Oligodendrocyte origin and development in the zebrafish visual system

Abstract Oligodendrocytes are the myelinating cells in the central nervous system. In birds and mammals, the oligodendrocyte progenitor cells (OPCs) originate in the preoptic area (POA) of the hypothalamus. However, it remains unclear in other vertebrates such as fish. Thus, we have studied the early progression of OPCs during zebrafish visual morphogenesis from 2 days post fertilization (dpf) until 11 dpf using the olig2:EGFP transgenic line; and we have analyzed the differential expression of transcription factors involved in oligodendrocyte differentiation: Sox2 (using immunohistochemistry) and Sox10 (using the transgenic line sox10:tagRFP). The first OPCs (olig2:EGFP/Sox2) were found at 2 dpf in the POA. From 3 dpf onwards, these olig2:EGFP/Sox2 cells migrate to the optic chiasm, where they invade the optic nerve (ON), extending toward the retina. At 5 dpf, olig2:EGFP/Sox2 cells in the ON also colocalize with sox10:tagRFP. When olig2:EGFP cells differentiate and present more projections, they become positive only for sox10:tagRFP. olig2:EGFP/sox10: tagRFP cells ensheath the ON by 5 dpf when they also become positive for a myelin marker, based on the mbpa:tagRFPt transgenic line. We also found olig2:EGFP cells in other regions of the visual system. In the central retina at 2 dpf, they are positive for Sox2 but later become restricted to the proliferative germinal zone without this marker. In the ventricular areas of the optic tectum, olig2:EGFP cells present Sox2 but arborized ones sox10:tagRFP instead. Our data matches with other models, where OPCs are specified in the POA and migrate to the ON through the optic chiasm.


INTRODUCTION
Oligodendrocytes are the myelinating cells in the central nervous system (CNS) of vertebrates. Among other functions, they form the myelin sheath required for the fast saltatory conduction of nerve impulse (Baumann & Pham-Dinh, 2001;Czopka, 2016;Hines, 2021).
The visual system, as an intrinsic part of the CNS, is not an exception to myelination (Reichenbach et al., 1988), although there are some variations between species. For example, the ganglion cells axons that form the optic nerve (ON) are myelinated within the retina in fish (Fujita et al., 2000;Lillo et al., 2002;Münzel et al., 2012;Parrilla et al., 2016), reptiles (Santos et al., 2006), birds (Nakazawa et al., 1993), and some mammals like rabbits (Morcos & Chan-Ling, 1997).
However, in other mammals, including humans, the interior of the retina is not myelinated and its aberrant myelination is associated with age and causes several conditions (Berry-Brincat & Shafquat, 2008;FitzGibbon & Nestorovski, 1997;Perry & Lund, 1990). Deficient myelination specifically in the visual system can also lead to several diseases known as Neuromyelitis Optica Spectrum Disorder (Berry-Brincat & Shafquat, 2008). Thus, it is crucial to understand the first moments of myelination. Zebrafish is a well-established model to study development and oligodendrocytes share many key transcriptional factors and specification routes between mammals and fish (Buckley et al., 2008;Lyons & Talbot, 2014;Mathews & Appel, 2016;Preston & Macklin, 2015).
Oligodendrocytes arise from the oligodendrocyte progenitor cells (OPCs), which are characterized by several transcription factors, including Olig2, Sox2, and Sox10 (Park et al., 2002;. OPCs are highly mobile and proliferative cells, which migrate and search for their target axons along the nervous tracts until they ensheath them. In that moment, OPCs become myelinating oligodendrocytes and present, along with Olig2 and Sox10, myelin proteins such as Mbpa, Mpz, or Claudin Κ (Jung et al., 2010;Münzel et al., 2014;Nawaz et al., 2013). In vertebrates like chickens and mice, OPCs in the visual system are originated in the preoptic area (POA), from where they migrate to the optic chiasm (Bribián et al., 2006;Gao & Miller, 2006;Klionsky et al., 2021;Ono et al., 2017). However, the place where these OPCs originate in teleosts is not clear (Tian et al., 2016). Furthermore, the initiation of the myelination process during development is not fully understood. For example, although the first evidence of myelination (mbpa expression) is reported from 2 days post fertilization (dpf), the entrance of mature oligodendrocytes in the retina does not occur until 12 dpf (Brösamle & Halpern, 2002;Buckley et al., 2008). To complicate this story, typical oligodendrocyte markers like Olig2 and Sox10 are present in other cells such as neurons (Sagner et al., 2018) and regions outside the CNS (Santos-Ledo et al., 2017). More interestingly, OPCs produce other members from the Sox family. For example, Sox2 has been implicated in the development of the visual system (Graham et al., 2003;Mercurio et al.,

Embryo manipulation
For tissue sections, embryos/larvae were hand-dechorionated when necessary and fixed in 4% paraformaldehyde in 0. For life imaging, embryos at the proper stage were anesthetized as usual and embedded in 1.5% low melting agarose (ThermoFisher Scientific R0801) in E3 medium.

Immunohistochemistry
Sections were washed several times in PBS and incubated for 90 min in 5% normal donkey (DK) serum in PBS with 0.2% Triton X-100 at room temperature. After that, primary antibodies (

Image acquisition
All images were obtained with a LeicaStellaris (inverted DMI8) microscope. Living embryos tg(olig2:EGFP;mbpa:tagRFPt) were imaged using a 20× objective from the dorsal side. Sections were imaged using a 40× oil immersion objective. In the case of the sections, four tiles were acquired and automatically assembled by the software. Acquired z-stacks were transformed into maximum intensity projections using the LAS X software from Leica. Images were later cropped or rotated in ImageJ. Bright and contrast were only adjusted for better visualization.
Finally, figures were built using Photoshop CS5.

Quantification and statistics
Colocalization of tg(olig2:EGFP; mbpa:tagRFPt) was quantified using the manual Cell Counter plugin included in (Fiji is Just) ImageJ. Six embryos were used per stage. First, the total number of mbpa:tagRFPt cells in the ON chiasm area was counted. Then, we also quantified the number of olig2:EGFP cells in this region and analyzed how many of them presented a total or partially overlapping mbpa:tagRFPt. ANOVA test with a Bonferroni post-test statistics was performed using Graphpad Prism.

OPCs markers are detected from 2 dpf onwards
OPCs have been studied for a long time and their role in health and disease has been pointed out by several groups (Clayton & Tesar, 2021).
However, we lack a full picture of their maturation and the beginning of myelination. Thus, to identify OPCs, we used the transgenic line tg(olig2:EGFP). Our protocol quenched the EGFP fluorescence, so we amplified it by immunohistochemistry. Then, to characterize these olig2:EGFP cells we detected two transcription factors that have been implicated in OPCs differentiation : Sox2 by immunohistochemistry and Sox10 by breeding the double transgenic line tg(olig2:EGFP; sox10:tagRFP).
Olig2:EGFP was present from early somitogenesis, but the first colocalization events with Sox2 and sox10:tagRFP were detected at 2 dpf ( Figure 1a and inset). Based on the evidence in other models (Bribián et al., 2006;Ono et al., 2017) and their position, we identify these olig2:EGFP/Sox2 cells as the first OPCs that will invade the ON.
Since we detected many olig2:EGFP cells negative for Sox2 in the mesencephalon, we wondered if they presented other members of this family. Sox10 is an important transcription factor during oligodendrocyte differentiation (Krasnow et al., 2018;Modzelewska et al., 2016).
So the colocalization between Olig2 and Sox10 was analyzed using the Based on their position, far from the ventricular proliferative zones but close to the POA, and their arborized morphology, we identified these cells as OPCs differentiating into oligodendrocytes. As expected, we also observed single labeled sox10:tagRFP cells in the mesencephalon since this transcription factor is expressed in cells other than oligodendrocytes ( Figure 1f) (Santos-Ledo et al., 2017).

From 3 dpf onwards olig2:EGFP cells spread throughout the visual system
At 3 dpf, when retinal lamination is evident in zebrafish,  (Figure 2m,n,p) and were negative for Sox2 (described later in Figure 5). The most striking change was found in the ON, olig2:EGFP/sox10:tagRFP cells around the ON showed projections colocalizing with CR suggesting that they are differentiated oligodendrocytes and the ensheathing process is starting ( Figure 2o).

Olig2 is also involved in the differentiation of other glial cells in the retina
Our data indicated that OPCs (olig2:EGFP/Sox2 cells) originated in the POA and then extended toward the ON. However, we also observed this colocalization in the retina (Figure 1 and 2). The olig2:EGFP/Sox2 cells in the INL presented elongated morphologies characteristic of Múller glia (Figure 1 and 2), which are very important for the retinal maintenance and structure (Thummel et al., 2008). Olig2 have been implicated in the differentiation of other glial cells (Cai et al., 2007). Thus, we used typical markers for Müller cells (GFAP and GS)  (Figure 2b), we wondered if they colocalized with Pax2, the typical marker for these astrocytes. They did not but they were close to the Pax2+ astrocytes (Figure 3g-i).

Olig2:EGFP cells are observed differentiated from 7 dpf in the ON
The colocalization of olig2:EGFP and Sox2 changed drastically at 7 dpf At 11 dpf, the pattern of olig2:EGFP/Sox2 cells was identical to 7 dpf (Figure 4i,j,l). Sox2 colocalized very rarely with olig2:EGFP in the ON (Figure 4k). In relation to the differentiated oligodendrocytes (olig2:EGFP/sox10:tagRFP), the most important difference compared to 7 dpf is the increased abundance of these cells in the ON and OT with greater arborization (Figure 4 m-p).

3.5
Oligodendrocytes switch from Sox2 to sox10:tagRFP at 5 dpf in the ON Our data pointed to an abundance of olig2:EGFP/Sox2 cells at 3 dpf in the ON (Figure 3). As the embryo matured, we observed a reduction in these cells but an increase in arborized olig2:EGFP/sox10:tagRFP ( Figure 4). To clarify if these cells were the same cells changing their profile, we wondered if they ever presented Sox2 and sox10:tagRFP at the same time.
At 3 dpf in the ON region, olig2:EGFP cells with round morphologies or slightly arborized colocalized with Sox2 but not with sox10:tagRFP In case of the zebrafish ON, this transition first occurs at 5 dpf.

3.6
The first evidence of the ON myelination is detected at 5 dpf An important function of oligodendrocytes is the myelination of axons within the CNS. To follow up the relation between Olig2 and the myelination process, we established a double transgenic line(olig2:EGFP/mbpa:tagRFPt) and analyze the distribution of the transgenes in vivo at 5, 7, and 11 dpf. Mbpa protein binds to myelin and thus can be useful to understand when myelination is occurring (Hughes & Appel, 2020).
The first mbpa:tagRFPt cells of the ON were detected at 5 dpf in the optic chiasm area (Figure 6a,a′). We counted 2 or 3 mbpa:tagRFPt cells per embryo (quantified in figure 6d). This number increased to 5 cells at 7 dpf ( Figure 6b′) and to 8-10 cells at 11 dpf (Figure 6c′; quantified in Figure 6d).
We previously identified key morphological changes of the olig2:EGFP cells starting at 5 dpf. These included cellular elongation and increased arborization. We wondered if this timeline was coincidental with the detection of mbpa:tagRFPt. olig2:EGFP cells in the OT with full arborization colocalized with mbpa:tagRFPt (insets in Figure 6b,c). Interestingly, in the ON, only 40% of olig2:EGFP cells showed this colocalization (inset in Figure 6a′, quantified in Figure 6e).
These colocalization events significantly increased to more than 60% at 7 dpf (inset in Figure 6b′ and quantified in Figure 6e). We observed no further changes between 7 and 11 dpf in relation to the colocalization (inset in Figure 6c′ and quantified in Figure 6e). These data F I G U R E 3 olig2:EGFP cells present other glial markers in the retina; GFAP and GS. olig2:EGFP cells also colocalized with Müller markers: GFAP (a, b) and GS (d, e) in the retina but not in the OT (c, f). Colocalization was detected mostly in the central area of the retina (arrows in band e), while cells in the periphery were just olig2:EGFP (arrowheads in b and e). olig2:EGFP cells in the periphery of the ONH do not colocalize with Pax2, typical maker for reticular astrocytes (g-i), although these two populations are very close (arrow in h). D: dorsal; L: lateral; H: hypothalamus; ONH: optic nerve head; OT: optic tectum. Scale bar in a, d, g: 100 µm; in b, c, e, f, h, i: 50 µm would again suggest that crucial changes are occurring between 5 and 7 dpf.

DISCUSSION
Our analysis show that the visual OPCs (olig2:EGFP/Sox2) are located in the POA at 2 dpf. Olig2 is a key transcription factor during oligodendrocyte differentiation (Zou & Hu, 2021) and Sox2 maintain stem cell properties (Wegner & Stolt, 2005). At 3 dpf, we can find olig2:EGFP/Sox2 cells also in the chiasmatic region of the ON where they start acquiring a more elongated morphology. Five dpf is a key stage since we have observed triple colocalization events (olig2:EGFP/Sox2/sox10:tagRFP) in the ON and increased cellular arborization. Since these cells maintain Sox2 but also colocalize with sox10:tagRFP, a mature oligodendrocytic marker (Parrilla et al., 2016), we identified them as differentiating oligodendrocytes. Finally, from 7 dpf onwards, most of the olig2:EGFP cells in the ON colocalize with sox10:tagRFP but not with Sox2. We have identified these cells as fully differentiated oligodendrocytes. From 7 dpf, the three different populations (olig2:EGFP/Sox2;olig2:EGFP/Sox2/sox10:tagRFP; olig2:EGFP/sox10:tagRFP) coexist in the ON. Tian et al. (2016) suggest an extraocular origin of the oligodendrocytes that envelop the ON but start their description at 7 dpf. Other studies that use ablation or demyelination experiments also describe the formation of oligodendrocytes and their differentiation, but they F I G U R E 4 olig2:EGFP cells are differentiated from 7 dpf. In the retina at 7 dpf, olig2:EGFP cells are restricted to the PGZ and the TZ (a, b). But only the cells in the TZ colocalize with Sox2 (arrows in b). Sox2 that do not show olig2:EGFP are abundant in the proliferative areas of the brain (c, d). olig2:EGFP occasionally colocalize with Sox2 in the optic nerve chiasm at 7 dpf (arrow in c). At 7 dpf olig2:EGFP/sox10:tagRFP cells were found in the ON chiasm (e, arrow in g) and in other parts of the brain (arrows in h). Differentiated part of the retina was empty of olig2:EGFP/sox10:tagRFP cells (f). At 11 dpf, Sox2 keeps its pattern in the retina (i, j) and in the ON (arrow k). In the OT, Sox2 cells are restricted to the ventricular area (l).  , e, i, m: 100 µm; in b, c, d, f, g, h, j, k, l, n, o, p: 50 µm do not pinpoint their origin (Chung et al., 2013;Fang et al., 2014).
Our evidence supports the extraocular origin hypothesis, but indicate that the first OPCs (olig2:EGFP/Sox2) originate in the POA at 2 dpf and then move to the ON arriving to the optic chiasm at 3 dpf. These OPCs will then colonize pre and postchiasmatic regions. Since we do not detect evidence of myelin-related proteins until 5 dpf, this would suggest an earlier contribution of these cells to the development of the ON where they could have more functions besides myelination. In fact, very recently, Xiao et al. (2022) show that OPCs also contribute to axon remodeling during zebrafish ON development as early as 5 dpf. , and 11 (c′) dpf. A 25 µm thick stack is shown as max intensity projection in the OT area (a, b, and c) and the optic nerve chiasm (a′, b′, and c′). Detection of mbpa:tagRFPt starts at 5 dpf in the ON (a′). The number of mbpa:tagRFPt cells increase significantly from 3-4 cells at 5 dpf to 8-10 cells at 11 dpf (a′, b′, c′, quantified in d). A total of 40% olig2:EGFP colocalize with mbpa:tagRFPt at 5 dpf. This percentage increases to 60% from 5 dpf to 7 dpf (quantified in e). E: eye: L: lens; MBH; midbrain hindbrain boundary; ON: optic nerve; OT: optic tectum; PGZ: proliferative germinal zone; SC: spinal cord. Scale bar: 100 µm The timeline described by us (graphical abstract) coincides with data from mice and chicken, where OPCs migrate through the brain until the optic chiasm, from where they finally colonize the rest of the ON (Bribián et al., 2006;Gao & Miller, 2006;Merchán et al., 2007). In mouse and chicken, the OPCs that populate the ON are originated in the third ventricle, but with some differences. In mouse, they are dorsal to the developing optic chiasm (Gao & Miller, 2006), while in chicken they are in the ventral midline region (Ono et al., 1997 We have also found olig2:EGFP cells in the retina. At early stages (2 to 5 dpf) together with Sox2, a progenitor marker (Bylund et al., 2003) and later restricted to the proliferative growth zone where retinal progenitors are . At 3 dpf, olig2:EGFP cells also present markers for Müller glia (GS+). This expression has been associated with developmental and regenerative processes since Müller cells act as retinal neural progenitors (Fimbel et al., 2007;Nakamura et al., 2006;Shibasaki et al., 2007;Thummel et al., 2008). Surrounding the ONH, we have found also olig2:EGFP/Sox2 cells at 3 dpf that lose the olig2:EGFP expression by 5 dpf. Based on our previous experience and since Olig2 is involved in the differentiation of other glial cells (Cai et al., 2007), we hypothesized that they could be the reticular astrocytes that form the optic stalk. However, they lack the characteristic marker Pax2 (Parrilla et al., 2012(Parrilla et al., , 2013Tiwari et al., 2014).
Thus, our data indicate that Olig2 and Sox2 might play a role during the differentiation of the retinal glial cells (DeOliveira-Mello et al., 2019).
To unravel the specific role of these transcription factors would require further experiments.
While the onset of myelination in the spinal cord has been deeply studied (Hines, 2021;Kordes et al., 2005;, there is not much known regarding the myelination in the zebrafish visual system. To study the relationship between oligodendrocytes and the first steps of myelination in vivo, we used the transgenic line mbpa:tagRFPt that allow us to observe the activation of myelin binding protein a gene (Hughes & Appel, 2020;Xiao et al., 2022). In zebrafish CNS, the Mauthner axons are the first ones to become myelinated by 3 dpf (Buckley et al., 2008). Mbpa, and other genes related to myelination, are clearly expressed in some brain regions by 4 dpf (Bai et al., 2011;Brösamle & Halpern, 2002;Jung et al., 2010;Pinzon-Olejua et al., 2017). These authors do not find myelinated axons in the ON by electron microscopy until 7 dpf (Brösamle & Halpern, 2002). However, we detected the first olig2:EGFP/mbpa:tagRFPt cells in the ON at 5 dpf.
All together this data indicate that myelination is a progressive process that might be triggered in different places simultaneously and that there is a lag between the expression of myelin-related proteins and myelination. In fact, it has been shown that OPCs undergo complex morphological changes over time in response to neural activity before myelination can occur (Krasnow et al., 2018). Our results match with these data. The first projections from olig2:EGFP/Sox2/sox10:tagRFP cells that envelop the ON are detected at 5 dpf, as well as the first mbpa:tagRFPt. By 11 dpf, projections are becoming sheaths that envelope the ON (labeled with CR) and mbpa:tagRFPt cells increase significantly. We did not find a full colocalization between mbpa:tagRFPt and olig2:EFGP transgenes, but a close relationship between them. As previously suggested by Hughes and Appel (2020) using the same transgenic line, we are detecting processes associated with myelin rather than oligodendrocytes bodies. Concomitantly to the detection of mbpa:tagRFPt and to the morphological changes, olig2:EGFP cells switch from Sox2 to sox10:tagRFP. This transition is obvious in the ON between 5 and 7 dpf. This might be a consequence of the transition from the proliferative state of OPCs to more mature oligodendrocytes (Ono et al., 2017). This view is reinforced by our description of the early olig2:EGFP/Sox2 cells in the midline, a typical neurogenic zone, that will remain just as Sox2 (Germanà et al., 2011) and by the exclusive presence of sox10:tagRFP in the fully differentiated oligodendrocytes in other areas such as the OT.
Our results show that OPCs in the zebrafish visual system are generated in similar brain areas to other groups of vertebrates, at earlier time points than previously described. We have also found that myelin-related markers can be detected at 5 dpf in the optic chiasm, and that oligodendrocytes extend both dorsally and ventrally in the visual pathway. Therefore, the visual system myelination of zebrafish resembles other vertebrates despite the morphological differences and validate this model to study human diseases related to aberrant myelination. Furthermore, it would be interesting to investigate in the future whether the changes we described during development are conserved in adulthood and during regeneration. For example, oligodendrocytes expressing sox10 have been described to be involved in mouse regeneration (Mendonça et al., 2021). Zebrafish, an animal with continuous growth and where multiple de and remyelination events occur could be an ideal model for this type of research (Chung et al., 2013;Fang et al., 2014;Zou & Hu, 2021).