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Investigative Ophthalmology & Visual Science. Vol. 31. No. 8. August 1990
Copyright © Association lor Research in Vision ;wd Ophthalmology
Localization of Insulin-Like Growth Factor-1 Binding
Sites in the Embryonic Chicken Eye
Steven Dossnerr and Dovid C. Deebe
Insulin-like growth factor-1 (IGF-1) binding sites were localized in the embryonic chicken lens, retina,
and retinal pigmented epithelium (RPE) with the use of autoradiography. Each of the ocular tissues
exhibited specific binding of radiolabeled ligand. Labeling occurred over the entire surface of the lens
from 6-day-old embryos, including the epithelium, basal ends of the fiber cells, and the annular pad.
There was relatively little labeling over the lens nucleus. A similar pattern was seen in lenses from
19-day-old embryos. In isolated retinas from embryos of this age, the inner and outer plcxiform layers
were most heavily labeled. In the 19-day-old RPE, only the apical surface of the cells was heavily
labeled. Electron microscopic studies revealed an apical layer of membranous material that may
represent outer photoreceptor segments that remained attached to the RPE during dissection. It was
uncertain, therefore, whether the IGF-1 was binding to sites on the RPE or to these membrane
fragments. Invest Ophthalmol Vis Sci 31:1637-1643, 1990
High-affinity receptors for insulin-like growth factor-1 (IGF-1) have been identified in a number of
embryonic tissues,1"3 and IGF-1 has been shown to
stimulate growth and differentiation in both whole
embryos4 and cultured cell lines.56 Recently, IGF-1
has been implicated in the control of cellular differentiation in the lens of the eye.7 We now report on the
distribution of IGF-1 binding sites in the embryonic
chicken lens and other ocular tissues.
The lens of the vertebrate eye consists of two cell
types: elongated fiber cells, which make up the bulk
of the lens, and an anterior layer of epithelial cells,
from which the fiber cells are derived. Fiber differentiation is characterized by extensive cellular and biochemical specialization, including cell elongation,
synthesis and accumulation of lens crystallin pro-
From the Department of Anatomy, Uniformed Services University of the Health Sciences, Bethcsda. Maryland.
Supported by National Institutes of Health grant EY04853 and
Biomedical Research Support Grant 0996-700-0678 administered
through the Henry M. Jackson Foundation for the Advancement
of Military Medicine.
The opinions or assertions contained herein are the private ones
of the authors and arc not to be construed as official or reflecting
the views of the Department of Defense or the Uniformed Services
University of the Health Sciences.
The experiments reported herein were conducted according to
the principles set forth in the Guide for Care and Use of Laboratory
Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23.
Submitted for publication: September 8, 1989; accepted November 27, 1989.
Reprint requests: Steven Bassnctt, PhD, Department of Anatomy, Uniformed Services University of the Health Sciences. 4301
Jones Bridge Road, Bcthesda. MD 20814-4799.
teins, cessation of cell division, and the degradation
of membrane-bound organelles. In the adult, lens cell
differentiation is restricted to the lens equator, where
new fiber cells are continually produced from the mitotically active cells at the margin of the epithelium.
This process continues throughout life, with older
fiber cells being covered by layers of newer fibers.
The embryonic chicken lens has provided a useful
model with which to study the factors that control
lens fiber cell differentiation. Primary explains of epithelial cells from the lenses of 6-day-old embryos undergo many of the changes characteristic of fiber cell
formation when exposed to IGF-1,7 insulin,89 vitreous humor,10 or fetal bovine serum.1112 A factor(s)
that promotes lens cell differentiation in vitro has
been identified in vitreous humor and termed lentropin.10 Recently, Beebe et al7 used a monoclonal antibody raised against human IGF-1 to prepare affinity
columns that completely removed lentropin activity
from chicken embryo vitreous humor. In addition,
they showed that IGF-1 could substitute for lentropin
in the chick lens explant bioassay, raising the possibility that lentropin may be identical to, or closely related to, IGF-1. It has generally been hypothesized
that intraocular concentration gradients of lentropin
are responsible for the spatial patterns of cell differentiation observed in both embryonic and adult
lenses in vivo. A simple hypothesis, suggested initially
by the elegant lens reversal experiments of Coulombre and Coulombre,1314 is that a sharp vitreousto-aqueous concentration gradient might specifically
promote cell differentiation in the epithelial cells immediately anterior to the lens equator. Another possibility is that lentropin receptors are restricted to cer-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1990
tain cell populations and that it is the distribution of
receptors rather than gradients of lentropin that is
responsible for the patterns of growth and differentiation observed. In their experiments, the Coulombres were able to demonstrate that the entire anterior epithelium of a 6-day lens was capable of differentiating into fiber cells when the orientation of
the lens was surgically reversed in the embryonic
eye.13 This indicates that the epithelial cells from
6-day-old lenses possess lentropin receptors. However, unlike epithelial explants from 6-day-old lenses,
explants from 19-day-old embryonic lenses or adult
lenses do not differentiate in vitro in response to the
stimuli listed above.15"17 This could be because of the
lack of receptors for lentropin or changes in the way
these cells respond to receptor occupancy. If we make
the assumption that IGF-1 and lentropin are identical, or closely related, then we can differentiate between these alternatives, using 125I-IGF-1 autoradiography to map the distribution of binding sites in
lenses from 6- or 19-day-old chicken embryos. During these experiments we also localized IGF-1 binding sites in retina and retinal pigmented epithelium
(RPE). Although previous studies have identified specific IGF-1 receptors in embryonic chicken lenses18
and in rat19 and bovine20 retinas, this is the first study
to describe the distribution of binding sites at the
cellular level.
Materials and Methods
Peptides
Recombinant, HPLC-purified human. (3-[l25I]iodotyrosyl) IGF-1 [Thr59] was purchased from Amersham (Arlington Heights, IL) at a specific activity of
74 TBq/mmol. Unlabeled recombinant human
IGF-1 was from AMGen (Thousand Oaks, CA). Both
peptides were reconstituted directly into the incubation media.
Tissue Preparation
Fertile chicken eggs were obtained from Truslow
Farms (Chestertown, MD) and incubated at 38°C in
a forced draft incubator (Humidaire Model 55, Humidaire. New Madison, WI). Tissue was removed
from the eyes of 6- or 19-day-old embryos and placed
in warm Ringer's solution of the following composition: NaCl, 133 mM; KC1, 4.5 mM; MgCl2, 1 mM;
CaCl2, 1.5 mM; glucose, 6 mM; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 mM
(pH 7.3). With the use of fine forceps, the adhering
ciliary epithelium and vitreous humor were carefully
removed from the lenses. After the vitreous was removed, the RPE and retina were easily accessible,
Vol. 31
although they tended to become detached from each
other during removal. The two layers were cut into
small pieces (5 to 10 mm2) before incubation with the
peptides.
Autoradiography
After a 30-minute equilibration period in Ringer's
solution, tissue was placed in 200 n\ of incubation
medium in a humidified chamber. The incubation
medium consisted of Ringer's solution containing either 5 nM 125I-labeled IGF-1 or 5 nM l25I-labeled
IGF-1 and 500 nM unlabeled IGF-1. Lenses were
incubated for 5 or 15 hr in one of the two incubation
media. To prevent receptor internalization and recycling, incubations were performed at 4°C. Pieces of
retina and RPE were incubated for 5 hr in a similar
fashion. For the binding competition studies, contralateral lenses, or, in the case of retina and RPE, pieces
of tissue from the same eye, were used. After incubation, tissue was briefly washed in Ringer's solution
(4°C) and then fixed in 2% glutaraldehyde/0.05 M
phosphate buffer (pH 7.4). For the RPE and retinal
tissue, overnight fixation at 4°C gave satisfactory results, but for the lenses 3 or 4 days of fixation was
necessary. After this period, the yellow "fixation
band" had progressed to the center of the older lenses,
and subsequent histologic examination revealed good
tissue preservation. After fixation, the tissues were
washed in phosphate buffer (2 X 10 min), dehydrated
through a graded series of alcohols and propylene
oxide, and embedded in Epon/Araldile (Electron Microscopy Sciences, Ft. Washington, PA). Midsagittal
sections were cut at 1 ^m and collected onto cleaned
glass slides. Some sections were stained with methylene blue for orientation and photographic purposes.
Adjacent sections were dipped in Nuclear Track
emulsion (NTB2; Eastman Kodak, Rochester, NY)
and exposed for 2-3 weeks at -20°C. The autoradiographs were developed for 5 min at 15°C in halfstrength D19 developer (Eastman Kodak) and fixed
for 5 min in general purpose fix (Eastman Kodak).
After 2 X 10 min washes in deionized water, the autoradiographs were allowed to air dry before being coverslipped and viewed with the use of dark-field microscopy. Cohort tissue, which had not been incubated with iodinated peptide, was also processed for
autoradiography, to control for chemographic alteration of the final autoradiographic image. For electron microscopic examination, sections were cut at
50-75 nm, stained with uranyl acetate and lead citrate, and viewed on a JEOL 100CX microscope
operated at 80 kV. The autoradiographs presented
here are representative of at least three labeling experiments for each tissue type using two or more
pieces of tissue in each experiment.
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CHICKEN IGF-1 BINDING 5ITE5 / Dossnerr ond Deebe
No. 8
This study was performed in accordance with the
ARVO Resolution on the Use of Animals in Research.
Results
Lens
After a 5-hr incubation period in 125 I-IGF-1,
6-day-old chicken lenses had a normal appearance,
except for vacuole formation in the anterior tips of
the primary fibers (Fig. 1 A). Autoradiographs of these
lenses showed abundant silver grains over the anterior epithelium and equatorial region, with more diffuse labeling over the basal ends of the fibers. The
distribution of label was best seen in dark-field micrographs (Fig. 1B). The central (nuclear) regions of
the lenses showed very few grains. In the presence of a
100-fold excess of unlabeled IGF-1, the silver grains
were reduced to near background levels over the entire lens (Fig. 1C). The cortical regions of lenses from
19-day-old embryos were not well labeled after a 5-hr
incubation, so the period was increased to 15 hr to
allow greater penetration of the ligand. Figure 2A
shows the typical morphologic characteristics of a
19-day-old lens after a 15-hour incubation in 1251-
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IGF-1. Vacuoles appeared in the annular pad region,
but the remainder of the lens was relatively unaffected by this prolonged treatment at 4°C. The darkfield autoradiograph of an adjacent section is shown
in Figure 2B. Silver grains were present across the
epithelium and annular pad and in the posterior
(basal) ends of the lens fibers. In the presence of excess unlabeled IGF-1, most of the labeling was prevented, leaving only light labeling over the lens capsule and adhering vitreous humor (Fig. 2C). It is interesting that, in lenses of both ages, the heaviest
IGF-1 labeling was often seen along the apical (inward facing) surface of the epithelium.
Retina
Despite prolonged incubation at 4°C, the 19-dayold chicken retina had normal morphologic characteristics and the layers of the differentiated retina
were clearly visible (Fig. 3A). In chicken embryos of
this age, the retina usually became detached from the
RPE during dissection of the tissues from the eye and
the inner segments of the photoreceptors generally
remained associated with the retina. The binding of
t25
I-IGF-l to the 19-day chicken retina is shown in
' " I V 1 . ' rii,
A
Fig. 1. IGF-1 autoradiographs of the 6-day-old chicken lens.
(A) Methylene blue-stained section of lens after incubation at 4°C
for 5 hr with I251-1GF-1. The epithelium (E) and annular pad (AP)
are clearly visible. There was some vacuole formation (V) in the
apical ends on the fiber cells. (B) Dark-field image of a nearby
section processed forautoradiography. The silver grains appeared
bright against a dark background. Note the heavy labeling over
the lens epithelium and annular pad region. (C) Dark-field autoradiograph of lens incubated with 125I-IGF-I in the presence of
excess unlabeled IGF-1. (Bar = 250 ixm.)
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Augusr 1990
Vol. 01
•<•
Fig. 2. IGF-1 autoradiographs of the 19-day-old chicken lens. (A)
Montage of a methylene blue-stained section of lens after incubation at
4°C for 15 hr in 1251-IGF-1. The epithelium (E) and annular pad (AP)
are visible, although there is some vacuole formation (V) in the latter.
(B) Dark-field montage of a nearby section processed for autoradiography. Note the heavy labeling over the epithelium and annular pad. (C)
Montage of a dark-field autoradiograph of lens incubated with l2i lJGF-1 in the presence of excess unlabeled IGF-1. (Bar = 500 ftm.)
Fig. 3. IGF-1 autoradiographs of 19-day-old chicken retina. (A) Interference contrast image of a methylene blue-stained section of retina
after a 5-hr incubation at 4°C in l25I-IGF-l. The retinal layers are clearly visible. N, nervefiberlayer; G, ganglion cell layer; IP, inner plexiform
layer; IN, inner nuclear layer; OP, outer plexiform layer; ON, outer nuclear layer; PR, photoreceptors. (B) Dark-field image of a nearby
section processed for autoradiography. Note the heavy labeling over the plexiform layers. (C) Dark-field autoradiograph of retina incubated
with '"I-IGF-I in the presence of unlabeled IGF-1. (Bar = 50 urn.)
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CHICKEN IGF-1 BINDING 5ITE5 / Oossnerr and Deebe
Figure 3B. Although I25I-IGF-1 bound to all the
layers of the isolated retina, the greatest binding appeared to be to the two plexiform layers. In the presence of the unlabeled peptide, labeling was significantly reduced in all layers (Fig. 3C). At day 6 of
embryonic development, the presumptive retina was
present as a pseudostratified epithelium. Studies on
tissue of this age identified a low level of specific binding uniformly across the entire width of the undifferentiated epithelium (data not shown).
Retinal Pigmented Epithelium
At day 19 of development, the chicken RPE is
composed of heavily pigmented cuboidal epithelial
cells (Fig. 4A). Light microscopic examination of
methylene blue-stained sections revealed an amor-
- .7-'-v^*/*
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phous layer of lightly stained material associated with
the apical surface of the RPE cells. At the electron
microscopic level, this layer was seen to consist of
vesicular membrane fragments and may represent
the remains of the developing outer segments of the
retinal photoreceptors (Fig. 4B). The binding of I2SIIGF-1 to the RPE and surrounding tissue is shown in
Figure 4C. The chemical processing of the autoradiograph caused the melanin pigment in the RPE to
bleach and allowed clear visualization of the silver
grains over the tissue. The apical surface of the RPE
was heavily labeled. There was a diffuse distribution
of grains over the connective tissue and blood vessels
of the adjacent choroid. In the presence of competing
peptide, the labeling over the apical surface of the
epithelium was reduced to near background levels
(Fig. 4D). It appeared that most of the specific label-
....-.
Fig. 4. IGF-1 autoradiographs of 19-day-old RPE and adjacent choroidat tissue. (A) Bright-field image of methylene blue-stained tissue after
incubation for 5 hr at 4°C in I25I-IGF-1. The RPE is visible as a layer of densely pigmented cells overlying the vascular and connective tissue of
the choroid. A layer of lightly stained material (arrow) is visible along the apical surface of the RPE layer. This region is shown in the electron
micrograph in (B) where it can be seen to consist of vesicular membranous debris (arrows). (C) Bright-field light micrograph of an unstained
section processed for autoradiography. The melanin pigment was bleached by the photographic processing, enabling visualization of the silver
grains, which are dark against the bright background. In this unstained section the nuclei (n) of the RPE appear as gaps in the pigment layer.
(D) Bright-field autoradiograph of tissue incubated in 12SI-IGF-I in the presence of unlabeled IGF-1. (Bar = 50 pm (A) and 2 p.™ (B).)
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1990
ing at the apical end of the RPE cells was associated
with the membrane fragments, not the apical membrane of the RPE itself. The presence of excess unlabeled peptide caused a small reduction in the labeling
of the choroid. Therefore, it appears that there was
relatively little specific binding to this tissue.
Discussion
Quantitation of ligand binding in autoradiographic
studies of bulk tissue generally requires the use of
serially sectioned frozen material. Unfortunately,
high-quality frozen sections of eye tissue and lens in
particular are difficult to obtain. The approach taken
in this study was to incubate fresh tissue with labeled
peptide and then to use conventional fixation and
plastic embedding to visualize tissue morphologic
characteristics and ligand binding sites. This approach precludes the ready use of densitometric
image analysis for precise quantitation but docs permit autoradiography of high spatial resolution. Although IGF-1 binding studies on whole lenses require
prolonged incubation at 4°C, tissue prepared in this
way retained excellent morphologic characteristics.
The vacuole formation observed in some of the lens
preparations may result from the mechanical removal of the vitreous rather than the lengthy incubation.21
In the current study the identity of the IGF-1 binding site was not determined. Although it was tempting to think that the radiolabeled ligand was binding
to an authentic IGF-1 receptor, it was possible that
some of the ligand was bound to an IGF-2 receptor or
even to an immobilized carrier protein. It should be
noted, however, that previous biochemical studies
have identified high-affinity IGF-1 receptors in the
lens18 and retina.1920 Many of these studies have also
indicated the presence of insulin receptors in the ocular tissues. In preliminary experiments, we have also
detected the binding of 125I-insulin to the tissues studied here (data not shown).
The technique of incubating fresh pieces of tissue
with radiolabeled ligand, followed by fixation and
plastic embedding, provides excellent spatial resolution and morphologic characteristics, but rather poor
penetration. This is particularly true in tissue with
little extracellular space, such as the lens. It is possible, therefore, that the lack of binding of I25I-IGF-1 to
the central regions of the 6- or 19-day-old lenses was a
consequence of the inability of labeled ligand to diffuse between the tightly packed fiber cells to the innermost membranes. Although increasing the incubation time for 19-day lenses allowed for better penetration, to clarify this point further one would have to
prepare frozen sections of lens, in which all fiber
Vol. 31
membranes had equal access to the ligand. It should
be noted that Bassas et al18 found fewer receptors in
membranes prepared from lens fiber cells than from
epithelial cells. Therefore, the absence of binding
over the central fibers in our studies may reflect the
absence of receptors in this region. Clearly, however,
ligand penetration was not a problem in the outer
layers of the lens. This allowed us to address the hypothesis of whether changes in IGF-1 binding site
distribution during development were responsible for
the spatial patterns of growth and differentiation seen
in the lens. There was no evidence in the current
study that the binding sites became preferentially localized during embryonic development to the equatorial zone, where fiber cell differentiation occurs.
Thus, it appears that the topography of lens growth
and differentiation does not simply reflect IGF-1
binding site distribution. The inability of central epithelial cells to differentiate into fibers indicates a
change in the way cells respond to binding site occupancy, not simply the absence of the binding site.
Waldbillig et al20 identified high-affinity IGF-1 receptors in bovine retina. Their studies on isolated rod
inner and outer segment membranes and whole retina suggested that a significant fraction of the IGF-1
receptors must be present in nonphotoreceptor regions. In the current work, we demonstrated binding
to all retinal layers, with enhanced binding to the
plexiform layers. The high level of binding to the
plexiform layers may result from a selective concentration of binding sites in these synaptic layers or may
simply reflect a higher density of membrane surface
area that may be present in this region. Recent work
by Ocrant et al22 has reported the use of autoradiography to localize IGF-1 receptors in frozen sections of
mammalian retina. In contrast to our findings in the
embryonic avian retina, these authors found the
greatest binding of IGF-1 to be to the nuclear and
pholoreceptor layers.
Because of the detachment of the retina from the
RPE during dissection, the outer portions of the photoreceptors were largely lost from our retinal preparations. Binding of IGF-1 to the inner portions of the
photoreceptors appeared to be no greater than the
binding to any of the other cellular layers of the retina. However, the dense labeling of the apical surface
of the RPE cells may represent binding of IGF-1 to
photoreceptor membrane fragments that remained
attached to the RPE during dissection. The basolateral surface of the RPE and the tissue immediately
adjacent to it showed little sign of IGF-1 binding.
This study has confirmed that many, possibly all,
embryonic ocular tissues possess IGF-1 binding sites
that, by analogy with other better-characterized systems,3"6 probably play a role in metabolism and tis-
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CHICKEN IGF-1 BINDING SITES / Dossnerr and Deebe
No. 8
sue differentiation. In the lens, where a role in differentiation is suspected,7 the distribution of IGF-1
binding sites in the outer cell layers is apparently uniform. This suggests that spatial differentiation in this
tissue may result from other factors, such as external
concentration gradients. If this is the case, then it is
important to identify the source of ocular IGF-1. In
human fetuses, the sclera, which is adjacent to the
RPE, was the only abundant source of IGF-1 mRNA
in the posterior globe.23 If IGF-1 is lentropin, then its
transport across the blood-ocular barrier from some
external site of synthesis into the vitreous humor will
be a crucial area for future study.
Key words: IGF-1 receptor, chicken embryo, autoradiography, lens, development
8.
9.
10.
11.
12.
13.
14.
Acknowledgments
The authors thank Dr. Phil Smith for advice on the autoradiography and many useful discussions during the course
of this project.
15.
16.
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