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Differential Expression of Fibroblast Growth Factor Receptors During Rat Lens Morphogenesis and Growth Robbert U. de Iongh, Frank J. Lovicu, Coral G. Chamberlain, and John W. McAvoy Purpose. Fibroblast growth factors (FGF) play important roles in the developmental biology of the lens. Recently, it was shown that the expression of one of the FGF receptors, FGFR1 (fig; fibroblast growth factor receptor 1), was closely associated with the onset of lens fiber differentiation. In this study, the expression patterns of three other members of the FGF receptor family were analyzed and compared. Methods. The expression patterns of FGFR2 (bek and keratinocyte growth factor receptor [KGFR] variants) and FGFR3 were analyzed by in situ hybridization during embryonic and postnatal lens development. Results. In the ocular primordia, both FGFR2 variants were detected on embryonic day 12 (E12) and FGFR3 was detected on E14. From E16 to E20, distinct spatial expression patterns became evident within the lens; FGFR3 showed an anteroposterior increase in expression, with strongest expression in the outer cortical fibers. In contrast, bek showed uniform expression throughout the lens epithelium (including the central and germinative zones) and the transitional zone, with a subsequent decline in maturing fibers. The KGFR variant of FGFR2 showed strongest expression in the earlyfibersof the transitional zone; its expression in the epithelium was weaker in the germinative zone of embryonic and neonatal rats. There was an age-related decline in expression of FGFRs after birth—an effect that was more marked for FGFR3 than for the FGFR2 variants. Conclusions. Combined with those in a previous study, these results indicate that the FGFR1, bek, KGFR, and FGFR3 genes exhibit different, yet overlapping, patterns of expression throughout lens development and differentiation. The distinct spatiotemporal patterns of expression of FGF receptors may play an important role in regulating anteroposterior patterns of lens cell behavior. Invest Ophthalmol Vis Sci. 1997;38:1688-1699. Jr ibroblast growth factors (FGFs) constitute a family of at least 10 structurally related polypeptides, which are highly conserved between species and induce a wide range of responses in various cell and tissue types, including proliferation, differentiation, matrix deposition, and cell migration. The FGFs show distinct spatial and temporal expression patterns in embryos and adults and are involved in many key developmental processes, including From the Department of Anatomy and Histology and Institute for Biomedical Research (FB), The University of Sydney, Australia. Supported by grant RO1 EY03177 from the National Eye Institute, US Department of Health and Human Services, Public Health Service; by a grant from the National Health and Medical Research Council (NHMRC), Australia; and by an NHMRC Biomedical Research Scholarship and a traveling scholarship from the Faculty of Medicine (Rdel) and a Postdoctoral Research Fellowship from the Medical Foundation (FJL), University of Sydney, Australia. Submitted for publication November 20, 1996; revised March 27, 1997; accepted March 31, 1997. Proprietary interest category: N. Reprint requests: fohn W. McAvoy, Department of Anatomy and Histology (F13), University of Sydney, Sydney NSW, Australia 2006. 1688 determination of the anteroposterior axis and induction of mesoderm during early embryonic development.' Fibroblast growth factors play a pivotal role in lens differentiation.2 The differentiated lens has a highly ordered cellular architecture. It is composed of two distinct forms of lens cell: elongated fibers, aligned in an anteroposterior axis, make up the bulk of the lens; and a monolayer of cuboidal epithelial cells covers the anterior surface of the fibers. The lens grows by proliferation of epithelial cells in the germinative zone of the lens, just anterior to the lens equator; and progeny of these divisions move posteriorly into the transitional zone of the lens where they elongate and differentiate into fibers. This process continues to add fiber cells to the lens mass throughout life, so that the position of a fiber cell within the fiber mass (from outer cortex to nucleus) reflects its state of differentiation. Results of previous studies in vitro have shown that FGF induces lens epithelial cells to un- Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 FGF Receptor Expression in Lens Development dergo proliferation, migration, and fiber differentiation in a progressive dose-dependent manner.3 There is increasing evidence that an anteroposterior gradient of FGF stimulation plays an important role in regulating lens polarity and growth patterns.2 This hypothesis is supported by results of recent transgenic studies, which indicate that overexpression and inappropriate secretion of FGFs in the lens induce differentiation of the anterior epithelium and abolish lens polarity.4"7 The FGFs bind two distinct types of cell-surface receptors, high affinity tyrosine kinase receptors (FGFR)8 and lower affinity heparan sulfate proteoglycans, both of which are required for biologic activities of FGF.910 There are at least four FGFR genes, and alternative splicing of their messenger RNAs (mRNAs) gives rise to several variants that differ in their affinities for members of the FGF family.811 Ligand binding to the extracellular domain of the FGFR induces receptor dimerization, which results in transphosphorylation of tyrosine (s) in the intracellular tyrosine kinase domains of the receptors in the dimer.12 Activation of the kinase domain permits binding and phosphorylation of intracellular signaling proteins and activation of specific intracellular signaling pathways. The importance of FGF signaling through FGFRs for lens development has been demonstrated by findings in transgenic studies in which a dominant-negative FGF receptor was expressed in the lens fibers to inhibit FGF signaling.1314 These findings showed that lens fibers are dependent on FGF signaling through FGFRs for normal differentiation and survival. Recently, results of detailed studies of the spatiotemporal expression patterns of FGFRl during lens development produced results that established that a high level of expression of FGFRl was associated with the onset of lens fiber differentiation.15 In other studies, results have indicated that lens cells also express variants of FGFR216 and FGFR317; but, because these studies were restricted to a single early-stage embryo, information on the spatial expression patterns in the lens was limited, and there was no information on the temporal expression patterns during lens development. This report presents a detailed, in situ hybridization analysis of the expression patterns of FGFR2 and FGFR3 throughout embryonic and postnatal lens development. Specific probes for bek and keratinocyte growth factor receptor (KGFR) allowed detailed analysis of the expression of these alternatively spliced variants of FGFR2. The results of this study establish that mRNAs for these FGFRs have unique, yet overlapping, patterns of expression during lens development and that they are present in different proportions in the different zones of cellular activity. METHODS Animal and Tissue Preparation All procedures involving animals were in accordance with the National Health and Medical Research Coun- 1689 cil (Australia) guidelines, the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH; Bethesda, MD) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Embryos at various stages of gestation (embryonic days 11 to 20; Ell to E20) and eyes from neonatal (postnatal day three; P3), weanling (P21), and adult (PI00) rats were immersed in Tissue-Tek OCT compound (Miles, Elkhart, IN) and frozen in iso-pentane cooled by liquid nitrogen. Specimens were stored in liquid nitrogen until sectioned. Complementary DNA Probes Two complementary DNAs (cDNAs) for the alternatively spliced forms of murine FGFR2 (bek and KGFR: Orr-Urtreger et al16) and coding for the variable regions of the third immunoglobulin domain were obtained from Dr. M. Bedford and Dr. P. Lonai (Weizmann Institute of Science, Rehovot, Israel). The murine bek cDNA was a 119-bp fragment (PPum I-Eco RV), which was proved by sequence analysis (BLAST analysis at the National Center for Biotechnology Information, NIH) to be 99% homologous with the published rat bek cDNA.18 The murine KGFR cDNA was a 160-bp fragment (PPum I-Hae II), of which the first 158 bp were 100% homologous with the published rat KGFR cDNA.18 Both variants of FGFR2 were subcloned into pBluescript (Stratagene, Lajolla, CA). A 487-bp cDNA encoding the complete third immunoglobulin domain, including exon Illb, of murine FGFR3, subcloned into pBluescript KS+, was obtained from Dr. L.T. Williams (University of California, San Francisco). The first 291 bp and the last 44 bp of this cDNA code for common regions in the Illb and IIIc exon forms of FGFR3,19 whereas nucleotides 292 to 443 are specific for exon Illb only. In sequence analysis, this sequence proved to have regions with high levels of homology (99%) with human FGFR3 sequences but only moderate homology with rat FGFRl (72%), FGFR2 (72%), or FGFR4 (73%). RNA probes transcribed from these cDNAs were used for in situ hybridization experiments under high-stringency conditions, as described previously.15"2021 In Situ Hybridization In situ hybridization was performed on paraformaldehyde-fixed frozen sections of rat ocular tissues, using SP6, T3, or T7 RNA polymerase-derived RNA probes, labeled with 35S-UTP (Amersham, Sydney, Australia). After hybridization and final high-stringency washing (0.1 X SSPE at 65°C; 1 X SSPE = 15 mM NaCl, 1 mM NaH2PO4, 1 mM EDTA), sections were dehydrated and exposed for 5 days to autoradiograph film (/3-Max Hyperfilm, Amersham), which was developed according to manufacturer's instructions. Slides were Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 1690 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 then coated with NTB-2 emulsion (Kodak, Sydney, Australia) and stored in light-tight boxes with desiccant at 4°C for 3 to 5 weeks, after which they were developed (D-19, Kodak), rinsed, and stained with hematoxylin. Sections were photographed using a Leitz Dialux 20 microscope (Wetzlar, Germany) and 400 ASA film (T-Max, Kodak) processed according to manufacturer's instructions. Image Analysis of Fibroblast Growth FactorReceptor Hybridization Signal Image analysis was used to compare the density of FGFR expression of different regions of the fetal (E20) lens, as previously described.15 Briefly, dark-field images were captured directly from slides by video camera (slow scan, Dage/MTI, Michigan City, IN) and the optical density of hybridization signals was measured in six distinct regions (Figure 3) of lens sections, using a Tracor Northern Image Analysis system (Tracor Northern, Middleton, WI, USA). For each region, the perinuclear area that contained the specific signal was delineated, and the optical density of that region was measured. The regions measured were consistent for each of the different FGFR probes analyzed. To control for variability in hybridization and autoradiography conditions between experiments, data for each experiment were first corrected for nonspecific background and then normalized for the mean count for region I. Values obtained were compared using oneway analysis of variance and Student's t-test. This approach allowed comparison of the relative expression of each of the FGFR mRNAs in the various regions of the lens. However, it does not necessarily reflect changes in cellular expression for a given receptor, as dramatic changes in cell shape and size occur as lens cells differentiate into fibers. The changes in expression of each FGFR with age were compared by laser densitometry. As described previously,15 sections of whole eyes from P3, P21, and PI00 rats, which had been hybridized with the same probe under identical conditions, were exposed to an autoradiograph film (/?-Max Hyperfilm, Amersham) for 5 days. The film was scanned using a HeNe laser densitometer (Molecular Dynamics, Sunnyvale, CA), and the density of signals in region III of P3, P21, and P100 lenses was quantified using image analysis software (ImageQuant; Molecular Dynamics). After correction for background, the data were compared using one-way analysis of variance and Student's Rest, as described. RESULTS Transcripts for two alternatively spliced variants of FGFR2 {bek and KGFR) and for FGFR3 were detected at various stages of lens development. At no stage were distinct signals detected with the respective sense probes (not shown). No distinct hybridization signals for bek, KGFR, or FGFR3 were detected at Ell (data not shown). At El2, there was detectable expression of bek transcripts in the lens pit and surrounding mesenchyme (Fig. ID). FIGURE l. Expression of FGFR during lens morphogenesis. Expression of bek (D to F), KGFR (G to I) and FGFR3 (J to L) in E12 (A,D,GJ), E14 (B,E,H,K), and E16 (C,F,I,L) embryos. A, B, and C are hematoxylin-stained sagittal sections through the ocular primordia, as shown in D, E, and F by dark-field microscopy. The section shown in B and E is slightly tangential to the sagittal plane. At El 2, distinct expression for bek (D) was detected in the lens pit (lp), anterior parts of the optic cup (oc), diencephalon (d), and undifferendated extraocular mesenchyme (m), whereas only weak KGFR expression was found in ectoderm in the area around the lens pit {arrowheads, G), and FGFR3 was undetectable (J). At E14, the lens is comprised of an epidielium {arrowhead) and a differentiating fiber mass (If). Bek (E) and KGFR (H) were expressed in the epithelium and fibers of the lens vesicle, whereas FGFR3 (K) was expressed only in die fibers. All three receptors were detected in presumptive choroid sclera (cs) and in the cartilaginous mesenchymal condensations representing bone precursors {solid arroius), albeit weakly for FGFR3 (K). KGFR and, to a lesser extent, FGFR3 were strongly expressed in the invaginating ectoderm of the presumptive eyelids {curved arroius, H,K). In the lens (1) at E16, bek (F) and KGFR (I) were expressed in lens epithelium {small arrowheads) and in the early fibers at the equator {large arrowheads), whereas FGFR3 (L) was expressed in all lens cells but was particularly strong in the elongating fibers. Bek transcripts were also found in the developing cornea (c), ganglion cell layer of the neural retina (nr), the peripheral redna (pr; presumptive ciliary body and iris), and in the choroid sclera (cs). KGFR expression was detected in the peripheral retina, the choroid sclera and the invaginating ectoderm of the presumptive eyelids {curved arroiu). FGFR3 was also detected in the eyelid ectoderm {curved arroiu) and developing bone of the skull {solid arrow). Scale bar = 50 //m except for A, D, G, and J, for which scale bar = 20 /xm. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 FGF Receptor Expression in Lens Development Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 1691 1692 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 Although little or no signal was detected in the optic cup, it was detectable in the diencephalon. Weak signals for KGFR transcripts were only detected in the ectoderm in the area around the lens pit (Fig. 1G). No discernible signals for KGFR were present in the lens pit or optic cup. Distinct signaling for FGFR3 was not detected in the eye primordia at this stage (Fig. u>- At El4, strong signals for bek were detected in the epithelial cells and in early fibers of the lens vesicle (Fig. IE). Signals were also detected in the peripheral optic cup, in condensing mesenchyme of the choroid sclera and in presumptive bone (Fig. IE), but not in the central optic cup or in regions of undifferentiated mesenchyme (Fig. IE). With the KGFR probe, signals were detected in the lens vesicle, particularly in the early-elongating primary fibers (Fig. 1H). Other tissues that showed signals for KGFR included condensing mesenchyme of the choroid sclera, presumptive bone (Fig. 1H) and the imaginations of the ectoderm that give rise to the eyelids and conjunctival epithelium (Fig. 1H). At this stage, transcripts for FGFR3 werefirstdetected in the elongated primary lens fibers (Fig. IK), but not in the epithelium. Strong signals were detected in the invaginating ectoderm of the presumptive eyelids (Fig. IK). Weak signals for FGFR3 were detectable in the mesenchyme of the choroid sclera and in the presumptive bone (Fig. IK). With further differentiation of die lens at E16, the hybridization signals for the three FGFR probes showed more distinct spatial expression patterns. Strong signals for bek transcripts were detected in the anterior epithelium and equatorial regions of the lens (Fig. IF). Strong signals were also present in the cornea and regions of the peripheral retina destined to form the ciliary body and iris (Fig. IF). Weaker signals were found in the developing ganglion cell layer and in the choroid sclera (Fig. IF). For KGFR, strong signals were found in the equatorial lens, presumptive ciliai'y body, and iris, with strongest signals in the ectodermal invaginations of the eyelids. Weaker signals were found in the anterior lens epithelium and in the choroid sclera (Fig. II). Transcripts of FGFR3 were found throughout the E16 lens but were most strongly localized in the elongating lens fibers. Signals were also detected in the eyelid ectoderm imagination and in developing bone (Fig. 1L). In the elongating fibers, the signal for all three FGFR probes was predominantly perinuclear (Figs. IF, II, 1L). At E20, distinct spatial expression patterns for the three FGFRs in the lens were well established. In the lens, a uniform signal was detected for bek transcripts from the central epithelium, through the germinative zone, to the transitional zone, which is located posterior to the lens equator (Fig. 2B). The signal diminished in die cortical fibers that had undergone further FIGURE 2. Expression of FGFRs in the lens at £20. (A) Hematoxylin-slained section of E20 eye showing lens epithelium (le) lens fibers (If), cornea (c), iris (i), ciliary body (cb), neural retina (nr), choroid sclera (cs), eyelid epidermis (e), eyelid suture (s), and eyelid mesenchyme (em). Dark-field micrographs show expression of bek (B), KGFR (C) and FGFR3 (D) in the E20 lens. (B) Transcripts for bek at E20 showed a uniform distribution throughout the epithelium, (including the central and germinative zones) and transitional zone. Signals decreased progressively in the inner and outer cortical fibers and in the mature fibers (asterisk). See Figure 3A for definition of lens regions. (C) Signals for KGFR appeared uniform in the anterior central lens epithelium, decreased slightly in the germinative zone, and then increased in the transitional zone. No significant signals above background were detected in the maturefibers(asterisk). (D) Signals for FGFR3 was detected anteriorly in the central epithelium and increased in the germinative and transitional zones. Strongest signals were detected in the cortical fibers (arroioheads). No significant signals above background were detected in the mature fibers (asterisk). Scale bar = 50 /im. elongation and differentiation and was virtually absent in the most mature fibers in the center of the lens (Fig. 2B). In contrast, the signals for KGFR and FGFR3 showed an anteroposterior increase widi weaker signals in the central epithelium than in the more posterior regions (Figs. 2C, 2D). Signals for FGFR3 were Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 FGF Receptor Expression in Lens Development particularly strong in the more mature fibers of the outer cortex of the lens (Fig. 2D). For KGFR and FGFR3, the signals diminished with further differentiation of lens fibers in deeper regions of the lens (Figs. 2C, 2D). As in E16 lenses, the signal for all three FGFR probes was predominantly perinuclear: This was particularly evident in the elongated fibers of the lens cortex (Figs. 2B, 2C, 2D). Outside the lens, strong signals for bek were detected in the ciliary body, the iris, and the epidermis and hair follicles of the eyelids, with weaker signals in the corneal stroma, neural retina, and mesenchyme of the eyelid and choroid sclera. For KGFR, strong signals were detected in the basal layer of the eyelid epidermis (including the eyelid suture and hair follicles) and were continuous with signals in the conjunctival and corneal epithelia; relatively weak signals were detected in the ciliary body and iris. For FGFR3, although signals were not detected in the surface epidermis, strong signals were present in the epidermal cells at the eyelid sutures and in the conjunctival and corneal epithelia. In the lens, quantitative analysis of the hybridization signals confirmed that the signal density for bek was uniform from the anterior epithelium to the germinative zone (Fig. 3B), whereas signals for KGFR and FGFR3 increased in the more posterior regions of the lens and subsequently decreased in fibers undergoing later stages of fiber differentiation (Figs. 3C, 3D). Peak expression for KGFR occurred in region III (transitional zone) and for FGFR3 in region IV (early cortical fibers). Interestingly, the signal for KGFR showed a small but significant decrease in region II (germinative zone) of E20 lenses (Fig. 3C). A slight decrease in region II was also observed in P3 (P < 0.05), but not in P21 (see Fig. 4E) or in P100 lenses. In postnatal rats, the patterns of FGFR expression established at E20 persisted, with bek showing a uniform distribution in the lens epithelium from anterior to posterior regions, whereas KGFR and FGFR3 signals increased in the transitional zone and outer cortex, respectively (Fig. 4). This anteroposterior increase was most pronounced for FGFR3 (Figs. 4C, 4F). With increasing postnatal age (P21 and P100), the signal for KGFR and FGFR3 in the cortical fibers became more restricted, and strongest expression for FGFR3 was often found in the posterior parts of the transitional zone, rather than in the cortical fibers (Fig. 4F). Signals for bek and KGFR were detectable in the ciliary body, iris, and cornea at P3 (Figs. 4A, 4B) and P21 (Figs. 4D, 4E), but no distinct expression of FGFR3 was detected outside the lens (Fig. 4). In the neural retina at P21, bek, and KGFR showed essentially similar patterns of expression, with weak signals present in the ganglion cell and inner nuclear layers and along the outer edge of the outer nuclear layer (Figs. 1693 5B, 5C). Distinct signals for bek, but not for KGFR, were present in the pigmented epithelium and weak signals for both were detected in the choroid sclera. Densitometric measurement of signal intensity in lens region III from autoradiographs of P3, P21, and PI00 eye sections showed that there were age-related declines in expression for all three receptors, but this was most evident for FGFR3 (Fig. 6). For bek and KGFR forms of FGFR2, there were significant declines in signals from days P3 to P21 (P < 0.005), but not from days P21 to P100 of postnatal development. For FGFR3, there was a progressive, significant decline in expression during both periods of postnatal development (P= 0.0001). DISCUSSION The findings in our investigation have established that two-splice variants of FGFR2 (bek and KGFR) and FGFR3 are each expressed in different patterns in ocular tissues, including the lens, throughout embryonic and postnatal life in the rat. Results of previous studies from this laboratory showed that FGFR1 also has distinct spatiotemporal expression patterns during rat lens development.15 These findings are consistent with those in studies that show that FGFR genes and their isoforms have different patterns of expression in other tissues during embryonic and adult life.22 During rat lens morphogenesis, FGFR1 is the first FGF receptor to appear. At Ell, it becomes detectable in the ocular anlage, with distinct expression in presumptive lens ectoderm and optic vesicle.15 Messenger RNAs for both members of the FGFR2 family (bek and KGFR) only become clearly detectable at El2: Bek is expressed in the ectoderm and lens pit, whereas KGFR is expressed only in the ectoderm in the area of the forming eye. FGFR3 appears later at E14, with formation of the primary lens fibers in the lens vesicle. During these stages of development, each of these receptors shows distinct patterns of expression in various ocular tissues. Once the lens has acquired its distinct polarity, differences in the spatial expression patterns of FGF receptors become more evident. FGFR1 is expressed weakly in the anterior epithelium, increases toward the lens equator in the germinative zone, and reaches a maximum in the transitional zone of the lens (region III) where fiber differentiation begins.15 The two variants of FGFR2 show strikingly different patterns of expression. Bek is uniformly expressed throughout the lens epithelium (including the central and germinative zones) and transitional zone, with a subsequent decline in the maturing fibers; KGFR expression is detectable in the anterior central epithelium, but tends to decline in the germinative zone, at least in younger animals. Strongest expression occurs in the Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 1694 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 FIGURE 3. Quantitative analysis of FGFR expression in different regions of the E20 lens. (A) Micrographs of the E20 lens were divided into six regions for image analysis: I, the anterior epithelium; II, the germinative zone; III, the transitional zone; IV, the outer cortical fibers; V, the inner cortex; and VI, the mature fibers. The density of hybridization signals for bek (B), KGFR (C), and FGFR3 (D) were assessed. Values were normalized for the mean signal for each probe in lens region I. Each bar represents the mean ± SEM of determinations from 13 to 17 separate sections on 4 to 7 separate slides. (B) For bek, the same level of signal density was found in regions I to III, but a progressive decline in signal occurred in regions IV to VI (P < 0.05). (C) For KGFR, expression density was lower in region II than in regions I, III, and TV (P< 0.05), and maximal signal density was observed in region III, with a significant decline in regions IV to VI. (D) FGFR3 expression showed a progressive increase from region I to region IV, with almost a twofold increase between regions I and TV (P < 0.005) and subsequent decrease between regions IV and VI (P < 0.0001). 0.0 i II in iv v vi Lens regions early fibers of the transitional zone. Similar to FGFR1, FGFR3 shows an anteroposterior increase in expression; however, strongest expression occurs in the outer cortical fibers. Thus peak expression of FGFR3 occurs later in the fiber differentiation process than does peak expression of FGFR1. For all probes, only low levels of signal are present in the more mature nuclear fibers. Notably, FGFR3 is more highly expressed in the lens than in other ocular tissues and, with postnatal development, is almost exclusively expressed in the lens. It is of interest that all four FGFR probes studied have shown a predominandy perinuclear localization of FGFR transcripts, particularly evident in elongating and differentiating fibers of the lens cortex. Because FGFRs are membrane proteins, their actively translated mRNAs would be expected to be associated with the rough, endoplasmic reticulum of the cell that, in chick and rat lens cells, forms a dense meshwork around the nuclei.23 Thus, perinuclear localization may reflect active translation of the FGFRs. Consistent with this hypothesis is the finding that there is strong correlation between FGFR1 mRNA and protein expression during lens development.15 However, perinuclear localization may also indicate that these mRNAs have short half-lives. A quantitative in situ hybridization approach, using image analysis techniques, confirmed tfiat the different FGFRs are expressed in distinct, yet overlapping, patterns in the developing and maturing lens. Results of additional studies show that these expression patterns are distinct from that of glyceraldehyde3-phosphate dehydrogenase (GAPDH; de Iongh, unpublished data), which is generally considered to be a "house-keeping" gene. The pattern of GAPDH is uniform throughout the anterior epithelium and region III, with a slight rise in the germinative zone (region II). Distinct from the FGFR genes, the expression of GAPDH decreases more rapidly after region III and is negligible in regions V and VI. The distinct spatial patterns of FGFR expression are suggestive of their involvement in regulating spatial patterns of lens cell behavior. For example, FGFR1 and FGFR3 show major increases in expression at various stages of fiber differentiation, whereas KGFR expression declines in the germinative zone. In lens epithelial explants, FGF induces different responses at different concentrations; proliferation, migration, and fiber differentiation are Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 FGF Receptor Expression in Lens Development 1695 FIGURE 4. Expression of bek, KGFR, and FGFR3 in the postnatal lens. Sagittal sections of eyes from P3 (A to C) and P21 (D to F) rats. For bek (A, D), there was uniform expression in the lens epithelium, including the central epithelium (arrowhead), germinative (open arroio), and transitional (solid arroio) zones, in P3 and P21 lenses. Signals diminished beyond this region, and no significant signals above background were detected in the mature fibers (asterisk). Strong signals were also evident in the ciliary body (cb) and iris (i), with weaker signals in the cornea (c). (B) KGFR signals were detected anteriorly in the P3 central lens epithelium (arrowhead), diminished slightly in the germinative zone (open arrow), but increased in the transitional zone (solid arrow). Signals decreased in the cortical region, and no significant signals above background were detected in the central lens (asterisk). Signals were also detected in the ciliary body <cb) and iris (i). (E) AT P21, uniformly weak signals for KGFR were detected throughout the lens epithelium. Signals were also detected in the ciliary body (cb), iris (i), and cornea (c). For FGFR3 (C,F), expression was detected only in the lens. It was weak in the anterior epithelium (arrowhead) but increasingly strong in the germinative (open arrow) and transitional (solid arrow) zones. Signals were also strong in the outer cortical fibers, particularly at P3 and were diminished in the inner cortical region, with no signals above background evident in the mature fibers (asterisk). There is a refraction artifact at the outer edge of the cornea in B, E, and F. Scale bar = 50 fj.ni. induced in a progressive, dose-dependent manner.3 These cellular behaviors occur in the same sequence in the lens in an anteroposterior pattern (that is, from region I to region VI; Fig. 3). The underlying mechanism that enables lens cells to vary their response to FGF is not clear at present; however, it is possible that their primary response may depend on the predominant form(s) of FGFRs present on the lens cells, either as homo- or heterodimers. For example, the patterns of FGFR1 and FGFR3 expression suggest that stimulation of these receptors may be associated with events in fiber differentiation. In contrast, bek may have a more general role in epithelial maintenance, proliferation, and early fiber differ- entiation, whereas increased KGFR expression just beyond the germinative zone may be linked with withdrawal from the cell cycle and commencement of fiber differentiation. The suggestion that different receptors may be involved in influencing the primary response of lens cells to FGF is supported by findings in studies of FGFR signaling pathways in other cellular systems. These indicate that activation of different FGFRs by FGF can induce different patterns of intracellular protein phosphorylation and mediate different cellular responses.24'25 The temporal expression patterns for FGFRs throughout development are also consistent with the above suggestion that differences in cellular responses Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 1696 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 FIGURE 5. Expression of FGFRs in the P21 retina. (A) Hematoxylin-stained section of P21 retina showing the cellular layers of the retina: ganglion cell layer (gel), inner nuclear layer (inl), outer nuclear layer (onl), retinal pigmented epithelium (rpe), and the choroid sclera (cs). Dark-field micrographs show expression of bek (B), KGFR (C), and FGFR3 (D). Bek and KGFR probes showed similar patterns of expression with signals detectable in the ganglion cell and inner nuclear layers. Most of the outer nuclear layer was not labeled but, in each case, distinct signals were detected along the outer edge of this layer, corresponding to the outer segments of the photoreceptors. Distinct signals for bek but not for KGFR were found in the pigmented epithelium, and weak signals for both were found in the choroid sclera. Signals for FGFR3 were not detected in the neural retina. There is a prominent refraction artifact at the outer edge of the retina in C and D. Scale bar = 20 fj,m. to FGF are related to differences in cellular populations of FGFRs. During embryonic development, there is a distinct sequence of FGFR expression: first FGFR1, then FGFR2, andfinally,FGFR3. Furthermore, FGFR1 and FGFR3, which are consistendy expressed most strongly during periods of active fiber differentiation, show a marked age-related, postnatal decline in expression (Fig. 6),15 similar to the documented decline in lens fiber differentiation with age.26"28 In contrast, bek and KGFR show a less marked decline in expression with age, indicating a role more in line with cell maintenance than with fiber differentiation. Lens fibers are clearly dependent on FGF for survival and differentiation, as indicated by findings in recent transgenic studies in which a dominant negative FGFR was overexpressed in the mouse lens (see introduction).1314 However, because the dominant-negative construct inhibits all FGF receptors,29'30 these data do not provide information about whether multiple or individual FGFRs are involved in eliciting specific cellular responses. Recendy, mutations of three of the FGFR genes have been identified as responsible for several autosomal dominant human skeletal disorders.31 These mu- tations are associated with unique skeletal phenorypes that can be correlated with the expression patterns of the FGFRs but cannot be explained simply in terms of loss of gene function.31"33 For instance, FGFR gene knockout studies in transgenic mice show that heterozygous animals have normal "wild-type" phenotype whereas homozygous animals either are not viable embryonically, as in the case of FGFR1M'35 or have a phenotype distinct from the human syndromes, as in the case of FGFR3.33 Comparison of the different patterns of bone growth in the homozygous mouse FGFR3 gene knockout and the human FGFR3 mutant phenotype (achondroplasia) led Deng et al33 to conclude that in the human syndrome there is a gain rather than a loss of function: the mutant FGFR3 is constitutively active and, during normal terminal bone differentiation, FGFR3 may have a negative regulatory role by inhibiting proliferation and terminal differentiation of chondrocytes. Because peak expression of FGFR3 occurs in the lens in terminally differentiating fibers, it is possible that it has an analogous, negative regulatory role during terminal differentiation of lens fibers. Characteristic features of the human craniosy- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 FGF Receptor Expression in Lens Development A bek P3 P21 P100 Age of rat 6. Age-related decline of FGFR expression in the lens. Sections of whole eyes from rats at P3, P21, and P100 were hybridized with probes for bek (A), KGFR (B), and FGFR3 (C) under identical conditions and exposed to the same autoradiographic film for 5 days. The density of the hybridization signal in region III of lenses (Fig. 3) was measured by laser densitometry. Each column represents the mean ± SEM of measurements from 5 to 8 lenses. Density of hybridization signal for bek and KGFR probes declined significantly between P3 and P21 (P < 0.005) but not between P21 and P100. Signal density for FGFR3 declined progressively from P3 to P100 (P < 0.005). FIGURE nostosis syndromes, which result from mutations of FGFR1, FGFR2, or both, include ocular defects; however, although not specifically studied, there are only rare accounts of such lens abnormalities as cataract.36'37 Preliminary examinations of eyes from chimeric FGFR1 gene knockout mice (Dr. J. Rossant, personal communication, 1996) and of the FGFR3 homozygous mutant (Dr. C. X. Deng, personal communication, 1996) similarly suggest that eye development proceeds normally, but detailed studies of lens differentiation have not yet been conducted. In contrast, inhibition of all FGFR signaling in transgenic mice using a dominant-negative strategy produced significant lens abnormalities. 1 3 1 4 Although these results do not exclude the possibility of individual receptors having different functional 1697 roles in lens development, they do suggest there may be some redundancy of FGFR function. The four FGFRs expressed in the lens have different affinities for FGF family members. FGFR1 binds FGF-1 and FGF-2 equally and with high affinity, but binds FGF-4 with a lower affinity.38"40 FGFR2 (bek) binds FGF-1, FGF-2, and FGF-4 with similar high affinity, but does not bind FGF-5 or FGF-738'41; KGFR binds FGF-7 and FGF-1 with equal high affinity, but binds FGF-2 with a much lower affinity.42'43 FGFR3 binds FGF-1 with high affinity and FGF-2 with low affinity,44 and it has been shown that the mitogenic effects of FGF-1 and FGF-4, mediated by FGFR3, are 10-fold higher than the effect of FGF-2, whereas FGF-5 elicits no response. 44 FGF-1 and FGF-2 can induce lens cell proliferation, migration, and fiber differentiation in vitro, but FGF-2 is more potent than FGF-1.2 Findings in preliminary studies in which FGFs are overexpressed in lens fibers of transgenic mice indicate that FGF-3, FGF-4, FGF-5, FGF-7, and FGF-8 also elicit fiberdifferentiation-like responses in lens epithelium 4 " 7 but their relative potency in eliciting responses in lens cells is unknown. To date, expression of 5 of the 10 FGF family members (FGF-1, FGF-2, FGF-3, FGF-5, and FGF-7) has been detected in the developing eye. FGF-1 and FGF-2 mRNA and protein are expressed widely in the eye 2145 " 48 and are detected in the ocular media that bathe the lens, with more of both forms in the vitreous humor than in the aqueous humor. 2 ' 49 FGF-3, FGF-5, and FGF-7 have more restricted expression patterns. FGF-3 is expressed in the neural retina during development but is restricted to the peripheral neural retina at birth; it declines after birth and is undetectable in the mature retina. 50 FGF-5 expression appears to be restricted to the neural retina and pigmented epithelium, 5152 and FGF-7 is expressed in periocular mesenchyme during embryonic development 53 ; but in the mature eye, expression appears to be restricted to the cornea and conjunctiva.54'55 Clearly, cellular responses to FGF in various regions of the lens in situ will depend not only on the types and abundance of FGFRs present but also on the types and abundance of FGF present. Such responses may also depend on the capacity of the FGFRs present to form particular homo- and heterodimer combinations on ligand binding (see introduction). The current results establish that FGFRs are expressed in diverse temporal and spatial patterns during lens morphogenesis, differentiation, and growth and highlight the complexity of the regulation of lens cell behavior by FGF. There are many potential ways in which the responses of lens cells to FGF may be regulated in situ.2 Although the availability of ligands in the extracellular milieu is a major consideration, the current findings suggest that the regulation of Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 1698 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 the spatiotemporal expression patterns of the various FGFRs, with their capacity to form homodimers and heterodimers, as well as their differing ligand binding and signaling properties, may also play an important role in regulating anteroposterior patterns of cell behavior in the lens. Key Words 12. 13. 14. fiber differentiation, fibroblast growth factor, fibroblast growth factor receptor, lens development Acknowledgments The authors thank Dr. M. Bedford and Dr. P. Lonai (Weizmann Institute of Science, Rehovot, Israel) for the murine bek and KGFR cDNAs, Dr. L. T. Williams (University of California, San Francisco, CA, USA) for the FGFR3 cDNA, Roland Smith for technical assistance with photography, and staff of The University of Sydney Electron Microscope Unit for the use of their image analysis facilities. 15. 16. 17. References 1. Yamaguchi TP, RossantJ. Fibroblast growth factors in mammalian development. Curr Opin Genet Dev. 1995; 5:485-491. 2. Chamberlain CG, McAvoy JW. Fibre differentiation and polarity in the mammalian lens: A key role for FGF. Prog Retina Eye Res. 1996; (In press). 3. McAvoy JW, Chamberlain CG. Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development. 1989; 107:221-228. 4. Chepelinsky AB, Robinson ML, Ash J, Parker-Wilson DM, Ohtaka-Maruyama C, Overbeek, PA. Secreted FGF-3 induces cell cycle withdrawal and differentiation of lens epithelia in transgenic mice. ARVO Abstracts. Invest Ophthalviol Vis Sci. 1996;37:S924. 5. Lovicu FJ, Srinivasan Y, Overbeek PA. Developmental changes induced by lens-specific expression of different FGFs in transgenic mice. ICER Abracts. Exp Eye Res. 1997;63(Suppl):S16. 6. Robinson ML, Overbeek PA, Verran DJ, et al. Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice. Development. 1995;'121:505-514. 7. Srinivasan Y, Overbeek PA. Expression of lens specific fibroblast growth factor (FGF5) causes altered epithelial cell morphology and degeneration of fiber cells. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1996; 37:S924. 8. Johnson DE, Williams LT. Structural and functional diversity in the FGF receptor multigene family. Adv Cancel-Res. 1993; 60:1-41. 9. Klagsbrun M. Mediators of angiogenesis: The biological significance of basic fibroblast growth factor (bFGF)-heparin and heparan sulfate interactions. Semin Cancer Biol. 1992;3:81-87. 10. McKeehan WL, Kan M. Heparan sulfate fibroblast growdi factor receptor complex: Structure-function relationships. Mol Reprod Dev. 1994;39:69-81. 11. Jaye M, Schlessinger J, Dionne CA. Fibroblast growth 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. factor receptor tyrosine kinases: Molecular analysis and signal transduction. Biochim Biophys Ada. 1992; 1135:185-199. Schlessinger J, Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron. 1992;9:383-391. Chow RL, Roux GD, Roghani M, et al. FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens. Development. 1995; 121:4383-4393. Robinson ML, MacMillan-Crow LA, Thompson JA, Overbeek PA. Expression of a truncated FGF receptor results in defective lens development in transgenic mice. Development. 1995; 121:3959-3967. de Iongh RU, Lovicu FJ, Hanneken A, Baird A, McAvoy JW. FGF receptor-1 (fig) expression is correlated with fibre differentiation during rat lens morphogenesis and growth. DevDynam. 1996;206:412-426. Orr-Urtreger A, Bedford MT, Burakova T, et al. Developmental localization of die splicing alternatives of the fibroblast growth factor receptor-2. Dev Biol. 1993; 158:475-486. Peters K, Ornitz D, Werner S, Williams LT. Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol. 1993; 155:423-430. Yan G, McBride G, McKeeban WL. Exon skipping causes alteration of the COOH-terminus and deletion of the phospholipase-C-gamma 1 interaction site in the FGF receptor 2 kinase in normal prostate epithelial cells. Biochem Biophys Res Commun. 1993; 194:512518. Chellaiah AT, McEwan DG, Werner S, Xu J, Omitz DM. Alternative splicing in immunoglobulin-like domain III creates a receptor highly specific for acidic FGF/FGF-1./.BioZ Chem. 1994;269:11620-11627. Simmons DM, Arriza JL, Swanson LW. A complete protocol of in situ hybridization of messenger RNA in brain and tissues with radiolabelled single-stranded RNA probes. /Histotechnol. 1989; 12:169-181. Lovicu FJ, de Iongh R, McAvoy JW. Expression of FGF1 and FGF-2 mRNA during lens morphogenesis, differentiation and growth. Curr Eye Res. 1997; 16:222223. Wilkie AOM, Morris-Kay GM, Jones EY, Health JK. Functions of fibroblast growth factors and their receptors. Curr Biol. 1995;5:500-507. Bassnett S. The fate of the golgi apparatus and the endoplasmic reticulum during lens fiber differentiation. Invest Ophthalmol Vis Sci. 1995;36:1793-1803. Vainikka S, Partanen J, Bellosta P, et al. Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction. EMBOJ. 1992; 11:4273-4280. WangJK, Gao G, Goldfarb M. Fibroblast growth factor receptors have different signalling and mitogenic potentials. Mol Cell Biol. 1994; 14:181-188. Cenedella RJ. Aging and rates of lens cell differentiation in vivo, measured by a chemical approach. Invest Ophthalmol Vis Sci. 1989; 30:575-579. Lovicu FJ, McAvoy JW. The age of rats affects the response of lens epithelial explants to fibroblast growth factor: An ultrastructural analysis. Invest Ophthalmol Vis Sci. 1992;33:2629-2278. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017 FGF Receptor Expression in Lens Development 28. Richardson NA, McAvoy JW, Chamberlain CG. Age of rats affects response of lens epithelial explants to fibroblast growth factor. Exp Eye Res. 1992; 55:649656. 29. Amaya E, Musci TJ, Kirschner M. Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell. 1991; 66:257-270. 30. Ueno H, Gunn M, Dell K, Tseng A, Williams L. A truncated form of fibroblast growth factor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor. / Biol Chem. 1992; 267:14701476. 31. Muenke M, Schell U. Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet. 1995; 11:308-313. 32. Jabs EW, Li X, Scott AF, et al. Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet. 1994; 8:275-279. 33. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell. 1996;84:911-921. 34. Deng C, Wynshaw-Boris A, Shen MM, Daugherty C, Ornitz DM, Leder P. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 1994;8:3045-3057. 35. Yamaguchi TP, Harpal K, Henkemeyer M, Rossant J. fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 1994;8:3032-3044. 36. Howell SC. Craniostenosis. Report of 22 cases. Am J Ophthalmol. 1954; 37:359-367. 37. Menashe Y, Ben Baruch G, Rabinovitch O, Shalev Y, Katzenlson MBM, Shalev E. Exophthalmus—prenatal ultrasonic features for diagnosis of Crouzon syndrome. PrenatDiagn. 1989;9:805-808. 38. Dionne CA, Crumley G, Bellot F, et al. Cloning and expression of two distinct high-affinity receptors crossreacting with acidic and basic fibroblast growth factors. EMBOj. 1990; 9:2685-2692. 39. Johnson DE, Lee PL, Lu J, Williams LT. Diverse forms of a receptor for acidic and basic fibroblast growth factors. Mol Cell Biol. 1990; 10:4728-4736. 40. Mansukhani A, Dell'Era P, Moscatelli D, Kornbluth S, Hanafusa H, Basilico C. Characterization of the murine BEKfibroblastgrowth factor (FGF) receptor: Activation by three members of the FGF family and requirement for heparin. Proc Nail Acad Sci USA. 1992;89:3305-3309. 41. Mansukhani A, Moscatelli D, Talarico D, Levytska V, Basilico C. A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF. Proc Natl Acad Sci USA. 1990;87:4378-4382 1699 42. Bottaro DP, Rubin JS, Ron D, Finch PW, Florio C, Aaronson SA. Characterization of the receptor for keratinocyte growth factor—evidence for multiple fibroblast growth factors. /Biol Chem. 1990;265:1276712770. 43. Miki T, Bottaro DP, Fleming TP, et al. Determination of the ligand-binding specificity by alternative splicing. Two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci USA. 1992; 89:246-250. 44. Ornitz DM, Leder P. Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3. J Biol Chem. 1992; 267:16305-16311. 45. de Iongh R, McAvoy JW. Distribution of acidic and basic fibroblast growth factors (FGF) in the foetal rat eye: Implications for lens development. Growth Factors. 1992;6:159-177. 46. de Iongh R, McAvoy JW. Spatio-temporal distribution of acidic and basic FGF indicates a role for FGF in rat lens morphogenesis. Dev Dynam. 1993; 198:190-202. 47. Noji S, Matsuo T, Koyama E, et al. Expression pattern of acidic and basic fibroblast growth factor genes in adult rat eyes. Biochem Biophys Res Commun. 1990; 168:343-349. 48. Lovicu FJ, McAvoy JW. Immunolocalization of acidic FGF, basic FGF and heparan sulfate proteoglycan in the rat lens: Implications for lens polarity and growth patterns. Invest Ophthalmol Vis Sci. 1993; 34:3355-3365. 49. Schulz M, Chamberlain CG, de Iongh RU, McAvoy JW. Acidic and basic FGF in ocular media: Implications for lens polarity and growth patterns. Development. 1993; 118:117-126. 50. Wilkinson DG, Bhatt S, Kennedy AP. Expression of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal development. Development. 1989; 105:131-136. 51. Bost LM, Aotaki-Keen AE, Hjelmeland LM. Coexpression of FGF-5 and bFGF by the retinal pigment epithelium in vitro. Exp Eye Res. 1992; 55:727-34. 52. Kitaoka T, Aotaki-Keen AE, Hjelmeland LM. Distribution of FGF-5 in the rhesus macaque retina. Invest Ophthalmol Vis Sci. 1994;35:3189-3198. 53. Finch PW, Cunha GR, Rubin JS, WongJ, Ron D. Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal-epithelial interactions. Dev Dynam. 1995; 203:223-240. 54. Wilson SE, Walker JW, Chwang EL, He YG. Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor-2, and the cells of die cornea. Invest Ophthalmol Vis Sci. 1993; 34:2544-2561. 55. Li DQ, Tseng SC. Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. / Cell Physiol. 1995; 163:61-79. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 05/04/2017