Sox10 Expression in Goldfish Retina and Optic Nerve Head in Controls and after the Application of Two Different Lesion Paradigms

The mammalian central nervous system (CNS) is unable to regenerate. In contrast, the CNS of fish, including the visual system, is able to regenerate after damage. Moreover, the fish visual system grows continuously throughout the life of the animal, and it is therefore an excellent model to analyze processes of myelination and re-myelination after an injury. Here we analyze Sox10+ oligodendrocytes in the goldfish retina and optic nerve in controls and after two kinds of injuries: cryolesion of the peripheral growing zone and crushing of the optic nerve. We also analyze changes in a major component of myelin, myelin basic protein (MBP), as a marker for myelinated axons. Our results show that Sox10+ oligodendrocytes are located in the retinal nerve fiber layer and along the whole length of the optic nerve. MBP was found to occupy a similar location, although its loose appearance in the retina differed from the highly organized MBP+ axon bundles in the optic nerve. After optic nerve crushing, the number of Sox10+ cells decreased in the crushed area and in the optic nerve head. Consistent with this, myelination was highly reduced in both areas. In contrast, after cryolesion we did not find changes in the Sox10+ population, although we did detect some MBP- degenerating areas. We show that these modifications in Sox10+ oligodendrocytes are consistent with their role in oligodendrocyte identity, maintenance and survival, and we propose the optic nerve head as an excellent area for research aimed at better understanding of de- and remyelination processes.


Introduction
The transcription factor Sox10 belongs to the Sox family and is characterized by the highmobility DNA-binding HMG domain. Sox10, together with Sox8 and Sox9, form the SoxE subfamily. During the development of the central nervous system (CNS) SoxE proteins promote the formation, differentiation and survival of the oligodendrocyte lineage, from OPCs to myelinating oligodendrocytes [1,2]. Studies performed in Sox10 mutant mice and mutant zebrafish have shown that Sox10 is necessary for oligodendrocyte differentiation during development [3,4]. Moreover, highly conserved enhancer elements of SOX10 have allowed the identification of human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells in cell cultures [5]. Sox10 also promotes the expression of some myelin genes [1,2], such as myelin basic protein (MBP), the second most abundant protein in the CNS [6], which plays a fundamental role in myelin formation [7,8]. Sox10 is thought to promote the gene expression of factors that mediate oligodendrocyte-axon interactions, which are necessary for oligodendrocyte survival during development [4]. Finally, Sox10 is also involved in the myelination of the peripheral nerve system (PNS) during development [2]. In the adult CNS, Sox10 continues to be expressed by mature oligodendrocytes, maintaining their oligodendroglial phenotype [2].
The visual system of adult fish grows continuously due to the addition of retinal cells from the periphery of the retina, called the peripheral growth zone (PGZ). Moreover, the fish visual system can regenerate after suffering a lesion [29,30], in contrast to mammals, whose axons have a very low capacity for regeneration [31]. Thus, nascent axons from the new RGCs can be detected constantly, growing from the PGZ towards the ONH, where they converge to form the ON. When the PGZ is destroyed [32,33], Pax2 astrocytes in the ONH react and proliferate after the arrival of newly born axons from the PGZ [33]. In addition, when the ON is crushed and severed RGC axons are re-growing, the astrocytes and oligodendrocytes of the ONH are respectively responsible for their guidance and packaging [34] and their myelination once they have reached the optic tectum [35,36].
Despite the large body of information available regarding fish ONH astrocytes, little is known about retinal and ONH oligodendrocytes. Thus, the present work aims to clarify the identity of these cells. Moreover, we analyze the response of oligodendrocytes in the absence of newly formed RGCs when the PGZ is destroyed and the new axonal input is lost, comparing this with a well-established model for axonal degeneration, ON crushing.

Material and Methods Animals
We used adult 8-12-cm long goldfish (Carassius auratus) obtained from commercial suppliers. All animals were kept in aquaria at 18±1°C on a 12 h light/dark cycle; they were daily fed and, prior to analysis, were deeply anesthetized with 0.03% tricaine methanesulfonate in water (MS-222; Sigma). All experimental procedures were in accordance with the guidelines of the European Union Council Directive (2010/63/EU) and current Spanish legislation for the use and care of animals (RD 53/2013). Also, all procedures involving animals were assessed and approved by the Animal Ethical Committee of the University of Salamanca before commencing the experimentation in the centres associated with the Experimentation Service.
used to induce the lesion in the peripheral part of the retina of the right eye . In the ON crush  experiments, the lesion was performed in the left ON, 1 mm away from the eyeball with a fine  watchmaker`s forceps for about 3 seconds.  A total number of 100 fish were sacrificed at 2, 7, 15, 21, 30, 60, 90, 120, 180 and 210 days post-injury (d).

Immunohistochemistry
Four animals from each time-point analyzed were perfused transcardially with a NaCl solution followed by 4% paraformaldehyde (PFA) and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4 (PB). Their eyes were dissected out and post-fixed for 2 h at room temperature (RT) in the same fixative solution, then washed in PB, cryoprotected in 50% sucrose, and 14-μm transverse sections were obtained in a cryostat.
For whole-mount retinal staining, fish were dark-adapted for 2 h and then sacrificed. Retinas were dissected out maintaining their orientation from 3 animals per group, fixed for 4h in 4% PFA, and thoroughly washed in PBS. Immunostaining was performed as described above, except that the primary antibodies were incubated for 4 days at 4°C and secondary antibodies were incubated overnight at 4°C.

Image analysis
ONH cell quantification was performed as previously described [33,34]. We counted cells from 2-6 tissue sections randomly selected from 4 control and 4 injured animals from each survival time (2-210d). We counted all labeled cells present in the ONH, which is the area located between the two edges of the neural retina, excluding the central artery [39]. In whole-mount retinas, cells were counted from 3 retinas from both control and injured animals. All cell counts were performed manually with ImageJ software. Statistical analysis were carried out with the Graph Prism software. ANOVA test was run after checking data normality and homoscedasticity. Dunnet's post hoc test was performed to show the differences between control and injured groups.

Electron microscopy
Two animals from each time-point analyzed were perfused transcardially with NaCl solution followed by 4% PFA, 0.2% picric acid and 0.25% glutaraldehyde in PB. The ONH was dissected out and post-fixed with 4% PFA and 0.25% glutaraldehyde in PB for 2 h at RT. After several washes in PB, the samples were fixed with 0.5% aqueous osmium tetroxide, dehydrated and flat-embedded in Epon 12 resin, as described previously [33,39]. Ultrathin sections were stained with 2% aqueous uranyl acetate and lead citrate.

Photomicrographs
Some of the light microscopy and fluorescence images were obtained with an Olympus AX-70 microscope and the other fluorescence images were obtained with a laser scanning spectral confocal microscope (Leica TCS SP2). Ultrathin sections were observed under a Zeiss EM900 electron microscope and pictures were taken with a digital camera connected to the microscope using ImageSP software. The brightness and contrast of the original images were further processed with Adobe Photoshop CS4 software.

Location and distribution of Sox10 + cells in the goldfish retina and ONH
We found that Sox10 immunolabeling was located exclusively in the nuclei of the cells, which were distributed from the retina (Fig 1A-1C) along the whole length of the ON (Fig 1E, see section 2.5).
In the retina, Sox10 + cells were located in the nerve fiber layer (NFL) from regions close to the PGZ ( Fig 1A) to the central retina (Fig 1B and 1C). However, the number of Sox10 + cells in the peripheral retina was much lower than in the central retina (Fig 1A and 1B). In the central retina they were located close to the RGCs in the ganglion cell layer (GCL) and also in vitreal parts of the NFL ( Fig 1B). Similar results were found for the myelin protein marker MBP. We detected a low amount of MBP + labeling in the PGZ, where young, newly formed Zn8 + RGC axons were located (S1 Fig). However, in the central retina MBP + labeling was higher and was distributed throughout the whole thickness of the NFL, whereas the Zn8 + RGC axons remained in the vitreal part of this layer ( Fig 1D).
In the ONH, Sox10 + cells were numerous and were organized in groups and in rows ( Fig 1C  and 1E). They were distributed throughout the whole ONH, but they did not form part of the glia limitans. This organization and distribution differed from Pax2 + astrocytes, which were mostly located in the glia limitans of the central artery (CA) and in the retina-ONH transition ( Fig 1F). By contrast, MBP was located throughout the ONH, with a much more intense staining and organization than in the retina ( Fig 1G and S1 Fig). While Zn8 + /MBP -RGC axons were located close to the CA in the growing edge (Fig 1H), well-organized and compacted MBP + myelin sheaths were located in the mature ONH ( Fig 1I). Fig 1J summarizes the distribution of both Pax2 + astrocytes and Sox10 + cells in the goldfish ONH.
In semithin sections we found these oligodendrocytes arranged in rows, and we analyzed their ultrastructure by electron microscopy (Fig 2A and 2B). They showed a heterochromatic round nucleus and scarce cytoplasm, both classical characteristics of oligodendrocytes. They never showed the typical features of astrocytes, i.e., intermediate filaments or desmosomes ( Fig  2B). Moreover, the oligodendrocytes were enveloped by astrocyte intermediate filament-filled processes, and were located close to myelinated and unmyelinated axons ( Fig 2B).

Sox10 + ONH oligodendrocytes after PGZ cryolesion
Previous studies carried out at our laboratory revealed the relevance of the ONH Pax2 + astrocytes when the PGZ was eliminated. In the present study, we wished to analyze the effect of the loss of this axonal input on Sox10 + ONH cells. To investigate their behavior, we removed this area by cryolesion. The damage and recovery of the PGZ was followed using anti-PCNA antibody, a marker of dividing cells, as previously described by our group [32] (data not shown). At no point of the regenerative process did we detect any significant change in the Sox10 + ONH cell population (p>0.05, Fig 3A) or in its distribution in the ONH (Fig 3B-3D).
In contrast, after 7d we found MBPareas in the ONH. This coincided with the observation that none or very few Zn8 + RGC axons were detected at this regenerative stage (Fig 3E). At 15-21d we detected larger MBPareas, which were mostly located in the ONH (Fig 3F). At the same time, the PGZ regenerated and new Zn8 regenerating RGC axons reached the ONH ( Fig  3F). After 30-60d, the ONH recovered the MBP + labeling, leaving the Zn8 + growing edge unmyelinated (Fig 3G).
We found similar results in both semithin and ultrathin sections. At 7d, we noted the presence of small degenerative areas in the ONH in semithin sections (Fig 4A), which increased at 21d (Fig 4B). After 7d, in ultrathin sections the tissue surrounding these small degenerating areas displayed a well-organized appearance (Fig 4C). In these regions we also detected some cells featuring the typical characteristics of microglial cells, such as heterochromatic and irregular nuclei and cytoplasmic vesicles. We also found cells showing a different pattern of  Sox10 Expression in Goldfish Retina and Optic Nerve Head chromatin packaging and a rich cytoplasm, suggesting oligodendrocytes in different stages of maturation (Fig 4C). In contrast to 7d, after 21d the tissue was much more disorganized with a larger number of degenerating areas (Fig 4D). We found more microglia and blood cells incorporated to the nerve tissue. Similar to 7d, we also observed cells in different stages of maturation (Fig 4D). Sox10 + ONH oligodendrocytes after ON crushing In previous work carried out by us we have shown that Pax2 + astrocytes in the ONH may have a relevant function in axon guidance when ON crushing was performed [34]. In contrast to PGZ cryolesion, after ON crushing most of the damaged RGCs survive, although they have to regenerate their axons and re-establish their retinotectal connections [40]. In order to investigate the role of Sox10 + cells in the ONH in a de-and regenerative environment, we performed ON crushing as a second lesion paradigm.
We detected a highly significant decrease in the number of Sox10 + cells ( ÃÃ p<0.01, Figs 5A and 6A) in the ONH sections after 7d. We found a marked decrease in MBP + immunolabeling at the same degenerative stage (Fig 6B). The remaining weak MBP + labeling in the ONH appeared highly disorganized (Fig 6C). There were no changes in the retinal Sox10 + cells (Fig 5B).
After 15-21d, the number of Sox10 + cells was still significantly lower than in the control animals ( ÃÃ p<0.01, Figs 5A and 6D) and MBP + labeling was still very weak and disorganized ( Fig  6E). From 30d onwards, the number of Sox10 + cells was similar to control fish (p>0.05, Figs 5A, 6F and 6I) and we detected more extensive MBP + areas (Fig 6G). Also, the MBP + myelin sheaths began to look organized (Fig 6H). At 180d, we detected fully recovered MBP + labeling, revealing well-organized myelin sheaths (Fig 6J-6K).
In contrast to cryolesion, in semithin and ultrathin sections we found fewer areas undergoing degeneration in the ONH at 7 and 21d (Fig 7A, 7B, 7E and 7F). Interestingly, and consistent with the MBP + labeling described above, at 7d most RGC axons appeared unmyelinated, and the few that did have a thin myelin sheath were mainly located at a considerable distance from the CA (Fig 7B). Similar to the situation observed after cryolesion, we detected high numbers of microglia and blood cells (Fig 7B). Finally, at 7d we found some cells with a   Sox10 Expression in Goldfish Retina and Optic Nerve Head euchromatic nucleus and a nucleolus. These cells had a rich cytoplasm with an abundance of microtubules, a typical feature of immature oligodendrocytes (Fig 7C and 7D). We found similar results at 21d. Some immature cells were also detected ( Fig 7F).

Sox10 + cell proliferation in the ONH
In control ONH, we found scarce proliferating Sox10 + /PCNA + cells located among the RGC axons (Fig 8A and 8B). After both types of injury we detected a slight qualitative increase in Sox10 + /PCNA + cells in the ONH. While in cryolesioned animals we did not note this increase until 21d after injury (Fig 8D, 8D 1 and 8D 2 ), in the ON-crushed animals we detected it earlier, at 7d (Fig 8F). In both cases, we were able to identify these proliferating Sox10 + cells throughout the regenerative processes (Fig 8C-8H).

Localization and distribution of Sox10 + cells in the ON
Sox10 + cells were distributed throughout the whole ON (Fig 9A). The MBP labeling revealed that the ON was fully myelinated, leaving only the Zn8 + growing edge axons unlabeled ( Fig  10A-10C). In control ON, we also found scarce Sox10 + /PCNA + cells (Fig 9A-9D).

Sox10 + cells after ON crushing
In contrast to the cryolesioned animals, where Sox10 + cells did not change in the ON during regeneration (data not shown), we found important modifications in ON-crushed animals.
After 7d, we detected scarce Sox10 + cells proximal to the crushed area, some of them Sox10 + /PCNA + (Fig 9E-9G). At the same time-point, most of the region was unmyelinated and we only found some degenerating MBP + areas (Fig 10D). Furthermore, we failed to detect any growing Zn8 + axons (Fig 10E and 10F). In contrast, in the distal ON, Sox10 + cells were distributed as in the control animals and some of them were also Sox10 + /PCNA + (Fig 9H-9J). This region was highly myelinated, although some areas appeared with a less intense labeling ( Fig 10G). As in the crushed area, we did not find Zn8 + axons in this region (Fig 10H and 10I).
After 21d, we detected a higher amount of Sox10 + cells proximal to the injured area, some of them colocalizing with PCNA (Fig 9K-9M). Also, only small degenerating MBP + areas appeared in this region ( Fig 10J) and many Zn8 + /MBPregenerating RGC axons populated the proximal ON (Fig 10J-10L). In the distal ON, we found again Sox10 + cells distributed throughout the whole section, together with some Sox10 + / PCNA + cells (Fig 9N-9P). A larger unmyelinated area appeared in the distal ON, which was occupied by Zn8 + /MBPregenerating axons. After 21d, the remaining distal ON contained MBP + axons (Fig 10M-10O).
From 30d onwards, myelin sheaths began to appear organized and Zn8 + axons were restricted to the growth zone. At 180-210d, the ON had completely recovered (data not shown).

Discussion
Here we analyzed Sox10 + cells for the first time in the fish retina and ON, using the goldfish as an animal model. In the development of the CNS and PNS of both mammals and fish Sox10 is present in the oligodendrocyte lineage [1][2][3][4][5], and in mammals it has been demonstrated that Sox10 is expressed in mature CNS oligodendrocytes [2]. In this work we demonstrate that Sox10 is present in retinal and ON oligodendrocytes in adult goldfish. Moreover, we report the behavior of Sox10 + oligodendrocytes in the ONH and ON after retinal cryolesion and ON crush, and show that they behave in a similar way to other oligodendrocyte protein markers during ON regeneration [19,[41][42][43].   The transcription factor Sox10 is a highly conserved protein that has been described in zebrafish, mice and humans [1][2][3][4][5]. It participates actively in the specification of the oligodendrocyte lineage and the differentiation and survival of these cells, as it is expressed from OPCs to mature, myelinating oligodendrocytes [1][2][3][4]. In the latter ones, it is involved in the expression of some myelin genes, such as MBP; and in PNS myelination [1,2], and it is thought to mediate oligodendrocyte-axon interactions indirectly [4]. Here, we used an antibody designed to specifically recognize zebrafish Sox10 [4,44] in order to locate goldfish oligodendrocytes in the visual system. We also show that Sox10 is located in the nuclei of cells in the retina NFL, in the ONH, and in the ON. This location differs from that observed for astrocytes, characterized by the expression of the Pax2 transcription factor. Pax2 + astrocytes are not detected in the goldfish retina, and in the ONH they mainly form the glia limitans [39]. By contrast, Sox10 + cells were distributed from areas close to the PGZ to the whole length of the ON. Furthermore, Sox10 + cells were mainly organized in groups and rows. This particular organization has been described previously in tench oligodendrocytes using electron microscopy techniques [17,18]. Upon examining the ultrastructure of these rows in goldfish, they showed the same characteristics. Thus, we can affirm that goldfish Sox10 + cells are oligodendrocytes.

Oligodendrocyte location in the retina
In this study we detected the presence of both Sox10 + oligodendrocytes and MBP + axons in the NFL of the goldfish retina. Sox10 + cells were organized in groups and in rows, and they were distributed throughout the whole ONH and retina. We also found that retinal MBP + axon bundles had a loose appearance compared with the compact axon bundles detected in the ONH and ON. This is consistent with previous electron microscopy analyses carried out in other fish species [15,17,18,43]. Besides the expression of Sox10 and MBP described here, retinal oligodendrocytes express other proteins and genes related to the oligodendrocyte lineage in zebrafish. Examples of this are Contactin1a, only expressed in differentiating oligodendrocytes [43]; A20 antigen, which recognizes a meshwork surrounding myelinated axons [45]; Claudin k, located in the mesaxon, and the transcription factor Olig2 [19]. However, these oligodendrocytes fail to express P0, a marker of compact myelin [42].
The role of these retinal oligodendrocytes is still unclear, although different theories have been proposed. In birds, which also have oligodendrocytes in both the NFL and the GCL, oligodendrocytes are believed to participate not only in the myelination but also in the nutrition and protection of ganglion cells [22,24]. In lizards, these retinal oligodendrocytes seem to be involved in re-myelination processes after surgical lesion, and it has been proposed that the loose myelin sheaths help to maintain retinal transparency [21]. In fish, it has been suggested that environmental factors would prevent retinal oligodendrocytes from forming compact myelin [19]. All the data taken together indicate that goldfish retinal oligodendrocytes seem to maintain certain aspects of immature oligodendrocytes that allow the continuous growth of the visual system and its ability to regenerate.

Sox10 + oligodendrocytes in regeneration
We have previously shown that ONH Pax2 astrocytes play an important role not only in retinal regeneration [33] but also after ON crushing [34]. Since the fish retina contains mature myelinated axons and immature unmyelinated axons [15], we aimed to analyze the behavior of ONH oligodendrocytes when deprived of new unmyelinated axons (PGZ cryolesion) and when large numbers of myelinated axons and their myelin sheaths are destroyed (ON crushing). Many authors have analyzed the role of oligodendrocytes and myelin-related proteins in the de-and regeneration and remyelination of RGC axons in the ON after an injury [19,35,[41][42][43][46][47][48][49]; however, little attention has been paid to their behavior in the ONH.
Here we show that after PGZ cryolesion no changes occur in the number and organization of Sox10 + oligodendrocytes in the ONH, although we did detect degenerating myelin areas during the first days of regeneration. This was probably due to cell death phenomena, triggered by the elimination of the somata of some RGC in the PGZ. In contrast, after ON crushing, the number of ONH Sox10 + oligodendrocytes was markedly reduced during the first days of recovery, although their population did not change in the retina. Also, MBP + labeling decreased and, instead, a large number of regenerating Zn8 + axons was observed (data not shown) [40]. These data are especially interesting, since numerous studies have described a fast regenerative response of injured axons in the crushed area, which is distant from the ONH [50][51][52][53][54][55][56]. A retrograde degenerating process may first exist prior to regeneration, due to the strong demyelination undergone by the ONH during the first week after crushing. Another more plausible explanation is that the oligodendrocytes could lose contact with the RGC axons as a consequence of ON crushing. In support of this, it has been proposed that the loss of oligodendrocyte-axon contact leads to oligodendrocyte death [4,57,58]. However, whether the oligodendrocytes die, return to an OPC state or dedifferentiate into other type of progenitor, stopping the expression of Sox10, remains to be analyzed.
The results of our analyses of Sox10 + oligodendrocytes and MBP in both the crushed area and the distal ON are similar to those of other studies performed on oligodendrocyte-related proteins [19,[41][42][43]. After 7d, we detected few Sox10 + oligodendrocytes and few myelin MBP sheaths, which increased after 15-21d. The new myelinating cells that invade the regenerating nerve have been described as Schwann cells [49]. Both oligodendrocytes and Schwann cells express Sox10 [1,2,4], although recent studies support the notion that the cells responsible for myelinating the regenerating axons are oligodendrocytes [19,59].
In both the ONH and ON we found a slight increase in dividing Sox10 + /PCNA + cells, and our electron microscopy analyses revealed oligodendrocytes in different stages of maturation. It has previously been demonstrated that oligodendrocytes can be newly generated after ON crushing [19] or from oligodendrocytes that de-and re-differentiate during regeneration [41].
The role of Sox10 in ON regeneration has not yet been analyzed. In other parts of the nervous system, Sox10 has been shown to be involved in the synthesis of myelin proteins, such as MBP [1,2]. Integrins are also candidate targets for Sox10 regulation because they mediate axon-dependent oligodendrocyte survival [4]. Thus, it seems likely that Sox10 plays an active role in fish axon regeneration and remyelination. Further experiments need to be carried out, and as we have shown here the fish visual system, including the ONH, provides a versatile model for the study of oligodendrocytes.

Conclusions
We have shown that the adult goldfish retina contains Sox10 + oligodendrocytes and MBP + loose myelin axons. We also detected mature Sox10 + oligodendrocytes in the ONH and ON. We tested their response after two different lesion paradigms: PGZ cryolesion, which destroys the input of unmyelinated axons to the visual system, and ON crushing, which destroys stable, myelinated axons. Their different behavior reveals their extreme plasticity and suggests that the ONH would be an excellent area for further investigation of these cells.