Heterogeneity of glia in the retina and optic nerve of birds and mammals.

Abstract

We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the nestin-related intermediate filament transition. These changes in glial activity correspond with increased susceptibility of neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells. The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells. We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified numerous astrocytes that expressed GFAP, S100beta, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG cells.

Full text

Page 1

Heterogeneity of Glia in the Retina and Optic Nerve of
Birds and Mammals
Andy J. Fischer*, Christopher Zelinka, Melissa A. Scott
Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America
Abstract
We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These
cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the
nestin-related intermediate filament transitin. These changes in glial activity correspond with increased susceptibility of
neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells.
The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells.
We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were
distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of
adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified
numerous astrocytes that expressed GFAP, S100b, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that
were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in
the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and
NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are
present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG
cells.
Citation: Fischer AJ, Zelinka C, Scott MA (2010) Heterogeneity of Glia in the Retina and Optic Nerve of Birds and Mammals. PLoS ONE 5(6): e10774. doi:10.1371/
journal.pone.0010774
Editor: Karl-Wilhelm Koch, University of Oldenburg, Germany
Received March 9, 2010; Accepted May 4, 2010; Published June 17, 2010
Copyright: ? 2010 Fischer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health, National Eye Institute RO1 EY016043-04. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: fischer.412@osu.edu
Introduction
The retinas of vertebrates contain many different types of glial
cells. The primary activities of these glia include retinal
homeostasis and support of neuronal function. Across all
vertebrate species, retinal glia include Mu ¨ller glia, derived from
retinal stem cells, and microglia, derived from hematopoietic stem
cells. With some variations between species, retinal glia can
include astrocytes and oligodendrocytes. For example, the
avascular retinas of chickens, guinea pigs and rabbits contain
oligodendrocytes that myelinate the axons of ganglion cells in the
nerve fiber layer (NFL) [2,3,4]. Although vascular retinas contain
significant numbers of astrocytes that are closely associated with
the blood vessels [5,6,7], avascular retinas contain few, if any,
astrocytes [1,8].
In the chicken eye, we have recently identified a novel type of
glial cell that is scattered across inner retinal layer [1]. These cells
were termed Non-astrocytic Inner Retinal Glial (NIRG) cells. The
NIRG cells express vimentin, Sox2 and Sox9, similar to Mu ¨ller
glia and retinal progenitors, but these cells do not express other
glial markers such as TopAP, glutamine synthetase or high levels of
glial fibrilliary acidic protein (GFAP). We found that IGF1
stimulated the NIRG cells to proliferate, migrate distally into the
retina, and up-regulate transitin, an intermediate filament
orthologous to mammalian nestin. Further, IGF1 stimulated
microglia to acquire a reactive morphology and up-regulate
CD45 and lysosomal membrane glycoprotein. The IGF1 receptor
was expressed only by presumptive NIRG cells and microglia that
were scattered across inner retinal layers. With glial cells
stimulated by IGF1, there were elevated levels of cell death and
widespread focal retinal detachments in response to an excitotoxic
insult. The increased cell death was prominent within areas of
retinal detachment which were coincident with a stark loss of
Mu ¨ller glia and an accumulation of NIRG cells. Taken together,
these findings indicate that NIRG cells are a novel type of retinal
cell that is sensitive to IGF1 and whose activity impacts the
survival of retinal neurons and Mu ¨ller glia. At the time of
hatching, NIRG cells are scattered across all regions of the retina,
with greater abundance in central regions, and this distribution
remains unchanged during the first 4 weeks of postnatal
development [1].
There have been no studies that examine whether NIRG cells
are present in the retinas of non-avian vertebrates. Accordingly, in
this study we test the hypothesis that NIRG-like cells are present in
the retinas of mice, guinea pigs, dogs and non-human primates. In
addition, we probe for NIRG-like cells in the optic nerve and
nerve head of chickens, mice, guinea pigs, dogs and macaque
monkeys.
Results
For all results described herein, observations were made on
tissues obtained from mature animals with normal, healthy retinas.
Therefore, descriptions of the different types of glial cells represent
PLoS ONE | www.plosone.org1 June 2010 | Volume 5 | Issue 6 | e10774

Page 2

the phenotypes of stable, mature, non-reactive cells. In the retinas
of species examined in these studies, astrocytes were identified
based on expression of GFAP and other markers known to be
expressed by mature retinal glia including Sox2, Sox9 and S100b
[1,9,10,11,12,13,14]. We probed for NIRG cells immunolabeling
for Sox2, Sox9, Nkx2.2, vimentin and transitin/nestin, and an
absence of GFAP [1]. Although the homeodomain transcription
factors Sox2 and Sox9, and the intermediate filament transitin/
nestin are best-known to be expressed by neural and glial
progenitors, many recent studies have indicated that the
expression of these genes persists in mature, post-mitotic,
functional glial cells in the retina of birds and mammals
[1,9,10,11,12,13,14]. Nkx2.2,
factor, that is best-known to be expressed by oligodendrocyte
precursors in the developing spinal cord [15,16,17], and by
oligodendrocyte precursors in the developing chick visual system
[18].
ahomeodomain transcription
Glial cells in the optic nerve of chickens
Our previous work has demonstrated that NIRG cells are
scattered across central and peripheral regions of the retina; these
cells are predominantly found in the distal third of the IPL and in
the GCL [1]. We have observed that NIRG cells migrate into the
retina from the optic nerve (Zelinka, Scott and Fischer,
unpublished), similar to the astrocytes and oligodendrocytes that
are found in the avian retina [18,19]. However, the distribution of
NIRG cells in the optic nerve of post-hatch chickens has not been
reported. Accordingly, we assayed for NIRG cells and oligoden-
drocytes in the optic nerve and optic nerve head (ONH) of the
post-hatch chicken.
We found numerous NIRG cells within the chick optic nerve
and nerve head (Fig. 1). The NIRG cells were identified by co-
expression of Nkx2.2 and Sox2, and an absence of GFAP and
absence of transferrin binding protein (TFBP). Oligodendrocytes
in the avian central nervous system can be identified by
immunolabeling for TFBP [20,21,22]. Although a few of the
TFBP+oligodendrocytes within the GCL were Nkx2.2-positive
(Figs. 1a-d), none (n=312) of the TFBP+oligodendrocytes in the
optic nerve were immunoreactive for Nkx2.2 (Figs. 1a-g).
Consistent with the pattern of expression in the retina, a small
minority (8.261.9%) of the TFBP+cells in the optic nerve were
immunoreactive for Sox2; the majority of the Sox2+cells in the
optic nerve were negative for (or expressed very low levels of)
TFBP (Figs. 1e-g). Immunoreactivity for GFAP was elevated in
Mu ¨ller glia that were located within 200 mm of the ONH (Figs. 1e-
g). Within the optic nerve and nerve head numerous cells were
intensely immunoreactive for GFAP, and most of these cells
expressed Sox2 (Figs. 1h-j) and Pax2 (not shown). However, there
were numerous Sox2+cells in the optic nerve that were negative
for Nkx2.2 (Figs. 1h-j). The Sox2+/GFAP+/Nkx2.22cells were
likely to be optic nerve astrocytes. In addition, we identified
numerous cells in the optic nerve and nerve head that were
positive for Sox2/Nkx2.2 and negative for GFAP (Figs. 1h-j); these
cells were likely to be NIRG cells. See table 1 for a summary of
markers expressed by glial cells in the chick eye, and table 2 for a
summary of the types of glial cells in the retina, optic nerve and
nerve head.
Glial cell in the mouse eye
The NIRG cells in the chick retina express the transcription
factors Sox2, Sox9 and Nkx2.2. Thus, we began by assaying for
Sox2, Sox9 and Nkx2.2 in the adult mouse retina to test whether
NIRG cells are present in this tissue. Although we failed to detect
Nkx2.2 in the mouse retina, we found widespread expression of
Sox2 and Sox9; these factors were present in the nuclei of Mu ¨ller
glia (Figs. 2a and 2e), consistent with previous reports [12,13]. In
addition, Sox2 and Sox9 were present in the nuclei of S100b/
GFAP-positive astrocytes in inner retinal layers (Figs. 2a-g). All
(n=187) of the Sox9-positive nuclei within the ganglion cell layer
and NFL were co-labeled for GFAP. Similarly, all (n=127) of the
Sox2-positive nuclei within the GCL and NFL were labeled for
S100b. In addition, we observed Sox2-immunoreactivity in the
nuclei of cholinergic amacrine cells in the vitread INL and
displaced to the GCL; these cells were co-labeled for Islet1 (data
not shown). Co-labeling for Islet1 and Sox2 was used to distinguish
displaced cholinergic amacrine cells in the GCL; Islet1 is expressed
by cholinergic retinal amacrine cells in all birds and mammals
[23,24,25]. Within the optic nerve and nerve head of the mouse
eye, numerous astrocytes were labeled for Sox9 and GFAP or
Sox2 and S100b (Figs. 2h and 2i). We did not detect Nkx2.2-
positive cells in the ONH or optic nerve, within 1 mm of the
retina. Thus, NIRG-like cells may not be present in the eyes of
mice. See table 1 for a summary of markers expressed by glial cells
in the mouse eye, and table 2 for a summary of the types of glial
cells in the retina, optic nerve and nerve head.
Glial cells in the guinea pig eye
To assay for NIRG cells in the guinea pig retina we labeled
tissues with antibodies to Sox2, Sox9 and Islet1; presumptive
NIRG cells in the IPL or GCL should be positive for Sox2 and
Sox9, but not Islet1. Immunoreactivity for Islet1 was detected in
the nuclei of presumptive bipolar cells, ganglion cells and sparsely
distributed cholinergic amacrine cells (Figs. 3a and 3b). Immuno-
reactivity for Sox9 was found only in the nuclei of Sox2+Mu ¨ller
glia (Figs. 3c-e). In addition to the Mu ¨lller glia, antibodies to Sox2
labeled the nuclei of orthotopic and displaced cholinergic
amacrine cells that were positive for Islet1 (Figs. 3a-e). To further
probe for glial cells we labeled retinal sections for S100b. We
found that all of the fusiform Sox2-positive nuclei in the middle of
the INL were those of S100b-expressing Mu ¨ller glia (Figs. 3g-h).
There were no cells that were labeled for Sox2 and Sox9 in the
IPL, GCL or NFL, suggesting that NIRG cells are not present in
the guinea pig retina. GFAP-immunofluorescence was not
detected in the guinea pig retina (data not shown), suggesting
the absence of astrocytes within the neural retina. The absence of
GFAP-expressing cells in the guinea pig retina is consistent with
previous reports [14,26,27].
We next probed for glial cells in the optic nerve and nerve head
of the guinea pig eye. We found numerous cells that expressed
Sox2 and Sox9 within the ONH; these cells did not express GFAP
or Nkx2.2 (Figs. 4a-d). Interestingly, cells that expressed GFAP
and/or Nkx2.2 were observed within the optic nerve; these cells
were located approximately 300 mm posterior to the vitread
surface of the ONH (Figs. 4a-d). In the optic nerve, all (n=164) of
the Nkx2.2-positive cells were immunoreactive for Sox9. The
identity of the GFAP/Sox9/Nkx2.2-expressing cells in the optic
nerve of the guinea pig remains uncertain, but these cells are not
orthologous to the avian NIRG cells given the expression of
GFAP. Within the nerve head and the optic nerve, all of the Sox9-
positive cells were immunoreactive for S100b, consistent with the
hypothesis that these were glial cells. See table 1 for a summary of
markers expressed by glial cells in the guinea pig eye, and table 2
for a summary of the types of glial cells in the retina, optic nerve
and nerve head.
Glial cells in the dog eye
TheexpressionpatternsofIslet1,Sox2andSox9indogretinaand
optic nerve were similar to patterns seen in chick, mouse and guinea
Glial Cells in the Retina
PLoS ONE | www.plosone.org2 June 2010 | Volume 5 | Issue 6 | e10774

Page 3

pig eyes. For example, immunoreactivity for Islet1 was observed in
the nuclei of bipolar cells in the distal INL and in presumptive
cholinergic amacrine cells in the proximal INL and GCL that were
positive for Sox2 (Figs. 5a-d). We observed immunoreactivity for
Sox2 and Sox9 in the nuclei of Mu ¨ller glia and in the nuclei of cells
that were scattered across the GCL and NFL (Figs. 5a-d).
Approximately half of the Sox9+cells in the GCL and NFL were
immunoreactive for GFAP (Figs. 5e-h), indicating that these cells
were astrocytes. Levels of GFAP-immunoreactivity were not
detectable in the Mu ¨ller glia in normal, healthy dog retinas. More
than half (52.567.7%; n=225 cells) of the Sox9-positive cells in the
GCL were negative for GFAP. The identity of the Sox9+/GFAP2
cells in the GCL and NFL of the dog retina remains uncertain.
There was no labeling for Nkx2.2 in the dog retina (not shown). The
Sox2+/Sox9+cells in the dog retina were negative for PCNA (data
not shown), indicating that these cells were post-mitotic.
Figure 1. Glial cells in the chick optic nerve are immunoreactive for Nkx2.2, Sox2, Sox9, GFAP and TFBP. Longitudinal sections through
the optic nerve and nerve head were labeled with antibodies to Nkx2.2 (magenta in a, d, f and g; blue in e; green in h and j), Sox2 (red), TFBP (green
in c–e and g) and GFAP (grayscale; i and j). Images were obtained by using wide-field epifluoresence microscopy (a–g) or confocal microscopy (h–j).
The region indicated by the yellow box in panel d is enlarged 4-fold in panels e–g, and the region in panel j is enlarged 2-fold in the inset. Arrow-
heads indicate the nuclei of GFAP/Sox2-positive Mu ¨ller glia. Small double-arrows indicate TFBP-positive oligodendrocytes in the retina. Arrows
indicate Sox2/Nkx2.2-positive nuclei of NIRG cells in the optic nerve and retina. Carets indicate Sox2-positive nuclei of peripapillary glia. Hollow arrow-
heads indicate Sox2/Nkx2.2-negative, TFBP-positive oligodendrocytes in the optic nerve. Small double-arrows indicate TFBP-positive
oligodendrocytes in the retina. The scale bar (50 mm) in panel d applies to a–d, and the bar in j applies to h–j. Abbreviations: ONL – outer
nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer.
doi:10.1371/journal.pone.0010774.g001
Glial Cells in the Retina
PLoS ONE | www.plosone.org3June 2010 | Volume 5 | Issue 6 | e10774

Page 4

In the optic nerve and nerve head of dog eyes, we found
numerous cells that were immunoreactive for Sox2 and Sox9.
Nearly all (95.264.1%) of the Sox9-positive cells were immuno-
reactive for Sox2, and all of the Sox2-positive cells were
immunoreactive for Sox9 (Fig. 6). Nearly half (44.368.5%) of
the Sox9-positive cells were immunoreactive for Nkx2.2 (Figs. 6a-
f). Many of Sox2/Nkx2.2-positive cells in the optic nerve and
nerve head were negative for GFAP (not shown), suggesting that
these cells may have been NIRG cells. The peripapillary glia were
positive for Sox2 and Sox9, but negative for Nkx2.2 (Figs. 6c-f).
See table 1 for a summary of markers expressed by glial cells in the
dog eye, and table 2 for a summary of the types of glial cells in the
retina, optic nerve and nerve head.
Glial cells in the monkey eye
Patterns of expression for Islet1, Sox2 and Sox9 in monkey
retina were similar to those observed in the eyes of other species
that we examined. Islet1 was detected in the nuclei of bipolar cells,
ganglion cells and cholinergic amacrine cells (Figs. 7a and 7d).
Immunolabeling for Islet1, Sox2 and Sox9 revealed many cells
scattered across the GCL and NFL that were immunoreactive for
both Sox2 and Sox9, but negative for Islet1 (Figs. 7a-d). All
(n=271) of the Sox9-positive cells in the GCL or NFL were
immunoreactive for Sox2 (Figs. 7a-d). The Sox2+/Sox92cells in
the GCL were Islet1+, suggesting that these cells were displaced
cholinergic amacrine cells (Figs. 7a-d). We tested whether any of
the Sox9-positive cells in the GCL were some type of ganglion cell
by combining labeling for Sox9 and Brn3a. Brn3a is known to be
expressed by ,98% of ganglion cells [28]. There was no overlap
of labeling for Sox9 and Brn3a (Fig. 7a), suggesting that none of
the Sox9-positive cells in the GCL were ganglion cells.
More than half (58.369.4%; n=164 cells) of the Sox9-positive
cells in the GCL were positive for GFAP (Fig. 7f-l), indicating that
Sox9 is expressed by mature astrocytes. However, this finding also
suggests that the Sox9+/GFAP- cells in the GCL and NFL are
some type of non-astrocytic glial cell. To assess this possibility we
probed for additional glial markers. Since the astrocytes in the
mouse retina (Fig. 2) and dog retina (not shown) express S100b, we
examined whether the Sox2+/Sox9+cells in the GCL and NFL of
the monkey retina were positive for S100b. Surprisingly, we found
that the patterns of S100b expression in the monkey retina were
Table 1. Summary of the glial cell types in the eyes of
different vertebrate species.
Cell type ChickenMouse
Guinea
PigDog Monkey
Mu ¨ller glianumerous numerous numerous numerous numerous
Retinal astrocytes raremanynonemanymany
Retinal
oligodendrocytes
some none somenonenone
Retinal NIRG cells manynonenonemanymany
NIRG-like cells in
the ON
numerousnonenonemanymany
GFAP+ glia in the
ONH
numerousnumerous nonenumerous numerous
ONH – Optic Nerve Head, ON – optic nerve.
doi:10.1371/journal.pone.0010774.t001
Table 2. Summary of immunolabeling of glial cells in the retina and optic nerve of different vertebrate species.
RETINA ChickenMouse Guinea Pig DogMonkey
GFAPAstrocytesAstrocytes No labeling Astrocytes Astrocytes
S100b
No labelingAstrocytes Mu ¨ller gliaNo labeling Mu ¨ller glia ,half
vimentinMu ¨ller glia & NIRG cells Mu ¨ller glia end-feet &
astrocytes
Mu ¨ller glia end-feetMu ¨ller glia end-feet Mu ¨ller glia end-feet
astrocytes
Nestin/transitinNIRG cells No labeling Not doneNot done NIRG cells
Nkx2.2NIRG cells & Oligodendrocytes No labelingNo labeling No labeling No labeling
Sox2Mu ¨ller glia Cholinergic
amacrine cells & NIRG cells
Mu ¨ller glia, Cholinergic
amacrine cells & astrocytes
Mu ¨ller glia Cholinergic
amacrine cells
Mu ¨ller glia, Astrocytes,
Cholinergic amacrine
cells & NIRG cells
Mu ¨ller glia, Astrocytes,
Cholinergic amacrine
cells & NIRG cells
Sox9Mu ¨ller glia NIRG cells
Oligodendrocytes
Mu ¨ller glia & AstrocytesMu ¨ller glia Mu ¨ller glia, Astrocytes
& NIRG cells
Mu ¨ller glia, Astrocytes
& NIRG cells
OPTIC NERVE Chicken MouseGuinea Pig DogMonkey
GFAP AstrocytesPeripapillary glia &
Astrocytes
Astrocytes AstrocytesAstrocytes
S100b
No labelingPeripapillary glia &
Astrocytes
Peripapillary glia & optic
nerve glia
No labelingPeripapillary glia & optic
nerve glia
vimentinAstrocytes & Peripapillary glia,Not doneNot done Astrocytes Astrocytes
Nestin/transitin NIRG cells No labelingNot doneNot done Glia in the ONH
Nkx2.2Glia in the ONH & optic
nerve
No labelingOptic nerve glia Optic nerve glia &
ONH glia
Optic nerve glia
Sox2 Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Sox9 Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
Peripapillary glia & optic
nerve glia
ONH – Optic Nerve Head.
doi:10.1371/journal.pone.0010774.t002
Glial Cells in the Retina
PLoS ONE | www.plosone.org4 June 2010 | Volume 5 | Issue 6 | e10774

Page 5

not similar to those seen in the retinas of mice and dogs, but were
similar to those seen in the retinas of guinea pigs. Nearly half
(46.168.1%; n=201 cells) of the Sox2-positive Mu ¨ller glia were
co-labeled for S100b (Figs. 8a-g). Interestingly, not all of the
Mu ¨ller glia were labeled for S100b, and S100b was not detected
other types of glial cells in the primate retina.
Since the NIRG cells in the chick retina normally express
transitin, the avian ortholog of mammalian nestin, we examined
whether nestin was expressed in primate retina. Scattered across
the NFL in central and peripheral regions of the retina, we
detected many cells that were immunoreactive for Sox9 and nestin
(Figs. 8g-i). The processes of nestin-positive cells tended to project
parallel to the vitread surface of the retina (Figs. 8g and 8h). In
addition, we observed numerous cells within the ONH that were
immunoreactive for both nestin and Sox9 (Fig. 8f). A recent report
has demonstrated that Pax2 is expressed by glial cells in the adult
primate retina; nearly half of the Pax2-positive cells in the GCL or
NFL are GFAP-expressing astrocytes [14]. We found that more
than one-third (39.168.6%) of the Pax2+/Sox2+cells in the GCL
or NFL expressed nestin (Figs. 8l-s). The Sox2+/Pax22cells in the
GCL were putative displaced cholinergic amacrine cells (Figs. 8l-s).
The Sox2+/Sox9+cells in the primate retina were negative for
PCNA (data not shown), indicating that these cells were post-
mitotic.
Since the NIRG cells in the chick retina express Nkx2.2, we
probed for Nkx2.2 in the glial cells of the monkey retina. We failed
to detect Nkx2.2+cells within the monkey retina (not shown).
However, similar to the guinea pig eye, we detected numerous
Figure 2. Glial cells in the mouse retina and optic nerve are immunoreactive for Sox2, Sox9, GFAP, and S100b. Sections through the
retina and optic nerve head were labeled with antibodies to Sox9 (magenta; a–d and h), GFAP (green; a–d and h), Sox2 (magenta; e–g and i), and
S100b (green; e–g and i). Hollow arrow-heads indicate the nuclei of Mu ¨ller glia. Arrows indicate the nuclei of astrocytes. Small double-arrow-heads
indicate the nuclei of cholinergic amacrine cells that are labeled for Sox2. Asterisks indicate blood vessels. The scale bar (50 mm) in panel a applies to
a alone, the bar in d applies to b–d, the bar in g applies to e–g, and the bar in i applies to i and h. Abbreviations: ONL – outer nuclear layer, INL –
inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
doi:10.1371/journal.pone.0010774.g002
Glial Cells in the Retina
PLoS ONE | www.plosone.org5 June 2010 | Volume 5 | Issue 6 | e10774