(Investigative Ophthalmology and Visual Science. 2000;41:2019-2022.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Cone Properties of Retinal Margin Cells in the Monkey (Macaca mulatta)
Xiaoming Chen1,
Kenneth C. Wikler2 and
Peter R. MacLeish1
1 From the Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia; and
2 Division of Neurobiology, Yale School of Medicine, New Haven, Connecticut.
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Abstract
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PURPOSE. To characterize a cell population in the monkey retinal margin that was
labeled with a cone-specific antibody and to determine the presence of
additional markers.
METHODS. Retinal whole-mount preparations from infant and adult rhesus monkeys
(Macaca mulatta) were immunolabeled by incubation overnight
with the primary antibodies 7G6, a cone-specific antibody; SV2, a
synaptic-vesicle antibody; and opsin antibodies that recognize either
the short or long/middle wavelength–sensitive opsins.
RESULTS. The retinal margin cells labeled by 7G6 lay within 1 mm of the ora
serrata and differed from 7G6-labeled cones in the central retina. The
margin cells possessed a soma, a fiber process, and a terminal
enlargement that lay in the plane of the retina; no outer segment was
discernible. A total of 5400 and 7252 margin cones cells were found in
each of two monkeys. The terminal enlargement and soma of the labeled
margin cells also showed SV2 immunoreactivity. Surprisingly, opsin
immunoreactivity extended throughout the margin cell, which is
consistent with the absence of a discernible outer segment.
CONCLUSIONS. Cells with immunoreactive cone properties were found in the margin of
the monkey retina. The absence of an outer segment and the presence of
somatic opsin and SV2 are reminiscent of features observed in the
central cones of fetal monkey retinas. These results suggest that a
subpopulation of cones in the retinal margin might fail to mature
completely and thus retain juvenile characteristics into
adulthood.
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Introduction
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The margin of the retina in vertebrates is the last retinal area
to differentiate during development, and in a number of cold-blooded
vertebrates, the cells in this area retain embryonic features such as
the ability to proliferate.1
2
In fish, where eye growth
continues throughout adulthood, new cells are added at the retinal
margin. In adult newts, which are capable of retinal regeneration, a
major site of proliferation is the retinal margin.3
Thus,
in cold-blooded vertebrates the margin retains progenitor cells that
can be stimulated to divide in the adult. The developmental potential
of retinal margin cells in primates has received less attention. In
primates, as in fish and amphibians, the margin of the retina becomes
increasingly thin, and the characteristic laminar organization of the
retina is progressively degraded. In addition, the cellular composition
and interconnections of the margin remain poorly defined. In monkey
retinal whole mounts treated with a cone-selective antibody, we
detected a population of cells that possessed immunoreactive properties
of central cones but was oriented differently in that the cells lay in
the plane of the retina, had their long axis parallel to the retinal
surface, and lacked a distinct outer segment.4
The present
article reports the distribution of these conelike cells that we will
refer to as margin cones and the presence of other immunologic markers
found in central retinal cones.
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Materials and Methods
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Tissue Preparation
Eyes were obtained from 18 rhesus macaque monkeys (Macaca
mulatta) in adherence with the ARVO Statement on the Use of
Animals in Ophthalmic and Vision Research. The monkeys ranged in age
from 1 month to 19 years and were euthanatized after terminal
experiments at the Yerkes Regional Primate Center of Emory University.
No monkey was killed solely for the purposes of the experiments
reported here. The globes were removed within 10 to 15 minutes of death
and placed in Hanks’ balanced salt solution at 4°C. Retinas were
removed within 1 hour of enucleation, separated from the retinal
pigment epithelium, and fixed in 4% paraformaldehyde for at least 1
hour. Complete retinal whole mounts or retinal pieces were rinsed with
phosphate-buffered saline (PBS) and then labeled with the antibody 7G6
in the presence of 0.1% Triton X-100 at 4°C overnight. Antibody 7G6
is a mouse monoclonal antibody known to label cones in the central
retina.5
The tissue was then incubated with secondary
antibody from a Vectastain Elite ABC kit (Vector, Burlingame,
CA) and treated according to the manufacturer’s instructions to
visualize labeled cells. Other whole-mount preparations were
double-labeled, either with 7G6 plus SV2, a mouse monoclonal antibody
to synaptic vesicles6
or with 7G6 plus rabbit antibodies
that recognize the short wavelenth–sensitive (S) opsins or the
long/middle wavelength–sensitive (L/M) opsins.7
For
double labeling with the two mouse primary antibodies, 7G6 and SV2, the
7G6 antibody was directly conjugated with Cy2, a green fluorescent
probe (Amersham Pharmacia Biotech, Piscataway, NJ) following
the manufacturer’s instructions. The retinal whole mounts were first
incubated with SV2 in the presence of 0.1% Triton X-100 at 4°C
overnight and then with rabbit anti-mouse IgG conjugated to Texas red
for 2 hours. Next, the tissue was incubated with 7G6 directly
conjugated to Cy2 in the presence of 0.1% Triton X-100 at 4°C
overnight. For double labeling with 7G6 and the opsin antibodies,
conventional indirect methods were used because the primary antibodies
were from different species. The double-labeled retinal tissue was
mounted on slides with the photoreceptors side up and viewed with a
conventional microscope or with a confocal laser scanning microscope
(Multiprobe 2001; Molecular Dynamics, Sunnyvale, CA) in the presence of
antifade reagent (Bio-Rad, Hercules, CA).
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Results
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7G6-Labeled Cells at the Retinal Margin
The antibody 7G6 labeled a population of cells with unique
morphologic characteristics in the very far periphery or margin of the
retina, that is, within 1 mm of the ora serrata (Fig. 1)
. This distinct population of cells lay in the plane of the retina with
their long axis parallel to the retinal surface, and frequently an
entire cell was in the plane of focus within the whole-mount. This
contrasts with cones in posterior retina, which lie more or less
perpendicular to the retinal surface. Most of the labeled cells in the
margin possessed a soma with no obvious outer segment, an elongated
process, and a terminal enlargement. Other labeled cells seemed to
consist only of a soma. These cells were as heavily labeled as cones in
central retina. The intensity of the labeling of the margin cells
suggests that they were related to central cones. Therefore, we will
refer to these cells as margin cones.
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Figure 1. Whole-mount labeling of retinal margin cells with 7G6, a cone-specific
antibody. A population of heavily labeled cells can be seen at the
retinal margin. The cells lie in the plane of the retina with their
long axis parallel to the retinal surface. Most of the labeled cells
possess a soma without an obvious outer segment, a fiber process, and a
terminal enlargement. Some cells show only the soma. Calibration bar,
20 µm.
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Numbers and Distribution of Margin Cones
Margin cones were observed in all 18 retinas examined. These
retinas ranged in age from 1 month to 19 years. Typically, labeled cell
bodies were not contiguous to one another, and the process and terminal
enlargement lay in a position central to the cell body. Margin cones
were more prominent in some retinas than in others, but their
prominence did not seem to depend on age. We counted the 7G6-labeled
cells in the retinal margin from two animals. The retinas of one animal
(age 9 years) contained 2472 and 2928 margin cones, and of the other
(age 4.5 years) 3457 and 3795 margin cones (Fig. 2)
. The distribution of the margin cones within a single retina was not
uniform, as shown by the numbers of cells in each quadrant of the whole
mount (Fig. 2)
, but no obvious pattern was discerned.
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Figure 2. Distribution of 7G6-labeled margin cones. Whole retinas were labeled
with 7G6, cut as shown, and mounted on slides. The total number of
labeled cells is shown for each quadrant. Margin cones were found
within 1 mm of the periphery. Local variations in the density of cell
labeling was observed. F, fovea; O, optic nerve head.
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Double-Label Studies with 7G6 and SV2 Antibodies
The presence of other markers associated with cones in the central
retina was investigated with double-labeling experiments. The
double-labeling procedure with the two mouse antibodies, 7G6 and SV2,
gave excellent separation of labeling, as judged by the specificity of
labeling of central retinal sections (not shown). Confocal microscopic
images of double-labeled whole mounts of peripheral retina show the
pattern of 7G6 labeling (Fig. 3A
) and of SV2 labeling of the same field (Fig. 3B)
. The labeling with
SV2 antibody was present not only in the terminal region but extended
along the process and was even observed in the soma. When the labeled
images are superimposed (Fig. 3C)
and enlarged (Fig. 3D)
, both labels
overlapped most extensively in the terminal region. Therefore, the
margin cones, identified by 7G6 labeling, contain synaptic vesicle
protein detected by SV2.
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Figure 3. Confocal microscopic images of the same field of a retinal whole-mount
preparation showing margin cones double-labeled with 7G6 and SV2, an
antibody against synaptic vesicle protein. (A) Cells labeled
with 7G6 directly conjugated to Cy2 (green). (B)
Cells labeled with SV2 followed by rabbit anti-mouse secondary antibody
conjugated to Texas red. Heavy labeling is seen in the
terminal region and in the distal part of the fiber process. When
present, the labeling in the perinuclear region was moderate.
(C) The superimposed images from (A) and
(B). Areas that are double-labeled with 7G6 and SV2 appear
yellow. Calibration bar, 20 µm. (D) Enlargement
of the double-labeled cell shown inside the box in
(C). Calibration bar, 5 µm.
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Antibodies to Cone Opsins
The presence of cone opsins was investigated within the population
of margin cones by double labeling with 7G6 and cone opsin antibodies.
The vast majority of these cones expressed the L/M-opsin (Figs. 4A 4B)
. In a survey of 10 fields that contained L/M-labeled cells, 92%
(490/530) of the 7G6-labeled cells showed L/M antiopsin reactivity; no
cells labeled with L/M-opsin antibody were negative for 7G6. The
L/M-opsin antibody was clearly distributed throughout the soma,
process, and terminal enlargement. As reported earlier, the absence of
an outer segment from these margin cones was a consistent feature. In
central cones, the labeling with opsin antibody was highly localized in
the outer segment (not shown). In contrast, double labeling with 7G6
and S-opsin antibody indicated a small proportion of the margin cones
expressed S-opsin (Figs. 5A 5B)
. In a survey of 10 fields, 2.7% (11/401) of the 7G6-labeled margin
cells were also labeled with the S-opsin antibody. No cells positive
for the S-opsin antibody were negative for 7G6. Like the labeling with
the L/M-opsin antibody, the labeling with S-opsin antibody was found on
the soma, process, and terminal enlargement.
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Figure 4. Confocal microscopic images of a whole-mount preparation of retinal
margin tissue double-labeled with 7G6 and L/M-opsin antibody. Those two
antibodies were from different species and were applied at the same
time; they were followed by anti-mouse IgG conjugated to Texas red and
anti-rabbit IgG conjugated to FITC. L/M-opsin antibody labeling
(B) was seen in most of the 7G6-labeled cells
(A). Note that the entire cell is labeled with the L/M-opsin
antibody. The asterisk shows cells labeled with 7G6 but not
with L/M-opsin antibody. Calibration bar, 20 µm.
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Figure 5. Confocal microscopic images of a whole-mount preparation of retinal
margin tissue double-labeled with 7G6 and S-opsin antibody. The
double-labeling procedure was same as that described for Figure 4
. Labeling with S-opsin antibody (B) was seen in a
small subpopulation of 7G6-labeled cells (A). The
asterisk shows cells that were labeled with 7G6 but not with
S-opsin antibody. Calibration bar, 20 µm.
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Discussion
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The cone-selective antibody, 7G6, identified a population of cells
in the margin of the monkey retina that was specifically and intensely
labeled, as were cones in the central retina. Given the high
specificity of 7G6 for central cones, we conclude that these margin
cells are related to central cones. The long axis of the margin cone
lay in the plane of the retina and was orthogonal to that of central
cones. The presence of these margin cones was detected in retinas that
ranged in age from 1 month to 19 years. On the basis of this
observation, we conclude that these cells are present throughout the
life of the retina and do not represent an aging process,8
given their presence in young retinas. The high specificity of 7G6 for
cones facilitated the identification of the margin cones. In the
flat-mount preparations, these cells have a striking appearance and
seem healthy at the light microscopic level. The antigen is one of the
earliest found in cones in central retina,5
and efforts
are underway to identify it.
In addition to 7G6 labeling, margin cones shared other properties with
cones in central retina. Both cell populations are labeled with SV2,
but the labeling was not as highly localized in the margin cones. In
the central retina, SV2 labeling is confined to the terminal region. In
developing cones,9
10
in addition to labeling of the
terminal, diffuse labeling was observed in the soma. The finding of SV2
labeling in peripheral cones within the age group in this study, 1
month to 19 years, prompted the notion that the margin cones might be
halted at a stage of development.
The labeling of margin cells with the antibodies to cone opsins also
revealed similarities and differences with central cones. The main
difference was the diffuse pattern of labeling in the margin cones
compared with that in mature central cones, where labeling is almost
exclusively in the outer segment. The diffuse labeling by opsin
antibodies was, however, similar to that seen during retinal
development10
11
12
and reminiscent of SV2 labeling
described above. Diffuse visual pigment labeling has also been observed
in association with disease states. Milam et al.13
reported widespread delocalization of rhodopsin in rods obtained from
patients with retinitis pigmentosa. It seems that mutations in
rhodopsin and/or the absence of a mature or intact outer segment can
cause massive rerouting of visual pigment to compartments other than
the outer segment. Another similarity lay in the proportion of
peripheral cones expressing L/M- or S-opsin. The percentage of
7G6-labeled cells that showed L/M-opsin antibody labeling was similar
to that reported for the central retinal cones, roughly 90%. The
percentage of 7G6-labeled margin cones that showed S-opsin antibody
labeling was just under 3%, again roughly mirroring the proportion of
S-opsin central cones. Labeling with L/M- or S-opsin antibodies,
however, accounted for only approximately 95% of the 7G6-labeled
cones. A proportion of the remaining 5% might have arisen from failure
to count cells that were lightly labeled with the opsin antibodies or
from the presence of cones that were truly negative for opsins. If a
population of L/M- and S-opsin–negative cones did exist, the question
that arises is whether or not this population remained permanently
negative or represented progenitor cells that eventually expressed one
of the opsins.
Williams14
reported the presence of a band of cones in the
margin of the human retina in a similar position as the margin cones
that we identified in the monkey retina. We wondered whether the human
cells are counterparts of the monkey cells. Williams identified the
human margin cones on the basis of cell size and labeling with opsin
antibodies, whereas we relied on 7G6 labeling. Two major differences in
the two cell types are the presence of the outer segment and the lack
of opsin labeling in the soma of the human margin cones. With our
current information, it is difficult to reconcile these differences. It
might be necessary to immunolabel the human retina with 7G6 to provide
further insight into the relationship between the band of cones in
humans and the margin cones in monkeys.
We have shown that the margin cones possess essential parts of the
transduction machinery, the opsins and, possibly, the molecular
apparatus for neurotransmitter storage and release as detected by SV-2
labeling. Whether the margin cones are capable of transducing light and
transmitting light information to other retinal cells warrants further
investigation.
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Acknowledgements
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We thank the Yerkes Regional Primate Research Center of Emory
University for providing monkey eyes. We also thank Kathy Buckley, PhD,
for the kind gift of SV2 antibody and Jeremy Nathans, MD, PhD, for the
generous gift of the opsin antibodies.
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Footnotes
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Supported by grants RO1 NS-35510 (PRM), U54 NS-34194 (PRM), and RCMI grant 5G12RR03034.
Submitted for publication March 2, 1999; revised November 2, 1999 and January 11, 2000; accepted February 2, 2000.
Commercial relationships policy: N.
Corresponding author: Peter R. MacLeish, Neuroscience Institute, Morehouse School of Medicine, 720 Westview Drive, GA 30310. macleip{at}msm.edu
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Abstract
Introduction
