Gradients of cone differentiation and FGF expression duringdevelopment of the foveal depression in macaque retina
ELISA E. CORNISH,1 MICHELE C. MADIGAN,1 RICCARDO NATOLI,2 ANGELA HALES,1
ANITA E. HENDRICKSON,3 and JAN M. PROVIS2
1Department of Ophthalmology and Save Sight Institute, University of Sydney, NSW 2006, Australia2Research School of Biological Sciences, Bldg. 46, Biology Place, The Australian National University,Canberra, ACT 0200, Australia3Department of Biological Structure, University of Washington, Seattle
(Received December 9, 2004; Accepted January 18, 2005!
Abstract
Cones in the foveola of adult primate retina are narrower and more elongated than cones on the foveal rim, whichin turn, are narrower and more elongated than those located more eccentric. This gradient of cone morphology isdirectly correlated with cone density and acuity. Here we investigate the hypothesis that fibroblast growth factor~FGF! signaling mediates the morphological differentiation of foveal cones—in particular, the mechanism regulatingthe elongation of foveal cones. We used immunoreactivity to FGF receptor ~R! 4, and quantitative analysis to studycone elongation on the horizontal meridian of macaque retinae, aged between foetal day ~Fd!95 and 2.5 years postnatal ~P 2.5y!. We also used in situ hybridization and immunohistochemistry to investigatethe expression patterns of FGF2 and FGFR1–4 at the developing fovea, and three other sample locations on thehorizontal meridian. Labeled RNA was detected using the fluorescent marker “Fast Red” ~Roche! and levels ofexpression in cone inner segments and in the ganglion cell layer ~GCL! were compared using confocal microscopy,optical densitometry, and tested for statistical significance. Our results show that morphological differentiationof cones begins near the optic disc around Fd 95, progressing toward the developing fovea up until birth,approximately. Levels of FGF2 and FGFR4 mRNAs expression are low in foveal cones, compared with conescloser to the optic disc, during this period. There is no similar gradient of FGF2 mRNA expression in the ganglioncell layer of the same sections. Maturation of foveal cones is delayed until the postnatal period. The results suggestthat a wave of cone differentiation spreads from the disc region toward the developing fovea during the second halfof gestation in the macaque. A gradient of expression of FGFR4 and FGF2 associated with the wave ofdifferentiation suggests that FGF signalling mediates cone narrowing and elongation.
Vertebrate retina develops in a centroperipheral sequence. In pri-mates, the first cells to exit the cell cycle are located at theincipient fovea ~Rapaport & Stone, 1982; Provis et al., 1983!, withlater generated cells being positioned in more peripheral locations.Following a similar developmental pattern, synapses are formedinitially in the inner plexiform layer ~IPL! and outer plexiformlayer ~OPL! of central retina ~Okada et al., 1994; Crooks et al.,1995; Hendrickson, 1996! then in more peripheral locations untilthey are present at the peripheral edge. Waves of cell death in theganglion cell layer ~GCL! ~Provis & Penfold, 1988! and innernuclear layer ~INL! ~Georges et al., 1999! also first appear cen-
trally then progress peripherally. As a result of this developmentalgradient, more peripheral regions of the retina develop at later timepoints so that, in general, the central region is more mature thanany region peripheral to it.
Consistent with this pattern, cones express opsins in a centro-peripheral sequence ~Bumsted & Hendrickson, 1999; Xiao &Hendrickson, 2000! starting in central cones at around fetal day~Fd! 65–75 ~monkey! and fetal week ~Fw! 12–15 ~human!, reach-ing the periphery at Fd 135 in monkey and around birth in humans~Bumsted & Hendrickson, 1999; Xiao & Hendrickson, 2000!.Paradoxically, however, at birth human foveal cones are lessmature than cones in the parafovea and perifovea ~Abramov et al.,1982; Hendrickson & Drucker 1992!. Similar delayed develop-ment of foveal cones in macaque retina has been identified ~Hen-drickson & Kupfer, 1976; Springer & Hendrickson, 2004a!. Themorphological development of central cones has been describedpreviously; immature cones are cuboidal cells ~Hendrickson &Yuodelis, 1984!, which nonetheless make synaptic contacts on
Address correspondence and reprint requests to: Jan M. Provis, SeniorFellow, Research School of Biological Sciences, Bldg. 46, Biology Place,The Australian National University, Canberra, ACT 0200, Australia. E-mail:[email protected]
their inner aspect with horizontal, then bipolar cells, at an earlystage of development ~Linberg & Fisher, 1990!. The first feature ofmorphological specialization of cones is elongation of the axonalprocess. This elongation, forming the fiber of Henle, is needed toaccommodate the movement of INL cells away from the center ofthe foveal cone mosaic during development of the foveal depres-sion, and takes place over a protracted period, between about Fd105 and one year postnatal ~P 1y! in macaques and Fw25 and 2–3years postnatal in humans ~Hendrickson & Yuodelis, 1984; Yuode-lis & Hendrickson, 1986; Packer et al., 1990; Hendrickson, 1992!.While previous studies have noted that commencement of thismaturation process is somewhat delayed in foveal cones, ~Bach &Seefelder, 1911, 1912, 1914; Mann, 1964; Hendrickson & Kupfer,1976; Abramov et al., 1982; Hendrickson & Yuodelis, 1984;Hendrickson, 1988; Hendrickson, 1992; Provis et al., 1998! rela-tively little attention has been paid to the dynamics, or to theexpression of factors that may regulate this maturation.
The mammalian fibroblast growth factor ~FGF! family com-prises at least 23 structurally related ligands that interact withlow-affinity heparan sulfate proteoglycans molecules and high-affinity, transmembrane, tyrosine kinase receptors ~see Ornitz et al.,1996!. Recently, we reported that fibroblast growth factor recep-tors ~FGFR! have discrete distributions on the soma, fiber ofHenle, and synaptic pedicle of developing and adult cones ~Cor-nish et al., 2004!. We found that FGFR4 is one of the earliestmarkers expressed by cones and is distributed throughout thedeveloping soma and axonal process ~fiber of Henle!, but is absentfrom the pedicle. In contrast, FGFR1 is highly expressed on thepedicle but is absent from the axonal process. FGFR3 is expressedon the soma and proximal part of the axon, while all threereceptors are expressed in the inner segments. Here we report thepatterns of distribution of FGF2 mRNA in macaque retina. We alsoreport apparent regulation of expression of FGFR4 and FGF2 incones, which appears to be correlated with delayed onset ofmorphological maturation of foveal cones.
Materials and methods
Specimens
Macaque monkey retinae were obtained from Bogor AgricultureUniversity, Indonesia, with approval of the Ethics Committee ofThe University of Washington, Seattle. Fetuses were delivered byaseptic cesarean section and mothers returned to the breeding col-ony after a suitable recovery period. Fetuses were euthanased by anintravascular overdose of barbiturate, eyes were enucleated, theninjected with Carnoy’s fixative ~methyl alcohol: acetic acid: chlo-roform, 6:3:1! and fixed whole for 2–4 h. Eyes were paraffin em-bedded and sectioned in the horizontal plane at 8 µm. Only sectionsthrough the optic disc and fovea were analyzed quantitatively. Forthis study, 12 macaque retinae were grouped in three age ranges: ~1!before formation of the foveal depression ~Fd 90 and Fd 95!; ~2!during pit formation ~Fd 110, Fd 115, Fd 120, Fd 130, Fd 164, andFd 165!; and ~3! after the foveal depression has formed @postnatal~P! 3 weeks ~wks!, P4 months ~m!, P 2.5 years ~yrs!, and P 11yrs# .One section in every ten was Nissl stained using cresyl violet. Thesesections were used to identify sections containing the fovea or in-cipient fovea and to obtain details of cone morphology.
Riboprobes
We used riboprobes to FGFR1, R2, and FGF-2 made from ratcDNA donated by Andrew Baird, Scripps Institute, California,
USA. The sequences for these target RNAs in rat are better than90% homologous with sequences for the human RNAs. We gen-erated riboprobes to FGFR3 and FGFR4 from PCR products fromhuman fetal retina, because fresh macaque material was not avail-able ~Cornish et al., 2004!. We have not yet obtained fresh ma-caque retina to verify homology with the human sequences, but~based on high levels of homology between other, less closelyrelated species! they are assumed to be high ~�90%!. cDNAfragments were amplified by RT-PCR from purified, total RNAfrom human fetal retina, ligated to pGem�-T DNA vector ~Pro-mega #A3610, Madison, WI! and cloned in JM109 competentcells, using the Promega pGem�-T DNA vector system protocol.Antisense and sense probes were generated by in vitro transcrip-tion of linearized plasmid constructs containing the DNA frag-ments using bacteriophage RNA polymerases ~Promega!. Thetemplates were purified, sequenced, and riboprobes prepared usingDigoxigenin ~DIG!-labelled-11-UTP ~DIG RNA labelling kit; Roche#1175025, Nutley, NJ!.
Digoxigenin-11-UTP-labeled RNA was resuspended in hybrid-ization solution at 100–300 ng0mL. The prehybridization solutionconsisted of 50% deionized formamide, 20% dextran sulphate~Amresco #0198-250G!, 500 µg0mL poly-A ~Sigma #P9403, St.Louis, MO!, 50 µg0mL yeast t-RNA ~Sigma #R8759!, 50 µMDithiothreitol ~Sigma #D-9779! and a salt solution containing300 mM NaCl, 10 mM Tris-base ~Sigma #T-6066!, 10 mM sodiumphosphate ~NaH2PO4 Sigma #S-3264!, 5 mM NaEDTA, 0.2%Ficoll 400 ~Sigma #F-2637!, and 0.2% Polyvinylpyrrolidone ~Sigma#P-5288!. The hybridization solution comprises pre-hybridizationsolution plus the required concentration of DIG-labeled probe.
Paraffin sections were dewaxed through xylene ~Chem Supply#XA003! ~2 � 10 min! before being hydrated in graded ethanolsand rinsed in phosphate-buffered saline ~PBS!. Sections were fixedin 10% neutral buffered formalin ~NBF, BDH #90245.001! for20 min, washed in PBS for 5 min, and then placed in a solutioncontaining 20 mg0ml of Proteinase K ~Roche #745723! diluted inTE ~50 mM Tris-HCl, 5 mM EDTA, pH 8! for 7 min at 378C. Thesections were then rinsed in PBS, and re-fixed in NBF for 20 min.Slides were placed into a solution containing 0.1 M Triethanol-amine ~pH 8.0, Sigma #T-9534! and acetic anhydride ~2.5%,Sigma #A-6404! for 10 min, washed in PBS then 0.9% NaCl for5 min each. The slides were then dehydrated through gradedethanols and air dried.
The prehybridization solution was preheated to 658C on a heatblock, before being added to the sections under a coverslip,followed by an incubation at 558C for a minimum of 1 h. Follow-ing this, the coverslip and prehybridization solution was carefullyremoved and the preheated hybridization solution ~containing theprobe! was added to the sections and covered by a new coverslip.Sections were hybridized in 100–300 ng of probe0ml overnight, atoptimal temperature. Coverslips were removed and sections placedfor 5 min at room temperature ~RT! in 2� saline sodium citrate~SSC, pH 7.4!, 0.5� SSC and 0.1� SSC, followed by 0.1� SSCat the appropriate posthybridization wash temperature ~55–758C!for 2 h. The posthybridization temperature was determined bytesting a range of temperatures to find the temperature producingthe cleanest antisense and sense labeling. The slides were washedin 0.1� SSC at RT for 5 min. The slides were rinsed in washingbuffer, placed in blocking solution for 30 min, then incubated inanti-DIG antibody ~1:2000! for 1 h. Following antibody incuba-tion, slides were rinsed twice in washing buffer, then in detectionbuffer for 5 min ~Cornish et al., 2004!. Probes were visualizedusing an alkaline phosphatase antibody and fluorescent HNPP
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‘Fast Red’ ~Roche #1758888!. Sections were counterimmunola-baled using antibody to long0medium wavelength-sensitive opsin~anti-L0M opsin, 1:1000; donated by Jack Saari, University ofWashington!, anti-vimentin ~1:100, DAKO!, or anti-cellular retin-aldehyde binding protein ~CRALBP, 1:2500, donated by JackSaari!. After washing, sections were blocked in 10% normal serumfor 30 min, then incubated overnight at 48C. Antibody was de-tected using FiTC- or Alexa 488-conjugated secondary antibody,incubated at room temperature for 1 h. Sections were mounted inglycerol, coverslipped, and sealed with nail varnish.
Double immunolabeling
Sections passing through the fovea at Fd 85, 120, and 164 weredouble immunolabeled using antibodies to human FGFR1, R2, R3,or R4 ~Santa Cruz, goat anti-rabbit, 1:200! in combination withanti-vimentin ~mouse anti-swine, 1:100, DAKO! or anti-CRALBP~rabbit anti-swine, 1:2500, donated by Jack Saari!. Slides weredewaxed, blocked, and incubated in primary antibodies overnight.After rinsing, slides were incubated in biotinylated secondaryantibody for 1 h, rinsed, then streptavidin-conjugated Alexa ~488or 594! fluorchrome for 1 h. After rinsing, slides were incubated influorochrome Alexa ~488 or 594! tagged secondary antibody todetect the second primary label. Sections were imaged using aconfocal microscope and images were exported and processedusing Adobe Photoshop ~v. 6.1!. Optical densitometry was carriedout as described below ~Quantification!. Sections immunolabeledwith anti-FGFR4 were used to carry out a morphometric analysisof cones in and adjacent to the foveal region.
Analyses
The length of cones, from the external limiting membrane to thecone pedicle synapse, was measured in two sections through thecentral fovea at each of Fd 85, 120, and 164, including a samplefrom the center of the foveal cone mosaic0fovea. Sample locationswere evenly spaced ~;500 µm separating samples! temporal andnasal to the foveal cone mosaic. Files were imported into Adobe
Photoshop and individual cones were marked with either a singleline ~for less mature cones, showing no tilt in the fiber of Henle!or a segmented line to represent the somal and axonal portions,where the fiber of Henle was directed away from the fovea. Asingle measurement was made using NIH Image ~NIH—http:00www.scioncorp.com0!, the data entered into an Excel spreadsheet,then graphed. Lengths of the inner and outer segments were notincluded in the measurements.
Cresyl-violet sections were viewed using a Nikon Optiphot andphotographed with Kodak Ektachrome color film at a final mag-nification of 150�. Fluorescent sections were imaged using a scan-ning Leica Confocal Microscope and TCSNT software, v. 1.6.587.Images were captured using a 16 � ~field size 512 � 512 µm! or40 � objective ~field size 250 � 250 µm!; a selection of fieldswas magnified two or four times ~zoom 2, zoom 4! for opticaldensitometry.
Images were viewed and presented using Adobe Photoshop.Adjustments for color balance and contrast were made to double-immunolabeled sections, as required for the analyses.
Quantification
No adjustments of levels, color balance, or contrast were made toimages of the hybridized sections. Images of mRNA labelling wereconverted to greyscale, inverted using Photoshop for densitometricanalysis, and quantified using Scion Image. Mean optical densityestimates ~arbitrary units! were obtained from a standard rectanglesample with an area approximately the size of 1.5 cone cell nucleiof the outer nuclear layer ~ONL! ~Fig. 1A!. This sample area wasselected after testing statistically four different sample shapes andsizes ~rectangle 1, 750 pixels; rectangle 2350 pixels; oval, 300pixels; and circle, 276 pixels!. Using the Kruskal-Wallis test, a750-pixel rectangle was found to result in minimum standard errorand mean square error in optical density measurements. All quan-tification was performed using grey-scale pixilation from the “red”channel of the Adobe Photoshop image. The same protocol wasfollowed to determine the best sample area to measure FGF2mRNA expression in the ganglion cell layer ~GCL! control.
Fig. 1. Analysis of FGF labeling. ~A! Section of fetal retina showing fluorescent FGF2 mRNA labelling ~image converted to greyscaleand inverted using Photoshop!. All densitometry was carried out on images in this format. The size and shape of the samples for opticaldensitometry is shown. ~B! Schematic, showing the approximate locations of the four sampling regions referred to in this study. Samplelocations were defined relative to two landmarks, optic disc and developing fovea, to facilitate sampling at similar locations in differenteyes at different ages.
FGF gradients in developing fovea 449
Four locations between the foveal cone mosaic and optic discon the horizontal meridian were selected for optical density ana-lyzes. This region elongates relatively little during development,and the presence of two landmarks ~incipient fovea and optic disc!make it possible to define relative locations that can be identifiedat different ages, regardless of eye growth. Location 1—is definedas the central or incipient fovea; Location 2—on the edge of thefoveal cone mosaic, or foveal rim, on the nasal side; Location3—halfway between the fovea and the optic disc; and Location4—immediately temporal to the optic disc ~Fig. 1B!. For eachsection, the mean optical density was calculated from ten samplesin each region and the data summarized in a histogram. Differ-ences in the variance of mRNA mean optical density was testedamong the four retinal regions in each section using the Kruskal-Wallis test. A post-hoc Dunnett-like multiple comparison analysiswas then used to compare mean optical density in nonfovealsamples ~Locations 2, 3, and 4! with those from the central fovealregion ~Location 1!. All comparisons of levels of mRNA expres-
sion were made from the raw data between regions, in a singlesection; “between section” comparisons are not valid as, althoughevery attempt was made to standardize tissue processing, condi-tions cannot be assumed to be uniform between hybridizations ondifferent slides, different sections, or at different times. In somecases data showing expression levels was normalized to a singlelocation before graphing, in order to facilitate comparison of levelsof expression at different ages by the reader.
Results
Cone morphology
The morphology of cones along the horizontal meridian is illus-trated in Fig. 2. Four locations, comparable to those used formRNA optical densitometry, are shown at Fd 95, Fd 130, and 11months postnatal ~P 11 mo!. Cones closer to the optic disc ~Lo-cation 4! begin to morphologically differentiate ahead of more
Fig. 2. Morphological differentiation of cones at Locations 1–4 ~see Methods! on the horizontal meridian at three ages. A–D: Fd 95,before formation of the foveal depression, when there is relatively uniform morphology of cones at all locations. E–H; Fd 130, duringformation of the foveal depression. ~E! cones at the fovea ~Location 1! at Fd 130 are relatively undifferentiated compared with the rim~F! and perifovea ~G,H!. I–L: P 1 months, after formation of the fovea ~I! the foveal cones are narrower and more elongated than conesat all other locations ~J–L!. Scale bars � 20 µm.
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central cones. At Fd 95 at Location 4, cones are 25–30% longerthan those at locations 1–3, due to their elongated axons ~Fig. 2Dcf. 2A–2C!. With increased age cones closer to the foveal regionbegin to elongate and elaborate fibers of Henle. By Fd 130, coneson the foveal rim ~Fig. 2F! have started to elongate and are25–30% longer than those near the disc ~Fig. 2H!. Cones withinthe foveal depression are the last to differentiate morphologically~Fig. 2E!. At Fd 130, the centralmost cones have poorly differen-tiated fibers of Henle and small inner segments compared to thoseon the rim ~Fig. 2F! and near the optic ~Fig. 2H!, respectively. Incontrast, in mature retina central cones have the most elaboratefibers of Henle and longer, thinner inner segments compared withall locations ~Fig. 2I cf. 2J–2L!.
Morphometric data from cones at eight locations along thehorizontal meridian before ~Fd 85!, during ~Fd 120!, and after ~Fd164! formation of the foveal depression are shown in Fig. 3. Thegraph shows relatively uniform length of photoreceptors ~fromexternal limiting membrane to cone pedicle! at all locations at Fd85, while at Fd 120 cones in the developing fovea are significantlyshorter than those on the foveal rim ~n1 and t1! and at other nasallocations ~n2–4! ~P�0.0001; Student t-test!. By Fd 164, fovealcones have differentiated little compared with those at other loca-tions. In contrast, in adult macaque retina central cones haveaxonal processes measuring 200–500 µm ~Wässle et al., 1990!.The present data indicate delayed maturation of foveal cones,compared with adjacent regions, and suggest a wave of differen-tiation moving towards the foveal region during the second half ofgestation; the data also indicate that elaboration of fibers of Henleby foveal cones largely occurs in the postnatal period.
Cellular localization of FGF2: A gradient of FGF2mRNA in the ONL
In situ hybridization with FGF2 antisense probe showed intenseexpression of FGF–2 mRNA throughout the retina at all agesstudied, with somewhat lower levels expressed in cells in the
middle of the inner nuclear layer ~INL! ~Figs. 4A & 4B!. Nolabeling was found using the sense probe ~Fig. 4C!. At Fd 95~Fig. 4A!, FGF2 was present throughout the cytoplasm of cones inthe foveal cone mosaic, which were also immunoreactive to anti-body against L0M opsin. Later in gestation ~Fig. 4B, Fd 164! andin adult retina ~not shown! FGF2 mRNA was present in the myoidof the inner segment, sclerad to the external limiting membrane,and in the soma on the sclerad side of the nucleus.
The intensity of FGF2 mRNA labelling was not uniform through-out the ONL in all specimens. A montage showing the distributionof FGF2 mRNA across the developing fovea is shown in Fig. 5A;regions selected for high magnification imaging and optical den-sitometric analysis are indicated. A fourth sample region waspositioned immediately adjacent to the optic disc ~see Fig. 1!, thatis to the right of the image in Fig 5A. Control samples were takenfrom the GCL immediately inwards from the ONL sample locations.
High-power images of the ONL at Locations 1 ~fovea!, 2 ~edgeof fovea 0 rim!, and 4 ~near optic disc! are shown in Figs. 5B–5P.At Fd 95, the intensity of Fast Red labelling of FGF2 mRNA isapproximately equivalent at the three sample locations ~Figs. 5B–5D!. However, at Fd 115, 130, and 164, labeling of mRNA in thefovea samples ~Location 1! is less intense than on the emergingfoveal rim ~Location 2!, or near the optic disc ~Location 4!~Figs. 5E, 5H, 5K compared with 5F–5G, 5I–5J, 5L–5M, respec-tively!; labeling at Location 3 was similar to Location 4 in all cases~data not shown!. Similar observations were made in retinas at Fd110, 120, and 150. At P 2.5 years, the intensity of FGF2 labellingis uniform across all locations ~Figs. 5N–5P!.
Optical densitometric data for the four sample locations fromsix retinas is shown in Fig. 6. The histograms indicate lower levelsof FGF2 mRNA expression in the ONL at Location 1 ~fovealcenter! at Fd 115, 130, and 164—during formation of the fovealdepression—but not before ~Fd 95! or after ~P 4 mo, 2.5y!~Fig. 6A!. Densitometric data from the GCL, at similar locations inthe same retinas, does not show lower levels of expression atLocation 1 at the same ages, indicating that this is not a general
Fig. 3. Estimates of cone soma–axonal process length along the horizontal meridian at three ages, at the fovea and at three locationstemporal ~t! and nasal ~n! to the fovea. Approximately 500 µm separates sample locations. Cone morphology at Fd 85 is relativelyuniform. By Fd 120 there is significant elongation of cones on the foveal rim ~t1 and n1—Student t-test, t � 9.9 and 22.0, respectively!and throughout the nasal samples, compared with the fovea. At Fd 164 foveal cones have not started to elongate, while elongation ofcones on the foveal rim ~t1 and n1! is well advanced. Foveal cones in adult retina have axonal processes 200–500 µm in length.
FGF gradients in developing fovea 451
feature of this region of the retina, but is specific to the ONL~Fig. 6B!. We used the Kruskal-Wallis test and post-hoc Dunnett-like multiple comparison analysis to test for significance in thesedifferences. The final values ~F � 15.18, 32.06 and 34.14 at Fd115, 130, and 164, respectively! showed significantly lower levelsof FGF2 mRNA expression ~P�0.01, n � 40 for each sample! atLocation 1, compared with data averaged from the other threeLocations, at Fd 115, 130, and 164.
FGF receptors
ImmunolabellingWe have shown previously that all four FGF receptor ~FGFR1–4!
proteins are immunoreactive ~IR! in adult and developing macaqueretina ~Cornish et al., 2004!. Here we report that both FGFR1 andFGFR4 appear to be regulated in the foveal cone mosaic duringformation of the foveal depression. Photomicrographs showing thepatterns of immunoreactivity in the ONL for FGFR1 and R4receptors are shown at an early stage of formation of the fovealdepression ~Fd 115, Locations 1, 2, and 4! in Figs. 7 ~A–F!.Comparable patterns of immunoreactivity were observed at Loca-tions 3 and 4 in all cases. The images suggest a downregulation atthe fovea of FGFR1 protein ~Fig. 7A! and FGFR4 ~Fig. 7D!compared with samples from Locations 2 and 4 ~Figs. 7B,C and7E,F, respectively!, which was confirmed by optical densitometry~data not shown!. We also sought to correlate these patterns ofimmunoreactivity with patterns of mRNA expression at Locations1–4 by in situ hybridization for FGFR1, FGFR2, FGFR3 ~notshown!, and FGFR4 ~Figs. 7G–7I!.
In situ hybridizationThe general expression patterns for FGFR1, R2, R3, and R4
mRNA in monkey retina have been reported previously ~Cornishet al., 2004!. Here we investigate relative levels of mRNA expres-sion in the ONL across the fovea in sections on the horizontalmeridian at different stages of development. Photomicrographsshowing levels of FGFR4 mRNA expression at Locations 1, 2, and4 at Fd 120 are shown in Figs. 7G–7I. The images indicate lowerlevels of FGFR4 mRNA at Location 1 compared with Locations 2and 4. The optical densitometry data are shown in Fig. 8. We foundno evidence for regulation of FGFR2 ~Fig. 8B!, or FGFR3 ~datanot shown! across the foveal region at any stage of development.There was some suggestion of downregulation of FGFR1 in Lo-cation 1 ~fovea! at Fd 130, but there was no consistent trend acrossthe age group ~“during”! for FGFR1 ~Fig. 8A!. We analyzedintensity of FGFR4 mRNA labeling at Fd 115, Fd 120, and at P11yrs only, due to unavailability of sections through the centralfovea at other ages ~Fig. 8C!. The intensity of FGFR4 mRNAexpression at Location 1 was significantly less than at Locations 3and 4 at Fd 115 ~Kruskal-Wallis F � 95.42, P � 0.01, n � 40;Dunnett-like: P � 0.01!. At Fd 120, the intensity of mRNAexpression at Location 1 was significantly less than at all otherlocations ~2–4! ~F � 137.23; P � 0.01; n � 40!. These differenceswere not detected in adult ~P 11yrs! retina ~Fig. 8C!.
Discussion
This study presents two new findings regarding primate retinaldevelopment. First, we describe a pattern of morphological differ-
Fig. 4. FGF2 mRNA expression ~red! in macaque retina. A: Fd 95 showing colocalization with anti-L0M opsin immunoreactivity~green!. B: Fd 164, FGF2 mRNA antisense ~red!, counter immunolabeled with vimentin antibody ~green!, showing intense expressionin the inner segments of cones, and in the GCL. C: Sense probe counterlabeled with vimentin antibody ~green!. Scale bars � 20 µm.FH, fiber of Henle; IS, inner segment; OS, outer segment.
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entiation of cones that progresses from the disc region towards thefoveal region—the reverse of the usual centro-peripheral matura-tion gradient of retinal development. Second, we document agradient of FGF2 mRNA expression in the ONL, along with one ofits high-affinity receptors, FGFR4, in which the developing foveahas lower levels of mRNA expression. These gradients correlatewith the delayed morphological differentiation of foveal cones.
FGF2 expression in the retina
Expression of a number of the FGF ligands has been reported inmammalian retina, including FGF1 ~Caruelle et al., 1989; Bau-douin et al., 1990; Noji et al., 1990!, FGF2 ~Noji et al., 1990;
Hageman et al., 1991; Connolly et al., 1992; Gao & Hollyfield,1995; Mervin et al., 1999!, FGF5 ~Kitaoka et al., 1994!, FGF8~Vogel-Hopker et al. 2000!, FGF9 ~Colvin et al., 1999!, andFGF19 ~Xie et al., 1999!. Of these, FGF1, FGF2, FGF5, andFGF19 have been described in human and0or monkey retina~Caruelle et al., 1989; Baudouin et al., 1990; Kitaoka et al.,1994; Li et al., 1997; Xie et al., 1999!. Further, we haveconfirmed expression of those ligands in human retina by RT-PCR and established expression of FGF3, FGF8, and FGF17 inhuman retina ~E. Chao, R. Natoli and J. Provis, unpublisheddata!.
An earlier report investigating immunoreactivity to FGF2 inhuman retina, suggested that a gradient of FGF2 in the adult
Fig. 5. FGF2 mRNA gradients in the ONL. ~A! A montage of a Fd 115 retina showing the early foveal depression ~double-headedarrow!, the emerging foveal rim ~asterisks!, and the position of ONL samples at Locations 1–3 ~small boxes!. Location 4 is positionedoff the montage to the right, near the optic disc. FGF2mRNA expression ~red! is shown at Locations 1, 2 and 4 at ~B–D! Fd 95—beforeformation of the foveal depression; ~E–G! Fd 115, ~H–J! Fd 130 ~K-M! Fd 164—during formation of the foveal depression; and ~N–P!postnatal 2.5 yrs, after formation of the foveal depression. Note the low levels of FGF2 mRNA at Location 1 at Fd 115 ~E!, Fd 130~H! and Fd 164 ~K! ~the “during” age groups!, compared with Locations 2 and 4 at the same ages. Levels of FGF2mRNA expressionare uniform at all locations at Fd 85 ~“before”! and P 2.5 years ~“after”!. Levels of expression of FGF2 mRNA at Locations 3 and 4were similar at all ages. Scale bar ~B–P! 20 µm.
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human retina correlates with rod density ~Li et al., 1997!. Thosefindings are not confirmed in the present study of macaque retina.Rather, we found high levels of FGF2 mRNA expressed by adultcones. The differences between the present results and those of ~Liet al., 1997! are likely due to the differences in approach ~in situhybridization vs. immunoreactivity! and condition of the tissuesinvestigated. In this study, we used retinas fixed in methyl Car-noy’s immediately post mortem, while in the previous study humandonor retinas were obtained hours after death and stored for sometime prior to immunohistochemistry. In this study, we foundimmunoreactivity for FGF2 to be unreliable in retinal sections,although we obtained strong and reliable staining from antibodiesto the FGFRs. We relied on in situ hybridization to investigateexpression of the FGF2 and conclude that in situ hybridization isa more reliable method than immunohistochemistry for this pur-pose. While in situ hybridization does not permit localization ofthe FGF2 ligand ~protein!, which may be remote from the source
of its mRNA, it is not confounded by the possibility that FGFprotein may be differentially metabolized or degraded artefactuallyduring processing.
Gradients of cone maturation and FGF expression
In general, mammalian retinal development progresses in centro-peripheral sequence. In primates, foveal cones are amongst thefirst cells to differentiate, and expression of opsins is also detectedcentro-peripherally. Despite this, it has been recognized for sometime that morphogenesis of foveal cones is delayed during the thirdtrimester of human development when cones found in the parafo-vea are more mature, that is, narrower and more elongated thanfoveal cones ~Bach & Seefelder 1911, 1912, 1914; Mann, 1964;Abramov et al., 1982; Hendrickson & Yuodelis, 1984; Hendrick-son & Drucker, 1992!. We also know that the human fovealdepression is not fully formed at birth ~Mann, 1964; Abramov
Fig. 6. Quantitative analysis of FGF2 mRNA expression in specimens from the three age groups—before ~formation of the fovealdepression!, Fd 95; during, Fds 115, 130, and 164; and after, P 4 mo. and 2.5 yrs. ~A! Optical density data at four locations in the ONL,normalized to expression levels at Location 4. Significantly lower levels of FGF2 mRNA were detected in the raw ONL data at Location1 ~*Kruskal-Wallace test!, compared with other locations during formation of the foveal depression ~Fds 115, 130, and 164!. Thiscorresponds to the period when foveal cone morphology is immature, or appears stunted. Levels of mRNA are comparable across alllocations at Fd 95 ~“before”) and at postnatal ages ~“after”!. ~B! Optical density data at the same four locations, but in the GCL ~controlsamples!, normalized to expression levels at Location 3. Levels of mRNA were comparable at all locations sampled in the GCL,including sections where gradients were present in the ONL of the same section. “§” sample not measured from Location 4 for technicalreasons; “†” there is no GCL at Location 1 at Fd 164.
454 E.E. Cornish et al.
et al. 1982; Hendrickson & Yuodelis, 1984; Hendrickson, 1992,1994; Provis et al., 1998! and is similar to the monkey fovea at Fd131–155, based on the morphology of the inner retina and theproportion of peak adult cone density ~Hendrickson, 1992!. The
slow maturation of central retina is reflected in the poor visualacuity of newborn human and macaque infants ~Dobson & Teller,1978; Williams & Booth, 1981!. In newborn humans, contrastsensitivity is higher in the parafovea and near the periphery than in
Fig. 7. Photomicrographs showing observed gradients of FGFR1 and R4 expression at Fd 120. Double arrowheads in each panelindicate the length of the cone soma � axonal process. A–C: Immunoreactivity to FGFR1 was lower in the region of the developinginner segments and at the level of the synapses at Location 1 ~A! compared with Locations 2 ~B!, 3 ~not shown! and 4 ~C!. D–F:Immunoreactivity to FGFR4 was somewhat lower at Location 1 ~D! compared with the Locations 2 ~E!, 3 ~not shown! and 4 ~F!. G–I:In situ hybridization for FGFR4 showed lower levels of FGFR4 mRNA at Location 1 ~G! compared with the Locations 2 ~H!, 3 ~notshown! and 4 ~I!. Scale bars � 20 µm.
FGF gradients in developing fovea 455
the fovea ~Candy et al., 1998!, consistent with the more advanceddifferentiation of parafoveal cones.
The present results confirm other studies, showing that inmacaques the foveal cones have not reached adult proportions atbirth and that cones surrounding the fovea are more advancedmorphologically than foveal cones at least until birth ~Springer& Hendrickson, in press!. The data also suggest a “reverse”wave of cone morphogenesis, such that cones remote from thecenter become narrower, elaborate axonal processes, and de-velop inner segments ahead of those positioned more centrally.Based on the present data, a “wave” of cone differentiation isdetectable at the optic disc at Fd 90, passes through the fovealrim at approximately Fd 130, and reaches the foveal conessometime after birth.
Our findings also show downregulation of FGF2 expression infoveal cones over a period that correlates with the delay in their
maturation. The youngest animal in which we detected lowerlevels of FGF2 expression in foveal cones was at Fd 110; theoldest at Fd 165. This time-frame corresponds to the period duringwhich foveal cones exhibit “delayed maturation”, compared withcones positioned closer to the optic disc ~present study!. It alsocorrelates closely with the period during which the foveal de-pression forms and reaches its greatest depth, between Fd 105 andbirth at Fd 172 ~Hendrickson, 1992; Kirby & Steineke, 1996;Provis et al., 2000; Springer & Hendrickson, 2004b!. We alsodetected a significant downregulation of FGFR4—a high-affinityreceptor for FGF2 ~Ornitz et al., 1996!—during this interval.Although fewer animals were available for investigation using theFGFR4 probe, we detected lower levels of FGFR4 mRNA infoveal cones at Fd 115 and Fd 120, but not in adult retina. Thesedata implicate FGF2 and FGFR4 in a critical period of conemorphological development.
Fig. 8. Optical densitometry data showing levels of FGF-R1,FGF-R2, and, FGF-R4 mRNAs in the ONL at Locations 1–4 onthe horizontal meridian, before ~Fd 95!, during ~Fds 115, 130,and 164!, and after ~P 2.5 yrs! formation of the foveal depression.~A! A significantly lower level of FGFR1 mRNA was detected inthe ONL at Fd 130 but this trend was not observed throughout theage group ~“during”!, and therefore not tested for significance.~B! There were no consistent trends in the levels of FGFR2mRNA measured, in any of the age groups. Similar data wereobtained for FGFR3 ~not shown!. ~C! Two specimens in the‘during’ age group ~Fd 115 and Fd 120! showed significantlylower levels of FGFR4 mRNA in the foveal cones ~Location 1!compared with Locations 3 and 4; in one of these there was alsoa significant difference between Locations 1 and 2. These agesrepresent the period in which foveal cone morphology is imma-ture, and appears stunted, compared with other locations. Therewas no comparable pattern of FGFR4 mRNA levels in the adult~P 11yrs!. Data from Fd 95 and Fd 164 is not shown, as we hadno sections through the central fovea for analysis.
456 E.E. Cornish et al.
Significance
A role for FGF signalling in cone morphogenesis?FGF2 is a high-affinity ligand of FGFR1 ~IIIc!, FGFR3 ~IIIc!,
and FGFR4 ~Ornitz et al., 1996! having diverse roles, includingneurotrophic effects, and roles in mitogenesis, morphogenesis, andcell-fate determination. The effects of FGF2 are mediated bydistinct signalling pathways ~Kay et al., 1998!, and in the lenspromote the differentiation, elongation, and migration of epithelialcells into lens fiber cells ~McAvoy et al., 1991!. These regulatoryeffects take place in the presence of gradients of FGF2 expres-sion across the antero-posterior axis of the lens, in combinationwith spatio-temporal patterns of FGFR expression ~Lovicu &McAvoy, 1993; de Iongh et al., 1996, 1997!. The morphologicaldifferentiation of cuboidal lens epithelial cells into densely packed,elongated lens fiber cells has some similarities with the morpho-logical changes that take place in foveal cones during fetal andearly postnatal life, raising the possibility that similar mechanismsmay be activated during the morphological differentiation of bothcell types.
We recently described discrete distributions of FGFR on conesin developing and adult primate retina ~Cornish et al., 2004!. Inthat study we found FGFR4 to be an early marker of cones, but notrods, expressed ahead of any of the other known markers of cones,including cone-specific alpha-transducin. FGFR4 is distributedthroughout the soma and along the axonal process, or fiber ofHenle, during morphological differentiation and in adult cones. Incontrast, FGFR1 is absent from the axonal process and localizedonly to the synaptic region of the cone pedicle, while FGFR3 islocalized to the soma and in adult retina, to the proximal part of thefiber of Henle. Each of these receptors was localized to the innersegment ~Cornish et al., 2004!. The distinct distribution of FGFRin cones implies a role for FGF signalling in the morphogenesis ofthese cells. Based on their distributions, we hypothesize thatsignalling via FGFR4 mediates narrowing of the cone soma andelongation of the axonal process, along with the possible involve-ment of FGFR3. FGFR1, on the other hand, is more likely to havea role in formation and maintenance of the cone synapses.
Cone narrowing and elongation is associated with early in-creases in cone density ~Diaz-Araya & Provis, 1992! and withcone packing in the fovea, after birth ~Packer et al., 1990!. Theincrease in FGF2 and FGFR4 mRNA expression in foveal conesafter birth, described in the present study, is consistent with thisprocess. Similarly, the observed downregulation of FGF2 andFGFR4 mRNAs in foveal cones, during the period that theirmorphological differentiation is suspended, is consistent with theconcept that morphological differentiation of cones is mediated byFGF signaling.
The gradient of cone differentiationThe significance of the gradient of cone maturation is not
understood. We note a striking coincidence in the timing of thedelay in foveal cone morphogenesis and formation of the fovealdepression, which begins around Fd 105–110 in macaques ~Hen-drickson, 1994; Kirby & Steineke, 1996; Provis et al., 2000!.Initially, the fovea is a simple depression in the GCL, but over timecells of the inner retinal layers are displaced radially ~centrifu-gally! so that by birth the somata of cones comprise the innermostlayer of cells at the fovea ~Hendrickson & Yuodelis, 1984; Proviset al., 1998; Springer & Hendrickson, 2004b!. It is during thisperiod that the foveal cones retain their immature morphology,while cones on the foveal rim, and in the parafovea continue to
mature ~present study!. The relationship between prolongation ofthe immature morphology of the foveal cones and formation of thedepression itself, however, is not understood. Indeed, it is not clearthat the two phenomena are directly related. They may, however,both be linked to formation of the perifoveal capillary plexus,which appears to be a significant factor affecting formation of thefoveal depression ~Provis et al., 1998, 2000; Springer & Hendrick-son, 2004a,b!.
The perifoveal capillary plexus comprises three layers of ves-sels. Innermost is the “GCL plexus” ~or GCP! at the interfacebetween the GCL and IPL; a middle layer is present in thesuperficial INL ~SINL!; and a third stratum is deep in the INL~DINL! ~Gariano et al., 1994; Provis et al., 2000; Provis, 2001!.All of these are delayed in their development, compared with otherretinal vessels ~Engerman, 1976; Provis et al., 2000; Provis, 2001!.The GCP forms near the optic disc at around Fd 80–85 and growstowards the foveal region to form a ring of vessels around thefuture fovea by Fd 105, creating a foveal avascular zone. Thefoveal depression then forms within this clearly demarked region,with the perifoveal “ring” marking the rim of the early depression~Provis et al., 2000; Provis, 2001!. The deep perifoveal plexusbegins to form in the vicinity of the optic disc at around Fd110–115; the DINL stratum reaches the developing foveal depres-sion ahead of the SINL, at around birth, so that anastomosesaround the foveal depression form in the early weeks postnatal~P1–3 wks! but never invade the foveal avascular zone ~Proviset al., 2000; Provis, 2001!. The appearance of the outer plexusaround the fovea is coincident with both a period of quite rapidelongation of the foveal cones and acceleration of cone packing~Provis et al., 2000; Provis, 2001; Springer & Hendrickson,2004a,b!. One possibility, therefore, is that additional nutrientsdelivered to the photoreceptors by the developing DINL influencestheir development, by driving inherent developmental mechanismsat a higher rate. Conversely, it is possible that the delayed matu-ration of the foveal cones results from the significant delay invascularization of the central region of the retina, compared toregions closer to the disc and farther out in temporal retina.
Relationship to other proposed mechanisms
Finite-element analysis suggests that mechanical factors, includingretinal stretch and intraocular pressure, have a major role indevelopment of the foveal region ~Springer & Hendrickson, 2004a;Springer & Hendrickson, 2004b; Springer & Hendrickson, 2005!.The present findings complement the suggested influences of thesemechanical factors on photoreceptor crowding and formation ofthe foveal depression. Our studies strongly suggest that molecularmechanisms involving the FGF family of ligands and receptors arefundamental to morphological differentiation of primate cones,being expressed early in development ~present study!, well beforechanges in the foveal region—that may be induced by retinalstretch—have commenced. In this early phase of development~before Fd 105!, FGF signalling may mediate the morphologicaladaptations of cones ~that is, narrowing and elongation! that areassociated with increased density in the foveal cone mosaic in thefirst0early phase of photoreceptor accumulations ~Diaz-Araya &Provis, 1992; see also, Springer & Hendrickson, 2005!. Further,the molecular mechanisms proposed here might, in later develop-ment, be considered as processes through which retinal cells~particularly cones! might adapt to mechanical forces acting onthem. We suggest that in later development ~after Fd 105! molec-ular mechanisms, including the ones alluded to in this study, are
FGF gradients in developing fovea 457
likely to be regulated by a wide range of factors, including eyegrowth and retinal stretch, along with the factors that regulateperifoveal vascular development.
Further studies
This is the first study to identify molecular factors that appear tohave a role in regulating differentiation of foveal cones, and whichmay be involved in formation of the foveal depression. Furtherstudies investigating patterns of FGF expression in other species,FGF signalling via FGFR4, and in vitro or in vivo studies inves-tigating the effects of blocking or silencing FGFR are required totest the correlations described here.
Acknowledgments
We thank Dr. Dondin Sajuthi and staff at the Bogor Primate Center,Indonesia, for their expertise in obtaining timed macaque fotuses andDayat Djajadi for his expert skills in preparing retinal sections.
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