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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- 1637 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017 1638 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017 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- 1639 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.) Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017 1640 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.) Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017 - No. 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^*/* 1641 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).) Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017 1642 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- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017 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. References 1. Bassas L, De Pablo F, Lesniak MA. and Roth J: The insulin receptors of chick embryo show tissue-specific structural differences which parallel those of the insuli'n-likc growth factor I receptors. Endocrinology 121:1468, 1987. 2. Bhaumick B and Bala RM: Receptors for insulin-like growth factors I and II in developing embryonic mouse limb bud. Biochim Biophys Acta 927:1 17, 1987. 3. Akahane K, Tojo A, Tobc K, Kasuga M. Urabe A. and Takaku F: Binding properties and proliferative potency of insulin-like growth factor I in fetal mouse liver cells. Exp Hcmatol 15:1068. 1987. 4. Girbau M, Gomez JA, Lesniak MA, and De Pablo F: Insulin and insulin like growth factor I both stimulate metabolism, growth, and differentiation in the postneurula chick embryo. Endocrinology 121:1477, 1987. 5. Sancto RP, Low KG, Mclncr MH, and De Vellis J: Insulin/insulin-likc growth factor I and other cpigenetic modulators of myclin basic protein expression in isolated oligodendrocytc progenitor cells. J Neurosci Res 21:210, 1988. 6. Florini JR and Magri KA: Effects of growth factors on myogenic differentiation. Am J Physiol 256:C70l, 1989. 7. Beebc DC, Silver MH, Belcher KS, Van Wyk JJ, Svoboda ME. and Zclenka PS: Lentropin. a protein that controls lens fiber formation, is related functionally and immunologically to the 17. 18. 19. 20. 21. 22. 23. 1643 insulin-like growth factors. Proc Natl Acad Sci USA 84:2327. 1987. Piatigorsky J: Insulin stimulation of lens fiber differentiation in culture: Elongation of embryonic lens epithelial cells. Dcv Biol 30:214. 1973. Milstonc LM and Piatigorsky J: Delta crystallin gene expression in embryonic chick lens cpithelia cultured in the presence of insulin. Exp Cell Res 105:9, 1977. Beebc DC, Feagans DE, and Jcbcns AH: Lentropin: A factor in vitreous humor which promotes lens fiber cell differentiation. Proc Natl Acad Sci USA 77:490, 1980. Philpott GW and Coulombrc AJ: Lens development. The differentiation of embryonic chick lens epithelial cells /// vitro and in vivo. Exp Cell Res 38:635, 1965. Piatigorsky J. Webster HD, and Craig SP. Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers /// vivo and in vitro. Dcv Biol 27:176, 1972. Coulombre AJ and Coulbombrc L: Lens development. Fiber elongation and lens orientation. Science 142:1489, 1963. Coulombre JL and Coulombrc AJ: Lens development. IV. Size, shape, and orientation. Invest Ophthalmol 8:251, 1969. Piatigorsky J and Rothschild SS: Loss during development of the ability of chick embryonic lens cells to elongate in culture: Inverse relationship between cell division and cell elongation. Dcv Biol 28:382. 1972. Beebc DC and Piatigorsky J: The control of delta crystallin gene expression during lens cell development: Dissociation of lens cell elongation, cell division, delta crystallin synthesis, and delta crystallin mRNA accumulation. Dcv Biol 59:174, 1977. Beebe DC and Piatigorsky J: Translational regulation of delta crystallin synthesis during lens development in the chicken embryo. Dcv Biol 84:96, 1981. Bassas L, Zclenka PS. Serrano J. and De Pablo F: Insulin and IGF receptors are dcvelopmcntally regulated in the chick embryo eye lens. Exp Cell Res 168:561. 1987. Zick Y. Spiegel AM, and Sagi-Eisenbcrg R: Insulin-like growth factor 1 receptors in retinal rod outer segments. J Biol Chcm 262:10259. 1987. Waldbillig RJ. Fletcher RT, Somcrs RL, and Chader GJ: IGF-1 receptors in the bovine neural retina: Structure, kinasc activity and comparison with retinal insulin receptors. Exp Eye Res 47:587. 1988. Shinohara T, Robison WG, and Piatigorsky J: Delta crystallin synthesis and vacuole formation during induced opacification of cultured embryonic chick lenses. Invest Ophthalmol Visual Sci 17:515, 1978. Ocrant I, Valentino KL, King MG, Wimpy TH, Roscnlcld RG, and Baskin DG: Localization and structural characterization of insulin-like growth factor receptors in mammalian retina. Endocrinology 125:2407, 1989. Han VKM, D'Ercole AJ, and Lund PK: Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus..Science 236:193, 1987. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933156/ on 06/16/2017