Download Differential temporal and spatial expression of insulin-like

Differential Temporal and Spatial Expression of Insulinlike Growth Factor Binding Protein-2 in Developing Chick
Ocular Tissues
Timothy J. Schoen* Carolyn A. Bondy,\ Jian Zhou,\ Rita Dhawan,% Krzysztof Mazuruk*
Dagmar R. Arnold* Ignacio R. Rodriguez,* Robert J. Waldbillig* David C. Beebe,%
and Gerald J. Chader*
Purpose. To determine the developmental expression and localization of mRNA for insulinlike growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II,
in ocular tissues of the embryonic and early posthatched chick.
Methods. In situ hybridization and northern blot analysis were used to analyze the cellular
origin and relative expression of IGFBP-2 mRNA in ocular tissues.
Results. Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic
development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo,
including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb
buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at
this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and
endothelium. By El2, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in
several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina,
a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to
the nuclear layers by El2. Northern blot analysis confirms that a high level of IGFBP-2
expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA
expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern
blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of
IGFBP-2 mRNA expression, a value equal to that of brain and liver.
Conclusions. The early appearance, along with differential temporal and spatial expression of
IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of
growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.
Invest Ophthalmol Vis Sci. 1995;36:2652-2662.
A he insulin-like growth factors (IGF-I and IGF-II) are
polypeptides that exhibit mitogenic, metabolic, and
differentiative effects on a variety of cell types. Because
of the relative abundancy of both IGF-I and IGF-II
during development, the IGFs are thought to play an
especially important role in the proliferation and dif-
I'rom the * Laboratory of Retinal Cell and Molecular Biology, National Eye Institute;
the ^Developmental Endocrinology Branch, National Institute of Child Health and
Development, National Institutes of Health; and the %I)eparlment of Anatomy and
Cell Biology, Uniformed Services University of the Health Sciences, Belhesdn,
Maryland.
Submitted for publication January 18, 1995; revised June 23, 1995; accepted
August 16, 1995.
Proprietary interest category: N.
Reprint requests: Timothy /. Schoen, National Eye Institute, National Institutes of
Health, 9000 Rockville Pike, Building 6, Room 304. Bethesda, Ml) 20892.
2652
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ferentiation of embryonic tissues.1'2 As proof of IGF's
developmental importance, experimental mutation of
a single IGF-I allele in mice results in a reduction in
body size; 95% of animals homozygous for the mutation die soon after birth. 3 Animals with a single IGFII allele show a similar reduction in body size and
weight; however, knockout of both IGF-II alleles does
not result in increased morbidity.4
Insulin-like growth factors are found normally in
association with specific, high-affinity binding proteins
(IGFBPs). At present, six closely related, yet structurally different, IGFBPs have been cloned and sequenced. 5 Although their functions are only partly
understood, IGFBPs appear to be important not only
as carriers of IGF but also as modulators of IGF activity
Investigative Ophthalmology & Visual Science, December 1995, Vol. 36, No. 13
Copyright © Association for Research in Vision and Ophthalmology
IGFBP-2 Development in the Eye
by either inhibiting or facilitating ligand-receptor interactions.'1 Importantly, IGFBP-17 and an IGFBP produced by endothelial cells8 have been shown to exhibit
activities independent of IGF-I.
All the components of an IGF autocrine-paracrine system,'1"" including several different IGFBPs,
I2 M
" have been identified in ocular tissues. We15 and
others"1 have previously used chick embryos to show
that IGFBPs in the vitreous humor exhibit an expression pattern different from that observed for serum
IGFBPs. This suggests a local synthesis of IGFBPs in
the eye rather than uptake from the systemic circulation. However, relatively little is known regarding the
cellular origins of IGFBP synthesis and secretion in
the chick eye. Therefore, as a first step in identifying
the specific cell types containing IGFBP mRNA and
in investigating the temporal appearance of IGFBP-2
expression in developing ocular tissues, we recently
have isolated and characterized a cDNA and gene for
IGFBP-2 in ocular tissues of the chick embryo.17
IGFBP-2 was chosen because of its predominance in
ocular tissues.M In the current study, we now describe
the localization and expression of IGFBP-2 mRNA in
specific ocular tissues of the developing chick using
both northern blot analysis and in situ hybridization
and report that the onset of binding protein expression appears to be regulated temporally and spatially.
MATERIALS AND METHODS
In Situ Hybridization
Chicken eggs and newly hatched chicks were purchased from Truslow Farms (Chestertown, MD) and
staged according to Hamburger and Hamiliton.18
Treatment of chicks conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research. Wholemount in situ hybridization was performed on stage 22 embryos using a previously published technique.1'1 Embryos used for conventional autoradiography in situ were fixed for a minimum of 48
hours in 10% neutral buffered formalin (Polysciences,
Warrington, PA). Paraffin sections were taken and
placed on silanized slides (Oncor, Gaithersburg, MD).
After removal of the paraffin with xylene and rehydration through graded ethanols, slides were incubated
with proteinase K (1 //g/ml) for 30 minutes at 37°C,
then treated with 0.1 M triethanolamine for 10 minutes followed by 0.25% acetic anhydride in 0.1 M
triethanolamine for 10 minutes at room temperature,
washed, and dehydrated. Hybridization was performed with 107 dpm/ml (50 ng/ml) of the cRNA
probe described below in a hybridization buffer containing 50% formamide, 0.3 M NaCl, 20 mM Tris HC1
(pH 8.0), 5 mM EDTA, 500 fig tRNA/ml, 10% dextran
sulfate, 10 mM dithiothreitol, and 0.02% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone.
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2653
After incubation at 55°C for 14 to 16 hours in a
humidified chamber, slides were washed several times
in 4 X SSC followed by dehydration through a series
of graded ethanol solutions and immersed in 0.3 M
NaCl, 50% formamide, 20 mM Tris HC1 and 1 mM
EDTA at 60°C for 10 minutes. Sections were then
treated with RNase-A (20 /ig/ml) for 30 minutes at
room temperature, followed by a 15-minute wash in
0.1 X SSC at 55°C. After a final dehydration, sections
were air dried and coated with Kodak NTB3 nuclear
emulsion (Eastman Kodak, Rochester, NY) and stored
with desiccant for 6 to 12 days. Slides were then developed with Dektol (Eastman Kodak) and stained with
hematoxylin and eosin for microscopic evaluation.
The synthesis of sense and anti-sense A'S-labeled
riboprobes and the in situ hybridization procedure
was performed as previously described.20 The chicken
IGFBP-2 cDNA clone used for probe synthesis was isolated from an E18 retina cDNA library.17 35S-labeled
cRNA probes were synthesized in 10-/xl reactions containing 250 fj,Ci 35S-CTP and 250 //Ci MS-UTP, 10 mM
NaCl, 6 mM MgCl2, 40 mM Tris (pH 7.5), 2 mM spermidine, 10 mM dithiothreitol, 500 fjM each of unlabeled adenosine triphosphate and guanosine triphosphate, 25 (iM each of unlabeled uridine triphosphate
and cytidine triphosphate, 500 ng linearized template,
15 U of the appropriate polymerase (T3 for the control sense probe; T7 for the specific IGFBP-2 antisense
probe), and 15 U of RNasin. The reaction was incubated at 42°C for 1 hour, after which the DNA template was removed by digestion with DNase-I at 37°C
for 10 minutes. Labeled cRNA was purified by the
addition of 50 //g yeast tRNA, phenol-chloroform extraction, chloroform extraction, and ethanol precipitation. The pellet was resuspended in 200 /A 0.2%
sodium dodecyl sulfate, 2 mM EDTA, and 0.3 M ammonium acetate (pH 5.2) and precipitated with cold
ethanol. The average specific activity of probes generated in this protocol was 2 to 3 X 108 dpm//zg.
Because of the variable background encountered
with some of the sections, grain densities were evaluated for six different fields of view (100 fim2 each)
from both the control (sense) and specific (antisense)
probes. Student's t-test was performed on the mean
± SEM for each of the sections.
RNA Extraction and Northern Blot Analysis
Total RNA was isolated from different ocular tissues
at E8, El 2, E18, and P2 tissues using the RNAzol technique (Cinna/Biotecx Labs, Friendswood, TX). Three
separate batches of eggs were used in three separate
experiments to determine if there were individual
batch variations. Sixty embryos were used for E8, 40
for E12, 30 for E18, and 20 for P2. Five micrograms
of total RNA from selected tissues was electrophoresed
on a 1% agarose-formaldehyde gel at 25 V for 3
2654
Investigative Ophthalmology & Visual Science, December 1995, Vol. 36, No. 13
1. IGFBP-2 mRNA expression in the chick at embryonic day 3.5 (stage 22) as revealed
by wholemount in situ hybridization. (A) IGFBP-2 mRNA was visualized as a dark precipitate
in surface ectoderm covering the eye, specific regions of the brain (br.), pharyngeal arches
(P.a.), somites (s), wing buds (w.b.), and limb buds (l.b.)- (B) No reactive precipitate was
visible in the embryo treated with the sense probe.
FIGURE
hours. After electrophoresis, the gel was photographed and soaked in 20 X SSC for 15 minutes to
remove excess formaldehyde. RNA was then transferred to a nylon membrane (Nytran, Schleicher and
Schuell, Keene, NH) using a Vacuum Gene transfer
apparatus (Pharmacia, Uppsala, Sweden). After the
transfer, the blot was washed for 5 minutes in 20 X
SSC, blotted damp dry, and cross-linked using a UV
Stratalinker (Stratagene, Lajolla, CA). Northern blots
were probed with a 212 bp polymerase chain reaction
(PCR) product from exon-2 of the chicken IGFBP-217
that was labeled with :wP-dCTP using a PCR-labeling
technique.21 After washing (0.1 X SSC; 0.1% sodium
dodecyl sulfate for 15 minutes at 60°C), the blots were
placed in film cassettes and exposed to Kodak XAR
autoradiographic film (Eastman Kodak). The relative
amount of IGFBP-2 hybridization in each lane of the
autoradiograph was determined by scanning densitometry using a model 620 Video Densitometer with
1-D Analyst software (Biorad, Hercules, CA). To normalize for variations in loading, two different normalization techniques were used. In the first technique,
the ethidium-stained 28S and 18S bands in the gel
were photographed, and the negative was scanned and
quantified. In the second technique, the blots were
stripped after the first probing and reprobed with a
Hy
P-labeled 18S ribosomal PCR probe and subjected
to auto radiography. In this case, the relative hybridiza-
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tion to the 18S band was used as the denominator in
normalizing each lane. Both techniques were found
to yield similar results. Molecular weight estimation of
the hybridizing band was obtained by comparison with
the migration of RNA molecular weight standards
(Bethesda Research Lab, Gaithersburg, MD).
RESULTS
In situ Hybridization
Using a wholemount in situ hybridization technique,10
IGFBP-2 mRNA was observed in a number of different
tissues and organs at stage 22 (E3.5). These included
surface ectoderm, eye, regions of the brain, somites,
pharyngeal arches, wing buds, and limb buds (Fig.
1A). Embryos probed with the control sense strand
were negative for IGFBP-2 mRNA (Fig. IB). Hematoxylin and eosin-stained sections through regions of the
head containing the eye at E3.5 revealed die close
apposition of the lens and cornea (Figs. 2A, 2B). At
this very early stage of development, IGFBP-2 mRNA
was present only in the surface ectoderm, presumptive
corneal epithelium (Figs. 2C, 2D). Similar sections
from embryos probed with the control sense strand
showed only weak, nonspecific background staining
(Figs. 2E, 2F).
By E6, the cornea consisted of an epithelium, nar-
2655
IGFBP-2 Development in the Eye
A
«4
It
c.e.
1
lens
FIGURE 2. IGFBP-2 mRNA expression in the eye region of the chick at embryonic day 3.5
(stage 22). Low (A) and high <B) magnification of hematoxylin and eosin-stained ocular
tissues revealed the close apposition of the lens to the surface ectoderm destined to form
the corneal epithelium (c.e.)- Low (C) and high (D) magnification of unstained sections
showed precipitate indicating the presence of IGFBP-2 mRNA in presumptive corneal epithelium (c.e.). IGFBP-2 mRNA was undetectable in the retina (r) and surrounding mesenchyme
(m). Little or no precipitate was present in control sections treated with sense-strand probe
(E,F). Scale bar = 100 ym in A,C,E and 25 //m in B,D,F.
row stroma, and endothelium (Fig. 3A). Conventional
in situ hybridization studies revealed that, at this stage,
IGFBP-2 mRNA was present in the developing epithelium and endothelium but was undetectable in the
stroma (Fig. 3B). The control sense sections showed
little reactivity over the cornea in general, although a
weak band of silver grains could be seen in the corneal
epithelium (Fig. 3C). The variable degree of background reactivity observed in control sections was most
likely caused by the high guanosine + cytosine content
of probe. Average silver grain counts/100 \i\nl of the
corneal epithelium were 325 ± 32 using the antisense
probe (Fig. 3B) and 74.5 ± 14 using the control, sense
probe (Fig. 3C), a highly statistically significant difference (P < 0.0001). By E12, the corneal epithelium,
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stroma, and endothelium were almost fully developed
(Fig. 3D) and IGFBP-2 mRNA expression was observed
in all three layers as well as in other structures, such as
the eyelid and the nonpigmented layer of the ciliary
body (Fig. 3E). Average silver grain counts/100 ^m a
over the cornea at this time were 146.5 ± 22 using the
antisense probe and 34.7 ± 3.5 using the sense probe
(Fig. 3F), with P < 0.0001. The pigmented layer of the
El2 ciliary body contained abundant light-refracting
melanin granules in both antisense (Fig. 3E) and control sense sections (Fig. 3F). It was thus not possible to
determine by this method if the ciliary body contained
IGFBP-2 mRNA at this stage. Little nonspecific reactivity was observed with the sense probe at this stage of
development (Fig. 3F).
2656
Investicative Ophthalmology & Visual Science. Decemher 1995. Vol. 5tfi NTn
A
Epithelium
LID
COR
Endothelium
FIGURE 3. Hematoxylin and eosin bright-field sections of cornea at embryonic day 6 (A) and
embryonic day 12 (D) anterior segment. Dark-field illumination revealed IGFBP-2 mRNA
in the epithelium and endothelium of the E6 cornea (B). Reaction product was distributed
uniformly across the eyelid (lid), cornea (COR), and ciliary body (C.B.) of the E12 anterior
segment (E). No labeling was observed in control sections probed with the sense strand in
E6 cornea (C) or El2 anterior segment (F). A.C. = anterior chamber. Scale bar = 25 fim
(A,B,C), 100 tan (D,E,F).
The neural retina at E6 contained a single lamina
of undifferentiated neuronal cells (Fig. 4A). At this
stage of development, IGFBP-2 mRNA was diffusely
distributed across the tissue (Fig. 4B). A somewhat
higher level of IGFBP-2 mRNA expression was seen in
the sclera (Fig. 4B). Silver grain counts/100 fj,m2 over
the retina averaged 112 ± 60 using the antisense
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probe and 44 ± 11 using the sense probe with P <
0.02. The sense-strand control demonstrated the specificity of the labeling in both retina and sclera (Fig.
4C). As with the ciliary body (Fig. 3), it was difficult
to ascertain if specific labeling was present in the pigment epithelial layer. By E12, the retina has differentiated into distinct ganglion cell, inner plexiform, inner
2657
IGFBP-2 Development in the Eye
A
GCL
IPL
NR
«•'.-
IINL
SCL
OPL
ONL
FIGURE 4. Hematoxylin and eosin bright-field sections of retina and sclera at embryonic
day 6 (A) and embryonic day 12 (D). Dark-field illumination showed that IGFBP-2 mRNA
expression was distributed diffusely across the E6 neural retina (NR) (B) and was expressed
in the sclera (SCL). The apparent intense reactivity of the retinal pigmented epithelium
(RPE) was caused by light-scattering melanin granules and probably not by IGFBP-2 mRNA
because it was observed as well, to a similar extent, in the control sections using sense probe
(C). By E12 (E), IGFBP-2 mRNA expression was confined mostly to the ganglion cell layer
(GCL), inner nuclear layer (TNL), and outer nuclear layer (ONL). Little or no labeling was
observed in the inner plexiform layer (IPL). Background labeling with the sense-strand
probe was low (F). Scale bar = 25 £tm.
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2658
Investigative Ophthalmology & Visual Science, December 1995, Vol. 36, No. 13
CH
SCL
BV
FIGURE 5.
Bright-field illumination of an hematoxylin and eosin-stained section through an
embryonic day 12 posterior eye cup (A) showing the retinal pigment epithelium (RPE),
choroid (CH), cartilage (C), blood vessels (BV), and sclera (SCL). Dark-field illumination
showed a diffusely distributed expression of IGFBP-2 mRNA across the sclera (B). Intense
reactivity of the retinal pigment epithelium (RPE) (B) was caused mainly by light-scattering
melanin granules; it was observed also in the control sense-strand sections (C). Scale bar =
25 pm.
nuclear, outer plexiform, and outer nuclear layers,
although photoreceptor outer segments are yet to
form (Fig. 4D). At this stage, the expression of IGFBP2 mRNA is confined mainly to layers of the retina
containing the cell bodies, e.g., ganglion cell, inner
nuclear, and outer nuclear layers, and it was undetectable in the plexiform layers (Fig. 4E). Control sections
of the E12 retina probed with the sense strand showed
little labeling (Fig. 4F). Little or no specific staining
was observed in the lens under these conditions (figure not shown).
Figure 5A focuses on the histologic appearance
of the retinal pigment epithelium-choroid—sclera
complex, demonstrating that it was relatively well
formed and delineated at El2. At this midembryonic
stage of development, IGFBP-2 mRNA was diffusely
expressed across the choroid and sclera (Fig. 5B). It
was present in the cartilage, which forms part of the
eye wall in the chicken, but was absent in nucleated
red cells contained within blood vessels. Control,
sense sections were only weakly labeled (Fig. 5C). Silver grain counts/100 /im'2 averaged 88 ± 7.5 using the
antisense probe (Fig. 5B) and 19 ± 4 using the sense
probe (Fig. 5C) with P < 0.0001.
Northern Blot Analysis
To compare the relative abundance of mRNA in cornea, retina, and sclera, northern blot analysis was per-
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formed on each of the tissues at different stages of
development (Fig. 6A). IGFBP-2 autoradiographs
were normalized to the relative intensities of 28S and
18S bands stained with ethidium bromide (Fig. 6B).
The results of experiments using three separate
batches of animals are graphically represented in Figure 6C. Of the stages examined, IGFBP-2 mRNA expression in the cornea was highest at E8 (Fig. 6C, left
panel). By El2, IGFBP-2 mRNA expression decreased
by approximately 50% and then remained relatively
steady from E18 through hatching (P2). In contrast
to the cornea, expression of IGFBP-2 mRNA in the
retina was low at E8. This increased approximately
twofold by El 2 and peaked at El8 with an approximate fivefold increase over the E8 level (Fig. 6C, center panel). After hatching (P2), IGFBP-2 mRNA expression decreased to approximately 60% of that observed at El8. The developing sclera exhibits an
expression pattern somewhat similar to that observed
for the cornea. The highest level of IGFBP-2 mRNA
expression was observed at E8, early in ocular development (Fig. 6, right panel). Thereafter, IGFBP-2 mRNA
expression decreases and, at P2, is approximately 50%
of that observed at E8.
To compare more accurately the relative levels of
IGFBP-2 message between ocular tissues and with
those in other tissues, northern blot analyses were performed at P2 on cornea, retina, sclera, and, for com-
IGFBP-2 Development in the Eye
2659
Cornea
Retina
Sclera
IGFBP-2
(2.4kb)
B
28S -
c
_
E-8 E-12 E-18 P-2
AGE (days)
E-8 E-12 E-18 P-2
AGE (days)
E-8 E-12 E-18 P-2
AGE (days)
FIGURE 6. Northern blot analysis of IGFBP-2 mRNA expression in developing ocular tissues.
Samples of total RNA (5 (J,g) from cornea, retina, and sclera at embryonic days 8, 12,
and 18 and at posthatch day 2 were electrophoresed on a 1% agarose-formaldehyde gel,
transferred to a nylon membrane and probed for IGFBP-2 (A). Ethidium bromide staining
(B) demonstrated RNA integrity and the relative lack of variation in loading of individual
lanes. Autoradiograms were scanned densitometrically and normalized to the relative intensity of the 28S and 18S bands as shown in the histograms (C). Results are expressed as the
mean values ± SEM for three different sets of animals as described in Materials and Methods.
parison, brain and liver (Fig. 7A). Blots were normalized using an 18S ribosomal probe (Fig. 7B); a graphic
representation of the results is shown in Figure 7C.
As expected, liver and brain tissues from the newly
hatched animals exhibited a high level of IGFBP-2
mRNA expression. Surprisingly, however, the cornea
exhibited a level of IGFBP-2 mRNA expression equivalent to that observed in brain and liver tissues. The
neural retina and the sclera exhibited approximately
60% of the IGFBP-2 expression observed in the two
nonocular tissues. The lens (fibers and epithelium)
exhibited only a weak signal for IGFBP-2 mRNA in
agreement with the in situ hybridization data.
DISCUSSION
A high level of IGFBP-2 mRNA expression in several
embryonic tissues derived from ectoderm has been
reported for the midgestational rat embryo.22 The results of our study demonstrated that, at a much earlier
age of embryonic development (i.e., E3.5), IGFBP-2
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mRNA was expressed in a number of chick tissues,
including the presumptive corneal epithelium (surface ectoderm). Although no corneal stroina or endothelium was present at this stage, IGFBP-2 mRNA expression was detected readily in the presumptive corneal epithelium but not in other ocular tissues.
However, by E6, migrating neural crest cells, destined
to form head mesenchyme, invaded the space between
the corneal epithelium and lens and began to form
the corneal stroma and endothelium.^ The high level
of IGFBP-2 mRNA expression in the corneal epithelium during the formation of the stroma raised the
possibility that IGFBP-2 may be involved in regulating
the migration and differentiation of invading mesenchymal cells. In support of this hypothesis, it has been
shown that IGFBP-1 is able to stimulate fibroblast migration by its interaction with ab /?, integrin.8 Interestingly, we have found17 that the chick IGFBP-2 also
contains the RGD peptide, although it is unknown
whether IGFBP-2 binds to cell surfaces. A distinct spatial expression of IGFBP-2 mRNA is observed in limb
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Investigative Ophthalmology & Visual Science, December 1995, Vol. 36, No. 13
ern blot analysis, the developmental patterns of
IGFBP-2 mRNA expression in several important ocular
tissues are markedly different. This may indicate that
IGFBP-2 has separate roles in the different tissues.
IGFBP-2
Early in development (E3.5 to E8), for example, there
is a high level of IGFBP-2 mRNA expression in the
(2.4 kb)
cornea and the sclera at a time when the cells of these
tissues are proliferating rapidly.2b>27 This suggests a
role for IGFBP-2 in facilitating the proliferative effects
B
of IGF on these tissues during the early growdi phase
of the eye. In support of this idea, previous studies28
have shown that IGFBP-2 functions synergistically with
IGF-I to promote smooth muscle cell proliferation in
18Svivo. IGFBP-2 itself could be involved in this phase of
ocular growth because mounting evidence indicates
that IGFBPs have intrinsic biologic activity independent of tfieir interactions with the IGFs.7 8
In contrast to the cornea and the sclera, IGFBP2 mRNA expression in the undifferentiated retina
from E3 to E8 is low and increases only as neuronal
< S 0.8 differentiation progresses. In general, the proliferative
<
LLJ C
phase of retinal development is from E3 through E7,
rr
and terminal differentiation takes place from approximately Ell through E21 (hatching).12'1 IGFBP-2 mRNA
expression peaks in the late embryonic period on ap< •- 0 4 III
r>
W."T
proximately E18, a time when cell proliferation has
c5
ceased in the retina but photoreceptor cells are undergoing rapid structural differentiation (e.g., outer segment elongation).30 The high level of IGFBP-2 mRNA
0.0
expression at this stage of development thus suggests
a role for IGFBP-2 (alone or with IGF-I or IGF-II)
in mediating neuronal differentiation and maturation
rather than neuronal cell proliferation. In support of
FIGURE 7. Northern blot analysis of IGFBP-2 mRNA expres- this idea, IGF-I, its receptor, and IGFBP-5 have been
sion in tissues at posthatch day 2. Five-microgram samples
shown to be present in the developing retina.81 lA2 IGFof total RNA from cornea, retina, sclera, lens, brain, and
I also has been shown to be important for the differenliver were electrophoresed on a 1 % agarose-formaldelhyde tiation and survival of several neuronal cell types in
gel, transferred to a nylon membrane, and probed for
vitro,33 and, in combination with bFGF, it stimulates
IGFBP-2 (A). Individual loading variation was normalized
the in vitro differentiation of retinal precursor cells
by reprobing the blot with an 18S ribosomal probe (B). A
into
a rod photoreceptor-specific phenotype.:w Our
graphic representation of the results is shown <C). Densitoadditional finding that IGFBP-2 mRNA expression is
metric analysis was performed on nonsaturated autoradioconfined mainly to the nuclear layers of the retina
graphs; however, for illustrative purposes, autoradiographs
were overexposed to show that a low level of IGFBP-2 mRNA and is not observed in the plexiform layers supports
the concept that IGFBP-2 may be synthesized and reexpression could be detected in the lens.
leased locally by the neural cell soma, as opposed to
being transported to and released from dendrites.^
development, during which IGFBP-2 mRNA is concentrated in the apical ectodermal ridge and IGF-I and
Nordiern blot comparisons of ocular tissues with
IGF-II mRNAs are found in the adjacent mesoderm.^4
brain and liver at P2 reveal that, among the ocular
Although die localization of IGF mRNA was not investissues examined, cornea has the highest IGFBP-2
tigated in the current study, IGF-II mRNA has been
mRNA expression, although IGFBP-2 mRNA expresshown to be highly expressed by mesenchymal tissues
sion is maintained in the sclera and retina at relatively
forming the sclera and corneal stroma, and it is exhigh levels. It should be noted here that, although
pressed only weakly in the retina.0 Importandy, we15
ocular growth continues, the chick has a functional
and others25 have shown that IGFBP-2 binds IGF-II as
visual system at P2. Previous IGF binding studies from
well as IGF-I.
our laboratory have demonstrated that the adult cornea has the highest level of IGFBPs among ocular
As judged by both in situ hybridization and north-
1
*•*•#§
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IGFBP-2 Development in the Eye
tissues and fluids.14 The finding of a high level of
IGFBP-2 mRNA in the embryonic cornea, coupled
with evidence that both IGF-P and IGF-II36 are able
to stimulate the proliferation of several corneal cell
types, strongly suggests that IGFBP-2 is involved in the
growth of the cornea and the sclera as well. In this
regard, it would be interesting to investigate a possible
role for IGFBPs in myopia and other structural diseases of the eye. High levels of IGFBPs in the postnatal
and adult cornea and sclera point toward a continuing
role for IGFBPs in the maintenance of these mature
tissues. Similarly, in the retina, maintenance of IGFBP2 mRNA expression in the postnatal period, well after
cessation of cellular proliferation, could indicate an
important role of this IGFBP in attainment of full
differentiation and in mature retinal cell functioning.
In summary, the results of the current study reveal
a differential expression of IGFBP-2 mRNA in several
tissues of the developing chick eye with distinct temporal and spatial gradients observed (e.g., cornea).
These gradients are reminiscent of those extensively
studied by a number of investigators37'38 for the cellular retinoic acid-binding protein-I (CRABP-I). CRABPI mRNA is expressed in a well-defined gradient in
developing limb buds, for example, suggesting that it
mediates the inductive effects of retinoic acid during
pattern formation of the limb. Similarly, complementary expression of IGFBP-2 mRNA, along with those
for IGF-I and IGF-II in rat fetal limbs,24 indicates that
the binding protein probably contributes to limb outgrowth and patterning. The unique temporal and spatial expression of IGFBP-2 in the eye suggests that it
could be involved in the regulation of ocular growth
and differentiation as well as in homeostasis in the
mature eye.
Key Words
development, eye, growth, insulin-like growth factor, insulinlike growth factor binding protein
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