 sc-56 Mouse 1:300 P3088 ab-97959 zn8 Source Jackson Jackson Jackson Jackson Mouse Rabbit Mouse Host Donkey Donkey Donkey Donkey 1:500 1:400 1:100 Dilution 1:400 1:250 1:400 1:250 24 Anti-Rabbit Anti-Goat Cy3 Cy3 Jackson Jackson Donkey Donkey 1:400 1:250 Highlights: In the fish retina, Sox2 is mainly expressed in proliferative, amacrine and glial cells. Some, but not all glial cell types in the optic nerve of fish express Sox2. No Sox2 positive cells are found in the optic nerve head. In the optic nerve, some Sox2+ cells align with bundles of growing axons. Differences in expression suggest different functions for Sox2 in the adult CNS. 25 26 27 28 29 30 31 32 33