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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 Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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. Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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- Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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, Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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 Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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. Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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- Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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 Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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 2660 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 *•*•#§ Downloaded From: http://iovs.arvojournals.org/ on 05/08/2017 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 References 1. dePablo F, Perez-Villami B, Serna J, et al. IGF-I and the IGF-I receptor in development of nonmammalian vertebrates. Mol Reprod Dev. 1993; 35:427-433. 2. Heyner S, Shi C, Garside WT, Smith RM. Functions of the IGFs in early mammalian development. Mol Reprod Dev. 1993; 35:421-426. 3. Powell-Braxton L, Hollingshead P, Giltinan D, PittsMeek S, Stewart T. Inactivation of the IGF-I gene in mice results in perinatal lethality. Ann NY Acad Sci. 1993; 692:300-301. 4. 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